BRCA1	NN	O	O
is	NN	O	O
secreted	NN	O	O
and	NN	O	O
exhibits	NN	O	O
properties	NN	O	O
of	NN	O	O
a	NN	O	O
granin	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
are	NN	O	O
responsible	NN	O	O
for	NN	O	O
most	NN	O	O
cases	NN	O	O
of	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

However	NN	O	O
,	NN	O	O
the	NN	O	O
function	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
protein	NN	O	O
has	NN	O	O
remained	NN	O	O
elusive	NN	O	O
.	NN	O	O

We	NN	O	O
now	NN	O	O
show	NN	O	O
that	NN	O	O
BRCA1	NN	O	O
encodes	NN	O	O
a	NN	O	O
190	NN	O	O
-	NN	O	O
kD	NN	O	O
protein	NN	O	O
with	NN	O	O
sequence	NN	O	O
homology	NN	O	O
and	NN	O	O
biochemical	NN	O	O
analogy	NN	O	O
to	NN	O	O
the	NN	O	O
granin	NN	O	O
protein	NN	O	O
family	NN	O	O
.	NN	O	O

Interestingly	NN	O	O
,	NN	O	O
BRCA2	NN	O	O
also	NN	O	O
includes	NN	O	O
a	NN	O	O
motif	NN	O	O
similar	NN	O	O
to	NN	O	O
the	NN	O	O
granin	NN	O	O
consensus	NN	O	O
at	NN	O	O
the	NN	O	O
C	NN	O	O
terminus	NN	O	O
of	NN	O	O
the	NN	O	O
protein	NN	O	O
.	NN	O	O

Both	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
the	NN	O	O
granins	NN	O	O
localize	NN	O	O
to	NN	O	O
secretory	NN	O	O
vesicles	NN	O	O
,	NN	O	O
are	NN	O	O
secreted	NN	O	O
by	NN	O	O
a	NN	O	O
regulated	NN	O	O
pathway	NN	O	O
,	NN	O	O
are	NN	O	O
post	NN	O	O
-	NN	O	O
translationally	NN	O	O
glycosylated	NN	O	O
and	NN	O	O
are	NN	O	O
responsive	NN	O	O
to	NN	O	O
hormones	NN	O	O
.	NN	O	O

As	NN	O	O
a	NN	O	O
regulated	NN	O	O
secretory	NN	O	O
protein	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
appears	NN	O	O
to	NN	O	O
function	NN	O	O
by	NN	O	O
a	NN	O	O
mechanism	NN	O	O
not	NN	O	O
previously	NN	O	O
described	NN	O	O
for	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
products	NN	O	O
.	NN	O	O
.	NN	O	O

Ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
risk	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
carriers	NN	O	O
is	NN	O	O
modified	NN	O	O
by	NN	O	O
the	NN	O	O
HRAS1	NN	O	O
variable	NN	O	O
number	NN	O	O
of	NN	O	O
tandem	NN	O	O
repeat	NN	O	O
(	NN	O	O
VNTR	NN	O	O
)	NN	O	O
locus	NN	O	O
.	NN	O	O

Women	NN	O	O
who	NN	O	O
carry	NN	O	O
a	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
(	NN	O	O
on	NN	O	O
chromosome	NN	O	O
17q21	NN	O	O
)	NN	O	O
,	NN	O	O
have	NN	O	O
an	NN	O	O
80	NN	O	O
%	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
a	NN	O	O
40	NN	O	O
%	NN	O	O
risk	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
by	NN	O	O
the	NN	O	O
age	NN	O	O
of	NN	O	O
70	NN	O	O
(	NN	O	O
ref	NN	O	O
.	NN	O	O
1	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
variable	NN	O	O
penetrance	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
suggests	NN	O	O
that	NN	O	O
other	NN	O	O
genetic	NN	O	O
and	NN	O	O
non	NN	O	O
-	NN	O	O
genetic	NN	O	O
factors	NN	O	O
play	NN	O	O
a	NN	O	O
role	NN	O	O
in	NN	O	O
tumourigenesis	NN	O	O
in	NN	O	O
these	NN	O	O
individuals	NN	O	O
.	NN	O	O

The	NN	O	O
HRAS1	NN	O	O
variable	NN	O	O
number	NN	O	O
of	NN	O	O
tandem	NN	O	O
repeats	NN	O	O
(	NN	O	O
VNTR	NN	O	O
)	NN	O	O
polymorphism	NN	O	O
,	NN	O	O
located	NN	O	O
1	NN	O	O
kilobase	NN	O	O
(	NN	O	O
kb	NN	O	O
)	NN	O	O
downstream	NN	O	O
of	NN	O	O
the	NN	O	O
HRAS1	NN	O	O
proto	NN	O	O
-	NN	O	O
oncogene	NN	O	O
(	NN	O	O
chromosome	NN	O	O
11p15	NN	O	O
.	NN	O	O
5	NN	O	O
)	NN	O	O
is	NN	O	O
one	NN	O	O
possible	NN	O	O
genetic	NN	O	O
modifier	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
penetrance	NN	O	O
.	NN	O	O

Individuals	NN	O	O
who	NN	O	O
have	NN	O	O
rare	NN	O	O
alleles	NN	O	O
of	NN	O	O
the	NN	O	O
VNTR	NN	O	O
have	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
of	NN	O	O
certain	NN	O	O
types	NN	O	O
of	NN	O	O
cancers	NN	O	B-Disease
,	NN	O	O
including	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
(	NN	O	O
2	NN	O	O
-	NN	O	O
4	NN	O	O
)	NN	O	O
.	NN	O	O

To	NN	O	O
investigate	NN	O	O
whether	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
rare	NN	O	O
HRAS1	NN	O	O
alleles	NN	O	O
increases	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
have	NN	O	O
typed	NN	O	O
a	NN	O	O
panel	NN	O	O
of	NN	O	O
307	NN	O	O
female	NN	O	O
BRCA1	NN	O	O
carriers	NN	O	O
at	NN	O	O
this	NN	O	O
locus	NN	O	O
using	NN	O	O
a	NN	O	O
PCR	NN	O	O
-	NN	O	O
based	NN	O	O
technique	NN	O	O
.	NN	O	O

The	NN	O	O
risk	NN	O	O
for	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
was	NN	O	O
2	NN	O	O
.	NN	O	O

11	NN	O	O
times	NN	O	O
greater	NN	O	O
for	NN	O	O
BRCA1	NN	O	O
carriers	NN	O	O
harbouring	NN	O	O
one	NN	O	O
or	NN	O	O
two	NN	O	O
rare	NN	O	O
HRAS1	NN	O	O
alleles	NN	O	O
,	NN	O	O
compared	NN	O	O
to	NN	O	O
carriers	NN	O	O
with	NN	O	O
only	NN	O	O
common	NN	O	O
alleles	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
015	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
magnitude	NN	O	O
of	NN	O	O
the	NN	O	O
relative	NN	O	O
risk	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
rare	NN	O	O
HRAS1	NN	O	O
allele	NN	O	O
was	NN	O	O
not	NN	O	O
altered	NN	O	O
by	NN	O	O
adjusting	NN	O	O
for	NN	O	O
the	NN	O	O
other	NN	O	O
known	NN	O	O
risk	NN	O	O
factors	NN	O	O
for	NN	O	O
hereditary	NN	O	B-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
(	NN	O	O
5	NN	O	O
)	NN	O	O
.	NN	O	O

Susceptibility	NN	O	O
to	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
did	NN	O	O
not	NN	O	O
appear	NN	O	O
to	NN	O	O
be	NN	O	O
affected	NN	O	O
by	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
rare	NN	O	O
HRAS1	NN	O	O
alleles	NN	O	O
.	NN	O	O

This	NN	O	O
study	NN	O	O
is	NN	O	O
the	NN	O	O
first	NN	O	O
to	NN	O	O
show	NN	O	O
the	NN	O	O
effect	NN	O	O
of	NN	O	O
a	NN	O	O
modifying	NN	O	O
gene	NN	O	O
on	NN	O	O
the	NN	O	O
penetrance	NN	O	O
of	NN	O	O
an	NN	O	O
inherited	NN	O	B-Disease
cancer	NN	O	I-Disease
syndrome	NN	O	I-Disease

A	NN	O	O
novel	NN	O	O
homeodomain	NN	O	O
-	NN	O	O
encoding	NN	O	O
gene	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
large	NN	O	O
CpG	NN	O	O
island	NN	O	O
interrupted	NN	O	O
by	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
unstable	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
-	NN	O	O
untranslated	NN	O	O
region	NN	O	O
of	NN	O	O
a	NN	O	O
protein	NN	O	O
kinase	NN	O	O
-	NN	O	O
encoding	NN	O	O
gene	NN	O	O
,	NN	O	O
DMPK	NN	O	O
,	NN	O	O
which	NN	O	O
maps	NN	O	O
to	NN	O	O
chromosome	NN	O	O
19q13	NN	O	O
.	NN	O	O

3	NN	O	O
.	NN	O	O

Characterisation	NN	O	O
of	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
this	NN	O	O
gene	NN	O	O
in	NN	O	O
patient	NN	O	O
tissues	NN	O	O
has	NN	O	O
thus	NN	O	O
far	NN	O	O
generated	NN	O	O
conflicting	NN	O	O
data	NN	O	O
on	NN	O	O
alterations	NN	O	O
in	NN	O	O
the	NN	O	O
steady	NN	O	O
state	NN	O	O
levels	NN	O	O
of	NN	O	O
DMPK	NN	O	O
mRNA	NN	O	O
,	NN	O	O
and	NN	O	O
on	NN	O	O
the	NN	O	O
final	NN	O	O
DMPK	NN	O	O
protein	NN	O	O
levels	NN	O	O
in	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
the	NN	O	O
expansion	NN	O	O
.	NN	O	O

The	NN	O	O
DM	NN	O	B-Disease
region	NN	O	O
of	NN	O	O
chromosome	NN	O	O
19	NN	O	O
is	NN	O	O
gene	NN	O	O
rich	NN	O	O
,	NN	O	O
and	NN	O	O
it	NN	O	O
is	NN	O	O
possible	NN	O	O
that	NN	O	O
the	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
may	NN	O	O
lead	NN	O	O
to	NN	O	O
dysfunction	NN	O	O
of	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
transcription	NN	O	O
units	NN	O	O
in	NN	O	O
the	NN	O	O
vicinity	NN	O	O
,	NN	O	O
perhaps	NN	O	O
as	NN	O	O
a	NN	O	O
consequence	NN	O	O
of	NN	O	O
chromatin	NN	O	O
disruption	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
searched	NN	O	O
for	NN	O	O
genes	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
CpG	NN	O	O
island	NN	O	O
at	NN	O	O
the	NN	O	O
3	NN	O	O
end	NN	O	O
of	NN	O	O
DMPK	NN	O	O
.	NN	O	O

Sequencing	NN	O	O
of	NN	O	O
this	NN	O	O
region	NN	O	O
shows	NN	O	O
that	NN	O	O
the	NN	O	O
island	NN	O	O
extends	NN	O	O
over	NN	O	O
3	NN	O	O
.	NN	O	O

5	NN	O	O
kb	NN	O	O
and	NN	O	O
is	NN	O	O
interrupted	NN	O	O
by	NN	O	O
the	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
.	NN	O	O

Comparison	NN	O	O
of	NN	O	O
genomic	NN	O	O
sequences	NN	O	O
downstream	NN	O	O
(	NN	O	O
centromeric	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
repeat	NN	O	O
in	NN	O	O
human	NN	O	O
and	NN	O	O
mouse	NN	O	O
identified	NN	O	O
regions	NN	O	O
of	NN	O	O
significant	NN	O	O
homology	NN	O	O
.	NN	O	O

These	NN	O	O
correspond	NN	O	O
to	NN	O	O
exons	NN	O	O
of	NN	O	O
a	NN	O	O
gene	NN	O	O
predicted	NN	O	O
to	NN	O	O
encode	NN	O	O
a	NN	O	O
homeodomain	NN	O	O
protein	NN	O	O
.	NN	O	O

RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
analysis	NN	O	O
shows	NN	O	O
that	NN	O	O
this	NN	O	O
gene	NN	O	O
,	NN	O	O
which	NN	O	O
we	NN	O	O
have	NN	O	O
called	NN	O	O
DM	NN	O	B-Disease
locus	NN	O	O
-	NN	O	O
associated	NN	O	O
homeodomain	NN	O	O
protein	NN	O	O
(	NN	O	O
DMAHP	NN	O	O
)	NN	O	O
,	NN	O	O
is	NN	O	O
expressed	NN	O	O
in	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
human	NN	O	O
tissues	NN	O	O
,	NN	O	O
including	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
,	NN	O	O
heart	NN	O	O
and	NN	O	O
brain	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
hereditary	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
analyzed	NN	O	O
the	NN	O	O
27	NN	O	O
exons	NN	O	O
and	NN	O	O
the	NN	O	O
promoter	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
in	NN	O	O
familial	NN	O	B-Disease
or	NN	O	I-Disease
sporadic	NN	O	I-Disease
bilateral	NN	O	I-Disease
retinoblastoma	NN	O	I-Disease
by	NN	O	O
using	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
analysis	NN	O	O
.	NN	O	O

For	NN	O	O
improvement	NN	O	O
over	NN	O	O
previous	NN	O	O
studies	NN	O	O
,	NN	O	O
a	NN	O	O
new	NN	O	O
set	NN	O	O
of	NN	O	O
primers	NN	O	O
has	NN	O	O
been	NN	O	O
designed	NN	O	O
,	NN	O	O
which	NN	O	O
allow	NN	O	O
for	NN	O	O
amplification	NN	O	O
of	NN	O	O
the	NN	O	O
coding	NN	O	O
and	NN	O	O
splicing	NN	O	O
sequences	NN	O	O
only	NN	O	O
.	NN	O	O

The	NN	O	O
positioning	NN	O	O
of	NN	O	O
the	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
(	NN	O	O
PCR	NN	O	O
)	NN	O	O
primers	NN	O	O
was	NN	O	O
such	NN	O	O
that	NN	O	O
the	NN	O	O
resulting	NN	O	O
PCR	NN	O	O
products	NN	O	O
were	NN	O	O
of	NN	O	O
different	NN	O	O
sizes	NN	O	O
,	NN	O	O
which	NN	O	O
enabled	NN	O	O
us	NN	O	O
to	NN	O	O
analyze	NN	O	O
two	NN	O	O
different	NN	O	O
exons	NN	O	O
simultaneously	NN	O	O
and	NN	O	O
still	NN	O	O
distinguish	NN	O	O
between	NN	O	O
the	NN	O	O
banding	NN	O	O
profiles	NN	O	O
for	NN	O	O
both	NN	O	O
(	NN	O	O
biplex	NN	O	O
analysis	NN	O	O
)	NN	O	O
.	NN	O	O

By	NN	O	O
using	NN	O	O
this	NN	O	O
approach	NN	O	O
,	NN	O	O
we	NN	O	O
were	NN	O	O
able	NN	O	O
to	NN	O	O
identify	NN	O	O
mutation	NN	O	O
in	NN	O	O
22	NN	O	O
new	NN	O	O
patients	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
overall	NN	O	O
efficiency	NN	O	O
of	NN	O	O
the	NN	O	O
procedure	NN	O	O
when	NN	O	O
we	NN	O	O
used	NN	O	O
a	NN	O	O
single	NN	O	O
-	NN	O	O
pass	NN	O	O
regimen	NN	O	O
was	NN	O	O
only	NN	O	O
48	NN	O	O
%	NN	O	O
.	NN	O	O

The	NN	O	O
mutations	NN	O	O
were	NN	O	O
small	NN	O	O
insertions	NN	O	O
and	NN	O	O
deletions	NN	O	O
and	NN	O	O
point	NN	O	O
mutations	NN	O	O
in	NN	O	O
roughly	NN	O	O
equal	NN	O	O
proportions	NN	O	O
.	NN	O	O
.	NN	O	O

Type	NN	O	B-Disease
II	NN	O	I-Disease
human	NN	O	I-Disease
complement	NN	O	I-Disease
C2	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Allele	NN	O	O
-	NN	O	O
specific	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitutions	NN	O	O
(	NN	O	O
Ser189	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Phe	NN	O	O
;	NN	O	O
Gly444	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Arg	NN	O	O
)	NN	O	O
cause	NN	O	O
impaired	NN	O	O
C2	NN	O	O
secretion	NN	O	O
.	NN	O	O

Type	NN	O	B-Disease
II	NN	O	I-Disease
complement	NN	O	I-Disease
protein	NN	O	I-Disease
C2	NN	O	I-Disease
deficiency	NN	O	I-Disease
is	NN	O	O
characterized	NN	O	O
by	NN	O	O
a	NN	O	O
selective	NN	O	O
block	NN	O	O
in	NN	O	O
C2	NN	O	O
secretion	NN	O	O
.	NN	O	O

The	NN	O	O
Type	NN	O	O
II	NN	O	O
C2	NN	O	O
null	NN	O	O
allele	NN	O	O
(	NN	O	O
C2Q0	NN	O	O
)	NN	O	O
is	NN	O	O
linked	NN	O	O
to	NN	O	O
two	NN	O	O
major	NN	O	O
histocompatibility	NN	O	O
haplotypes	NN	O	O
(	NN	O	O
MHC	NN	O	O
)	NN	O	O
that	NN	O	O
differ	NN	O	O
from	NN	O	O
the	NN	O	O
MHC	NN	O	O
of	NN	O	O
the	NN	O	O
more	NN	O	O
common	NN	O	O
Type	NN	O	B-Disease
I	NN	O	I-Disease
C2	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
determine	NN	O	O
the	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
Type	NN	O	B-Disease
II	NN	O	I-Disease
deficiency	NN	O	I-Disease
the	NN	O	O
two	NN	O	O
Type	NN	O	O
II	NN	O	O
C2Q0	NN	O	O
genes	NN	O	O
were	NN	O	O
isolated	NN	O	O
and	NN	O	O
transfected	NN	O	O
separately	NN	O	O
into	NN	O	O
L	NN	O	O
-	NN	O	O
cells	NN	O	O
.	NN	O	O

Subsequent	NN	O	O
molecular	NN	O	O
biology	NN	O	O
,	NN	O	O
biosynthetic	NN	O	O
,	NN	O	O
and	NN	O	O
immunofluorescence	NN	O	O
studies	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
C2	NN	O	O
secretion	NN	O	O
is	NN	O	O
impaired	NN	O	O
in	NN	O	O
Type	NN	O	B-Disease
II	NN	O	I-Disease
C2	NN	O	I-Disease
deficiency	NN	O	I-Disease
because	NN	O	O
of	NN	O	O
different	NN	O	O
missense	NN	O	O
mutations	NN	O	O
at	NN	O	O
highly	NN	O	O
conserved	NN	O	O
residues	NN	O	O
in	NN	O	O
each	NN	O	O
of	NN	O	O
the	NN	O	O
C2Q0	NN	O	O
alleles	NN	O	O
.	NN	O	O

One	NN	O	O
is	NN	O	O
in	NN	O	O
exon	NN	O	O
5	NN	O	O
(	NN	O	O
nucleotide	NN	O	O
C566	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
;	NN	O	O
Ser189	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Phe	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
C2Q0	NN	O	O
gene	NN	O	O
linked	NN	O	O
to	NN	O	O
the	NN	O	O
MHC	NN	O	O
haplotype	NN	O	O
A11	NN	O	O
,	NN	O	O
B35	NN	O	O
,	NN	O	O
DRw1	NN	O	O
,	NN	O	O
BFS	NN	O	O
,	NN	O	O
C4A0B1	NN	O	O
.	NN	O	O

The	NN	O	O
other	NN	O	O
is	NN	O	O
in	NN	O	O
exon	NN	O	O
11	NN	O	O
(	NN	O	O
G1930	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
;	NN	O	O
Gly444	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Arg	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
C2Q0	NN	O	O
gene	NN	O	O
linked	NN	O	O
to	NN	O	O
the	NN	O	O
MHC	NN	O	O
haplotype	NN	O	O
A2	NN	O	O
,	NN	O	O
B5	NN	O	O
,	NN	O	O
DRw4	NN	O	O
,	NN	O	O
BFS	NN	O	O
,	NN	O	O
C4A3B1	NN	O	O
.	NN	O	O

Each	NN	O	O
mutant	NN	O	O
C2	NN	O	O
gene	NN	O	O
product	NN	O	O
is	NN	O	O
retained	NN	O	O
early	NN	O	O
in	NN	O	O
the	NN	O	O
secretory	NN	O	O
pathway	NN	O	O
.	NN	O	O

These	NN	O	O
mutants	NN	O	O
provide	NN	O	O
models	NN	O	O
for	NN	O	O
elucidating	NN	O	O
the	NN	O	O
C2	NN	O	O
secretory	NN	O	O
pathway	NN	O	O
.	NN	O	O
.	NN	O	O

Defective	NN	O	O
dimerization	NN	O	O
of	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
factor	NN	O	O
subunits	NN	O	O
due	NN	O	O
to	NN	O	O
a	NN	O	O
Cys	NN	O	O
-	NN	O	O
>	NN	O	O
Arg	NN	O	O
mutation	NN	O	O
in	NN	O	O
type	NN	O	B-Disease
IID	NN	O	I-Disease
von	NN	O	I-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
same	NN	O	O
heterozygous	NN	O	O
T	NN	O	O
-	NN	O	O
>	NN	O	O
C	NN	O	O
transition	NN	O	O
at	NN	O	O
nt	NN	O	O
8567	NN	O	O
of	NN	O	O
the	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
factor	NN	O	O
(	NN	O	O
vWF	NN	O	O
)	NN	O	O
transcript	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
two	NN	O	O
unrelated	NN	O	O
patients	NN	O	O
with	NN	O	O
type	NN	O	B-Disease
IID	NN	O	I-Disease
von	NN	O	I-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
,	NN	O	O
with	NN	O	O
no	NN	O	O
other	NN	O	O
apparent	NN	O	O
abnormality	NN	O	O
.	NN	O	O

In	NN	O	O
one	NN	O	O
family	NN	O	O
,	NN	O	O
both	NN	O	O
alleles	NN	O	O
were	NN	O	O
normal	NN	O	O
in	NN	O	O
the	NN	O	O
parents	NN	O	O
and	NN	O	O
one	NN	O	O
sister	NN	O	O
;	NN	O	O
thus	NN	O	O
,	NN	O	O
the	NN	O	O
mutation	NN	O	O
originated	NN	O	O
de	NN	O	O
novo	NN	O	O
in	NN	O	O
the	NN	O	O
proposita	NN	O	O
.	NN	O	O

The	NN	O	O
second	NN	O	O
patient	NN	O	O
also	NN	O	O
had	NN	O	O
asymptomatic	NN	O	O
parents	NN	O	O
who	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
were	NN	O	O
not	NN	O	O
available	NN	O	O
for	NN	O	O
study	NN	O	O
.	NN	O	O

The	NN	O	O
structural	NN	O	O
consequences	NN	O	O
of	NN	O	O
the	NN	O	O
identified	NN	O	O
mutation	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
the	NN	O	O
CyS2010	NN	O	O
-	NN	O	O
>	NN	O	O
Arg	NN	O	O
substitution	NN	O	O
,	NN	O	O
were	NN	O	O
evaluated	NN	O	O
by	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
vWF	NN	O	O
carboxyl	NN	O	O
-	NN	O	O
terminal	NN	O	O
domain	NN	O	O
containing	NN	O	O
residues	NN	O	O
1366	NN	O	O
-	NN	O	O
2050	NN	O	O
.	NN	O	O

Insect	NN	O	O
cells	NN	O	O
infected	NN	O	O
with	NN	O	O
recombinant	NN	O	O
baculovirus	NN	O	O
expressing	NN	O	O
normal	NN	O	O
vWF	NN	O	O
sequence	NN	O	O
secreted	NN	O	O
a	NN	O	O
disulfide	NN	O	O
linked	NN	O	O
dimeric	NN	O	O
molecule	NN	O	O
with	NN	O	O
an	NN	O	O
apparent	NN	O	O
molecular	NN	O	O
mass	NN	O	O
of	NN	O	O
150	NN	O	O
kDa	NN	O	O
before	NN	O	O
reduction	NN	O	O
,	NN	O	O
yielding	NN	O	O
a	NN	O	O
single	NN	O	O
band	NN	O	O
of	NN	O	O
80	NN	O	O
kDa	NN	O	O
after	NN	O	O
disulfide	NN	O	O
bond	NN	O	O
reduction	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
cells	NN	O	O
expressing	NN	O	O
the	NN	O	O
mutant	NN	O	O
fragment	NN	O	O
secreted	NN	O	O
a	NN	O	O
monomeric	NN	O	O
molecule	NN	O	O
of	NN	O	O
apparent	NN	O	O
molecular	NN	O	O
mass	NN	O	O
of	NN	O	O
80	NN	O	O
kDa	NN	O	O
,	NN	O	O
which	NN	O	O
remained	NN	O	O
unchanged	NN	O	O
after	NN	O	O
reduction	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
CyS2010	NN	O	O
is	NN	O	O
essential	NN	O	O
for	NN	O	O
normal	NN	O	O
dimerization	NN	O	O
of	NN	O	O
vWF	NN	O	O
subunits	NN	O	O
through	NN	O	O
disulfide	NN	O	O
bonding	NN	O	O
of	NN	O	O
carboxyl	NN	O	O
-	NN	O	O
terminal	NN	O	O
domains	NN	O	O
and	NN	O	O
that	NN	O	O
a	NN	O	O
heterozygous	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
corresponding	NN	O	O
codon	NN	O	O
is	NN	O	O
responsible	NN	O	O
for	NN	O	O
defective	NN	O	O
multimer	NN	O	O
formation	NN	O	O
in	NN	O	O
type	NN	O	B-Disease
IID	NN	O	I-Disease
von	NN	O	I-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
protein	NN	O	O
,	NN	O	O
a	NN	O	O
novel	NN	O	O
effector	NN	O	O
for	NN	O	O
the	NN	O	O
GTPase	NN	O	O
CDC42Hs	NN	O	O
,	NN	O	O
is	NN	O	O
implicated	NN	O	O
in	NN	O	O
actin	NN	O	O
polymerization	NN	O	O
.	NN	O	O

The	NN	O	O
Rho	NN	O	O
family	NN	O	O
of	NN	O	O
GTPases	NN	O	O
control	NN	O	O
diverse	NN	O	O
biological	NN	O	O
processes	NN	O	O
,	NN	O	O
including	NN	O	O
cell	NN	O	O
morphology	NN	O	O
and	NN	O	O
mitogenesis	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
identified	NN	O	O
WASP	NN	O	O
,	NN	O	O
the	NN	O	O
protein	NN	O	O
that	NN	O	O
is	NN	O	O
defective	NN	O	O
in	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
WAS	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
as	NN	O	O
a	NN	O	O
novel	NN	O	O
effector	NN	O	O
for	NN	O	O
CDC42Hs	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
for	NN	O	O
the	NN	O	O
other	NN	O	O
Rho	NN	O	O
family	NN	O	O
members	NN	O	O
,	NN	O	O
Rac	NN	O	O
and	NN	O	O
Rho	NN	O	O
.	NN	O	O

This	NN	O	O
interaction	NN	O	O
is	NN	O	O
dependent	NN	O	O
on	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
the	NN	O	O
G	NN	O	O
protein	NN	O	O
-	NN	O	O
binding	NN	O	O
domain	NN	O	O
.	NN	O	O

Cellular	NN	O	O
expression	NN	O	O
of	NN	O	O
epitope	NN	O	O
-	NN	O	O
tagged	NN	O	O
WASP	NN	O	O
produces	NN	O	O
clusters	NN	O	O
of	NN	O	O
WASP	NN	O	O
that	NN	O	O
are	NN	O	O
highly	NN	O	O
enriched	NN	O	O
in	NN	O	O
polymerized	NN	O	O
actin	NN	O	O
.	NN	O	O

This	NN	O	O
clustering	NN	O	O
is	NN	O	O
not	NN	O	O
observed	NN	O	O
with	NN	O	O
a	NN	O	O
C	NN	O	O
-	NN	O	O
terminally	NN	O	O
deleted	NN	O	O
WASP	NN	O	O
and	NN	O	O
is	NN	O	O
inhibited	NN	O	O
by	NN	O	O
coexpression	NN	O	O
with	NN	O	O
dominant	NN	O	O
negative	NN	O	O
CDC42Hs	NN	O	O
-	NN	O	O
N17	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
with	NN	O	O
dominant	NN	O	O
negative	NN	O	O
forms	NN	O	O
of	NN	O	O
Rac	NN	O	O
or	NN	O	O
Rho	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
WASP	NN	O	O
provides	NN	O	O
a	NN	O	O
novel	NN	O	O
link	NN	O	O
between	NN	O	O
CDC42Hs	NN	O	O
and	NN	O	O
the	NN	O	O
actin	NN	O	O
cytoskeleton	NN	O	O
,	NN	O	O
which	NN	O	O
suggests	NN	O	O
a	NN	O	O
molecular	NN	O	O
mechanism	NN	O	O
for	NN	O	O
many	NN	O	O
of	NN	O	O
the	NN	O	O
cellular	NN	O	O
abnormalities	NN	O	O
in	NN	O	O
WAS	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
WASP	NN	O	O
sequence	NN	O	O
contains	NN	O	O
two	NN	O	O
novel	NN	O	O
domains	NN	O	O
that	NN	O	O
are	NN	O	O
homologous	NN	O	O
to	NN	O	O
other	NN	O	O
proteins	NN	O	O
involved	NN	O	O
in	NN	O	O
action	NN	O	O
organization	NN	O	O
.	NN	O	O
.	NN	O	O

X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
adrenoleukodystrophy	NN	O	I-Disease
is	NN	O	O
a	NN	O	O
frequent	NN	O	O
cause	NN	O	O
of	NN	O	O
idiopathic	NN	O	O
Addison	NN	O	B-Disease
'	NN	O	I-Disease
s	NN	O	I-Disease
disease	NN	O	I-Disease
in	NN	O	O
young	NN	O	O
adult	NN	O	O
male	NN	O	O
patients	NN	O	O
.	NN	O	O

X	NN	O	B-Disease
-	NN	O	I-Disease
Linked	NN	O	I-Disease
adrenoleukodystrophy	NN	O	I-Disease
(	NN	O	O
ALD	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
a	NN	O	O
genetic	NN	O	B-Disease
disease	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
demyelination	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
central	NN	O	I-Disease
nervous	NN	O	I-Disease
system	NN	O	I-Disease
,	NN	O	O
adrenal	NN	O	B-Disease
insufficiency	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
accumulation	NN	O	O
of	NN	O	O
very	NN	O	O
long	NN	O	O
chain	NN	O	O
fatty	NN	O	O
acids	NN	O	O
in	NN	O	O
tissue	NN	O	O
and	NN	O	O
body	NN	O	O
fluids	NN	O	O
.	NN	O	O

ALD	NN	O	B-Disease
is	NN	O	O
due	NN	O	O
to	NN	O	O
mutation	NN	O	O
of	NN	O	O
a	NN	O	O
gene	NN	O	O
located	NN	O	O
in	NN	O	O
Xq28	NN	O	O
that	NN	O	O
encodes	NN	O	O
a	NN	O	O
peroxisomal	NN	O	O
transporter	NN	O	O
protein	NN	O	O
of	NN	O	O
unknown	NN	O	O
function	NN	O	O
.	NN	O	O

The	NN	O	O
most	NN	O	O
common	NN	O	O
phenotype	NN	O	O
of	NN	O	O
ALD	NN	O	B-Disease
is	NN	O	O
the	NN	O	O
cerebral	NN	O	O
form	NN	O	O
(	NN	O	O
45	NN	O	O
%	NN	O	O
)	NN	O	O
that	NN	O	O
develops	NN	O	O
in	NN	O	O
boys	NN	O	O
between	NN	O	O
5	NN	O	O
-	NN	O	O
12	NN	O	O
yr	NN	O	O
.	NN	O	O

Adrenomyeloneuropathy	NN	O	B-Disease
(	NN	O	O
AMN	NN	O	B-Disease
)	NN	O	O
involves	NN	O	O
the	NN	O	O
spinal	NN	O	O
cord	NN	O	O
and	NN	O	O
peripheral	NN	O	O
nerves	NN	O	O
in	NN	O	O
young	NN	O	O
adults	NN	O	O
(	NN	O	O
35	NN	O	O
%	NN	O	O
)	NN	O	O
.	NN	O	O

Adrenal	NN	O	B-Disease
insufficiency	NN	O	I-Disease
(	NN	O	O
Addisons	NN	O	B-Disease
disease	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
frequently	NN	O	O
associated	NN	O	O
with	NN	O	O
AMN	NN	O	B-Disease
or	NN	O	O
cerebral	NN	O	B-Disease
ALD	NN	O	I-Disease
and	NN	O	O
may	NN	O	O
remain	NN	O	O
the	NN	O	O
only	NN	O	O
clinical	NN	O	O
expression	NN	O	O
of	NN	O	O
ALD	NN	O	B-Disease
(	NN	O	O
8	NN	O	O
%	NN	O	O
of	NN	O	O
cases	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
prevalence	NN	O	O
of	NN	O	O
ALD	NN	O	B-Disease
among	NN	O	O
adults	NN	O	O
with	NN	O	O
Addisons	NN	O	B-Disease
disease	NN	O	I-Disease
remains	NN	O	O
unknown	NN	O	O
.	NN	O	O

To	NN	O	O
evaluate	NN	O	O
this	NN	O	O
prevalence	NN	O	O
,	NN	O	O
we	NN	O	O
performed	NN	O	O
biochemical	NN	O	O
analysis	NN	O	O
of	NN	O	O
very	NN	O	O
long	NN	O	O
chain	NN	O	O
fatty	NN	O	O
acids	NN	O	O
in	NN	O	O
14	NN	O	O
male	NN	O	O
patients	NN	O	O
(	NN	O	O
age	NN	O	O
ranging	NN	O	O
from	NN	O	O
12	NN	O	O
-	NN	O	O
45	NN	O	O
yr	NN	O	O
at	NN	O	O
diagnosis	NN	O	O
)	NN	O	O
previously	NN	O	O
diagnosed	NN	O	O
as	NN	O	O
having	NN	O	O
primary	NN	O	O
idiopathic	NN	O	O
adrenocortical	NN	O	B-Disease
insufficiency	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
5	NN	O	O
of	NN	O	O
14	NN	O	O
patients	NN	O	O
(	NN	O	O
35	NN	O	O
%	NN	O	O
)	NN	O	O
,	NN	O	O
elevated	NN	O	O
plasma	NN	O	O
concentrations	NN	O	O
of	NN	O	O
very	NN	O	O
long	NN	O	O
chain	NN	O	O
fatty	NN	O	O
acids	NN	O	O
were	NN	O	O
detected	NN	O	O
.	NN	O	O

None	NN	O	O
of	NN	O	O
these	NN	O	O
patients	NN	O	O
had	NN	O	O
adrenocortical	NN	O	O
antibodies	NN	O	O
.	NN	O	O

By	NN	O	O
electrophysiological	NN	O	O
tests	NN	O	O
and	NN	O	O
magnetic	NN	O	O
resonance	NN	O	O
imaging	NN	O	O
it	NN	O	O
was	NN	O	O
determined	NN	O	O
that	NN	O	O
two	NN	O	O
patients	NN	O	O
had	NN	O	O
cerebral	NN	O	B-Disease
ALD	NN	O	I-Disease
,	NN	O	O
one	NN	O	O
had	NN	O	O
adrenomyeloneuropathy	NN	O	B-Disease
with	NN	O	O
cerebral	NN	O	O
involvement	NN	O	O
,	NN	O	O
and	NN	O	O
two	NN	O	O
had	NN	O	O
preclinical	NN	O	O
AMN	NN	O	B-Disease
.	NN	O	O

Our	NN	O	O
data	NN	O	O
support	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
ALD	NN	O	B-Disease
is	NN	O	O
a	NN	O	O
frequent	NN	O	O
cause	NN	O	O
of	NN	O	O
idiopathic	NN	O	O
Addisons	NN	O	B-Disease
disease	NN	O	I-Disease
in	NN	O	O
children	NN	O	O
and	NN	O	O
adults	NN	O	O
.	NN	O	O
.	NN	O	O

Tumor	NN	O	B-Disease
suppression	NN	O	O
and	NN	O	O
apoptosis	NN	O	O
of	NN	O	O
human	NN	O	O
prostate	NN	O	B-Disease
carcinoma	NN	O	I-Disease
mediated	NN	O	O
by	NN	O	O
a	NN	O	O
genetic	NN	O	O
locus	NN	O	O
within	NN	O	O
human	NN	O	O
chromosome	NN	O	O
10pter	NN	O	O
-	NN	O	O
q11	NN	O	O
.	NN	O	O

Prostate	NN	O	B-Disease
cancer	NN	O	I-Disease
is	NN	O	O
the	NN	O	O
second	NN	O	O
leading	NN	O	O
cause	NN	O	O
of	NN	O	O
male	NN	O	O
cancer	NN	O	B-Disease
deaths	NN	O	O
in	NN	O	O
the	NN	O	O
United	NN	O	O
States	NN	O	O
.	NN	O	O

Yet	NN	O	O
,	NN	O	O
despite	NN	O	O
a	NN	O	O
large	NN	O	O
international	NN	O	O
effort	NN	O	O
,	NN	O	O
little	NN	O	O
is	NN	O	O
known	NN	O	O
about	NN	O	O
the	NN	O	O
molecular	NN	O	O
mechanisms	NN	O	O
that	NN	O	O
underlie	NN	O	O
this	NN	O	O
devastating	NN	O	O
disease	NN	O	O
.	NN	O	O

Prostate	NN	O	O
secretory	NN	O	O
epithelial	NN	O	O
cells	NN	O	O
and	NN	O	O
androgen	NN	O	O
-	NN	O	O
dependent	NN	O	O
prostate	NN	O	B-Disease
carcinomas	NN	O	I-Disease
undergo	NN	O	O
apoptosis	NN	O	O
in	NN	O	O
response	NN	O	O
to	NN	O	O
androgen	NN	O	O
deprivation	NN	O	O
and	NN	O	O
,	NN	O	O
furthermore	NN	O	O
,	NN	O	O
most	NN	O	O
prostate	NN	O	B-Disease
carcinomas	NN	O	I-Disease
become	NN	O	O
androgen	NN	O	O
independent	NN	O	O
and	NN	O	O
refractory	NN	O	O
to	NN	O	O
further	NN	O	O
therapeutic	NN	O	O
manipulations	NN	O	O
during	NN	O	O
disease	NN	O	O
progression	NN	O	O
.	NN	O	O

Definition	NN	O	O
of	NN	O	O
the	NN	O	O
genetic	NN	O	O
events	NN	O	O
that	NN	O	O
trigger	NN	O	O
apoptosis	NN	O	O
in	NN	O	O
the	NN	O	O
prostate	NN	O	O
could	NN	O	O
provide	NN	O	O
important	NN	O	O
insights	NN	O	O
into	NN	O	O
critical	NN	O	O
pathways	NN	O	O
in	NN	O	O
normal	NN	O	O
development	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
elucidate	NN	O	O
the	NN	O	O
perturbations	NN	O	O
of	NN	O	O
those	NN	O	O
key	NN	O	O
pathways	NN	O	O
in	NN	O	O
neoplastic	NN	O	O
transformation	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
the	NN	O	O
functional	NN	O	O
definition	NN	O	O
of	NN	O	O
a	NN	O	O
novel	NN	O	O
genetic	NN	O	O
locus	NN	O	O
within	NN	O	O
human	NN	O	O
chromosome	NN	O	O
10pter	NN	O	O
-	NN	O	O
q11	NN	O	O
that	NN	O	O
mediates	NN	O	O
both	NN	O	O
in	NN	O	O
vivo	NN	O	O
tumor	NN	O	O
suppression	NN	O	O
and	NN	O	O
in	NN	O	O
vitro	NN	O	O
apoptosis	NN	O	O
of	NN	O	O
prostatic	NN	O	B-Disease
adenocarcinoma	NN	O	I-Disease
cells	NN	O	O
.	NN	O	O

A	NN	O	O
defined	NN	O	O
fragment	NN	O	O
of	NN	O	O
human	NN	O	O
chromosome	NN	O	O
10	NN	O	O
was	NN	O	O
transferred	NN	O	O
via	NN	O	O
microcell	NN	O	O
fusion	NN	O	O
into	NN	O	O
a	NN	O	O
prostate	NN	O	B-Disease
adenocarcinoma	NN	O	I-Disease
cell	NN	O	O
line	NN	O	O
.	NN	O	O

Microcell	NN	O	O
hybrids	NN	O	O
containing	NN	O	O
only	NN	O	O
the	NN	O	O
region	NN	O	O
10pter	NN	O	O
-	NN	O	O
q11	NN	O	O
were	NN	O	O
suppressed	NN	O	O
for	NN	O	O
tumorigenicity	NN	O	O
following	NN	O	O
injection	NN	O	O
of	NN	O	O
microcell	NN	O	O
hybrids	NN	O	O
into	NN	O	O
nude	NN	O	O
mice	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
the	NN	O	O
complemented	NN	O	O
hybrids	NN	O	O
undergo	NN	O	O
programmed	NN	O	O
cell	NN	O	O
death	NN	O	O
in	NN	O	O
vitro	NN	O	O
via	NN	O	O
a	NN	O	O
mechanism	NN	O	O
that	NN	O	O
does	NN	O	O
not	NN	O	O
require	NN	O	O
nuclear	NN	O	O
localization	NN	O	O
of	NN	O	O
p53	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
functionally	NN	O	O
define	NN	O	O
a	NN	O	O
novel	NN	O	O
genetic	NN	O	O
locus	NN	O	O
,	NN	O	O
designated	NN	O	O
PAC1	NN	O	O
,	NN	O	O
for	NN	O	O
prostate	NN	O	O
adenocarcinoma	NN	O	O
1	NN	O	O
,	NN	O	O
involved	NN	O	O
in	NN	O	O
tumor	NN	O	O
suppression	NN	O	O
of	NN	O	O
human	NN	O	O
prostate	NN	O	B-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
furthermore	NN	O	O
strongly	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
cell	NN	O	O
death	NN	O	O
pathway	NN	O	O
can	NN	O	O
be	NN	O	O
functionally	NN	O	O
restored	NN	O	O
in	NN	O	O
prostatic	NN	O	B-Disease
adenocarcinoma	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Low	NN	O	O
incidence	NN	O	O
of	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
other	NN	O	O
cancers	NN	O	B-Disease
.	NN	O	O

Inherited	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
of	NN	O	O
familial	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
genes	NN	O	O
predispose	NN	O	O
individuals	NN	O	O
to	NN	O	O
particular	NN	O	O
types	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
addition	NN	O	O
to	NN	O	O
an	NN	O	O
involvement	NN	O	O
in	NN	O	O
inherited	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
cancer	NN	O	B-Disease
,	NN	O	O
these	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
genes	NN	O	O
are	NN	O	O
targets	NN	O	O
for	NN	O	O
somatic	NN	O	O
mutations	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
cancers	NN	O	I-Disease
of	NN	O	O
the	NN	O	O
same	NN	O	O
type	NN	O	O
found	NN	O	O
in	NN	O	O
the	NN	O	O
familial	NN	O	O
forms	NN	O	O
.	NN	O	O

An	NN	O	O
exception	NN	O	O
is	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
which	NN	O	O
contributes	NN	O	O
to	NN	O	O
a	NN	O	O
significant	NN	O	O
fraction	NN	O	O
of	NN	O	O
familial	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
undergoes	NN	O	O
mutation	NN	O	O
at	NN	O	O
very	NN	O	O
low	NN	O	O
rates	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O

This	NN	O	O
finding	NN	O	O
suggests	NN	O	O
that	NN	O	O
other	NN	O	O
genes	NN	O	O
may	NN	O	O
be	NN	O	O
the	NN	O	O
principal	NN	O	O
targets	NN	O	O
for	NN	O	O
somatic	NN	O	O
mutation	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
carcinoma	NN	O	I-Disease
.	NN	O	O

A	NN	O	O
second	NN	O	O
,	NN	O	O
recently	NN	O	O
identified	NN	O	O
familial	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
gene	NN	O	O
,	NN	O	O
BRCA2	NN	O	O
(	NN	O	O
refs	NN	O	O
5	NN	O	O
-	NN	O	O
8	NN	O	O
)	NN	O	O
,	NN	O	O
accounts	NN	O	O
for	NN	O	O
a	NN	O	O
proportion	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
roughly	NN	O	O
equal	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

Like	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
BRCA2	NN	O	O
behaves	NN	O	O
as	NN	O	O
a	NN	O	O
dominantly	NN	O	O
inherited	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
.	NN	O	O

Individuals	NN	O	O
who	NN	O	O
inherit	NN	O	O
one	NN	O	O
mutant	NN	O	O
allele	NN	O	O
are	NN	O	O
at	NN	O	O
increased	NN	O	O
risk	NN	O	O
for	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
the	NN	O	O
tumours	NN	O	B-Disease
they	NN	O	O
develop	NN	O	O
lose	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
allele	NN	O	O
by	NN	O	O
heterozygous	NN	O	O
deletion	NN	O	O
.	NN	O	O

The	NN	O	O
BRCA2	NN	O	O
coding	NN	O	O
sequence	NN	O	O
is	NN	O	O
huge	NN	O	O
,	NN	O	O
composed	NN	O	O
of	NN	O	O
26	NN	O	O
exons	NN	O	O
that	NN	O	O
span	NN	O	O
10	NN	O	O
,	NN	O	O
443	NN	O	O
bp	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
investigate	NN	O	O
the	NN	O	O
rate	NN	O	O
of	NN	O	O
BRCA2	NN	O	O
mutation	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
breast	NN	O	I-Disease
cancers	NN	O	I-Disease
and	NN	O	O
in	NN	O	O
a	NN	O	O
set	NN	O	O
of	NN	O	O
cell	NN	O	O
lines	NN	O	O
that	NN	O	O
represent	NN	O	O
twelve	NN	O	O
other	NN	O	O
tumour	NN	O	B-Disease
types	NN	O	O
.	NN	O	O

Surprisingly	NN	O	O
,	NN	O	O
mutations	NN	O	O
in	NN	O	O
BRCA2	NN	O	O
are	NN	O	O
infrequent	NN	O	O
in	NN	O	O
cancers	NN	O	B-Disease
including	NN	O	O
breast	NN	O	B-Disease
carcinoma	NN	O	I-Disease
.	NN	O	O

However	NN	O	O
,	NN	O	O
a	NN	O	O
probable	NN	O	O
germline	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
pancreatic	NN	O	B-Disease
tumour	NN	O	I-Disease
cell	NN	O	O
line	NN	O	O
suggests	NN	O	O
a	NN	O	O
role	NN	O	O
for	NN	O	O
BRCA2	NN	O	O
in	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
pancreatic	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Founding	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
in	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
southern	NN	O	O
Sweden	NN	O	O
.	NN	O	O

Nine	NN	O	O
different	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
susceptibility	NN	O	O
gene	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
15	NN	O	O
of	NN	O	O
47	NN	O	O
kindreds	NN	O	O
from	NN	O	O
southern	NN	O	O
Sweden	NN	O	O
,	NN	O	O
by	NN	O	O
use	NN	O	O
of	NN	O	O
SSCP	NN	O	O
and	NN	O	O
heteroduplex	NN	O	O
analysis	NN	O	O
of	NN	O	O
all	NN	O	O
exons	NN	O	O
and	NN	O	O
flanking	NN	O	O
intron	NN	O	O
region	NN	O	O
and	NN	O	O
by	NN	O	O
a	NN	O	O
protein	NN	O	O
-	NN	O	O
truncation	NN	O	O
test	NN	O	O
for	NN	O	O
exon	NN	O	O
11	NN	O	O
,	NN	O	O
followed	NN	O	O
by	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
.	NN	O	O

All	NN	O	O
but	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
are	NN	O	O
predicted	NN	O	O
to	NN	O	O
give	NN	O	O
rise	NN	O	O
to	NN	O	O
premature	NN	O	O
translation	NN	O	O
termination	NN	O	O
and	NN	O	O
include	NN	O	O
seven	NN	O	O
frameshift	NN	O	O
insertions	NN	O	O
or	NN	O	O
deletions	NN	O	O
,	NN	O	O
a	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
,	NN	O	O
and	NN	O	O
a	NN	O	O
splice	NN	O	O
acceptor	NN	O	O
site	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
remaining	NN	O	O
mutation	NN	O	O
is	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
(	NN	O	O
Cys61Gly	NN	O	O
)	NN	O	O
in	NN	O	O
the	NN	O	O
zinc	NN	O	O
-	NN	O	O
binding	NN	O	O
motif	NN	O	O
.	NN	O	O

Four	NN	O	O
novel	NN	O	O
Swedish	NN	O	O
founding	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
the	NN	O	O
nucleotide	NN	O	O
2595	NN	O	O
deletion	NN	O	O
A	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
five	NN	O	O
families	NN	O	O
,	NN	O	O
the	NN	O	O
C	NN	O	O
1806	NN	O	O
T	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
three	NN	O	O
families	NN	O	O
,	NN	O	O
the	NN	O	O
3166	NN	O	O
insertion	NN	O	O
TGAGA	NN	O	O
in	NN	O	O
three	NN	O	O
families	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
nucleotide	NN	O	O
1201	NN	O	O
deletion	NN	O	O
11	NN	O	O
in	NN	O	O
two	NN	O	O
families	NN	O	O
.	NN	O	O

Analysis	NN	O	O
of	NN	O	O
the	NN	O	O
intragenic	NN	O	O
polymorphism	NN	O	O
D17S855	NN	O	O
supports	NN	O	O
common	NN	O	O
origins	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
.	NN	O	O

Eleven	NN	O	O
of	NN	O	O
the	NN	O	O
15	NN	O	O
kindreds	NN	O	O
manifesting	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
were	NN	O	O
breast	NN	O	B-Disease
-	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
,	NN	O	O
several	NN	O	O
of	NN	O	O
them	NN	O	O
with	NN	O	O
a	NN	O	O
predominant	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
phenotype	NN	O	O
.	NN	O	O

The	NN	O	O
set	NN	O	O
of	NN	O	O
32	NN	O	O
families	NN	O	O
in	NN	O	O
which	NN	O	O
no	NN	O	O
BRCA1	NN	O	O
alterations	NN	O	O
were	NN	O	O
detected	NN	O	O
included	NN	O	O
1	NN	O	O
breast	NN	O	B-Disease
-	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
kindred	NN	O	O
manifesting	NN	O	O
clear	NN	O	O
linkage	NN	O	O
to	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
region	NN	O	O
and	NN	O	O
loss	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
chromosome	NN	O	O
in	NN	O	O
associated	NN	O	O
tumors	NN	O	B-Disease
.	NN	O	O

Other	NN	O	O
tumor	NN	O	B-Disease
types	NN	O	O
found	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
/	NN	O	O
haplotype	NN	O	O
carriers	NN	O	O
included	NN	O	O
prostatic	NN	O	B-Disease
,	NN	O	I-Disease
pancreas	NN	O	I-Disease
,	NN	O	I-Disease
skin	NN	O	I-Disease
,	NN	O	I-Disease
and	NN	O	I-Disease
lung	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
a	NN	O	O
malignant	NN	O	B-Disease
melanoma	NN	O	I-Disease
,	NN	O	O
an	NN	O	O
oligodendroglioma	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
a	NN	O	O
carcinosarcoma	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
all	NN	O	O
,	NN	O	O
12	NN	O	O
of	NN	O	O
16	NN	O	O
kindreds	NN	O	O
manifesting	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
or	NN	O	O
linkage	NN	O	O
contained	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
as	NN	O	O
compared	NN	O	O
with	NN	O	O
only	NN	O	O
6	NN	O	O
of	NN	O	O
the	NN	O	O
remaining	NN	O	O
31	NN	O	O
families	NN	O	O
(	NN	O	O
P	NN	O	O
<	NN	O	O
.	NN	O	O
001	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
present	NN	O	O
study	NN	O	O
confirms	NN	O	O
the	NN	O	O
involvement	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
in	NN	O	O
disease	NN	O	O
predisposition	NN	O	O
for	NN	O	O
a	NN	O	O
subset	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
often	NN	O	O
characterized	NN	O	O
by	NN	O	O
ovarian	NN	O	B-Disease
cancers	NN	O	I-Disease
.	NN	O	O

Rapid	NN	O	O
detection	NN	O	O
of	NN	O	O
regionally	NN	O	O
clustered	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
by	NN	O	O
multiplex	NN	O	O
heteroduplex	NN	O	O
analysis	NN	O	O
.	NN	O	O

UKCCCR	NN	O	O
Familial	NN	O	O
Ovarian	NN	O	B-Disease
Cancer	NN	O	I-Disease
Study	NN	O	O
Group	NN	O	O
.	NN	O	O

Germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
are	NN	O	O
responsible	NN	O	O
for	NN	O	O
a	NN	O	O
substantial	NN	O	O
proportion	NN	O	O
of	NN	O	O
families	NN	O	O
with	NN	O	O
multiple	NN	O	O
cases	NN	O	O
of	NN	O	O
early	NN	O	O
-	NN	O	O
onset	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Since	NN	O	O
the	NN	O	O
isolation	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
last	NN	O	O
year	NN	O	O
,	NN	O	O
>	NN	O	O
65	NN	O	O
distinct	NN	O	O
mutations	NN	O	O
scattered	NN	O	O
throughout	NN	O	O
the	NN	O	O
coding	NN	O	O
region	NN	O	O
have	NN	O	O
been	NN	O	O
detected	NN	O	O
,	NN	O	O
making	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
time	NN	O	O
consuming	NN	O	O
and	NN	O	O
technically	NN	O	O
challenging	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
developed	NN	O	O
a	NN	O	O
multiplex	NN	O	O
heteroduplex	NN	O	O
analysis	NN	O	O
that	NN	O	O
is	NN	O	O
designed	NN	O	O
to	NN	O	O
analyze	NN	O	O
one	NN	O	O
-	NN	O	O
quarter	NN	O	O
of	NN	O	O
the	NN	O	O
coding	NN	O	O
sequence	NN	O	O
in	NN	O	O
a	NN	O	O
single	NN	O	O
-	NN	O	O
step	NN	O	O
screening	NN	O	O
procedure	NN	O	O
and	NN	O	O
that	NN	O	O
will	NN	O	O
detect	NN	O	O
approximately	NN	O	O
50	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
so	NN	O	O
far	NN	O	O
reported	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
used	NN	O	O
this	NN	O	O
technique	NN	O	O
to	NN	O	O
analyze	NN	O	O
BRCA1	NN	O	O
in	NN	O	O
162	NN	O	O
families	NN	O	O
with	NN	O	O
a	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
identified	NN	O	O
12	NN	O	O
distinct	NN	O	O
mutations	NN	O	O
in	NN	O	O
35	NN	O	O
families	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
previously	NN	O	O
undescribed	NN	O	O
mutation	NN	O	O
within	NN	O	O
the	NN	O	O
tetramerisation	NN	O	O
domain	NN	O	O
of	NN	O	O
TP53	NN	O	O
in	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
Li	NN	O	B-Disease
-	NN	O	I-Disease
Fraumeni	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
details	NN	O	O
of	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
classic	NN	O	O
Li	NN	O	B-Disease
-	NN	O	I-Disease
Fraumeni	NN	O	I-Disease
syndrome	NN	O	I-Disease
in	NN	O	O
which	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
mutation	NN	O	O
in	NN	O	O
codon	NN	O	O
344	NN	O	O
of	NN	O	O
the	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
TP53	NN	O	O
.	NN	O	O

Codon	NN	O	O
344	NN	O	O
is	NN	O	O
a	NN	O	O
key	NN	O	O
residue	NN	O	O
within	NN	O	O
the	NN	O	O
tetramerisation	NN	O	O
domain	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitution	NN	O	O
of	NN	O	O
a	NN	O	O
proline	NN	O	O
for	NN	O	O
a	NN	O	O
leucine	NN	O	O
is	NN	O	O
predicted	NN	O	O
to	NN	O	O
have	NN	O	O
profound	NN	O	O
implications	NN	O	O
for	NN	O	O
tetramerisation	NN	O	O
and	NN	O	O
potentially	NN	O	O
DNA	NN	O	O
binding	NN	O	O
.	NN	O	O

This	NN	O	O
is	NN	O	O
the	NN	O	O
first	NN	O	O
report	NN	O	O
of	NN	O	O
a	NN	O	O
mutation	NN	O	O
at	NN	O	O
this	NN	O	O
residue	NN	O	O
in	NN	O	O
either	NN	O	O
sporadic	NN	O	B-Disease
tumours	NN	O	I-Disease
or	NN	O	O
in	NN	O	O
the	NN	O	O
germline	NN	O	O
and	NN	O	O
the	NN	O	O
first	NN	O	O
report	NN	O	O
of	NN	O	O
a	NN	O	O
germline	NN	O	O
mutation	NN	O	O
within	NN	O	O
the	NN	O	O
tetramerisation	NN	O	O
domain	NN	O	O
.	NN	O	O

The	NN	O	O
family	NN	O	O
does	NN	O	O
not	NN	O	O
appear	NN	O	O
to	NN	O	O
be	NN	O	O
remarkable	NN	O	O
in	NN	O	O
the	NN	O	O
spectrum	NN	O	O
of	NN	O	O
tumours	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
there	NN	O	O
is	NN	O	O
loss	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
allele	NN	O	O
in	NN	O	O
a	NN	O	O
leiomyosarcoma	NN	O	B-Disease
from	NN	O	O
the	NN	O	O
proband	NN	O	O
.	NN	O	O

A	NN	O	O
cell	NN	O	O
line	NN	O	O
has	NN	O	O
been	NN	O	O
established	NN	O	O
from	NN	O	O
the	NN	O	O
tumour	NN	O	B-Disease
of	NN	O	O
the	NN	O	O
proband	NN	O	O
and	NN	O	O
cytogenetic	NN	O	O
and	NN	O	O
molecular	NN	O	O
studies	NN	O	O
carried	NN	O	O
out	NN	O	O
,	NN	O	O
providing	NN	O	O
an	NN	O	O
extensive	NN	O	O
analysis	NN	O	O
in	NN	O	O
this	NN	O	O
family	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
spectrum	NN	O	O
of	NN	O	O
RB1	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
in	NN	O	O
hereditary	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
searched	NN	O	O
for	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
RB1	NN	O	O
mutations	NN	O	O
in	NN	O	O
119	NN	O	O
patients	NN	O	O
with	NN	O	O
hereditary	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

Previous	NN	O	O
investigations	NN	O	O
by	NN	O	O
Southern	NN	O	O
blot	NN	O	O
hybridization	NN	O	O
and	NN	O	O
PCR	NN	O	O
fragment	NN	O	O
-	NN	O	O
length	NN	O	O
analysis	NN	O	O
had	NN	O	O
revealed	NN	O	O
mutations	NN	O	O
in	NN	O	O
48	NN	O	O
patients	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
report	NN	O	O
on	NN	O	O
the	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
remaining	NN	O	O
71	NN	O	O
patients	NN	O	O
.	NN	O	O

By	NN	O	O
applying	NN	O	O
heteroduplex	NN	O	O
analysis	NN	O	O
,	NN	O	O
nonisotopic	NN	O	O
SSCP	NN	O	O
,	NN	O	O
and	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
,	NN	O	O
we	NN	O	O
detected	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
resulting	NN	O	O
in	NN	O	O
premature	NN	O	O
termination	NN	O	O
codons	NN	O	O
or	NN	O	O
disruption	NN	O	O
of	NN	O	O
splice	NN	O	O
signals	NN	O	O
in	NN	O	O
51	NN	O	O
(	NN	O	O
72	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
71	NN	O	O
patients	NN	O	O
.	NN	O	O

Four	NN	O	O
patients	NN	O	O
also	NN	O	O
showed	NN	O	O
rare	NN	O	O
sequence	NN	O	O
variants	NN	O	O
.	NN	O	O

No	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
was	NN	O	O
preferentially	NN	O	O
involved	NN	O	O
in	NN	O	O
single	NN	O	O
base	NN	O	O
substitutions	NN	O	O
.	NN	O	O

Recurrent	NN	O	O
transitions	NN	O	O
were	NN	O	O
observed	NN	O	O
at	NN	O	O
most	NN	O	O
of	NN	O	O
the	NN	O	O
14	NN	O	O
codons	NN	O	O
within	NN	O	O
the	NN	O	O
RB1	NN	O	O
.	NN	O	O

No	NN	O	O
mutation	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
exons	NN	O	O
25	NN	O	O
-	NN	O	O
27	NN	O	O
,	NN	O	O
although	NN	O	O
this	NN	O	O
region	NN	O	O
contains	NN	O	O
two	NN	O	O
CGA	NN	O	O
codons	NN	O	O
.	NN	O	O

This	NN	O	O
suggests	NN	O	O
that	NN	O	O
mutations	NN	O	O
within	NN	O	O
the	NN	O	O
3	NN	O	O
-	NN	O	O
terminal	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
may	NN	O	O
not	NN	O	O
be	NN	O	O
oncogenic	NN	O	O
.	NN	O	O

When	NN	O	O
these	NN	O	O
data	NN	O	O
were	NN	O	O
combined	NN	O	O
with	NN	O	O
the	NN	O	O
results	NN	O	O
of	NN	O	O
our	NN	O	O
previous	NN	O	O
investigations	NN	O	O
,	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
a	NN	O	O
total	NN	O	O
of	NN	O	O
99	NN	O	O
(	NN	O	O
83	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
119	NN	O	O
patients	NN	O	O
.	NN	O	O

The	NN	O	O
spectrum	NN	O	O
comprises	NN	O	O
15	NN	O	O
%	NN	O	O
large	NN	O	O
deletions	NN	O	O
,	NN	O	O
26	NN	O	O
%	NN	O	O
small	NN	O	O
length	NN	O	O
alterations	NN	O	O
,	NN	O	O
and	NN	O	O
42	NN	O	O
%	NN	O	O
base	NN	O	O
substitutions	NN	O	O
.	NN	O	O

No	NN	O	O
correlation	NN	O	O
between	NN	O	O
the	NN	O	O
location	NN	O	O
of	NN	O	O
frameshift	NN	O	O
or	NN	O	O
nonsense	NN	O	O
mutations	NN	O	O
and	NN	O	O
phenotypic	NN	O	O
features	NN	O	O
,	NN	O	O
including	NN	O	O
age	NN	O	O
at	NN	O	O
diagnosis	NN	O	O
,	NN	O	O
the	NN	O	O
number	NN	O	O
of	NN	O	O
tumor	NN	O	B-Disease
foci	NN	O	O
,	NN	O	O
and	NN	O	O
manifestation	NN	O	O
of	NN	O	O
nonocular	NN	O	B-Disease
tumors	NN	O	I-Disease
was	NN	O	O
observed	NN	O	O
.	NN	O	O
.	NN	O	O

Phenotypic	NN	O	O
characterization	NN	O	O
of	NN	O	O
individuals	NN	O	O
with	NN	O	O
30	NN	O	O
-	NN	O	O
40	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
in	NN	O	O
the	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
HD	NN	O	B-Disease
)	NN	O	O
gene	NN	O	O
reveals	NN	O	O
HD	NN	O	B-Disease
cases	NN	O	O
with	NN	O	O
36	NN	O	O
repeats	NN	O	O
and	NN	O	O
apparently	NN	O	O
normal	NN	O	O
elderly	NN	O	O
individuals	NN	O	O
with	NN	O	O
36	NN	O	O
-	NN	O	O
39	NN	O	O
repeats	NN	O	O
.	NN	O	O

Abnormal	NN	O	O
CAG	NN	O	O
expansions	NN	O	O
in	NN	O	O
the	NN	O	O
IT	NN	O	O
-	NN	O	O
15	NN	O	O
gene	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
HD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
diagnostic	NN	O	O
setting	NN	O	O
it	NN	O	O
is	NN	O	O
necessary	NN	O	O
to	NN	O	O
define	NN	O	O
the	NN	O	O
limits	NN	O	O
of	NN	O	O
the	NN	O	O
CAG	NN	O	O
size	NN	O	O
ranges	NN	O	O
on	NN	O	O
normal	NN	O	O
and	NN	O	O
HD	NN	O	B-Disease
-	NN	O	O
associated	NN	O	O
chromosomes	NN	O	O
.	NN	O	O

Most	NN	O	O
large	NN	O	O
analyses	NN	O	O
that	NN	O	O
defined	NN	O	O
the	NN	O	O
limits	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
and	NN	O	O
pathological	NN	O	O
size	NN	O	O
ranges	NN	O	O
employed	NN	O	O
PCR	NN	O	O
assays	NN	O	O
,	NN	O	O
which	NN	O	O
included	NN	O	O
the	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
and	NN	O	O
a	NN	O	O
CCG	NN	O	O
repeat	NN	O	O
tract	NN	O	O
that	NN	O	O
was	NN	O	O
thought	NN	O	O
to	NN	O	O
be	NN	O	O
invariant	NN	O	O
.	NN	O	O

Many	NN	O	O
of	NN	O	O
these	NN	O	O
experiments	NN	O	O
found	NN	O	O
an	NN	O	O
overlap	NN	O	O
between	NN	O	O
the	NN	O	O
normal	NN	O	O
and	NN	O	O
disease	NN	O	O
size	NN	O	O
ranges	NN	O	O
.	NN	O	O

Subsequent	NN	O	O
findings	NN	O	O
that	NN	O	O
the	NN	O	O
CCG	NN	O	O
repeats	NN	O	O
vary	NN	O	O
by	NN	O	O
8	NN	O	O
trinucleotide	NN	O	O
lengths	NN	O	O
suggested	NN	O	O
that	NN	O	O
the	NN	O	O
limits	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
and	NN	O	O
disease	NN	O	O
size	NN	O	O
ranges	NN	O	O
should	NN	O	O
be	NN	O	O
reevaluated	NN	O	O
with	NN	O	O
assays	NN	O	O
that	NN	O	O
exclude	NN	O	O
the	NN	O	O
CCG	NN	O	O
polymorphism	NN	O	O
.	NN	O	O

Since	NN	O	O
patients	NN	O	O
with	NN	O	O
between	NN	O	O
30	NN	O	O
and	NN	O	O
40	NN	O	O
repeats	NN	O	O
are	NN	O	O
rare	NN	O	O
,	NN	O	O
a	NN	O	O
consortium	NN	O	O
was	NN	O	O
assembled	NN	O	O
to	NN	O	O
collect	NN	O	O
such	NN	O	O
individuals	NN	O	O
.	NN	O	O

All	NN	O	O
178	NN	O	O
samples	NN	O	O
were	NN	O	O
reanalyzed	NN	O	O
in	NN	O	O
Cambridge	NN	O	O
by	NN	O	O
using	NN	O	O
assays	NN	O	O
specific	NN	O	O
for	NN	O	O
the	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
optimized	NN	O	O
methods	NN	O	O
for	NN	O	O
reliable	NN	O	O
sizing	NN	O	O
of	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
and	NN	O	O
show	NN	O	O
cases	NN	O	O
that	NN	O	O
demonstrate	NN	O	O
the	NN	O	O
dangers	NN	O	O
of	NN	O	O
using	NN	O	O
PCR	NN	O	O
assays	NN	O	O
that	NN	O	O
include	NN	O	O
both	NN	O	O
the	NN	O	O
CAG	NN	O	O
and	NN	O	O
CCG	NN	O	O
polymorphisms	NN	O	O
.	NN	O	O

Seven	NN	O	O
HD	NN	O	B-Disease
patients	NN	O	O
had	NN	O	O
36	NN	O	O
repeats	NN	O	O
,	NN	O	O
which	NN	O	O
confirms	NN	O	O
that	NN	O	O
this	NN	O	O
allele	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
disease	NN	O	O
.	NN	O	O

Individuals	NN	O	O
without	NN	O	O
apparent	NN	O	O
symptoms	NN	O	O
or	NN	O	O
signs	NN	O	O
of	NN	O	O
HD	NN	O	B-Disease
were	NN	O	O
found	NN	O	O
at	NN	O	O
36	NN	O	O
repeats	NN	O	O
(	NN	O	O
aged	NN	O	O
74	NN	O	O
,	NN	O	O
78	NN	O	O
,	NN	O	O
79	NN	O	O
,	NN	O	O
and	NN	O	O
87	NN	O	O
years	NN	O	O
)	NN	O	O
,	NN	O	O
37	NN	O	O
repeats	NN	O	O
(	NN	O	O
aged	NN	O	O
69	NN	O	O
years	NN	O	O
)	NN	O	O
,	NN	O	O
38	NN	O	O
repeats	NN	O	O
(	NN	O	O
aged	NN	O	O
69	NN	O	O
and	NN	O	O
90	NN	O	O
years	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
39	NN	O	O
repeats	NN	O	O
(	NN	O	O
aged	NN	O	O
67	NN	O	O
,	NN	O	O
90	NN	O	O
,	NN	O	O
and	NN	O	O
95	NN	O	O
years	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
detailed	NN	O	O
case	NN	O	O
histories	NN	O	O
of	NN	O	O
an	NN	O	O
exceptional	NN	O	O
case	NN	O	O
from	NN	O	O
this	NN	O	O
series	NN	O	O
will	NN	O	O
be	NN	O	O
presented	NN	O	O
a	NN	O	O
95	NN	O	O
-	NN	O	O
year	NN	O	O
-	NN	O	O
old	NN	O	O
man	NN	O	O
with	NN	O	O
39	NN	O	O
repeats	NN	O	O
who	NN	O	O
did	NN	O	O
not	NN	O	O
have	NN	O	O
classical	NN	O	O
features	NN	O	O
of	NN	O	O
HD	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
apparently	NN	O	O
healthy	NN	O	O
survival	NN	O	O
into	NN	O	O
old	NN	O	O
age	NN	O	O
of	NN	O	O
some	NN	O	O
individuals	NN	O	O
with	NN	O	O
36	NN	O	O
-	NN	O	O
39	NN	O	O
repeats	NN	O	O
suggests	NN	O	O
that	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
mutation	NN	O	O
may	NN	O	O
not	NN	O	O
always	NN	O	O
be	NN	O	O
fully	NN	O	O
penetrant	NN	O	O
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
and	NN	O	O
expression	NN	O	O
of	NN	O	O
eight	NN	O	O
novel	NN	O	O
mutations	NN	O	O
among	NN	O	O
non	NN	O	O
-	NN	O	O
Jewish	NN	O	O
patients	NN	O	O
with	NN	O	O
Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
is	NN	O	O
inherited	NN	O	O
as	NN	O	O
an	NN	O	O
autosomal	NN	O	O
recessive	NN	O	O
trait	NN	O	O
that	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
the	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
aspartoacylase	NN	O	I-Disease
(	NN	O	O
ASPA	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
majority	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
are	NN	O	O
from	NN	O	O
an	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
background	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
ASPA	NN	O	O
that	NN	O	O
lead	NN	O	O
to	NN	O	O
loss	NN	O	O
of	NN	O	O
enzymatic	NN	O	O
activity	NN	O	O
have	NN	O	O
been	NN	O	O
identified	NN	O	O
,	NN	O	O
and	NN	O	O
E285A	NN	O	O
and	NN	O	O
Y231X	NN	O	O
are	NN	O	O
the	NN	O	O
two	NN	O	O
predominant	NN	O	O
mutations	NN	O	O
that	NN	O	O
account	NN	O	O
for	NN	O	O
97	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
chromosomes	NN	O	O
in	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
patients	NN	O	O
.	NN	O	O

The	NN	O	O
current	NN	O	O
study	NN	O	O
was	NN	O	O
aimed	NN	O	O
at	NN	O	O
finding	NN	O	O
the	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
in	NN	O	O
25	NN	O	O
independent	NN	O	O
patients	NN	O	O
of	NN	O	O
non	NN	O	O
-	NN	O	O
Jewish	NN	O	O
background	NN	O	O
.	NN	O	O

Eight	NN	O	O
novel	NN	O	O
and	NN	O	O
three	NN	O	O
previously	NN	O	O
characterized	NN	O	O
mutations	NN	O	O
accounted	NN	O	O
for	NN	O	O
80	NN	O	O
%	NN	O	O
(	NN	O	O
40	NN	O	O
/	NN	O	O
50	NN	O	O
)	NN	O	O
of	NN	O	O
mutant	NN	O	O
chromosomes	NN	O	O
.	NN	O	O

The	NN	O	O
A305E	NN	O	O
missense	NN	O	O
mutation	NN	O	O
accounted	NN	O	O
for	NN	O	O
48	NN	O	O
%	NN	O	O
(	NN	O	O
24	NN	O	O
/	NN	O	O
50	NN	O	O
)	NN	O	O
of	NN	O	O
mutant	NN	O	O
chromosomes	NN	O	O
in	NN	O	O
patients	NN	O	O
of	NN	O	O
western	NN	O	O
European	NN	O	O
descent	NN	O	O
,	NN	O	O
while	NN	O	O
the	NN	O	O
two	NN	O	O
predominant	NN	O	O
Jewish	NN	O	O
mutations	NN	O	O
each	NN	O	O
accounted	NN	O	O
for	NN	O	O
a	NN	O	O
single	NN	O	O
mutant	NN	O	O
chromosome	NN	O	O
.	NN	O	O

The	NN	O	O
eight	NN	O	O
novel	NN	O	O
mutations	NN	O	O
identified	NN	O	O
included	NN	O	O
1	NN	O	O
-	NN	O	O
and	NN	O	O
4	NN	O	O
-	NN	O	O
bp	NN	O	O
deletions	NN	O	O
(	NN	O	O
32	NN	O	O
deltaT	NN	O	O
and	NN	O	O
876	NN	O	O
deltaAGAA	NN	O	O
,	NN	O	O
respectively	NN	O	O
)	NN	O	O
and	NN	O	O
I16T	NN	O	O
,	NN	O	O
G27R	NN	O	O
,	NN	O	O
D114E	NN	O	O
,	NN	O	O
G123E	NN	O	O
,	NN	O	O
C152Y	NN	O	O
,	NN	O	O
and	NN	O	O
R168C	NN	O	O
missense	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
homozygous	NN	O	O
32	NN	O	O
deltaT	NN	O	O
deletion	NN	O	O
was	NN	O	O
identified	NN	O	O
in	NN	O	O
the	NN	O	O
only	NN	O	O
known	NN	O	O
patient	NN	O	O
of	NN	O	O
African	NN	O	O
-	NN	O	O
American	NN	O	O
origin	NN	O	O
with	NN	O	O
Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
heterozygosity	NN	O	O
for	NN	O	O
876	NN	O	O
deltaAGAA	NN	O	O
mutation	NN	O	O
was	NN	O	O
identified	NN	O	O
in	NN	O	O
three	NN	O	O
independent	NN	O	O
patients	NN	O	O
from	NN	O	O
England	NN	O	O
.	NN	O	O

Six	NN	O	O
single	NN	O	O
-	NN	O	O
base	NN	O	O
changes	NN	O	O
leading	NN	O	O
to	NN	O	O
missense	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
patients	NN	O	O
from	NN	O	O
Turkey	NN	O	O
(	NN	O	O
D114E	NN	O	O
,	NN	O	O
R168C	NN	O	O
)	NN	O	O
,	NN	O	O
The	NN	O	O
Netherlands	NN	O	O
(	NN	O	O
I16T	NN	O	O
)	NN	O	O
,	NN	O	O
Germany	NN	O	O
(	NN	O	O
G27R	NN	O	O
)	NN	O	O
,	NN	O	O
Ireland	NN	O	O
(	NN	O	O
C152Y	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
Canada	NN	O	O
(	NN	O	O
G123E	NN	O	O
)	NN	O	O
.	NN	O	O

A	NN	O	O
PCR	NN	O	O
-	NN	O	O
based	NN	O	O
protocol	NN	O	O
is	NN	O	O
described	NN	O	O
that	NN	O	O
was	NN	O	O
used	NN	O	O
to	NN	O	O
introduce	NN	O	O
mutations	NN	O	O
in	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
cDNA	NN	O	O
.	NN	O	O

In	NN	O	O
vitro	NN	O	O
expression	NN	O	O
of	NN	O	O
mutant	NN	O	O
cDNA	NN	O	O
clones	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
all	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
led	NN	O	O
to	NN	O	O
a	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
ASPA	NN	O	I-Disease
and	NN	O	O
should	NN	O	O
therefore	NN	O	O
result	NN	O	O
in	NN	O	O
Canavan	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
and	NN	O	O
chromosomal	NN	O	O
localization	NN	O	O
of	NN	O	O
Atm	NN	O	O
,	NN	O	O
the	NN	O	O
mouse	NN	O	O
homolog	NN	O	O
of	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
gene	NN	O	O
.	NN	O	O

Atm	NN	O	O
,	NN	O	O
the	NN	O	O
mouse	NN	O	O
homolog	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
ATM	NN	O	O
gene	NN	O	O
defective	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
identified	NN	O	O
.	NN	O	O

The	NN	O	O
entire	NN	O	O
coding	NN	O	O
sequence	NN	O	O
of	NN	O	O
the	NN	O	O
Atm	NN	O	O
transcript	NN	O	O
was	NN	O	O
cloned	NN	O	O
and	NN	O	O
found	NN	O	O
to	NN	O	O
contain	NN	O	O
an	NN	O	O
open	NN	O	O
reading	NN	O	O
frame	NN	O	O
encoding	NN	O	O
a	NN	O	O
protein	NN	O	O
of	NN	O	O
3066	NN	O	O
amino	NN	O	O
acids	NN	O	O
with	NN	O	O
84	NN	O	O
%	NN	O	O
overall	NN	O	O
identity	NN	O	O
and	NN	O	O
91	NN	O	O
%	NN	O	O
similarity	NN	O	O
to	NN	O	O
the	NN	O	O
human	NN	O	O
ATM	NN	O	O
protein	NN	O	O
.	NN	O	O

Variable	NN	O	O
levels	NN	O	O
of	NN	O	O
expression	NN	O	O
of	NN	O	O
Atm	NN	O	O
were	NN	O	O
observed	NN	O	O
in	NN	O	O
different	NN	O	O
tissues	NN	O	O
.	NN	O	O

Fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
and	NN	O	O
linkage	NN	O	O
analysis	NN	O	O
located	NN	O	O
the	NN	O	O
Atm	NN	O	O
gene	NN	O	O
on	NN	O	O
mouse	NN	O	O
chromosome	NN	O	O
9	NN	O	O
,	NN	O	O
band	NN	O	O
9C	NN	O	O
,	NN	O	O
in	NN	O	O
a	NN	O	O
region	NN	O	O
homologous	NN	O	O
to	NN	O	O
the	NN	O	O
ATM	NN	O	O
region	NN	O	O
on	NN	O	O
human	NN	O	O
chromosome	NN	O	O
11q22	NN	O	O
-	NN	O	O
q23	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
mouse	NN	O	O
homolog	NN	O	O
of	NN	O	O
the	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
protein	NN	O	O
(	NN	O	O
WASP	NN	O	O
)	NN	O	O
gene	NN	O	O
is	NN	O	O
highly	NN	O	O
conserved	NN	O	O
and	NN	O	O
maps	NN	O	O
near	NN	O	O
the	NN	O	O
scurfy	NN	O	O
(	NN	O	O
sf	NN	O	O
)	NN	O	O
mutation	NN	O	O
on	NN	O	O
the	NN	O	O
X	NN	O	O
chromosome	NN	O	O
.	NN	O	O

The	NN	O	O
mouse	NN	O	O
WASP	NN	O	O
gene	NN	O	O
,	NN	O	O
the	NN	O	O
homolog	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
mutated	NN	O	O
in	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
has	NN	O	O
been	NN	O	O
isolated	NN	O	O
and	NN	O	O
sequenced	NN	O	O
.	NN	O	O
the	NN	O	O
predicted	NN	O	O
amino	NN	O	O
acid	NN	O	O
sequence	NN	O	O
is	NN	O	O
86	NN	O	O
%	NN	O	O
identical	NN	O	O
to	NN	O	O
the	NN	O	O
human	NN	O	O
WASP	NN	O	O
sequence	NN	O	O
.	NN	O	O

A	NN	O	O
distinct	NN	O	O
feature	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
gene	NN	O	O
is	NN	O	O
an	NN	O	O
expanded	NN	O	O
polymorphic	NN	O	O
GGA	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
that	NN	O	O
codes	NN	O	O
for	NN	O	O
polyglycine	NN	O	O
and	NN	O	O
varies	NN	O	O
from	NN	O	O
15	NN	O	O
to	NN	O	O
17	NN	O	O
triplets	NN	O	O
in	NN	O	O
different	NN	O	O
Mus	NN	O	O
musculus	NN	O	O
strains	NN	O	O
.	NN	O	O

The	NN	O	O
genomic	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
WASP	NN	O	O
gene	NN	O	O
is	NN	O	O
expressed	NN	O	O
as	NN	O	O
an	NN	O	O
approximately	NN	O	O
2	NN	O	O
.	NN	O	O

4	NN	O	O
-	NN	O	O
kb	NN	O	O
mRNA	NN	O	O
in	NN	O	O
thymus	NN	O	O
and	NN	O	O
spleen	NN	O	O
.	NN	O	O

Chromosomal	NN	O	O
mapping	NN	O	O
in	NN	O	O
an	NN	O	O
interspecific	NN	O	O
M	NN	O	O
.	NN	O	O

Musculus	NN	O	O
/	NN	O	O
M	NN	O	O
.	NN	O	O
spretus	NN	O	O
backcross	NN	O	O
placed	NN	O	O
the	NN	O	O
Wasp	NN	O	O
locus	NN	O	O
near	NN	O	O
the	NN	O	O
centromere	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
X	NN	O	O
chromosome	NN	O	O
,	NN	O	O
inseparable	NN	O	O
from	NN	O	O
Gata1	NN	O	O
,	NN	O	O
Tcfe3	NN	O	O
,	NN	O	O
and	NN	O	O
scurfy	NN	O	O
(	NN	O	O
sf	NN	O	O
)	NN	O	O
.	NN	O	O

This	NN	O	O
localization	NN	O	O
makes	NN	O	O
Wasp	NN	O	O
a	NN	O	O
candidate	NN	O	O
for	NN	O	O
involvement	NN	O	O
in	NN	O	O
scurfy	NN	O	O
,	NN	O	O
a	NN	O	O
T	NN	O	O
cell	NN	O	O
-	NN	O	O
mediated	NN	O	O
fatal	NN	O	B-Disease
lymphoreticular	NN	O	I-Disease
disease	NN	O	I-Disease
of	NN	O	O
mice	NN	O	O
that	NN	O	O
has	NN	O	O
previously	NN	O	O
been	NN	O	O
proposed	NN	O	O
as	NN	O	O
a	NN	O	O
mouse	NN	O	O
homolog	NN	O	O
of	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Northern	NN	O	O
analysis	NN	O	O
of	NN	O	O
sf	NN	O	O
tissue	NN	O	O
samples	NN	O	O
indicated	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
WASP	NN	O	O
mRNA	NN	O	O
in	NN	O	O
liver	NN	O	O
and	NN	O	O
skin	NN	O	O
,	NN	O	O
presumably	NN	O	O
as	NN	O	O
a	NN	O	O
consequence	NN	O	O
of	NN	O	O
lymphocytic	NN	O	O
infiltration	NN	O	O
,	NN	O	O
but	NN	O	O
non	NN	O	O
abnormalities	NN	O	O
in	NN	O	O
the	NN	O	O
amount	NN	O	O
or	NN	O	O
size	NN	O	O
of	NN	O	O
mRNA	NN	O	O
present	NN	O	O
.	NN	O	O

Colchicine	NN	O	O
in	NN	O	O
breast	NN	O	O
milk	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
familial	NN	O	B-Disease
Mediterranean	NN	O	I-Disease
fever	NN	O	I-Disease
.	NN	O	O

OBJECTIVE	NN	O	O
.	NN	O	O

To	NN	O	O
clarify	NN	O	O
whether	NN	O	O
colchicine	NN	O	O
is	NN	O	O
excreted	NN	O	O
in	NN	O	O
breast	NN	O	O
milk	NN	O	O
,	NN	O	O
and	NN	O	O
to	NN	O	O
compare	NN	O	O
its	NN	O	O
concentrations	NN	O	O
in	NN	O	O
the	NN	O	O
serum	NN	O	O
and	NN	O	O
breast	NN	O	O
milk	NN	O	O
of	NN	O	O
lactating	NN	O	O
women	NN	O	O
who	NN	O	O
have	NN	O	O
familial	NN	O	B-Disease
Mediterranean	NN	O	I-Disease
fever	NN	O	I-Disease
(	NN	O	O
FMF	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

METHODS	NN	O	O
.	NN	O	O

Using	NN	O	O
a	NN	O	O
specific	NN	O	O
radioimmunoassay	NN	O	O
,	NN	O	O
we	NN	O	O
determined	NN	O	O
colchicine	NN	O	O
concentrations	NN	O	O
in	NN	O	O
the	NN	O	O
serum	NN	O	O
and	NN	O	O
breast	NN	O	O
milk	NN	O	O
of	NN	O	O
4	NN	O	O
patients	NN	O	O
at	NN	O	O
various	NN	O	O
time	NN	O	O
points	NN	O	O
,	NN	O	O
following	NN	O	O
oral	NN	O	O
administration	NN	O	O
of	NN	O	O
the	NN	O	O
drug	NN	O	O
.	NN	O	O

The	NN	O	O
study	NN	O	O
evaluated	NN	O	O
4	NN	O	O
patients	NN	O	O
with	NN	O	O
FMF	NN	O	B-Disease
who	NN	O	O
had	NN	O	O
been	NN	O	O
taking	NN	O	O
colchicine	NN	O	O
on	NN	O	O
a	NN	O	O
long	NN	O	O
-	NN	O	O
term	NN	O	O
basis	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
.	NN	O	O

Colchicine	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
excreted	NN	O	O
in	NN	O	O
breast	NN	O	O
milk	NN	O	O
.	NN	O	O

Its	NN	O	O
levels	NN	O	O
ranged	NN	O	O
between	NN	O	O
1	NN	O	O
.	NN	O	O

9	NN	O	O
and	NN	O	O
8	NN	O	O
.	NN	O	O

6	NN	O	O
ng	NN	O	O
/	NN	O	O
ml	NN	O	O
,	NN	O	O
which	NN	O	O
were	NN	O	O
similar	NN	O	O
to	NN	O	O
those	NN	O	O
found	NN	O	O
in	NN	O	O
the	NN	O	O
serum	NN	O	O
(	NN	O	O
parallel	NN	O	O
concentration	NN	O	O
time	NN	O	O
curves	NN	O	O
)	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
there	NN	O	O
appeared	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
considerable	NN	O	O
variation	NN	O	O
in	NN	O	O
colchicine	NN	O	O
milk	NN	O	O
concentration	NN	O	O
among	NN	O	O
the	NN	O	O
different	NN	O	O
patients	NN	O	O
,	NN	O	O
which	NN	O	O
might	NN	O	O
be	NN	O	O
related	NN	O	O
to	NN	O	O
individual	NN	O	O
breast	NN	O	O
milk	NN	O	O
composition	NN	O	O
and	NN	O	O
,	NN	O	O
possibly	NN	O	O
,	NN	O	O
to	NN	O	O
other	NN	O	O
nutritional	NN	O	O
or	NN	O	O
metabolic	NN	O	O
factors	NN	O	O
.	NN	O	O

CONCLUSION	NN	O	O
.	NN	O	O

The	NN	O	O
extensive	NN	O	O
peripheral	NN	O	O
tissue	NN	O	O
binding	NN	O	O
and	NN	O	O
relatively	NN	O	O
low	NN	O	O
concentration	NN	O	O
of	NN	O	O
colchicine	NN	O	O
in	NN	O	O
breast	NN	O	O
milk	NN	O	O
suggests	NN	O	O
that	NN	O	O
the	NN	O	O
amount	NN	O	O
ingested	NN	O	O
by	NN	O	O
the	NN	O	O
infant	NN	O	O
is	NN	O	O
small	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
based	NN	O	O
on	NN	O	O
our	NN	O	O
clinical	NN	O	O
experience	NN	O	O
,	NN	O	O
nursing	NN	O	O
appears	NN	O	O
to	NN	O	O
be	NN	O	O
safe	NN	O	O
for	NN	O	O
lactating	NN	O	O
women	NN	O	O
with	NN	O	O
FMF	NN	O	B-Disease
who	NN	O	O
continue	NN	O	O
to	NN	O	O
take	NN	O	O
colchicine	NN	O	O
.	NN	O	O

Abnormal	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
protein	NN	O	O
kinase	NN	O	O
levels	NN	O	O
produce	NN	O	O
only	NN	O	O
mild	NN	O	O
myopathy	NN	O	B-Disease
in	NN	O	O
mice	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
commonly	NN	O	O
associated	NN	O	O
with	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
expansions	NN	O	O
within	NN	O	O
the	NN	O	O
gene	NN	O	O
for	NN	O	O
DM	NN	O	O
-	NN	O	O
protein	NN	O	O
kinase	NN	O	O
(	NN	O	O
DMPK	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
effect	NN	O	O
of	NN	O	O
altered	NN	O	O
expression	NN	O	O
levels	NN	O	O
of	NN	O	O
DMPK	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
ubiquitously	NN	O	O
expressed	NN	O	O
in	NN	O	O
all	NN	O	O
muscle	NN	O	O
cell	NN	O	O
lineages	NN	O	O
during	NN	O	O
development	NN	O	O
,	NN	O	O
was	NN	O	O
examined	NN	O	O
by	NN	O	O
disrupting	NN	O	O
the	NN	O	O
endogenous	NN	O	O
Dmpk	NN	O	O
gene	NN	O	O
and	NN	O	O
overexpressing	NN	O	O
a	NN	O	O
normal	NN	O	O
human	NN	O	O
DMPK	NN	O	O
transgene	NN	O	O
in	NN	O	O
mice	NN	O	O
.	NN	O	O

Nullizygous	NN	O	O
(	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
)	NN	O	O
mice	NN	O	O
showed	NN	O	O
only	NN	O	O
inconsistent	NN	O	O
and	NN	O	O
minor	NN	O	O
size	NN	O	O
changes	NN	O	O
in	NN	O	O
head	NN	O	O
and	NN	O	O
neck	NN	O	O
muscle	NN	O	O
fibres	NN	O	O
at	NN	O	O
older	NN	O	O
age	NN	O	O
,	NN	O	O
animals	NN	O	O
with	NN	O	O
the	NN	O	O
highest	NN	O	O
DMPK	NN	O	O
transgene	NN	O	O
expression	NN	O	O
showed	NN	O	O
hypertrophic	NN	O	B-Disease
cardiomyopathy	NN	O	I-Disease
and	NN	O	O
enhanced	NN	O	O
neonatal	NN	O	O
mortality	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
both	NN	O	O
models	NN	O	O
lack	NN	O	O
other	NN	O	O
frequent	NN	O	O
DM	NN	O	B-Disease
symptoms	NN	O	O
including	NN	O	O
the	NN	O	O
fibre	NN	O	O
-	NN	O	O
type	NN	O	O
dependent	NN	O	O
atrophy	NN	O	B-Disease
,	NN	O	O
myotonia	NN	O	B-Disease
,	NN	O	O
cataract	NN	O	B-Disease
and	NN	O	O
male	NN	O	B-Disease
-	NN	O	I-Disease
infertility	NN	O	I-Disease
.	NN	O	O

These	NN	O	O
results	NN	O	O
strengthen	NN	O	O
the	NN	O	O
contention	NN	O	O
that	NN	O	O
simple	NN	O	O
loss	NN	O	O
-	NN	O	O
or	NN	O	O
gain	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
expression	NN	O	O
of	NN	O	O
DMPK	NN	O	O
is	NN	O	O
not	NN	O	O
the	NN	O	O
only	NN	O	O
crucial	NN	O	O
requirement	NN	O	O
for	NN	O	O
development	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O
.	NN	O	O

Mice	NN	O	O
lacking	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
protein	NN	O	O
kinase	NN	O	O
develop	NN	O	O
a	NN	O	O
late	NN	O	O
onset	NN	O	O
progressive	NN	O	O
myopathy	NN	O	B-Disease
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disorder	NN	O	I-Disease
resulting	NN	O	O
from	NN	O	O
the	NN	O	O
expansion	NN	O	O
of	NN	O	O
a	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
untranslated	NN	O	O
region	NN	O	O
of	NN	O	O
a	NN	O	O
putative	NN	O	O
protein	NN	O	O
kinase	NN	O	O
(	NN	O	O
DMPK	NN	O	O
)	NN	O	O
.	NN	O	O

To	NN	O	O
elucidate	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
DMPK	NN	O	O
in	NN	O	O
DM	NN	O	B-Disease
pathogenesis	NN	O	O
we	NN	O	O
have	NN	O	O
developed	NN	O	O
Dmpk	NN	O	B-Disease
deficient	NN	O	I-Disease
(	NN	O	O
Dmpk	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
)	NN	O	O
mice	NN	O	O
.	NN	O	O

Dmpk	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
develop	NN	O	O
a	NN	O	O
late	NN	O	O
-	NN	O	O
onset	NN	O	O
,	NN	O	O
progressive	NN	O	B-Disease
skeletal	NN	O	I-Disease
myopathy	NN	O	I-Disease
that	NN	O	O
shares	NN	O	O
some	NN	O	O
pathological	NN	O	O
features	NN	O	O
with	NN	O	O
DM	NN	O	B-Disease
.	NN	O	O

Muscles	NN	O	O
from	NN	O	O
mature	NN	O	O
mice	NN	O	O
show	NN	O	O
variation	NN	O	O
in	NN	O	O
fibre	NN	O	O
size	NN	O	O
,	NN	O	O
increased	NN	O	O
fibre	NN	O	B-Disease
degeneration	NN	O	I-Disease
and	NN	O	O
fibrosis	NN	O	B-Disease
.	NN	O	O

Adult	NN	O	O
Dmpk	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
show	NN	O	O
ultrastructural	NN	O	O
changes	NN	O	O
in	NN	O	O
muscle	NN	O	O
and	NN	O	O
a	NN	O	O
50	NN	O	O
%	NN	O	O
decrease	NN	O	O
in	NN	O	O
force	NN	O	O
generation	NN	O	O
compared	NN	O	O
to	NN	O	O
young	NN	O	O
mice	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
DMPK	NN	O	O
may	NN	O	O
be	NN	O	O
necessary	NN	O	O
for	NN	O	O
the	NN	O	O
maintenance	NN	O	O
of	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
structure	NN	O	O
and	NN	O	O
function	NN	O	O
and	NN	O	O
suggest	NN	O	O
that	NN	O	O
a	NN	O	O
decrease	NN	O	O
in	NN	O	O
DMPK	NN	O	O
levels	NN	O	O
may	NN	O	O
contribute	NN	O	O
to	NN	O	O
DM	NN	O	B-Disease
pathology	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
Brca1	NN	O	O
is	NN	O	O
required	NN	O	O
for	NN	O	O
embryonic	NN	O	O
cellular	NN	O	O
proliferation	NN	O	O
in	NN	O	O
the	NN	O	O
mouse	NN	O	O
.	NN	O	O

Mutations	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gone	NN	O	O
in	NN	O	O
humans	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
predisposition	NN	O	O
to	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
show	NN	O	O
here	NN	O	O
that	NN	O	O
Brca1	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
are	NN	O	O
normal	NN	O	O
and	NN	O	O
fertile	NN	O	O
and	NN	O	O
lack	NN	O	O
tumors	NN	O	B-Disease
by	NN	O	O
age	NN	O	O
eleven	NN	O	O
months	NN	O	O
.	NN	O	O

Homozygous	NN	O	O
Brca1	NN	O	O
(	NN	O	O
5	NN	O	O
-	NN	O	O
6	NN	O	O
)	NN	O	O
mutant	NN	O	O
mice	NN	O	O
die	NN	O	O
before	NN	O	O
day	NN	O	O
7	NN	O	O
.	NN	O	O

5	NN	O	O
of	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

Mutant	NN	O	O
embryos	NN	O	O
are	NN	O	O
poorly	NN	O	O
developed	NN	O	O
,	NN	O	O
with	NN	O	O
no	NN	O	O
evidence	NN	O	O
of	NN	O	O
mesoderm	NN	O	O
formation	NN	O	O
.	NN	O	O

The	NN	O	O
extraembryonic	NN	O	O
region	NN	O	O
is	NN	O	O
abnormal	NN	O	O
,	NN	O	O
but	NN	O	O
aggregation	NN	O	O
with	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
tetraploid	NN	O	O
embryos	NN	O	O
does	NN	O	O
not	NN	O	O
rescue	NN	O	O
the	NN	O	O
lethality	NN	O	O
.	NN	O	O

In	NN	O	O
vivo	NN	O	O
,	NN	O	O
mutant	NN	O	O
embryos	NN	O	O
do	NN	O	O
not	NN	O	O
exhibit	NN	O	O
increased	NN	O	O
apoptosis	NN	O	O
but	NN	O	O
show	NN	O	O
reduced	NN	O	O
cell	NN	O	O
proliferation	NN	O	O
accompanied	NN	O	O
by	NN	O	O
decreased	NN	O	O
expression	NN	O	O
of	NN	O	O
cyclin	NN	O	O
E	NN	O	O
and	NN	O	O
mdm	NN	O	O
-	NN	O	O
2	NN	O	O
,	NN	O	O
a	NN	O	O
regulator	NN	O	O
of	NN	O	O
p53	NN	O	O
activity	NN	O	O
.	NN	O	O

The	NN	O	O
expression	NN	O	O
of	NN	O	O
cyclin	NN	O	O
-	NN	O	O
dependent	NN	O	O
kinase	NN	O	O
inhibitor	NN	O	O
p21	NN	O	O
is	NN	O	O
dramatically	NN	O	O
increased	NN	O	O
in	NN	O	O
the	NN	O	O
mutant	NN	O	O
embryos	NN	O	O
.	NN	O	O

Buttressing	NN	O	O
these	NN	O	O
in	NN	O	O
vivo	NN	O	O
observations	NN	O	O
is	NN	O	O
the	NN	O	O
fact	NN	O	O
that	NN	O	O
mutant	NN	O	O
blastocyst	NN	O	O
growth	NN	O	O
is	NN	O	O
grossly	NN	O	O
impaired	NN	O	O
in	NN	O	O
vitro	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
the	NN	O	O
death	NN	O	O
of	NN	O	O
Brca1	NN	O	O
(	NN	O	O
5	NN	O	O
-	NN	O	O
6	NN	O	O
)	NN	O	O
mutant	NN	O	O
embryos	NN	O	O
prior	NN	O	O
to	NN	O	O
gastrulation	NN	O	O
may	NN	O	O
be	NN	O	O
due	NN	O	O
to	NN	O	O
a	NN	O	O
failure	NN	O	O
of	NN	O	O
the	NN	O	O
proliferative	NN	O	O
burst	NN	O	O
required	NN	O	O
for	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
the	NN	O	O
different	NN	O	O
germ	NN	O	O
layers	NN	O	O
.	NN	O	O

Increased	NN	O	O
coronary	NN	O	B-Disease
heart	NN	O	I-Disease
disease	NN	O	I-Disease
in	NN	O	O
Japanese	NN	O	O
-	NN	O	O
American	NN	O	O
men	NN	O	O
with	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
cholesteryl	NN	O	O
ester	NN	O	O
transfer	NN	O	O
protein	NN	O	O
gene	NN	O	O
despite	NN	O	O
increased	NN	O	O
HDL	NN	O	O
levels	NN	O	O
.	NN	O	O

Plasma	NN	O	O
high	NN	O	O
density	NN	O	O
lipoprotein	NN	O	O
(	NN	O	O
HDL	NN	O	O
)	NN	O	O
levels	NN	O	O
are	NN	O	O
strongly	NN	O	O
genetically	NN	O	O
determined	NN	O	O
and	NN	O	O
show	NN	O	O
a	NN	O	O
general	NN	O	O
inverse	NN	O	O
relationship	NN	O	O
with	NN	O	O
coronary	NN	O	B-Disease
heart	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
CHD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

The	NN	O	O
cholesteryl	NN	O	O
ester	NN	O	O
transfer	NN	O	O
protein	NN	O	O
(	NN	O	O
CETP	NN	O	O
)	NN	O	O
mediates	NN	O	O
the	NN	O	O
transfer	NN	O	O
of	NN	O	O
cholesteryl	NN	O	O
esters	NN	O	O
from	NN	O	O
HDL	NN	O	O
to	NN	O	O
other	NN	O	O
lipoproteins	NN	O	O
and	NN	O	O
is	NN	O	O
a	NN	O	O
key	NN	O	O
participant	NN	O	O
in	NN	O	O
the	NN	O	O
reverse	NN	O	O
transport	NN	O	O
of	NN	O	O
cholesterol	NN	O	O
from	NN	O	O
the	NN	O	O
periphery	NN	O	O
to	NN	O	O
the	NN	O	O
liver	NN	O	O
.	NN	O	O

A	NN	O	O
high	NN	O	O
prevalence	NN	O	O
of	NN	O	O
two	NN	O	O
different	NN	O	O
CETP	NN	O	O
gene	NN	O	O
mutations	NN	O	O
(	NN	O	O
D442G	NN	O	O
,	NN	O	O
5	NN	O	O
.	NN	O	O
1	NN	O	O
%	NN	O	O
;	NN	O	O
intron	NN	O	O
14G	NN	O	O
A	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
5	NN	O	O
%	NN	O	O
)	NN	O	O
,	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
3	NN	O	O
,	NN	O	O
469	NN	O	O
men	NN	O	O
of	NN	O	O
Japanese	NN	O	O
ancestry	NN	O	O
in	NN	O	O
the	NN	O	O
Honolulu	NN	O	O
Heart	NN	O	O
Program	NN	O	O
and	NN	O	O
mutations	NN	O	O
were	NN	O	O
associated	NN	O	O
with	NN	O	O
decreased	NN	O	O
CETP	NN	O	O
(	NN	O	O
-	NN	O	O
35	NN	O	O
%	NN	O	O
)	NN	O	O
and	NN	O	O
increased	NN	O	O
HDL	NN	O	O
chol	NN	O	O
levels	NN	O	O
(	NN	O	O
+	NN	O	O
10	NN	O	O
%	NN	O	O
for	NN	O	O
D442G	NN	O	O
)	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
the	NN	O	O
overall	NN	O	O
prevalence	NN	O	O
of	NN	O	O
definite	NN	O	O
CHD	NN	O	B-Disease
was	NN	O	O
21	NN	O	O
%	NN	O	O
in	NN	O	O
men	NN	O	O
with	NN	O	O
mutations	NN	O	O
and	NN	O	O
16	NN	O	O
%	NN	O	O
in	NN	O	O
men	NN	O	O
without	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
relative	NN	O	O
risk	NN	O	O
(	NN	O	O
RR	NN	O	O
)	NN	O	O
of	NN	O	O
CHD	NN	O	B-Disease
was	NN	O	O
1	NN	O	O
.	NN	O	O

43	NN	O	O
in	NN	O	O
men	NN	O	O
with	NN	O	O
mutations	NN	O	O
(	NN	O	O
P	NN	O	O
<	NN	O	O
.	NN	O	O
05	NN	O	O
)	NN	O	O
;	NN	O	O
after	NN	O	O
adjustment	NN	O	O
for	NN	O	O
CHD	NN	O	B-Disease
risk	NN	O	O
factors	NN	O	O
,	NN	O	O
the	NN	O	O
RR	NN	O	O
was	NN	O	O
1	NN	O	O
.	NN	O	O

55	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
.	NN	O	O
02	NN	O	O
)	NN	O	O
;	NN	O	O
after	NN	O	O
additional	NN	O	O
adjustment	NN	O	O
for	NN	O	O
HDL	NN	O	O
levels	NN	O	O
,	NN	O	O
the	NN	O	O
RR	NN	O	O
was	NN	O	O
1	NN	O	O
.	NN	O	O

68	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
.	NN	O	O
008	NN	O	O
)	NN	O	O
.	NN	O	O

Similar	NN	O	O
RR	NN	O	O
values	NN	O	O
were	NN	O	O
obtained	NN	O	O
for	NN	O	O
the	NN	O	O
D442G	NN	O	O
mutation	NN	O	O
alone	NN	O	O
.	NN	O	O

Increased	NN	O	O
CHD	NN	O	B-Disease
in	NN	O	O
men	NN	O	O
with	NN	O	O
mutations	NN	O	O
was	NN	O	O
primarily	NN	O	O
observed	NN	O	O
for	NN	O	O
HDL	NN	O	O
chol	NN	O	O
41	NN	O	O
-	NN	O	O
60	NN	O	O
mg	NN	O	O
/	NN	O	O
dl	NN	O	O
;	NN	O	O
for	NN	O	O
HDL	NN	O	O
chol	NN	O	O
>	NN	O	O
60	NN	O	O
mg	NN	O	O
/	NN	O	O
dl	NN	O	O
men	NN	O	O
with	NN	O	O
and	NN	O	O
without	NN	O	O
mutations	NN	O	O
had	NN	O	O
low	NN	O	O
CHD	NN	O	B-Disease
prevalence	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
genetic	NN	O	O
CETP	NN	O	B-Disease
deficiency	NN	O	I-Disease
appears	NN	O	O
to	NN	O	O
be	NN	O	O
an	NN	O	O
independent	NN	O	O
risk	NN	O	O
factor	NN	O	O
for	NN	O	O
CHD	NN	O	B-Disease
,	NN	O	O
primarily	NN	O	O
due	NN	O	O
to	NN	O	O
increased	NN	O	O
CHD	NN	O	B-Disease
prevalence	NN	O	O
in	NN	O	O
men	NN	O	O
with	NN	O	O
the	NN	O	O
D442G	NN	O	O
mutation	NN	O	O
and	NN	O	O
HDL	NN	O	O
cholesterol	NN	O	O
between	NN	O	O
41	NN	O	O
and	NN	O	O
60	NN	O	O
mg	NN	O	O
/	NN	O	O
dl	NN	O	O
.	NN	O	O

The	NN	O	O
findings	NN	O	O
suggest	NN	O	O
that	NN	O	O
both	NN	O	O
HDL	NN	O	O
concentration	NN	O	O
and	NN	O	O
the	NN	O	O
dynamics	NN	O	O
of	NN	O	O
cholesterol	NN	O	O
transport	NN	O	O
through	NN	O	O
HDL	NN	O	O
(	NN	O	O
i	NN	O	O
.	NN	O	O
e	NN	O	O
.	NN	O	O
,	NN	O	O
reverse	NN	O	O
cholesterol	NN	O	O
transport	NN	O	O
)	NN	O	O
determine	NN	O	O
the	NN	O	O
anti	NN	O	O
-	NN	O	O
atherogenicity	NN	O	O
of	NN	O	O
the	NN	O	O
HDL	NN	O	O
fraction	NN	O	O
.	NN	O	O

Mapping	NN	O	O
the	NN	O	O
homolog	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
Rb1	NN	O	O
gene	NN	O	O
to	NN	O	O
chromosome	NN	O	O
14	NN	O	O
of	NN	O	O
higher	NN	O	O
primates	NN	O	O
.	NN	O	O

The	NN	O	O
Rb1	NN	O	O
gene	NN	O	O
has	NN	O	O
been	NN	O	O
implicated	NN	O	O
with	NN	O	O
retinoblastoma	NN	O	B-Disease
and	NN	O	O
is	NN	O	O
located	NN	O	O
on	NN	O	O
human	NN	O	O
Chromosome	NN	O	O
(	NN	O	O
Chr	NN	O	O
)	NN	O	O
13q14	NN	O	O
.	NN	O	O

2	NN	O	O
2	NN	O	O
.	NN	O	O

A	NN	O	O
unique	NN	O	O
sequence	NN	O	O
human	NN	O	O
Rb1	NN	O	O
cosmid	NN	O	O
DNA	NN	O	O
probe	NN	O	O
has	NN	O	O
been	NN	O	O
used	NN	O	O
to	NN	O	O
localize	NN	O	O
this	NN	O	O
region	NN	O	O
on	NN	O	O
apes	NN	O	O
Chr	NN	O	O
14	NN	O	O
by	NN	O	O
the	NN	O	O
FISH	NN	O	O
technique	NN	O	O
.	NN	O	O

The	NN	O	O
conservation	NN	O	O
of	NN	O	O
the	NN	O	O
Rb1	NN	O	O
gene	NN	O	O
in	NN	O	O
higher	NN	O	O
primates	NN	O	O
at	NN	O	O
the	NN	O	O
corresponding	NN	O	O
equivalent	NN	O	O
chromosome	NN	O	O
locus	NN	O	O
(	NN	O	O
14q14	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
may	NN	O	O
serve	NN	O	O
as	NN	O	O
a	NN	O	O
phylogenetic	NN	O	O
marker	NN	O	O
to	NN	O	O
further	NN	O	O
trace	NN	O	O
the	NN	O	O
evolutionary	NN	O	O
pathway	NN	O	O
of	NN	O	O
human	NN	O	O
descent	NN	O	O
.	NN	O	O

Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
:	NN	O	O
no	NN	O	O
strict	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
but	NN	O	O
clustering	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
amino	NN	O	O
-	NN	O	O
terminal	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
WASP	NN	O	O
gene	NN	O	O
product	NN	O	O
.	NN	O	O

The	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
protein	NN	O	O
(	NN	O	O
WASP	NN	O	O
)	NN	O	O
gene	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
mutated	NN	O	O
in	NN	O	O
patients	NN	O	O
presenting	NN	O	O
with	NN	O	O
WAS	NN	O	B-Disease
and	NN	O	O
in	NN	O	O
patients	NN	O	O
showing	NN	O	O
X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
thrombocytopenia	NN	O	I-Disease
.	NN	O	O

Mutation	NN	O	O
analysis	NN	O	O
in	NN	O	O
19	NN	O	O
families	NN	O	O
of	NN	O	O
German	NN	O	O
,	NN	O	O
Swiss	NN	O	O
and	NN	O	O
Turkish	NN	O	O
descent	NN	O	O
by	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
and	NN	O	O
sequencing	NN	O	O
resulted	NN	O	O
in	NN	O	O
the	NN	O	O
detection	NN	O	O
of	NN	O	O
seven	NN	O	O
novel	NN	O	O
and	NN	O	O
10	NN	O	O
known	NN	O	O
mutations	NN	O	O
.	NN	O	O

A	NN	O	O
striking	NN	O	O
clustering	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
first	NN	O	O
four	NN	O	O
exons	NN	O	O
contrasted	NN	O	O
with	NN	O	O
a	NN	O	O
random	NN	O	O
distribution	NN	O	O
of	NN	O	O
nonsense	NN	O	O
mutations	NN	O	O
.	NN	O	O

More	NN	O	O
than	NN	O	O
85	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
known	NN	O	O
missense	NN	O	O
mutations	NN	O	O
were	NN	O	O
localized	NN	O	O
in	NN	O	O
the	NN	O	O
amino	NN	O	O
-	NN	O	O
terminal	NN	O	O
stretch	NN	O	O
of	NN	O	O
the	NN	O	O
WASP	NN	O	O
gene	NN	O	O
product	NN	O	O
;	NN	O	O
this	NN	O	O
region	NN	O	O
contained	NN	O	O
a	NN	O	O
mutational	NN	O	O
hot	NN	O	O
spot	NN	O	O
at	NN	O	O
codon	NN	O	O
86	NN	O	O
.	NN	O	O

No	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlation	NN	O	O
emerged	NN	O	O
after	NN	O	O
a	NN	O	O
comparison	NN	O	O
of	NN	O	O
the	NN	O	O
identified	NN	O	O
mutations	NN	O	O
with	NN	O	O
the	NN	O	O
resulting	NN	O	O
clinical	NN	O	O
picture	NN	O	O
for	NN	O	O
a	NN	O	O
classical	NN	O	O
WAS	NN	O	B-Disease
phenotype	NN	O	O
.	NN	O	O

A	NN	O	O
substitution	NN	O	O
at	NN	O	O
codon	NN	O	O
86	NN	O	O
resulted	NN	O	O
in	NN	O	O
an	NN	O	O
extremely	NN	O	O
variable	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
in	NN	O	O
a	NN	O	O
large	NN	O	O
Swiss	NN	O	O
family	NN	O	O
.	NN	O	O

An	NN	O	O
extended	NN	O	O
homology	NN	O	O
search	NN	O	O
revealed	NN	O	O
a	NN	O	O
distant	NN	O	O
relationship	NN	O	O
of	NN	O	O
this	NN	O	O
stretch	NN	O	O
to	NN	O	O
the	NN	O	O
vasodilator	NN	O	O
-	NN	O	O
stimulated	NN	O	O
phosphoprotein	NN	O	O
(	NN	O	O
VASP	NN	O	O
)	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
maintenance	NN	O	O
of	NN	O	O
cyto	NN	O	O
-	NN	O	O
architecture	NN	O	O
by	NN	O	O
interacting	NN	O	O
with	NN	O	O
actin	NN	O	O
-	NN	O	O
like	NN	O	O
filaments	NN	O	O
.	NN	O	O
.	NN	O	O

Influence	NN	O	O
of	NN	O	O
PAX6	NN	O	O
gene	NN	O	O
dosage	NN	O	O
on	NN	O	O
development	NN	O	O
:	NN	O	O
overexpression	NN	O	O
causes	NN	O	O
severe	NN	O	O
eye	NN	O	B-Disease
abnormalities	NN	O	I-Disease
.	NN	O	O

Aniridia	NN	O	B-Disease
in	NN	O	O
man	NN	O	O
and	NN	O	O
Small	NN	O	O
eye	NN	O	O
in	NN	O	O
mice	NN	O	O
are	NN	O	O
semidominant	NN	O	B-Disease
developmental	NN	O	I-Disease
disorders	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
within	NN	O	O
the	NN	O	O
paired	NN	O	O
box	NN	O	O
gene	NN	O	O
PAX6	NN	O	O
.	NN	O	O

Whereas	NN	O	O
heterozygotes	NN	O	O
suffer	NN	O	O
from	NN	O	O
iris	NN	O	B-Disease
hypoplasia	NN	O	I-Disease
,	NN	O	O
homozygous	NN	O	O
mice	NN	O	O
lack	NN	O	O
eyes	NN	O	O
and	NN	O	O
nasal	NN	O	O
cavities	NN	O	O
and	NN	O	O
exhibit	NN	O	O
brain	NN	O	B-Disease
abnormalities	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
investigate	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
gene	NN	O	O
dosage	NN	O	O
in	NN	O	O
more	NN	O	O
detail	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
generated	NN	O	O
yeast	NN	O	O
artificial	NN	O	O
chromosome	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
carrying	NN	O	O
the	NN	O	O
human	NN	O	O
PAX6	NN	O	O
locus	NN	O	O
.	NN	O	O

When	NN	O	O
crossed	NN	O	O
onto	NN	O	O
the	NN	O	O
Small	NN	O	O
eye	NN	O	O
background	NN	O	O
,	NN	O	O
the	NN	O	O
transgene	NN	O	O
rescues	NN	O	O
the	NN	O	O
mutant	NN	O	O
phenotype	NN	O	O
.	NN	O	O

Strikingly	NN	O	O
,	NN	O	O
mice	NN	O	O
carrying	NN	O	O
multiple	NN	O	O
copies	NN	O	O
on	NN	O	O
a	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
background	NN	O	O
show	NN	O	O
specific	NN	O	O
developmental	NN	O	B-Disease
abnormalities	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
eye	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
not	NN	O	O
of	NN	O	O
other	NN	O	O
tissues	NN	O	O
expressing	NN	O	O
the	NN	O	O
gene	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
at	NN	O	O
least	NN	O	O
five	NN	O	O
different	NN	O	O
eye	NN	O	O
phenotypes	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
changes	NN	O	O
in	NN	O	O
PAX6	NN	O	O
expression	NN	O	O
.	NN	O	O

We	NN	O	O
provide	NN	O	O
evidence	NN	O	O
that	NN	O	O
not	NN	O	O
only	NN	O	O
reduced	NN	O	O
,	NN	O	O
but	NN	O	O
also	NN	O	O
increased	NN	O	O
levels	NN	O	O
of	NN	O	O
transcriptional	NN	O	O
regulators	NN	O	O
can	NN	O	O
cause	NN	O	O
developmental	NN	O	B-Disease
defects	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Heterodimer	NN	O	O
formation	NN	O	O
and	NN	O	O
activity	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
enzyme	NN	O	O
galactose	NN	O	O
-	NN	O	O
1	NN	O	O
-	NN	O	O
phosphate	NN	O	O
uridylyltransferase	NN	O	O
.	NN	O	O

One	NN	O	O
of	NN	O	O
the	NN	O	O
fundamental	NN	O	O
questions	NN	O	O
concerning	NN	O	O
expression	NN	O	O
and	NN	O	O
function	NN	O	O
of	NN	O	O
dimeric	NN	O	O
enzymes	NN	O	O
involves	NN	O	O
the	NN	O	O
impact	NN	O	O
of	NN	O	O
naturally	NN	O	O
occurring	NN	O	O
mutations	NN	O	O
on	NN	O	O
subunit	NN	O	O
assembly	NN	O	O
and	NN	O	O
heterodimer	NN	O	O
activity	NN	O	O
.	NN	O	O

This	NN	O	O
question	NN	O	O
is	NN	O	O
of	NN	O	O
particular	NN	O	O
interest	NN	O	O
for	NN	O	O
the	NN	O	O
human	NN	O	O
enzyme	NN	O	O
galactose	NN	O	O
-	NN	O	O
l	NN	O	O
-	NN	O	O
phosphate	NN	O	O
uridylyl	NN	O	O
-	NN	O	O
transferase	NN	O	O
(	NN	O	O
GALT	NN	O	O
)	NN	O	O
,	NN	O	O
impairment	NN	O	O
of	NN	O	O
which	NN	O	O
results	NN	O	O
in	NN	O	O
the	NN	O	O
inherited	NN	O	B-Disease
metabolic	NN	O	I-Disease
disorder	NN	O	I-Disease
galactosemia	NN	O	B-Disease
,	NN	O	O
because	NN	O	O
many	NN	O	O
if	NN	O	O
not	NN	O	O
most	NN	O	O
patients	NN	O	O
studied	NN	O	O
to	NN	O	O
date	NN	O	O
are	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
rather	NN	O	O
than	NN	O	O
true	NN	O	O
molecular	NN	O	O
homozygotes	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
the	NN	O	O
broad	NN	O	O
range	NN	O	O
of	NN	O	O
phenotypic	NN	O	O
severity	NN	O	O
observed	NN	O	O
in	NN	O	O
these	NN	O	O
patients	NN	O	O
raises	NN	O	O
the	NN	O	O
possibility	NN	O	O
that	NN	O	O
allelic	NN	O	O
combination	NN	O	O
,	NN	O	O
not	NN	O	O
just	NN	O	O
allelic	NN	O	O
constitution	NN	O	O
,	NN	O	O
may	NN	O	O
play	NN	O	O
some	NN	O	O
role	NN	O	O
in	NN	O	O
determining	NN	O	O
outcome	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
work	NN	O	O
described	NN	O	O
herein	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
selected	NN	O	O
two	NN	O	O
distinct	NN	O	O
naturally	NN	O	O
occurring	NN	O	O
null	NN	O	O
mutations	NN	O	O
of	NN	O	O
GALT	NN	O	O
,	NN	O	O
Q188R	NN	O	O
and	NN	O	O
R333W	NN	O	O
,	NN	O	O
and	NN	O	O
asked	NN	O	O
the	NN	O	O
questions	NN	O	O
(	NN	O	O
i	NN	O	O
)	NN	O	O
what	NN	O	O
are	NN	O	O
the	NN	O	O
impacts	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
on	NN	O	O
subunit	NN	O	O
assembly	NN	O	O
,	NN	O	O
and	NN	O	O
(	NN	O	O
ii	NN	O	O
)	NN	O	O
if	NN	O	O
heterodimers	NN	O	O
do	NN	O	O
form	NN	O	O
,	NN	O	O
are	NN	O	O
they	NN	O	O
active	NN	O	O
?	NN	O	O

To	NN	O	O
answer	NN	O	O
these	NN	O	O
questions	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
established	NN	O	O
a	NN	O	O
yeast	NN	O	O
system	NN	O	O
for	NN	O	O
the	NN	O	O
coexpression	NN	O	O
of	NN	O	O
epitope	NN	O	O
-	NN	O	O
tagged	NN	O	O
alleles	NN	O	O
of	NN	O	O
human	NN	O	O
GALT	NN	O	O
and	NN	O	O
investigated	NN	O	O
both	NN	O	O
the	NN	O	O
extent	NN	O	O
of	NN	O	O
specific	NN	O	O
GALT	NN	O	O
subunit	NN	O	O
interactions	NN	O	O
and	NN	O	O
the	NN	O	O
activity	NN	O	O
of	NN	O	O
defined	NN	O	O
heterodimer	NN	O	O
pools	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
found	NN	O	O
that	NN	O	O
both	NN	O	O
homodimers	NN	O	O
and	NN	O	O
heterodimers	NN	O	O
do	NN	O	O
form	NN	O	O
involving	NN	O	O
each	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
subunits	NN	O	O
tested	NN	O	O
and	NN	O	O
that	NN	O	O
both	NN	O	O
heterodimer	NN	O	O
pools	NN	O	O
retain	NN	O	O
substantial	NN	O	O
enzymatic	NN	O	O
activity	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
are	NN	O	O
significant	NN	O	O
not	NN	O	O
only	NN	O	O
in	NN	O	O
terms	NN	O	O
of	NN	O	O
their	NN	O	O
implications	NN	O	O
for	NN	O	O
furthering	NN	O	O
our	NN	O	O
understanding	NN	O	O
of	NN	O	O
galactosemia	NN	O	B-Disease
and	NN	O	O
GALT	NN	O	O
holoenzyme	NN	O	O
structure	NN	O	O
-	NN	O	O
function	NN	O	O
relationships	NN	O	O
but	NN	O	O
also	NN	O	O
because	NN	O	O
the	NN	O	O
system	NN	O	O
described	NN	O	O
may	NN	O	O
serve	NN	O	O
as	NN	O	O
a	NN	O	O
model	NN	O	O
for	NN	O	O
similar	NN	O	O
studies	NN	O	O
of	NN	O	O
other	NN	O	O
complexes	NN	O	O
composed	NN	O	O
of	NN	O	O
multiple	NN	O	O
subunits	NN	O	O
.	NN	O	O
.	NN	O	O

Cleavage	NN	O	O
of	NN	O	O
huntingtin	NN	O	O
by	NN	O	O
apopain	NN	O	O
,	NN	O	O
a	NN	O	O
proapoptotic	NN	O	O
cysteine	NN	O	O
protease	NN	O	O
,	NN	O	O
is	NN	O	O
modulated	NN	O	O
by	NN	O	O
the	NN	O	O
polyglutamine	NN	O	O
tract	NN	O	O
.	NN	O	O

Apoptosis	NN	O	O
has	NN	O	O
recently	NN	O	O
been	NN	O	O
recognized	NN	O	O
as	NN	O	O
a	NN	O	O
mode	NN	O	O
of	NN	O	O
cell	NN	O	O
death	NN	O	O
in	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
HD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Apopain	NN	O	O
,	NN	O	O
a	NN	O	O
human	NN	O	O
counterpart	NN	O	O
of	NN	O	O
the	NN	O	O
nematode	NN	O	O
cysteine	NN	O	O
protease	NN	O	O
death	NN	O	O
-	NN	O	O
gene	NN	O	O
product	NN	O	O
,	NN	O	O
CED	NN	O	O
-	NN	O	O
3	NN	O	O
,	NN	O	O
has	NN	O	O
a	NN	O	O
key	NN	O	O
role	NN	O	O
in	NN	O	O
proteolytic	NN	O	O
events	NN	O	O
leading	NN	O	O
to	NN	O	O
apoptosis	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
show	NN	O	O
that	NN	O	O
apoptotic	NN	O	O
extracts	NN	O	O
and	NN	O	O
apopain	NN	O	O
itself	NN	O	O
specifically	NN	O	O
cleave	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
gene	NN	O	O
product	NN	O	O
,	NN	O	O
huntingtin	NN	O	O
.	NN	O	O

The	NN	O	O
rate	NN	O	O
of	NN	O	O
cleavage	NN	O	O
increases	NN	O	O
with	NN	O	O
the	NN	O	O
length	NN	O	O
of	NN	O	O
the	NN	O	O
huntingtin	NN	O	O
polyglutamine	NN	O	O
tract	NN	O	O
,	NN	O	O
providing	NN	O	O
an	NN	O	O
explanation	NN	O	O
for	NN	O	O
the	NN	O	O
gain	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
function	NN	O	O
associated	NN	O	O
with	NN	O	O
CAG	NN	O	O
expansion	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
show	NN	O	O
that	NN	O	O
huntingtin	NN	O	O
is	NN	O	O
cleaved	NN	O	O
by	NN	O	O
cysteine	NN	O	O
proteases	NN	O	O
and	NN	O	O
suggest	NN	O	O
that	NN	O	O
HD	NN	O	B-Disease
might	NN	O	O
be	NN	O	O
a	NN	O	O
disorder	NN	O	B-Disease
of	NN	O	I-Disease
inappropriate	NN	O	I-Disease
apoptosis	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

The	NN	O	O
5	NN	O	O
'	NN	O	O
end	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
lies	NN	O	O
within	NN	O	O
a	NN	O	O
duplicated	NN	O	O
region	NN	O	O
of	NN	O	O
human	NN	O	O
chromosome	NN	O	O
17q21	NN	O	O
.	NN	O	O

To	NN	O	O
begin	NN	O	O
to	NN	O	O
address	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
abnormal	NN	O	O
regulation	NN	O	O
of	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
susceptibility	NN	O	O
gene	NN	O	O
BRCA1	NN	O	O
is	NN	O	O
a	NN	O	O
critical	NN	O	O
step	NN	O	O
in	NN	O	O
sporadic	NN	O	O
breast	NN	O	O
/	NN	O	O
ovarian	NN	O	O
tumorigenesis	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
determined	NN	O	O
the	NN	O	O
detailed	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
genomic	NN	O	O
region	NN	O	O
.	NN	O	O

We	NN	O	O
show	NN	O	O
that	NN	O	O
this	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
genome	NN	O	O
contains	NN	O	O
a	NN	O	O
tandem	NN	O	O
duplication	NN	O	O
of	NN	O	O
approximately	NN	O	O
30	NN	O	O
kilobases	NN	O	O
,	NN	O	O
which	NN	O	O
results	NN	O	O
in	NN	O	O
two	NN	O	O
copies	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
exons	NN	O	O
1	NN	O	O
and	NN	O	O
2	NN	O	O
,	NN	O	O
of	NN	O	O
exons	NN	O	O
1	NN	O	O
and	NN	O	O
3	NN	O	O
of	NN	O	O
the	NN	O	O
adjacent	NN	O	O
1A1	NN	O	O
-	NN	O	O
3B	NN	O	O
gene	NN	O	O
and	NN	O	O
of	NN	O	O
the	NN	O	O
previously	NN	O	O
reported	NN	O	O
295	NN	O	O
base	NN	O	O
pair	NN	O	O
intergenic	NN	O	O
region	NN	O	O
.	NN	O	O

Sequence	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
duplicated	NN	O	O
exons	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
1A1	NN	O	O
-	NN	O	O
3B	NN	O	O
and	NN	O	O
flanking	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
reveals	NN	O	O
maintenance	NN	O	O
of	NN	O	O
the	NN	O	O
intron	NN	O	O
-	NN	O	O
exon	NN	O	O
structure	NN	O	O
and	NN	O	O
a	NN	O	O
high	NN	O	O
degree	NN	O	O
of	NN	O	O
nucleotide	NN	O	O
sequence	NN	O	O
identity	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
these	NN	O	O
are	NN	O	O
non	NN	O	O
-	NN	O	O
processed	NN	O	O
pseudogenes	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
duplication	NN	O	O
is	NN	O	O
a	NN	O	O
recent	NN	O	O
event	NN	O	O
in	NN	O	O
evolutionary	NN	O	O
terms	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
show	NN	O	O
that	NN	O	O
a	NN	O	O
processed	NN	O	O
pseudogene	NN	O	O
of	NN	O	O
the	NN	O	O
acidic	NN	O	O
ribosomal	NN	O	O
phosphoprotein	NN	O	O
P1	NN	O	O
(	NN	O	O
ARPP1	NN	O	O
)	NN	O	O
is	NN	O	O
inserted	NN	O	O
directly	NN	O	O
upstream	NN	O	O
of	NN	O	O
pseudo	NN	O	O
-	NN	O	O
BRCA1	NN	O	O
exon	NN	O	O
1A	NN	O	O
.	NN	O	O

We	NN	O	O
believe	NN	O	O
that	NN	O	O
these	NN	O	O
findings	NN	O	O
could	NN	O	O
not	NN	O	O
only	NN	O	O
confound	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
,	NN	O	O
but	NN	O	O
could	NN	O	O
have	NN	O	O
implications	NN	O	O
for	NN	O	O
the	NN	O	O
normal	NN	O	O
and	NN	O	O
abnormal	NN	O	O
regulation	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
transcription	NN	O	O
,	NN	O	O
translation	NN	O	O
and	NN	O	O
function	NN	O	O
.	NN	O	O
.	NN	O	O

Deletion	NN	O	O
of	NN	O	O
small	NN	O	O
nuclear	NN	O	O
ribonucleoprotein	NN	O	O
polypeptide	NN	O	O
N	NN	O	O
(	NN	O	O
SNRPN	NN	O	O
)	NN	O	O
in	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
detected	NN	O	O
by	NN	O	O
fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
:	NN	O	O
two	NN	O	O
sibs	NN	O	O
with	NN	O	O
the	NN	O	O
typical	NN	O	O
phenotype	NN	O	O
without	NN	O	O
a	NN	O	O
cytogenetic	NN	O	O
deletion	NN	O	O
in	NN	O	O
chromosome	NN	O	O
15q	NN	O	O
.	NN	O	O

The	NN	O	O
small	NN	O	O
nuclear	NN	O	O
ribonucleoprotein	NN	O	O
polypeptide	NN	O	O
N	NN	O	O
(	NN	O	O
SNRPN	NN	O	O
)	NN	O	O
gene	NN	O	O
is	NN	O	O
regarded	NN	O	O
as	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
candidates	NN	O	O
for	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
PWS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

We	NN	O	O
describe	NN	O	O
two	NN	O	O
sibs	NN	O	O
with	NN	O	O
typical	NN	O	O
PWS	NN	O	B-Disease
presenting	NN	O	O
deletion	NN	O	O
of	NN	O	O
SNRPN	NN	O	O
detected	NN	O	O
by	NN	O	O
fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
(	NN	O	O
FISH	NN	O	O
)	NN	O	O
.	NN	O	O

Neither	NN	O	O
a	NN	O	O
cytogenetically	NN	O	O
detectable	NN	O	O
15q12	NN	O	O
deletion	NN	O	O
nor	NN	O	O
a	NN	O	O
deletion	NN	O	O
for	NN	O	O
the	NN	O	O
D15S11	NN	O	O
,	NN	O	O
D15S10	NN	O	O
,	NN	O	O
and	NN	O	O
GABRB3	NN	O	O
cosmid	NN	O	O
probes	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
either	NN	O	O
patient	NN	O	O
.	NN	O	O

This	NN	O	O
implies	NN	O	O
a	NN	O	O
smaller	NN	O	O
deletion	NN	O	O
limited	NN	O	O
to	NN	O	O
the	NN	O	O
PWS	NN	O	B-Disease
critical	NN	O	O
region	NN	O	O
.	NN	O	O

FISH	NN	O	O
with	NN	O	O
a	NN	O	O
SNRPN	NN	O	O
probe	NN	O	O
will	NN	O	O
permit	NN	O	O
analysis	NN	O	O
of	NN	O	O
PWS	NN	O	B-Disease
patients	NN	O	O
with	NN	O	O
limited	NN	O	O
deletions	NN	O	O
not	NN	O	O
detectable	NN	O	O
with	NN	O	O
other	NN	O	O
probes	NN	O	O
.	NN	O	O
.	NN	O	O

Expression	NN	O	O
of	NN	O	O
the	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
disease	NN	O	I-Disease
tumour	NN	O	I-Disease
suppressor	NN	O	O
gene	NN	O	O
during	NN	O	O
human	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

The	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
(	NN	O	I-Disease
VHL	NN	O	I-Disease
)	NN	O	I-Disease
disease	NN	O	I-Disease
product	NN	O	O
is	NN	O	O
thought	NN	O	O
to	NN	O	O
down	NN	O	O
-	NN	O	O
regulate	NN	O	O
transcription	NN	O	O
by	NN	O	O
antagonizing	NN	O	O
elongin	NN	O	O
-	NN	O	O
enhanced	NN	O	O
transcriptional	NN	O	O
elongation	NN	O	O
.	NN	O	O

Germline	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
mutations	NN	O	O
predispose	NN	O	O
to	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
retinal	NN	O	B-Disease
,	NN	O	I-Disease
cerebellar	NN	O	I-Disease
and	NN	O	I-Disease
spinal	NN	O	I-Disease
haemangioblastomas	NN	O	I-Disease
,	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
phaeochromocytoma	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
somatic	NN	O	O
Inactivation	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
is	NN	O	O
frequent	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
haemangioblastoma	NN	O	B-Disease
.	NN	O	O

Regulation	NN	O	O
of	NN	O	O
transcript	NN	O	O
elongation	NN	O	O
is	NN	O	O
an	NN	O	O
important	NN	O	O
control	NN	O	O
mechanism	NN	O	O
for	NN	O	O
gene	NN	O	O
expression	NN	O	O
and	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
might	NN	O	O
modify	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
proto	NN	O	O
-	NN	O	O
oncogenes	NN	O	O
and	NN	O	O
growth	NN	O	O
suppressor	NN	O	O
genes	NN	O	O
during	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

We	NN	O	O
therefore	NN	O	O
investigated	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
VHL	NN	O	B-Disease
mRNA	NN	O	O
during	NN	O	O
human	NN	O	O
embryogenesis	NN	O	O
by	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
studies	NN	O	O
at	NN	O	O
4	NN	O	O
,	NN	O	O
6	NN	O	O
and	NN	O	O
10	NN	O	O
weeks	NN	O	O
post	NN	O	O
conception	NN	O	O
.	NN	O	O

Although	NN	O	O
VHL	NN	O	B-Disease
mRNA	NN	O	O
was	NN	O	O
expressed	NN	O	O
in	NN	O	O
all	NN	O	O
three	NN	O	O
germ	NN	O	O
layers	NN	O	O
,	NN	O	O
strong	NN	O	O
expression	NN	O	O
was	NN	O	O
noted	NN	O	O
in	NN	O	O
the	NN	O	O
central	NN	O	O
nervous	NN	O	O
system	NN	O	O
,	NN	O	O
kidneys	NN	O	O
,	NN	O	O
testis	NN	O	O
and	NN	O	O
lung	NN	O	O
.	NN	O	O

Within	NN	O	O
the	NN	O	O
kidney	NN	O	O
,	NN	O	O
VHL	NN	O	B-Disease
mRNA	NN	O	O
was	NN	O	O
differentially	NN	O	O
expressed	NN	O	O
within	NN	O	O
renal	NN	O	O
tubules	NN	O	O
suggesting	NN	O	O
that	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
product	NN	O	O
may	NN	O	O
have	NN	O	O
a	NN	O	O
specific	NN	O	O
role	NN	O	O
in	NN	O	O
kidney	NN	O	O
development	NN	O	O
.	NN	O	O

Two	NN	O	O
alternatively	NN	O	O
spliced	NN	O	O
VHL	NN	O	B-Disease
mRNAs	NN	O	O
characterized	NN	O	O
by	NN	O	O
inclusion	NN	O	O
(	NN	O	O
isoform	NN	O	O
I	NN	O	O
)	NN	O	O
or	NN	O	O
exclusion	NN	O	O
(	NN	O	O
isoform	NN	O	O
II	NN	O	O
)	NN	O	O
of	NN	O	O
exon	NN	O	O
2	NN	O	O
are	NN	O	O
transcribed	NN	O	O
in	NN	O	O
adult	NN	O	O
tissues	NN	O	O
.	NN	O	O

To	NN	O	O
investigate	NN	O	O
if	NN	O	O
the	NN	O	O
two	NN	O	O
isoforms	NN	O	O
are	NN	O	O
differentially	NN	O	O
expressed	NN	O	O
during	NN	O	O
embryogenesis	NN	O	O
,	NN	O	O
VHL	NN	O	B-Disease
mRNA	NN	O	O
was	NN	O	O
reverse	NN	O	O
transcribed	NN	O	O
from	NN	O	O
13	NN	O	O
fetal	NN	O	O
tissues	NN	O	O
(	NN	O	O
8	NN	O	O
-	NN	O	O
10	NN	O	O
weeks	NN	O	O
gestation	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
quantitative	NN	O	O
distribution	NN	O	O
of	NN	O	O
VHL	NN	O	B-Disease
mRNA	NN	O	O
within	NN	O	O
fetal	NN	O	O
tissues	NN	O	O
reflected	NN	O	O
that	NN	O	O
seen	NN	O	O
by	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
and	NN	O	O
the	NN	O	O
ratio	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
VHL	NN	O	B-Disease
isoforms	NN	O	O
was	NN	O	O
similar	NN	O	O
between	NN	O	O
tissues	NN	O	O
.	NN	O	O

Although	NN	O	O
the	NN	O	O
genes	NN	O	O
regulated	NN	O	O
by	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
product	NN	O	O
have	NN	O	O
not	NN	O	O
yet	NN	O	O
been	NN	O	O
identified	NN	O	O
,	NN	O	O
our	NN	O	O
findings	NN	O	O
are	NN	O	O
compatible	NN	O	O
with	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
VHL	NN	O	B-Disease
-	NN	O	O
mediated	NN	O	O
control	NN	O	O
of	NN	O	O
transcriptional	NN	O	O
elongation	NN	O	O
may	NN	O	O
have	NN	O	O
a	NN	O	O
role	NN	O	O
in	NN	O	O
normal	NN	O	O
human	NN	O	O
development	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
heterogeneity	NN	O	O
in	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
:	NN	O	O
role	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

The	NN	O	O
common	NN	O	O
hereditary	NN	O	O
forms	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
have	NN	O	O
been	NN	O	O
largely	NN	O	O
attributed	NN	O	O
to	NN	O	O
the	NN	O	O
inheritance	NN	O	O
of	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
genes	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
it	NN	O	O
is	NN	O	O
not	NN	O	O
yet	NN	O	O
clear	NN	O	O
what	NN	O	O
proportion	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
is	NN	O	O
explained	NN	O	O
by	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
or	NN	O	O
by	NN	O	O
some	NN	O	O
other	NN	O	O
unidentified	NN	O	O
susceptibility	NN	O	O
gene	NN	O	O
(	NN	O	O
s	NN	O	O
)	NN	O	O
.	NN	O	O

We	NN	O	O
describe	NN	O	O
the	NN	O	O
proportion	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
explained	NN	O	O
by	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
in	NN	O	O
a	NN	O	O
sample	NN	O	O
of	NN	O	O
North	NN	O	O
American	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancers	NN	O	I-Disease
and	NN	O	O
assess	NN	O	O
the	NN	O	O
evidence	NN	O	O
for	NN	O	O
additional	NN	O	O
susceptibility	NN	O	O
genes	NN	O	O
that	NN	O	O
may	NN	O	O
confer	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
risk	NN	O	O
.	NN	O	O

Twenty	NN	O	O
-	NN	O	O
three	NN	O	O
families	NN	O	O
were	NN	O	O
identified	NN	O	O
through	NN	O	O
two	NN	O	O
high	NN	O	O
-	NN	O	O
risk	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
research	NN	O	O
programs	NN	O	O
.	NN	O	O

Genetic	NN	O	O
analysis	NN	O	O
was	NN	O	O
undertaken	NN	O	O
to	NN	O	O
establish	NN	O	O
linkage	NN	O	O
between	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
and	NN	O	O
markers	NN	O	O
on	NN	O	O
chromosomes	NN	O	O
17q	NN	O	O
(	NN	O	O
BRCA1	NN	O	O
)	NN	O	O
and	NN	O	O
13q	NN	O	O
(	NN	O	O
BRCA2	NN	O	O
)	NN	O	O
.	NN	O	O

Mutation	NN	O	O
analysis	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
genes	NN	O	O
was	NN	O	O
also	NN	O	O
undertaken	NN	O	O
in	NN	O	O
all	NN	O	O
families	NN	O	O
.	NN	O	O

The	NN	O	O
pattern	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
14	NN	O	O
(	NN	O	O
61	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
23	NN	O	O
families	NN	O	O
studied	NN	O	O
was	NN	O	O
attributed	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
by	NN	O	O
a	NN	O	O
combination	NN	O	O
of	NN	O	O
linkage	NN	O	O
and	NN	O	O
mutation	NN	O	O
analyses	NN	O	O
.	NN	O	O

No	NN	O	O
families	NN	O	O
were	NN	O	O
attributed	NN	O	O
to	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

Five	NN	O	O
families	NN	O	O
(	NN	O	O
22	NN	O	O
%	NN	O	O
)	NN	O	O
provided	NN	O	O
evidence	NN	O	O
against	NN	O	O
linkage	NN	O	O
to	NN	O	O
both	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

No	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
these	NN	O	O
five	NN	O	O
families	NN	O	O
.	NN	O	O

The	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
status	NN	O	O
of	NN	O	O
four	NN	O	O
families	NN	O	O
(	NN	O	O
17	NN	O	O
%	NN	O	O
)	NN	O	O
could	NN	O	O
not	NN	O	O
be	NN	O	O
determined	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
probably	NN	O	O
explain	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
that	NN	O	O
exists	NN	O	O
in	NN	O	O
the	NN	O	O
North	NN	O	O
American	NN	O	O
population	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
one	NN	O	O
or	NN	O	O
more	NN	O	O
additional	NN	O	O
genes	NN	O	O
may	NN	O	O
yet	NN	O	O
be	NN	O	O
found	NN	O	O
that	NN	O	O
explain	NN	O	O
some	NN	O	O
proportion	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

An	NN	O	O
intronic	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
lariat	NN	O	O
branchpoint	NN	O	O
sequence	NN	O	O
is	NN	O	O
a	NN	O	O
direct	NN	O	O
cause	NN	O	O
of	NN	O	O
an	NN	O	O
inherited	NN	O	B-Disease
human	NN	O	I-Disease
disorder	NN	O	I-Disease
(	NN	O	O
fish	NN	O	B-Disease
-	NN	O	I-Disease
eye	NN	O	I-Disease
disease	NN	O	I-Disease
)	NN	O	O
.	NN	O	O

The	NN	O	O
first	NN	O	O
step	NN	O	O
in	NN	O	O
the	NN	O	O
splicing	NN	O	O
of	NN	O	O
an	NN	O	O
intron	NN	O	O
from	NN	O	O
nuclear	NN	O	O
precursors	NN	O	O
of	NN	O	O
mRNA	NN	O	O
results	NN	O	O
in	NN	O	O
the	NN	O	O
formation	NN	O	O
of	NN	O	O
a	NN	O	O
lariat	NN	O	O
structure	NN	O	O
.	NN	O	O

A	NN	O	O
distinct	NN	O	O
intronic	NN	O	O
nucleotide	NN	O	O
sequence	NN	O	O
,	NN	O	O
known	NN	O	O
as	NN	O	O
the	NN	O	O
branchpoint	NN	O	O
region	NN	O	O
,	NN	O	O
plays	NN	O	O
a	NN	O	O
central	NN	O	O
role	NN	O	O
in	NN	O	O
this	NN	O	O
process	NN	O	O
.	NN	O	O

We	NN	O	O
here	NN	O	O
describe	NN	O	O
a	NN	O	O
point	NN	O	O
mutation	NN	O	O
in	NN	O	O
such	NN	O	O
a	NN	O	O
sequence	NN	O	O
.	NN	O	O

Three	NN	O	O
sisters	NN	O	O
were	NN	O	O
shown	NN	O	O
to	NN	O	O
suffer	NN	O	O
from	NN	O	O
fish	NN	O	B-Disease
-	NN	O	I-Disease
eye	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
FED	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
a	NN	O	O
disorder	NN	O	O
which	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
gene	NN	O	O
coding	NN	O	O
for	NN	O	O
lecithin	NN	O	O
cholesterol	NN	O	O
acyltransferase	NN	O	O
(	NN	O	O
LCAT	NN	O	O
)	NN	O	O
.	NN	O	O

Sequencing	NN	O	O
of	NN	O	O
the	NN	O	O
LCAT	NN	O	O
gene	NN	O	O
of	NN	O	O
all	NN	O	O
three	NN	O	O
probands	NN	O	O
revealed	NN	O	O
compound	NN	O	O
heterozygosity	NN	O	O
for	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
which	NN	O	O
is	NN	O	O
reported	NN	O	O
to	NN	O	O
underlie	NN	O	O
the	NN	O	O
FED	NN	O	B-Disease
phenotype	NN	O	O
,	NN	O	O
and	NN	O	O
a	NN	O	O
point	NN	O	O
mutation	NN	O	O
located	NN	O	O
in	NN	O	O
intron	NN	O	O
4	NN	O	O
(	NN	O	O
IVS4	NN	O	O
T	NN	O	O
-	NN	O	O
22C	NN	O	O
)	NN	O	O
.	NN	O	O

By	NN	O	O
performing	NN	O	O
in	NN	O	O
vitro	NN	O	O
expression	NN	O	O
of	NN	O	O
LCAT	NN	O	O
minigenes	NN	O	O
and	NN	O	O
reverse	NN	O	O
transcriptase	NN	O	O
PCR	NN	O	O
on	NN	O	O
mRNA	NN	O	O
isolated	NN	O	O
from	NN	O	O
leukocytes	NN	O	O
of	NN	O	O
the	NN	O	O
patient	NN	O	O
,	NN	O	O
this	NN	O	O
gene	NN	O	O
defect	NN	O	O
was	NN	O	O
shown	NN	O	O
to	NN	O	O
cause	NN	O	O
a	NN	O	O
null	NN	O	O
allele	NN	O	O
as	NN	O	O
the	NN	O	O
result	NN	O	O
of	NN	O	O
complete	NN	O	O
intron	NN	O	O
retention	NN	O	O
.	NN	O	O

In	NN	O	O
conclusion	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
a	NN	O	O
point	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
lariat	NN	O	O
branchpoint	NN	O	O
consensus	NN	O	O
sequence	NN	O	O
causes	NN	O	O
a	NN	O	O
null	NN	O	O
allele	NN	O	O
in	NN	O	O
a	NN	O	O
patient	NN	O	O
with	NN	O	O
FED	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
our	NN	O	O
finding	NN	O	O
illustrates	NN	O	O
the	NN	O	O
importance	NN	O	O
of	NN	O	O
this	NN	O	O
sequence	NN	O	O
for	NN	O	O
normal	NN	O	O
human	NN	O	O
mRNA	NN	O	O
processing	NN	O	O
.	NN	O	O

Finally	NN	O	O
,	NN	O	O
this	NN	O	O
report	NN	O	O
provides	NN	O	O
a	NN	O	O
widely	NN	O	O
applicable	NN	O	O
strategy	NN	O	O
which	NN	O	O
ensures	NN	O	O
fast	NN	O	O
and	NN	O	O
effective	NN	O	O
screening	NN	O	O
for	NN	O	O
intronic	NN	O	O
defects	NN	O	O
that	NN	O	O
underlie	NN	O	O
differential	NN	O	O
gene	NN	O	O
expression	NN	O	O
.	NN	O	O
.	NN	O	O

Mutations	NN	O	O
associated	NN	O	O
with	NN	O	O
variant	NN	O	O
phenotypes	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
identified	NN	O	O
14	NN	O	O
families	NN	O	O
with	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
in	NN	O	O
which	NN	O	O
mutation	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
less	NN	O	O
severe	NN	O	O
clinical	NN	O	O
and	NN	O	O
cellular	NN	O	O
phenotype	NN	O	O
(	NN	O	O
approximately	NN	O	O
10	NN	O	O
%	NN	O	O
-	NN	O	O
15	NN	O	O
%	NN	O	O
of	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
families	NN	O	O
identified	NN	O	O
in	NN	O	O
the	NN	O	O
United	NN	O	O
Kingdom	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
10	NN	O	O
of	NN	O	O
these	NN	O	O
families	NN	O	O
,	NN	O	O
all	NN	O	O
the	NN	O	O
homozygotes	NN	O	O
have	NN	O	O
a	NN	O	O
137	NN	O	O
-	NN	O	O
bp	NN	O	O
insertion	NN	O	O
in	NN	O	O
their	NN	O	O
cDNA	NN	O	O
caused	NN	O	O
by	NN	O	O
a	NN	O	O
point	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
sequence	NN	O	O
resembling	NN	O	O
a	NN	O	O
splice	NN	O	O
-	NN	O	O
donor	NN	O	O
site	NN	O	O
.	NN	O	O

The	NN	O	O
second	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
allele	NN	O	O
has	NN	O	O
a	NN	O	O
different	NN	O	O
mutation	NN	O	O
in	NN	O	O
each	NN	O	O
patient	NN	O	O
.	NN	O	O

We	NN	O	O
show	NN	O	O
that	NN	O	O
the	NN	O	O
less	NN	O	O
severe	NN	O	O
phenotype	NN	O	O
in	NN	O	O
these	NN	O	O
patients	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
some	NN	O	O
degree	NN	O	O
of	NN	O	O
normal	NN	O	O
splicing	NN	O	O
,	NN	O	O
which	NN	O	O
occurs	NN	O	O
as	NN	O	O
an	NN	O	O
alternative	NN	O	O
product	NN	O	O
from	NN	O	O
the	NN	O	O
insertion	NN	O	O
-	NN	O	O
containing	NN	O	O
allele	NN	O	O
.	NN	O	O

The	NN	O	O
level	NN	O	O
of	NN	O	O
the	NN	O	O
137	NN	O	O
-	NN	O	O
bp	NN	O	O
PCR	NN	O	O
product	NN	O	O
containing	NN	O	O
the	NN	O	O
insertion	NN	O	O
was	NN	O	O
lowest	NN	O	O
in	NN	O	O
two	NN	O	O
patients	NN	O	O
who	NN	O	O
showed	NN	O	O
a	NN	O	O
later	NN	O	O
onset	NN	O	O
of	NN	O	O
cerebellar	NN	O	B-Disease
ataxia	NN	O	I-Disease
.	NN	O	O

A	NN	O	O
further	NN	O	O
four	NN	O	O
families	NN	O	O
who	NN	O	O
do	NN	O	O
not	NN	O	O
have	NN	O	O
this	NN	O	O
insertion	NN	O	O
have	NN	O	O
been	NN	O	O
identified	NN	O	O
.	NN	O	O

Mutations	NN	O	O
detected	NN	O	O
in	NN	O	O
two	NN	O	O
of	NN	O	O
four	NN	O	O
of	NN	O	O
these	NN	O	O
are	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
normally	NN	O	O
rare	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

The	NN	O	O
demonstration	NN	O	O
of	NN	O	O
mutations	NN	O	O
giving	NN	O	O
rise	NN	O	O
to	NN	O	O
a	NN	O	O
slightly	NN	O	O
milder	NN	O	O
phenotype	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
raises	NN	O	O
the	NN	O	O
interesting	NN	O	O
question	NN	O	O
of	NN	O	O
what	NN	O	O
range	NN	O	O
of	NN	O	O
phenotypes	NN	O	O
might	NN	O	O
occur	NN	O	O
in	NN	O	O
individuals	NN	O	O
in	NN	O	O
whom	NN	O	O
both	NN	O	O
mutations	NN	O	O
are	NN	O	O
milder	NN	O	O
.	NN	O	O

One	NN	O	O
possibility	NN	O	O
might	NN	O	O
be	NN	O	O
that	NN	O	O
individuals	NN	O	O
who	NN	O	O
are	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
are	NN	O	O
more	NN	O	O
common	NN	O	O
than	NN	O	O
we	NN	O	O
realize	NN	O	O
.	NN	O	O
.	NN	O	O

Mutation	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	O
gene	NN	O	O
is	NN	O	O
associated	NN	O	O
exclusively	NN	O	O
with	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
papillary	NN	O	I-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
define	NN	O	O
the	NN	O	O
possible	NN	O	O
role	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
in	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
sporadic	NN	O	B-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
91	NN	O	O
different	NN	O	O
parenchymal	NN	O	B-Disease
tumours	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
kidney	NN	O	I-Disease
have	NN	O	O
been	NN	O	O
investigated	NN	O	O
for	NN	O	O
mutation	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
by	NN	O	O
single	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
(	NN	O	O
SSCP	NN	O	O
)	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
heteroduplex	NN	O	O
(	NN	O	O
HD	NN	O	O
)	NN	O	O
techniques	NN	O	O
.	NN	O	O

Chromosome	NN	O	O
3p	NN	O	O
deletion	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
98	NN	O	O
per	NN	O	O
cent	NN	O	O
of	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
papillary	NN	O	I-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
and	NN	O	O
in	NN	O	O
25	NN	O	O
per	NN	O	O
cent	NN	O	O
of	NN	O	O
chromophobe	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
22	NN	O	O
of	NN	O	O
the	NN	O	O
43	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
papillary	NN	O	I-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
abnormally	NN	O	O
migrating	NN	O	O
DNA	NN	O	O
bands	NN	O	O
were	NN	O	O
detected	NN	O	O
by	NN	O	O
SSCP	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
HD	NN	O	O
analysis	NN	O	O
.	NN	O	O

No	NN	O	O
mobility	NN	O	O
shift	NN	O	O
was	NN	O	O
seen	NN	O	O
in	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
23	NN	O	O
chromophobe	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
15	NN	O	O
papillary	NN	O	B-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
tumours	NN	O	I-Disease
and	NN	O	O
ten	NN	O	O
renal	NN	O	B-Disease
oncocytomas	NN	O	I-Disease
,	NN	O	O
which	NN	O	O
are	NN	O	O
characterized	NN	O	O
by	NN	O	O
genetic	NN	O	O
changes	NN	O	O
other	NN	O	O
than	NN	O	O
loss	NN	O	O
of	NN	O	O
chromosome	NN	O	O
3p	NN	O	O
sequences	NN	O	O
,	NN	O	O
were	NN	O	O
analysed	NN	O	O
for	NN	O	O
mutation	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

None	NN	O	O
of	NN	O	O
these	NN	O	O
tumours	NN	O	B-Disease
showed	NN	O	O
abnormal	NN	O	O
migration	NN	O	O
patterns	NN	O	O
.	NN	O	O

The	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
mutation	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
is	NN	O	O
associated	NN	O	O
exclusively	NN	O	O
with	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
papillary	NN	O	I-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

The	NN	O	O
Emery	NN	O	B-Disease
-	NN	O	I-Disease
Dreifuss	NN	O	I-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
protein	NN	O	O
,	NN	O	O
emerin	NN	O	O
,	NN	O	O
is	NN	O	O
a	NN	O	O
nuclear	NN	O	O
membrane	NN	O	O
protein	NN	O	O
.	NN	O	O

A	NN	O	O
large	NN	O	O
fragment	NN	O	O
of	NN	O	O
emerin	NN	O	O
cDNA	NN	O	O
was	NN	O	O
prepared	NN	O	O
by	NN	O	O
PCR	NN	O	O
and	NN	O	O
expressed	NN	O	O
as	NN	O	O
a	NN	O	O
recombinant	NN	O	O
protein	NN	O	O
in	NN	O	O
Escherichia	NN	O	O
coli	NN	O	O
.	NN	O	O

Using	NN	O	O
this	NN	O	O
as	NN	O	O
immunogen	NN	O	O
,	NN	O	O
we	NN	O	O
prepared	NN	O	O
a	NN	O	O
panel	NN	O	O
of	NN	O	O
12	NN	O	O
monoclonal	NN	O	O
antibodies	NN	O	O
which	NN	O	O
recognise	NN	O	O
at	NN	O	O
least	NN	O	O
four	NN	O	O
different	NN	O	O
epitopes	NN	O	O
on	NN	O	O
emerin	NN	O	O
in	NN	O	O
order	NN	O	O
to	NN	O	O
ensure	NN	O	O
that	NN	O	O
emerin	NN	O	O
can	NN	O	O
be	NN	O	O
distinguished	NN	O	O
from	NN	O	O
non	NN	O	O
-	NN	O	O
specific	NN	O	O
cross	NN	O	O
-	NN	O	O
reacting	NN	O	O
proteins	NN	O	O
.	NN	O	O

All	NN	O	O
the	NN	O	O
mAbs	NN	O	O
recognised	NN	O	O
a	NN	O	O
34	NN	O	O
kDa	NN	O	O
protein	NN	O	O
in	NN	O	O
all	NN	O	O
tissues	NN	O	O
tested	NN	O	O
,	NN	O	O
though	NN	O	O
minor	NN	O	O
emerin	NN	O	O
-	NN	O	O
related	NN	O	O
bands	NN	O	O
were	NN	O	O
also	NN	O	O
detected	NN	O	O
in	NN	O	O
some	NN	O	O
tissues	NN	O	O
.	NN	O	O

Immunofluorescence	NN	O	O
microscopy	NN	O	O
showed	NN	O	O
that	NN	O	O
emerin	NN	O	O
is	NN	O	O
located	NN	O	O
at	NN	O	O
the	NN	O	O
nuclear	NN	O	O
rim	NN	O	O
in	NN	O	O
all	NN	O	O
tissues	NN	O	O
examined	NN	O	O
.	NN	O	O

A	NN	O	O
muscle	NN	O	O
biopsy	NN	O	O
from	NN	O	O
an	NN	O	O
Emery	NN	O	B-Disease
-	NN	O	I-Disease
Dreifuss	NN	O	I-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
EMDM	NN	O	B-Disease
)	NN	O	O
patient	NN	O	O
showed	NN	O	O
complete	NN	O	O
absence	NN	O	O
of	NN	O	O
emerin	NN	O	O
by	NN	O	O
both	NN	O	O
Western	NN	O	O
blotting	NN	O	O
and	NN	O	O
immunohistochemistry	NN	O	O
,	NN	O	O
suggesting	NN	O	O
a	NN	O	O
simple	NN	O	O
diagnostic	NN	O	O
antibody	NN	O	O
test	NN	O	O
for	NN	O	O
EDMD	NN	O	B-Disease
families	NN	O	O
.	NN	O	O

Biochemical	NN	O	O
fractionation	NN	O	O
of	NN	O	O
brain	NN	O	O
and	NN	O	O
liver	NN	O	O
tissues	NN	O	O
showed	NN	O	O
that	NN	O	O
emerin	NN	O	O
was	NN	O	O
present	NN	O	O
in	NN	O	O
nuclei	NN	O	O
purified	NN	O	O
by	NN	O	O
centrifugation	NN	O	O
through	NN	O	O
65	NN	O	O
%	NN	O	O
sucrose	NN	O	O
and	NN	O	O
was	NN	O	O
absent	NN	O	O
from	NN	O	O
soluble	NN	O	O
fractions	NN	O	O
(	NN	O	O
post	NN	O	O
-	NN	O	O
100	NN	O	O
,	NN	O	O
000	NN	O	O
g	NN	O	O
)	NN	O	O
.	NN	O	O

From	NN	O	O
these	NN	O	O
results	NN	O	O
,	NN	O	O
together	NN	O	O
with	NN	O	O
sequence	NN	O	O
and	NN	O	O
structural	NN	O	O
homologies	NN	O	O
between	NN	O	O
emerin	NN	O	O
,	NN	O	O
thymopoietins	NN	O	O
and	NN	O	O
the	NN	O	O
nuclear	NN	O	O
lamina	NN	O	O
-	NN	O	O
associated	NN	O	O
protein	NN	O	O
,	NN	O	O
LAP2	NN	O	O
,	NN	O	O
we	NN	O	O
suggest	NN	O	O
that	NN	O	O
emerin	NN	O	O
will	NN	O	O
prove	NN	O	O
to	NN	O	O
be	NN	O	O
one	NN	O	O
member	NN	O	O
of	NN	O	O
a	NN	O	O
family	NN	O	O
of	NN	O	O
inner	NN	O	O
nuclear	NN	O	O
membrane	NN	O	O
proteins	NN	O	O
.	NN	O	O
.	NN	O	O

Mutation	NN	O	O
of	NN	O	O
MSH3	NN	O	O
in	NN	O	O
endometrial	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
evidence	NN	O	O
for	NN	O	O
its	NN	O	O
functional	NN	O	O
role	NN	O	O
in	NN	O	O
heteroduplex	NN	O	O
repair	NN	O	O
.	NN	O	O

Many	NN	O	O
human	NN	O	O
tumours	NN	O	B-Disease
have	NN	O	O
length	NN	O	O
alterations	NN	O	O
in	NN	O	O
repetitive	NN	O	O
sequence	NN	O	O
elements	NN	O	O
.	NN	O	O

Although	NN	O	O
this	NN	O	O
microsatellite	NN	O	B-Disease
instability	NN	O	I-Disease
has	NN	O	O
been	NN	O	O
attributed	NN	O	O
to	NN	O	O
mutations	NN	O	O
in	NN	O	O
four	NN	O	O
DNA	NN	O	O
mismatch	NN	O	O
repair	NN	O	O
genes	NN	O	O
in	NN	O	O
hereditary	NN	O	B-Disease
nonpolyposis	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
(	NN	O	O
HNPCC	NN	O	B-Disease
)	NN	O	O
kindreds	NN	O	O
,	NN	O	O
many	NN	O	O
sporadic	NN	O	B-Disease
tumours	NN	O	I-Disease
exhibit	NN	O	O
instability	NN	O	O
but	NN	O	O
no	NN	O	O
detectable	NN	O	O
mutations	NN	O	O
in	NN	O	O
these	NN	O	O
genes	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
therefore	NN	O	O
of	NN	O	O
interest	NN	O	O
to	NN	O	O
identify	NN	O	O
other	NN	O	O
genes	NN	O	O
that	NN	O	O
contribute	NN	O	O
to	NN	O	O
this	NN	O	O
instability	NN	O	O
.	NN	O	O

In	NN	O	O
yeast	NN	O	O
,	NN	O	O
mutations	NN	O	O
in	NN	O	O
several	NN	O	O
genes	NN	O	O
,	NN	O	O
including	NN	O	O
RTH	NN	O	O
and	NN	O	O
MSH3	NN	O	O
,	NN	O	O
cause	NN	O	O
microsatellite	NN	O	O
instability	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
we	NN	O	O
screened	NN	O	O
16	NN	O	O
endometrial	NN	O	B-Disease
carcinomas	NN	O	I-Disease
with	NN	O	O
microsatellite	NN	O	O
instability	NN	O	O
for	NN	O	O
alterations	NN	O	O
in	NN	O	O
FEN1	NN	O	O
(	NN	O	O
the	NN	O	O
human	NN	O	O
homolog	NN	O	O
of	NN	O	O
RTH	NN	O	O
)	NN	O	O
and	NN	O	O
in	NN	O	O
MSH3	NN	O	O
(	NN	O	O
refs	NN	O	O
12	NN	O	O
-	NN	O	O
14	NN	O	O
)	NN	O	O
.	NN	O	O

Although	NN	O	O
we	NN	O	O
found	NN	O	O
no	NN	O	O
FEN1	NN	O	O
mutations	NN	O	O
,	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
in	NN	O	O
MSH3	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
an	NN	O	O
endometrial	NN	O	B-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
in	NN	O	O
an	NN	O	O
endometrial	NN	O	B-Disease
carcinoma	NN	O	I-Disease
cell	NN	O	O
line	NN	O	O
.	NN	O	O

Extracts	NN	O	O
of	NN	O	O
the	NN	O	O
cell	NN	O	O
line	NN	O	O
were	NN	O	O
deficient	NN	O	O
in	NN	O	O
repair	NN	O	O
of	NN	O	O
DNA	NN	O	O
substrates	NN	O	O
containing	NN	O	O
mismatches	NN	O	O
or	NN	O	O
extra	NN	O	O
nucleotides	NN	O	O
.	NN	O	O

Introducing	NN	O	O
chromosome	NN	O	O
5	NN	O	O
,	NN	O	O
encoding	NN	O	O
the	NN	O	O
MSH3	NN	O	O
gene	NN	O	O
,	NN	O	O
into	NN	O	O
the	NN	O	O
mutant	NN	O	O
cell	NN	O	O
line	NN	O	O
increased	NN	O	O
the	NN	O	O
stability	NN	O	O
of	NN	O	O
some	NN	O	O
but	NN	O	O
not	NN	O	O
all	NN	O	O
microsatellites	NN	O	O
.	NN	O	O

Extracts	NN	O	O
of	NN	O	O
these	NN	O	O
cells	NN	O	O
repaired	NN	O	O
certain	NN	O	O
substrates	NN	O	O
containing	NN	O	O
extra	NN	O	O
nucleotides	NN	O	O
,	NN	O	O
but	NN	O	O
were	NN	O	O
deficient	NN	O	O
in	NN	O	O
repair	NN	O	O
of	NN	O	O
those	NN	O	O
containing	NN	O	O
mismatches	NN	O	O
or	NN	O	O
other	NN	O	O
extra	NN	O	O
nucleotides	NN	O	O
.	NN	O	O

A	NN	O	O
subsequent	NN	O	O
search	NN	O	O
revealed	NN	O	O
a	NN	O	O
second	NN	O	O
gene	NN	O	O
mutation	NN	O	O
in	NN	O	O
HHUA	NN	O	O
cells	NN	O	O
,	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
MSH6	NN	O	O
gene	NN	O	O
.	NN	O	O

Together	NN	O	O
the	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
MSH3	NN	O	O
gene	NN	O	O
encodes	NN	O	O
a	NN	O	O
product	NN	O	O
that	NN	O	O
functions	NN	O	O
in	NN	O	O
repair	NN	O	O
of	NN	O	O
some	NN	O	O
but	NN	O	O
not	NN	O	O
all	NN	O	O
pre	NN	O	O
-	NN	O	O
mutational	NN	O	O
intermediates	NN	O	O
,	NN	O	O
its	NN	O	O
mutation	NN	O	O
in	NN	O	O
tumours	NN	O	B-Disease
can	NN	O	O
result	NN	O	O
in	NN	O	O
genomic	NN	O	O
instability	NN	O	O
and	NN	O	O
,	NN	O	O
as	NN	O	O
in	NN	O	O
yeast	NN	O	O
,	NN	O	O
MSH3	NN	O	O
and	NN	O	O
MSH6	NN	O	O
are	NN	O	O
partially	NN	O	O
redundant	NN	O	O
for	NN	O	O
mismatch	NN	O	O
repair	NN	O	O
.	NN	O	O
.	NN	O	O

Comparative	NN	O	O
genome	NN	O	O
mapping	NN	O	O
of	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
region	NN	O	O
in	NN	O	O
mouse	NN	O	O
,	NN	O	O
rat	NN	O	O
,	NN	O	O
and	NN	O	O
Syrian	NN	O	O
hamster	NN	O	O
.	NN	O	O

Chromosomal	NN	O	O
locations	NN	O	O
of	NN	O	O
the	NN	O	O
Atm	NN	O	O
(	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
AT	NN	O	B-Disease
)	NN	O	O
-	NN	O	O
mutated	NN	O	O
)	NN	O	O
and	NN	O	O
Acat1	NN	O	O
(	NN	O	O
mitochondrial	NN	O	O
acetoacetyl	NN	O	O
-	NN	O	O
CoA	NN	O	O
thiolase	NN	O	O
)	NN	O	O
genes	NN	O	O
in	NN	O	O
mouse	NN	O	O
,	NN	O	O
rat	NN	O	O
,	NN	O	O
and	NN	O	O
Syrian	NN	O	O
hamster	NN	O	O
were	NN	O	O
determined	NN	O	O
by	NN	O	O
direct	NN	O	O
R	NN	O	O
-	NN	O	O
banding	NN	O	O
FISH	NN	O	O
.	NN	O	O

Both	NN	O	O
genes	NN	O	O
were	NN	O	O
colocalized	NN	O	O
to	NN	O	O
the	NN	O	O
C	NN	O	O
-	NN	O	O
D	NN	O	O
band	NN	O	O
of	NN	O	O
mouse	NN	O	O
chromosome	NN	O	O
9	NN	O	O
,	NN	O	O
the	NN	O	O
proximal	NN	O	O
end	NN	O	O
of	NN	O	O
q24	NN	O	O
.	NN	O	O

1	NN	O	O
of	NN	O	O
rat	NN	O	O
chromosome	NN	O	O
8	NN	O	O
,	NN	O	O
and	NN	O	O
qa4	NN	O	O
-	NN	O	O
qa5	NN	O	O
of	NN	O	O
Syrian	NN	O	O
hamster	NN	O	O
chromosome	NN	O	O
12	NN	O	O
.	NN	O	O

The	NN	O	O
regions	NN	O	O
in	NN	O	O
the	NN	O	O
mouse	NN	O	O
and	NN	O	O
rat	NN	O	O
were	NN	O	O
homologous	NN	O	O
to	NN	O	O
human	NN	O	O
chromosome	NN	O	O
11q	NN	O	O
.	NN	O	O

Fine	NN	O	O
genetic	NN	O	O
linkage	NN	O	O
mapping	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
AT	NN	O	B-Disease
region	NN	O	O
was	NN	O	O
performed	NN	O	O
using	NN	O	O
the	NN	O	O
interspecific	NN	O	O
backcross	NN	O	O
mice	NN	O	O
.	NN	O	O

Atm	NN	O	O
,	NN	O	O
Acat1	NN	O	O
,	NN	O	O
and	NN	O	O
Npat	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
a	NN	O	O
new	NN	O	O
gene	NN	O	O
isolated	NN	O	O
from	NN	O	O
the	NN	O	O
AT	NN	O	O
region	NN	O	O
,	NN	O	O
and	NN	O	O
12	NN	O	O
flanking	NN	O	O
microsatellite	NN	O	O
DNA	NN	O	O
markers	NN	O	O
were	NN	O	O
examined	NN	O	O
.	NN	O	O

No	NN	O	O
recombinations	NN	O	O
were	NN	O	O
found	NN	O	O
among	NN	O	O
the	NN	O	O
Atm	NN	O	O
,	NN	O	O
Npat	NN	O	O
,	NN	O	O
Acat1	NN	O	O
,	NN	O	O
and	NN	O	O
D9Mit6	NN	O	O
loci	NN	O	O
,	NN	O	O
and	NN	O	O
these	NN	O	O
loci	NN	O	O
were	NN	O	O
mapped	NN	O	O
2	NN	O	O
.	NN	O	O

0	NN	O	O
cM	NN	O	O
distal	NN	O	O
to	NN	O	O
D9Mit99	NN	O	O
and	NN	O	O
1	NN	O	O
.	NN	O	O

3	NN	O	O
cM	NN	O	O
proximal	NN	O	O
to	NN	O	O
D9Mit102	NN	O	O
.	NN	O	O

Comparison	NN	O	O
of	NN	O	O
the	NN	O	O
linkage	NN	O	O
map	NN	O	O
of	NN	O	O
mouse	NN	O	O
chromosome	NN	O	O
9	NN	O	O
(	NN	O	O
MMU9	NN	O	O
)	NN	O	O
and	NN	O	O
that	NN	O	O
of	NN	O	O
human	NN	O	O
chromosome	NN	O	O
11	NN	O	O
(	NN	O	O
HSA11	NN	O	O
)	NN	O	O
indicates	NN	O	O
that	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
chromosomal	NN	O	O
rearrangement	NN	O	O
due	NN	O	O
to	NN	O	O
an	NN	O	O
inversion	NN	O	O
between	NN	O	O
Ets1	NN	O	O
and	NN	O	O
Atm	NN	O	O
-	NN	O	O
Npat	NN	O	O
-	NN	O	O
Acat1	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
inversion	NN	O	O
of	NN	O	O
MMU9	NN	O	O
originated	NN	O	O
from	NN	O	O
the	NN	O	O
chromosomal	NN	O	O
breakage	NN	O	O
at	NN	O	O
the	NN	O	O
boundary	NN	O	O
between	NN	O	O
Gria4	NN	O	O
and	NN	O	O
Atm	NN	O	O
-	NN	O	O
Npat	NN	O	O
-	NN	O	O
Acat1	NN	O	O
on	NN	O	O
HSA11	NN	O	O
.	NN	O	O

This	NN	O	O
type	NN	O	O
of	NN	O	O
inversion	NN	O	O
appeared	NN	O	O
to	NN	O	O
be	NN	O	O
conserved	NN	O	O
in	NN	O	O
the	NN	O	O
three	NN	O	O
rodent	NN	O	O
species	NN	O	O
,	NN	O	O
mouse	NN	O	O
,	NN	O	O
rat	NN	O	O
,	NN	O	O
and	NN	O	O
Syrian	NN	O	O
hamster	NN	O	O
,	NN	O	O
using	NN	O	O
additional	NN	O	O
comparative	NN	O	O
mapping	NN	O	O
data	NN	O	O
with	NN	O	O
the	NN	O	O
Rck	NN	O	O
gene	NN	O	O

An	NN	O	O
animal	NN	O	O
model	NN	O	O
for	NN	O	O
Norrie	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
ND	NN	O	B-Disease
)	NN	O	O
:	NN	O	O
gene	NN	O	O
targeting	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
ND	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
elucidate	NN	O	O
the	NN	O	O
cellular	NN	O	O
and	NN	O	O
molecular	NN	O	O
processes	NN	O	O
which	NN	O	O
are	NN	O	O
involved	NN	O	O
in	NN	O	O
Norrie	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
ND	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
used	NN	O	O
gene	NN	O	O
targeting	NN	O	O
technology	NN	O	O
to	NN	O	O
generate	NN	O	O
ND	NN	O	B-Disease
mutant	NN	O	O
mice	NN	O	O
.	NN	O	O

The	NN	O	O
murine	NN	O	O
homologue	NN	O	O
of	NN	O	O
the	NN	O	O
ND	NN	O	B-Disease
gene	NN	O	O
was	NN	O	O
cloned	NN	O	O
and	NN	O	O
shown	NN	O	O
to	NN	O	O
encode	NN	O	O
a	NN	O	O
polypeptide	NN	O	O
that	NN	O	O
shares	NN	O	O
94	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
amino	NN	O	O
acid	NN	O	O
sequence	NN	O	O
with	NN	O	O
its	NN	O	O
human	NN	O	O
counterpart	NN	O	O
.	NN	O	O

RNA	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
revealed	NN	O	O
expression	NN	O	O
in	NN	O	O
retina	NN	O	O
,	NN	O	O
brain	NN	O	O
and	NN	O	O
the	NN	O	O
olfactory	NN	O	O
bulb	NN	O	O
and	NN	O	O
epithelium	NN	O	O
of	NN	O	O
2	NN	O	O
week	NN	O	O
old	NN	O	O
mice	NN	O	O
.	NN	O	O

Hemizygous	NN	O	O
mice	NN	O	O
carrying	NN	O	O
a	NN	O	O
replacement	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
2	NN	O	O
of	NN	O	O
the	NN	O	O
ND	NN	O	B-Disease
gene	NN	O	O
developed	NN	O	O
retrolental	NN	O	O
structures	NN	O	O
in	NN	O	O
the	NN	O	O
vitreous	NN	O	O
body	NN	O	O
and	NN	O	O
showed	NN	O	O
an	NN	O	O
overall	NN	O	O
disorganization	NN	O	O
of	NN	O	O
the	NN	O	O
retinal	NN	O	O
ganglion	NN	O	O
cell	NN	O	O
layer	NN	O	O
.	NN	O	O

The	NN	O	O
outer	NN	O	O
plexiform	NN	O	O
layer	NN	O	O
disappears	NN	O	O
occasionally	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
a	NN	O	O
juxtaposed	NN	O	O
inner	NN	O	O
and	NN	O	O
outer	NN	O	O
nuclear	NN	O	O
layer	NN	O	O
.	NN	O	O

At	NN	O	O
the	NN	O	O
same	NN	O	O
regions	NN	O	O
,	NN	O	O
the	NN	O	O
outer	NN	O	O
segments	NN	O	O
of	NN	O	O
the	NN	O	O
photoreceptor	NN	O	O
cell	NN	O	O
layer	NN	O	O
are	NN	O	O
no	NN	O	O
longer	NN	O	O
present	NN	O	O
.	NN	O	O

These	NN	O	O
ocular	NN	O	O
findings	NN	O	O
are	NN	O	O
consistent	NN	O	O
with	NN	O	O
observations	NN	O	O
in	NN	O	O
ND	NN	O	B-Disease
patients	NN	O	O
and	NN	O	O
the	NN	O	O
generated	NN	O	O
mouse	NN	O	O
line	NN	O	O
provides	NN	O	O
a	NN	O	O
faithful	NN	O	O
model	NN	O	O
for	NN	O	O
study	NN	O	O
of	NN	O	O
early	NN	O	O
pathogenic	NN	O	O
events	NN	O	O
in	NN	O	O
this	NN	O	O
severe	NN	O	O
X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
recessive	NN	O	I-Disease
neurological	NN	O	I-Disease
disorder	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

The	NN	O	O
hybrid	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
fusion	NN	O	O
protein	NN	O	O
of	NN	O	O
alveolar	NN	O	B-Disease
rhabdomyosarcoma	NN	O	I-Disease
transforms	NN	O	O
fibroblasts	NN	O	O
in	NN	O	O
culture	NN	O	O
.	NN	O	O

Pediatric	NN	O	B-Disease
alveolar	NN	O	I-Disease
rhabdomyosarcoma	NN	O	I-Disease
is	NN	O	O
characterized	NN	O	O
by	NN	O	O
a	NN	O	O
chromosomal	NN	O	O
translocation	NN	O	O
that	NN	O	O
fuses	NN	O	O
parts	NN	O	O
of	NN	O	O
the	NN	O	O
PAX3	NN	O	O
and	NN	O	O
FKHR	NN	O	O
genes	NN	O	O
.	NN	O	O

PAX3	NN	O	O
codes	NN	O	O
for	NN	O	O
a	NN	O	O
transcriptional	NN	O	O
regulator	NN	O	O
that	NN	O	O
controls	NN	O	O
developmental	NN	O	O
programs	NN	O	O
,	NN	O	O
and	NN	O	O
FKHR	NN	O	O
codes	NN	O	O
for	NN	O	O
a	NN	O	O
forkhead	NN	O	O
-	NN	O	O
winged	NN	O	O
helix	NN	O	O
protein	NN	O	O
,	NN	O	O
also	NN	O	O
a	NN	O	O
likely	NN	O	O
transcription	NN	O	O
factor	NN	O	O
.	NN	O	O

The	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
fusion	NN	O	O
product	NN	O	O
retains	NN	O	O
the	NN	O	O
DNA	NN	O	O
binding	NN	O	O
domains	NN	O	O
of	NN	O	O
the	NN	O	O
PAX3	NN	O	O
protein	NN	O	O
and	NN	O	O
the	NN	O	O
putative	NN	O	O
activator	NN	O	O
domain	NN	O	O
of	NN	O	O
the	NN	O	O
FKHR	NN	O	O
protein	NN	O	O
.	NN	O	O

The	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
protein	NN	O	O
has	NN	O	O
been	NN	O	O
shown	NN	O	O
to	NN	O	O
function	NN	O	O
as	NN	O	O
a	NN	O	O
transcriptional	NN	O	O
activator	NN	O	O
.	NN	O	O

Using	NN	O	O
the	NN	O	O
RCAS	NN	O	O
retroviral	NN	O	O
vector	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
introduced	NN	O	O
the	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
gene	NN	O	O
into	NN	O	O
chicken	NN	O	O
embryo	NN	O	O
fibroblasts	NN	O	O
.	NN	O	O

Expression	NN	O	O
of	NN	O	O
the	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
protein	NN	O	O
in	NN	O	O
these	NN	O	O
cells	NN	O	O
leads	NN	O	O
to	NN	O	O
transformation	NN	O	O
the	NN	O	O
cells	NN	O	O
become	NN	O	O
enlarged	NN	O	O
,	NN	O	O
grow	NN	O	O
tightly	NN	O	O
packed	NN	O	O
and	NN	O	O
in	NN	O	O
multiple	NN	O	O
layers	NN	O	O
,	NN	O	O
and	NN	O	O
acquire	NN	O	O
the	NN	O	O
ability	NN	O	O
for	NN	O	O
anchorage	NN	O	O
-	NN	O	O
independent	NN	O	O
growth	NN	O	O
.	NN	O	O

This	NN	O	O
cellular	NN	O	O
transformation	NN	O	O
in	NN	O	O
vitro	NN	O	O
will	NN	O	O
facilitate	NN	O	O
studies	NN	O	O
on	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
-	NN	O	O
induced	NN	O	O
oncogenesis	NN	O	O
.	NN	O	O
.	NN	O	O

Somatic	NN	O	O
-	NN	O	O
cell	NN	O	O
selection	NN	O	O
is	NN	O	O
a	NN	O	O
major	NN	O	O
determinant	NN	O	O
of	NN	O	O
the	NN	O	O
blood	NN	O	O
-	NN	O	O
cell	NN	O	O
phenotype	NN	O	O
in	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
glucose	NN	O	O
-	NN	O	O
6	NN	O	O
-	NN	O	O
phosphate	NN	O	O
dehydrogenase	NN	O	O
mutations	NN	O	O
causing	NN	O	O
severe	NN	O	O
enzyme	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

X	NN	O	O
-	NN	O	O
chromosome	NN	O	O
inactivation	NN	O	O
in	NN	O	O
mammals	NN	O	O
is	NN	O	O
regarded	NN	O	O
as	NN	O	O
an	NN	O	O
essentially	NN	O	O
random	NN	O	O
process	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
resulting	NN	O	O
somatic	NN	O	O
-	NN	O	O
cell	NN	O	O
mosaicism	NN	O	O
creates	NN	O	O
the	NN	O	O
opportunity	NN	O	O
for	NN	O	O
cell	NN	O	O
selection	NN	O	O
.	NN	O	O

In	NN	O	O
most	NN	O	O
people	NN	O	O
with	NN	O	O
red	NN	O	O
-	NN	O	O
blood	NN	O	O
-	NN	O	O
cell	NN	O	O
glucose	NN	O	B-Disease
-	NN	O	I-Disease
6	NN	O	I-Disease
-	NN	O	I-Disease
phosphate	NN	O	I-Disease
dehydrogenase	NN	O	I-Disease
(	NN	O	I-Disease
G6PD	NN	O	I-Disease
)	NN	O	I-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
enzyme	NN	O	O
-	NN	O	O
deficient	NN	O	O
phenotype	NN	O	O
is	NN	O	O
only	NN	O	O
moderately	NN	O	O
expressed	NN	O	O
in	NN	O	O
nucleated	NN	O	O
cells	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
in	NN	O	O
a	NN	O	O
small	NN	O	O
subset	NN	O	O
of	NN	O	O
hemizygous	NN	O	O
males	NN	O	O
who	NN	O	O
suffer	NN	O	O
from	NN	O	O
chronic	NN	O	B-Disease
nonspherocytic	NN	O	I-Disease
hemolytic	NN	O	I-Disease
anemia	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
underlying	NN	O	O
mutations	NN	O	O
(	NN	O	O
designated	NN	O	O
class	NN	O	O
I	NN	O	O
)	NN	O	O
cause	NN	O	O
more	NN	O	O
-	NN	O	O
severe	NN	O	O
G6PD	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
this	NN	O	O
might	NN	O	O
provide	NN	O	O
an	NN	O	O
opportunity	NN	O	O
for	NN	O	O
selection	NN	O	O
in	NN	O	O
heterozygous	NN	O	O
females	NN	O	O
during	NN	O	O
development	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
test	NN	O	O
this	NN	O	O
possibility	NN	O	O
we	NN	O	O
have	NN	O	O
analyzed	NN	O	O
four	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
class	NN	O	O
I	NN	O	O
G6PD	NN	O	O
mutations	NN	O	O
two	NN	O	O
with	NN	O	O
G6PD	NN	O	O
Portici	NN	O	O
(	NN	O	O
1178G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
)	NN	O	O
and	NN	O	O
two	NN	O	O
with	NN	O	O
G6PD	NN	O	O
Bari	NN	O	O
(	NN	O	O
1187C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
)	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
that	NN	O	O
in	NN	O	O
fractionated	NN	O	O
blood	NN	O	O
cell	NN	O	O
types	NN	O	O
(	NN	O	O
including	NN	O	O
erythroid	NN	O	O
,	NN	O	O
myeloid	NN	O	O
,	NN	O	O
and	NN	O	O
lymphoid	NN	O	O
cell	NN	O	O
lineages	NN	O	O
)	NN	O	O
there	NN	O	O
was	NN	O	O
a	NN	O	O
significant	NN	O	O
excess	NN	O	O
of	NN	O	O
G6PD	NN	O	O
-	NN	O	O
normal	NN	O	O
cells	NN	O	O
.	NN	O	O

The	NN	O	O
significant	NN	O	O
concordance	NN	O	O
that	NN	O	O
we	NN	O	O
have	NN	O	O
observed	NN	O	O
in	NN	O	O
the	NN	O	O
degree	NN	O	O
of	NN	O	O
imbalance	NN	O	O
in	NN	O	O
the	NN	O	O
different	NN	O	O
blood	NN	O	O
-	NN	O	O
cell	NN	O	O
lineages	NN	O	O
indicates	NN	O	O
that	NN	O	O
a	NN	O	O
selective	NN	O	O
mechanism	NN	O	O
is	NN	O	O
likely	NN	O	O
to	NN	O	O
operate	NN	O	O
at	NN	O	O
the	NN	O	O
level	NN	O	O
of	NN	O	O
pluripotent	NN	O	O
blood	NN	O	O
stem	NN	O	O
cells	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
it	NN	O	O
appears	NN	O	O
that	NN	O	O
severe	NN	O	O
G6PD	NN	O	B-Disease
deficiency	NN	O	I-Disease
affects	NN	O	O
adversely	NN	O	O
the	NN	O	O
proliferation	NN	O	O
or	NN	O	O
the	NN	O	O
survival	NN	O	O
of	NN	O	O
nucleated	NN	O	O
blood	NN	O	O
cells	NN	O	O
and	NN	O	O
that	NN	O	O
this	NN	O	O
phenotypic	NN	O	O
characteristic	NN	O	O
is	NN	O	O
critical	NN	O	O
during	NN	O	O
hematopoiesis	NN	O	O
.	NN	O	O
.	NN	O	O

Analysis	NN	O	O
of	NN	O	O
meiotic	NN	O	O
segregation	NN	O	O
,	NN	O	O
using	NN	O	O
single	NN	O	O
-	NN	O	O
sperm	NN	O	O
typing	NN	O	O
:	NN	O	O
meiotic	NN	O	O
drive	NN	O	O
at	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
locus	NN	O	O
.	NN	O	O

Meiotic	NN	O	O
drive	NN	O	O
at	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
locus	NN	O	O
has	NN	O	O
recently	NN	O	O
been	NN	O	O
suggested	NN	O	O
as	NN	O	O
being	NN	O	O
responsible	NN	O	O
for	NN	O	O
maintaining	NN	O	O
the	NN	O	O
frequency	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
population	NN	O	O
,	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
chromosomes	NN	O	O
capable	NN	O	O
of	NN	O	O
expansion	NN	O	O
to	NN	O	O
the	NN	O	O
disease	NN	O	O
state	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
test	NN	O	O
this	NN	O	O
hypothesis	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
studied	NN	O	O
samples	NN	O	O
of	NN	O	O
single	NN	O	O
sperm	NN	O	O
from	NN	O	O
three	NN	O	O
individuals	NN	O	O
heterozygous	NN	O	O
at	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
locus	NN	O	O
,	NN	O	O
each	NN	O	O
with	NN	O	O
one	NN	O	O
allele	NN	O	O
larger	NN	O	O
and	NN	O	O
one	NN	O	O
allele	NN	O	O
smaller	NN	O	O
than	NN	O	O
19	NN	O	O
CTG	NN	O	O
repeats	NN	O	O
.	NN	O	O

To	NN	O	O
guard	NN	O	O
against	NN	O	O
the	NN	O	O
possible	NN	O	O
problem	NN	O	O
of	NN	O	O
differential	NN	O	O
PCR	NN	O	O
amplification	NN	O	O
rates	NN	O	O
based	NN	O	O
on	NN	O	O
the	NN	O	O
lengths	NN	O	O
of	NN	O	O
the	NN	O	O
alleles	NN	O	O
,	NN	O	O
the	NN	O	O
sperm	NN	O	O
were	NN	O	O
also	NN	O	O
typed	NN	O	O
at	NN	O	O
another	NN	O	O
closely	NN	O	O
linked	NN	O	O
marker	NN	O	O
whose	NN	O	O
allele	NN	O	O
size	NN	O	O
was	NN	O	O
unrelated	NN	O	O
to	NN	O	O
the	NN	O	O
allele	NN	O	O
size	NN	O	O
at	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
locus	NN	O	O
.	NN	O	O

Using	NN	O	O
statistical	NN	O	O
models	NN	O	O
specifically	NN	O	O
designed	NN	O	O
to	NN	O	O
study	NN	O	O
single	NN	O	O
-	NN	O	O
sperm	NN	O	O
segregation	NN	O	O
data	NN	O	O
,	NN	O	O
we	NN	O	O
find	NN	O	O
no	NN	O	O
evidence	NN	O	O
of	NN	O	O
meiotic	NN	O	O
segregation	NN	O	O
distortion	NN	O	O
.	NN	O	O

The	NN	O	O
upper	NN	O	O
limit	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
-	NN	O	O
sided	NN	O	O
95	NN	O	O
%	NN	O	O
confidence	NN	O	O
interval	NN	O	O
for	NN	O	O
the	NN	O	O
estimate	NN	O	O
of	NN	O	O
the	NN	O	O
common	NN	O	O
segregation	NN	O	O
probability	NN	O	O
for	NN	O	O
the	NN	O	O
three	NN	O	O
donors	NN	O	O
is	NN	O	O
at	NN	O	O
or	NN	O	O
below	NN	O	O
.	NN	O	O

515	NN	O	O
for	NN	O	O
all	NN	O	O
models	NN	O	O
considered	NN	O	O
,	NN	O	O
and	NN	O	O
no	NN	O	O
statistically	NN	O	O
significant	NN	O	O
difference	NN	O	O
from	NN	O	O
.	NN	O	O

5	NN	O	O
is	NN	O	O
detected	NN	O	O
in	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
models	NN	O	O
.	NN	O	O

This	NN	O	O
suggests	NN	O	O
that	NN	O	O
any	NN	O	O
greater	NN	O	O
amount	NN	O	O
of	NN	O	O
segregation	NN	O	O
distortion	NN	O	O
at	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
locus	NN	O	O
must	NN	O	O
result	NN	O	O
from	NN	O	O
events	NN	O	O
following	NN	O	O
sperm	NN	O	O
ejaculation	NN	O	O
.	NN	O	O

LPP	NN	O	O
,	NN	O	O
the	NN	O	O
preferred	NN	O	O
fusion	NN	O	O
partner	NN	O	O
gene	NN	O	O
of	NN	O	O
HMGIC	NN	O	O
in	NN	O	O
lipomas	NN	O	B-Disease
,	NN	O	O
is	NN	O	O
a	NN	O	O
novel	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
LIM	NN	O	O
protein	NN	O	O
gene	NN	O	O
family	NN	O	O
.	NN	O	O

A	NN	O	O
major	NN	O	O
cytogenetic	NN	O	O
subgroup	NN	O	O
of	NN	O	O
lipomas	NN	O	B-Disease
is	NN	O	O
characterized	NN	O	O
by	NN	O	O
recurrent	NN	O	O
chromosome	NN	O	O
aberrations	NN	O	O
,	NN	O	O
mainly	NN	O	O
translocations	NN	O	O
,	NN	O	O
that	NN	O	O
involve	NN	O	O
chromosome	NN	O	O
segment	NN	O	O
12q13	NN	O	O
-	NN	O	O
q15	NN	O	O
.	NN	O	O

Multiple	NN	O	O
chromosomes	NN	O	O
have	NN	O	O
been	NN	O	O
found	NN	O	O
as	NN	O	O
the	NN	O	O
translocation	NN	O	O
partners	NN	O	O
of	NN	O	O
chromosome	NN	O	O
12	NN	O	O
but	NN	O	O
3q27	NN	O	O
-	NN	O	O
q28	NN	O	O
is	NN	O	O
preferentially	NN	O	O
involved	NN	O	O
.	NN	O	O

In	NN	O	O
previous	NN	O	O
studies	NN	O	O
,	NN	O	O
it	NN	O	O
has	NN	O	O
been	NN	O	O
shown	NN	O	O
that	NN	O	O
the	NN	O	O
high	NN	O	O
mobility	NN	O	O
group	NN	O	O
(	NN	O	O
HMG	NN	O	O
)	NN	O	O
protein	NN	O	O
gene	NN	O	O
HMGIC	NN	O	O
at	NN	O	O
12q15	NN	O	O
is	NN	O	O
consistently	NN	O	O
rearranged	NN	O	O
as	NN	O	O
a	NN	O	O
consequence	NN	O	O
of	NN	O	O
these	NN	O	O
translocations	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
we	NN	O	O
report	NN	O	O
the	NN	O	O
identification	NN	O	O
and	NN	O	O
characterization	NN	O	O
of	NN	O	O
the	NN	O	O
chromosome	NN	O	O
3	NN	O	O
-	NN	O	O
derived	NN	O	O
translocation	NN	O	O
partner	NN	O	O
gene	NN	O	O
,	NN	O	O
which	NN	O	O
we	NN	O	O
have	NN	O	O
designated	NN	O	O
LPP	NN	O	O
(	NN	O	O
lipoma	NN	O	B-Disease
preferred	NN	O	O
partner	NN	O	O
gene	NN	O	O
)	NN	O	O
.	NN	O	O

Using	NN	O	O
3	NN	O	O
-	NN	O	O
RACE	NN	O	O
analysis	NN	O	O
of	NN	O	O
HMGIC	NN	O	O
fusion	NN	O	O
transcripts	NN	O	O
in	NN	O	O
lipoma	NN	O	B-Disease
cell	NN	O	O
line	NN	O	O
Li	NN	O	O
-	NN	O	O
501	NN	O	O
/	NN	O	O
SV40	NN	O	O
,	NN	O	O
ectopic	NN	O	O
genetic	NN	O	O
sequences	NN	O	O
were	NN	O	O
obtained	NN	O	O
,	NN	O	O
which	NN	O	O
by	NN	O	O
CASH	NN	O	O
(	NN	O	O
chromosome	NN	O	O
assignment	NN	O	O
using	NN	O	O
somatic	NN	O	O
cell	NN	O	O
hybrids	NN	O	O
)	NN	O	O
and	NN	O	O
FISH	NN	O	O
(	NN	O	O
fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
)	NN	O	O
analysis	NN	O	O
were	NN	O	O
found	NN	O	O
to	NN	O	O
originate	NN	O	O
from	NN	O	O
chromosome	NN	O	O
segment	NN	O	O
3q27	NN	O	O
-	NN	O	O
q28	NN	O	O
.	NN	O	O

In	NN	O	O
Northern	NN	O	O
blot	NN	O	O
analysis	NN	O	O
,	NN	O	O
an	NN	O	O
mRNA	NN	O	O
of	NN	O	O
over	NN	O	O
10	NN	O	O
kb	NN	O	O
was	NN	O	O
detected	NN	O	O
by	NN	O	O
these	NN	O	O
ectopic	NN	O	O
sequences	NN	O	O
in	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
human	NN	O	O
tissues	NN	O	O
but	NN	O	O
not	NN	O	O
in	NN	O	O
brain	NN	O	O
and	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
leukocytes	NN	O	O
.	NN	O	O

Upon	NN	O	O
partial	NN	O	O
cDNA	NN	O	O
cloning	NN	O	O
,	NN	O	O
features	NN	O	O
of	NN	O	O
the	NN	O	O
genetic	NN	O	O
organization	NN	O	O
of	NN	O	O
LPP	NN	O	O
were	NN	O	O
established	NN	O	O
.	NN	O	O

The	NN	O	O
gene	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
span	NN	O	O
a	NN	O	O
genomic	NN	O	O
region	NN	O	O
of	NN	O	O
over	NN	O	O
400	NN	O	O
kb	NN	O	O
.	NN	O	O

Nucleotide	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
of	NN	O	O
a	NN	O	O
composite	NN	O	O
cDNA	NN	O	O
of	NN	O	O
LPP	NN	O	O
revealed	NN	O	O
an	NN	O	O
open	NN	O	O
reading	NN	O	O
frame	NN	O	O
of	NN	O	O
1836	NN	O	O
nucleotides	NN	O	O
encoding	NN	O	O
a	NN	O	O
proline	NN	O	O
-	NN	O	O
rich	NN	O	O
protein	NN	O	O
containing	NN	O	O
a	NN	O	O
leucine	NN	O	O
-	NN	O	O
zipper	NN	O	O
motif	NN	O	O
in	NN	O	O
its	NN	O	O
amino	NN	O	O
-	NN	O	O
terminal	NN	O	O
region	NN	O	O
and	NN	O	O
three	NN	O	O
LIM	NN	O	O
domains	NN	O	O
in	NN	O	O
its	NN	O	O
carboxy	NN	O	O
-	NN	O	O
terminal	NN	O	O
region	NN	O	O
.	NN	O	O

The	NN	O	O
LPP	NN	O	O
-	NN	O	O
encoded	NN	O	O
protein	NN	O	O
should	NN	O	O
be	NN	O	O
classified	NN	O	O
as	NN	O	O
a	NN	O	O
novel	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
group	NN	O	O
3	NN	O	O
proteins	NN	O	O
of	NN	O	O
the	NN	O	O
LIM	NN	O	O
protein	NN	O	O
gene	NN	O	O
family	NN	O	O
.	NN	O	O

Using	NN	O	O
reverse	NN	O	O
transcriptase	NN	O	O
combined	NN	O	O
with	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reactions	NN	O	O
in	NN	O	O
the	NN	O	O
analysis	NN	O	O
of	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
lipoma	NN	O	B-Disease
cell	NN	O	O
lines	NN	O	O
and	NN	O	O
primary	NN	O	B-Disease
lipomas	NN	O	I-Disease
,	NN	O	O
it	NN	O	O
appeared	NN	O	O
that	NN	O	O
LPP	NN	O	O
is	NN	O	O
frequently	NN	O	O
rearranged	NN	O	O
also	NN	O	O
in	NN	O	O
cases	NN	O	O
without	NN	O	O
a	NN	O	O
cytogenetically	NN	O	O
detectable	NN	O	O
involvement	NN	O	O
of	NN	O	O
3q27	NN	O	O
-	NN	O	O
q28	NN	O	O
.	NN	O	O

Two	NN	O	O
alternative	NN	O	O
HMGIC	NN	O	O
/	NN	O	O
LPP	NN	O	O
hybrid	NN	O	O
transcripts	NN	O	O
have	NN	O	O
been	NN	O	O
detected	NN	O	O
;	NN	O	O
the	NN	O	O
difference	NN	O	O
between	NN	O	O
them	NN	O	O
is	NN	O	O
mainly	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
either	NN	O	O
two	NN	O	O
or	NN	O	O
three	NN	O	O
LIM	NN	O	O
domains	NN	O	O
in	NN	O	O
the	NN	O	O
predicted	NN	O	O
HMGI	NN	O	O
-	NN	O	O
C	NN	O	O
/	NN	O	O
LPP	NN	O	O
fusion	NN	O	O
proteins	NN	O	O
.	NN	O	O
.	NN	O	O

Absence	NN	O	O
of	NN	O	O
disease	NN	O	O
phenotype	NN	O	O
and	NN	O	O
intergenerational	NN	O	O
stability	NN	O	O
of	NN	O	O
the	NN	O	O
CAG	NN	O	O
repeat	NN	O	O
in	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
expressing	NN	O	O
the	NN	O	O
human	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
transcript	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
underlying	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
HD	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
CAG	NN	O	O
expansion	NN	O	O
in	NN	O	O
the	NN	O	O
first	NN	O	O
exon	NN	O	O
of	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
CAG	NN	O	O
expansion	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
HD	NN	O	B-Disease
,	NN	O	O
we	NN	O	O
have	NN	O	O
produced	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
containing	NN	O	O
the	NN	O	O
full	NN	O	O
length	NN	O	O
human	NN	O	O
HD	NN	O	B-Disease
cDNA	NN	O	O
with	NN	O	O
44	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
.	NN	O	O

By	NN	O	O
1	NN	O	O
year	NN	O	O
,	NN	O	O
these	NN	O	O
mice	NN	O	O
have	NN	O	O
no	NN	O	O
behavioral	NN	O	B-Disease
abnormalities	NN	O	I-Disease
and	NN	O	O
morphometric	NN	O	O
analysis	NN	O	O
at	NN	O	O
6	NN	O	O
(	NN	O	O
one	NN	O	O
animal	NN	O	O
)	NN	O	O
and	NN	O	O
9	NN	O	O
(	NN	O	O
two	NN	O	O
animals	NN	O	O
)	NN	O	O
months	NN	O	O
age	NN	O	O
revealed	NN	O	O
no	NN	O	O
changes	NN	O	O
.	NN	O	O

Despite	NN	O	O
high	NN	O	O
levels	NN	O	O
of	NN	O	O
mRNA	NN	O	O
expression	NN	O	O
,	NN	O	O
there	NN	O	O
was	NN	O	O
no	NN	O	O
evidence	NN	O	O
of	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
gene	NN	O	O
product	NN	O	O
in	NN	O	O
any	NN	O	O
of	NN	O	O
these	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
.	NN	O	O

In	NN	O	O
vitro	NN	O	O
transfection	NN	O	O
studies	NN	O	O
indicated	NN	O	O
that	NN	O	O
the	NN	O	O
inclusion	NN	O	O
of	NN	O	O
120	NN	O	O
bp	NN	O	O
of	NN	O	O
the	NN	O	O
5	NN	O	O
UTR	NN	O	O
in	NN	O	O
the	NN	O	O
cDNA	NN	O	O
construct	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
2349	NN	O	O
prevented	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
cDNA	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
HD	NN	O	B-Disease
is	NN	O	O
not	NN	O	O
mediated	NN	O	O
through	NN	O	O
DNA	NN	O	O
-	NN	O	O
protein	NN	O	O
interaction	NN	O	O
and	NN	O	O
that	NN	O	O
presence	NN	O	O
of	NN	O	O
the	NN	O	O
RNA	NN	O	O
transcript	NN	O	O
with	NN	O	O
an	NN	O	O
expanded	NN	O	O
CAG	NN	O	O
repeat	NN	O	O
is	NN	O	O
insufficient	NN	O	O
to	NN	O	O
cause	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O

Rather	NN	O	O
,	NN	O	O
translation	NN	O	O
of	NN	O	O
the	NN	O	O
CAG	NN	O	O
is	NN	O	O
crucial	NN	O	O
for	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
HD	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
to	NN	O	O
that	NN	O	O
seen	NN	O	O
in	NN	O	O
humans	NN	O	O
,	NN	O	O
the	NN	O	O
CAG	NN	O	O
repeat	NN	O	O
in	NN	O	O
these	NN	O	O
mice	NN	O	O
was	NN	O	O
remarkably	NN	O	O
stable	NN	O	O
in	NN	O	O
97	NN	O	O
meioses	NN	O	O
.	NN	O	O

This	NN	O	O
suggests	NN	O	O
that	NN	O	O
genomic	NN	O	O
sequences	NN	O	O
may	NN	O	O
play	NN	O	O
a	NN	O	O
critical	NN	O	O
role	NN	O	O
in	NN	O	O
influencing	NN	O	O
repeat	NN	O	O
instability	NN	O	O
.	NN	O	O
.	NN	O	O

FISH	NN	O	O
studies	NN	O	O
in	NN	O	O
a	NN	O	O
patient	NN	O	O
with	NN	O	O
sporadic	NN	O	B-Disease
aniridia	NN	O	I-Disease
and	NN	O	O
t	NN	O	O
(	NN	O	O
7	NN	O	O
;	NN	O	O
11	NN	O	O
)	NN	O	O
(	NN	O	O
q31	NN	O	O
.	NN	O	O
2	NN	O	O
;	NN	O	O
p13	NN	O	O
)	NN	O	O
.	NN	O	O

A	NN	O	O
2	NN	O	O
year	NN	O	O
old	NN	O	O
female	NN	O	O
presenting	NN	O	O
with	NN	O	O
bilateral	NN	O	B-Disease
sporadic	NN	O	I-Disease
aniridia	NN	O	I-Disease
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
an	NN	O	O
apparently	NN	O	O
balanced	NN	O	O
reciprocal	NN	O	O
translocation	NN	O	O
with	NN	O	O
a	NN	O	O
chromosome	NN	O	O
11	NN	O	O
breakpoint	NN	O	O
within	NN	O	O
band	NN	O	O
p13	NN	O	O
.	NN	O	O

Fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridisation	NN	O	O
(	NN	O	O
FISH	NN	O	O
)	NN	O	O
studies	NN	O	O
with	NN	O	O
distal	NN	O	O
11p13	NN	O	O
specific	NN	O	O
cosmids	NN	O	O
showed	NN	O	O
that	NN	O	O
the	NN	O	O
chromosome	NN	O	O
11	NN	O	O
breakpoint	NN	O	O
lay	NN	O	O
between	NN	O	O
the	NN	O	O
aniridia	NN	O	B-Disease
(	NN	O	O
PAX6	NN	O	O
)	NN	O	O
locus	NN	O	O
and	NN	O	O
a	NN	O	O
region	NN	O	O
approximately	NN	O	O
100	NN	O	O
kb	NN	O	O
distal	NN	O	O
to	NN	O	O
PAX6	NN	O	O
defined	NN	O	O
by	NN	O	O
the	NN	O	O
cosmid	NN	O	O
FO2121	NN	O	O
.	NN	O	O

Although	NN	O	O
this	NN	O	O
patient	NN	O	O
did	NN	O	O
not	NN	O	O
have	NN	O	O
a	NN	O	O
detectable	NN	O	O
deletion	NN	O	O
within	NN	O	O
PAX6	NN	O	O
,	NN	O	O
her	NN	O	O
aniridia	NN	O	B-Disease
may	NN	O	O
have	NN	O	O
resulted	NN	O	O
from	NN	O	O
a	NN	O	O
disruption	NN	O	O
of	NN	O	O
the	NN	O	O
distal	NN	O	O
chromatin	NN	O	O
domain	NN	O	O
containing	NN	O	O
either	NN	O	O
enhancers	NN	O	O
or	NN	O	O
regulators	NN	O	O
for	NN	O	O
PAX6	NN	O	O
.	NN	O	O

This	NN	O	O
case	NN	O	O
may	NN	O	O
therefore	NN	O	O
be	NN	O	O
another	NN	O	O
example	NN	O	O
of	NN	O	O
aniridia	NN	O	B-Disease
caused	NN	O	O
by	NN	O	O
a	NN	O	O
position	NN	O	O
effect	NN	O	O
as	NN	O	O
recently	NN	O	O
described	NN	O	O
in	NN	O	O
two	NN	O	O
familial	NN	O	B-Disease
aniridia	NN	O	I-Disease
patients	NN	O	O
in	NN	O	O
which	NN	O	O
the	NN	O	O
phenotype	NN	O	O
cosegregated	NN	O	O
with	NN	O	O
chromosome	NN	O	B-Disease
abnormalities	NN	O	I-Disease
with	NN	O	O
11p13	NN	O	O
breakpoints	NN	O	O
.	NN	O	O
.	NN	O	O

Muscle	NN	O	O
expression	NN	O	O
of	NN	O	O
glucose	NN	O	B-Disease
-	NN	O	I-Disease
6	NN	O	I-Disease
-	NN	O	I-Disease
phosphate	NN	O	I-Disease
dehydrogenase	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
different	NN	O	O
variants	NN	O	O
.	NN	O	O

Muscle	NN	O	O
expression	NN	O	O
of	NN	O	O
G6PD	NN	O	B-Disease
deficiency	NN	O	I-Disease
has	NN	O	O
been	NN	O	O
investigated	NN	O	O
in	NN	O	O
Mediterranean	NN	O	O
,	NN	O	O
Seattle	NN	O	O
-	NN	O	O
like	NN	O	O
and	NN	O	O
A	NN	O	O
-	NN	O	O
variants	NN	O	O
.	NN	O	O

G6PD	NN	O	O
activity	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
samples	NN	O	O
obtained	NN	O	O
from	NN	O	O
biopsies	NN	O	O
on	NN	O	O
the	NN	O	O
quadriceps	NN	O	O
muscle	NN	O	O
of	NN	O	O
seven	NN	O	O
males	NN	O	O
and	NN	O	O
one	NN	O	O
female	NN	O	O
.	NN	O	O

The	NN	O	O
type	NN	O	O
of	NN	O	O
genetic	NN	O	O
variant	NN	O	O
was	NN	O	O
determined	NN	O	O
by	NN	O	O
molecular	NN	O	O
analysis	NN	O	O
of	NN	O	O
DNA	NN	O	O
,	NN	O	O
extracted	NN	O	O
from	NN	O	O
blood	NN	O	O
samples	NN	O	O
.	NN	O	O

All	NN	O	O
variants	NN	O	O
showed	NN	O	O
the	NN	O	O
enzyme	NN	O	O
defect	NN	O	O
in	NN	O	O
muscle	NN	O	O
.	NN	O	O

A	NN	O	O
statistically	NN	O	O
significant	NN	O	O
relationship	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
the	NN	O	O
activity	NN	O	O
of	NN	O	O
G6PD	NN	O	O
between	NN	O	O
erythrocytes	NN	O	O
and	NN	O	O
muscle	NN	O	O
of	NN	O	O
the	NN	O	O
male	NN	O	O
subjects	NN	O	O
(	NN	O	O
r	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
968	NN	O	O
;	NN	O	O
p	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
00008	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
equation	NN	O	O
for	NN	O	O
the	NN	O	O
best	NN	O	O
fit	NN	O	O
line	NN	O	O
was	NN	O	O
Y	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O

390X	NN	O	O
+	NN	O	O
0	NN	O	O
.	NN	O	O

198	NN	O	O
198	NN	O	O
.	NN	O	O

The	NN	O	O
results	NN	O	O
suggest	NN	O	O
that	NN	O	O
,	NN	O	O
for	NN	O	O
a	NN	O	O
given	NN	O	O
variant	NN	O	O
,	NN	O	O
the	NN	O	O
extent	NN	O	O
of	NN	O	O
the	NN	O	O
enzyme	NN	O	O
defect	NN	O	O
in	NN	O	O
muscle	NN	O	O
may	NN	O	O
be	NN	O	O
determined	NN	O	O
,	NN	O	O
using	NN	O	O
this	NN	O	O
equation	NN	O	O
,	NN	O	O
from	NN	O	O
the	NN	O	O
G6PD	NN	O	O
activity	NN	O	O
of	NN	O	O
erythrocytes	NN	O	O

Gene	NN	O	O
therapy	NN	O	O
for	NN	O	O
phenylketonuria	NN	O	B-Disease
.	NN	O	O

Classical	NN	O	O
phenylketonuria	NN	O	B-Disease
(	NN	O	O
PKU	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
disorder	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
a	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
hepatic	NN	O	I-Disease
phenylalanine	NN	O	I-Disease
hydroxylase	NN	O	I-Disease
(	NN	O	O
PAH	NN	O	O
)	NN	O	O
.	NN	O	O

Limitations	NN	O	O
of	NN	O	O
the	NN	O	O
current	NN	O	O
dietary	NN	O	O
treatment	NN	O	O
for	NN	O	O
PKU	NN	O	B-Disease
have	NN	O	O
led	NN	O	O
to	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
potential	NN	O	O
treatments	NN	O	O
based	NN	O	O
on	NN	O	O
somatic	NN	O	O
gene	NN	O	O
transfer	NN	O	O
.	NN	O	O

Three	NN	O	O
different	NN	O	O
vector	NN	O	O
systems	NN	O	O
have	NN	O	O
been	NN	O	O
examined	NN	O	O
.	NN	O	O

Vectors	NN	O	O
derived	NN	O	O
from	NN	O	O
a	NN	O	O
recombinant	NN	O	O
retrovirus	NN	O	O
or	NN	O	O
a	NN	O	O
DNA	NN	O	O
/	NN	O	O
protein	NN	O	O
complex	NN	O	O
can	NN	O	O
efficiently	NN	O	O
transduce	NN	O	O
the	NN	O	O
PAH	NN	O	O
cDNA	NN	O	O
into	NN	O	O
PAH	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
hepatocytes	NN	O	O
in	NN	O	O
vitro	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
application	NN	O	O
of	NN	O	O
these	NN	O	O
vector	NN	O	O
systems	NN	O	O
is	NN	O	O
presently	NN	O	O
limited	NN	O	O
by	NN	O	O
their	NN	O	O
low	NN	O	O
transduction	NN	O	O
efficiency	NN	O	O
in	NN	O	O
vivo	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
a	NN	O	O
vector	NN	O	O
derived	NN	O	O
from	NN	O	O
a	NN	O	O
recombinant	NN	O	O
adenovirus	NN	O	O
can	NN	O	O
restore	NN	O	O
10	NN	O	O
%	NN	O	O
-	NN	O	O
80	NN	O	O
%	NN	O	O
of	NN	O	O
normal	NN	O	O
hepatic	NN	O	O
PAH	NN	O	O
activity	NN	O	O
into	NN	O	O
PAH	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
,	NN	O	O
which	NN	O	O
completely	NN	O	O
normalizes	NN	O	O
serum	NN	O	O
phenylalanine	NN	O	O
levels	NN	O	O
.	NN	O	O

This	NN	O	O
treatment	NN	O	O
is	NN	O	O
transient	NN	O	O
and	NN	O	O
cannot	NN	O	O
be	NN	O	O
effectively	NN	O	O
re	NN	O	O
-	NN	O	O
administered	NN	O	O
due	NN	O	O
to	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
neutralizing	NN	O	O
antibodies	NN	O	O
directed	NN	O	O
against	NN	O	O
the	NN	O	O
recombinant	NN	O	O
adenoviral	NN	O	O
vector	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
these	NN	O	O
findings	NN	O	O
suggest	NN	O	O
that	NN	O	O
PKU	NN	O	B-Disease
can	NN	O	O
be	NN	O	O
completely	NN	O	O
corrected	NN	O	O
by	NN	O	O
somatic	NN	O	O
gene	NN	O	O
therapy	NN	O	O
,	NN	O	O
and	NN	O	O
provide	NN	O	O
some	NN	O	O
direction	NN	O	O
for	NN	O	O
the	NN	O	O
future	NN	O	O
development	NN	O	O
of	NN	O	O
adenoviral	NN	O	O
vectors	NN	O	O
.	NN	O	O
.	NN	O	O

Exon	NN	O	O
-	NN	O	O
intron	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
neuronal	NN	O	O
nicotinic	NN	O	O
acetylcholine	NN	O	O
receptor	NN	O	O
alpha	NN	O	O
4	NN	O	O
subunit	NN	O	O
(	NN	O	O
CHRNA4	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
human	NN	O	O
neuronal	NN	O	O
nicotinic	NN	O	O
acetylcholine	NN	O	O
receptor	NN	O	O
alpha	NN	O	O
4	NN	O	O
subunit	NN	O	O
gene	NN	O	O
(	NN	O	O
CHRNA4	NN	O	O
)	NN	O	O
is	NN	O	O
located	NN	O	O
in	NN	O	O
the	NN	O	O
candidate	NN	O	O
region	NN	O	O
for	NN	O	O
three	NN	O	O
different	NN	O	O
phenotypes	NN	O	O
benign	NN	O	B-Disease
familial	NN	O	I-Disease
neonatal	NN	O	I-Disease
convulsions	NN	O	I-Disease
,	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
nocturnal	NN	O	I-Disease
frontal	NN	O	I-Disease
lobe	NN	O	I-Disease
epilepsy	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
low	NN	O	O
-	NN	O	O
voltage	NN	O	O
EEG	NN	O	O
.	NN	O	O

Recently	NN	O	O
,	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
in	NN	O	O
transmembrane	NN	O	O
domain	NN	O	O
2	NN	O	O
of	NN	O	O
CHRNA4	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
nocturnal	NN	O	I-Disease
frontal	NN	O	I-Disease
lobe	NN	O	I-Disease
epilepsy	NN	O	I-Disease
in	NN	O	O
one	NN	O	O
extended	NN	O	O
pedigree	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
determined	NN	O	O
the	NN	O	O
genomic	NN	O	O
organization	NN	O	O
of	NN	O	O
CHRNA4	NN	O	O
,	NN	O	O
which	NN	O	O
consists	NN	O	O
of	NN	O	O
six	NN	O	O
exons	NN	O	O
distributed	NN	O	O
over	NN	O	O
approximately	NN	O	O
17	NN	O	O
kb	NN	O	O
of	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
.	NN	O	O

The	NN	O	O
nucleotide	NN	O	O
sequence	NN	O	O
obtained	NN	O	O
from	NN	O	O
the	NN	O	O
genomic	NN	O	O
regions	NN	O	O
adjacent	NN	O	O
to	NN	O	O
the	NN	O	O
exon	NN	O	O
boundaries	NN	O	O
enabled	NN	O	O
us	NN	O	O
to	NN	O	O
develop	NN	O	O
a	NN	O	O
set	NN	O	O
of	NN	O	O
primer	NN	O	O
pairs	NN	O	O
for	NN	O	O
PCR	NN	O	O
amplification	NN	O	O
of	NN	O	O
the	NN	O	O
complete	NN	O	O
coding	NN	O	O
region	NN	O	O
.	NN	O	O

The	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
provides	NN	O	O
the	NN	O	O
basis	NN	O	O
for	NN	O	O
a	NN	O	O
comprehensive	NN	O	O
mutation	NN	O	O
screening	NN	O	O
of	NN	O	O
CHRNA4	NN	O	O
in	NN	O	O
the	NN	O	O
above	NN	O	O
-	NN	O	O
mentioned	NN	O	O
phenotypes	NN	O	O
and	NN	O	O
possibly	NN	O	O
in	NN	O	O
other	NN	O	O
types	NN	O	O
of	NN	O	O
idiopathic	NN	O	B-Disease
epilepsies	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Ashkenazi	NN	O	O
Jewish	NN	O	O
population	NN	O	O
frequencies	NN	O	O
for	NN	O	O
common	NN	O	O
mutations	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
are	NN	O	O
the	NN	O	O
two	NN	O	O
major	NN	O	O
identified	NN	O	O
causes	NN	O	O
of	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
either	NN	O	O
gene	NN	O	O
conferring	NN	O	O
up	NN	O	O
to	NN	O	O
80	NN	O	O
-	NN	O	O
90	NN	O	O
%	NN	O	O
lifetime	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
carrier	NN	O	O
females	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
account	NN	O	O
for	NN	O	O
approximately	NN	O	O
45	NN	O	O
%	NN	O	O
of	NN	O	O
familial	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
whereas	NN	O	O
mutations	NN	O	O
in	NN	O	O
BRCA2	NN	O	O
account	NN	O	O
for	NN	O	O
a	NN	O	O
comparable	NN	O	O
percentage	NN	O	O
of	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
.	NN	O	O

Over	NN	O	O
85	NN	O	O
distinct	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
and	NN	O	O
a	NN	O	O
growing	NN	O	O
list	NN	O	O
of	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
have	NN	O	O
been	NN	O	O
identified	NN	O	O
,	NN	O	O
with	NN	O	O
the	NN	O	O
majority	NN	O	O
resulting	NN	O	O
in	NN	O	O
protein	NN	O	O
truncation	NN	O	O
.	NN	O	O

A	NN	O	O
specific	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
,	NN	O	O
185delAG	NN	O	O
,	NN	O	O
has	NN	O	O
a	NN	O	O
reported	NN	O	O
increased	NN	O	O
carrier	NN	O	O
frequency	NN	O	O
of	NN	O	O
approximately	NN	O	O
0	NN	O	O
.	NN	O	O

9	NN	O	O
%	NN	O	O
in	NN	O	O
the	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
population	NN	O	O
,	NN	O	O
but	NN	O	O
is	NN	O	O
also	NN	O	O
found	NN	O	O
in	NN	O	O
rare	NN	O	O
non	NN	O	O
-	NN	O	O
Jewish	NN	O	O
patients	NN	O	O
with	NN	O	O
a	NN	O	O
different	NN	O	O
haplotype	NN	O	O
.	NN	O	O

The	NN	O	O
6174delT	NN	O	O
mutation	NN	O	O
in	NN	O	O
BRCA2	NN	O	O
was	NN	O	O
recently	NN	O	O
identified	NN	O	O
as	NN	O	O
a	NN	O	O
frequent	NN	O	O
mutation	NN	O	O
in	NN	O	O
8	NN	O	O
out	NN	O	O
of	NN	O	O
107	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
women	NN	O	O
diagnosed	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
by	NN	O	O
age	NN	O	O
50	NN	O	O
(	NN	O	O
ref	NN	O	O
.	NN	O	O
8	NN	O	O
)	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
in	NN	O	O
three	NN	O	O
Ashkenazi	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
conducted	NN	O	O
a	NN	O	O
large	NN	O	O
-	NN	O	O
scale	NN	O	O
population	NN	O	O
study	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
prevalence	NN	O	O
of	NN	O	O
specific	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
in	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
individuals	NN	O	O
who	NN	O	O
were	NN	O	O
unselected	NN	O	O
for	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

BRCA1	NN	O	O
mutation	NN	O	O
screening	NN	O	O
on	NN	O	O
approximately	NN	O	O
3	NN	O	O
,	NN	O	O
000	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
samples	NN	O	O
determined	NN	O	O
a	NN	O	O
carrier	NN	O	O
frequency	NN	O	O
of	NN	O	O
1	NN	O	O
.	NN	O	O

09	NN	O	O
%	NN	O	O
for	NN	O	O
the	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
and	NN	O	O
0	NN	O	O
.	NN	O	O

13	NN	O	O
%	NN	O	O
for	NN	O	O
the	NN	O	O
5382insC	NN	O	O
mutation	NN	O	O
.	NN	O	O

BRCA2	NN	O	O
analysis	NN	O	O
on	NN	O	O
3	NN	O	O
,	NN	O	O
085	NN	O	O
individuals	NN	O	O
from	NN	O	O
the	NN	O	O
same	NN	O	O
population	NN	O	O
showed	NN	O	O
a	NN	O	O
carrier	NN	O	O
frequency	NN	O	O
of	NN	O	O
1	NN	O	O
.	NN	O	O

52	NN	O	O
%	NN	O	O
for	NN	O	O
the	NN	O	O
6174delT	NN	O	O
mutation	NN	O	O
.	NN	O	O

This	NN	O	O
expanded	NN	O	O
population	NN	O	O
-	NN	O	O
based	NN	O	O
study	NN	O	O
confirms	NN	O	O
that	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
and	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
6174delT	NN	O	O
mutation	NN	O	O
constitute	NN	O	O
the	NN	O	O
two	NN	O	O
most	NN	O	O
frequent	NN	O	O
mutation	NN	O	O
alleles	NN	O	O
predisposing	NN	O	O
to	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
among	NN	O	O
the	NN	O	O
Ashkenazim	NN	O	O
,	NN	O	O
and	NN	O	O
suggests	NN	O	O
a	NN	O	O
relatively	NN	O	O
lower	NN	O	O
penetrance	NN	O	O
for	NN	O	O
the	NN	O	O
6174delT	NN	O	O
mutation	NN	O	O
in	NN	O	O
BRCA2	NN	O	O

Dual	NN	O	O
roles	NN	O	O
of	NN	O	O
ATM	NN	O	O
in	NN	O	O
the	NN	O	O
cellular	NN	O	O
response	NN	O	O
to	NN	O	O
radiation	NN	O	O
and	NN	O	O
in	NN	O	O
cell	NN	O	O
growth	NN	O	O
control	NN	O	O
.	NN	O	O

The	NN	O	O
gene	NN	O	O
mutated	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
AT	NN	O	B-Disease
)	NN	O	O
patients	NN	O	O
,	NN	O	O
denoted	NN	O	O
ATM	NN	O	O
,	NN	O	O
encodes	NN	O	O
a	NN	O	O
putative	NN	O	O
protein	NN	O	O
or	NN	O	O
lipid	NN	O	O
kinase	NN	O	O
.	NN	O	O

To	NN	O	O
elucidate	NN	O	O
the	NN	O	O
functions	NN	O	O
of	NN	O	O
ATM	NN	O	O
,	NN	O	O
we	NN	O	O
disrupted	NN	O	O
the	NN	O	O
mouse	NN	O	O
ATM	NN	O	O
gene	NN	O	O
through	NN	O	O
homologous	NN	O	O
recombination	NN	O	O
in	NN	O	O
mice	NN	O	O
.	NN	O	O

Consistent	NN	O	O
with	NN	O	O
cellular	NN	O	O
defects	NN	O	O
of	NN	O	O
AT	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
the	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
cells	NN	O	O
are	NN	O	O
hypersensitive	NN	O	O
to	NN	O	O
gamma	NN	O	O
-	NN	O	O
irradiation	NN	O	O
and	NN	O	O
defective	NN	O	O
in	NN	O	O
cell	NN	O	O
-	NN	O	O
cycle	NN	O	O
arrest	NN	O	O
following	NN	O	O
radiation	NN	O	O
,	NN	O	O
correlating	NN	O	O
with	NN	O	O
a	NN	O	O
defective	NN	O	O
up	NN	O	O
-	NN	O	O
regulation	NN	O	O
of	NN	O	O
p53	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mouse	NN	O	O
thymocytes	NN	O	O
are	NN	O	O
more	NN	O	O
resistant	NN	O	O
to	NN	O	O
apoptosis	NN	O	O
induced	NN	O	O
by	NN	O	O
gamma	NN	O	O
-	NN	O	O
irradiation	NN	O	O
than	NN	O	O
normal	NN	O	O
thymocytes	NN	O	O
.	NN	O	O

ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
fibroblasts	NN	O	O
are	NN	O	O
inefficient	NN	O	O
in	NN	O	O
G1	NN	O	O
to	NN	O	O
S	NN	O	O
-	NN	O	O
phase	NN	O	O
progression	NN	O	O
following	NN	O	O
serum	NN	O	O
stimulation	NN	O	O
and	NN	O	O
senesce	NN	O	O
after	NN	O	O
only	NN	O	O
a	NN	O	O
few	NN	O	O
passages	NN	O	O
in	NN	O	O
culture	NN	O	O
.	NN	O	O

They	NN	O	O
have	NN	O	O
an	NN	O	O
increased	NN	O	O
constitutive	NN	O	O
level	NN	O	O
of	NN	O	O
p21CP1	NN	O	O
/	NN	O	O
WAF1	NN	O	O
.	NN	O	O

The	NN	O	O
ATM	NN	O	O
protein	NN	O	O
is	NN	O	O
therefore	NN	O	O
critical	NN	O	O
both	NN	O	O
for	NN	O	O
cellular	NN	O	O
responses	NN	O	O
to	NN	O	O
ionizing	NN	O	O
radiation	NN	O	O
and	NN	O	O
for	NN	O	O
normal	NN	O	O
cell	NN	O	O
-	NN	O	O
cycle	NN	O	O
progression	NN	O	O
.	NN	O	O

ATM	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
fibroblasts	NN	O	O
and	NN	O	O
thymocytes	NN	O	O
showed	NN	O	O
intermediately	NN	O	O
defective	NN	O	O
responses	NN	O	O
to	NN	O	O
irradiation	NN	O	O
but	NN	O	O
no	NN	O	O
growth	NN	O	O
defect	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
the	NN	O	O
increased	NN	O	O
cancer	NN	O	B-Disease
risk	NN	O	O
of	NN	O	O
AT	NN	O	B-Disease
heterozygotes	NN	O	O
could	NN	O	O
be	NN	O	O
attributable	NN	O	O
to	NN	O	O
poor	NN	O	O
checkpoint	NN	O	O
function	NN	O	O
.	NN	O	O
.	NN	O	O

Targeted	NN	O	O
disruption	NN	O	O
of	NN	O	O
ATM	NN	O	O
leads	NN	O	O
to	NN	O	O
growth	NN	O	B-Disease
retardation	NN	O	I-Disease
,	NN	O	O
chromosomal	NN	O	B-Disease
fragmentation	NN	O	I-Disease
during	NN	O	I-Disease
meiosis	NN	O	I-Disease
,	NN	O	O
immune	NN	O	B-Disease
defects	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
thymic	NN	O	B-Disease
lymphoma	NN	O	I-Disease
.	NN	O	O

ATM	NN	O	O
,	NN	O	O
the	NN	O	O
gene	NN	O	O
mutated	NN	O	O
in	NN	O	O
the	NN	O	O
inherited	NN	O	B-Disease
human	NN	O	I-Disease
disease	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
,	NN	O	O
is	NN	O	O
a	NN	O	O
member	NN	O	O
of	NN	O	O
a	NN	O	O
family	NN	O	O
of	NN	O	O
kinases	NN	O	O
involved	NN	O	O
in	NN	O	O
DNA	NN	O	O
metabolism	NN	O	O
and	NN	O	O
cell	NN	O	O
-	NN	O	O
cycle	NN	O	O
checkpoint	NN	O	O
control	NN	O	O
.	NN	O	O

To	NN	O	O
help	NN	O	O
clarify	NN	O	O
the	NN	O	O
physiological	NN	O	O
roles	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
protein	NN	O	O
,	NN	O	O
we	NN	O	O
disrupted	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
in	NN	O	O
mice	NN	O	O
through	NN	O	O
homologous	NN	O	O
recombination	NN	O	O
.	NN	O	O

Initial	NN	O	O
evaluation	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
knockout	NN	O	O
animals	NN	O	O
indicates	NN	O	O
that	NN	O	O
inactivation	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
ATM	NN	O	O
gene	NN	O	O
recreates	NN	O	O
much	NN	O	O
of	NN	O	O
the	NN	O	O
phenotype	NN	O	O
of	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
homozygous	NN	O	O
mutant	NN	O	O
(	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
)	NN	O	O
mice	NN	O	O
are	NN	O	O
viable	NN	O	O
,	NN	O	O
growth	NN	O	O
-	NN	O	O
retarded	NN	O	O
,	NN	O	O
and	NN	O	O
infertile	NN	O	O
.	NN	O	O

The	NN	O	O
infertility	NN	O	B-Disease
of	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
results	NN	O	O
from	NN	O	O
meiotic	NN	O	O
failure	NN	O	O
.	NN	O	O

Meiosis	NN	O	O
is	NN	O	O
arrested	NN	O	O
at	NN	O	O
the	NN	O	O
zygotene	NN	O	O
/	NN	O	O
pachytene	NN	O	O
stage	NN	O	O
of	NN	O	O
prophase	NN	O	O
I	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
abnormal	NN	O	O
chromosomal	NN	O	O
synapsis	NN	O	O
and	NN	O	O
subsequent	NN	O	O
chromosome	NN	O	O
fragmentation	NN	O	O
.	NN	O	O

Immune	NN	O	B-Disease
defects	NN	O	I-Disease
also	NN	O	O
are	NN	O	O
evident	NN	O	O
in	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
,	NN	O	O
including	NN	O	O
reduced	NN	O	O
numbers	NN	O	O
of	NN	O	O
B220	NN	O	O
+	NN	O	O
CD43	NN	O	O
-	NN	O	O
pre	NN	O	O
-	NN	O	O
B	NN	O	O
cells	NN	O	O
,	NN	O	O
thymocytes	NN	O	O
,	NN	O	O
and	NN	O	O
peripheral	NN	O	O
T	NN	O	O
cells	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
functional	NN	O	O
impairment	NN	O	O
of	NN	O	O
T	NN	O	O
-	NN	O	O
cell	NN	O	O
-	NN	O	O
dependent	NN	O	O
immune	NN	O	O
responses	NN	O	O
.	NN	O	O

The	NN	O	O
cerebella	NN	O	O
of	NN	O	O
ATM	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
mice	NN	O	O
appear	NN	O	O
normal	NN	O	O
by	NN	O	O
histologic	NN	O	O
examination	NN	O	O
at	NN	O	O
3	NN	O	O
to	NN	O	O
4	NN	O	O
months	NN	O	O
and	NN	O	O
the	NN	O	O
mice	NN	O	O
have	NN	O	O
no	NN	O	O
gross	NN	O	O
behavioral	NN	O	B-Disease
abnormalities	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
majority	NN	O	O
of	NN	O	O
mutant	NN	O	O
mice	NN	O	O
rapidly	NN	O	O
develop	NN	O	O
thymic	NN	O	B-Disease
lymphomas	NN	O	I-Disease
and	NN	O	O
die	NN	O	O
before	NN	O	O
4	NN	O	O
months	NN	O	O
of	NN	O	O
age	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
product	NN	O	O
plays	NN	O	O
an	NN	O	O
essential	NN	O	O
role	NN	O	O
in	NN	O	O
a	NN	O	O
diverse	NN	O	O
group	NN	O	O
of	NN	O	O
cellular	NN	O	O
processes	NN	O	O
,	NN	O	O
including	NN	O	O
meiosis	NN	O	O
,	NN	O	O
the	NN	O	O
normal	NN	O	O
growth	NN	O	O
of	NN	O	O
somatic	NN	O	O
tissues	NN	O	O
,	NN	O	O
immune	NN	O	O
development	NN	O	O
,	NN	O	O
and	NN	O	O
tumor	NN	O	B-Disease
suppression	NN	O	O
.	NN	O	O
.	NN	O	O

Cloning	NN	O	O
and	NN	O	O
characterization	NN	O	O
of	NN	O	O
human	NN	O	O
very	NN	O	O
-	NN	O	O
long	NN	O	O
-	NN	O	O
chain	NN	O	O
acyl	NN	O	O
-	NN	O	O
CoA	NN	O	O
dehydrogenase	NN	O	O
cDNA	NN	O	O
,	NN	O	O
chromosomal	NN	O	O
assignment	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
and	NN	O	O
identification	NN	O	O
in	NN	O	O
four	NN	O	O
patients	NN	O	O
of	NN	O	O
nine	NN	O	O
different	NN	O	O
mutations	NN	O	O
within	NN	O	O
the	NN	O	O
VLCAD	NN	O	O
gene	NN	O	O
.	NN	O	O

Very	NN	O	O
-	NN	O	O
long	NN	O	O
-	NN	O	O
chain	NN	O	O
acyl	NN	O	O
-	NN	O	O
CoA	NN	O	O
dehydrogenase	NN	O	O
(	NN	O	O
VLCAD	NN	O	O
)	NN	O	O
is	NN	O	O
one	NN	O	O
of	NN	O	O
four	NN	O	O
straight	NN	O	O
-	NN	O	O
chain	NN	O	O
acyl	NN	O	O
-	NN	O	O
CoA	NN	O	O
dehydrogenase	NN	O	O
(	NN	O	O
ACD	NN	O	O
)	NN	O	O
enzymes	NN	O	O
,	NN	O	O
which	NN	O	O
are	NN	O	O
all	NN	O	O
nuclear	NN	O	O
encoded	NN	O	O
mitochondrial	NN	O	O
flavoproteins	NN	O	O
catalyzing	NN	O	O
the	NN	O	O
initial	NN	O	O
step	NN	O	O
in	NN	O	O
fatty	NN	O	O
acid	NN	O	O
beta	NN	O	O
-	NN	O	O
oxidation	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
used	NN	O	O
the	NN	O	O
very	NN	O	O
fast	NN	O	O
,	NN	O	O
Rapid	NN	O	O
Amplification	NN	O	O
of	NN	O	O
cDNA	NN	O	O
Ends	NN	O	O
(	NN	O	O
RACE	NN	O	O
)	NN	O	O
based	NN	O	O
strategy	NN	O	O
to	NN	O	O
obtain	NN	O	O
the	NN	O	O
sequence	NN	O	O
of	NN	O	O
cDNAs	NN	O	O
encoding	NN	O	O
human	NN	O	O
VLCAD	NN	O	O
from	NN	O	O
placenta	NN	O	O
and	NN	O	O
fibroblasts	NN	O	O
.	NN	O	O

Alignment	NN	O	O
of	NN	O	O
the	NN	O	O
predicted	NN	O	O
amino	NN	O	O
acid	NN	O	O
sequence	NN	O	O
of	NN	O	O
human	NN	O	O
VLCAD	NN	O	O
with	NN	O	O
those	NN	O	O
of	NN	O	O
the	NN	O	O
other	NN	O	O
human	NN	O	O
ACD	NN	O	O
enzymes	NN	O	O
revealed	NN	O	O
extensive	NN	O	O
sequence	NN	O	O
homology	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
human	NN	O	O
VLCAD	NN	O	O
and	NN	O	O
human	NN	O	O
acyl	NN	O	O
-	NN	O	O
CoA	NN	O	O
oxidase	NN	O	O
showed	NN	O	O
extensive	NN	O	O
sequence	NN	O	O
homology	NN	O	O
corroborating	NN	O	O
the	NN	O	O
notion	NN	O	O
that	NN	O	O
these	NN	O	O
genes	NN	O	O
are	NN	O	O
evolutionarily	NN	O	O
related	NN	O	O
.	NN	O	O

Southern	NN	O	O
blot	NN	O	O
analysis	NN	O	O
of	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
from	NN	O	O
hybrid	NN	O	O
cell	NN	O	O
lines	NN	O	O
was	NN	O	O
used	NN	O	O
to	NN	O	O
localize	NN	O	O
the	NN	O	O
VLCAD	NN	O	O
gene	NN	O	O
to	NN	O	O
human	NN	O	O
chromosome	NN	O	O
17p11	NN	O	O
.	NN	O	O

2	NN	O	O
-	NN	O	O
p11	NN	O	O
.	NN	O	O

13105	NN	O	O
.	NN	O	O

Using	NN	O	O
Northern	NN	O	O
and	NN	O	O
Western	NN	O	O
blot	NN	O	O
analysis	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
tissue	NN	O	O
specific	NN	O	O
distribution	NN	O	O
of	NN	O	O
VLCAD	NN	O	O
mRNA	NN	O	O
and	NN	O	O
protein	NN	O	O
in	NN	O	O
several	NN	O	O
human	NN	O	O
tissues	NN	O	O
we	NN	O	O
showed	NN	O	O
that	NN	O	O
VLCAD	NN	O	O
is	NN	O	O
most	NN	O	O
abundant	NN	O	O
in	NN	O	O
heart	NN	O	O
and	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
.	NN	O	O

This	NN	O	O
agrees	NN	O	O
well	NN	O	O
with	NN	O	O
the	NN	O	O
fact	NN	O	O
that	NN	O	O
cardiac	NN	O	O
and	NN	O	O
muscle	NN	O	O
symptoms	NN	O	O
are	NN	O	O
characteristic	NN	O	O
for	NN	O	O
patients	NN	O	O
with	NN	O	O
VLCAD	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Northern	NN	O	O
blot	NN	O	O
analysis	NN	O	O
and	NN	O	O
sequencing	NN	O	O
of	NN	O	O
cloned	NN	O	O
PCR	NN	O	O
amplified	NN	O	O
VLCAD	NN	O	O
cDNA	NN	O	O
from	NN	O	O
four	NN	O	O
unrelated	NN	O	O
patients	NN	O	O
with	NN	O	O
VLCAD	NN	O	B-Disease
deficiency	NN	O	I-Disease
showed	NN	O	O
that	NN	O	O
VLCAD	NN	O	O
mRNA	NN	O	O
was	NN	O	O
undetectable	NN	O	O
in	NN	O	O
one	NN	O	O
patient	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
other	NN	O	O
three	NN	O	O
have	NN	O	O
mutations	NN	O	O
in	NN	O	O
both	NN	O	O
VLCAD	NN	O	O
alleles	NN	O	O
.	NN	O	O

Western	NN	O	O
blot	NN	O	O
analysis	NN	O	O
of	NN	O	O
patient	NN	O	O
fibroblasts	NN	O	O
showed	NN	O	O
that	NN	O	O
the	NN	O	O
identified	NN	O	O
mutations	NN	O	O
result	NN	O	O
in	NN	O	O
severely	NN	O	O
reduced	NN	O	O
amounts	NN	O	O
of	NN	O	O
VLCAD	NN	O	O
protein	NN	O	O
.	NN	O	O

Molecular	NN	O	O
bases	NN	O	O
of	NN	O	O
combined	NN	O	B-Disease
subtotal	NN	O	I-Disease
deficiencies	NN	O	I-Disease
of	NN	O	I-Disease
C6	NN	O	I-Disease
and	NN	O	I-Disease
C7	NN	O	I-Disease
:	NN	O	O
their	NN	O	O
effects	NN	O	O
in	NN	O	O
combination	NN	O	O
with	NN	O	O
other	NN	O	O
C6	NN	O	B-Disease
and	NN	O	I-Disease
C7	NN	O	I-Disease
deficiencies	NN	O	I-Disease
.	NN	O	O

Combined	NN	O	B-Disease
subtotal	NN	O	I-Disease
deficiency	NN	O	I-Disease
of	NN	O	I-Disease
C6	NN	O	I-Disease
and	NN	O	I-Disease
C7	NN	O	I-Disease
,	NN	O	O
in	NN	O	O
which	NN	O	O
both	NN	O	O
proteins	NN	O	O
are	NN	O	O
expressed	NN	O	O
at	NN	O	O
very	NN	O	O
low	NN	O	O
levels	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
observed	NN	O	O
in	NN	O	O
homozygous	NN	O	O
form	NN	O	O
in	NN	O	O
two	NN	O	O
families	NN	O	O
.	NN	O	O

A	NN	O	O
defect	NN	O	O
at	NN	O	O
the	NN	O	O
5	NN	O	O
splice	NN	O	O
donor	NN	O	O
site	NN	O	O
of	NN	O	O
intron	NN	O	O
15	NN	O	O
of	NN	O	O
the	NN	O	O
C6	NN	O	O
gene	NN	O	O
explains	NN	O	O
the	NN	O	O
low	NN	O	O
molecular	NN	O	O
weight	NN	O	O
of	NN	O	O
the	NN	O	O
C6	NN	O	O
protein	NN	O	O
and	NN	O	O
is	NN	O	O
probably	NN	O	O
responsible	NN	O	O
for	NN	O	O
its	NN	O	O
low	NN	O	O
expressed	NN	O	O
concentration	NN	O	O
.	NN	O	O

The	NN	O	O
C7	NN	O	O
defect	NN	O	O
is	NN	O	O
more	NN	O	O
enigmatic	NN	O	O
the	NN	O	O
protein	NN	O	O
is	NN	O	O
of	NN	O	O
normal	NN	O	O
molecular	NN	O	O
weight	NN	O	O
,	NN	O	O
low	NN	O	O
circulating	NN	O	O
concentration	NN	O	O
,	NN	O	O
and	NN	O	O
altered	NN	O	O
isoelectric	NN	O	O
point	NN	O	O
.	NN	O	O

An	NN	O	O
Arg	NN	O	O
>	NN	O	O
Ser	NN	O	O
codon	NN	O	O
substitution	NN	O	O
in	NN	O	O
exon	NN	O	O
11	NN	O	O
is	NN	O	O
the	NN	O	O
only	NN	O	O
molecular	NN	O	O
alteration	NN	O	O
within	NN	O	O
the	NN	O	O
mature	NN	O	O
C7	NN	O	O
protein	NN	O	O
.	NN	O	O

These	NN	O	O
defects	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
characteristic	NN	O	O
set	NN	O	O
of	NN	O	O
polymorphic	NN	O	O
DNA	NN	O	O
markers	NN	O	O
in	NN	O	O
the	NN	O	O
C6	NN	O	O
/	NN	O	O
C7	NN	O	O
region	NN	O	O
,	NN	O	O
forming	NN	O	O
a	NN	O	O
distinct	NN	O	O
haplotype	NN	O	O
.	NN	O	O

The	NN	O	O
haplotype	NN	O	O
has	NN	O	O
been	NN	O	O
found	NN	O	O
in	NN	O	O
combination	NN	O	O
with	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
other	NN	O	O
haplotypes	NN	O	O
containing	NN	O	O
defective	NN	O	O
genes	NN	O	O
that	NN	O	O
lead	NN	O	O
either	NN	O	O
to	NN	O	O
C6	NN	O	B-Disease
or	NN	O	I-Disease
C7	NN	O	I-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
with	NN	O	O
different	NN	O	O
consequences	NN	O	O
.	NN	O	O

Where	NN	O	O
it	NN	O	O
is	NN	O	O
combined	NN	O	O
with	NN	O	O
a	NN	O	O
C6	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
gene	NN	O	O
,	NN	O	O
the	NN	O	O
serum	NN	O	O
C7	NN	O	O
levels	NN	O	O
can	NN	O	O
be	NN	O	O
surprisingly	NN	O	O
high	NN	O	O
,	NN	O	O
possibly	NN	O	O
because	NN	O	O
there	NN	O	O
is	NN	O	O
no	NN	O	O
C6	NN	O	O
generating	NN	O	O
C56	NN	O	O
to	NN	O	O
consume	NN	O	O
the	NN	O	O
C7	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
where	NN	O	O
the	NN	O	O
C7	NN	O	O
genes	NN	O	O
are	NN	O	O
both	NN	O	O
defective	NN	O	O
(	NN	O	O
but	NN	O	O
still	NN	O	O
partially	NN	O	O
functional	NN	O	O
)	NN	O	O
,	NN	O	O
there	NN	O	O
may	NN	O	O
be	NN	O	O
a	NN	O	O
profound	NN	O	O
deficit	NN	O	O
of	NN	O	O
circulating	NN	O	O
C7	NN	O	O
because	NN	O	O
there	NN	O	O
is	NN	O	O
ample	NN	O	O
C6	NN	O	O
to	NN	O	O
produce	NN	O	O
C56	NN	O	O
and	NN	O	O
consume	NN	O	O
the	NN	O	O
already	NN	O	O
small	NN	O	O
amount	NN	O	O
of	NN	O	O
C7	NN	O	O
.	NN	O	O

Each	NN	O	O
molecular	NN	O	O
defect	NN	O	O
has	NN	O	O
also	NN	O	O
been	NN	O	O
found	NN	O	O
in	NN	O	O
isolation	NN	O	O
and	NN	O	O
has	NN	O	O
the	NN	O	O
expected	NN	O	O
effect	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
bases	NN	O	O
of	NN	O	O
human	NN	O	O
complement	NN	O	B-Disease
C7	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Complement	NN	O	B-Disease
C7	NN	O	I-Disease
deficiency	NN	O	I-Disease
(	NN	O	O
C7D	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
associated	NN	O	O
frequently	NN	O	O
with	NN	O	O
recurrent	NN	O	O
bacterial	NN	O	B-Disease
infections	NN	O	I-Disease
,	NN	O	O
especially	NN	O	O
meningitis	NN	O	B-Disease
caused	NN	O	O
by	NN	O	O
Neisseria	NN	O	B-Disease
meningitidis	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
in	NN	O	O
this	NN	O	O
work	NN	O	O
the	NN	O	O
molecular	NN	O	O
bases	NN	O	O
of	NN	O	O
C7D	NN	O	B-Disease
in	NN	O	O
two	NN	O	O
unrelated	NN	O	O
Japanese	NN	O	O
males	NN	O	O
.	NN	O	O

We	NN	O	O
used	NN	O	O
exon	NN	O	O
-	NN	O	O
specific	NN	O	O
PCR	NN	O	O
/	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
analysis	NN	O	O
as	NN	O	O
a	NN	O	O
screening	NN	O	O
step	NN	O	O
for	NN	O	O
mutations	NN	O	O
.	NN	O	O

Subsequent	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
of	NN	O	O
the	NN	O	O
target	NN	O	O
exons	NN	O	O
identified	NN	O	O
homozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
exon	NN	O	O
16	NN	O	O
of	NN	O	O
case	NN	O	O
1	NN	O	O
and	NN	O	O
in	NN	O	O
exon	NN	O	O
15	NN	O	O
of	NN	O	O
case	NN	O	O
2	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
of	NN	O	O
case	NN	O	O
1	NN	O	O
was	NN	O	O
a	NN	O	O
homozygous	NN	O	O
T	NN	O	O
to	NN	O	O
A	NN	O	O
transversion	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
2250	NN	O	O
,	NN	O	O
the	NN	O	O
third	NN	O	O
nucleotide	NN	O	O
of	NN	O	O
the	NN	O	O
codon	NN	O	O
TGT	NN	O	O
for	NN	O	O
Cys728	NN	O	O
,	NN	O	O
leading	NN	O	O
to	NN	O	O
a	NN	O	O
stop	NN	O	O
codon	NN	O	O
TGA	NN	O	O
(	NN	O	O
C728X	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
case	NN	O	O
2	NN	O	O
,	NN	O	O
a	NN	O	O
homozygous	NN	O	O
2	NN	O	O
-	NN	O	O
bp	NN	O	O
deletion	NN	O	O
(	NN	O	O
2137delTG	NN	O	O
/	NN	O	O
2138delGT	NN	O	O
/	NN	O	O
2139delTG	NN	O	O
)	NN	O	O
caused	NN	O	O
a	NN	O	O
frameshift	NN	O	O
,	NN	O	O
generating	NN	O	O
a	NN	O	O
premature	NN	O	O
termination	NN	O	O
codon	NN	O	O
4	NN	O	O
to	NN	O	O
6	NN	O	O
nucleotides	NN	O	O
downstream	NN	O	O
.	NN	O	O

Family	NN	O	O
study	NN	O	O
in	NN	O	O
case	NN	O	O
1	NN	O	O
confirmed	NN	O	O
the	NN	O	O
genetic	NN	O	O
nature	NN	O	O
of	NN	O	O
the	NN	O	O
defect	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
we	NN	O	O
detected	NN	O	O
a	NN	O	O
novel	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
intron	NN	O	O
11	NN	O	O
that	NN	O	O
presumably	NN	O	O
is	NN	O	O
linked	NN	O	O
to	NN	O	O
the	NN	O	O
mutation	NN	O	O
responsible	NN	O	O
for	NN	O	O
C7D	NN	O	B-Disease
in	NN	O	O
case	NN	O	O
1	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
C7D	NN	O	B-Disease
is	NN	O	O
heterogeneous	NN	O	O
like	NN	O	O
most	NN	O	O
of	NN	O	O
the	NN	O	O
other	NN	O	O
deficiencies	NN	O	B-Disease
of	NN	O	I-Disease
complement	NN	O	I-Disease
components	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

HPRT	NN	O	B-Disease
-	NN	O	I-Disease
APRT	NN	O	I-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
are	NN	O	O
not	NN	O	O
a	NN	O	O
model	NN	O	O
for	NN	O	O
lesch	NN	O	B-Disease
-	NN	O	I-Disease
nyhan	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Complete	NN	O	B-Disease
hypoxanthine	NN	O	I-Disease
-	NN	O	I-Disease
guanine	NN	O	I-Disease
phosphoribosyl	NN	O	I-Disease
-	NN	O	I-Disease
transferase	NN	O	I-Disease
(	NN	O	I-Disease
HPRT	NN	O	I-Disease
)	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
humans	NN	O	O
results	NN	O	O
in	NN	O	O
the	NN	O	O
Lesch	NN	O	B-Disease
-	NN	O	I-Disease
Nyhan	NN	O	I-Disease
syndrome	NN	O	I-Disease
which	NN	O	O
is	NN	O	O
characterized	NN	O	O
,	NN	O	O
among	NN	O	O
other	NN	O	O
features	NN	O	O
,	NN	O	O
by	NN	O	O
compulsive	NN	O	O
self	NN	O	O
-	NN	O	O
injurious	NN	O	O
behavior	NN	O	O
.	NN	O	O

HPRT	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
generated	NN	O	O
using	NN	O	O
mouse	NN	O	O
embryonic	NN	O	O
stem	NN	O	O
cells	NN	O	O
exhibit	NN	O	O
none	NN	O	O
of	NN	O	O
the	NN	O	O
behavioral	NN	O	O
symptoms	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
Lesch	NN	O	B-Disease
-	NN	O	I-Disease
Nyhan	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Administration	NN	O	O
of	NN	O	O
drugs	NN	O	O
that	NN	O	O
inhibit	NN	O	O
adenine	NN	O	O
phosphoribosyltransferase	NN	O	O
(	NN	O	O
APRT	NN	O	O
)	NN	O	O
in	NN	O	O
HPRT	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
has	NN	O	O
produced	NN	O	O
the	NN	O	O
suggestion	NN	O	O
that	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
APRT	NN	O	I-Disease
in	NN	O	O
combination	NN	O	O
with	NN	O	O
HPRT	NN	O	B-Disease
-	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
mice	NN	O	O
may	NN	O	O
lead	NN	O	O
to	NN	O	O
self	NN	O	O
-	NN	O	O
mutilation	NN	O	O
behavior	NN	O	O
[	NN	O	O
C	NN	O	O
.	NN	O	O
L	NN	O	O
.	NN	O	O
Wu	NN	O	O
and	NN	O	O
D	NN	O	O
.	NN	O	O
W	NN	O	O
.	NN	O	O
Melton	NN	O	O
(	NN	O	O
1993	NN	O	O
)	NN	O	O
Nature	NN	O	O
Genet	NN	O	O
.	NN	O	O
3	NN	O	O
,	NN	O	O
235	NN	O	O
-	NN	O	O
240	NN	O	O
]	NN	O	O
.	NN	O	O

To	NN	O	O
test	NN	O	O
this	NN	O	O
proposition	NN	O	O
,	NN	O	O
we	NN	O	O
bred	NN	O	O
HPRT	NN	O	B-Disease
-	NN	O	I-Disease
APRT	NN	O	I-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
.	NN	O	O

Although	NN	O	O
the	NN	O	O
doubly	NN	O	O
-	NN	O	O
deficient	NN	O	O
mice	NN	O	O
excrete	NN	O	O
adenine	NN	O	O
and	NN	O	O
its	NN	O	O
highly	NN	O	O
insoluble	NN	O	O
derivative	NN	O	O
,	NN	O	O
2	NN	O	O
,	NN	O	O
8	NN	O	O
-	NN	O	O
dihydroxyadenine	NN	O	O
,	NN	O	O
which	NN	O	O
are	NN	O	O
also	NN	O	O
associated	NN	O	O
with	NN	O	O
human	NN	O	O
APRT	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
additional	NN	O	O
abnormalities	NN	O	O
or	NN	O	O
any	NN	O	O
self	NN	O	O
-	NN	O	O
injurious	NN	O	O
behavior	NN	O	O
were	NN	O	O
not	NN	O	O
detected	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
APRT	NN	O	B-Disease
-	NN	O	I-Disease
HPRT	NN	O	I-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
,	NN	O	O
which	NN	O	O
are	NN	O	O
devoid	NN	O	O
of	NN	O	O
any	NN	O	O
purine	NN	O	O
salvage	NN	O	O
pathways	NN	O	O
,	NN	O	O
show	NN	O	O
no	NN	O	O
novel	NN	O	O
phenotype	NN	O	O
and	NN	O	O
are	NN	O	O
not	NN	O	O
a	NN	O	O
model	NN	O	O
for	NN	O	O
the	NN	O	O
behavioral	NN	O	B-Disease
abnormalities	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
the	NN	O	O
Lesch	NN	O	B-Disease
-	NN	O	I-Disease
Nyhan	NN	O	I-Disease
syndrome	NN	O	I-Disease
as	NN	O	O
previously	NN	O	O
suggested	NN	O	O

Somatic	NN	O	O
alterations	NN	O	O
of	NN	O	O
the	NN	O	O
DPC4	NN	O	O
gene	NN	O	O
in	NN	O	O
human	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
in	NN	O	O
vivo	NN	O	O
.	NN	O	O

BACKGROUND	NN	O	O
&	NN	O	O
AIMS	NN	O	O
The	NN	O	O
chromosome	NN	O	O
region	NN	O	O
18q21	NN	O	O
has	NN	O	O
been	NN	O	O
shown	NN	O	O
to	NN	O	O
be	NN	O	O
frequently	NN	O	O
deleted	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
such	NN	O	O
frequent	NN	O	O
allelic	NN	O	O
loss	NN	O	O
is	NN	O	O
a	NN	O	O
hallmark	NN	O	O
of	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
tumor	NN	O	O
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
DPC4	NN	O	O
gene	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
located	NN	O	O
at	NN	O	O
18q21	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
identified	NN	O	O
as	NN	O	O
a	NN	O	O
tumor	NN	O	O
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
from	NN	O	O
examination	NN	O	O
of	NN	O	O
pancreatic	NN	O	B-Disease
cancers	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
aim	NN	O	O
of	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
was	NN	O	O
to	NN	O	O
determine	NN	O	O
if	NN	O	O
it	NN	O	O
might	NN	O	O
also	NN	O	O
be	NN	O	O
altered	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
.	NN	O	O

METHODS	NN	O	O
Mutation	NN	O	O
analyses	NN	O	O
of	NN	O	O
the	NN	O	O
DPC4	NN	O	O
gene	NN	O	O
were	NN	O	O
performed	NN	O	O
on	NN	O	O
complementary	NN	O	O
DNA	NN	O	O
samples	NN	O	O
from	NN	O	O
31	NN	O	O
primary	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
specimens	NN	O	O
using	NN	O	O
a	NN	O	O
combination	NN	O	O
of	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
,	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
,	NN	O	O
and	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
Four	NN	O	O
missense	NN	O	O
mutations	NN	O	O
producing	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitutions	NN	O	O
and	NN	O	O
a	NN	O	O
somatic	NN	O	O
12	NN	O	O
-	NN	O	O
base	NN	O	O
pair	NN	O	O
deletion	NN	O	O
in	NN	O	O
the	NN	O	O
coding	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
DPC4	NN	O	O
gene	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
31	NN	O	O
cancers	NN	O	B-Disease
(	NN	O	O
16	NN	O	O
%	NN	O	O
;	NN	O	O
5	NN	O	O
of	NN	O	O
31	NN	O	O
)	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
The	NN	O	O
DPC4	NN	O	O
gene	NN	O	O
may	NN	O	O
play	NN	O	O
a	NN	O	O
role	NN	O	O
as	NN	O	O
a	NN	O	O
tumor	NN	O	O
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
in	NN	O	O
a	NN	O	O
fraction	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
;	NN	O	O
however	NN	O	O
,	NN	O	O
while	NN	O	O
allelic	NN	O	O
loss	NN	O	O
at	NN	O	O
18q21	NN	O	O
is	NN	O	O
very	NN	O	O
often	NN	O	O
seen	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
,	NN	O	O
only	NN	O	O
a	NN	O	O
minority	NN	O	O
show	NN	O	O
DPC4	NN	O	O
mutations	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
there	NN	O	O
might	NN	O	O
be	NN	O	O
another	NN	O	O
tumor	NN	O	O
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
in	NN	O	O
this	NN	O	O
chromosome	NN	O	O
region	NN	O	O
.	NN	O	O
.	NN	O	O

Pleiotropic	NN	O	O
defects	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
protein	NN	O	O
-	NN	O	O
deficient	NN	O	O
mice	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
generated	NN	O	O
a	NN	O	O
mouse	NN	O	O
model	NN	O	O
for	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
by	NN	O	O
using	NN	O	O
gene	NN	O	O
targeting	NN	O	O
to	NN	O	O
generate	NN	O	O
mice	NN	O	O
that	NN	O	O
do	NN	O	O
not	NN	O	O
express	NN	O	O
the	NN	O	O
Atm	NN	O	O
protein	NN	O	O
.	NN	O	O

Atm	NN	O	O
-	NN	O	O
deficient	NN	O	O
mice	NN	O	O
are	NN	O	O
retarded	NN	O	O
in	NN	O	O
growth	NN	O	O
,	NN	O	O
do	NN	O	O
not	NN	O	O
produce	NN	O	O
mature	NN	O	O
sperm	NN	O	O
,	NN	O	O
and	NN	O	O
exhibit	NN	O	O
severe	NN	O	O
defects	NN	O	O
in	NN	O	O
T	NN	O	O
cell	NN	O	O
maturation	NN	O	O
while	NN	O	O
going	NN	O	O
on	NN	O	O
to	NN	O	O
develop	NN	O	O
thymomas	NN	O	B-Disease
.	NN	O	O

Atm	NN	O	O
-	NN	O	O
deficient	NN	O	O
fibroblasts	NN	O	O
grow	NN	O	O
poorly	NN	O	O
in	NN	O	O
culture	NN	O	O
and	NN	O	O
display	NN	O	O
a	NN	O	O
high	NN	O	O
level	NN	O	O
of	NN	O	O
double	NN	O	O
-	NN	O	O
stranded	NN	O	O
chromosome	NN	O	O
breaks	NN	O	O
.	NN	O	O

Atm	NN	O	O
-	NN	O	O
deficient	NN	O	O
thymocytes	NN	O	O
undergo	NN	O	O
spontaneous	NN	O	O
apoptosis	NN	O	O
in	NN	O	O
vitro	NN	O	O
significantly	NN	O	O
more	NN	O	O
than	NN	O	O
controls	NN	O	O
.	NN	O	O

Atm	NN	O	O
-	NN	O	O
deficient	NN	O	O
mice	NN	O	O
then	NN	O	O
exhibit	NN	O	O
many	NN	O	O
of	NN	O	O
the	NN	O	O
same	NN	O	O
symptoms	NN	O	O
found	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
patients	NN	O	O
and	NN	O	O
in	NN	O	O
cells	NN	O	O
derived	NN	O	O
from	NN	O	O
them	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
the	NN	O	O
Atm	NN	O	O
protein	NN	O	O
exists	NN	O	O
as	NN	O	O
two	NN	O	O
discrete	NN	O	O
molecular	NN	O	O
species	NN	O	O
,	NN	O	O
and	NN	O	O
that	NN	O	O
loss	NN	O	O
of	NN	O	O
one	NN	O	O
or	NN	O	O
of	NN	O	O
both	NN	O	O
of	NN	O	O
these	NN	O	O
can	NN	O	O
lead	NN	O	O
to	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
DCC	NN	O	O
protein	NN	O	O
and	NN	O	O
prognosis	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

BACKGROUND	NN	O	O
Allelic	NN	O	O
loss	NN	O	O
of	NN	O	O
chromosome	NN	O	O
18q	NN	O	O
predicts	NN	O	O
a	NN	O	O
poor	NN	O	O
outcome	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
stage	NN	O	B-Disease
II	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Although	NN	O	O
the	NN	O	O
specific	NN	O	O
gene	NN	O	O
inactivated	NN	O	O
by	NN	O	O
this	NN	O	O
allelic	NN	O	O
loss	NN	O	O
has	NN	O	O
not	NN	O	O
been	NN	O	O
elucidated	NN	O	O
,	NN	O	O
the	NN	O	O
DCC	NN	O	O
(	NN	O	O
deleted	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
)	NN	O	O
gene	NN	O	O
is	NN	O	O
a	NN	O	O
candidate	NN	O	O
.	NN	O	O

We	NN	O	O
investigated	NN	O	O
whether	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
DCC	NN	O	O
protein	NN	O	O
in	NN	O	O
tumor	NN	O	B-Disease
cells	NN	O	O
is	NN	O	O
a	NN	O	O
prognostic	NN	O	O
marker	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
carcinoma	NN	O	I-Disease
.	NN	O	O

METHODS	NN	O	O
The	NN	O	O
expression	NN	O	O
of	NN	O	O
DCC	NN	O	O
was	NN	O	O
evaluated	NN	O	O
immunohistochemically	NN	O	O
in	NN	O	O
132	NN	O	O
paraffin	NN	O	O
-	NN	O	O
embedded	NN	O	O
samples	NN	O	O
from	NN	O	O
patients	NN	O	O
with	NN	O	O
curatively	NN	O	O
resected	NN	O	O
stage	NN	O	B-Disease
II	NN	O	I-Disease
and	NN	O	I-Disease
III	NN	O	I-Disease
colorectal	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
Cox	NN	O	O
proportional	NN	O	O
-	NN	O	O
hazards	NN	O	O
model	NN	O	O
was	NN	O	O
used	NN	O	O
to	NN	O	O
adjust	NN	O	O
for	NN	O	O
covariates	NN	O	O
including	NN	O	O
age	NN	O	O
,	NN	O	O
sex	NN	O	O
,	NN	O	O
tumor	NN	O	B-Disease
site	NN	O	O
,	NN	O	O
degree	NN	O	O
of	NN	O	O
tumor	NN	O	B-Disease
differentiation	NN	O	O
,	NN	O	O
and	NN	O	O
use	NN	O	O
of	NN	O	O
adjuvant	NN	O	O
therapy	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
The	NN	O	O
expression	NN	O	O
of	NN	O	O
DCC	NN	O	O
was	NN	O	O
a	NN	O	O
strong	NN	O	O
positive	NN	O	O
predictive	NN	O	O
factor	NN	O	O
for	NN	O	O
survival	NN	O	O
in	NN	O	O
both	NN	O	O
stage	NN	O	B-Disease
II	NN	O	I-Disease
and	NN	O	I-Disease
stage	NN	O	I-Disease
III	NN	O	I-Disease
colorectal	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
patients	NN	O	O
with	NN	O	O
stage	NN	O	O
II	NN	O	O
disease	NN	O	O
whose	NN	O	O
tumors	NN	O	B-Disease
expressed	NN	O	O
DCC	NN	O	O
,	NN	O	O
the	NN	O	O
five	NN	O	O
-	NN	O	O
year	NN	O	O
survival	NN	O	O
rate	NN	O	O
was	NN	O	O
94	NN	O	O
.	NN	O	O

3	NN	O	O
percent	NN	O	O
,	NN	O	O
whereas	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
DCC	NN	O	B-Disease
-	NN	O	I-Disease
negative	NN	O	I-Disease
tumors	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
survival	NN	O	O
rate	NN	O	O
was	NN	O	O
61	NN	O	O
.	NN	O	O

6	NN	O	O
percent	NN	O	O
(	NN	O	O
P	NN	O	O
<	NN	O	O
0	NN	O	O
.	NN	O	O
001	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
patients	NN	O	O
with	NN	O	O
stage	NN	O	B-Disease
III	NN	O	I-Disease
disease	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
respective	NN	O	O
survival	NN	O	O
rates	NN	O	O
were	NN	O	O
59	NN	O	O
.	NN	O	O

3	NN	O	O
percent	NN	O	O
and	NN	O	O
33	NN	O	O
.	NN	O	O

2	NN	O	O
percent	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
03	NN	O	O
)	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
DCC	NN	O	O
is	NN	O	O
a	NN	O	O
prognostic	NN	O	O
marker	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
stage	NN	O	B-Disease
II	NN	O	I-Disease
or	NN	O	I-Disease
stage	NN	O	I-Disease
III	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
stage	NN	O	B-Disease
II	NN	O	I-Disease
colorectal	NN	O	I-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
absence	NN	O	O
of	NN	O	O
DCC	NN	O	O
identifies	NN	O	O
a	NN	O	O
subgroup	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
lesions	NN	O	O
that	NN	O	O
behave	NN	O	O
like	NN	O	O
stage	NN	O	B-Disease
III	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O

These	NN	O	O
findings	NN	O	O
may	NN	O	O
thus	NN	O	O
have	NN	O	O
therapeutic	NN	O	O
implications	NN	O	O
in	NN	O	O
this	NN	O	O
group	NN	O	O
of	NN	O	O
patients	NN	O	O

Association	NN	O	O
of	NN	O	O
anxiety	NN	O	B-Disease
-	NN	O	I-Disease
related	NN	O	I-Disease
traits	NN	O	I-Disease
with	NN	O	O
a	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
the	NN	O	O
serotonin	NN	O	O
transporter	NN	O	O
gene	NN	O	O
regulatory	NN	O	O
region	NN	O	O
.	NN	O	O

Transporter	NN	O	O
-	NN	O	O
facilitated	NN	O	O
uptake	NN	O	O
of	NN	O	O
serotonin	NN	O	O
(	NN	O	O
5	NN	O	O
-	NN	O	O
hydroxytryptamine	NN	O	O
or	NN	O	O
5	NN	O	O
-	NN	O	O
HT	NN	O	O
)	NN	O	O
has	NN	O	O
been	NN	O	O
implicated	NN	O	O
in	NN	O	O
anxiety	NN	O	B-Disease
in	NN	O	O
humans	NN	O	O
and	NN	O	O
animal	NN	O	O
models	NN	O	O
and	NN	O	O
is	NN	O	O
the	NN	O	O
site	NN	O	O
of	NN	O	O
action	NN	O	O
of	NN	O	O
widely	NN	O	O
used	NN	O	O
uptake	NN	O	O
-	NN	O	O
inhibiting	NN	O	O
antidepressant	NN	O	O
and	NN	O	O
antianxiety	NN	O	O
drugs	NN	O	O
.	NN	O	O

Human	NN	O	O
5	NN	O	O
-	NN	O	O
HT	NN	O	O
transporter	NN	O	O
(	NN	O	O
5	NN	O	O
-	NN	O	O
HTT	NN	O	O
)	NN	O	O
gene	NN	O	O
transcription	NN	O	O
is	NN	O	O
modulated	NN	O	O
by	NN	O	O
a	NN	O	O
common	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
its	NN	O	O
upstream	NN	O	O
regulatory	NN	O	O
region	NN	O	O
.	NN	O	O

The	NN	O	O
short	NN	O	O
variant	NN	O	O
of	NN	O	O
the	NN	O	O
polymorphism	NN	O	O
reduces	NN	O	O
the	NN	O	O
transcriptional	NN	O	O
efficiency	NN	O	O
of	NN	O	O
the	NN	O	O
5	NN	O	O
-	NN	O	O
HTT	NN	O	O
gene	NN	O	O
promoter	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
decreased	NN	O	O
5	NN	O	O
-	NN	O	O
HTT	NN	O	O
expression	NN	O	O
and	NN	O	O
5	NN	O	O
-	NN	O	O
HT	NN	O	O
uptake	NN	O	O
in	NN	O	O
lymphoblasts	NN	O	O
.	NN	O	O

Association	NN	O	O
studies	NN	O	O
in	NN	O	O
two	NN	O	O
independent	NN	O	O
samples	NN	O	O
totaling	NN	O	O
505	NN	O	O
individuals	NN	O	O
revealed	NN	O	O
that	NN	O	O
the	NN	O	O
5	NN	O	O
-	NN	O	O
HTT	NN	O	O
polymorphism	NN	O	O
accounts	NN	O	O
for	NN	O	O
3	NN	O	O
to	NN	O	O
4	NN	O	O
percent	NN	O	O
of	NN	O	O
total	NN	O	O
variation	NN	O	O
and	NN	O	O
7	NN	O	O
to	NN	O	O
9	NN	O	O
percent	NN	O	O
of	NN	O	O
inherited	NN	O	O
variance	NN	O	O
in	NN	O	O
anxiety	NN	O	B-Disease
-	NN	O	I-Disease
related	NN	O	I-Disease
personality	NN	O	I-Disease
traits	NN	O	I-Disease
in	NN	O	O
individuals	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
sibships	NN	O	O
.	NN	O	O
.	NN	O	O

Phenotypic	NN	O	O
and	NN	O	O
genotypic	NN	O	O
overlap	NN	O	O
between	NN	O	O
atelosteogenesis	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
and	NN	O	O
diastrophic	NN	O	B-Disease
dysplasia	NN	O	I-Disease
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
diastrophic	NN	O	B-Disease
dysplasia	NN	O	I-Disease
sulfate	NN	O	O
transporter	NN	O	O
gene	NN	O	O
DTDST	NN	O	O
have	NN	O	O
been	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
family	NN	O	O
of	NN	O	O
chondrodysplasias	NN	O	B-Disease
that	NN	O	O
comprises	NN	O	O
,	NN	O	O
in	NN	O	O
order	NN	O	O
of	NN	O	O
increasing	NN	O	O
severity	NN	O	O
,	NN	O	O
diastrophic	NN	O	B-Disease
dysplasia	NN	O	I-Disease
(	NN	O	O
DTD	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
atelosteogenesis	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
(	NN	O	O
AO2	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
and	NN	O	O
achondrogenesis	NN	O	B-Disease
type	NN	O	I-Disease
1B	NN	O	I-Disease
(	NN	O	O
ACG1B	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

To	NN	O	O
learn	NN	O	O
more	NN	O	O
about	NN	O	O
the	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
DTDST	NN	O	B-Disease
chondrodysplasias	NN	O	I-Disease
and	NN	O	O
about	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
,	NN	O	O
we	NN	O	O
studied	NN	O	O
fibroblast	NN	O	O
cultures	NN	O	O
of	NN	O	O
three	NN	O	O
new	NN	O	O
patients	NN	O	O
one	NN	O	O
with	NN	O	O
AO	NN	O	B-Disease
-	NN	O	I-Disease
2	NN	O	I-Disease
,	NN	O	O
one	NN	O	O
with	NN	O	O
DTD	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
one	NN	O	O
with	NN	O	O
an	NN	O	O
intermediate	NN	O	O
phenotype	NN	O	O
(	NN	O	O
AO2	NN	O	B-Disease
/	NN	O	O
DTD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Reduced	NN	O	O
incorporation	NN	O	O
of	NN	O	O
inorganic	NN	O	O
sulfate	NN	O	O
into	NN	O	O
macromolecules	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
all	NN	O	O
three	NN	O	O
.	NN	O	O

Each	NN	O	O
of	NN	O	O
the	NN	O	O
three	NN	O	O
patients	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
a	NN	O	O
c862t	NN	O	O
transition	NN	O	O
predicting	NN	O	O
a	NN	O	O
R279W	NN	O	O
substitution	NN	O	O
in	NN	O	O
the	NN	O	O
third	NN	O	O
extracellular	NN	O	O
loop	NN	O	O
of	NN	O	O
DTDST	NN	O	O
.	NN	O	O

In	NN	O	O
two	NN	O	O
patients	NN	O	O
(	NN	O	O
DTD	NN	O	B-Disease
and	NN	O	O
AO2	NN	O	B-Disease
/	NN	O	O
DTD	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
no	NN	O	O
other	NN	O	O
structural	NN	O	O
mutation	NN	O	O
was	NN	O	O
found	NN	O	O
,	NN	O	O
but	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
amplification	NN	O	O
and	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
analysis	NN	O	O
of	NN	O	O
fibroblast	NN	O	O
cDNA	NN	O	O
showed	NN	O	O
reduced	NN	O	O
mRNA	NN	O	O
levels	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
DTDST	NN	O	O
allele	NN	O	O
these	NN	O	O
two	NN	O	O
patients	NN	O	O
may	NN	O	O
be	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
the	NN	O	O
"	NN	O	O
Finnish	NN	O	O
"	NN	O	O
mutation	NN	O	O
(	NN	O	O
as	NN	O	O
yet	NN	O	O
uncharacterized	NN	O	O
at	NN	O	O
the	NN	O	O
DNA	NN	O	O
level	NN	O	O
)	NN	O	O
,	NN	O	O
which	NN	O	O
causes	NN	O	O
reduced	NN	O	O
expression	NN	O	O
of	NN	O	O
DTDST	NN	O	O
.	NN	O	O

The	NN	O	O
third	NN	O	O
patient	NN	O	O
(	NN	O	O
with	NN	O	O
AO2	NN	O	B-Disease
)	NN	O	O
had	NN	O	O
the	NN	O	O
R279W	NN	O	O
mutation	NN	O	O
compounded	NN	O	O
with	NN	O	O
a	NN	O	O
novel	NN	O	O
mutation	NN	O	O
,	NN	O	O
the	NN	O	O
deletion	NN	O	O
of	NN	O	O
cytosine	NN	O	O
418	NN	O	O
(	NN	O	O
delta	NN	O	O
c418	NN	O	O
)	NN	O	O
,	NN	O	O
predicting	NN	O	O
a	NN	O	O
frameshift	NN	O	O
with	NN	O	O
premature	NN	O	O
termination	NN	O	O
.	NN	O	O

Also	NN	O	O
the	NN	O	O
delta	NN	O	O
c418	NN	O	O
allele	NN	O	O
was	NN	O	O
underrepresented	NN	O	O
in	NN	O	O
the	NN	O	O
cDNA	NN	O	O
,	NN	O	O
in	NN	O	O
accordance	NN	O	O
with	NN	O	O
previous	NN	O	O
observations	NN	O	O
that	NN	O	O
premature	NN	O	O
stop	NN	O	O
codons	NN	O	O
reduce	NN	O	O
mRNA	NN	O	O
levels	NN	O	O
.	NN	O	O

The	NN	O	O
presence	NN	O	O
of	NN	O	O
the	NN	O	O
DTDST	NN	O	O
R279W	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
total	NN	O	O
of	NN	O	O
11	NN	O	O
patients	NN	O	O
with	NN	O	O
AO2	NN	O	B-Disease
or	NN	O	O
DTD	NN	O	B-Disease
emphasizes	NN	O	O
the	NN	O	O
overlap	NN	O	O
between	NN	O	O
these	NN	O	O
conditions	NN	O	O
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
has	NN	O	O
not	NN	O	O
been	NN	O	O
found	NN	O	O
so	NN	O	O
far	NN	O	O
in	NN	O	O
8	NN	O	O
analyzed	NN	O	O
ACG1B	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
it	NN	O	O
allows	NN	O	O
some	NN	O	O
residual	NN	O	O
activity	NN	O	O
of	NN	O	O
the	NN	O	O
sulfate	NN	O	O
transporter	NN	O	O
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
of	NN	O	O
WASP	NN	O	O
mutations	NN	O	O
,	NN	O	O
mutation	NN	O	O
hotspots	NN	O	O
and	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
disparities	NN	O	O
in	NN	O	O
24	NN	O	O
patients	NN	O	O
with	NN	O	O
the	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
Wiskott	NN	O	B-Disease
-	NN	O	I-Disease
Aldrich	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
WAS	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
an	NN	O	O
X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
immunodeficiency	NN	O	I-Disease
disease	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
recently	NN	O	O
isolated	NN	O	O
gene	NN	O	O
encoding	NN	O	O
WAS	NN	O	B-Disease
protein	NN	O	O
(	NN	O	O
WASP	NN	O	O
)	NN	O	O
,	NN	O	O
is	NN	O	O
known	NN	O	O
to	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
extensive	NN	O	O
clinical	NN	O	O
heterogeneity	NN	O	O
.	NN	O	O

Cumulative	NN	O	O
mutation	NN	O	O
data	NN	O	O
have	NN	O	O
revealed	NN	O	O
that	NN	O	O
WASP	NN	O	O
genotypes	NN	O	O
are	NN	O	O
also	NN	O	O
highly	NN	O	O
variable	NN	O	O
among	NN	O	O
WAS	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
relationship	NN	O	O
of	NN	O	O
phenotype	NN	O	O
with	NN	O	O
genotype	NN	O	O
in	NN	O	O
this	NN	O	O
disease	NN	O	O
remains	NN	O	O
unclear	NN	O	O
.	NN	O	O

To	NN	O	O
address	NN	O	O
this	NN	O	O
issue	NN	O	O
we	NN	O	O
characterized	NN	O	O
WASP	NN	O	O
mutations	NN	O	O
in	NN	O	O
24	NN	O	O
unrelated	NN	O	O
WAS	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
including	NN	O	O
18	NN	O	O
boys	NN	O	O
with	NN	O	O
severe	NN	O	O
classical	NN	O	O
WAS	NN	O	B-Disease
and	NN	O	O
6	NN	O	O
boys	NN	O	O
expressing	NN	O	O
mild	NN	O	O
forms	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
,	NN	O	O
and	NN	O	O
then	NN	O	O
examined	NN	O	O
the	NN	O	O
degree	NN	O	O
of	NN	O	O
correlation	NN	O	O
of	NN	O	O
these	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
all	NN	O	O
previously	NN	O	O
published	NN	O	O
WASP	NN	O	O
mutations	NN	O	O
with	NN	O	O
disease	NN	O	O
severity	NN	O	O
.	NN	O	O

By	NN	O	O
analysis	NN	O	O
of	NN	O	O
these	NN	O	O
compiled	NN	O	O
mutation	NN	O	O
data	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrated	NN	O	O
clustering	NN	O	O
of	NN	O	O
WASP	NN	O	O
mutations	NN	O	O
within	NN	O	O
the	NN	O	O
four	NN	O	O
most	NN	O	O
N	NN	O	O
-	NN	O	O
terminal	NN	O	O
exons	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
and	NN	O	O
also	NN	O	O
identified	NN	O	O
several	NN	O	O
sites	NN	O	O
within	NN	O	O
this	NN	O	O
region	NN	O	O
as	NN	O	O
hotspots	NN	O	O
for	NN	O	O
WASP	NN	O	O
mutation	NN	O	O
.	NN	O	O

These	NN	O	O
characteristics	NN	O	O
were	NN	O	O
observed	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
in	NN	O	O
both	NN	O	O
severe	NN	O	O
and	NN	O	O
mild	NN	O	O
cases	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O

Similarly	NN	O	O
,	NN	O	O
while	NN	O	O
the	NN	O	O
cumulative	NN	O	O
data	NN	O	O
revealed	NN	O	O
a	NN	O	O
predominance	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
among	NN	O	O
the	NN	O	O
WASP	NN	O	O
gene	NN	O	O
lesions	NN	O	O
observed	NN	O	O
in	NN	O	O
boys	NN	O	O
with	NN	O	O
isolated	NN	O	B-Disease
thrombocytopenia	NN	O	I-Disease
,	NN	O	O
missense	NN	O	O
mutations	NN	O	O
were	NN	O	O
not	NN	O	O
exclusively	NN	O	O
associated	NN	O	O
with	NN	O	O
milder	NN	O	O
WAS	NN	O	B-Disease
phenotypes	NN	O	O
,	NN	O	O
but	NN	O	O
also	NN	O	O
comprised	NN	O	O
a	NN	O	O
substantial	NN	O	O
portion	NN	O	O
(	NN	O	O
38	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
WASP	NN	O	O
gene	NN	O	O
defects	NN	O	O
found	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
severe	NN	O	O
disease	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
the	NN	O	O
detection	NN	O	O
of	NN	O	O
identical	NN	O	O
WASP	NN	O	O
mutations	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
disparate	NN	O	O
phenotypes	NN	O	O
,	NN	O	O
reveal	NN	O	O
a	NN	O	O
lack	NN	O	O
of	NN	O	O
phenotype	NN	O	O
concordance	NN	O	O
with	NN	O	O
genotype	NN	O	O
in	NN	O	O
WAS	NN	O	B-Disease
and	NN	O	O
thus	NN	O	O
imply	NN	O	O
that	NN	O	O
phenotypic	NN	O	O
outcome	NN	O	O
in	NN	O	O
this	NN	O	O
disease	NN	O	O
cannot	NN	O	O
be	NN	O	O
reliably	NN	O	O
predicted	NN	O	O
solely	NN	O	O
on	NN	O	O
the	NN	O	O
basis	NN	O	O
of	NN	O	O
WASP	NN	O	O
genotypes	NN	O	O
.	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
'	NN	O	O
part	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
exon	NN	O	O
15	NN	O	O
do	NN	O	O
not	NN	O	O
result	NN	O	O
in	NN	O	O
truncated	NN	O	O
proteins	NN	O	O
and	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
attenuated	NN	O	O
adenomatous	NN	O	B-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
.	NN	O	O

Familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
(	NN	O	O
FAP	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
inherited	NN	O	O
predisposition	NN	O	O
to	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
characterized	NN	O	O
by	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
numerous	NN	O	O
adenomatous	NN	O	B-Disease
polyps	NN	O	I-Disease
predominantly	NN	O	O
in	NN	O	O
the	NN	O	O
colorectal	NN	O	O
region	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
adenomatous	NN	O	B-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
(	NN	O	O
APC	NN	O	B-Disease
)	NN	O	O
gene	NN	O	O
are	NN	O	O
responsible	NN	O	O
for	NN	O	O
most	NN	O	O
cases	NN	O	O
of	NN	O	O
FAP	NN	O	B-Disease
.	NN	O	O

Mutations	NN	O	O
at	NN	O	O
the	NN	O	O
5	NN	O	O
end	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
are	NN	O	O
known	NN	O	O
to	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
relatively	NN	O	O
mild	NN	O	O
form	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
,	NN	O	O
called	NN	O	O
attenuated	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
(	NN	O	O
AAPC	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
part	NN	O	O
of	NN	O	O
exon	NN	O	O
15	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
a	NN	O	O
stop	NN	O	O
codon	NN	O	O
at	NN	O	O
1862	NN	O	O
,	NN	O	O
in	NN	O	O
a	NN	O	O
large	NN	O	O
Dutch	NN	O	O
kindred	NN	O	O
with	NN	O	O
AAPC	NN	O	B-Disease
.	NN	O	O

Western	NN	O	O
blot	NN	O	O
analysis	NN	O	O
of	NN	O	O
lymphoblastoid	NN	O	O
cell	NN	O	O
lines	NN	O	O
derived	NN	O	O
from	NN	O	O
affected	NN	O	O
family	NN	O	O
members	NN	O	O
from	NN	O	O
this	NN	O	O
kindred	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
from	NN	O	O
a	NN	O	O
previously	NN	O	O
reported	NN	O	O
Swiss	NN	O	O
family	NN	O	O
carrying	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
at	NN	O	O
codon	NN	O	O
1987	NN	O	O
and	NN	O	O
displaying	NN	O	O
a	NN	O	O
similar	NN	O	O
attenuated	NN	O	O
phenotype	NN	O	O
,	NN	O	O
showed	NN	O	O
only	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
APC	NN	O	B-Disease
protein	NN	O	O
.	NN	O	O

Our	NN	O	O
study	NN	O	O
indicates	NN	O	O
that	NN	O	O
chain	NN	O	O
-	NN	O	O
terminating	NN	O	O
mutations	NN	O	O
located	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
part	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
do	NN	O	O
not	NN	O	O
result	NN	O	O
in	NN	O	O
detectable	NN	O	O
truncated	NN	O	O
polypeptides	NN	O	O
and	NN	O	O
we	NN	O	O
hypothesize	NN	O	O
that	NN	O	O
this	NN	O	O
is	NN	O	O
likely	NN	O	O
to	NN	O	O
be	NN	O	O
the	NN	O	O
basis	NN	O	O
for	NN	O	O
the	NN	O	O
observed	NN	O	O
AAPC	NN	O	B-Disease
phenotype	NN	O	O
.	NN	O	O
.	NN	O	O

Complete	NN	O	O
genomic	NN	O	O
sequence	NN	O	O
and	NN	O	O
analysis	NN	O	O
of	NN	O	O
117	NN	O	O
kb	NN	O	O
of	NN	O	O
human	NN	O	O
DNA	NN	O	O
containing	NN	O	O
the	NN	O	O
gene	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

Over	NN	O	O
100	NN	O	O
distinct	NN	O	O
disease	NN	O	O
-	NN	O	O
associated	NN	O	O
mutations	NN	O	O
have	NN	O	O
been	NN	O	O
identified	NN	O	O
in	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
-	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
susceptibility	NN	O	O
gene	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

Loss	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
allele	NN	O	O
in	NN	O	O
>	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
tumors	NN	O	B-Disease
from	NN	O	O
patients	NN	O	O
with	NN	O	O
inherited	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
indicates	NN	O	O
tumor	NN	O	B-Disease
suppressive	NN	O	O
function	NN	O	O
.	NN	O	O

The	NN	O	O
low	NN	O	O
incidence	NN	O	O
of	NN	O	O
somatic	NN	O	O
mutations	NN	O	O
suggests	NN	O	O
that	NN	O	O
BRCA1	NN	O	O
inactivation	NN	O	O
in	NN	O	O
sporadic	NN	O	O
tumors	NN	O	B-Disease
occurs	NN	O	O
by	NN	O	O
alternative	NN	O	O
mechanisms	NN	O	O
,	NN	O	O
such	NN	O	O
as	NN	O	O
interstitial	NN	O	O
chromosomal	NN	O	O
deletion	NN	O	O
or	NN	O	O
reduced	NN	O	O
transcription	NN	O	O
.	NN	O	O

To	NN	O	O
identify	NN	O	O
possible	NN	O	O
features	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
genomic	NN	O	O
region	NN	O	O
that	NN	O	O
may	NN	O	O
contribute	NN	O	O
to	NN	O	O
chromosomal	NN	O	O
instability	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
potential	NN	O	O
transcriptional	NN	O	O
regulatory	NN	O	O
elements	NN	O	O
,	NN	O	O
a	NN	O	O
117	NN	O	O
,	NN	O	O
143	NN	O	O
-	NN	O	O
bp	NN	O	O
DNA	NN	O	O
sequence	NN	O	O
encompassing	NN	O	O
BRCA1	NN	O	O
was	NN	O	O
obtained	NN	O	O
by	NN	O	O
random	NN	O	O
sequencing	NN	O	O
of	NN	O	O
four	NN	O	O
cosmids	NN	O	O
identified	NN	O	O
from	NN	O	O
a	NN	O	O
human	NN	O	O
chromosome	NN	O	O
17	NN	O	O
specific	NN	O	O
library	NN	O	O
.	NN	O	O

The	NN	O	O
24	NN	O	O
exons	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
span	NN	O	O
an	NN	O	O
81	NN	O	O
-	NN	O	O
kb	NN	O	O
region	NN	O	O
that	NN	O	O
has	NN	O	O
an	NN	O	O
unusually	NN	O	O
high	NN	O	O
density	NN	O	O
of	NN	O	O
Alu	NN	O	O
repetitive	NN	O	O
DNA	NN	O	O
(	NN	O	O
41	NN	O	O
.	NN	O	O
5	NN	O	O
%	NN	O	O
)	NN	O	O
,	NN	O	O
but	NN	O	O
relatively	NN	O	O
low	NN	O	O
density	NN	O	O
(	NN	O	O
4	NN	O	O
.	NN	O	O
8	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
other	NN	O	O
repetitive	NN	O	O
sequences	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
intron	NN	O	O
lengths	NN	O	O
range	NN	O	O
in	NN	O	O
size	NN	O	O
from	NN	O	O
403	NN	O	O
bp	NN	O	O
to	NN	O	O
9	NN	O	O
.	NN	O	O

2	NN	O	O
kb	NN	O	O
and	NN	O	O
contain	NN	O	O
the	NN	O	O
intragenic	NN	O	O
microsatellite	NN	O	O
markers	NN	O	O
D17S1323	NN	O	O
,	NN	O	O
D17S1322	NN	O	O
,	NN	O	O
and	NN	O	O
D17S855	NN	O	O
,	NN	O	O
which	NN	O	O
localize	NN	O	O
to	NN	O	O
introns	NN	O	O
12	NN	O	O
,	NN	O	O
19	NN	O	O
,	NN	O	O
and	NN	O	O
20	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
the	NN	O	O
contig	NN	O	O
contains	NN	O	O
two	NN	O	O
complete	NN	O	O
genes	NN	O	O
Rho7	NN	O	O
,	NN	O	O
a	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
rho	NN	O	O
family	NN	O	O
of	NN	O	O
GTP	NN	O	O
binding	NN	O	O
proteins	NN	O	O
,	NN	O	O
and	NN	O	O
VAT1	NN	O	O
,	NN	O	O
an	NN	O	O
abundant	NN	O	O
membrane	NN	O	O
protein	NN	O	O
of	NN	O	O
cholinergic	NN	O	O
synaptic	NN	O	O
vesicles	NN	O	O
.	NN	O	O

Partial	NN	O	O
sequences	NN	O	O
of	NN	O	O
the	NN	O	O
1A1	NN	O	O
-	NN	O	O
3B	NN	O	O
B	NN	O	O
-	NN	O	O
box	NN	O	O
protein	NN	O	O
pseudogene	NN	O	O
and	NN	O	O
IFP	NN	O	O
35	NN	O	O
,	NN	O	O
an	NN	O	O
interferon	NN	O	O
induced	NN	O	O
leucine	NN	O	O
zipper	NN	O	O
protein	NN	O	O
,	NN	O	O
reside	NN	O	O
within	NN	O	O
the	NN	O	O
contig	NN	O	O
.	NN	O	O

An	NN	O	O
L21	NN	O	O
ribosomal	NN	O	O
protein	NN	O	O
pseudogene	NN	O	O
is	NN	O	O
embedded	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
intron	NN	O	O
13	NN	O	O
.	NN	O	O

The	NN	O	O
order	NN	O	O
of	NN	O	O
genes	NN	O	O
on	NN	O	O
the	NN	O	O
chromosome	NN	O	O
is	NN	O	O
centromere	NN	O	O
-	NN	O	O
1FP	NN	O	O
35	NN	O	O
-	NN	O	O
VAT1	NN	O	O
-	NN	O	O
Rho7	NN	O	O
-	NN	O	O
BRCA1	NN	O	O
-	NN	O	O
1A1	NN	O	O
-	NN	O	O
3B	NN	O	O
-	NN	O	O
telomere	NN	O	O

Identification	NN	O	O
of	NN	O	O
a	NN	O	O
RING	NN	O	O
protein	NN	O	O
that	NN	O	O
can	NN	O	O
interact	NN	O	O
in	NN	O	O
vivo	NN	O	O
with	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
product	NN	O	O
.	NN	O	O

The	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
gene	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
encodes	NN	O	O
a	NN	O	O
large	NN	O	O
polypeptide	NN	O	O
that	NN	O	O
contains	NN	O	O
the	NN	O	O
cysteine	NN	O	O
-	NN	O	O
rich	NN	O	O
RING	NN	O	O
motif	NN	O	O
,	NN	O	O
a	NN	O	O
zinc	NN	O	O
-	NN	O	O
binding	NN	O	O
domain	NN	O	O
found	NN	O	O
in	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
regulatory	NN	O	O
proteins	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
describe	NN	O	O
a	NN	O	O
novel	NN	O	O
protein	NN	O	O
that	NN	O	O
interacts	NN	O	O
in	NN	O	O
vivo	NN	O	O
with	NN	O	O
the	NN	O	O
N	NN	O	O
-	NN	O	O
terminal	NN	O	O
region	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

This	NN	O	O
BRCA1	NN	O	O
-	NN	O	O
associated	NN	O	O
RING	NN	O	O
domain	NN	O	O
(	NN	O	O
BARD1	NN	O	O
)	NN	O	O
protein	NN	O	O
contains	NN	O	O
an	NN	O	O
N	NN	O	O
-	NN	O	O
terminal	NN	O	O
RING	NN	O	O
motif	NN	O	O
,	NN	O	O
three	NN	O	O
tandem	NN	O	O
ankyrin	NN	O	O
repeats	NN	O	O
,	NN	O	O
and	NN	O	O
a	NN	O	O
C	NN	O	O
-	NN	O	O
terminal	NN	O	O
sequence	NN	O	O
with	NN	O	O
significant	NN	O	O
homology	NN	O	O
to	NN	O	O
the	NN	O	O
phylogenetically	NN	O	O
conserved	NN	O	O
BRCT	NN	O	O
domains	NN	O	O
that	NN	O	O
lie	NN	O	O
near	NN	O	O
the	NN	O	O
C	NN	O	O
terminus	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

The	NN	O	O
BARD1	NN	O	O
/	NN	O	O
BRCA1	NN	O	O
interaction	NN	O	O
is	NN	O	O
disrupted	NN	O	O
by	NN	O	O
BRCA1	NN	O	O
missense	NN	O	O
mutations	NN	O	O
that	NN	O	O
segregate	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
susceptibility	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
BARD1	NN	O	O
may	NN	O	O
be	NN	O	O
involved	NN	O	O
in	NN	O	O
mediating	NN	O	O
tumour	NN	O	B-Disease
suppression	NN	O	O
by	NN	O	O
BRCA1	NN	O	O
.	NN	O	O
.	NN	O	O

Detection	NN	O	O
of	NN	O	O
heterozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
using	NN	O	O
high	NN	O	O
density	NN	O	O
oligonucleotide	NN	O	O
arrays	NN	O	O
and	NN	O	O
two	NN	O	O
-	NN	O	O
colour	NN	O	O
fluorescence	NN	O	O
analysis	NN	O	O
.	NN	O	O

The	NN	O	O
ability	NN	O	O
to	NN	O	O
scan	NN	O	O
a	NN	O	O
large	NN	O	O
gene	NN	O	O
rapidly	NN	O	O
and	NN	O	O
accurately	NN	O	O
for	NN	O	O
all	NN	O	O
possible	NN	O	O
heterozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
large	NN	O	O
numbers	NN	O	O
of	NN	O	O
patient	NN	O	O
samples	NN	O	O
will	NN	O	O
be	NN	O	O
critical	NN	O	O
for	NN	O	O
the	NN	O	O
future	NN	O	O
of	NN	O	O
medicine	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
designed	NN	O	O
high	NN	O	O
-	NN	O	O
density	NN	O	O
arrays	NN	O	O
consisting	NN	O	O
of	NN	O	O
over	NN	O	O
96	NN	O	O
,	NN	O	O
600	NN	O	O
oligonucleotides	NN	O	O
20	NN	O	O
-	NN	O	O
nucleotides	NN	O	O
(	NN	O	O
nt	NN	O	O
)	NN	O	O
in	NN	O	O
length	NN	O	O
to	NN	O	O
screen	NN	O	O
for	NN	O	O
a	NN	O	O
wide	NN	O	O
range	NN	O	O
of	NN	O	O
heterozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
.	NN	O	O

45	NN	O	O
-	NN	O	O
kilobases	NN	O	O
(	NN	O	O
kb	NN	O	O
)	NN	O	O
exon	NN	O	O
11	NN	O	O
of	NN	O	O
the	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
gene	NN	O	O
BRCA1	NN	O	O
.	NN	O	O

Reference	NN	O	O
and	NN	O	O
test	NN	O	O
samples	NN	O	O
were	NN	O	O
co	NN	O	O
-	NN	O	O
hybridized	NN	O	O
to	NN	O	O
these	NN	O	O
arrays	NN	O	O
and	NN	O	O
differences	NN	O	O
in	NN	O	O
hybridization	NN	O	O
patterns	NN	O	O
quantitated	NN	O	O
by	NN	O	O
two	NN	O	O
-	NN	O	O
colour	NN	O	O
analysis	NN	O	O
.	NN	O	O

Fourteen	NN	O	O
of	NN	O	O
fifteen	NN	O	O
patient	NN	O	O
samples	NN	O	O
with	NN	O	O
known	NN	O	O
mutations	NN	O	O
were	NN	O	O
accurately	NN	O	O
diagnosed	NN	O	O
,	NN	O	O
and	NN	O	O
no	NN	O	O
false	NN	O	O
positive	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
20	NN	O	O
control	NN	O	O
samples	NN	O	O
.	NN	O	O

Eight	NN	O	O
single	NN	O	O
nucleotide	NN	O	O
polymorphisms	NN	O	O
were	NN	O	O
also	NN	O	O
readily	NN	O	O
detected	NN	O	O
.	NN	O	O

DNA	NN	O	O
chip	NN	O	O
-	NN	O	O
based	NN	O	O
assays	NN	O	O
may	NN	O	O
provide	NN	O	O
a	NN	O	O
valuable	NN	O	O
new	NN	O	O
technology	NN	O	O
for	NN	O	O
high	NN	O	O
-	NN	O	O
throughput	NN	O	O
cost	NN	O	O
-	NN	O	O
efficient	NN	O	O
detection	NN	O	O
of	NN	O	O
genetic	NN	O	O
alterations	NN	O	O
.	NN	O	O

Autosomal	NN	O	O
dominant	NN	O	O
primary	NN	O	O
hyperparathyroidism	NN	O	B-Disease
and	NN	O	I-Disease
jaw	NN	O	I-Disease
tumor	NN	O	I-Disease
syndrome	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
renal	NN	O	B-Disease
hamartomas	NN	O	I-Disease
and	NN	O	O
cystic	NN	O	B-Disease
kidney	NN	O	I-Disease
disease	NN	O	I-Disease
:	NN	O	O
linkage	NN	O	O
to	NN	O	O
1q21	NN	O	O
-	NN	O	O
q32	NN	O	O
and	NN	O	O
loss	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
type	NN	O	O
allele	NN	O	O
in	NN	O	O
renal	NN	O	B-Disease
hamartomas	NN	O	I-Disease
.	NN	O	O

Hereditary	NN	O	B-Disease
hyperparathyroidism	NN	O	I-Disease
-	NN	O	I-Disease
jaw	NN	O	I-Disease
tumor	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
HPT	NN	O	B-Disease
-	NN	O	I-Disease
JT	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
OMIM	NN	O	O
145001	NN	O	O
)	NN	O	O
that	NN	O	O
has	NN	O	O
recently	NN	O	O
been	NN	O	O
mapped	NN	O	O
to	NN	O	O
chromosomal	NN	O	O
region	NN	O	O
1q21	NN	O	O
-	NN	O	O
q32	NN	O	O
(	NN	O	O
HRPT2	NN	O	O
)	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
report	NN	O	O
two	NN	O	O
families	NN	O	O
with	NN	O	O
HPT	NN	O	B-Disease
-	NN	O	I-Disease
JT	NN	O	I-Disease
syndrome	NN	O	I-Disease
in	NN	O	O
which	NN	O	O
adult	NN	O	B-Disease
renal	NN	O	I-Disease
hamartomas	NN	O	I-Disease
or	NN	O	O
cystic	NN	O	B-Disease
kidney	NN	O	I-Disease
disease	NN	O	I-Disease
were	NN	O	O
prominent	NN	O	O
associated	NN	O	O
features	NN	O	O
,	NN	O	O
possibly	NN	O	O
representing	NN	O	O
a	NN	O	O
new	NN	O	O
phenotypic	NN	O	O
variant	NN	O	O
of	NN	O	O
the	NN	O	O
HPT	NN	O	B-Disease
-	NN	O	I-Disease
JT	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
the	NN	O	O
first	NN	O	O
family	NN	O	O
,	NN	O	O
renal	NN	O	B-Disease
lesions	NN	O	I-Disease
were	NN	O	O
present	NN	O	O
in	NN	O	O
five	NN	O	O
out	NN	O	O
of	NN	O	O
six	NN	O	O
affected	NN	O	O
individuals	NN	O	O
,	NN	O	O
whereas	NN	O	O
HPT	NN	O	B-Disease
and	NN	O	O
JT	NN	O	B-Disease
were	NN	O	O
seen	NN	O	O
in	NN	O	O
four	NN	O	O
and	NN	O	O
two	NN	O	O
cases	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
second	NN	O	O
family	NN	O	O
,	NN	O	O
JT	NN	O	B-Disease
was	NN	O	O
found	NN	O	O
in	NN	O	O
three	NN	O	O
of	NN	O	O
the	NN	O	O
five	NN	O	O
affected	NN	O	O
individuals	NN	O	O
and	NN	O	O
two	NN	O	O
affected	NN	O	O
members	NN	O	O
also	NN	O	O
exhibited	NN	O	O
polycystic	NN	O	B-Disease
kidney	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
possibility	NN	O	O
of	NN	O	O
the	NN	O	O
latter	NN	O	O
cosegregating	NN	O	O
as	NN	O	O
a	NN	O	O
separate	NN	O	O
autosomal	NN	O	O
dominant	NN	O	O
gene	NN	O	O
can	NN	O	O
not	NN	O	O
be	NN	O	O
ruled	NN	O	O
out	NN	O	O
.	NN	O	O

A	NN	O	O
sex	NN	O	O
-	NN	O	O
dependent	NN	O	O
penetrance	NN	O	O
of	NN	O	O
primary	NN	O	B-Disease
HPT	NN	O	I-Disease
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
predominantly	NN	O	O
male	NN	O	O
-	NN	O	O
affected	NN	O	O
cases	NN	O	O
was	NN	O	O
evident	NN	O	O
in	NN	O	O
the	NN	O	O
two	NN	O	O
families	NN	O	O
.	NN	O	O

Twenty	NN	O	O
microsatellite	NN	O	O
markers	NN	O	O
in	NN	O	O
the	NN	O	O
HRPT2	NN	O	O
region	NN	O	O
were	NN	O	O
typed	NN	O	O
,	NN	O	O
in	NN	O	O
addition	NN	O	O
to	NN	O	O
markers	NN	O	O
in	NN	O	O
the	NN	O	O
multiple	NN	O	B-Disease
endocrine	NN	O	I-Disease
neoplasia	NN	O	I-Disease
(	NN	O	I-Disease
MEN	NN	O	I-Disease
)	NN	O	I-Disease
types	NN	O	I-Disease
1	NN	O	I-Disease
and	NN	O	I-Disease
2	NN	O	I-Disease
regions	NN	O	O
at	NN	O	O
11q13	NN	O	O
and	NN	O	O
10q11	NN	O	O
.	NN	O	O

The	NN	O	O
disease	NN	O	O
in	NN	O	O
these	NN	O	O
two	NN	O	O
kindreds	NN	O	O
was	NN	O	O
linked	NN	O	O
to	NN	O	O
five	NN	O	O
markers	NN	O	O
in	NN	O	O
the	NN	O	O
1q21	NN	O	O
-	NN	O	O
q32	NN	O	O
region	NN	O	O
(	NN	O	O
logarithm	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
odds	NN	O	O
scores	NN	O	O
3	NN	O	O
.	NN	O	O
2	NN	O	O
-	NN	O	O
4	NN	O	O
2	NN	O	O
-	NN	O	O
4	NN	O	O
.	NN	O	O
2	NN	O	O
)	NN	O	O
,	NN	O	O
whereas	NN	O	O
linkage	NN	O	O
to	NN	O	O
the	NN	O	O
MEN1	NN	O	B-Disease
and	NN	O	O
MEN2	NN	O	B-Disease
regions	NN	O	O
was	NN	O	O
excluded	NN	O	O
.	NN	O	O

Meiotic	NN	O	O
recombinations	NN	O	O
detected	NN	O	O
in	NN	O	O
affected	NN	O	O
individuals	NN	O	O
placed	NN	O	O
the	NN	O	O
locus	NN	O	O
telomeric	NN	O	O
of	NN	O	O
D1S215	NN	O	O
,	NN	O	O
thus	NN	O	O
narrowing	NN	O	O
the	NN	O	O
HRPT2	NN	O	O
region	NN	O	O
from	NN	O	O
>	NN	O	O
60	NN	O	O
to	NN	O	O
approximately	NN	O	O
34	NN	O	O
centimorgans	NN	O	O
.	NN	O	O

Loss	NN	O	O
of	NN	O	O
heterozygosity	NN	O	O
was	NN	O	O
studied	NN	O	O
in	NN	O	O
seven	NN	O	O
renal	NN	O	B-Disease
hamartomas	NN	O	I-Disease
from	NN	O	O
two	NN	O	O
affected	NN	O	O
individuals	NN	O	O
in	NN	O	O
the	NN	O	O
first	NN	O	O
family	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
in	NN	O	O
a	NN	O	O
jaw	NN	O	B-Disease
tumor	NN	O	I-Disease
and	NN	O	O
a	NN	O	O
parathyroid	NN	O	B-Disease
tumor	NN	O	I-Disease
from	NN	O	O
the	NN	O	O
second	NN	O	O
family	NN	O	O
.	NN	O	O

All	NN	O	O
renal	NN	O	B-Disease
hamartomas	NN	O	I-Disease
showed	NN	O	O
loss	NN	O	O
of	NN	O	O
heterozygosity	NN	O	O
at	NN	O	O
the	NN	O	O
1q21	NN	O	O
-	NN	O	O
q32	NN	O	O
region	NN	O	O
.	NN	O	O

The	NN	O	O
losses	NN	O	O
invariably	NN	O	O
involved	NN	O	O
the	NN	O	O
wild	NN	O	O
type	NN	O	O
allele	NN	O	O
derived	NN	O	O
from	NN	O	O
the	NN	O	O
unaffected	NN	O	O
parent	NN	O	O
,	NN	O	O
suggesting	NN	O	O
the	NN	O	O
inactivation	NN	O	O
of	NN	O	O
a	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
in	NN	O	O
this	NN	O	O
region	NN	O	O

Independent	NN	O	O
origin	NN	O	O
of	NN	O	O
single	NN	O	O
and	NN	O	O
double	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
glucose	NN	O	O
6	NN	O	O
-	NN	O	O
phosphate	NN	O	O
dehydrogenase	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
vast	NN	O	O
majority	NN	O	O
of	NN	O	O
both	NN	O	O
polymorphic	NN	O	O
and	NN	O	O
sporadic	NN	O	O
G6PD	NN	O	O
variants	NN	O	O
are	NN	O	O
due	NN	O	O
to	NN	O	O
single	NN	O	O
missense	NN	O	O
mutations	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
four	NN	O	O
polymorphic	NN	O	O
variants	NN	O	O
that	NN	O	O
have	NN	O	O
two	NN	O	O
point	NN	O	O
mutations	NN	O	O
,	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
is	NN	O	O
always	NN	O	O
376	NN	O	O
A	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
(	NN	O	O
126	NN	O	O
Asn	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Asp	NN	O	O
)	NN	O	O
,	NN	O	O
which	NN	O	O
on	NN	O	O
its	NN	O	O
own	NN	O	O
gives	NN	O	O
rise	NN	O	O
to	NN	O	O
the	NN	O	O
nondeficient	NN	O	O
polymorphic	NN	O	O
variant	NN	O	O
,	NN	O	O
G6PD	NN	O	O
A	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
study	NN	O	O
of	NN	O	O
G6PD	NN	O	B-Disease
deficient	NN	O	I-Disease
patients	NN	O	O
who	NN	O	O
presented	NN	O	O
with	NN	O	O
clinical	NN	O	O
favism	NN	O	B-Disease
in	NN	O	O
Spain	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
found	NN	O	O
a	NN	O	O
new	NN	O	O
polymorphic	NN	O	O
variant	NN	O	O
that	NN	O	O
we	NN	O	O
have	NN	O	O
called	NN	O	O
G6PD	NN	O	O
Malaga	NN	O	O
,	NN	O	O
whose	NN	O	O
only	NN	O	O
abnormality	NN	O	O
is	NN	O	O
a	NN	O	O
542	NN	O	O
A	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
(	NN	O	O
181	NN	O	O
Asp	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Val	NN	O	O
)	NN	O	O
mutation	NN	O	O
.	NN	O	O

This	NN	O	O
is	NN	O	O
the	NN	O	O
same	NN	O	O
mutation	NN	O	O
as	NN	O	O
previously	NN	O	O
found	NN	O	O
in	NN	O	O
association	NN	O	O
with	NN	O	O
the	NN	O	O
mutation	NN	O	O
of	NN	O	O
G6PD	NN	O	O
A	NN	O	O
in	NN	O	O
the	NN	O	O
double	NN	O	O
mutant	NN	O	O
,	NN	O	O
G6PD	NN	O	O
Santamaria	NN	O	O
.	NN	O	O

G6PD	NN	O	O
Malaga	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
enzyme	NN	O	B-Disease
deficiency	NN	O	I-Disease
(	NN	O	O
class	NN	O	O
III	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
enzymic	NN	O	O
properties	NN	O	O
of	NN	O	O
G6PD	NN	O	O
Malaga	NN	O	O
and	NN	O	O
G6PD	NN	O	O
Santamaria	NN	O	O
are	NN	O	O
quite	NN	O	O
similar	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
in	NN	O	O
this	NN	O	O
case	NN	O	O
the	NN	O	O
effects	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
mutations	NN	O	O
are	NN	O	O
additive	NN	O	O
rather	NN	O	O
than	NN	O	O
synergistic	NN	O	O
.	NN	O	O

G6PD	NN	O	O
Santamaria	NN	O	O
might	NN	O	O
have	NN	O	O
been	NN	O	O
produced	NN	O	O
by	NN	O	O
recombination	NN	O	O
between	NN	O	O
G6PD	NN	O	O
A	NN	O	O
and	NN	O	O
G6PD	NN	O	O
Malaga	NN	O	O
;	NN	O	O
however	NN	O	O
haplotype	NN	O	O
analysis	NN	O	O
,	NN	O	O
including	NN	O	O
the	NN	O	O
use	NN	O	O
of	NN	O	O
a	NN	O	O
new	NN	O	O
silent	NN	O	O
polymorphism	NN	O	O
,	NN	O	O
suggests	NN	O	O
that	NN	O	O
the	NN	O	O
same	NN	O	O
542	NN	O	O
A	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
mutation	NN	O	O
has	NN	O	O
taken	NN	O	O
place	NN	O	O
independently	NN	O	O
in	NN	O	O
a	NN	O	O
G6PD	NN	O	O
B	NN	O	O
gene	NN	O	O
to	NN	O	O
give	NN	O	O
G6PD	NN	O	O
Malaga	NN	O	O
and	NN	O	O
in	NN	O	O
a	NN	O	O
G6PD	NN	O	O
A	NN	O	O
gene	NN	O	O
to	NN	O	O
give	NN	O	O
G6PD	NN	O	O
Santamaria	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
help	NN	O	O
to	NN	O	O
outline	NN	O	O
the	NN	O	O
relationship	NN	O	O
and	NN	O	O
evolution	NN	O	O
of	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
G6PD	NN	O	O
locus	NN	O	O
.	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
R841W	NN	O	O
:	NN	O	O
a	NN	O	O
strong	NN	O	O
candidate	NN	O	O
for	NN	O	O
a	NN	O	O
common	NN	O	O
mutation	NN	O	O
with	NN	O	O
moderate	NN	O	O
phenotype	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
mutations	NN	O	O
cause	NN	O	O
increased	NN	O	O
risk	NN	O	O
for	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
,	NN	O	O
frequently	NN	O	O
of	NN	O	O
early	NN	O	O
onset	NN	O	O
.	NN	O	O

Many	NN	O	O
different	NN	O	O
mutations	NN	O	O
occur	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
including	NN	O	O
several	NN	O	O
examples	NN	O	O
of	NN	O	O
recurrent	NN	O	O
mutations	NN	O	O
,	NN	O	O
each	NN	O	O
of	NN	O	O
which	NN	O	O
accounts	NN	O	O
for	NN	O	O
a	NN	O	O
significant	NN	O	O
number	NN	O	O
of	NN	O	O
families	NN	O	O
with	NN	O	O
heritable	NN	O	O
cancer	NN	O	B-Disease
predisposition	NN	O	O
.	NN	O	O

These	NN	O	O
common	NN	O	O
mutations	NN	O	O
have	NN	O	O
an	NN	O	O
etiological	NN	O	O
role	NN	O	O
in	NN	O	O
many	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
and	NN	O	O
provide	NN	O	O
the	NN	O	O
opportunity	NN	O	O
to	NN	O	O
examine	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
and	NN	O	O
genotype	NN	O	O
-	NN	O	O
environment	NN	O	O
interactions	NN	O	O
in	NN	O	O
individuals	NN	O	O
with	NN	O	O
the	NN	O	O
identical	NN	O	O
BRCA1	NN	O	O
lesion	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
a	NN	O	O
novel	NN	O	O
missense	NN	O	O
change	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
2640	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
(	NN	O	O
R841W	NN	O	O
)	NN	O	O
,	NN	O	O
found	NN	O	O
in	NN	O	O
3	NN	O	O
cases	NN	O	O
from	NN	O	O
a	NN	O	O
subject	NN	O	O
group	NN	O	O
of	NN	O	O
305	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
79	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
from	NN	O	O
Orange	NN	O	O
County	NN	O	O
,	NN	O	O
CA	NN	O	O
.	NN	O	O

These	NN	O	O
are	NN	O	O
consecutive	NN	O	O
,	NN	O	O
population	NN	O	O
-	NN	O	O
based	NN	O	O
cases	NN	O	O
not	NN	O	O
selected	NN	O	O
for	NN	O	O
age	NN	O	O
or	NN	O	O
family	NN	O	O
history	NN	O	O
.	NN	O	O

In	NN	O	O
all	NN	O	O
three	NN	O	O
cases	NN	O	O
,	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
strong	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
,	NN	O	I-Disease
ovarian	NN	O	I-Disease
,	NN	O	I-Disease
or	NN	O	I-Disease
other	NN	O	I-Disease
cancers	NN	O	I-Disease
possibly	NN	O	O
related	NN	O	O
to	NN	O	O
a	NN	O	O
BRCA1	NN	O	O
defect	NN	O	O
and	NN	O	O
family	NN	O	O
members	NN	O	O
showed	NN	O	O
a	NN	O	O
high	NN	O	O
concordance	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
incidence	NN	O	O
with	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
R841W	NN	O	O
.	NN	O	O

The	NN	O	O
age	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
onset	NN	O	O
was	NN	O	O
not	NN	O	O
always	NN	O	O
distinct	NN	O	O
from	NN	O	O
typical	NN	O	O
sporadic	NN	O	O
cases	NN	O	O
.	NN	O	O

Testing	NN	O	O
of	NN	O	O
a	NN	O	O
sample	NN	O	O
of	NN	O	O
413	NN	O	O
unrelated	NN	O	O
individuals	NN	O	O
to	NN	O	O
examine	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
R841W	NN	O	O
might	NN	O	O
be	NN	O	O
a	NN	O	O
rare	NN	O	O
polymorphism	NN	O	O
detected	NN	O	O
one	NN	O	O
additional	NN	O	O
instance	NN	O	O
in	NN	O	O
a	NN	O	O
woman	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
diagnosed	NN	O	O
at	NN	O	O
age	NN	O	O
77	NN	O	O
years	NN	O	O
,	NN	O	O
and	NN	O	O
cancer	NN	O	B-Disease
in	NN	O	O
one	NN	O	O
parent	NN	O	O
.	NN	O	O

R841W	NN	O	O
is	NN	O	O
likely	NN	O	O
to	NN	O	O
be	NN	O	O
an	NN	O	O
etiologically	NN	O	O
significant	NN	O	O
lesion	NN	O	O
with	NN	O	O
involvement	NN	O	O
in	NN	O	O
close	NN	O	O
to	NN	O	O
1	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
confidence	NN	O	O
interval	NN	O	O
of	NN	O	O
0	NN	O	O
-	NN	O	O
1	NN	O	O
.	NN	O	O
7	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
all	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancers	NN	O	I-Disease
in	NN	O	O
this	NN	O	O
population	NN	O	O
.	NN	O	O

Ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
:	NN	O	O
founder	NN	O	O
effect	NN	O	O
among	NN	O	O
north	NN	O	O
African	NN	O	O
Jews	NN	O	O
.	NN	O	O

The	NN	O	O
ATM	NN	O	O
gene	NN	O	O
is	NN	O	O
responsible	NN	O	O
for	NN	O	O
the	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
disorder	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
characterized	NN	O	O
by	NN	O	O
cerebellar	NN	O	B-Disease
degeneration	NN	O	I-Disease
,	NN	O	O
immunodeficiency	NN	O	B-Disease
and	NN	O	O
cancer	NN	O	B-Disease
predisposition	NN	O	I-Disease
.	NN	O	O

A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
carriers	NN	O	O
were	NN	O	O
reported	NN	O	O
to	NN	O	O
be	NN	O	O
moderately	NN	O	O
cancer	NN	O	B-Disease
-	NN	O	I-Disease
prone	NN	O	I-Disease
.	NN	O	O

A	NN	O	O
wide	NN	O	O
variety	NN	O	O
of	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
mutations	NN	O	O
,	NN	O	O
most	NN	O	O
of	NN	O	O
which	NN	O	O
are	NN	O	O
unique	NN	O	O
to	NN	O	O
single	NN	O	O
families	NN	O	O
,	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
various	NN	O	O
ethnic	NN	O	O
groups	NN	O	O
,	NN	O	O
precluding	NN	O	O
carrier	NN	O	O
screening	NN	O	O
with	NN	O	O
mutation	NN	O	O
-	NN	O	O
specific	NN	O	O
assays	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
a	NN	O	O
single	NN	O	O
mutation	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
32	NN	O	O
/	NN	O	O
33	NN	O	O
defective	NN	O	O
ATM	NN	O	O
alleles	NN	O	O
in	NN	O	O
Jewish	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
families	NN	O	O
of	NN	O	O
North	NN	O	O
African	NN	O	O
origin	NN	O	O
,	NN	O	O
coming	NN	O	O
from	NN	O	O
various	NN	O	O
regions	NN	O	O
of	NN	O	O
Morocco	NN	O	O
and	NN	O	O
Tunisia	NN	O	O
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
,	NN	O	O
103C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
,	NN	O	O
results	NN	O	O
in	NN	O	O
a	NN	O	O
stop	NN	O	O
codon	NN	O	O
at	NN	O	O
position	NN	O	O
35	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
protein	NN	O	O
.	NN	O	O

In	NN	O	O
keeping	NN	O	O
with	NN	O	O
the	NN	O	O
nature	NN	O	O
of	NN	O	O
this	NN	O	O
mutation	NN	O	O
,	NN	O	O
various	NN	O	O
antibodies	NN	O	O
directed	NN	O	O
against	NN	O	O
the	NN	O	O
ATM	NN	O	O
protein	NN	O	O
failed	NN	O	O
to	NN	O	O
defect	NN	O	O
this	NN	O	O
protein	NN	O	O
in	NN	O	O
patient	NN	O	O
cells	NN	O	O
.	NN	O	O

A	NN	O	O
rapid	NN	O	O
carrier	NN	O	O
detection	NN	O	O
assay	NN	O	O
detected	NN	O	O
this	NN	O	O
mutation	NN	O	O
in	NN	O	O
three	NN	O	O
out	NN	O	O
of	NN	O	O
488	NN	O	O
ATM	NN	O	O
alleles	NN	O	O
of	NN	O	O
Jewish	NN	O	O
Moroccan	NN	O	O
or	NN	O	O
Tunisian	NN	O	O
origin	NN	O	O
.	NN	O	O

This	NN	O	O
founder	NN	O	O
effect	NN	O	O
provides	NN	O	O
a	NN	O	O
unique	NN	O	O
opportunity	NN	O	O
for	NN	O	O
population	NN	O	O
-	NN	O	O
based	NN	O	O
screening	NN	O	O
for	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
carriers	NN	O	O
in	NN	O	O
a	NN	O	O
large	NN	O	O
Jewish	NN	O	O
community	NN	O	O
.	NN	O	O
.	NN	O	O

Mutation	NN	O	O
analysis	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
in	NN	O	O
a	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
population	NN	O	O
.	NN	O	O

A	NN	O	O
population	NN	O	O
-	NN	O	O
based	NN	O	O
series	NN	O	O
of	NN	O	O
54	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
from	NN	O	O
Southern	NN	O	O
California	NN	O	O
were	NN	O	O
analyzed	NN	O	O
for	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
genes	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

Nine	NN	O	O
(	NN	O	O
17	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
had	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
at	NN	O	O
least	NN	O	O
one	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relative	NN	O	O
.	NN	O	O

A	NN	O	O
further	NN	O	O
seven	NN	O	O
(	NN	O	O
13	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
reported	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
at	NN	O	O
least	NN	O	O
one	NN	O	O
second	NN	O	O
-	NN	O	O
degree	NN	O	O
relative	NN	O	O
and	NN	O	O
in	NN	O	O
no	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relatives	NN	O	O
.	NN	O	O

No	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
.	NN	O	O

Two	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
(	NN	O	O
4	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
total	NN	O	O
)	NN	O	O
were	NN	O	O
found	NN	O	O
to	NN	O	O
carry	NN	O	O
novel	NN	O	O
truncating	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
gene	NN	O	O
.	NN	O	O

Only	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
carrying	NN	O	O
a	NN	O	O
BRCA2	NN	O	O
mutation	NN	O	O
had	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
,	NN	O	O
with	NN	O	O
one	NN	O	O
case	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
a	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relative	NN	O	O
.	NN	O	O

The	NN	O	O
remaining	NN	O	O
eight	NN	O	O
cases	NN	O	O
(	NN	O	O
89	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
male	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
with	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relatives	NN	O	O
remain	NN	O	O
unaccounted	NN	O	O
for	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
either	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
or	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
gene	NN	O	O
.	NN	O	O
.	NN	O	O

Molecular	NN	O	O
basis	NN	O	O
for	NN	O	O
Duarte	NN	O	B-Disease
and	NN	O	I-Disease
Los	NN	O	I-Disease
Angeles	NN	O	I-Disease
variant	NN	O	I-Disease
galactosemia	NN	O	I-Disease
.	NN	O	O

Human	NN	O	O
orythrocytes	NN	O	O
that	NN	O	O
are	NN	O	O
homozygous	NN	O	O
for	NN	O	O
the	NN	O	O
Duarte	NN	O	B-Disease
enzyme	NN	O	I-Disease
variant	NN	O	I-Disease
of	NN	O	I-Disease
galactosemia	NN	O	I-Disease
(	NN	O	O
D	NN	O	O
/	NN	O	O
D	NN	O	O
)	NN	O	O
have	NN	O	O
a	NN	O	O
characteristic	NN	O	O
isoform	NN	O	O
on	NN	O	O
isoelectric	NN	O	O
focusing	NN	O	O
and	NN	O	O
50	NN	O	O
%	NN	O	O
reduction	NN	O	O
in	NN	O	O
galactose	NN	O	O
-	NN	O	O
1	NN	O	O
-	NN	O	O
phosphate	NN	O	O
uridyltransferase	NN	O	O
(	NN	O	O
GALT	NN	O	O
)	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
.	NN	O	O

The	NN	O	O
Duarte	NN	O	O
biochemical	NN	O	O
phenotype	NN	O	O
has	NN	O	O
a	NN	O	O
molecular	NN	O	O
genotype	NN	O	O
of	NN	O	O
N314D	NN	O	O
/	NN	O	O
N314D	NN	O	O
.	NN	O	O

The	NN	O	O
characteristic	NN	O	O
Duarte	NN	O	O
isoform	NN	O	O
is	NN	O	O
also	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
variant	NN	O	O
called	NN	O	O
the	NN	O	O
"	NN	O	O
Los	NN	O	O
Angeles	NN	O	O
(	NN	O	O
LA	NN	O	O
)	NN	O	O
phenotype	NN	O	O
,	NN	O	O
"	NN	O	O
which	NN	O	O
has	NN	O	O
increased	NN	O	O
GALT	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
.	NN	O	O

We	NN	O	O
evaluated	NN	O	O
GALT	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
and	NN	O	O
screened	NN	O	O
the	NN	O	O
GALT	NN	O	O
genes	NN	O	O
of	NN	O	O
145	NN	O	O
patients	NN	O	O
with	NN	O	O
one	NN	O	O
or	NN	O	O
more	NN	O	O
N314D	NN	O	O
-	NN	O	O
containing	NN	O	O
alleles	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
seven	NN	O	O
with	NN	O	O
the	NN	O	O
LA	NN	O	O
biochemical	NN	O	O
phenotype	NN	O	O
,	NN	O	O
and	NN	O	O
all	NN	O	O
had	NN	O	O
a	NN	O	O
1721C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
transition	NN	O	O
in	NN	O	O
exon	NN	O	O
7	NN	O	O
in	NN	O	O
cis	NN	O	O
with	NN	O	O
the	NN	O	O
N314D	NN	O	O
missense	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
1721C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
transition	NN	O	O
is	NN	O	O
a	NN	O	O
neutral	NN	O	O
polymorphism	NN	O	O
for	NN	O	O
leucine	NN	O	O
at	NN	O	O
amino	NN	O	O
acid	NN	O	O
218	NN	O	O
(	NN	O	O
L218L	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
pedigree	NN	O	O
analyses	NN	O	O
,	NN	O	O
this	NN	O	O
1721C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
transition	NN	O	O
segregated	NN	O	O
with	NN	O	O
the	NN	O	O
LA	NN	O	O
phenotype	NN	O	O
of	NN	O	O
increased	NN	O	O
GALT	NN	O	O
activity	NN	O	O
in	NN	O	O
three	NN	O	O
different	NN	O	O
biochemical	NN	O	O
phenotypes	NN	O	O
(	NN	O	O
LA	NN	O	O
/	NN	O	O
N	NN	O	O
,	NN	O	O
LA	NN	O	O
/	NN	O	O
G	NN	O	O
,	NN	O	O
and	NN	O	O
LA	NN	O	O
/	NN	O	O
D	NN	O	O
)	NN	O	O
.	NN	O	O

To	NN	O	O
determine	NN	O	O
the	NN	O	O
mechanism	NN	O	O
for	NN	O	O
increased	NN	O	O
activity	NN	O	O
of	NN	O	O
the	NN	O	O
LA	NN	O	O
variant	NN	O	O
,	NN	O	O
we	NN	O	O
compared	NN	O	O
GALT	NN	O	O
mRNA	NN	O	O
,	NN	O	O
protein	NN	O	O
abundance	NN	O	O
,	NN	O	O
and	NN	O	O
enzyme	NN	O	O
thermal	NN	O	O
stability	NN	O	O
in	NN	O	O
lymphoblast	NN	O	O
cell	NN	O	O
lines	NN	O	O
of	NN	O	O
D	NN	O	O
and	NN	O	O
LA	NN	O	O
phenotypes	NN	O	O
with	NN	O	O
comparable	NN	O	O
genotypes	NN	O	O
.	NN	O	O

GALT	NN	O	O
protein	NN	O	O
abundance	NN	O	O
was	NN	O	O
increased	NN	O	O
in	NN	O	O
LA	NN	O	O
compared	NN	O	O
to	NN	O	O
D	NN	O	O
alleles	NN	O	O
,	NN	O	O
but	NN	O	O
mRNA	NN	O	O
was	NN	O	O
similar	NN	O	O
among	NN	O	O
all	NN	O	O
genotypes	NN	O	O
.	NN	O	O

When	NN	O	O
LA	NN	O	O
/	NN	O	O
D	NN	O	O
and	NN	O	O
D	NN	O	O
/	NN	O	O
D	NN	O	O
GALT	NN	O	O
biochemical	NN	O	O
phenotypes	NN	O	O
were	NN	O	O
compared	NN	O	O
to	NN	O	O
N	NN	O	O
/	NN	O	O
N	NN	O	O
GALT	NN	O	O
phenotypes	NN	O	O
,	NN	O	O
both	NN	O	O
had	NN	O	O
50	NN	O	O
%	NN	O	O
,	NN	O	O
as	NN	O	O
compared	NN	O	O
to	NN	O	O
21	NN	O	O
%	NN	O	O
,	NN	O	O
reduction	NN	O	O
in	NN	O	O
GALT	NN	O	O
activity	NN	O	O
in	NN	O	O
the	NN	O	O
wild	NN	O	O
type	NN	O	O
(	NN	O	O
N	NN	O	O
/	NN	O	O
N	NN	O	O
)	NN	O	O
after	NN	O	O
exposure	NN	O	O
at	NN	O	O
identical	NN	O	O
initial	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
to	NN	O	O
50	NN	O	O
degrees	NN	O	O
C	NN	O	O
for	NN	O	O
15	NN	O	O
min	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
codon	NN	O	O
change	NN	O	O
N314D	NN	O	O
in	NN	O	O
cis	NN	O	O
with	NN	O	O
the	NN	O	O
base	NN	O	O
-	NN	O	O
pair	NN	O	O
transition	NN	O	O
1721C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
produces	NN	O	O
the	NN	O	O
LA	NN	O	B-Disease
variant	NN	O	I-Disease
of	NN	O	I-Disease
galactosemia	NN	O	I-Disease
and	NN	O	O
that	NN	O	O
this	NN	O	O
nucleotide	NN	O	O
change	NN	O	O
increases	NN	O	O
GALT	NN	O	O
activity	NN	O	O
by	NN	O	O
increasing	NN	O	O
GALT	NN	O	O
protein	NN	O	O
abundance	NN	O	O
without	NN	O	O
increasing	NN	O	O
transcription	NN	O	O
or	NN	O	O
decreasing	NN	O	O
thermal	NN	O	O
lability	NN	O	O
.	NN	O	O

A	NN	O	O
favorable	NN	O	O
codon	NN	O	O
bias	NN	O	O
for	NN	O	O
the	NN	O	O
mutated	NN	O	O
codon	NN	O	O
with	NN	O	O
consequently	NN	O	O
increased	NN	O	O
translation	NN	O	O
rates	NN	O	O
is	NN	O	O
postulated	NN	O	O
as	NN	O	O
the	NN	O	O
mechanism	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
TSG101	NN	O	O
tumor	NN	O	B-Disease
susceptibility	NN	O	O
gene	NN	O	O
is	NN	O	O
located	NN	O	O
in	NN	O	O
chromosome	NN	O	O
11	NN	O	O
band	NN	O	O
p15	NN	O	O
and	NN	O	O
is	NN	O	O
mutated	NN	O	O
in	NN	O	O
human	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Recent	NN	O	O
work	NN	O	O
has	NN	O	O
identified	NN	O	O
a	NN	O	O
mouse	NN	O	O
gene	NN	O	O
(	NN	O	O
tsg101	NN	O	O
)	NN	O	O
whose	NN	O	O
inactivation	NN	O	O
in	NN	O	O
fibroblasts	NN	O	O
results	NN	O	O
in	NN	O	O
cellular	NN	O	O
transformation	NN	O	O
and	NN	O	O
the	NN	O	O
ability	NN	O	O
to	NN	O	O
produce	NN	O	O
metastatic	NN	O	B-Disease
tumors	NN	O	I-Disease
in	NN	O	O
nude	NN	O	O
mice	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
we	NN	O	O
report	NN	O	O
that	NN	O	O
the	NN	O	O
human	NN	O	O
homolog	NN	O	O
,	NN	O	O
TSG101	NN	O	O
,	NN	O	O
which	NN	O	O
we	NN	O	O
isolated	NN	O	O
and	NN	O	O
mapped	NN	O	O
to	NN	O	O
chromosome	NN	O	O
11	NN	O	O
,	NN	O	O
bands	NN	O	O
15	NN	O	O
.	NN	O	O

1	NN	O	O
-	NN	O	O
15	NN	O	O
1	NN	O	O
-	NN	O	O
15	NN	O	O
.	NN	O	O

2	NN	O	O
,	NN	O	O
a	NN	O	O
region	NN	O	O
proposed	NN	O	O
to	NN	O	O
contain	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
(	NN	O	O
s	NN	O	O
)	NN	O	O
,	NN	O	O
is	NN	O	O
mutated	NN	O	O
at	NN	O	O
high	NN	O	O
frequency	NN	O	O
in	NN	O	O
human	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
7	NN	O	O
of	NN	O	O
15	NN	O	O
uncultured	NN	O	O
primary	NN	O	O
human	NN	O	O
breast	NN	O	B-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
intragenic	NN	O	O
deletions	NN	O	O
were	NN	O	O
shown	NN	O	O
in	NN	O	O
TSG101	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
and	NN	O	O
transcripts	NN	O	O
by	NN	O	O
gel	NN	O	O
and	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
,	NN	O	O
and	NN	O	O
mutations	NN	O	O
affecting	NN	O	O
two	NN	O	O
TSG101	NN	O	O
alleles	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
four	NN	O	O
of	NN	O	O
these	NN	O	O
cancers	NN	O	B-Disease
.	NN	O	O

No	NN	O	O
TSG101	NN	O	O
defects	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
matched	NN	O	O
normal	NN	O	O
breast	NN	O	O
tissue	NN	O	O
from	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
strongly	NN	O	O
implicate	NN	O	O
TSG101	NN	O	O
mutations	NN	O	O
in	NN	O	O
human	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease

Moderate	NN	O	O
intergenerational	NN	O	O
and	NN	O	O
somatic	NN	O	O
instability	NN	O	O
of	NN	O	O
a	NN	O	O
55	NN	O	O
-	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
in	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
expansion	NN	O	O
of	NN	O	O
a	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
untranslated	NN	O	O
region	NN	O	O
(	NN	O	O
UTR	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
protein	NN	O	O
kinase	NN	O	O
gene	NN	O	O
(	NN	O	O
DMPK	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
is	NN	O	O
polymorphic	NN	O	O
and	NN	O	O
varies	NN	O	O
in	NN	O	O
size	NN	O	O
between	NN	O	O
5	NN	O	O
and	NN	O	O
37	NN	O	O
repeats	NN	O	O
in	NN	O	O
unaffected	NN	O	O
individuals	NN	O	O
whereas	NN	O	O
in	NN	O	O
affected	NN	O	O
patients	NN	O	O
there	NN	O	O
are	NN	O	O
between	NN	O	O
50	NN	O	O
and	NN	O	O
4	NN	O	O
,	NN	O	O
000	NN	O	O
CTGs	NN	O	O
.	NN	O	O

The	NN	O	O
size	NN	O	O
of	NN	O	O
the	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
,	NN	O	O
which	NN	O	O
increases	NN	O	O
through	NN	O	O
generations	NN	O	O
,	NN	O	O
generally	NN	O	O
correlates	NN	O	O
with	NN	O	O
clinical	NN	O	O
severity	NN	O	O
and	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
.	NN	O	O

The	NN	O	O
instability	NN	O	O
of	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
appears	NN	O	O
to	NN	O	O
depend	NN	O	O
on	NN	O	O
its	NN	O	O
size	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
on	NN	O	O
the	NN	O	O
sex	NN	O	O
of	NN	O	O
the	NN	O	O
transmitting	NN	O	O
parent	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
mitotic	NN	O	O
instability	NN	O	O
analysis	NN	O	O
of	NN	O	O
different	NN	O	O
human	NN	O	O
DM	NN	O	B-Disease
tissues	NN	O	O
shows	NN	O	O
length	NN	O	O
mosaicism	NN	O	O
between	NN	O	O
different	NN	O	O
cell	NN	O	O
lineages	NN	O	O
.	NN	O	O

The	NN	O	O
molecular	NN	O	O
mechanisms	NN	O	O
of	NN	O	O
triplet	NN	O	O
instability	NN	O	O
remain	NN	O	O
elusive	NN	O	O
.	NN	O	O

To	NN	O	O
investigate	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
genomic	NN	O	O
sequences	NN	O	O
in	NN	O	O
instability	NN	O	O
,	NN	O	O
we	NN	O	O
produced	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
containing	NN	O	O
a	NN	O	O
45	NN	O	O
-	NN	O	O
kb	NN	O	O
genomic	NN	O	O
segment	NN	O	O
with	NN	O	O
a	NN	O	O
55	NN	O	O
-	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
cloned	NN	O	O
from	NN	O	O
a	NN	O	O
mildly	NN	O	O
affected	NN	O	O
patient	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
to	NN	O	O
other	NN	O	O
mouse	NN	O	O
models	NN	O	O
containing	NN	O	O
CAG	NN	O	O
repeats	NN	O	O
within	NN	O	O
cDNAs	NN	O	O
,	NN	O	O
these	NN	O	O
mice	NN	O	O
showed	NN	O	O
both	NN	O	O
intergenerational	NN	O	O
and	NN	O	O
somatic	NN	O	O
repeat	NN	O	O
instability	NN	O	O
.	NN	O	O
.	NN	O	O

Missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
Fas	NN	O	O
gene	NN	O	O
resulting	NN	O	O
in	NN	O	O
autoimmune	NN	O	B-Disease
lymphoproliferative	NN	O	I-Disease
syndrome	NN	O	I-Disease
:	NN	O	O
a	NN	O	O
molecular	NN	O	O
and	NN	O	O
immunological	NN	O	O
analysis	NN	O	O
.	NN	O	O

Programmed	NN	O	O
cell	NN	O	O
death	NN	O	O
(	NN	O	O
or	NN	O	O
apoptosis	NN	O	O
)	NN	O	O
is	NN	O	O
a	NN	O	O
physiological	NN	O	O
process	NN	O	O
essential	NN	O	O
to	NN	O	O
the	NN	O	O
normal	NN	O	O
development	NN	O	O
and	NN	O	O
homeostatic	NN	O	O
maintenance	NN	O	O
of	NN	O	O
the	NN	O	O
immune	NN	O	O
system	NN	O	O
.	NN	O	O

The	NN	O	O
Fas	NN	O	O
/	NN	O	O
Apo	NN	O	O
-	NN	O	O
1	NN	O	O
receptor	NN	O	O
plays	NN	O	O
a	NN	O	O
crucial	NN	O	O
role	NN	O	O
in	NN	O	O
the	NN	O	O
regulation	NN	O	O
of	NN	O	O
apoptosis	NN	O	O
,	NN	O	O
as	NN	O	O
demonstrated	NN	O	O
by	NN	O	O
lymphoproliferation	NN	O	O
in	NN	O	O
MRL	NN	O	O
-	NN	O	O
lpr	NN	O	O
/	NN	O	O
lpr	NN	O	O
mice	NN	O	O
and	NN	O	O
by	NN	O	O
the	NN	O	O
recently	NN	O	O
described	NN	O	O
autoimmune	NN	O	B-Disease
lymphoproliferative	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
ALPS	NN	O	B-Disease
)	NN	O	O
in	NN	O	O
humans	NN	O	O
,	NN	O	O
both	NN	O	O
of	NN	O	O
which	NN	O	O
are	NN	O	O
due	NN	O	O
to	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
Fas	NN	O	O
gene	NN	O	O
.	NN	O	O

We	NN	O	O
describe	NN	O	O
a	NN	O	O
novel	NN	O	O
family	NN	O	O
with	NN	O	O
ALPS	NN	O	B-Disease
in	NN	O	O
which	NN	O	O
three	NN	O	O
affected	NN	O	O
siblings	NN	O	O
carry	NN	O	O
two	NN	O	O
distinct	NN	O	O
missense	NN	O	O
mutations	NN	O	O
on	NN	O	O
both	NN	O	O
the	NN	O	O
Fas	NN	O	O
gene	NN	O	O
alleles	NN	O	O
and	NN	O	O
show	NN	O	O
lack	NN	O	O
of	NN	O	O
Fas	NN	O	O
-	NN	O	O
induced	NN	O	O
apoptosis	NN	O	O
.	NN	O	O

The	NN	O	O
children	NN	O	O
share	NN	O	O
common	NN	O	O
clinical	NN	O	O
features	NN	O	O
including	NN	O	O
splenomegaly	NN	O	B-Disease
and	NN	O	O
lymphadenopathy	NN	O	B-Disease
,	NN	O	O
but	NN	O	O
only	NN	O	O
one	NN	O	O
developed	NN	O	O
severe	NN	O	O
autoimmune	NN	O	B-Disease
manifestations	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
all	NN	O	O
three	NN	O	O
siblings	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrated	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
anergic	NN	O	O
CD3	NN	O	O
+	NN	O	O
CD4	NN	O	O
-	NN	O	O
CD8	NN	O	O
-	NN	O	O
(	NN	O	O
double	NN	O	O
negative	NN	O	O
,	NN	O	O
[	NN	O	O
DN	NN	O	O
]	NN	O	O
)	NN	O	O
T	NN	O	O
cells	NN	O	O
;	NN	O	O
moreover	NN	O	O
,	NN	O	O
a	NN	O	O
chronic	NN	O	O
lymphocyte	NN	O	O
activation	NN	O	O
was	NN	O	O
found	NN	O	O
,	NN	O	O
as	NN	O	O
demonstrated	NN	O	O
by	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
high	NN	O	O
levels	NN	O	O
of	NN	O	O
HLA	NN	O	O
-	NN	O	O
DR	NN	O	O
expression	NN	O	O
on	NN	O	O
peripheral	NN	O	O
CD3	NN	O	O
+	NN	O	O
cells	NN	O	O
and	NN	O	O
by	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
high	NN	O	O
levels	NN	O	O
of	NN	O	O
serum	NN	O	O
activation	NN	O	O
markers	NN	O	O
such	NN	O	O
as	NN	O	O
soluble	NN	O	O
interleukin	NN	O	O
-	NN	O	O
2	NN	O	O
receptor	NN	O	O
(	NN	O	O
slL	NN	O	O
-	NN	O	O
2R	NN	O	O
)	NN	O	O
and	NN	O	O
soluble	NN	O	O
CD30	NN	O	O
(	NN	O	O
sCD30	NN	O	O
)	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
gene	NN	O	O
product	NN	O	O
,	NN	O	O
a	NN	O	O
constitutively	NN	O	O
expressed	NN	O	O
nuclear	NN	O	O
protein	NN	O	O
that	NN	O	O
is	NN	O	O
not	NN	O	O
up	NN	O	O
-	NN	O	O
regulated	NN	O	O
following	NN	O	O
genome	NN	O	O
damage	NN	O	O
.	NN	O	O

The	NN	O	O
product	NN	O	O
of	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
gene	NN	O	O
(	NN	O	O
ATM	NN	O	O
)	NN	O	O
was	NN	O	O
identified	NN	O	O
by	NN	O	O
using	NN	O	O
an	NN	O	O
antiserum	NN	O	O
developed	NN	O	O
to	NN	O	O
a	NN	O	O
peptide	NN	O	O
corresponding	NN	O	O
to	NN	O	O
the	NN	O	O
deduced	NN	O	O
amino	NN	O	O
acid	NN	O	O
sequence	NN	O	O
.	NN	O	O

The	NN	O	O
ATM	NN	O	O
protein	NN	O	O
is	NN	O	O
a	NN	O	O
single	NN	O	O
,	NN	O	O
high	NN	O	O
-	NN	O	O
molecular	NN	O	O
weight	NN	O	O
protein	NN	O	O
predominantly	NN	O	O
confined	NN	O	O
to	NN	O	O
the	NN	O	O
nucleus	NN	O	O
of	NN	O	O
human	NN	O	O
fibroblasts	NN	O	O
,	NN	O	O
but	NN	O	O
is	NN	O	O
present	NN	O	O
in	NN	O	O
both	NN	O	O
nuclear	NN	O	O
and	NN	O	O
microsomal	NN	O	O
fractions	NN	O	O
from	NN	O	O
human	NN	O	O
lymphoblast	NN	O	O
cells	NN	O	O
and	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
lymphocytes	NN	O	O
.	NN	O	O

ATM	NN	O	O
protein	NN	O	O
levels	NN	O	O
and	NN	O	O
localization	NN	O	O
remain	NN	O	O
constant	NN	O	O
throughout	NN	O	O
all	NN	O	O
stages	NN	O	O
of	NN	O	O
the	NN	O	O
cell	NN	O	O
cycle	NN	O	O
.	NN	O	O

Truncated	NN	O	O
ATM	NN	O	O
protein	NN	O	O
was	NN	O	O
not	NN	O	O
detected	NN	O	O
in	NN	O	O
lymphoblasts	NN	O	O
from	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
patients	NN	O	O
homozygous	NN	O	O
for	NN	O	O
mutations	NN	O	O
leading	NN	O	O
to	NN	O	O
premature	NN	O	O
protein	NN	O	O
termination	NN	O	O
.	NN	O	O

Exposure	NN	O	O
of	NN	O	O
normal	NN	O	O
human	NN	O	O
cells	NN	O	O
to	NN	O	O
gamma	NN	O	O
-	NN	O	O
irradiation	NN	O	O
and	NN	O	O
the	NN	O	O
radiomimetic	NN	O	O
drug	NN	O	O
neocarzinostatin	NN	O	O
had	NN	O	O
no	NN	O	O
effect	NN	O	O
on	NN	O	O
ATM	NN	O	O
protein	NN	O	O
levels	NN	O	O
,	NN	O	O
in	NN	O	O
contrast	NN	O	O
to	NN	O	O
a	NN	O	O
noted	NN	O	O
rise	NN	O	O
in	NN	O	O
p53	NN	O	O
levels	NN	O	O
over	NN	O	O
the	NN	O	O
same	NN	O	O
time	NN	O	O
interval	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
are	NN	O	O
consistent	NN	O	O
with	NN	O	O
a	NN	O	O
role	NN	O	O
for	NN	O	O
the	NN	O	O
ATM	NN	O	O
protein	NN	O	O
in	NN	O	O
ensuring	NN	O	O
the	NN	O	O
fidelity	NN	O	O
of	NN	O	O
DNA	NN	O	O
repair	NN	O	O
and	NN	O	O
cell	NN	O	O
cycle	NN	O	O
regulation	NN	O	O
following	NN	O	O
genome	NN	O	O
damage	NN	O	O
.	NN	O	O
.	NN	O	O

Type	NN	O	O
III	NN	O	O
collagen	NN	O	O
is	NN	O	O
crucial	NN	O	O
for	NN	O	O
collagen	NN	O	O
I	NN	O	O
fibrillogenesis	NN	O	O
and	NN	O	O
for	NN	O	O
normal	NN	O	O
cardiovascular	NN	O	O
development	NN	O	O
.	NN	O	O

Type	NN	O	O
III	NN	O	O
collagen	NN	O	O
is	NN	O	O
a	NN	O	O
fibrillar	NN	O	O
forming	NN	O	O
collagen	NN	O	O
comprising	NN	O	O
three	NN	O	O
alpha1	NN	O	O
(	NN	O	O
III	NN	O	O
)	NN	O	O
chains	NN	O	O
and	NN	O	O
is	NN	O	O
expressed	NN	O	O
in	NN	O	O
early	NN	O	O
embryos	NN	O	O
and	NN	O	O
throughout	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
adult	NN	O	O
,	NN	O	O
type	NN	O	O
III	NN	O	O
collagen	NN	O	O
is	NN	O	O
a	NN	O	O
major	NN	O	O
component	NN	O	O
of	NN	O	O
the	NN	O	O
extracellular	NN	O	O
matrix	NN	O	O
in	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
internal	NN	O	O
organs	NN	O	O
and	NN	O	O
skin	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
COL3A1	NN	O	O
gene	NN	O	O
have	NN	O	O
been	NN	O	O
implicated	NN	O	O
as	NN	O	O
a	NN	O	O
cause	NN	O	O
of	NN	O	O
type	NN	O	B-Disease
IV	NN	O	I-Disease
Ehlers	NN	O	I-Disease
-	NN	O	I-Disease
Danlos	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
a	NN	O	O
disease	NN	O	O
leading	NN	O	O
to	NN	O	O
aortic	NN	O	B-Disease
rupture	NN	O	I-Disease
in	NN	O	O
early	NN	O	O
adult	NN	O	O
life	NN	O	O
.	NN	O	O

To	NN	O	O
directly	NN	O	O
study	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
Col3a1	NN	O	O
in	NN	O	O
development	NN	O	O
and	NN	O	O
disease	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
inactivated	NN	O	O
the	NN	O	O
Col3a1	NN	O	O
gene	NN	O	O
in	NN	O	O
embryonic	NN	O	O
stem	NN	O	O
cells	NN	O	O
by	NN	O	O
homologous	NN	O	O
recombination	NN	O	O
.	NN	O	O

The	NN	O	O
mutated	NN	O	O
allele	NN	O	O
was	NN	O	O
transmitted	NN	O	O
through	NN	O	O
the	NN	O	O
mouse	NN	O	O
germ	NN	O	O
line	NN	O	O
and	NN	O	O
homozygous	NN	O	O
mutant	NN	O	O
animals	NN	O	O
were	NN	O	O
derived	NN	O	O
from	NN	O	O
heterozygous	NN	O	O
intercrosses	NN	O	O
.	NN	O	O

About	NN	O	O
10	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
homozygous	NN	O	O
mutant	NN	O	O
animals	NN	O	O
survived	NN	O	O
to	NN	O	O
adulthood	NN	O	O
but	NN	O	O
have	NN	O	O
a	NN	O	O
much	NN	O	O
shorter	NN	O	O
life	NN	O	O
span	NN	O	O
compared	NN	O	O
with	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
mice	NN	O	O
.	NN	O	O

The	NN	O	O
major	NN	O	O
cause	NN	O	O
of	NN	O	O
death	NN	O	O
of	NN	O	O
mutant	NN	O	O
mice	NN	O	O
was	NN	O	O
rupture	NN	O	O
of	NN	O	O
the	NN	O	O
major	NN	O	O
blood	NN	O	O
vessels	NN	O	O
,	NN	O	O
similar	NN	O	O
to	NN	O	O
patients	NN	O	O
with	NN	O	O
type	NN	O	B-Disease
IV	NN	O	I-Disease
Ehlers	NN	O	I-Disease
-	NN	O	I-Disease
Danlos	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Ultrastructural	NN	O	O
analysis	NN	O	O
of	NN	O	O
tissues	NN	O	O
from	NN	O	O
mutant	NN	O	O
mice	NN	O	O
revealed	NN	O	O
that	NN	O	O
type	NN	O	O
III	NN	O	O
collagen	NN	O	O
is	NN	O	O
essential	NN	O	O
for	NN	O	O
normal	NN	O	O
collagen	NN	O	O
I	NN	O	O
fibrillogenesis	NN	O	O
in	NN	O	O
the	NN	O	O
cardiovascular	NN	O	O
system	NN	O	O
and	NN	O	O
other	NN	O	O
organs	NN	O	O
.	NN	O	O
.	NN	O	O

Nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
3	NN	O	O
of	NN	O	O
the	NN	O	O
proteolipid	NN	O	O
protein	NN	O	O
gene	NN	O	O
(	NN	O	O
PLP	NN	O	O
)	NN	O	O
in	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
an	NN	O	O
unusual	NN	O	O
form	NN	O	O
of	NN	O	O
Pelizaeus	NN	O	B-Disease
-	NN	O	I-Disease
Merzbacher	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
a	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
transition	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
431	NN	O	O
of	NN	O	O
the	NN	O	O
proteolipid	NN	O	O
protein	NN	O	O
gene	NN	O	O
(	NN	O	O
PLP	NN	O	O
)	NN	O	O
results	NN	O	O
in	NN	O	O
a	NN	O	O
nonsense	NN	O	O
codon	NN	O	O
in	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
an	NN	O	O
unusual	NN	O	O
form	NN	O	O
of	NN	O	O
Pelizaeus	NN	O	B-Disease
-	NN	O	I-Disease
Merzbacher	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
PMD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
,	NN	O	O
which	NN	O	O
creates	NN	O	O
a	NN	O	O
second	NN	O	O
AluI	NN	O	O
restriction	NN	O	O
site	NN	O	O
,	NN	O	O
results	NN	O	O
in	NN	O	O
a	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
PLP	NN	O	O
.	NN	O	O

The	NN	O	O
clinical	NN	O	O
picture	NN	O	O
resembles	NN	O	O
somewhat	NN	O	O
that	NN	O	O
of	NN	O	O
X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
spastic	NN	O	I-Disease
paraplegia	NN	O	I-Disease
(	NN	O	O
SPG	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

It	NN	O	O
differs	NN	O	O
from	NN	O	O
this	NN	O	O
and	NN	O	O
both	NN	O	O
the	NN	O	O
classical	NN	O	O
and	NN	O	O
connatal	NN	O	O
forms	NN	O	O
of	NN	O	O
PMD	NN	O	B-Disease
in	NN	O	O
that	NN	O	O
it	NN	O	O
is	NN	O	O
relatively	NN	O	O
mild	NN	O	O
in	NN	O	O
form	NN	O	O
,	NN	O	O
onset	NN	O	O
is	NN	O	O
delayed	NN	O	O
beyond	NN	O	O
age	NN	O	O
2	NN	O	O
years	NN	O	O
,	NN	O	O
nystagmus	NN	O	B-Disease
is	NN	O	O
absent	NN	O	O
,	NN	O	O
tremors	NN	O	B-Disease
are	NN	O	O
prominent	NN	O	O
,	NN	O	O
mental	NN	O	B-Disease
retardation	NN	O	I-Disease
is	NN	O	O
not	NN	O	O
severe	NN	O	O
,	NN	O	O
some	NN	O	O
patients	NN	O	O
show	NN	O	O
dementia	NN	O	B-Disease
or	NN	O	O
personality	NN	O	B-Disease
disorders	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
disease	NN	O	O
is	NN	O	O
progressive	NN	O	O
rather	NN	O	O
than	NN	O	O
static	NN	O	O
in	NN	O	O
some	NN	O	O
,	NN	O	O
and	NN	O	O
several	NN	O	O
females	NN	O	O
show	NN	O	O
signs	NN	O	O
of	NN	O	O
disease	NN	O	O
.	NN	O	O

The	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
in	NN	O	O
exon	NN	O	O
3B	NN	O	O
,	NN	O	O
should	NN	O	O
block	NN	O	O
the	NN	O	O
synthesis	NN	O	O
of	NN	O	O
normal	NN	O	O
PLP	NN	O	O
but	NN	O	O
spare	NN	O	O
DM20	NN	O	O
,	NN	O	O
the	NN	O	O
isoform	NN	O	O
whose	NN	O	O
persistence	NN	O	O
has	NN	O	O
been	NN	O	O
associated	NN	O	O
with	NN	O	O
mild	NN	O	O
forms	NN	O	O
of	NN	O	O
PLP	NN	O	B-Disease
-	NN	O	I-Disease
associated	NN	O	I-Disease
disease	NN	O	I-Disease
in	NN	O	O
both	NN	O	O
humans	NN	O	O
and	NN	O	O
mice	NN	O	O
.	NN	O	O
.	NN	O	O

Common	NN	O	O
BRCA1	NN	O	O
variants	NN	O	O
and	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
general	NN	O	O
population	NN	O	O
.	NN	O	O

Most	NN	O	O
multiple	NN	O	O
case	NN	O	O
families	NN	O	O
of	NN	O	O
young	NN	O	O
onset	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
are	NN	O	O
thought	NN	O	O
to	NN	O	O
be	NN	O	O
due	NN	O	O
to	NN	O	O
highly	NN	O	O
penetrant	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
predisposing	NN	O	O
genes	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
these	NN	O	O
mutations	NN	O	O
are	NN	O	O
uncommon	NN	O	O
in	NN	O	O
the	NN	O	O
population	NN	O	O
and	NN	O	O
they	NN	O	O
probably	NN	O	O
account	NN	O	O
for	NN	O	O
only	NN	O	O
a	NN	O	O
few	NN	O	O
percent	NN	O	O
of	NN	O	O
all	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
incidence	NN	O	O
.	NN	O	O

A	NN	O	O
much	NN	O	O
larger	NN	O	O
fraction	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
might	NN	O	O
,	NN	O	O
in	NN	O	O
principle	NN	O	O
,	NN	O	O
be	NN	O	O
due	NN	O	O
to	NN	O	O
common	NN	O	O
variants	NN	O	O
which	NN	O	O
confer	NN	O	O
more	NN	O	O
modest	NN	O	O
individual	NN	O	O
risks	NN	O	O
.	NN	O	O

There	NN	O	O
are	NN	O	O
several	NN	O	O
common	NN	O	O
polymorphisms	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
which	NN	O	O
generate	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitutions	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
examined	NN	O	O
the	NN	O	O
frequency	NN	O	O
of	NN	O	O
four	NN	O	O
of	NN	O	O
these	NN	O	O
polymorphisms	NN	O	O
Gln356Arg	NN	O	O
,	NN	O	O
Pro871Leu	NN	O	O
,	NN	O	O
Glu1038Gly	NN	O	O
and	NN	O	O
Ser1613Gly	NN	O	O
in	NN	O	O
large	NN	O	O
series	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
and	NN	O	O
matched	NN	O	O
controls	NN	O	O
.	NN	O	O

Due	NN	O	O
to	NN	O	O
strong	NN	O	O
linkage	NN	O	O
disequilibrium	NN	O	O
,	NN	O	O
these	NN	O	O
four	NN	O	O
sites	NN	O	O
generate	NN	O	O
only	NN	O	O
three	NN	O	O
haplotypes	NN	O	O
with	NN	O	O
a	NN	O	O
frequency	NN	O	O
>	NN	O	O
1	NN	O	O
.	NN	O	O

3	NN	O	O
%	NN	O	O
.	NN	O	O

The	NN	O	O
most	NN	O	O
common	NN	O	O
haplotypes	NN	O	O
,	NN	O	O
defined	NN	O	O
by	NN	O	O
the	NN	O	O
alleles	NN	O	O
Gln356Pro871Glu1038Ser1613	NN	O	O
and	NN	O	O
Gln356Leu871Gly1038Gly1613	NN	O	O
,	NN	O	O
have	NN	O	O
frequencies	NN	O	O
of	NN	O	O
0	NN	O	O
.	NN	O	O

57	NN	O	O
and	NN	O	O
0	NN	O	O
.	NN	O	O

32	NN	O	O
respectively	NN	O	O
,	NN	O	O
and	NN	O	O
these	NN	O	O
frequencies	NN	O	O
do	NN	O	O
not	NN	O	O
differ	NN	O	O
significantly	NN	O	O
between	NN	O	O
patient	NN	O	O
and	NN	O	O
control	NN	O	O
groups	NN	O	O
.	NN	O	O

Thus	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
polymorphisms	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
do	NN	O	O
not	NN	O	O
make	NN	O	O
a	NN	O	O
significant	NN	O	O
contribution	NN	O	O
to	NN	O	O
breast	NN	O	B-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
risk	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
our	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
Arg356	NN	O	O
allele	NN	O	O
may	NN	O	O
have	NN	O	O
a	NN	O	O
different	NN	O	O
genotype	NN	O	O
distribution	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
from	NN	O	O
that	NN	O	O
in	NN	O	O
controls	NN	O	O
(	NN	O	O
Arg356	NN	O	O
homozygotes	NN	O	O
are	NN	O	O
more	NN	O	O
frequent	NN	O	O
in	NN	O	O
the	NN	O	O
control	NN	O	O
groups	NN	O	O
,	NN	O	O
P	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
01	NN	O	O
)	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
it	NN	O	O
may	NN	O	O
be	NN	O	O
protective	NN	O	O
against	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

If	NN	O	O
this	NN	O	O
finding	NN	O	O
can	NN	O	O
be	NN	O	O
confirmed	NN	O	O
,	NN	O	O
it	NN	O	O
may	NN	O	O
provide	NN	O	O
an	NN	O	O
insight	NN	O	O
into	NN	O	O
the	NN	O	O
structural	NN	O	O
features	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
protein	NN	O	O
that	NN	O	O
are	NN	O	O
important	NN	O	O
for	NN	O	O
its	NN	O	O
function	NN	O	O
.	NN	O	O

Kniest	NN	O	B-Disease
dysplasia	NN	O	I-Disease
:	NN	O	O
Dr	NN	O	O
.	NN	O	O
W	NN	O	O
.	NN	O	O

Kniest	NN	O	O
,	NN	O	O
his	NN	O	O
patient	NN	O	O
,	NN	O	O
the	NN	O	O
molecular	NN	O	O
defect	NN	O	O
.	NN	O	O

Kniest	NN	O	B-Disease
dysplasia	NN	O	I-Disease
is	NN	O	O
a	NN	O	O
severe	NN	O	O
chondrodysplasia	NN	O	B-Disease
caused	NN	O	O
by	NN	O	O
the	NN	O	O
defective	NN	O	B-Disease
formation	NN	O	I-Disease
of	NN	O	I-Disease
type	NN	O	I-Disease
II	NN	O	I-Disease
collagen	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
about	NN	O	O
Dr	NN	O	O
.	NN	O	O
Kniest	NN	O	O
,	NN	O	O
who	NN	O	O
first	NN	O	O
described	NN	O	O
the	NN	O	O
condition	NN	O	O
in	NN	O	O
1952	NN	O	O
,	NN	O	O
and	NN	O	O
his	NN	O	O
patient	NN	O	O
,	NN	O	O
who	NN	O	O
,	NN	O	O
at	NN	O	O
the	NN	O	O
age	NN	O	O
of	NN	O	O
50	NN	O	O
years	NN	O	O
is	NN	O	O
severely	NN	O	B-Disease
handicapped	NN	O	I-Disease
with	NN	O	O
short	NN	O	B-Disease
stature	NN	O	I-Disease
,	NN	O	O
restricted	NN	O	B-Disease
joint	NN	O	I-Disease
mobility	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
blindness	NN	O	B-Disease
but	NN	O	O
is	NN	O	O
mentally	NN	O	O
alert	NN	O	O
and	NN	O	O
leads	NN	O	O
an	NN	O	O
active	NN	O	O
life	NN	O	O
.	NN	O	O

Molecular	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
DNA	NN	O	O
showed	NN	O	O
a	NN	O	O
single	NN	O	O
base	NN	O	O
(	NN	O	O
G	NN	O	O
)	NN	O	O
deletion	NN	O	O
involving	NN	O	O
the	NN	O	O
GT	NN	O	O
dinucleotide	NN	O	O
at	NN	O	O
the	NN	O	O
start	NN	O	O
of	NN	O	O
intron	NN	O	O
18	NN	O	O
destroying	NN	O	O
a	NN	O	O
splice	NN	O	O
site	NN	O	O
of	NN	O	O
the	NN	O	O
COL2A1	NN	O	O
gene	NN	O	O
.	NN	O	O

This	NN	O	O
is	NN	O	O
in	NN	O	O
accordance	NN	O	O
with	NN	O	O
molecular	NN	O	O
findings	NN	O	O
in	NN	O	O
other	NN	O	O
patients	NN	O	O
with	NN	O	O
Kniest	NN	O	B-Disease
dysplasia	NN	O	I-Disease
and	NN	O	O
confirms	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
original	NN	O	O
patient	NN	O	O
,	NN	O	O
that	NN	O	O
the	NN	O	O
disorder	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
small	NN	O	O
inframe	NN	O	O
deletions	NN	O	O
often	NN	O	O
due	NN	O	O
to	NN	O	O
exon	NN	O	O
skipping	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
COL2A1	NN	O	O
splice	NN	O	O
site	NN	O	O
mutations	NN	O	O
.	NN	O	O
.	NN	O	O

Cloning	NN	O	O
of	NN	O	O
the	NN	O	O
homogentisate	NN	O	O
1	NN	O	O
,	NN	O	O
2	NN	O	O
-	NN	O	O
dioxygenase	NN	O	O
gene	NN	O	O
,	NN	O	O
the	NN	O	O
key	NN	O	O
enzyme	NN	O	O
of	NN	O	O
alkaptonuria	NN	O	B-Disease
in	NN	O	O
mouse	NN	O	O
.	NN	O	O

We	NN	O	O
determined	NN	O	O
48	NN	O	O
amino	NN	O	O
acid	NN	O	O
residues	NN	O	O
from	NN	O	O
five	NN	O	O
peptides	NN	O	O
from	NN	O	O
the	NN	O	O
homogeneous	NN	O	O
monomer	NN	O	O
of	NN	O	O
homogentisate	NN	O	O
1	NN	O	O
,	NN	O	O
2	NN	O	O
-	NN	O	O
dioxygenase	NN	O	O
(	NN	O	O
HGO	NN	O	O
;	NN	O	O
E	NN	O	O
.	NN	O	O
C	NN	O	O
.	NN	O	O
1	NN	O	O
.	NN	O	O
13	NN	O	O
.	NN	O	O
11	NN	O	O
.	NN	O	O
15	NN	O	O
)	NN	O	O
of	NN	O	O
mouse	NN	O	O
liver	NN	O	O
.	NN	O	O

After	NN	O	O
digestion	NN	O	O
with	NN	O	O
trypsin	NN	O	O
,	NN	O	O
peptides	NN	O	O
were	NN	O	O
separated	NN	O	O
by	NN	O	O
reversed	NN	O	O
phase	NN	O	O
chromatography	NN	O	O
and	NN	O	O
amino	NN	O	O
acid	NN	O	O
sequenced	NN	O	O
.	NN	O	O

The	NN	O	O
deduced	NN	O	O
codon	NN	O	O
sequence	NN	O	O
of	NN	O	O
three	NN	O	O
peptides	NN	O	O
was	NN	O	O
used	NN	O	O
to	NN	O	O
derive	NN	O	O
degenerated	NN	O	O
oligomeres	NN	O	O
.	NN	O	O

By	NN	O	O
combining	NN	O	O
these	NN	O	O
oligos	NN	O	O
,	NN	O	O
we	NN	O	O
were	NN	O	O
able	NN	O	O
to	NN	O	O
amplify	NN	O	O
fragments	NN	O	O
from	NN	O	O
100	NN	O	O
to	NN	O	O
300	NN	O	O
bases	NN	O	O
(	NN	O	O
b	NN	O	O
)	NN	O	O
from	NN	O	O
mouse	NN	O	O
liver	NN	O	O
cDNA	NN	O	O
by	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
after	NN	O	O
reverse	NN	O	O
transcription	NN	O	O
(	NN	O	O
RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
)	NN	O	O
.	NN	O	O

A	NN	O	O
fragment	NN	O	O
of	NN	O	O
200	NN	O	O
b	NN	O	O
was	NN	O	O
cloned	NN	O	O
and	NN	O	O
used	NN	O	O
as	NN	O	O
a	NN	O	O
probe	NN	O	O
to	NN	O	O
screen	NN	O	O
a	NN	O	O
mouse	NN	O	O
liver	NN	O	O
cDNA	NN	O	O
library	NN	O	O
.	NN	O	O

One	NN	O	O
clone	NN	O	O
from	NN	O	O
this	NN	O	O
library	NN	O	O
contained	NN	O	O
the	NN	O	O
complete	NN	O	O
cDNA	NN	O	O
-	NN	O	O
insert	NN	O	O
for	NN	O	O
HGO	NN	O	O
as	NN	O	O
determined	NN	O	O
by	NN	O	O
sequencing	NN	O	O
.	NN	O	O

The	NN	O	O
cDNA	NN	O	O
encodes	NN	O	O
for	NN	O	O
a	NN	O	O
protein	NN	O	O
of	NN	O	O
50	NN	O	O
kDa	NN	O	O
,	NN	O	O
as	NN	O	O
predicted	NN	O	O
.	NN	O	O

The	NN	O	O
cDNA	NN	O	O
of	NN	O	O
mouse	NN	O	O
HGO	NN	O	O
has	NN	O	O
an	NN	O	O
overall	NN	O	O
identity	NN	O	O
of	NN	O	O
41	NN	O	O
%	NN	O	O
to	NN	O	O
the	NN	O	O
corresponding	NN	O	O
gene	NN	O	O
hmgA	NN	O	O
from	NN	O	O
Aspergillus	NN	O	O
.	NN	O	O

Sequence	NN	O	O
similarities	NN	O	O
to	NN	O	O
human	NN	O	O
expressed	NN	O	O
sequence	NN	O	O
tags	NN	O	O
(	NN	O	O
EST	NN	O	O
)	NN	O	O
clones	NN	O	O
ranged	NN	O	O
from	NN	O	O
70	NN	O	O
%	NN	O	O
to	NN	O	O
20	NN	O	O
%	NN	O	O
.	NN	O	O

The	NN	O	O
positions	NN	O	O
of	NN	O	O
122	NN	O	O
conserved	NN	O	O
amino	NN	O	O
acids	NN	O	O
could	NN	O	O
be	NN	O	O
determined	NN	O	O
by	NN	O	O
multiple	NN	O	O
sequence	NN	O	O
alignment	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
one	NN	O	O
first	NN	O	O
intron	NN	O	O
of	NN	O	O
928	NN	O	O
b	NN	O	O
in	NN	O	O
the	NN	O	O
mouse	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
gene	NN	O	O
for	NN	O	O
HGO	NN	O	O
seems	NN	O	O
to	NN	O	O
be	NN	O	O
expressed	NN	O	O
in	NN	O	O
various	NN	O	O
tissues	NN	O	O
,	NN	O	O
as	NN	O	O
shown	NN	O	O
by	NN	O	O
RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
on	NN	O	O
different	NN	O	O
cDNAs	NN	O	O
.	NN	O	O

FISH	NN	O	O
experiments	NN	O	O
with	NN	O	O
the	NN	O	O
whole	NN	O	O
murine	NN	O	O
cDNA	NN	O	O
as	NN	O	O
probe	NN	O	O
clearly	NN	O	O
revealed	NN	O	O
signals	NN	O	O
at	NN	O	O
the	NN	O	O
human	NN	O	O
chromosomal	NN	O	O
band	NN	O	O
3q13	NN	O	O
.	NN	O	O

3	NN	O	O
-	NN	O	O
q21	NN	O	O
.	NN	O	O

This	NN	O	O
corresponds	NN	O	O
well	NN	O	O
to	NN	O	O
the	NN	O	O
previous	NN	O	O
assignment	NN	O	O
of	NN	O	O
the	NN	O	O
locus	NN	O	O
for	NN	O	O
the	NN	O	O
human	NN	O	O
alkaptonuria	NN	O	B-Disease
gene	NN	O	O
(	NN	O	O
AKU	NN	O	O
)	NN	O	O
to	NN	O	O
the	NN	O	O
same	NN	O	O
chromosomal	NN	O	O
region	NN	O	O
by	NN	O	O
multipoint	NN	O	O
linkage	NN	O	O
analysis	NN	O	O
.	NN	O	O

We	NN	O	O
therefore	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
HGO	NN	O	O
cDNA	NN	O	O
encodes	NN	O	O
the	NN	O	O
gene	NN	O	O
responsible	NN	O	O
for	NN	O	O
alkaptonuria	NN	O	B-Disease
.	NN	O	O

PTEN	NN	O	O
,	NN	O	O
a	NN	O	O
putative	NN	O	O
protein	NN	O	O
tyrosine	NN	O	O
phosphatase	NN	O	O
gene	NN	O	O
mutated	NN	O	O
in	NN	O	O
human	NN	O	O
brain	NN	O	B-Disease
,	NN	O	I-Disease
breast	NN	O	I-Disease
,	NN	O	I-Disease
and	NN	O	I-Disease
prostate	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Mapping	NN	O	O
of	NN	O	O
homozygous	NN	O	O
deletions	NN	O	O
on	NN	O	O
human	NN	O	O
chromosome	NN	O	O
10q23	NN	O	O
has	NN	O	O
led	NN	O	O
to	NN	O	O
the	NN	O	O
isolation	NN	O	O
of	NN	O	O
a	NN	O	O
candidate	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
,	NN	O	O
PTEN	NN	O	O
,	NN	O	O
that	NN	O	O
appears	NN	O	O
to	NN	O	O
be	NN	O	O
mutated	NN	O	O
at	NN	O	O
considerable	NN	O	O
frequency	NN	O	O
in	NN	O	O
human	NN	O	O
cancers	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
preliminary	NN	O	O
screens	NN	O	O
,	NN	O	O
mutations	NN	O	O
of	NN	O	O
PTEN	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
31	NN	O	O
%	NN	O	O
(	NN	O	O
13	NN	O	O
/	NN	O	O
42	NN	O	O
)	NN	O	O
of	NN	O	O
glioblastoma	NN	O	B-Disease
cell	NN	O	O
lines	NN	O	O
and	NN	O	O
xenografts	NN	O	O
,	NN	O	O
100	NN	O	O
%	NN	O	O
(	NN	O	O
4	NN	O	O
/	NN	O	O
4	NN	O	O
)	NN	O	O
of	NN	O	O
prostate	NN	O	B-Disease
cancer	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
,	NN	O	O
6	NN	O	O
%	NN	O	O
(	NN	O	O
4	NN	O	O
/	NN	O	O
65	NN	O	O
)	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
and	NN	O	O
xenografts	NN	O	O
,	NN	O	O
and	NN	O	O
17	NN	O	O
%	NN	O	O
(	NN	O	O
3	NN	O	O
/	NN	O	O
18	NN	O	O
)	NN	O	O
of	NN	O	O
primary	NN	O	B-Disease
glioblastomas	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
predicted	NN	O	O
PTEN	NN	O	O
product	NN	O	O
has	NN	O	O
a	NN	O	O
protein	NN	O	O
tyrosine	NN	O	O
phosphatase	NN	O	O
domain	NN	O	O
and	NN	O	O
extensive	NN	O	O
homology	NN	O	O
to	NN	O	O
tensin	NN	O	O
,	NN	O	O
a	NN	O	O
protein	NN	O	O
that	NN	O	O
interacts	NN	O	O
with	NN	O	O
actin	NN	O	O
filaments	NN	O	O
at	NN	O	O
focal	NN	O	O
adhesions	NN	O	O
.	NN	O	O

These	NN	O	O
homologies	NN	O	O
suggest	NN	O	O
that	NN	O	O
PTEN	NN	O	O
may	NN	O	O
suppress	NN	O	O
tumor	NN	O	B-Disease
cell	NN	O	O
growth	NN	O	O
by	NN	O	O
antagonizing	NN	O	O
protein	NN	O	O
tyrosine	NN	O	O
kinases	NN	O	O
and	NN	O	O
may	NN	O	O
regulate	NN	O	O
tumor	NN	O	B-Disease
cell	NN	O	O
invasion	NN	O	O
and	NN	O	O
metastasis	NN	O	B-Disease
through	NN	O	O
interactions	NN	O	O
at	NN	O	O
focal	NN	O	O
adhesions	NN	O	O
.	NN	O	O
.	NN	O	O

Heterogeneity	NN	O	O
in	NN	O	O
Schwartz	NN	O	B-Disease
-	NN	O	I-Disease
Jampel	NN	O	I-Disease
chondrodystrophic	NN	O	I-Disease
myotonia	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
Schwartz	NN	O	B-Disease
-	NN	O	I-Disease
Jampel	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
SJS	NN	O	B-Disease
;	NN	O	O
chondrodystrophic	NN	O	B-Disease
myotonia	NN	O	I-Disease
;	NN	O	O
McK	NN	O	O
255	NN	O	O
,	NN	O	O
800	NN	O	O
)	NN	O	O
is	NN	O	O
a	NN	O	O
recessively	NN	O	B-Disease
inherited	NN	O	I-Disease
condition	NN	O	I-Disease
defined	NN	O	O
by	NN	O	O
myotonia	NN	O	B-Disease
,	NN	O	O
short	NN	O	B-Disease
stature	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
.	NN	O	O

Genetic	NN	O	O
linkage	NN	O	O
between	NN	O	O
SJS	NN	O	B-Disease
and	NN	O	O
chromosomal	NN	O	O
region	NN	O	O
1q36	NN	O	O
-	NN	O	O
34	NN	O	O
has	NN	O	O
been	NN	O	O
observed	NN	O	O
in	NN	O	O
several	NN	O	O
families	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
gene	NN	O	O
has	NN	O	O
not	NN	O	O
yet	NN	O	O
been	NN	O	O
identified	NN	O	O
.	NN	O	O

We	NN	O	O
studied	NN	O	O
the	NN	O	O
clinical	NN	O	O
and	NN	O	O
radiological	NN	O	O
features	NN	O	O
in	NN	O	O
81	NN	O	O
patients	NN	O	O
from	NN	O	O
the	NN	O	O
literature	NN	O	O
and	NN	O	O
5	NN	O	O
own	NN	O	O
patients	NN	O	O
trying	NN	O	O
to	NN	O	O
identify	NN	O	O
distinct	NN	O	O
subgroups	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
we	NN	O	O
tested	NN	O	O
genetic	NN	O	O
linkage	NN	O	O
to	NN	O	O
the	NN	O	O
SJS	NN	O	B-Disease
locus	NN	O	O
on	NN	O	O
chromosome	NN	O	O
1	NN	O	O
in	NN	O	O
one	NN	O	O
family	NN	O	O
with	NN	O	O
two	NN	O	O
affected	NN	O	O
sibs	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
that	NN	O	O
a	NN	O	O
group	NN	O	O
of	NN	O	O
patients	NN	O	O
have	NN	O	O
mild	NN	O	O
skeletal	NN	O	O
changes	NN	O	O
which	NN	O	O
may	NN	O	O
be	NN	O	O
secondary	NN	O	O
consequences	NN	O	O
of	NN	O	O
myotonia	NN	O	B-Disease
,	NN	O	O
while	NN	O	O
another	NN	O	O
group	NN	O	O
of	NN	O	O
patients	NN	O	O
appear	NN	O	O
to	NN	O	O
have	NN	O	O
primary	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
with	NN	O	O
myotonia	NN	O	B-Disease
.	NN	O	O

Within	NN	O	O
this	NN	O	O
latter	NN	O	O
group	NN	O	O
,	NN	O	O
there	NN	O	O
are	NN	O	O
differences	NN	O	O
in	NN	O	O
age	NN	O	O
of	NN	O	O
manifestation	NN	O	O
,	NN	O	O
clinical	NN	O	O
course	NN	O	O
and	NN	O	O
pattern	NN	O	O
of	NN	O	O
bone	NN	O	O
changes	NN	O	O
.	NN	O	O

We	NN	O	O
tentatively	NN	O	O
isolate	NN	O	O
three	NN	O	O
different	NN	O	O
types	NN	O	O
of	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
1A	NN	O	I-Disease
,	NN	O	O
usually	NN	O	O
recognized	NN	O	O
in	NN	O	O
childhood	NN	O	O
,	NN	O	O
with	NN	O	O
moderate	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
,	NN	O	O
corresponding	NN	O	O
to	NN	O	O
the	NN	O	O
original	NN	O	O
descriptions	NN	O	O
of	NN	O	O
Schwartz	NN	O	O
,	NN	O	O
Jampel	NN	O	O
and	NN	O	O
Aberfeld	NN	O	O
;	NN	O	O
type	NN	O	O
1B	NN	O	O
,	NN	O	O
similar	NN	O	O
to	NN	O	O
type	NN	O	O
1A	NN	O	O
but	NN	O	O
recognizable	NN	O	O
at	NN	O	O
birth	NN	O	O
,	NN	O	O
with	NN	O	O
more	NN	O	O
pronounced	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
resembling	NN	O	O
Kniest	NN	O	B-Disease
dysplasia	NN	O	I-Disease
;	NN	O	O
and	NN	O	O
type	NN	O	O
2	NN	O	O
,	NN	O	O
manifest	NN	O	O
at	NN	O	O
birth	NN	O	O
,	NN	O	O
with	NN	O	O
increased	NN	O	O
mortality	NN	O	O
and	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
resembling	NN	O	O
Pyle	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

Genetic	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
family	NN	O	O
with	NN	O	O
two	NN	O	O
sibs	NN	O	O
affected	NN	O	O
by	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
showed	NN	O	O
evidence	NN	O	O
against	NN	O	O
linkage	NN	O	O
to	NN	O	O
chromosome	NN	O	O
1p36	NN	O	O
-	NN	O	O
34	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
SJS	NN	O	B-Disease
is	NN	O	O
clinically	NN	O	O
and	NN	O	O
radiologically	NN	O	O
heterogeneous	NN	O	O
.	NN	O	O

The	NN	O	O
causes	NN	O	O
of	NN	O	O
heterogeneity	NN	O	O
are	NN	O	O
not	NN	O	O
known	NN	O	O
yet	NN	O	O
but	NN	O	O
are	NN	O	O
likely	NN	O	O
to	NN	O	O
include	NN	O	O
both	NN	O	O
different	NN	O	O
mutations	NN	O	O
at	NN	O	O
the	NN	O	O
SJS	NN	O	B-Disease
locus	NN	O	O
on	NN	O	O
chromosome	NN	O	O
1	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
second	NN	O	O
SJS	NN	O	B-Disease
locus	NN	O	O
.	NN	O	O

A	NN	O	O
tentative	NN	O	O
clinico	NN	O	O
-	NN	O	O
radiological	NN	O	O
classification	NN	O	O
can	NN	O	O
be	NN	O	O
useful	NN	O	O
for	NN	O	O
the	NN	O	O
characterization	NN	O	O
of	NN	O	O
patients	NN	O	O
and	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
clinical	NN	O	O
overview	NN	O	O
of	NN	O	O
WT1	NN	O	O
gene	NN	O	O
mutations	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
WT1	NN	O	O
gene	NN	O	O
were	NN	O	O
anticipated	NN	O	O
to	NN	O	O
explain	NN	O	O
the	NN	O	O
genetic	NN	O	O
basis	NN	O	O
of	NN	O	O
the	NN	O	O
childhood	NN	O	O
kidney	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
Wilms	NN	O	B-Disease
tumour	NN	O	I-Disease
(	NN	O	O
WT	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Six	NN	O	O
years	NN	O	O
on	NN	O	O
,	NN	O	O
we	NN	O	O
review	NN	O	O
100	NN	O	O
reports	NN	O	O
of	NN	O	O
intragenic	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
and	NN	O	O
examine	NN	O	O
the	NN	O	O
accompanying	NN	O	O
clinical	NN	O	O
phenotypes	NN	O	O
.	NN	O	O

While	NN	O	O
only	NN	O	O
5	NN	O	O
%	NN	O	O
of	NN	O	O
sporadic	NN	O	B-Disease
Wilms	NN	O	I-Disease
tumours	NN	O	I-Disease
have	NN	O	O
intragenic	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
,	NN	O	O
>	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
the	NN	O	O
Denys	NN	O	B-Disease
-	NN	O	I-Disease
Drash	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
renal	NN	O	B-Disease
nephropathy	NN	O	I-Disease
,	NN	O	O
gonadal	NN	O	B-Disease
anomaly	NN	O	I-Disease
,	NN	O	O
predisposition	NN	O	B-Disease
to	NN	O	I-Disease
WT	NN	O	I-Disease
)	NN	O	O
carry	NN	O	O
constitutional	NN	O	O
intragenic	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
.	NN	O	O

WT1	NN	O	O
mutations	NN	O	O
have	NN	O	O
also	NN	O	O
been	NN	O	O
reported	NN	O	O
in	NN	O	O
juvenile	NN	O	B-Disease
granulosa	NN	O	I-Disease
cell	NN	O	I-Disease
tumour	NN	O	I-Disease
,	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
asbestos	NN	O	I-Disease
related	NN	O	I-Disease
mesothelioma	NN	O	I-Disease
,	NN	O	O
desmoplastic	NN	O	B-Disease
small	NN	O	I-Disease
round	NN	O	I-Disease
cell	NN	O	I-Disease
tumour	NN	O	I-Disease
and	NN	O	O
,	NN	O	O
most	NN	O	O
recently	NN	O	O
,	NN	O	O
acute	NN	O	B-Disease
myeloid	NN	O	I-Disease
leukemia	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

A	NN	O	O
mutation	NN	O	O
in	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
myotonia	NN	O	I-Disease
congenita	NN	O	I-Disease
affects	NN	O	O
pore	NN	O	O
properties	NN	O	O
of	NN	O	O
the	NN	O	O
muscle	NN	O	O
chloride	NN	O	O
channel	NN	O	O
.	NN	O	O

Autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
myotonia	NN	O	I-Disease
congenita	NN	O	I-Disease
is	NN	O	O
an	NN	O	O
inherited	NN	O	B-Disease
disorder	NN	O	I-Disease
of	NN	O	I-Disease
skeletal	NN	O	I-Disease
muscle	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
a	NN	O	O
voltage	NN	O	O
-	NN	O	O
gated	NN	O	O
Cl	NN	O	O
-	NN	O	O
channel	NN	O	O
gene	NN	O	O
(	NN	O	O
CLCN1	NN	O	O
,	NN	O	O
7q35	NN	O	O
)	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
we	NN	O	O
report	NN	O	O
that	NN	O	O
a	NN	O	O
mutation	NN	O	O
predicting	NN	O	O
the	NN	O	O
substitution	NN	O	O
of	NN	O	O
Gly	NN	O	O
230	NN	O	O
by	NN	O	O
glutamic	NN	O	O
acid	NN	O	O
(	NN	O	O
G230E	NN	O	O
)	NN	O	O
between	NN	O	O
segments	NN	O	O
D3	NN	O	O
and	NN	O	O
D4	NN	O	O
dramatically	NN	O	O
alters	NN	O	O
the	NN	O	O
pore	NN	O	O
properties	NN	O	O
of	NN	O	O
a	NN	O	O
recombinant	NN	O	O
human	NN	O	O
muscle	NN	O	O
Cl	NN	O	O
-	NN	O	O
channel	NN	O	O
(	NN	O	O
hCIC	NN	O	O
-	NN	O	O
1	NN	O	O
)	NN	O	O
expressed	NN	O	O
in	NN	O	O
a	NN	O	O
mammalian	NN	O	O
cell	NN	O	O
line	NN	O	O
(	NN	O	O
tsA201	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
G230E	NN	O	O
mutation	NN	O	O
causes	NN	O	O
substantial	NN	O	O
changes	NN	O	O
in	NN	O	O
anion	NN	O	O
and	NN	O	O
cation	NN	O	O
selectivity	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
a	NN	O	O
fundamental	NN	O	O
change	NN	O	O
in	NN	O	O
rectification	NN	O	O
of	NN	O	O
the	NN	O	O
current	NN	O	O
-	NN	O	O
voltage	NN	O	O
relationship	NN	O	O
.	NN	O	O

Whereas	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
channels	NN	O	O
are	NN	O	O
characterized	NN	O	O
by	NN	O	O
pronounced	NN	O	O
inward	NN	O	O
rectification	NN	O	O
and	NN	O	O
a	NN	O	O
Cl	NN	O	O
>	NN	O	O
thiocyanate	NN	O	O
>	NN	O	O
Br	NN	O	O
>	NN	O	O
NO	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
>	NN	O	O
I	NN	O	O
>	NN	O	O
CH	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
SO	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
selectivity	NN	O	O
,	NN	O	O
G230E	NN	O	O
exhibits	NN	O	O
outward	NN	O	O
rectification	NN	O	O
at	NN	O	O
positive	NN	O	O
potentials	NN	O	O
and	NN	O	O
a	NN	O	O
thiocyanate	NN	O	O
>	NN	O	O
NO	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
>	NN	O	O
I	NN	O	O
>	NN	O	O
Br	NN	O	O
>	NN	O	O
Cl	NN	O	O
>	NN	O	O
CH	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
SO	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
selectivity	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
the	NN	O	O
cation	NN	O	O
-	NN	O	O
to	NN	O	O
-	NN	O	O
anion	NN	O	O
permeability	NN	O	O
ratio	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
is	NN	O	O
much	NN	O	O
greater	NN	O	O
than	NN	O	O
that	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
channel	NN	O	O
.	NN	O	O

Voltage	NN	O	O
-	NN	O	O
dependent	NN	O	O
blocks	NN	O	O
by	NN	O	O
intracellular	NN	O	O
and	NN	O	O
extracellular	NN	O	O
iodide	NN	O	O
help	NN	O	O
to	NN	O	O
distinguish	NN	O	O
two	NN	O	O
distinct	NN	O	O
ion	NN	O	O
binding	NN	O	O
sites	NN	O	O
within	NN	O	O
the	NN	O	O
hClC	NN	O	O
-	NN	O	O
1	NN	O	O
conduction	NN	O	O
pathway	NN	O	O
.	NN	O	O

Both	NN	O	O
binding	NN	O	O
sites	NN	O	O
are	NN	O	O
preserved	NN	O	O
in	NN	O	O
the	NN	O	O
mutant	NN	O	O
but	NN	O	O
have	NN	O	O
decreased	NN	O	O
affinities	NN	O	O
for	NN	O	O
iodide	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
suggest	NN	O	O
that	NN	O	O
Gly	NN	O	O
230	NN	O	O
is	NN	O	O
critical	NN	O	O
for	NN	O	O
normal	NN	O	O
ion	NN	O	O
conductance	NN	O	O
in	NN	O	O
hClC	NN	O	O
-	NN	O	O
1	NN	O	O
and	NN	O	O
that	NN	O	O
this	NN	O	O
residue	NN	O	O
resides	NN	O	O
within	NN	O	O
the	NN	O	O
channel	NN	O	O
pore	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
incidence	NN	O	O
of	NN	O	O
PAX6	NN	O	O
mutation	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
simple	NN	O	O
aniridia	NN	O	B-Disease
:	NN	O	O
an	NN	O	O
evaluation	NN	O	O
of	NN	O	O
mutation	NN	O	O
detection	NN	O	O
in	NN	O	O
12	NN	O	O
cases	NN	O	O
.	NN	O	O

Twelve	NN	O	O
aniridia	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
five	NN	O	O
with	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
and	NN	O	O
seven	NN	O	O
presumed	NN	O	O
to	NN	O	O
be	NN	O	O
sporadic	NN	O	O
,	NN	O	O
were	NN	O	O
exhaustively	NN	O	O
screened	NN	O	O
in	NN	O	O
order	NN	O	O
to	NN	O	O
test	NN	O	O
what	NN	O	O
proportion	NN	O	O
of	NN	O	O
people	NN	O	O
with	NN	O	O
aniridia	NN	O	B-Disease
,	NN	O	O
uncomplicated	NN	O	O
by	NN	O	O
associated	NN	O	O
anomalies	NN	O	O
,	NN	O	O
carry	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
PAX6	NN	O	O
gene	NN	O	O
.	NN	O	O

Mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
cases	NN	O	O
.	NN	O	O

Three	NN	O	O
mutation	NN	O	O
detection	NN	O	O
techniques	NN	O	O
were	NN	O	O
used	NN	O	O
to	NN	O	O
determine	NN	O	O
if	NN	O	O
one	NN	O	O
method	NN	O	O
was	NN	O	O
superior	NN	O	O
for	NN	O	O
this	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
protein	NN	O	O
truncation	NN	O	O
test	NN	O	O
(	NN	O	O
PTT	NN	O	O
)	NN	O	O
was	NN	O	O
used	NN	O	O
on	NN	O	O
RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
products	NN	O	O
,	NN	O	O
SSCP	NN	O	O
on	NN	O	O
genomic	NN	O	O
PCR	NN	O	O
amplifications	NN	O	O
,	NN	O	O
and	NN	O	O
chemical	NN	O	O
cleavage	NN	O	O
of	NN	O	O
mismatch	NN	O	O
on	NN	O	O
both	NN	O	O
RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
and	NN	O	O
genomic	NN	O	O
amplifications	NN	O	O
.	NN	O	O

For	NN	O	O
RT	NN	O	O
-	NN	O	O
PCR	NN	O	O
products	NN	O	O
,	NN	O	O
only	NN	O	O
the	NN	O	O
translated	NN	O	O
portion	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
was	NN	O	O
screened	NN	O	O
.	NN	O	O

On	NN	O	O
genomic	NN	O	O
products	NN	O	O
exons	NN	O	O
1	NN	O	O
to	NN	O	O
13	NN	O	O
(	NN	O	O
including	NN	O	O
740	NN	O	O
bp	NN	O	O
of	NN	O	O
the	NN	O	O
3	NN	O	O
untranslated	NN	O	O
sequence	NN	O	O
and	NN	O	O
all	NN	O	O
intron	NN	O	O
/	NN	O	O
exon	NN	O	O
boundaries	NN	O	O
)	NN	O	O
were	NN	O	O
screened	NN	O	O
,	NN	O	O
as	NN	O	O
was	NN	O	O
a	NN	O	O
neuroretina	NN	O	O
specific	NN	O	O
enhancer	NN	O	O
in	NN	O	O
intron	NN	O	O
4	NN	O	O
.	NN	O	O

Ten	NN	O	O
of	NN	O	O
the	NN	O	O
possible	NN	O	O
12	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
five	NN	O	O
familial	NN	O	O
cases	NN	O	O
and	NN	O	O
five	NN	O	O
of	NN	O	O
the	NN	O	O
sporadic	NN	O	O
patients	NN	O	O
were	NN	O	O
found	NN	O	O
,	NN	O	O
all	NN	O	O
of	NN	O	O
which	NN	O	O
conformed	NN	O	O
to	NN	O	O
a	NN	O	O
functional	NN	O	O
outcome	NN	O	O
of	NN	O	O
haploinsufficiency	NN	O	O
.	NN	O	O

Five	NN	O	O
were	NN	O	O
splice	NN	O	O
site	NN	O	O
mutations	NN	O	O
(	NN	O	O
one	NN	O	O
in	NN	O	O
the	NN	O	O
donor	NN	O	O
site	NN	O	O
of	NN	O	O
intron	NN	O	O
4	NN	O	O
,	NN	O	O
two	NN	O	O
in	NN	O	O
the	NN	O	O
donor	NN	O	O
site	NN	O	O
of	NN	O	O
intron	NN	O	O
6	NN	O	O
,	NN	O	O
one	NN	O	O
in	NN	O	O
each	NN	O	O
of	NN	O	O
the	NN	O	O
acceptor	NN	O	O
sites	NN	O	O
of	NN	O	O
introns	NN	O	O
8	NN	O	O
and	NN	O	O
9	NN	O	O
)	NN	O	O
and	NN	O	O
five	NN	O	O
were	NN	O	O
nonsense	NN	O	O
mutations	NN	O	O
in	NN	O	O
exons	NN	O	O
8	NN	O	O
,	NN	O	O
9	NN	O	O
,	NN	O	O
10	NN	O	O
,	NN	O	O
11	NN	O	O
,	NN	O	O
and	NN	O	O
12	NN	O	O
.	NN	O	O

SSCP	NN	O	O
analysis	NN	O	O
of	NN	O	O
individually	NN	O	O
amplified	NN	O	O
exons	NN	O	O
,	NN	O	O
with	NN	O	O
which	NN	O	O
nine	NN	O	O
of	NN	O	O
the	NN	O	O
10	NN	O	O
mutations	NN	O	O
were	NN	O	O
seen	NN	O	O
,	NN	O	O
was	NN	O	O
the	NN	O	O
most	NN	O	O
useful	NN	O	O
detection	NN	O	O
method	NN	O	O
for	NN	O	O
PAX6	NN	O	O
.	NN	O	O
.	NN	O	O

Insulin	NN	O	O
gene	NN	O	O
region	NN	O	O
contributes	NN	O	O
to	NN	O	O
genetic	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
,	NN	O	O
but	NN	O	O
may	NN	O	O
not	NN	O	O
to	NN	O	O
low	NN	O	O
incidence	NN	O	O
of	NN	O	O
,	NN	O	O
insulin	NN	O	B-Disease
-	NN	O	I-Disease
dependent	NN	O	I-Disease
diabetes	NN	O	I-Disease
mellitus	NN	O	I-Disease
in	NN	O	O
Japanese	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
Caucasian	NN	O	O
population	NN	O	O
,	NN	O	O
it	NN	O	O
has	NN	O	O
been	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
the	NN	O	O
insulin	NN	O	O
gene	NN	O	O
(	NN	O	O
INS	NN	O	O
)	NN	O	O
region	NN	O	O
contains	NN	O	O
the	NN	O	O
insulin	NN	O	B-Disease
-	NN	O	I-Disease
dependent	NN	O	I-Disease
diabetes	NN	O	I-Disease
mellitus	NN	O	I-Disease
locus	NN	O	O
(	NN	O	O
IDDM2	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
Japanese	NN	O	O
population	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
there	NN	O	O
has	NN	O	O
been	NN	O	O
no	NN	O	O
report	NN	O	O
demonstrating	NN	O	O
the	NN	O	O
contribution	NN	O	O
of	NN	O	O
IDDM2	NN	O	O
to	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
IDDM	NN	O	B-Disease
.	NN	O	O

We	NN	O	O
conducted	NN	O	O
an	NN	O	O
association	NN	O	O
study	NN	O	O
of	NN	O	O
IDDM	NN	O	B-Disease
in	NN	O	O
a	NN	O	O
large	NN	O	O
number	NN	O	O
of	NN	O	O
Japanese	NN	O	O
subjects	NN	O	O
with	NN	O	O
multiple	NN	O	O
polymorphisms	NN	O	O
in	NN	O	O
INS	NN	O	O
region	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
a	NN	O	O
significant	NN	O	O
association	NN	O	O
of	NN	O	O
the	NN	O	O
INS	NN	O	O
region	NN	O	O
with	NN	O	O
IDDM	NN	O	B-Disease
.	NN	O	O

Alleles	NN	O	O
positively	NN	O	O
associated	NN	O	O
with	NN	O	O
IDDM	NN	O	B-Disease
in	NN	O	O
INS	NN	O	O
region	NN	O	O
were	NN	O	O
the	NN	O	O
same	NN	O	O
as	NN	O	O
those	NN	O	O
positively	NN	O	O
-	NN	O	O
associated	NN	O	O
with	NN	O	O
IDDM	NN	O	B-Disease
in	NN	O	O
Caucasian	NN	O	O
population	NN	O	O
,	NN	O	O
although	NN	O	O
positively	NN	O	O
-	NN	O	O
associated	NN	O	O
alleles	NN	O	O
are	NN	O	O
very	NN	O	O
common	NN	O	O
(	NN	O	O
allele	NN	O	O
frequencies	NN	O	O
>	NN	O	O
0	NN	O	O
.	NN	O	O
9	NN	O	O
)	NN	O	O
in	NN	O	O
the	NN	O	O
Japanese	NN	O	O
general	NN	O	O
population	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
IDDM2	NN	O	O
is	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
genetic	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
IDDM	NN	O	B-Disease
in	NN	O	O
Japanese	NN	O	O
.	NN	O	O

The	NN	O	O
high	NN	O	O
frequencies	NN	O	O
of	NN	O	O
disease	NN	O	O
-	NN	O	O
associated	NN	O	O
alleles	NN	O	O
in	NN	O	O
the	NN	O	O
general	NN	O	O
population	NN	O	O
suggest	NN	O	O
that	NN	O	O
IDDM2	NN	O	O
locus	NN	O	O
is	NN	O	O
not	NN	O	O
responsible	NN	O	O
for	NN	O	O
the	NN	O	O
low	NN	O	O
incidence	NN	O	O
of	NN	O	O
IDDM	NN	O	B-Disease
in	NN	O	O
Japanese	NN	O	O
.	NN	O	O

The	NN	O	O
human	NN	O	O
complement	NN	O	O
C9	NN	O	O
gene	NN	O	O
:	NN	O	O
identification	NN	O	O
of	NN	O	O
two	NN	O	O
mutations	NN	O	O
causing	NN	O	O
deficiency	NN	O	O
and	NN	O	O
revision	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
structure	NN	O	O
.	NN	O	O

The	NN	O	O
ninth	NN	O	O
component	NN	O	O
of	NN	O	O
human	NN	O	O
complement	NN	O	O
(	NN	O	O
C9	NN	O	O
)	NN	O	O
is	NN	O	O
the	NN	O	O
last	NN	O	O
of	NN	O	O
the	NN	O	O
terminal	NN	O	O
complement	NN	O	O
components	NN	O	O
creating	NN	O	O
the	NN	O	O
membrane	NN	O	O
attack	NN	O	O
complex	NN	O	O
.	NN	O	O

C9	NN	O	O
is	NN	O	O
a	NN	O	O
single	NN	O	O
-	NN	O	O
chain	NN	O	O
serum	NN	O	O
protein	NN	O	O
that	NN	O	O
is	NN	O	O
encoded	NN	O	O
by	NN	O	O
a	NN	O	O
gene	NN	O	O
located	NN	O	O
on	NN	O	O
chromosome	NN	O	O
5p	NN	O	O
.	NN	O	O

Deficiency	NN	O	B-Disease
of	NN	O	I-Disease
terminal	NN	O	I-Disease
complement	NN	O	I-Disease
components	NN	O	I-Disease
is	NN	O	O
generally	NN	O	O
associated	NN	O	O
with	NN	O	O
recurrent	NN	O	O
neisseria	NN	O	B-Disease
infections	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
studied	NN	O	O
a	NN	O	O
previously	NN	O	O
described	NN	O	O
Swiss	NN	O	O
family	NN	O	O
with	NN	O	O
inherited	NN	O	B-Disease
C9	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
identify	NN	O	O
the	NN	O	O
genetic	NN	O	O
basis	NN	O	O
of	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
developed	NN	O	O
an	NN	O	O
approach	NN	O	O
using	NN	O	O
exon	NN	O	O
-	NN	O	O
specific	NN	O	O
PCR	NN	O	O
and	NN	O	O
direct	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
.	NN	O	O

As	NN	O	O
a	NN	O	O
cause	NN	O	O
of	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
found	NN	O	O
two	NN	O	O
different	NN	O	O
point	NN	O	O
mutations	NN	O	O
,	NN	O	O
both	NN	O	O
generating	NN	O	O
TGA	NN	O	O
stop	NN	O	O
codons	NN	O	O
in	NN	O	O
the	NN	O	O
coding	NN	O	O
sequence	NN	O	O
.	NN	O	O

One	NN	O	O
mutation	NN	O	O
,	NN	O	O
a	NN	O	O
C	NN	O	O
to	NN	O	O
A	NN	O	O
exchange	NN	O	O
,	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
exon	NN	O	O
2	NN	O	O
at	NN	O	O
cDNA	NN	O	O
position	NN	O	O
166	NN	O	O
,	NN	O	O
the	NN	O	O
other	NN	O	O
,	NN	O	O
a	NN	O	O
C	NN	O	O
to	NN	O	O
T	NN	O	O
exchange	NN	O	O
,	NN	O	O
was	NN	O	O
located	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
(	NN	O	O
cDNA	NN	O	O
position	NN	O	O
464	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
family	NN	O	O
studies	NN	O	O
of	NN	O	O
three	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relatives	NN	O	O
with	NN	O	O
heterozygous	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
the	NN	O	O
two	NN	O	O
mutations	NN	O	O
are	NN	O	O
segregating	NN	O	O
independently	NN	O	O
.	NN	O	O

Therefore	NN	O	O
,	NN	O	O
these	NN	O	O
mutations	NN	O	O
are	NN	O	O
sufficient	NN	O	O
to	NN	O	O
explain	NN	O	O
the	NN	O	O
complete	NN	O	O
deficiency	NN	O	O
of	NN	O	O
both	NN	O	O
the	NN	O	O
probands	NN	O	O
studied	NN	O	O
.	NN	O	O

DNA	NN	O	O
sequencing	NN	O	O
of	NN	O	O
the	NN	O	O
exon	NN	O	O
-	NN	O	O
intron	NN	O	O
junctions	NN	O	O
revealed	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
revisions	NN	O	O
regarding	NN	O	O
the	NN	O	O
boundaries	NN	O	O
between	NN	O	O
exons	NN	O	O
4	NN	O	O
,	NN	O	O
5	NN	O	O
,	NN	O	O
and	NN	O	O
6	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
between	NN	O	O
exons	NN	O	O
10	NN	O	O
and	NN	O	O
11	NN	O	O
.	NN	O	O

No	NN	O	O
additional	NN	O	O
introns	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
exons	NN	O	O
6	NN	O	O
and	NN	O	O
10	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
DNA	NN	O	O
marker	NN	O	O
studies	NN	O	O
were	NN	O	O
conducted	NN	O	O
using	NN	O	O
known	NN	O	O
polymorphisms	NN	O	O
of	NN	O	O
the	NN	O	O
C6	NN	O	O
,	NN	O	O
C7	NN	O	O
,	NN	O	O
and	NN	O	O
C9	NN	O	O
genes	NN	O	O
,	NN	O	O
confirming	NN	O	O
the	NN	O	O
linkage	NN	O	O
of	NN	O	O
the	NN	O	O
observed	NN	O	O
C9	NN	O	O
mutations	NN	O	O
with	NN	O	O
defined	NN	O	O
haplotypes	NN	O	O
.	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
mutations	NN	O	O
in	NN	O	O
women	NN	O	O
attending	NN	O	O
clinics	NN	O	O
that	NN	O	O
evaluate	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

BACKGROUND	NN	O	O
To	NN	O	O
define	NN	O	O
the	NN	O	O
incidence	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
among	NN	O	O
patients	NN	O	O
seen	NN	O	O
in	NN	O	O
clinics	NN	O	O
that	NN	O	O
evaluate	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
analyzed	NN	O	O
DNA	NN	O	O
samples	NN	O	O
from	NN	O	O
women	NN	O	O
seen	NN	O	O
in	NN	O	O
this	NN	O	O
setting	NN	O	O
and	NN	O	O
constructed	NN	O	O
probability	NN	O	O
tables	NN	O	O
to	NN	O	O
provide	NN	O	O
estimates	NN	O	O
of	NN	O	O
the	NN	O	O
likelihood	NN	O	O
of	NN	O	O
finding	NN	O	O
a	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
in	NN	O	O
individual	NN	O	O
families	NN	O	O
.	NN	O	O

METHODS	NN	O	O
Clinical	NN	O	O
information	NN	O	O
,	NN	O	O
family	NN	O	O
histories	NN	O	O
,	NN	O	O
and	NN	O	O
blood	NN	O	O
for	NN	O	O
DNA	NN	O	O
analysis	NN	O	O
were	NN	O	O
obtained	NN	O	O
from	NN	O	O
263	NN	O	O
women	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Conformation	NN	O	O
-	NN	O	O
sensitive	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
and	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
were	NN	O	O
used	NN	O	O
to	NN	O	O
identify	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
16	NN	O	O
percent	NN	O	O
of	NN	O	O
women	NN	O	O
with	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Only	NN	O	O
7	NN	O	O
percent	NN	O	O
of	NN	O	O
women	NN	O	O
from	NN	O	O
families	NN	O	O
with	NN	O	O
a	NN	O	O
history	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
but	NN	O	O
not	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
had	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
rates	NN	O	O
were	NN	O	O
higher	NN	O	O
among	NN	O	O
women	NN	O	O
from	NN	O	O
families	NN	O	O
with	NN	O	O
a	NN	O	O
history	NN	O	O
of	NN	O	O
both	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Among	NN	O	O
family	NN	O	O
members	NN	O	O
,	NN	O	O
an	NN	O	O
average	NN	O	O
age	NN	O	O
of	NN	O	O
less	NN	O	O
than	NN	O	O
55	NN	O	O
years	NN	O	O
at	NN	O	O
the	NN	O	O
diagnosis	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
same	NN	O	O
woman	NN	O	O
,	NN	O	O
and	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
ancestry	NN	O	O
were	NN	O	O
all	NN	O	O
associated	NN	O	O
with	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
of	NN	O	O
detecting	NN	O	O
a	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
.	NN	O	O

No	NN	O	O
association	NN	O	O
was	NN	O	O
found	NN	O	O
between	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
bilateral	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
or	NN	O	O
the	NN	O	O
number	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancers	NN	O	I-Disease
in	NN	O	O
a	NN	O	O
family	NN	O	O
and	NN	O	O
the	NN	O	O
detection	NN	O	O
of	NN	O	O
a	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
,	NN	O	O
or	NN	O	O
between	NN	O	O
the	NN	O	O
position	NN	O	O
of	NN	O	O
the	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
a	NN	O	O
family	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
Among	NN	O	O
women	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
,	NN	O	O
the	NN	O	O
percentage	NN	O	O
with	NN	O	O
BRCA1	NN	O	O
coding	NN	O	O
-	NN	O	O
region	NN	O	O
mutations	NN	O	O
is	NN	O	O
less	NN	O	O
than	NN	O	O
the	NN	O	O
45	NN	O	O
percent	NN	O	O
predicted	NN	O	O
by	NN	O	O
genetic	NN	O	O
-	NN	O	O
linkage	NN	O	O
analysis	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
suggest	NN	O	O
that	NN	O	O
even	NN	O	O
in	NN	O	O
a	NN	O	O
referral	NN	O	O
clinic	NN	O	O
specializing	NN	O	O
in	NN	O	O
screening	NN	O	O
women	NN	O	O
from	NN	O	O
high	NN	O	O
-	NN	O	O
risk	NN	O	O
families	NN	O	O
,	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
tests	NN	O	O
for	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
will	NN	O	O
be	NN	O	O
negative	NN	O	O
and	NN	O	O
therefore	NN	O	O
uninformative	NN	O	O
.	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
arginine	NN	O	O
-	NN	O	O
rich	NN	O	O
protein	NN	O	O
gene	NN	O	O
(	NN	O	O
ARP	NN	O	O
)	NN	O	O
in	NN	O	O
pancreatic	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
ARP	NN	O	O
gene	NN	O	O
encodes	NN	O	O
a	NN	O	O
highly	NN	O	O
conserved	NN	O	O
arginine	NN	O	O
-	NN	O	O
rich	NN	O	O
protein	NN	O	O
from	NN	O	O
chromosomal	NN	O	O
band	NN	O	O
3p21	NN	O	O
.	NN	O	O

1	NN	O	O
1	NN	O	O
.	NN	O	O

At	NN	O	O
the	NN	O	O
cytogenetic	NN	O	O
level	NN	O	O
this	NN	O	O
region	NN	O	O
is	NN	O	O
frequently	NN	O	O
deleted	NN	O	O
in	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
different	NN	O	O
solid	NN	O	B-Disease
tumors	NN	O	I-Disease
,	NN	O	O
although	NN	O	O
not	NN	O	O
in	NN	O	O
pancreatic	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
reported	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
specific	NN	O	O
mutation	NN	O	O
(	NN	O	O
ATG50	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
AGG	NN	O	O
)	NN	O	O
or	NN	O	O
deletion	NN	O	O
of	NN	O	O
codon	NN	O	O
50	NN	O	O
of	NN	O	O
the	NN	O	O
ARP	NN	O	O
gene	NN	O	O
in	NN	O	O
different	NN	O	O
tumor	NN	O	B-Disease
types	NN	O	I-Disease
(	NN	O	O
Shridhar	NN	O	O
et	NN	O	O
al	NN	O	O
.	NN	O	O
,	NN	O	O
1996	NN	O	O
,	NN	O	O
1996a	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
observed	NN	O	O
mutations	NN	O	O
involving	NN	O	O
codon	NN	O	O
50	NN	O	O
in	NN	O	O
11	NN	O	O
of	NN	O	O
37	NN	O	O
pancreatic	NN	O	B-Disease
tumors	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
frequency	NN	O	O
of	NN	O	O
codon	NN	O	O
50	NN	O	O
mutation	NN	O	O
is	NN	O	O
roughly	NN	O	O
the	NN	O	O
same	NN	O	O
in	NN	O	O
pancreatic	NN	O	B-Disease
tumors	NN	O	I-Disease
as	NN	O	O
in	NN	O	O
the	NN	O	O
other	NN	O	O
types	NN	O	O
of	NN	O	O
tumors	NN	O	B-Disease
previously	NN	O	O
examined	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
detected	NN	O	O
mutations	NN	O	O
at	NN	O	O
codon	NN	O	O
51	NN	O	O
in	NN	O	O
multiple	NN	O	O
PCR	NN	O	O
subclones	NN	O	O
in	NN	O	O
two	NN	O	O
other	NN	O	O
pancreatic	NN	O	B-Disease
tumors	NN	O	I-Disease
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ARP	NN	O	O
gene	NN	O	O
are	NN	O	O
thus	NN	O	O
commonly	NN	O	O
observed	NN	O	O
in	NN	O	O
pancreatic	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
many	NN	O	O
other	NN	O	O
cancers	NN	O	B-Disease
.	NN	O	O

Difficulties	NN	O	O
in	NN	O	O
the	NN	O	O
ascertainment	NN	O	O
of	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
:	NN	O	O
lessons	NN	O	O
to	NN	O	O
be	NN	O	O
drawn	NN	O	O
from	NN	O	O
a	NN	O	O
compound	NN	O	O
heterozygote	NN	O	O
C9	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
subject	NN	O	O
.	NN	O	O

A	NN	O	O
group	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
long	NN	O	O
-	NN	O	O
surviving	NN	O	O
mismatched	NN	O	O
kidney	NN	O	O
allografts	NN	O	O
were	NN	O	O
investigated	NN	O	O
for	NN	O	O
complement	NN	O	O
function	NN	O	O
using	NN	O	O
haemolytic	NN	O	O
assays	NN	O	O
in	NN	O	O
agarose	NN	O	O
gels	NN	O	O
.	NN	O	O

One	NN	O	O
patient	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
no	NN	O	O
alternative	NN	O	O
pathway	NN	O	O
activity	NN	O	O
but	NN	O	O
a	NN	O	O
low	NN	O	O
normal	NN	O	O
classical	NN	O	O
pathway	NN	O	O
.	NN	O	O

Surprisingly	NN	O	O
,	NN	O	O
investigation	NN	O	O
revealed	NN	O	O
that	NN	O	O
the	NN	O	O
patients	NN	O	O
complement	NN	O	O
was	NN	O	O
normal	NN	O	O
for	NN	O	O
all	NN	O	O
components	NN	O	O
except	NN	O	O
C9	NN	O	O
,	NN	O	O
which	NN	O	O
was	NN	O	O
functionally	NN	O	O
absent	NN	O	O
.	NN	O	O

The	NN	O	O
patient	NN	O	O
was	NN	O	O
shown	NN	O	O
to	NN	O	O
be	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
DNA	NN	O	O
markers	NN	O	O
in	NN	O	O
the	NN	O	O
C6	NN	O	O
,	NN	O	O
C7	NN	O	O
and	NN	O	O
C9	NN	O	O
region	NN	O	O
of	NN	O	O
chromosome	NN	O	O
5	NN	O	O
and	NN	O	O
therefore	NN	O	O
appears	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
compound	NN	O	O
heterozygote	NN	O	O
for	NN	O	O
two	NN	O	O
uncharacterized	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
genes	NN	O	O
.	NN	O	O

Serological	NN	O	O
analysis	NN	O	O
by	NN	O	O
ELISA	NN	O	O
revealed	NN	O	O
that	NN	O	O
he	NN	O	O
has	NN	O	O
trace	NN	O	O
concentrations	NN	O	O
of	NN	O	O
a	NN	O	O
non	NN	O	O
-	NN	O	O
functional	NN	O	O
C9	NN	O	O
molecule	NN	O	O
.	NN	O	O

Western	NN	O	O
blot	NN	O	O
analysis	NN	O	O
was	NN	O	O
not	NN	O	O
sufficiently	NN	O	O
sensitive	NN	O	O
to	NN	O	O
permit	NN	O	O
detection	NN	O	O
of	NN	O	O
this	NN	O	O
molecule	NN	O	O
.	NN	O	O

We	NN	O	O
hypothesize	NN	O	O
that	NN	O	O
the	NN	O	O
patient	NN	O	O
is	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
a	NN	O	O
complete	NN	O	B-Disease
deficiency	NN	O	I-Disease
of	NN	O	I-Disease
C9	NN	O	I-Disease
and	NN	O	O
for	NN	O	O
a	NN	O	O
gene	NN	O	O
directing	NN	O	O
hyposynthesis	NN	O	O
of	NN	O	O
a	NN	O	O
defective	NN	O	O
C9	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
suggest	NN	O	O
that	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
may	NN	O	O
be	NN	O	O
more	NN	O	O
common	NN	O	O
among	NN	O	O
Caucasians	NN	O	O
than	NN	O	O
has	NN	O	O
been	NN	O	O
reported	NN	O	O
.	NN	O	O
.	NN	O	O

Screening	NN	O	O
for	NN	O	O
ESR	NN	O	O
mutations	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
,	NN	O	O
leukocyte	NN	O	O
DNA	NN	O	O
from	NN	O	O
143	NN	O	O
patients	NN	O	O
with	NN	O	O
familial	NN	O	O
clustering	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
tumour	NN	O	B-Disease
DNA	NN	O	O
from	NN	O	O
96	NN	O	O
breast	NN	O	B-Disease
carcinomas	NN	O	I-Disease
were	NN	O	O
screened	NN	O	O
for	NN	O	O
base	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
estrogen	NN	O	O
receptor	NN	O	O
gene	NN	O	O
(	NN	O	O
ESR	NN	O	O
)	NN	O	O
.	NN	O	O

Three	NN	O	O
patients	NN	O	O
with	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
were	NN	O	O
carrying	NN	O	O
a	NN	O	O
Gly160Cys	NN	O	O
germline	NN	O	O
substitution	NN	O	O
.	NN	O	O

This	NN	O	O
alteration	NN	O	O
was	NN	O	O
also	NN	O	O
detected	NN	O	O
in	NN	O	O
eight	NN	O	O
(	NN	O	O
four	NN	O	O
females	NN	O	O
and	NN	O	O
four	NN	O	O
males	NN	O	O
)	NN	O	O
of	NN	O	O
729	NN	O	O
controls	NN	O	O
(	NN	O	O
366	NN	O	O
female	NN	O	O
,	NN	O	O
363	NN	O	O
males	NN	O	O
)	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
the	NN	O	O
substitution	NN	O	O
probably	NN	O	O
represents	NN	O	O
a	NN	O	O
polymorphism	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
229	NN	O	O
female	NN	O	O
controls	NN	O	O
in	NN	O	O
whom	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
was	NN	O	O
known	NN	O	O
,	NN	O	O
one	NN	O	O
of	NN	O	O
two	NN	O	O
who	NN	O	O
had	NN	O	O
a	NN	O	O
sister	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
was	NN	O	O
carrying	NN	O	O
the	NN	O	O
variant	NN	O	O
allele	NN	O	O
.	NN	O	O

Hence	NN	O	O
,	NN	O	O
a	NN	O	O
possible	NN	O	O
clinical	NN	O	O
significance	NN	O	O
of	NN	O	O
the	NN	O	O
glycine	NN	O	O
into	NN	O	O
cysteine	NN	O	O
cannot	NN	O	O
be	NN	O	O
completely	NN	O	O
ruled	NN	O	O
out	NN	O	O
and	NN	O	O
should	NN	O	O
be	NN	O	O
further	NN	O	O
investigated	NN	O	O
.	NN	O	O

Somatic	NN	O	O
mutations	NN	O	O
were	NN	O	O
not	NN	O	O
detected	NN	O	O
in	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
tumours	NN	O	B-Disease
studied	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
present	NN	O	O
data	NN	O	O
do	NN	O	O
not	NN	O	O
provide	NN	O	O
support	NN	O	O
for	NN	O	O
somatic	NN	O	O
ESR	NN	O	O
base	NN	O	O
mutations	NN	O	O
as	NN	O	O
an	NN	O	O
important	NN	O	O
mechanism	NN	O	O
for	NN	O	O
hormonal	NN	O	O
therapy	NN	O	O
resistance	NN	O	O
in	NN	O	O
estrogen	NN	O	B-Disease
receptor	NN	O	I-Disease
-	NN	O	I-Disease
positive	NN	O	I-Disease
breast	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Molecular	NN	O	O
bases	NN	O	O
of	NN	O	O
C7	NN	O	B-Disease
deficiency	NN	O	I-Disease
:	NN	O	O
three	NN	O	O
different	NN	O	O
defects	NN	O	O
.	NN	O	O

The	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
C7	NN	O	B-Disease
deficiency	NN	O	I-Disease
has	NN	O	O
been	NN	O	O
investigated	NN	O	O
in	NN	O	O
two	NN	O	O
Irish	NN	O	O
families	NN	O	O
and	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
Israeli	NN	O	O
families	NN	O	O
of	NN	O	O
Moroccan	NN	O	O
Sephardic	NN	O	O
Jewish	NN	O	O
origin	NN	O	O
.	NN	O	O

Exon	NN	O	O
PCR	NN	O	O
and	NN	O	O
sequencing	NN	O	O
revealed	NN	O	O
a	NN	O	O
heterozygous	NN	O	O
point	NN	O	O
mutation	NN	O	O
at	NN	O	O
the	NN	O	O
3	NN	O	O
splice	NN	O	O
acceptor	NN	O	O
site	NN	O	O
of	NN	O	O
intron	NN	O	O
1	NN	O	O
in	NN	O	O
one	NN	O	O
Irish	NN	O	O
family	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
other	NN	O	O
Irish	NN	O	O
family	NN	O	O
,	NN	O	O
exons	NN	O	O
7	NN	O	O
and	NN	O	O
8	NN	O	O
failed	NN	O	O
to	NN	O	O
amplify	NN	O	O
and	NN	O	O
they	NN	O	O
were	NN	O	O
shown	NN	O	O
to	NN	O	O
be	NN	O	O
deleted	NN	O	O
.	NN	O	O

Marker	NN	O	O
haplotype	NN	O	O
studies	NN	O	O
of	NN	O	O
the	NN	O	O
C6	NN	O	O
and	NN	O	O
C7	NN	O	O
gene	NN	O	O
region	NN	O	O
and	NN	O	O
Southern	NN	O	O
blots	NN	O	O
show	NN	O	O
that	NN	O	O
the	NN	O	O
Irish	NN	O	O
family	NN	O	O
with	NN	O	O
the	NN	O	O
splice	NN	O	O
defect	NN	O	O
also	NN	O	O
segregate	NN	O	O
for	NN	O	O
the	NN	O	O
deletion	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
not	NN	O	O
easily	NN	O	O
detected	NN	O	O
in	NN	O	O
heterozygotes	NN	O	O
.	NN	O	O

The	NN	O	O
Israeli	NN	O	O
C7	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
cases	NN	O	O
all	NN	O	O
share	NN	O	O
a	NN	O	O
C7	NN	O	O
haplotype	NN	O	O
and	NN	O	O
are	NN	O	O
homozygous	NN	O	O
for	NN	O	O
a	NN	O	O
mis	NN	O	O
-	NN	O	O
sense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
9	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
one	NN	O	O
individual	NN	O	O
is	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
markers	NN	O	O
at	NN	O	O
adjacent	NN	O	O
C6	NN	O	O
loci	NN	O	O
,	NN	O	O
showing	NN	O	O
that	NN	O	O
there	NN	O	O
has	NN	O	O
been	NN	O	O
an	NN	O	O
intergenic	NN	O	O
recombination	NN	O	O
and	NN	O	O
suggesting	NN	O	O
that	NN	O	O
the	NN	O	O
deficiency	NN	O	O
mutation	NN	O	O
is	NN	O	O
of	NN	O	O
appreciable	NN	O	O
antiquity	NN	O	O
.	NN	O	O
.	NN	O	O

Molecular	NN	O	O
heterogeneity	NN	O	O
of	NN	O	O
classical	NN	O	B-Disease
and	NN	O	I-Disease
Duarte	NN	O	I-Disease
galactosemia	NN	O	I-Disease
:	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
by	NN	O	O
denaturing	NN	O	O
gradient	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
.	NN	O	O

Classical	NN	O	B-Disease
galactosemia	NN	O	I-Disease
is	NN	O	O
caused	NN	O	O
by	NN	O	O
one	NN	O	O
common	NN	O	O
missense	NN	O	O
mutation	NN	O	O
(	NN	O	O
Q188R	NN	O	O
)	NN	O	O
and	NN	O	O
by	NN	O	O
several	NN	O	O
rare	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
galactose	NN	O	O
-	NN	O	O
1	NN	O	O
-	NN	O	O
phosphate	NN	O	O
uridyltransferase	NN	O	O
(	NN	O	O
GALT	NN	O	O
)	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
most	NN	O	O
common	NN	O	O
variant	NN	O	O
of	NN	O	O
GALT	NN	O	O
,	NN	O	O
the	NN	O	O
Duarte	NN	O	O
variant	NN	O	O
,	NN	O	O
occurs	NN	O	O
as	NN	O	O
two	NN	O	O
types	NN	O	O
,	NN	O	O
Duarte	NN	O	O
-	NN	O	O
1	NN	O	O
(	NN	O	O
D	NN	O	O
-	NN	O	O
1	NN	O	O
)	NN	O	O
and	NN	O	O
Duarte	NN	O	O
-	NN	O	O
2	NN	O	O
(	NN	O	O
D	NN	O	O
-	NN	O	O
2	NN	O	O
)	NN	O	O
,	NN	O	O
both	NN	O	O
of	NN	O	O
which	NN	O	O
carry	NN	O	O
the	NN	O	O
sequence	NN	O	O
change	NN	O	O
N314D	NN	O	O
.	NN	O	O

D	NN	O	O
-	NN	O	O
1	NN	O	O
increases	NN	O	O
,	NN	O	O
whereas	NN	O	O
D	NN	O	O
-	NN	O	O
2	NN	O	O
decreases	NN	O	O
GALT	NN	O	O
activity	NN	O	O
.	NN	O	O

To	NN	O	O
study	NN	O	O
the	NN	O	O
molecular	NN	O	O
genetics	NN	O	O
of	NN	O	O
classical	NN	O	B-Disease
and	NN	O	I-Disease
Duarte	NN	O	I-Disease
galactosemia	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
analyzed	NN	O	O
the	NN	O	O
GALT	NN	O	O
mutations	NN	O	O
in	NN	O	O
30	NN	O	O
families	NN	O	O
with	NN	O	O
classical	NN	O	B-Disease
galactosemia	NN	O	I-Disease
,	NN	O	O
in	NN	O	O
10	NN	O	O
families	NN	O	O
with	NN	O	O
the	NN	O	O
D	NN	O	O
-	NN	O	O
2	NN	O	O
variant	NN	O	O
and	NN	O	O
in	NN	O	O
3	NN	O	O
individuals	NN	O	O
carrying	NN	O	O
the	NN	O	O
D	NN	O	O
-	NN	O	O
1	NN	O	O
allele	NN	O	O
by	NN	O	O
denaturing	NN	O	O
gradient	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
(	NN	O	O
DGGE	NN	O	O
)	NN	O	O
.	NN	O	O

DGGE	NN	O	O
detected	NN	O	O
59	NN	O	O
of	NN	O	O
the	NN	O	O
60	NN	O	O
classical	NN	O	O
galactosemia	NN	O	B-Disease
alleles	NN	O	O
.	NN	O	O

Q188R	NN	O	O
accounted	NN	O	O
for	NN	O	O
60	NN	O	O
%	NN	O	O
,	NN	O	O
K285N	NN	O	O
accounted	NN	O	O
for	NN	O	O
28	NN	O	O
%	NN	O	O
of	NN	O	O
these	NN	O	O
alleles	NN	O	O
.	NN	O	O

Eight	NN	O	O
novel	NN	O	O
candidate	NN	O	O
galactosemia	NN	O	B-Disease
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
.	NN	O	O

On	NN	O	O
all	NN	O	O
D	NN	O	O
-	NN	O	O
2	NN	O	O
alleles	NN	O	O
N314D	NN	O	O
occurred	NN	O	O
in	NN	O	O
cis	NN	O	O
with	NN	O	O
two	NN	O	O
intronic	NN	O	O
sequence	NN	O	O
changes	NN	O	O
,	NN	O	O
on	NN	O	O
the	NN	O	O
D	NN	O	O
-	NN	O	O
1	NN	O	O
alleles	NN	O	O
in	NN	O	O
cis	NN	O	O
with	NN	O	O
a	NN	O	O
neutral	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
7	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
mutations	NN	O	O
causing	NN	O	O
galactosemia	NN	O	B-Disease
are	NN	O	O
highly	NN	O	O
heterogeneous	NN	O	O
and	NN	O	O
that	NN	O	O
K285N	NN	O	O
is	NN	O	O
a	NN	O	O
second	NN	O	O
common	NN	O	O
galactosemia	NN	O	B-Disease
mutation	NN	O	O
in	NN	O	O
our	NN	O	O
population	NN	O	O
.	NN	O	O
.	NN	O	O

Isolation	NN	O	O
of	NN	O	O
full	NN	O	O
-	NN	O	O
length	NN	O	O
ATM	NN	O	O
cDNA	NN	O	O
and	NN	O	O
correction	NN	O	O
of	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
cellular	NN	O	O
phenotype	NN	O	O
.	NN	O	O

A	NN	O	O
gene	NN	O	O
mutated	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
genetic	NN	O	B-Disease
disorder	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
ATM	NN	O	O
,	NN	O	O
was	NN	O	O
recently	NN	O	O
identified	NN	O	O
by	NN	O	O
positional	NN	O	O
cloning	NN	O	O
.	NN	O	O

ATM	NN	O	O
is	NN	O	O
a	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
phosphatidylinositol	NN	O	O
-	NN	O	O
3	NN	O	O
-	NN	O	O
kinase	NN	O	O
superfamily	NN	O	O
,	NN	O	O
some	NN	O	O
of	NN	O	O
which	NN	O	O
are	NN	O	O
protein	NN	O	O
kinases	NN	O	O
and	NN	O	O
appear	NN	O	O
to	NN	O	O
have	NN	O	O
important	NN	O	O
roles	NN	O	O
in	NN	O	O
cell	NN	O	O
cycle	NN	O	O
control	NN	O	O
and	NN	O	O
radiation	NN	O	O
signal	NN	O	O
transduction	NN	O	O
.	NN	O	O

We	NN	O	O
describe	NN	O	O
herein	NN	O	O
,	NN	O	O
to	NN	O	O
our	NN	O	O
knowledge	NN	O	O
,	NN	O	O
for	NN	O	O
the	NN	O	O
first	NN	O	O
time	NN	O	O
,	NN	O	O
the	NN	O	O
cloning	NN	O	O
of	NN	O	O
a	NN	O	O
full	NN	O	O
-	NN	O	O
length	NN	O	O
cDNA	NN	O	O
for	NN	O	O
ATM	NN	O	O
and	NN	O	O
correction	NN	O	O
of	NN	O	O
multiple	NN	O	O
aspects	NN	O	O
of	NN	O	O
the	NN	O	O
radio	NN	O	O
-	NN	O	O
sensitive	NN	O	O
phenotype	NN	O	O
of	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
cells	NN	O	O
by	NN	O	O
transfection	NN	O	O
with	NN	O	O
this	NN	O	O
cDNA	NN	O	O
.	NN	O	O

Overexpression	NN	O	O
of	NN	O	O
ATM	NN	O	O
cDNA	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
cells	NN	O	O
enhanced	NN	O	O
the	NN	O	O
survival	NN	O	O
of	NN	O	O
these	NN	O	O
cells	NN	O	O
in	NN	O	O
response	NN	O	O
to	NN	O	O
radiation	NN	O	O
exposure	NN	O	O
,	NN	O	O
decreased	NN	O	O
radiation	NN	O	O
-	NN	O	O
induced	NN	O	O
chromosome	NN	O	O
aberrations	NN	O	O
,	NN	O	O
reduced	NN	O	O
radio	NN	O	O
-	NN	O	O
resistant	NN	O	O
DNA	NN	O	O
synthesis	NN	O	O
,	NN	O	O
and	NN	O	O
partially	NN	O	O
corrected	NN	O	O
defective	NN	O	O
cell	NN	O	O
cycle	NN	O	O
checkpoints	NN	O	O
and	NN	O	O
induction	NN	O	O
of	NN	O	O
stress	NN	O	O
-	NN	O	O
activated	NN	O	O
protein	NN	O	O
kinase	NN	O	O
.	NN	O	O

This	NN	O	O
correction	NN	O	O
of	NN	O	O
the	NN	O	O
defects	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
cells	NN	O	O
provides	NN	O	O
further	NN	O	O
evidence	NN	O	O
of	NN	O	O
the	NN	O	O
multiplicity	NN	O	O
of	NN	O	O
effector	NN	O	O
functions	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
protein	NN	O	O
and	NN	O	O
suggests	NN	O	O
possible	NN	O	O
approaches	NN	O	O
to	NN	O	O
gene	NN	O	O
therapy	NN	O	O
.	NN	O	O
.	NN	O	O

Fusion	NN	O	O
genes	NN	O	O
resulting	NN	O	O
from	NN	O	O
alternative	NN	O	O
chromosomal	NN	O	O
translocations	NN	O	O
are	NN	O	O
overexpressed	NN	O	O
by	NN	O	O
gene	NN	O	O
-	NN	O	O
specific	NN	O	O
mechanisms	NN	O	O
in	NN	O	O
alveolar	NN	O	B-Disease
rhabdomyosarcoma	NN	O	I-Disease
.	NN	O	O

Chromosomal	NN	O	O
translocations	NN	O	O
identified	NN	O	O
in	NN	O	O
hematopoietic	NN	O	B-Disease
and	NN	O	I-Disease
solid	NN	O	I-Disease
tumors	NN	O	I-Disease
result	NN	O	O
in	NN	O	O
deregulated	NN	O	O
expression	NN	O	O
of	NN	O	O
protooncogenes	NN	O	O
or	NN	O	O
creation	NN	O	O
of	NN	O	O
chimeric	NN	O	O
proteins	NN	O	O
with	NN	O	O
tumorigenic	NN	O	O
potential	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
pediatric	NN	O	O
solid	NN	O	B-Disease
tumor	NN	O	I-Disease
alveolar	NN	O	B-Disease
rhabdomyosarcoma	NN	O	I-Disease
,	NN	O	O
a	NN	O	O
consistent	NN	O	O
t	NN	O	O
(	NN	O	O
2	NN	O	O
;	NN	O	O
13	NN	O	O
)	NN	O	O
(	NN	O	O
q35	NN	O	O
;	NN	O	O
q14	NN	O	O
)	NN	O	O
or	NN	O	O
variant	NN	O	O
t	NN	O	O
(	NN	O	O
1	NN	O	O
;	NN	O	O
13	NN	O	O
)	NN	O	O
(	NN	O	O
p36	NN	O	O
;	NN	O	O
q14	NN	O	O
)	NN	O	O
translocation	NN	O	O
generates	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
or	NN	O	O
PAX7	NN	O	O
-	NN	O	O
FKHR	NN	O	O
fusion	NN	O	O
proteins	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
report	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
in	NN	O	O
addition	NN	O	O
to	NN	O	O
functional	NN	O	O
alterations	NN	O	O
these	NN	O	O
translocations	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
fusion	NN	O	O
product	NN	O	O
overexpression	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
and	NN	O	O
PAX7	NN	O	O
-	NN	O	O
FKHR	NN	O	O
overexpression	NN	O	O
occurs	NN	O	O
by	NN	O	O
distinct	NN	O	O
mechanisms	NN	O	O
.	NN	O	O

Transcription	NN	O	O
of	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
is	NN	O	O
increased	NN	O	O
relative	NN	O	O
to	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
PAX3	NN	O	O
by	NN	O	O
a	NN	O	O
copy	NN	O	O
number	NN	O	O
-	NN	O	O
independent	NN	O	O
process	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
PAX7	NN	O	O
-	NN	O	O
FKHR	NN	O	O
overexpression	NN	O	O
results	NN	O	O
from	NN	O	O
fusion	NN	O	O
gene	NN	O	O
amplification	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
gene	NN	O	O
-	NN	O	O
specific	NN	O	O
mechanisms	NN	O	O
were	NN	O	O
selected	NN	O	O
to	NN	O	O
overexpress	NN	O	O
PAX3	NN	O	O
-	NN	O	O
FKHR	NN	O	O
and	NN	O	O
PAX7	NN	O	O
-	NN	O	O
FKHR	NN	O	O
in	NN	O	O
alveolar	NN	O	B-Disease
rhabdomyosarcoma	NN	O	I-Disease
,	NN	O	O
presumably	NN	O	O
due	NN	O	O
to	NN	O	O
differences	NN	O	O
in	NN	O	O
regulation	NN	O	O
between	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
loci	NN	O	O
.	NN	O	O

We	NN	O	O
postulate	NN	O	O
that	NN	O	O
these	NN	O	O
overexpression	NN	O	O
mechanisms	NN	O	O
ensure	NN	O	O
a	NN	O	O
critical	NN	O	O
level	NN	O	O
of	NN	O	O
gene	NN	O	O
product	NN	O	O
for	NN	O	O
the	NN	O	O
oncogenic	NN	O	O
effects	NN	O	O
of	NN	O	O
these	NN	O	O
fusions	NN	O	O
.	NN	O	O
.	NN	O	O

atm	NN	O	O
and	NN	O	O
p53	NN	O	O
cooperate	NN	O	O
in	NN	O	O
apoptosis	NN	O	O
and	NN	O	O
suppression	NN	O	O
of	NN	O	O
tumorigenesis	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
in	NN	O	O
resistance	NN	O	O
to	NN	O	O
acute	NN	O	B-Disease
radiation	NN	O	I-Disease
toxicity	NN	O	I-Disease
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
atm	NN	O	O
and	NN	O	O
p53	NN	O	O
cause	NN	O	O
the	NN	O	O
human	NN	O	O
cancer	NN	O	B-Disease
-	NN	O	I-Disease
associated	NN	O	I-Disease
diseases	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
and	NN	O	O
Li	NN	O	B-Disease
-	NN	O	I-Disease
Fraumeni	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

The	NN	O	O
two	NN	O	O
genes	NN	O	O
are	NN	O	O
believed	NN	O	O
to	NN	O	O
interact	NN	O	O
in	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
pathways	NN	O	O
,	NN	O	O
including	NN	O	O
regulation	NN	O	O
of	NN	O	O
DNA	NN	O	O
damage	NN	O	O
-	NN	O	O
induced	NN	O	O
cell	NN	O	O
-	NN	O	O
cycle	NN	O	O
checkpoints	NN	O	O
,	NN	O	O
apoptosis	NN	O	O
and	NN	O	O
radiation	NN	O	O
sensitivity	NN	O	O
,	NN	O	O
and	NN	O	O
cellular	NN	O	O
proliferation	NN	O	O
.	NN	O	O

Atm	NN	O	O
-	NN	O	O
null	NN	O	O
mice	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
those	NN	O	O
null	NN	O	O
for	NN	O	O
p53	NN	O	O
,	NN	O	O
develop	NN	O	O
mainly	NN	O	O
T	NN	O	B-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
lymphomas	NN	O	I-Disease
,	NN	O	O
supporting	NN	O	O
the	NN	O	O
view	NN	O	O
that	NN	O	O
these	NN	O	O
genes	NN	O	O
have	NN	O	O
similar	NN	O	O
roles	NN	O	O
in	NN	O	O
thymocyte	NN	O	O
development	NN	O	O
.	NN	O	O

To	NN	O	O
study	NN	O	O
the	NN	O	O
interactions	NN	O	O
of	NN	O	O
these	NN	O	O
two	NN	O	O
genes	NN	O	O
on	NN	O	O
an	NN	O	O
organismal	NN	O	O
level	NN	O	O
,	NN	O	O
we	NN	O	O
bred	NN	O	O
mice	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
null	NN	O	O
alleles	NN	O	O
of	NN	O	O
both	NN	O	O
atm	NN	O	O
and	NN	O	O
p53	NN	O	O
to	NN	O	O
produce	NN	O	O
all	NN	O	O
genotypic	NN	O	O
combinations	NN	O	O
.	NN	O	O

Mice	NN	O	O
doubly	NN	O	O
null	NN	O	O
for	NN	O	O
atm	NN	O	O
and	NN	O	O
p53	NN	O	O
exhibited	NN	O	O
a	NN	O	O
dramatic	NN	O	O
acceleration	NN	O	O
of	NN	O	O
tumour	NN	O	B-Disease
formation	NN	O	O
relative	NN	O	O
to	NN	O	O
singly	NN	O	O
null	NN	O	O
mice	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
both	NN	O	O
genes	NN	O	O
collaborate	NN	O	O
in	NN	O	O
a	NN	O	O
significant	NN	O	O
manner	NN	O	O
to	NN	O	O
prevent	NN	O	O
tumorigenesis	NN	O	O
.	NN	O	O

With	NN	O	O
respect	NN	O	O
to	NN	O	O
their	NN	O	O
roles	NN	O	O
in	NN	O	O
apoptosis	NN	O	O
,	NN	O	O
loss	NN	O	O
of	NN	O	O
atm	NN	O	O
rendered	NN	O	O
thymocytes	NN	O	O
only	NN	O	O
partly	NN	O	O
resistant	NN	O	O
to	NN	O	O
irradiation	NN	O	O
-	NN	O	O
induced	NN	O	O
apoptosis	NN	O	O
,	NN	O	O
whereas	NN	O	O
additional	NN	O	O
loss	NN	O	O
of	NN	O	O
p53	NN	O	O
engendered	NN	O	O
complete	NN	O	O
resistance	NN	O	O
.	NN	O	O

This	NN	O	O
implies	NN	O	O
that	NN	O	O
the	NN	O	O
irradiation	NN	O	O
-	NN	O	O
induced	NN	O	O
atm	NN	O	O
and	NN	O	O
p53	NN	O	O
apoptotic	NN	O	O
pathways	NN	O	O
are	NN	O	O
not	NN	O	O
completely	NN	O	O
congruent	NN	O	O
.	NN	O	O

Finally	NN	O	O
-	NN	O	O
and	NN	O	O
in	NN	O	O
contrast	NN	O	O
to	NN	O	O
prior	NN	O	O
predictions	NN	O	O
-	NN	O	O
atm	NN	O	O
and	NN	O	O
p53	NN	O	O
do	NN	O	O
not	NN	O	O
appear	NN	O	O
to	NN	O	O
interact	NN	O	O
in	NN	O	O
acute	NN	O	B-Disease
radiation	NN	O	I-Disease
toxicity	NN	O	I-Disease
,	NN	O	O
suggesting	NN	O	O
a	NN	O	O
separate	NN	O	O
atm	NN	O	O
effector	NN	O	O
pathway	NN	O	O
for	NN	O	O
this	NN	O	O
DNA	NN	O	O
damage	NN	O	O
response	NN	O	O
and	NN	O	O
having	NN	O	O
implications	NN	O	O
for	NN	O	O
the	NN	O	O
prognosis	NN	O	O
and	NN	O	O
treatment	NN	O	O
of	NN	O	O
human	NN	O	O
tumours	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Trinucleotide	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
at	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
locus	NN	O	O
reduces	NN	O	O
expression	NN	O	O
of	NN	O	O
DMAHP	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
,	NN	O	O
or	NN	O	O
dystrophia	NN	O	B-Disease
myotonica	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
multisystem	NN	O	I-Disease
disorder	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
the	NN	O	O
expansion	NN	O	O
of	NN	O	O
a	NN	O	O
CTG	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
untranslated	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
DMPK	NN	O	O
protein	NN	O	O
kinase	NN	O	O
gene	NN	O	O
on	NN	O	O
chromosome	NN	O	O
19q13	NN	O	O
.	NN	O	O

3	NN	O	O
(	NN	O	O
refs	NN	O	O
1	NN	O	O
-	NN	O	O
3	NN	O	O
)	NN	O	O
.	NN	O	O

Although	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
mutation	NN	O	O
was	NN	O	O
identified	NN	O	O
more	NN	O	O
than	NN	O	O
five	NN	O	O
years	NN	O	O
ago	NN	O	O
,	NN	O	O
the	NN	O	O
pathogenic	NN	O	O
mechanisms	NN	O	O
underlying	NN	O	O
this	NN	O	O
most	NN	O	O
prevalent	NN	O	O
form	NN	O	O
of	NN	O	O
hereditary	NN	O	O
adult	NN	O	O
neuromuscular	NN	O	B-Disease
disease	NN	O	I-Disease
remain	NN	O	O
elusive	NN	O	O
.	NN	O	O

Previous	NN	O	O
work	NN	O	O
from	NN	O	O
our	NN	O	O
laboratory	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
a	NN	O	O
DNase	NN	O	O
l	NN	O	O
-	NN	O	O
hypersensitive	NN	O	O
site	NN	O	O
located	NN	O	O
adjacent	NN	O	O
to	NN	O	O
the	NN	O	O
repeats	NN	O	O
on	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
allele	NN	O	O
is	NN	O	O
eliminated	NN	O	O
by	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
large	NN	O	O
CTG	NN	O	O
-	NN	O	O
repeat	NN	O	O
arrays	NN	O	O
may	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
local	NN	O	O
chromatin	NN	O	O
environment	NN	O	O
that	NN	O	O
represses	NN	O	O
gene	NN	O	O
expression	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
report	NN	O	O
that	NN	O	O
the	NN	O	O
hypersensitive	NN	O	O
site	NN	O	O
contains	NN	O	O
an	NN	O	O
enhancer	NN	O	O
element	NN	O	O
that	NN	O	O
regulates	NN	O	O
transcription	NN	O	O
of	NN	O	O
the	NN	O	O
adjacent	NN	O	O
DMAHP	NN	O	O
homeobox	NN	O	O
gene	NN	O	O
.	NN	O	O

Analysis	NN	O	O
of	NN	O	O
DMAHP	NN	O	O
expression	NN	O	O
in	NN	O	O
the	NN	O	O
cells	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
patients	NN	O	O
with	NN	O	O
loss	NN	O	O
of	NN	O	O
the	NN	O	O
hypersensitive	NN	O	O
site	NN	O	O
revealed	NN	O	O
a	NN	O	O
two	NN	O	O
-	NN	O	O
to	NN	O	O
fourfold	NN	O	O
reduction	NN	O	O
in	NN	O	O
steady	NN	O	O
-	NN	O	O
state	NN	O	O
DMAHP	NN	O	O
transcript	NN	O	O
levels	NN	O	O
relative	NN	O	O
to	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
controls	NN	O	O
.	NN	O	O

Allele	NN	O	O
-	NN	O	O
specific	NN	O	O
analysis	NN	O	O
of	NN	O	O
DMAHP	NN	O	O
expression	NN	O	O
showed	NN	O	O
that	NN	O	O
steady	NN	O	O
-	NN	O	O
state	NN	O	O
transcript	NN	O	O
levels	NN	O	O
from	NN	O	O
the	NN	O	O
expanded	NN	O	O
allele	NN	O	O
were	NN	O	O
greatly	NN	O	O
reduced	NN	O	O
in	NN	O	O
comparison	NN	O	O
to	NN	O	O
those	NN	O	O
from	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
allele	NN	O	O
.	NN	O	O

Together	NN	O	O
,	NN	O	O
these	NN	O	O
results	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
CTG	NN	O	O
-	NN	O	O
repeat	NN	O	O
expansions	NN	O	O
can	NN	O	O
suppress	NN	O	O
local	NN	O	O
gene	NN	O	O
expression	NN	O	O
and	NN	O	O
implicate	NN	O	O
DMAHP	NN	O	O
in	NN	O	O
DM	NN	O	B-Disease
pathogenesis	NN	O	O
.	NN	O	O

Constitutively	NN	O	O
methylated	NN	O	O
CpG	NN	O	O
dinucleotides	NN	O	O
as	NN	O	O
mutation	NN	O	O
hot	NN	O	O
spots	NN	O	O
in	NN	O	O
the	NN	O	O
retinoblastoma	NN	O	B-Disease
gene	NN	O	O
(	NN	O	O
RB1	NN	O	O
)	NN	O	O
.	NN	O	O

A	NN	O	O
wide	NN	O	O
spectrum	NN	O	O
of	NN	O	O
mutations	NN	O	O
,	NN	O	O
ranging	NN	O	O
from	NN	O	O
point	NN	O	O
mutations	NN	O	O
to	NN	O	O
large	NN	O	O
deletions	NN	O	O
,	NN	O	O
have	NN	O	O
been	NN	O	O
described	NN	O	O
in	NN	O	O
the	NN	O	O
retinoblastoma	NN	O	B-Disease
gene	NN	O	O
(	NN	O	O
RB1	NN	O	O
)	NN	O	O
.	NN	O	O

Mutations	NN	O	O
have	NN	O	O
been	NN	O	O
found	NN	O	O
throughout	NN	O	O
the	NN	O	O
gene	NN	O	O
;	NN	O	O
however	NN	O	O
,	NN	O	O
these	NN	O	O
genetic	NN	O	O
alterations	NN	O	O
do	NN	O	O
not	NN	O	O
appear	NN	O	O
to	NN	O	O
be	NN	O	O
homogeneously	NN	O	O
distributed	NN	O	O
.	NN	O	O

In	NN	O	O
particular	NN	O	O
,	NN	O	O
a	NN	O	O
significant	NN	O	O
proportion	NN	O	O
of	NN	O	O
disease	NN	O	O
-	NN	O	O
causing	NN	O	O
mutations	NN	O	O
results	NN	O	O
in	NN	O	O
the	NN	O	O
premature	NN	O	O
termination	NN	O	O
of	NN	O	O
protein	NN	O	O
synthesis	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
occur	NN	O	O
as	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
transitions	NN	O	O
at	NN	O	O
CpG	NN	O	O
dinucleotides	NN	O	O
(	NN	O	O
CpGs	NN	O	O
)	NN	O	O
.	NN	O	O

Such	NN	O	O
recurrent	NN	O	O
CpG	NN	O	O
mutations	NN	O	O
,	NN	O	O
including	NN	O	O
those	NN	O	O
found	NN	O	O
in	NN	O	O
RB1	NN	O	O
,	NN	O	O
are	NN	O	O
likely	NN	O	O
the	NN	O	O
result	NN	O	O
of	NN	O	O
the	NN	O	O
deamination	NN	O	O
of	NN	O	O
5	NN	O	O
-	NN	O	O
methylcytosine	NN	O	O
within	NN	O	O
these	NN	O	O
CpGs	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
,	NN	O	O
we	NN	O	O
used	NN	O	O
the	NN	O	O
sodiumbisulfite	NN	O	O
conversion	NN	O	O
method	NN	O	O
to	NN	O	O
detect	NN	O	O
cytosine	NN	O	O
methylation	NN	O	O
in	NN	O	O
representative	NN	O	O
exons	NN	O	O
of	NN	O	O
RB1	NN	O	O
.	NN	O	O

We	NN	O	O
analyzed	NN	O	O
DNA	NN	O	O
from	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
tissues	NN	O	O
and	NN	O	O
specifically	NN	O	O
targeted	NN	O	O
CGA	NN	O	O
codons	NN	O	O
in	NN	O	O
RB1	NN	O	O
,	NN	O	O
where	NN	O	O
recurrent	NN	O	O
premature	NN	O	O
termination	NN	O	O
mutations	NN	O	O
have	NN	O	O
been	NN	O	O
reported	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
that	NN	O	O
DNA	NN	O	O
methylation	NN	O	O
within	NN	O	O
RB1	NN	O	O
exons	NN	O	O
8	NN	O	O
,	NN	O	O
14	NN	O	O
,	NN	O	O
25	NN	O	O
,	NN	O	O
and	NN	O	O
27	NN	O	O
appeared	NN	O	O
to	NN	O	O
be	NN	O	O
restricted	NN	O	O
to	NN	O	O
CpGs	NN	O	O
,	NN	O	O
including	NN	O	O
six	NN	O	O
CGA	NN	O	O
codons	NN	O	O
.	NN	O	O

Other	NN	O	O
codons	NN	O	O
containing	NN	O	O
methylated	NN	O	O
cytosines	NN	O	O
have	NN	O	O
not	NN	O	O
been	NN	O	O
reported	NN	O	O
to	NN	O	O
be	NN	O	O
mutated	NN	O	O
.	NN	O	O

Therefore	NN	O	O
,	NN	O	O
disease	NN	O	O
-	NN	O	O
causing	NN	O	O
mutations	NN	O	O
at	NN	O	O
CpGs	NN	O	O
in	NN	O	O
RB1	NN	O	O
appear	NN	O	O
to	NN	O	O
be	NN	O	O
determined	NN	O	O
by	NN	O	O
several	NN	O	O
factors	NN	O	O
,	NN	O	O
including	NN	O	O
the	NN	O	O
constitutive	NN	O	O
presence	NN	O	O
of	NN	O	O
DNA	NN	O	O
methylation	NN	O	O
at	NN	O	O
cytosines	NN	O	O
within	NN	O	O
CpGs	NN	O	O
,	NN	O	O
the	NN	O	O
specific	NN	O	O
codon	NN	O	O
within	NN	O	O
which	NN	O	O
the	NN	O	O
methylated	NN	O	O
cytosine	NN	O	O
is	NN	O	O
located	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
particular	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
within	NN	O	O
which	NN	O	O
that	NN	O	O
codon	NN	O	O
resides	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
tumor	NN	O	I-Disease
suppressor	NN	O	O
gene	NN	O	O
product	NN	O	O
interacts	NN	O	O
with	NN	O	O
Sp1	NN	O	O
to	NN	O	O
repress	NN	O	O
vascular	NN	O	O
endothelial	NN	O	O
growth	NN	O	O
factor	NN	O	O
promoter	NN	O	O
activity	NN	O	O
.	NN	O	O

The	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
tumor	NN	O	I-Disease
suppressor	NN	O	O
gene	NN	O	O
(	NN	O	O
VHL	NN	O	O
)	NN	O	O
has	NN	O	O
a	NN	O	O
critical	NN	O	O
role	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
clear	NN	O	B-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
(	NN	O	O
RCC	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
as	NN	O	O
VHL	NN	O	O
mutations	NN	O	O
have	NN	O	O
been	NN	O	O
found	NN	O	O
in	NN	O	O
both	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
disease	NN	O	I-Disease
-	NN	O	I-Disease
associated	NN	O	I-Disease
and	NN	O	I-Disease
sporadic	NN	O	I-Disease
RCCs	NN	O	I-Disease
.	NN	O	O

Recent	NN	O	O
studies	NN	O	O
suggest	NN	O	O
that	NN	O	O
vascular	NN	O	O
endothelial	NN	O	O
growth	NN	O	O
factor	NN	O	O
(	NN	O	O
VEGF	NN	O	O
)	NN	O	O
mRNA	NN	O	O
is	NN	O	O
upregulated	NN	O	O
in	NN	O	O
RCC	NN	O	B-Disease
-	NN	O	I-Disease
and	NN	O	I-Disease
von	NN	O	I-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
disease	NN	O	I-Disease
-	NN	O	I-Disease
associated	NN	O	I-Disease
tumors	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
therefore	NN	O	O
assessed	NN	O	O
the	NN	O	O
effect	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	O
gene	NN	O	O
product	NN	O	O
on	NN	O	O
VEGF	NN	O	O
expression	NN	O	O
.	NN	O	O

VEGF	NN	O	O
promoter	NN	O	O
-	NN	O	O
luciferase	NN	O	O
constructs	NN	O	O
were	NN	O	O
transiently	NN	O	O
cotransfected	NN	O	O
with	NN	O	O
a	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	O
(	NN	O	O
wt	NN	O	O
-	NN	O	O
VHL	NN	O	O
)	NN	O	O
vector	NN	O	O
in	NN	O	O
several	NN	O	O
cell	NN	O	O
lines	NN	O	O
,	NN	O	O
including	NN	O	O
293	NN	O	O
embryonic	NN	O	O
kidney	NN	O	O
and	NN	O	O
RCC	NN	O	B-Disease
cell	NN	O	O
lines	NN	O	O
.	NN	O	O

wt	NN	O	O
-	NN	O	O
VHL	NN	O	O
protein	NN	O	O
inhibited	NN	O	O
VEGF	NN	O	O
promoter	NN	O	O
activity	NN	O	O
in	NN	O	O
a	NN	O	O
dose	NN	O	O
-	NN	O	O
dependent	NN	O	O
manner	NN	O	O
up	NN	O	O
to	NN	O	O
5	NN	O	O
-	NN	O	O
to	NN	O	O
10	NN	O	O
-	NN	O	O
fold	NN	O	O
.	NN	O	O

Deletion	NN	O	O
analysis	NN	O	O
defined	NN	O	O
a	NN	O	O
144	NN	O	O
-	NN	O	O
bp	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
VEGF	NN	O	O
promoter	NN	O	O
necessary	NN	O	O
for	NN	O	O
VHL	NN	O	O
repression	NN	O	O
.	NN	O	O

This	NN	O	O
VHL	NN	O	O
-	NN	O	O
responsive	NN	O	O
element	NN	O	O
is	NN	O	O
GC	NN	O	O
rich	NN	O	O
and	NN	O	O
specifically	NN	O	O
binds	NN	O	O
the	NN	O	O
transcription	NN	O	O
factor	NN	O	O
Sp1	NN	O	O
in	NN	O	O
crude	NN	O	O
nuclear	NN	O	O
extracts	NN	O	O
.	NN	O	O

In	NN	O	O
Drosophila	NN	O	O
cells	NN	O	O
,	NN	O	O
cotransfected	NN	O	O
VHL	NN	O	O
represses	NN	O	O
Sp1	NN	O	O
-	NN	O	O
mediated	NN	O	O
activation	NN	O	O
but	NN	O	O
not	NN	O	O
basal	NN	O	O
activity	NN	O	O
of	NN	O	O
the	NN	O	O
VEGF	NN	O	O
promoter	NN	O	O
.	NN	O	O

We	NN	O	O
next	NN	O	O
demonstrated	NN	O	O
in	NN	O	O
coimmunoprecipitates	NN	O	O
that	NN	O	O
VHL	NN	O	O
and	NN	O	O
Sp1	NN	O	O
were	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
same	NN	O	O
complex	NN	O	O
and	NN	O	O
,	NN	O	O
by	NN	O	O
using	NN	O	O
a	NN	O	O
glutathione	NN	O	O
-	NN	O	O
S	NN	O	O
-	NN	O	O
transferase	NN	O	O
-	NN	O	O
VHL	NN	O	O
fusion	NN	O	O
protein	NN	O	O
and	NN	O	O
purified	NN	O	O
Sp1	NN	O	O
,	NN	O	O
that	NN	O	O
VHL	NN	O	O
and	NN	O	O
Sp1	NN	O	O
directly	NN	O	O
interact	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
endogenous	NN	O	O
VEGF	NN	O	O
mRNA	NN	O	O
levels	NN	O	O
were	NN	O	O
suppressed	NN	O	O
in	NN	O	O
permanent	NN	O	O
RCC	NN	O	B-Disease
cell	NN	O	O
lines	NN	O	O
expressing	NN	O	O
wt	NN	O	O
-	NN	O	O
VHL	NN	O	O
,	NN	O	O
and	NN	O	O
nuclear	NN	O	O
run	NN	O	O
-	NN	O	O
on	NN	O	O
studies	NN	O	O
indicated	NN	O	O
that	NN	O	O
VHL	NN	O	O
regulation	NN	O	O
of	NN	O	O
VEGF	NN	O	O
occurs	NN	O	O
at	NN	O	O
least	NN	O	O
partly	NN	O	O
at	NN	O	O
the	NN	O	O
transcriptional	NN	O	O
level	NN	O	O
.	NN	O	O

These	NN	O	O
observations	NN	O	O
support	NN	O	O
a	NN	O	O
new	NN	O	O
mechanism	NN	O	O
for	NN	O	O
VHL	NN	O	O
-	NN	O	O
mediated	NN	O	O
transcriptional	NN	O	O
repression	NN	O	O
via	NN	O	O
a	NN	O	O
direct	NN	O	O
inhibitory	NN	O	O
action	NN	O	O
on	NN	O	O
Sp1	NN	O	O
and	NN	O	O
suggest	NN	O	O
that	NN	O	O
loss	NN	O	O
of	NN	O	O
Sp1	NN	O	O
inhibition	NN	O	O
may	NN	O	O
be	NN	O	O
important	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
disease	NN	O	I-Disease
and	NN	O	O
RCC	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Adult	NN	O	B-Disease
onset	NN	O	I-Disease
globoid	NN	O	I-Disease
cell	NN	O	I-Disease
leukodystrophy	NN	O	I-Disease
(	NN	O	O
Krabbe	NN	O	B-Disease
disease	NN	O	I-Disease
)	NN	O	O
:	NN	O	O
analysis	NN	O	O
of	NN	O	O
galactosylceramidase	NN	O	O
cDNA	NN	O	O
from	NN	O	O
four	NN	O	O
Japanese	NN	O	O
patients	NN	O	O
.	NN	O	O

We	NN	O	O
examined	NN	O	O
galactosylceramidase	NN	O	O
(	NN	O	O
GALC	NN	O	O
)	NN	O	O
cDNA	NN	O	O
in	NN	O	O
four	NN	O	O
Japanese	NN	O	O
patients	NN	O	O
with	NN	O	O
adult	NN	O	B-Disease
onset	NN	O	I-Disease
globoid	NN	O	I-Disease
cell	NN	O	I-Disease
leukodystrophy	NN	O	I-Disease
(	NN	O	O
Krabbe	NN	O	B-Disease
disease	NN	O	I-Disease
;	NN	O	O
AO	NN	O	B-Disease
-	NN	O	I-Disease
GLD	NN	O	I-Disease
)	NN	O	O
by	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
/	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
(	NN	O	O
PCR	NN	O	O
-	NN	O	O
SSCP	NN	O	O
)	NN	O	O
analysis	NN	O	O
,	NN	O	O
subsequent	NN	O	O
sequence	NN	O	O
determination	NN	O	O
,	NN	O	O
and	NN	O	O
restriction	NN	O	O
enzyme	NN	O	O
digestion	NN	O	O
of	NN	O	O
PCR	NN	O	O
products	NN	O	O
,	NN	O	O
initial	NN	O	O
symptoms	NN	O	O
were	NN	O	O
the	NN	O	O
onset	NN	O	O
of	NN	O	O
slowly	NN	O	O
progressive	NN	O	O
spastic	NN	O	B-Disease
paraplegia	NN	O	I-Disease
from	NN	O	O
the	NN	O	O
middle	NN	O	O
of	NN	O	O
the	NN	O	O
second	NN	O	O
decade	NN	O	O
,	NN	O	O
and	NN	O	O
all	NN	O	O
patients	NN	O	O
had	NN	O	O
diminished	NN	O	B-Disease
GALC	NN	O	I-Disease
activity	NN	O	I-Disease
in	NN	O	O
their	NN	O	O
leukocytes	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
three	NN	O	O
missense	NN	O	O
mutations	NN	O	O
(	NN	O	O
I66M	NN	O	O
,	NN	O	O
G270D	NN	O	O
,	NN	O	O
L618S	NN	O	O
)	NN	O	O
and	NN	O	O
one	NN	O	O
exon	NN	O	O
-	NN	O	O
6	NN	O	O
skipping	NN	O	O
(	NN	O	O
535	NN	O	O
-	NN	O	O
573del	NN	O	O
)	NN	O	O
.	NN	O	O

Two	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
had	NN	O	O
only	NN	O	O
the	NN	O	O
I66M	NN	O	O
mutant	NN	O	O
mRNA	NN	O	O
,	NN	O	O
and	NN	O	O
one	NN	O	O
only	NN	O	O
the	NN	O	O
G27OD	NN	O	O
mutant	NN	O	O
mRNA	NN	O	O
.	NN	O	O

The	NN	O	O
fourth	NN	O	O
patient	NN	O	O
carried	NN	O	O
a	NN	O	O
compound	NN	O	O
heterozygous	NN	O	O
mutation	NN	O	O
of	NN	O	O
535	NN	O	O
-	NN	O	O
573del	NN	O	O
and	NN	O	O
L618S	NN	O	O
.	NN	O	O

To	NN	O	O
determine	NN	O	O
the	NN	O	O
enzymatic	NN	O	O
activities	NN	O	O
produced	NN	O	O
by	NN	O	O
these	NN	O	O
mutations	NN	O	O
,	NN	O	O
we	NN	O	O
constructed	NN	O	O
mutated	NN	O	O
GALC	NN	O	O
cDNAs	NN	O	O
and	NN	O	O
expressed	NN	O	O
them	NN	O	O
in	NN	O	O
COS	NN	O	O
-	NN	O	O
1	NN	O	O
cells	NN	O	O
.	NN	O	O

Three	NN	O	O
mutations	NN	O	O
,	NN	O	O
viz	NN	O	O
.	NN	O	O
,	NN	O	O
G270D	NN	O	O
,	NN	O	O
L618S	NN	O	O
,	NN	O	O
and	NN	O	O
exon	NN	O	O
-	NN	O	O
6	NN	O	O
skipping	NN	O	O
(	NN	O	O
535	NN	O	O
-	NN	O	O
573del	NN	O	O
)	NN	O	O
,	NN	O	O
produced	NN	O	O
diminished	NN	O	B-Disease
GALC	NN	O	I-Disease
activity	NN	O	I-Disease
as	NN	O	O
expected	NN	O	O
.	NN	O	O

The	NN	O	O
I66M	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
GALC	NN	O	O
cDNA	NN	O	O
(	NN	O	O
I289	NN	O	O
)	NN	O	O
had	NN	O	O
normal	NN	O	O
activity	NN	O	O
,	NN	O	O
but	NN	O	O
when	NN	O	O
this	NN	O	O
mutation	NN	O	O
and	NN	O	O
the	NN	O	O
V289	NN	O	O
polymorphism	NN	O	O
were	NN	O	O
introduced	NN	O	O
into	NN	O	O
the	NN	O	O
same	NN	O	O
allele	NN	O	O
,	NN	O	O
it	NN	O	O
had	NN	O	O
decreased	NN	O	O
activity	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
the	NN	O	O
combination	NN	O	O
of	NN	O	O
a	NN	O	O
unique	NN	O	O
mutation	NN	O	O
and	NN	O	O
polymorphism	NN	O	O
causes	NN	O	O
conformational	NN	O	O
change	NN	O	O
in	NN	O	O
the	NN	O	O
GALC	NN	O	O
enzyme	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
low	NN	O	O
enzymatic	NN	O	O
activity	NN	O	O
.	NN	O	O

AO	NN	O	B-Disease
-	NN	O	I-Disease
GLD	NN	O	I-Disease
mutations	NN	O	O
,	NN	O	O
including	NN	O	O
those	NN	O	O
found	NN	O	O
here	NN	O	O
,	NN	O	O
are	NN	O	O
located	NN	O	O
in	NN	O	O
the	NN	O	O
N	NN	O	O
-	NN	O	O
terminus	NN	O	O
(	NN	O	O
I66M	NN	O	O
,	NN	O	O
G270D	NN	O	O
,	NN	O	O
535	NN	O	O
-	NN	O	O
573del	NN	O	O
)	NN	O	O
or	NN	O	O
C	NN	O	O
-	NN	O	O
terminus	NN	O	O
(	NN	O	O
L618S	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
GALC	NN	O	O
enzyme	NN	O	O
,	NN	O	O
whereas	NN	O	O
the	NN	O	O
reported	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
infantile	NN	O	O
form	NN	O	O
(	NN	O	O
IF	NN	O	B-Disease
-	NN	O	I-Disease
GLD	NN	O	I-Disease
)	NN	O	O
are	NN	O	O
in	NN	O	O
the	NN	O	O
central	NN	O	O
domain	NN	O	O
.	NN	O	O

This	NN	O	O
difference	NN	O	O
in	NN	O	O
mutation	NN	O	O
sites	NN	O	O
may	NN	O	O
affect	NN	O	O
the	NN	O	O
clinical	NN	O	O
features	NN	O	O
of	NN	O	O
GLD	NN	O	B-Disease
.	NN	O	O

