PAX genes and human neural tube defects: An amino acid substitution in PAX1 in a patient with spina bifida

Abstract

From studies in the mouse and from the clinical and molecular analysis of patients with type 1 Waardenburg syndrome, particular members of the PAX gene family are suspected factors in the aetiology of human neural tube defects (NTD). To investigate the role of PAX1, PAX3, PAX7, and PAX9, allelic association studies were performed in 79 sporadic and 38 familial NTD patients from the Dutch population. Sequence variation was studied by SSC analysis of the paired domain regions of the PAX1, PAX7, and PAX9 genes and of the complete PAX3 gene. In one patient with spina bifida, a mutation in the PAX1 gene was detected changing the conserved amino acid Gln to His at position 42 in the paired domain of the protein. The mutation was inherited through the maternal line from the unaffected grandmother and was not detected in 300 controls. In the PAX3 gene, variation was detected at several sites including a Thr/Lys amino acid substitution in exon 6. All alleles were present among patients and controls in about the same frequencies. However, an increased frequency of the rare allele of a silent polymorphism in exon 2 was found in NTD patients, but no significant association was observed (p = 0.06). No sequence variation was observed in the paired domain of the PAX7 and PAX9 genes. Our findings so far do not support a major role of the PAX genes examined in the aetiology of NTD. However, the detection of a mutation in PAX1 suggests that, in principle, this gene can act as a risk factor for human NTD.

Full text

JMed
Genet
1996;33:655-660
PAX
genes
and
human
neural
tube
defects:
an
amino
acid
substitution
in
PAX1
in
a
patient
with
spina
bifida
F
A
Hol,
M
P
A
Geurds,
S
Chatkupt,
Y Y
Shugart,
R
Balling,
C
T R
M
Schrander-Stumpel,
W
G
Johnson,
B
C
J
Hamel,
E
C
M
Mariman
Department
of
Human
Genetics,
University
Hospital
Nijmegen,
PO
Box
9101,
6500
HB
Nijmegen,
The
Netherlands
F
A
Hol
M
P
A
Geurds
B
C
J
Hamel
E
C
M
Mariman
Department
of
Neurosciences
and
Pediatrics,
UMDNJ-New
Jersey
Medical
School,
Newark,
New
Jersey,
USA
S
Chatkupt
Department
of
Genetics
and
Development,
Colombia
University,
New
York,
NY,
USA
Y
Y
Shugart
Institut
fuir
Saugetiergenetik,
GSF-Forschungszentrum
Neuherberg,
Oberschleissheim,
Germany
R
Balling
Department
of
Clinical
Genetics,
University
Hospital
Maastricht,
Maastricht,
The
Netherlands
CTRM
Schrander-Stumpel
Department
of
Neurology,
UMDNJ-Robert
Wood
Johnson
Medical
School,
New
Brunswick,
New
Jersey,
USA
W
G
Johnson
Correspondence
to:
Dr
Hol.
Received
27
October
1995
Revised
version
accepted
for
publication
22
March
1996
Abstract
From
studies
in
the
mouse
and
from
the
clinical
and
molecular
analysis
of
patients
with
type
1
Waardenburg
syndrome,
par-
ticular
members
of
the
PAX
gene
family
are
suspected
factors
in
the
aetiology
of
human
neural
tube
defects
(NTD).
To
investigate
the
role
of
PAXI,
PAX3, PAX7,
and
PAX9,
allelic
association
studies
were
performed
in
79
sporadic
and
38
familial
NTD
patients
from
the
Dutch
population.
Sequence
variation
was
studied
by
SSC
analysis
of
the
paired
domain
regions
of
the
PAXI,
PAX7,
and
PAX9
genes
and
of
the
complete
PAX3
gene.
In
one
patient
with
spina
bifida,
a
mutation
in
the
PAXI
gene
was
detected
changing
the
conserved
amino
acid
Gln
to
His
at
position
42
in
the
paired
domain
of
the
protein.
The
muta-
tion
was
inherited
through
the
maternal
line
from
the
unaffected
grandmother
and
was
not
detected
in
300
controls.
In
the
PAX3
gene,
variation
was
detected
at
sev-
eral
sites
including
a
Thr/Lys
amino
acid
substitution
in
exon
6.
All
alleles
were
present
among
patients
and
controls
in
about
the
same
frequencies.
However,
an
increased
frequency
of
the
rare
allele
of
a
silent
polymorphism
in
exon
2
was
found
in
NTD
patients,
but
no
significant
associ-
ation
was
observed
(p=0.06).
No
sequence
variation
was
observed
in
the
paired
domain
of
the
PAX7
and
PAX9
genes.
Our
findings
so
far
do
not
support
a
major
role
of
the
PAX
genes
examined
in
the
aetiology
of
NTD.
However,
the
detection
of
a
mutation
in
PAXI
suggests
that,
in
principle,
this
gene
can
act
as
a
risk
factor
for
human
NTD.
(JMed
Genet
1996;33:655-660)
Key
words:
neural
tube
defects;
spina
bifida;
PAX
genes.
Neural
tube
defects
(NTD)
constitute
a
major
group
of
congenital
malformations
with
an
incidence
of
approximately
1/1000
pregnan-
cies.
It
is
generally
accepted
that
they
represent
multifactorial
traits
with
genetic
and
environ-
mental
factors
contributing
to
the
aetiology.
Information
about
the
identity
of
the
genetic
factors
involved
is
scarce.
Induction
of
NTD
by
anti-epileptic
drugs'
and
prevention
of
NTD
by
folic
acid2
suggest
that
genes
for
drug
receptors
or
metabolic
enzymes
may
play
a
role.
Pedigree
analysis
and
linkage
data
suggest
that
the
human
X
chromosome
contains
one
or
more
contributing
genetic
factors.3
More
specifically,
several
members
of
the
PAX
gene
family,
encoding
a
class
of
related
embryonic
transcription
factors,4
have
been
proposed
as
candidate
genes
for
NTD.5
6
At
present
nine
members
have
been
identified
and
studies
in
the
mouse
have
shown
that
they
are
all
expressed
in
the
developing
brain,
the
neural
tube,
or
the
paraxial
mesoderm.
Mutations
in
the
gene
for
Pax-1
are
respon-
sible
for
the
phenotype
of
the
mouse
strain
undulated
with
various
vertebral
anomalies.7
8
Interestingly,
litters
resulting
from
a
cross
between
undulated
(un)
and
Patch
(Ph)
mice
have
a
high
incidence
of
lumbar
spina
bifida
occulta'
indicating
that
Pax-1
can
be
involved
in
specific
forms
of
NTD.
From
the
association
between
spina
bifida
and
Waardenburg
syndrome
type
1
(WS1),9-6
the
PAX3
gene
can
be
regarded
as
a
risk
factor
for
human
NTD.
Mutations
in
this
gene
have
been
detected
in
various
patients
with
both
spina
bifida
and
WS
1.17-1
Moreover,
mutations
in
the
murine
homologue
of
this
gene
cause
the
Splotch
phenotype.
Homozygous
Splotch
mice
exhibit
severe
NTD6
whereas
heterozygous
Pax-3
mutations
seem
to
increase
the
inci-
dence
of
NTD
in
other
predisposed
strains.20
Linkage
analysis
has
not
provided
genetic
evidence
for
a
major
role
of
PAX3
in
human
NTD.2'
The
above
findings
prompted
us
to
investi-
gate
further
the
role
of
PAX1
and
PAX3,
and
their
paralogues
PAX9
and
PAX7,
respectively,
by
searching
for
mutations
and
sequence
vari-
ants
in
association
with
non-syndromic
NTD.
Materials
and
methods
ASCERTAINMENT
OF
PATIENTS
Patients
with
non-syndromic
NTD
were
se-
lected
from
the
Dutch
population
in
collabora-
tion
with
the
Dutch
patient
organisation
BOSK
and
from
the
records
of
our
institute.
Multiple
case
families
were
selected
according
to
the
criteria
described
previously.22
In
short,
there
had
to
be
two
or
more
affected
members
in
each
family
with
a
close
degree
of
relation-
ship
(s<3).
In
this
way
38
multiple
case
families
were
selected.
One
affected
member
of
each
of
these
families
was
included
in
the
present
study.
The
types
of
NTD
in
this
group
were:
spina
bifida
(36),
encephalocele
(1),
and
craniorachischisis
(1).
In
addition,
a
group
of
655
group.bmj.com on July 14, 2011 - Published by jmg.bmj.comDownloaded from

Hol
et
al
1
2
3
4
5
6
7
8
PAX3
;
_
' . .
I
. . . .
4
.
2
3
4
PAX7
I
.-
.
PAX1
r~~
*
4
i=
PAX9
Figure1
Schematic
representation
of
parts
of
the
different
PAXgenes
that
were
subjected
to
SSC
analysis.
Arrowheads
with
connecting
bars
represent
the
amplification
primers
and
amplifiedfragments.
The
filled
region,
the
hatched
region,
and
the
double
hatched
region
represent
the
paired
domain,
the
octapeptide
sequence,
and
the
homeodomain,
respectively.
79
sporadic
patients
was
also
analysed,
which
included
patients
with
spina
bifida
(75),
with
anencephaly
(2),
and
with
encephalocele
(2).
Unaffected
and
unrelated
subjects
were
ran-
domly
chosen
from
the
Dutch
population
and
used
as
a
control
group
in
the
present
study.
Blood
was
sampled
and
DNA
was
extracted
according
to
the
procedure
of
Miller
et
al.23
SSC
ANALYSIS
PCR
amplification
was
performed
to
produce
the
appropriate
DNA
fragments
(fig
1).
Ampli-
fication
was
carried
out
in
a
total
volume
of
25
il
containing
50
ng
of
genomic
DNA,
0.45
mmol/l
of
each
primer
(table
1;
Isogen
Bioscience,
The
Netherlands),
0.1
mmol/l
dCTP,
0.4
mmol/l
dATP,
0.4
mmol/l
dGTP,
0.4
mmol/l
dTTP,
0.1
j1l
[a[32]P]dCTP
(Amersham)
in
PCR
buffer
(50
mmol/l
KC1,
10
mmol/l
TRIS-HCG,
pH
8.3,
1
mmol/l
DTE,
0.001%
gelatine,
1.5
-
6
mmol/l
MgCl2)
with
0.5
U
of
Taq
DNA
polymerase
(Boehringer,
Mannheim).
Samples
were
denatured
at
92°C
for
five
minutes
and
then
subjected
to
35
cycles
of
amplification:
92°C
for
50
seconds,
55°C
for
50
seconds,
72°C
for
one
minute
30
seconds.
Aliquots
of
the
amplified.
DNA
were
mixed
with
1
volume
formamide
dye
buffer,
dena-
tured
at
95°C
for
five
minutes,
and
placed
on
ice.
Samples
(4
,l)
were
loaded
on
a
5%
non-denaturing
polyacrylamide
gel
containing
10%
glycerol
and
on
a
similar
gel
without
glyc-
erol.
Electrophoresis
was
for
3-6
hours
at
40
W
and
4°C.
The
gels
were
dried
and
exposed
overnight
on
Kodak
X-omat
S
film.
SEQUENCING
OF
NORMAL
AND
VARIANT
ALLELES
To
determine
the
molecular
nature
of
the
shifted
bands
observed
by
SSC
analysis,
bands
representing
wild
type
and
variant
alleles
were
cut
out
of
the
gel.
DNA
was
eluted
from
each
of
the
gel
slices
in
50
gl
aquadest
for
one
hour
at
37°C
and
reamplified
under
the
conditions
described
above.
Subsequently,
the
amplified
DNA
fragments
were
purified
by
electrophore-
sis
on
a
1
%
agarose
gel
(one
hour,
10
V/cm),
Table
1
Primers
used
for
SSC
analysis'8
28-31
Size
(bp)
Forward
primer
Reverse
primer
PAX3
1
138
5'-CCTGGATATAATTTCCGAGCG-3'
5'-CGCTGAGGCCCTCCCTTA-3'
2A
266
5'-GAAGACTGCGAAATTACGTGCTGC-3'
5'-ACAGGATCTTGGAGACGCAGCC-3'
2B
208
5'-AACCACATCCGCCACAAGATCG-3'
5'-GACCACAGTCTGGGAGCCAGGAGG-3'
3
237
5'-CACCTGGCCCAGGGTACCGGGTAC-3'
5'-CGGGGTAATAGCGACTGACTGTC-3'
4
252
5'-AGCCCTGCTTGTCTCAACCATGTG-3'
5'-TGCCCTCCAAGTCACCCAGCAAGT-3'
5
304
5'-GACTTGGATCAATCTCAGT1--13'
5'-TAGGACACGGAGGTlTTGG-3'
6
250
5'-TTCATCAGTGAAATCCTTAAA1TT-3'
5'-CGCCTGGAAGTTACTTTCTA-3'
7
320
5'-GAACTTTCTCTGCTGGCCTA-3'
5'-TGGTTCTGGTATACAGCAAATC-3'
8
313
5'-GCTCGl-F-l--l-l-lAGGTAATGGGA-3'
5'-TGAGTTTATCTCCCTTCCAGG-3'
PAX7
2A
188
5'-TCCATCCTCACCCTGCACCT-3'
5'-CGGGAGATGACACAGGGCCG-3'
2B
214
5'-GCGACCCCTGCCTAACCACA-3'
5'-AAGCCAGCTGCCAGCCTCTGTG-3'
3
200
5'-CCCCATCCCATCTTTCCACTC-3'
5'-TGCCGGCTCAGCTGCC-TTCTCA-3'
4
189
5'-TTGCTCTTTGGCCTTTGAATTTCT-3'
5'-GCCTCGCAGCCCAGGGAA-3'
PAXI
1
194
5'-TCCGGCTCACTCTTGTCTGG-3'
5'-ATCTTGCTCACGCAGCCGTG-3'
2
201
5'-ACGCCATCCGCTTGCGCA1TT-3'
5'-AGTCCCGGATGTGCTTGACC-3'
3
200
5'-CCTGGCGCGCTACAACGAGA-3'
5'-TGGAGCTCACCGAAGGCACA-3'
4
200
5'-CTGGCATCTTTGCCTGGGAG-3'
5'-AGGGGTACTGGTAGATGTGG-3'
PAX9
1
200
5'-ATTTTGCAGAGCCAGCCTTC-3'
5'-CGAGCCCGTCTCGTTGTATC-3'
2
188
5'-GGCCCAACTGGGCATCCGAC-3'
5'-GGTCTCTCTGCTTGTAGGTC-3'
3
202
5'-ATCTTGCCAGGAGCCATCGG-3'
5'-TGCCGATCTTGTTGCGCAGAAT-3'
4
200
5'-ATCTTCGCCTGGGAGATCCGG-3'
5'-GGGTACGAGTAGATGTGGTTGT-3'
5
222
5'-ATTACGACTCATACAAGCAGCACC-3'
5'-CCCTACCTTGGTCGGTGATGG-3'
656
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PAX
genes
and
human
NTD
allowed
to
migrate
into
ultra
low
gelling
temperature
agarose
(Sigma),
and
sliced
out
of
the
gel.
This
material
served
as
substrate
for
direct
sequencing
using
a
cycle
sequence
kit
according
to
the
protocol
of
the
manufacturer
(BRL).
Sequences
were
determined
in
two
directions
with
the
forward
and
reverse
ampli-
fication
primers
after
5'
end
labelling
with
[32]
P.
Whenever
the
resolution
was
too
poor
to
cut
the
allelic
bands
out
of
the
SSC
gel
separately,
non-radioactive
PCR
was
per-
formed
on
genomic
DNA.
Amplification
prod-
ucts
were
purified
on
an
agarose
gel
and
cloned
into
the
SmaI
site
of
plasmid
vector
Bluescript
SK+
(Stratagene).
Dideoxy
DNA
sequencing
was
conducted
on
double
stranded
DNA
from
positive
clones
using
the
T7
sequencing
kit
(Pharmacia)
with
T7
and
T3
primers.
Se-
quences
were
obtained
from
at
least
three
independent
clones.
Results
SSC
ANALYSIS
OF
THE
PAXI
AND
PAX9
GENES
Double
mutant
mice
with
the
genotype
{un/
un;Ph/+}
exhibit
an
occult
form
of
spina
bifida5
indicating
that
mutations
in
Pax-1
can
influence
the
development
of
the
vertebral
arches.
In
order
to
assess
the
relevance
of
PAXI
for
human
NTD,
the
paired
domain
region,
which
is
the
only
part
of
the
gene
that
has
been
cloned
to
date,
was
subjected
to
SSC
analysis.
DNA
from
79
sporadic
and
38
famil-
ial
NTD
patients
from
the
Dutch
population
was
screened
for
the
presence
of
sequence
vari-
ation
(fig
1).
This
resulted
in
the
detection
of
a
shifted
band
in
a
single
sporadic
patient
with
spina
bifida
(fig
2A).
Direct
sequencing
of
the
shifted
fragment
showed
a
nucleotide
substitu-
tion
(G-*C,
fig
3A)
leading
to
the
exchange
of
glutamine
at
position
42
of
the
paired
domain
by
histidine.
This
nucleotide
substitution
disrupts
an
AluI
restriction
site
in
the
gene.
Therefore,
the
presence
of
the
base
change
could
be
confirmed
by
AluI
restriction
diges-
tion
of
the
PCR
product
leaving
the
DNA
from
this
patient
intact
(fig
4).
In
the
same
way
it
was
shown
that
the
base
change
was
absent
PAX1
A
P
C
C
B
P
C C
C
from
300
unaffected
controls
suggesting
that
this
alteration
is
aetiologically
related
to
the
NTD
of
this
person.
Moreover,
the
functional
importance
of
the
Gln
residue
at
this
position
is
corroborated
by
the
fact
that
it
has
been
con-
served
between
several
human
PAX
genes
as
well
as
the
murine
Pax-I
gene
(fig
5).
Finally,
the
predicted
structure
of
the
mutant
peptide
showed
the
loss
of
a
beta
sheet
conformation
surrounding
the
displaced
amino
acid
(fig
6).
Together,
these
results
argue
for
a
contribution
of
the
PAXI
mutation
to
the
development
of
spina
bifida
in
one
patient.
However,
the
same
mutation
was
also
found
in
the
unaffected
mother
and
grandmother
showing
that
this
factor
alone
is
not
sufficient
to
induce
a
NTD
during
embryogenesis.
In
addition,
we
have
examined
all
patients
for
sequence
variation
in
the
paired
domain
of
the
PAX9
gene
(fig
1),
which
is
highly
homolo-
gous
to
the
PAXl
gene.
No
band
shifts
were
detected.
SSC
ANALYSIS
OF
THE
PAX3
AND
PAx7
GENES
Although
linkage
studies
have
not
provided
any
indication
for
PAX3
being
a
major
aetiological
factor
for
familial
NTD,"
a
less
prominent
role
cannot
be
excluded
in
this
way,
in
particular
for
sporadic
cases.
Therefore,
a
similar
strategy
was
applied
as
performed
for
the
PAXl
gene,
that
is,
using
SSC
analysis
to
search
for
muta-
tions
or
rare
sequence
variants
primarily
occurring
among
patients.
The
complete
cod-
ing
sequence
of
the
PAX3
gene
contained
in
exons
1
to
8,
together
with
their
flanking
intron
sequences,
were
analysed
(fig
1).
The
most
dramatic
change
in
the
banding
patterns
was
observed
for
exon
5
in
a
familial
patient.
The
detailed
analysis
of
this
case,
which
turned
out
to
be
a
5
bp
deletion
causing
spina
bifida
and
WSl,
has
been
described
elsewhere.'9
Besides
the
normal
band
pattern,
shifted
bands
were
detected
for
exons
2,
6,
and
7
(fig
2).
The
nature
of
the
observed
shifts
was
determined
as
described
in
Materials
and
methods.
One
of
the
shifts
observed
for
the
exon
2
fragment
(fig
2B)
and
the
shift
observed
for
the
exon
7
frag-
-
PAX3
P
C
C
D
P
C
C
E
P
C
C
_
Paired
domain
exon
Exon
2
fragment
Exon
2
fragment
Exon
6
fragment
Exon
7
frag
inietit
Figure
2
Allelic
band
pattern
obtained
through
SSC
analysis
of
different
fragments
of
the
PAXI
gene
(A)
and
the
PAX3
gene
(B-E).
The
first
lane
shows
the
altered
pattern
(P)
which
was
occasionally
observed
in
patients
or
controls
or
both,
whereas
the
more
common
SSC
pattern
(C)
is
shown
in
lanes
2
and
3.
Arrowheads
mark
the
observed
shifted
allelic
bands.
657
)I
'to
-O'li-
';
group.bmj.com on July 14, 2011 - Published by jmg.bmj.comDownloaded from

Hol
et
al
Table
2
Frequencies
of
the
different
SSC
shifts
in
the
PAX3
gene
in
Dutch
patients
and
Table
3
Frequency
of
the
T
allele
chromosome
of
the
exon
controls
2
CITpolymorphism
in
Dutch
patients
and
controls
Patients
Controls
SSC
fragment
No
Frequency
No
Frequency
Exon
2
(5'
intron
polymorphism)
5
/
64
0.08
8
/
93
0.09
Exon
2
(silent
polymorphism)
42
/
113
0.37
60
/
229
0.26
Exon
6
(AA
polymorphism)
3
/
61
0.05
6
/
93
0.06
Exon
7
(3'
intron
polymorphism)
ND
-
ND
-
ment
(fig
2E)
were
shown
to
represent
single
nucleotide
substitutions
in
the
5'
and
3'
flanking
intronic
sequences
of
exon
2
(T->C,
fig
3B)
and
7
(G-*A,
fig
3E),
respectively.
Another
shift
of
the
same
fragment
of
exon
2
(fig
2C)
turned
out
to
be
the
result
of
a
single
nucleotide
substitution
(C-*T)
at
the
third
position
of
codon
43
representing
a
silent
base
change
(Gly43Gly,
fig
3C),
which
has
previ-
ously
been
reported
by
Tassabehji
et
al.24
In
the
3'
half
of
exon
6,
a
C-4A
change
was
identified
(fig
2D)
downstream
of
the
homeodomain
A
B
C
D
E
Exon
7
Intron
7
5
'-
CCTCTCACCTCAG
gtcagtcccgtgtttctagac
-3
'
Figure
3
Partial
DNA
and
protein
sequence
of
the
paired
domain
exon
of
PAXI
(A),
as
well
as
PAX3
exon
2
(B,
C),
exon
6
(D),
and
exon
7
(E).
The
nucleotide
substitutions
that
are
responsible
for
the
SSC
shifts
in
fig
2
are
shown.
When
the
nucleotide
substitution
gives
rise
to
an
amino
acid
substitution
in
the
deduced
peptide,
this
is
also
shown.
Patients
Controls
No
Frequency
No
Frequency
p
value
T
allele
46/226
0.20
67/458
0.15
0.06
resulting
in
an
amino
acid
substitution
(Thr315Lys,
fig
3D).
According
to
the
struc-
ture
prediction
analysis
(not
shown)
this
amino
acid
substitution
does
not
seem
to
be
influenc-
ing
the
protein
structure.
Genotyping
of
the
control
group
showed
that
all
the
sequence
variants
were
present
both
in
patients
and
con-
trols
with
approximately
equal
frequencies
in
both
groups
(table
2).
Interestingly,
the
T
allele
of
the
exon
2
silent
polymorphism
seemed
to
be
present
more
often
in
patients
than
in
con-
trols.
However,
a
thorough
analysis
of
the
data
(heterozygosity/homozygosity
scoring,
T
allele
frequency
determination)
applying
chi-square
statistics,
did
not
show
a
significant
association
between
the
T
allele
and
NTD
(p=0.06,
odds
ratio
=
1.49,
95%
confidence
interval
0.96-
2.30;
table
3).
Finally,
for
PAX7,
which
closely
resembles
PAX3
in
structure
and
expression,
the
same
groups
of
patients
and
controls
were
screened
for
the
presence
of
sequence
variation
in
the
paired
domain
(exons
2
to
4,
fig
1),
which
is
the
only
part
of
this
gene
cloned
so
far.
No
band
shifts
were
detected
in
the
SSC
analysis.
Discussion
PAX
genes
encode
a
class
of
highly
conserved
transcription
factors
with
a
characteristic
DNA
binding
paired
domain,
which
play
important
roles
in
embryonic
development.4
To
deter-
mine
more
accurately
the
extent
to
which
genetic
variation
in
the
PAX1
and
PAX3
genes,
and
their
paralogues
PAX9
and
PAX7,
might
predispose
to
NTD
we
have
performed
SSC
analysis
of
both
familial
and
sporadic
patients.
Analysis
of
the
paired
domain
of
PAXI
showed
a
missense
mutation
in
one
sporadic
NTD
patient.
The
NTD
was
detected
by
amniocen-
tesis
and
biochemical
analysis
of
amniotic
fluid,
indicated
by
the
fact
that
the
mother
was
a
known
carrier
of
a
balanced
translocation
t(7;20)(q22;ql3.2).
After
pregnancy
termina-
tion
at
19
weeks
of
gestation,
clinical
examin-
ation
showed
an
open
lumbar
spina
bifida
of
about
1.5
cm
in
size.
Uniparental
disomy
of
chromosome
7
or
20
in
the
fetus
was
excluded
by
the
analysis
of
genetic
markers.
Cytogenetic
analysis
showed
the
presence
of
the
same
balanced
translocation
in
the
fetus
and
the
maternal
grandmother.
Apparently,
the
muta-
tion
in
the
PAX1
gene,
which
is
located
at
20p1
l,
cosegregates
with
the
translocation
chromosomes.
Although
there
is
no
indication
for
a
major
pathological
effect
of
the
balanced
translocation,
an
influence
on
the
phenotype
of
the
fetus
cannot
be
excluded.
In
fact,
under
a
multifactorial
threshold
model
both
genetic
abnormalities,
that
is,
the
PAX1
mutation
and
the
translocation,
may
have
contributed
to
the
appearance
of
the
NTD.
C
5'-TGTGAGATCAGTCGGCA9CTCCGCGTATCCCAC-3'
CysAspIleSerArg
GjLeuArgValSerHis
t
His
C
I
5'-actgcgaaattacgtgctgctgttctttgctttt
tattttcctccagtgac
ttttcccttgcttctct
Intron
I
Exon
2
ttttcaccttcccacagTGTCCACTCCCCTCGGC-3'
Exon
2
T
I
5'-CAGGGCCGCGTCMACCAGCTCGGCGGCGTTTTTA-3'
GlnGlyArgValAsnGlnLeuGlyGlyValPhe
I
Gly
A
Exon
6
Intron
6
5'
-TACCAGCCCACATCTATTCCACMG
gtaccgagg
-3'
TyrGlnProThrSerIleProGln
Lys
658
group.bmj.com on July 14, 2011 - Published by jmg.bmj.comDownloaded from

659
PAX
genes
and
human
NTD
_
Mutant
allele
_
Wild
type
allele
Figure
4
Pedigree
of
the
family
in
which
the
PAXI
mutation
is
segregating.
Presence
of
the
mutation
could
be
confirmed
by
AluI
restriction
digestion
of
the
amplified
PAXI
paired
domain
fragment.
Heterozygous
carriers
show
a
mutant
allelic
band
in
addition
to
the
wild
type
band.
ELAQLGIRPCDISR
ELAQLGIRPCDISR
EMAHHGIRPCVISR
EMAHHGIRPCVISR
ELAQLGIRPCDISR
ELAQLGIRPCDISR
Q
LRVSHGCVSKILARY
Q
LRVSHGCVSKILARY
Q
LRVSHGCVSKILCRY
Q
LRVSHGCVSKILCRY
Q
LRVSHGCVSKILARY
H
LRVSHGCVSKILARY
Figure
5
Partial
protein
sequence
of
different
human
and
murine
PAXgenes
showing
that
the
glutamine
(Q)
residue
at
this
position
in
the
paired
domain
of
the
PAXI
gene
is
highly
conserved.
The
patient
carrying
the
PAXI
mutation
has
a
histidine
(H)
residue
at
this
position.
Based
on
data
from
the
Splotch
mouse
and
from
the
analysis
of
families
and
patients
with
Waardenburg
syndrome,
the
PAX3
gene
is
a
likely
candidate
for
neural
tube
defects.
Link-
age
analysis
of
multiple
case
families
with
the
dinucleotide
repeat
marker25
immediately
up-
stream
of
the
PAX3
gene
did
not
provide
any
indication
for
a
major
involvement
of
this
gene.2'
However,
situations
of
negative
linkage
but
positive
allelic
association
have
been
reported
for
other
genes
and
disorders,
such
as
the
TGFa
gene
in
cleft
lip
and
cleft
palate.26
27
With
respect
to
NTD,
no
significant
associ-
A
Turns
Alpha
helices
Beta
sheets
PAX1
Wild
type
LPNAIRLRIVELAQLGIRPCDISRtLRVSHGCVSKILARYNETGSI
t
B
Figure
6
Results
of
the
secondary
protein
structure
prediction,
of
the
wild
type
(A)
and
mutant
(B)
PAXI
peptide,
respectively.
Computer
analysis,
using
the
Chou-Fasman
algorithm,
shows
the
loss
of
a
sheet
conformation
at
the
position
of
the
mutation.
ation
(p>O.
1)
was
observed
with
any
of
the
alleles
of
the
dinucleotide
polymorphism
and
similar
results
were
obtained
using
patients
and
controls
from
the
US
population
(not
shown).
Moreover,
no
significant
association
was
observed
between
NTD
and
the
newly
detected
polymorphisms
described
in
this
study,
although
suggestive
results
were
ob-
tained
with
the
T
allele
of
a
C/T
silent
polymorphism
in
exon
2
(p=0.06).
The
T
at
the
polymorphic
site
by
itself
does
not
necessarily
have
to
be
pathogenic
to
explain
a
possible
association;
however,
one
could
imag-
ine
that
a
difference
in
codon
usage
or
pre-mRNA
processing
might
lead
to
a
slight
inequality
in
expression
efficiency
between
both
allelic
forms
of
the
PAX3
gene.
In
this
respect,
embryos
with
a
T
allele
would
have
a
minor
disadvantage
during
neurulation.
How-
ever,
at
this
point
the
evidence
for
association
is
not
convincing
and
additional
groups
of
patients
need
to
be
studied
to
determine
whether
the
increased
frequency
of
the
T
allele
in
our
patients
has
relevance
for
non-
syndromal
NTD.
In
this
study
we
have
investigated
whether
particular
members
of
the
PAX
gene
family
could
play
a
role
in
the
aetiology
of
human
NTD.
No
indications
for
an
involvement
of
PAX7
and
PAX9
have
been
obtained
so
far.
No
significant
association
was
detected
between
PAX3
and
non-syndromal
NTD.
On
the
other
hand,
when
considering
the
increased
fre-
quency
of
NTD
in
WS
1
families,'9
PAX3
mutations
do
seem
to
predispose
to
certain
syndromic
forms
of
NTD.
Our
present
results
argue
for
a
role
of
the
PAXI
gene
in
the
aetiol-
ogy
of
NTD
as
shown
by
the
detection
of
a
missense
mutation
in
the
paired
domain.
At
the
same
time
our
data
show
that
the
detected
mutation
in
PAX1
is
not
sufficient
to
cause
the
development
of
the
disorder.
Other
factors,
environmental
or
genetic
or
both,
would
have
to
exert
a
negative
influence
on
the
neurulation
process
to
increase
the
risk
further.
From
animal
studies5
the
gene
for
PDGFRax
underly-
ing
the
Patch
phenotype
is
a
suspected
candidate.
We
thank
the
Dutch
Working
Group
on
Hydrocephalus
and
Spina
Bifida
of
the
patient
organisation
BOSK
for
their
help
in
contacting
the
patients
and
families.
We
also
thank
Prof
Dr
H
H
Ropers
and
Professor
T
Strachan
for
helpful
discussions
and
S
van
der
Velde-Visser
and
E
van
Rossum-Boenders
for
cell
culture
and
EBV
transformations.
This
study
was
supported
by
the
Dutch
Prinses
Beatrix
Fonds
grant
No
93-005
and
95-0521.
The
cooperation
of
the
families
and
the
staff
of
spina
bifida
clinics
in
the
USA
is
gratefully
acknowledged.
The
support
of
NIH
grants
R29
NS29893
(SC)
and
HG00008
(YS)
and
the
March
of
Dimes
Birth
Defects
Foundation
(WJ,
SC)
is
gratefully
acknowledged.
We
also
thank
Q
Li,
C
Torigian,
A
Sarangi,
and
E
S
Stenroos
for
technical
assistance.
FH,
RB,
BH,
and
EM
are
members
of
INTEGER,
the
International
Neural
Tube
Embryology,
Genetics
and
Epidemiology
Research
consortium
to
identify
genes
which
predispose
to
neural
tube
defects.
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E,
Deichl
A,
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S,
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FA,
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T,
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Paxl
PAX3
PAX7
PAX9
PAX1
HUMAN
MOUSE
HUMAN
HUMAN
HUMAN
PATIENT
Turns
Alpha
helices
Beta
sheets
PAX1
Mutant
LPNAIRLRIVELAQLGIRPCDISRHLRVSHGCVSKILARYNETGSI
t
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59:263-5.
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Baldwin
CT,
Hoth
CF,
Amos
JA,
da-Silva
EO,
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exonic
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in
the
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paired
domain
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CF,
Milunski
A,
Lipsky
N,
Sheffer
R,
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SK,
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in
the
paired
domain
of
the
human
PAX3
gene
cause
Klein-Waardenburg
syndrome
(WS-III)
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as
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I
(WS-I).
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Hol
FA,
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doi: 10.1136/jmg.33.8.655
1996 33: 655-660J Med Genet
F A Hol, M P Geurds, S Chatkupt, et al.
patient with spina bifida.
an amino acid substitution in PAX1 in a
PAX genes and human neural tube defects:
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