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Candidate gene analysis in human neural tube defects.

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American Journal of Medical Genetics Part C (Semin. Med. Genet.) 135C:9 – 23 (2005)
A R T I C L E
Candidate Gene Analysis in Human Neural
Tube Defects
ABEE L. BOYLES, PRESTON HAMMOCK, AND MARCY C. SPEER*
Biochemical and developmental pathways, mouse models, and positional evidence have provided numerous
candidate genes for the study of human neural tube defects. In a survey of 80 studies on 38 candidate genes, few
found significant results in human populations through case-control or family-based association studies. While
the folate pathway has been explored extensively, only the MTHFR 677C > T polymorphism was significant, and
only in an Irish population. Developmental pathways such as the Wnt signaling pathway and Hox genes have also
been explored without positive results. More than 90 mouse candidates have been identified through
spontaneous and knockout mutations, but only the T locus (mouse Brachyury gene) showed association in an
initial study that was not confirmed on follow-up. Positional candidates have been derived from cytogenetic
evidence, but preliminary genomic screens have limited power due to small sample sizes. Future studies would
increase their power to detect association by using more samples. In addition a clarification of the phenotype
would be beneficial as many studies used different inclusion criteria. Incorporating several types of data could
highlight better candidates, as would looking beyond the traditional sources for candidate genes. Recent studies
of an energy metabolism gene (UCP2) and vitamin B metabolism (Transcoalbumin) have produced promising
results. Utilizing other model organisms may also be beneficial, as in a recent study from a chick model of NTDs in
NCAM1. New approaches combined with traditional methods and increased sample sizes will help prioritize
human NTD candidate genes and clarify the complex etiology of this condition. ß 2005 Wiley-Liss, Inc.
KEY WORDS: neural tube defects; candidate genes; gene mapping
INTRODUCTION
Candidate genes for human neural tube
defects have previously been supported
by one of three types of evidence:
biochemical pathways such as folate
metabolism, mouse model genes, and
positional candidates. The folate pathway provides candidates due to the
known decrease in NTD incidence in
Abee L. Boyles is a Ph.D. student in the
Duke University Program in Genetics and
Genomics. Her dissertation focuses on the
genetics of neural tube defects and Chiari
Malformation.
Preston Hammock, a former student
intern with the spina bifida genetic study at
Duke, is now enrolled in the XXX program at
the University of North Carolina at Chapel
Hill.
Marcy C. Speer is a genetic epidemiologist, board-certified as a Ph.D. medical
geneticist and genetic counselor. She heads
a national effort to identify genetic and
environmental contributions to spina bifida
and other neural tube defects.
*Correspondence to: Marcy C. Speer,
Ph.D., Duke University Medical Center, Box
3445, Durham, NC 27710.
E-mail: marcy@chg.duhs.duke.edu
DOI 10.1002/ajmg.c.30048
ß 2005 Wiley-Liss, Inc.
the children of mothers who take folic
acid periconceptionally. Mouse lines
with an increased rate of spontaneous
NTDs and mouse knockouts of specific
genes have implicated over 90 candidate
genes [Harris and Juriloff, 1999]. Positional candidates are derived from
regions identified through genomic
screens, which have been difficult to
conduct due to the limited availability of
families with multiple affected members.
Most screens to date have had little
power to detect major genes, but future
studies will improve as more samples
become available. Cytogenetic rearrangements and association of NTDs with
trisomy 13 and 18 have implicated genomic positions as well, albeit large ones.
Few of the candidate genes studied
in human neural tube defects have
proved to have significant impact on
the development of NTDs. Future
research is needed to combine the
strengths and weaknesses of traditional
approaches while incorporating new
sources of candidates. Other model
organisms such as the chick, zebrafish,
or sea urchin might clarify the numerous
candidates seen in mouse models and be
suitable organisms for testing intervention techniques in the future. Exploring
other developmental and metabolic
pathways, such as the retinoic acid
pathway, could provide new insight as
well. The definition of the neural tube
defect phenotype has varied widely in
these studies, with some utilizing only
lumbosacral myelomeningocele and
others a broad range of NTDs such as
other open defects like anencephaly as
well as closed defects like encephalocele
or lipomyelomeningocele.
Biochemical Pathways
Folic acid is known to reduce the
incidence of neural tube defects but the
exact mechanism is unclear [Milunsky
et al., 1991; MRC Vitamin Study
Research Group, 1991; Centers for
Disease Control and Prevention, 1992].
Folate is essential to the carbon transfer
necessary for DNA synthesis, cell division, and tissue growth [Botto and Yang,
2000]. It is also necessary for DNA
methylation, which plays an important
role in gene expression and chromatin
structure. Blood folate levels have a
10
AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.)
strong genetic component with an estimated heritability of 46% [Morrison
et al., 1998], yet maternal folate supplementation can only prevent 50%–70%
of NTDs [Chatkupt et al., 1994]. Folic
acid has also been demonstrated to
decrease the incidence of phenytoin
induced NTDs in developing chick embryos, with relevance to humans due to
the increased risk of NTDs associated
with maternal exposure to anti-epileptic
drugs [Guney et al., 2003]. Folate
deficiency is one contributor to the multifactorial etiology of NTDs, and genes
in this metabolic pathway have been the
basis for many candidate gene studies.
Folate deficiency is one
contributor to the multifactorial
etiology of NTDs, and genes
in this metabolic pathway
have been the basis for many
candidate gene studies.
Several potential genes have been
derived from the folic acid pathway
(outlined in Fig. 1): cystathionine-b-
Figure 1.
synthase (CBS), methionine synthase
(MS), and 5,10-methylenetetrahydrofolate reductase (MTHFR). CBS converts
homocysteine to cystathionine, thus
inefficiency in this enzyme may lead to
elevated homocysteine levels, as is often
observed in mothers of children with
NTDs [van der Put et al., 1995]. MS and
MTHFR are key enzymes in the
methylation cycle. To date, no association studies have found evidence to
support a role for MS or CBS alone in
NTD, although there is weak support
that MS might work in conjunction with
MTHFR [Morrison et al., 1998; Trembath et al., 1999].
Large doses of Vitamin A during
pregnancy have been associated with
congenital malformations including
NTDs in animals [Elwood et al., 1992].
Anti-epileptic agents, such as valproic
acid, are known to increase the risk of
NTDs and may interfere with retinoic
acid metabolism [Ross et al., 2000].
While the limited studies conducted to
date have failed to find a significant
association possibly due to small sample
sizes, this pathway may still prove to be a
fruitful avenue of research with candidates such as retinaldehyde dehydrogenase
or cellular retinoic acid binding proteins.
ARTICLE
Relatedmetabolicpathwayshavethepotential for involvement in NTDs as well.
Developmental Pathways
Experimental systems have long been
used to elucidate early developmental
pathways. From early physical ablation
experiments to later genetic disruptions,
these complex signaling systems have
been painstakingly deciphered. Interruption of several of these key pathways
can lead to neural tube defects in animal
models.
Wnt pathway signaling via b-catenin is essential in the development of
nematodes, Drosophila, and vertebrates.
Early in vertebrate development the
functions of this pathway include: dorsalization of the body, posteriorization of
the neural plate, midbrain development,
and somite dorsoventral organization
[National Research Council, 2000].
The Wnt pathway also acts in conjunction with Jun N-terminal Kinase (JNK)
to establish planar cell polarity. Members
of the Wnt pathway, such as Disheveled,
have been investigated as candidate
genes in human NTDs. Figures 2 and 3
depict the Wnt signaling pathways
[National Research Council, 2000].
Folic acid metabolism and the role of genes in the pathway [Sharp and Little, 2004].
ARTICLE
AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.)
11
Council, 2000]. These genes are necessary for proper development of vertebral
segments and have thus been investigated as candidates for human NTDs
[Volcik et al., 2002a].
Positional Candidates
Figure 2.
Wnt signaling via b-catenin [National Research Council, 2000].
The hedgehog pathway is also used
extensively in the early development of
Drosophila and vertebrates. Functions
such as notochord induction of the
neural tube floor plate, notochord and
floor plate induction of the somite sclerotome, and dorsoventral organization of
the neural tube make this pathway a
strong candidate for human NTDs
[National Research Council, 2000]. In
mice, null mutants of Sonic Hedgehog
and Patched receptor cause spinal cord
defects and an open neural tube, respectively, and these genes have been inves-
tigated as candidates in human NTDs as
well [Zhu et al., 2003a]. Figure 4 depicts
this pathway [National Research Council, 2000].
Homeobox (HOX) genes play a vital
role in the proper development anteriorposterior (A-P) segments in Drosophila
and vertebrates. The banded expression
patterns
determine
segmentation
boundaries along the A-P axis and these
patterns of expression are strikingly
similar between Drosophila and vertebrates, as is the organization of the genes
within the genome [National Research
Genomic screens can be a powerful tool
to locate regions that contain riskconferring genes for human neural tube
defects, but ascertaining samples from
families with multiple affected individuals is a difficult process. Recently a
sufficient number of families was collected through the NTD Collaborative
Group and the resulting genomic screen
has implicated several potential regions
[Speer et al., 2003]. The association of
NTDs with trisomies 13 and 18 has
implicated these chromosomes, and
several cytogenetic rearrangements have
provided positional candidates as well
[Melvin et al., 2000]. Expansion on
these types of studies can focus the
genomic regions that may contain
candidate genes.
HOW CANDIDATE GENES
ARE INVESTIGATED
Case-Control Studies
Retrospective case-control studies have
been classically used in epidemiology
and they are readily applied to genetic
studies. Typically a 2 2 table is constructed assessing the presence or absence
of a risk factor (an allele or genotype for
genetic studies) in a case population as
compared to a control population [Kahn
and Sempos, 1989]. Table I outlines the
construction of a 2 2 table for testing
the risk conferred by an allele, but this
example can be extended to a comparison of a risk genotype to other genotypes separately or as a group.
An odds ratio (OR) is constructed
to compare the frequency of the risk
allele in case to controls and confidence
intervals can be calculated to test for
significance.
a=ða þ bÞ
c=ðc þ dÞ
a=b
ffi
for a rare disease
c=d
OR ¼
Figure 3. Wnt signaling via JNK [National Research Council, 2000].
12
AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.)
ARTICLE
under study. In genetic studies this is
particularly problematic because underlying ethnic stratification within the
population can lead to inherit differences in allele frequencies. If case and
control populations are not truly interbreeding, any detected genetic differences may not be due to the analyzed
gene although this is a point of contention within the field [Thomas and Witte,
2002; Wacholder et al., 2002]. Casecontrol methods do not directly study
transmission from parent to child—the
key determining factor in genetics—so
alternative methods are necessary.
Family-Based Studies
Figure 4. Hedgehog signaling through the patched receptor [National Research
Council, 2000].
If the frequency of the risk allele is the
same in cases and controls, the OR will
be near 1. If the 95% confidence interval
does not contain 1, than the results are
significant with a P-value less than 0.05;
if this interval does contain 1, then the
results are not significant at the 0.05
level. However, using the traditional
0.05 P-value is not entirely appropriate
in these situations due to multiple testing
issues. In a genome-wide setting, it has
been proposed that several levels of
significance be used in publications:
suggestive, significant, and highly significant with point wise significance
levels of 7 104, 2 105, and
3 107, respectively [Lander and Kruglyak, 1995]. There are several variations
on these types of P-value adjustments to
account for smaller samples sizes or the
type of standard error used [Kahn and
Sempos, 1989].
For equivalent sample sizes, a casecontrol method will always have more
power than family-based methods, but
there are potential pitfalls to using this
method. If the case and control samples
are not taken from identical populations,
the measured differences between the
groups may not be due to the risk factor
If the case and control samples
are not taken from identical
populations, the measured differences between the groups may
not be due to the risk factor
under study. In genetic studies
this is particularly problematic
because underlying ethnic stratification within the population
can lead to inherit differences in
allele frequencies.
TABLE I. 2 2 Table for Construction an Odds Ratio
From Case-Control Data
Risk allele
Present
Absent
Total
Cases
Controls
Total
a
c
a þ c ¼ n1
b
d
b þ d ¼ n2
a þ b ¼ m1
c þ d ¼ m2
m1 þ m2 ¼ n1 þ n2 ¼ t
This score test is constructed from counts of the risk allele in case and control populations.
Family-based studies of candidate genes
offer a solution to population stratification issues by using unaffected members
of families or non-transmitted alleles in
place of the control population. While
these studies require more effort to
ascertain, considerable resources to genotype several members of a family, and
offer less power than case-control methods, they can improve the certainty of
the results.
The most common NTD families
available for research are small nuclear
families, which are ideally suited to
the transmission disequilibrium test
(TDT). The TDT has developed into
one of the most widely used tests of
association for candidate genes, and this
method sired several variations, such as
The TDT has developed
into one of the most widely
used tests of association for
candidate genes, and this
method sired several
variations, such as the PDT
and Sib-TDT that can
incorporate several types
of family structures
including missing parents
and unaffected siblings.
ARTICLE
AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.)
CANDIDATE GENES WITH
CONFIRMED ASSOCIATIONS
TABLE II. TDT 2 2 Table Construction
Non-transmitted allele
Transmitted allele
M1
M2
Total
M1
M2
Total
n11
n21
n1
n12
n22
n2
n1
n2
2n
The transmitted and non-transmitted alleles are counted for each sampled parent. Only
the heterozygous parents will contribute to the score test.
the PDT and Sib-TDT that can incorporate several types of family structures
including missing parents and unaffected
siblings [Spielman et al., 1993; Spielman
and Ewens, 1998; Martin et al., 2000].
The TDT test also uses a 2 2
matrix, but in a very different way (see
Table II). Within parent-child triads, the
transmitted and non-transmitted alleles
can be counted if the parents are
heterozygous at that particular locus.
The alleles transmitted to an affected
child are compared to the non-transmitted alleles across a large number of
samples to look for a deviation from the
50/50 expectation under Mendelian
segregation patterns assuming no linkage. If affected children have received
the disease-associated allele more often
than expected by chance, then there is
evidence for association of that allele and
susceptibility to disease. Population stratification is not an issue when potential
products of the same mating are used for
comparison.
Transmissions from homozygous
parents are not informative, and therefore n11 and n22 do not contribute to the
test statistics because they will always
transmit the same allele. The requirement of heterozygous parents makes
determining sample size a priori difficult
because it will vary according to the
heterozygosity of the marker in the
parental samples.
Multiallelic markers were one of the
first variants of the TDT to be developed
[Sham and Curtis, 1995]. Expansions on
the TDT have been made to utilize other
family structures, the PDT [Martin et al.,
2000], extended haplotypes, TDTHAP
13
[Clayton, 1999], and allowing for errors,
TDT-AE [Gordon et al., 2001]. TDTPC can be used to calculate the power of
a study given the samples size and other
marker parameters [Chen and Deng,
2001].
The TDT and TDT-like tests rely
on counts of transmissions and are
consequently considered score tests.
The Genotype Relative Risk (GRR)
model is a likelihood-based test of
association that also utilizes parent-child
triads and can test for parent-of-origin
effects as well as incorporate other
parameters such as environmental risk
factors [Schaid and Sommer, 1993]. The
GRR method has been expanded upon
in a TDT-based log-linear model that is
more accessible to researchers through a
SAS based program [Weinberg et al.,
1998].
Candidate Genes Studied
in Human Neural Tube Defects
Many candidate genes have been investigated in human NTD populations,
yielding few positive results. Table III
summarizes the results of these studies,
including why the candidate was investigated, what population was studied and
what type of test was used. The types of
NTDs included in the study sample are
also included when it was available.
While this table attempts to compare a
majority of work in the field, it cannot
capture every aspect of these publications, and the original literature should
always be consulted for details of these
studies.
Originally identified as a risk factor
for vascular disease, the 677C ! T
thermolabile isoform of MTHFR has
been associated with NTDs in some
populations. MTHFR reduces 5,10methylenetetrahydrofolate to 5-methylenetetrahydrofolate (the predominant
circulatory form of folate) for use as a
carbon donor for the re-methylation of
homocysteine to methionine [Frosst
et al., 1995]. Low MTHFR activity is
associated with low plasma folate and
high homocysteine levels. An early casecontrol study in 55 Dutch patients found
a higher incidence of the 677C > T
mutation in patients and parents of NTD
cases than in the control samples [van der
Put et al., 1995]. A much larger study of
218 Irish families supported this finding
and also found a modest additional risk
associated with the maternal TT genotype. ‘‘These results favor a biological
model of MTHFR-related NTD pathogenesis in which suboptimal maternal
folate status imposes biochemical stress
on the developing embryo, a stress it is ill
equipped to tolerate’’ if it is homozygous
for the thermolabile mutation [Shields
et al., 1999; Botto and Yang, 2000; Arole
et al., 2003].
Other studies failed to find evidence
of association with MTHFR in different
populations [Rampersaud et al., 2003].
Those studies that did find an association
could only attribute 11%–19% of NTDs
to this gene [Ou et al., 1996]. The
677C > T variant of the enzyme has
50%–60% lower activity than the wildtype, however homozygotes with a good
diet would have normal folate levels
[Botto and Yang, 2000]. The thermolabile form of MTHFR could be a risk
factor in populations with poor folate
nutrition, which would explain the
conflicting studies [Shields et al., 1999].
MTHFR contains another silent polymorphism, T1059C, that was associated
with NTDs in a small Iowa subset of a
larger study, but this association has not
been confirmed in other studies and it is
unclear what the functional significance
of this variant would be. Studies of other
folate metabolism genes have produced
17q 21
21q22.3
BRCA1
CBS (cystathionine
beta-synthase)
Mouse Model
15q23-q25
10q23-q24
17
CSK
CYP26 (cytochrome P450
retinoic acid-metabolizing
enzyme)
DVL2 (Disheveled)
FR-alpha (folate receptor alpha) 11q13.3-q13.5
Retinoic Acid Pathway
CRABP2 (cellular retinoic acid 1q21.3
binding protein 2)
Folate Metabolism
Mouse Model
Retinoic Acid Pathway
Retinoic Acid Pathway
CRABP1 (cellular retinoic acid 15
binding protein 1)
Folate Metabolism
Mouse Model
Mouse Model
14q22-q23
BMP4 (bone morphogenic
protein 4)
Type of candidate
Folate Metabolism
Human locus
BHMT (betaine-homocysteine 5q13.1-q13.2
methyltransferase)
Human gene
Exposure to high levels
or retinoic acid can
cause NTDs in
humans and
CRABP1 is highly
expressed in the
developing embryo
Exposure to high levels
or retinoic acid can
cause NTDs
Involved in the
organization of the
cytoskeleton; mice
deficient for CSK
have an increased
risk of NTDs
Exposure to high levels
or retinoic acid
can cause NTDs,
CYP26’s could
provide some
protection from
high levels of
retionoic acid
Essential for neural tube
closure (and other
functions) in mice
Primary folate receptor
responsible for
binding and
importing folate;
mutations may
reduce folate levels
Enzyme of the
homocysteine
methylation
pathway
Knockout mice exhibit
little mesoderm
differentiation
causing
disorganized
structures such as a
small neural plate
and open cranial
folds
Null mutation in mice
have an increased
frequency of NTD
Major enzyme
regulating
homocysteine levels
and elevated
maternal plasma
levels have been
observed in some
NTD pregnancies
Study rationale
Chinese
Zhao et al. [2000]
Trembath et al.
[1999]
Speer et al. [2003]
Dickerson et al.
[2002]
Klootwijk et al.
[2003]
Dickerson et al.
[2002]
Sample size
Diagnoses included
Spina bifida spina
bifida occulta, and
encephalocele
NTD (myelomeningocele,
anencephaly,
encephalocele)
L-S Myelomeningocele
NTD (myelomeningocele,
anencephaly,
encephalocele)
L-S Myelomeningocele
L-S Myelomeningocele
American Caucasian 477 sporadic and familial
L-S Myelomeningocele
cases and their families,
153 controls
Midwestern US
128 families from Iowa 35 from NTD (myelomeningocele,
NTD families
Minnesota, and 9 from
anencephaly,
Nebraska (96% Caucasian)
encephalocele)
American Caucasian 477 sporadic and familial
cases and their families,
153 controls
American Caucasian 477 sporadic and familial
cases and their families,
153 controls
Dutch
38 multiplex families;
79 case samples
Type of study
Frequency of the G742A
polymorphism
Summarized results
No transmission
disequilibrium
TDT (not sig)
No evidence for association
No evidence for association
No evidence for association
No evidence for association
No significant difference was Chi-square test (not sig)
No evidence for mothers’
found between NTDs
genotypes
mothers and non-NTDs
mothers
2 polymorphisms in both the PDT (not sig) FBAT (not sig) No evidence for association
gene and promoter region
did not show association
No transmission
disequilibrium
Odds Ratio with 95% CI
(not sig)
TDT (not sig)
Conclusion
No evidence for association
Screen for polymorphisms
Screen for polymorphisms
Screen for polymorphisms
8 novel SNPs identified, but n/a
none associated with
NTDs
No consistent polymorphisms n/a
found
No evidence for association
No evidence for association
Polymorphisms did not show PDT (not sig) FBAT (not sig) No evidence for association
significant association
3 polymorphisms did
PDT (not sig) FBAT (not sig) No evidence for association
not show significant
association
Screen for polymorphisms in 3 polymorphisms detected in Frequency too low for TDT No evidence for association
the coding region and part
both patients with NTDs
based log linear
of the introns
and controls with similar
frequencies
Screen for polymorphisms
Screen for polymorphisms
Screen for a novel
polymorphism and
T2199C
Frequency of T833C and
G919A in mothers of
NTD pregnancies
Test used (p-value)
Odds Ratio with 95%
CI (not sig)
Four mutations found in four Equal frequencies
case and unaffected
individuals
No significant difference was
found between cases and
controls
No increased frequency
Examined frequency of
G307S and I278T alleles as
well as a 68bp insertion
Screen for A4956G and
A1186G
Non-syndromic spina bifida Screen for mutations
aperta
including T455C
(meningocele and
polymorphism
myelomeningocele)
Spina bifida spina
bifida occulta, and
encephalocele
40 mothers of cases, 36 control NTD
mothers
79 cases and their families
83 cases, 79 mothers of cases,
201 control infants, 241
control mothers
79 cases and their families
179 cases, 161 controls
54 patients, 57 mothers of
Spina bifida
patients, 93 control children
and 86 control mothers
American Caucasian 477 sporadic and familial
cases and their families,
153 controls
Dutch and British
Morrison et al.
[1998]
Dickerson et al.
[2002]
Irish
Dutch and British
German
Canadian
Population
studied
Ramsbottom et al.
[1997]
Morrison et al.
[1998]
Felder et al. [2002]
Morin et al. [2003]
Reference
TABLE III. Candidate Gene Chapter
Four clusters: A on Mouse Models
7, B on 17,
C on 12, and
D on 2
1p34-1pter
1p34-1pter
HOX Gene Family
MACS (human homologue
of MARCKS)
MLP (MARCKS-like
protein)
Folate Metabolism
Folate Metabolism
MTHFD1 (methylenetetra14q24
hydrofolate dehydrogenase/
methenyltetrahydrofolatecyclohydrolase/
formyltetrahydrofolate
synthetase)
MTHFR (5,10methylenetetrahydrofolate
reductase)
1p36.3
Mouse Model
Msx2 (muscle segment homeo- 5q34-q35
box 2)
Mouse Model
Mouse Model
Folate Metabolism
11p11.2
GCPII (glutamate
carboxypeptidase II)
Folate Metabolism
11q13.3-q13.5
FR-beta (folate receptor beta)
128 Iowa families, 35 from
Minnesota, 9 from
Nebraska, 53 families from
the NTD Collaborative
Group, and 41 CEPH
families
Screen for polymorphisms
Individuals with NTDs and Tested for allelic variation
their immediate families
128 families from Iowa 35 from NTD (myelomeningocele,
Minnesota, and 9 from
anencephaly,
Nebraska (96% Caucasian)
encephalocele)
Dutch and British
Spina bifida spina bifida
occulta, and
encephalocele
79 cases and their families
Dutch
Irish
Brody et al. [2002]
38 familial cases, 79 sporadic
cases, and 300 controls
NTD (myelomeningocele,
anencephaly,
encephalocele)
Grouped into 3 groups:
1. Anencephaly
(including craniorachischisis)
2. Encephalocele
3. Spina Bifida Aperta
NTD (myelomeningocele,
anencephaly,
encephalocele)
L-S Myelomeningocele
L-S Myelomeningocele
319 complete triads, 22 cases,
NTD (spina bifida,
13 mothers of cases, 2
encephalocele,
fathers of cases, an additional
anencephaly, anence83 mothers of cases, and two
phaly plus spina bifida)
control populations of 699
individuals and 318
pregnant women
55 cases and controls
NTD
Dutch
204 cases (10 anencephaly,
8 encephalocele, and
183 spina bifida aperta)
and 222 German controls
38 multiplex families;
79 case samples
Dutch
German and Italian
43 simplex families
43 simplex families
Caucasian
Caucasian
Screen for C677T and
C1068T
Screen for variants
R653Q variant
SSCP screen for mutations
Mutation screen
2-sided Fisher exact test
(not sig)
n/a
TDT (not sig)
TDT (not sig)
No evidence for association
No evidence for association
No evidence for association
No evidence for association
No evidence for association
No evidence for association
No evidence for association
Lack of association is
consistent with
biochemical mechanisms
of folate deficiency
Odds Ratio (p < 0.05)
Equal transmission, but
TDT (not sig)
approaches significance in
conjunction with MS
C677T found to be
significantly different in
cases than controls
(Continued )
Possible association in
conjunction with MS
The C677T mutation is a
genetic risk factor for
spina bifida
One polymorphism found in n/a
No evidence for a major role
3 members of one family
however the identification
(NTD, SBO, and
of a mutation in one
unaffected) two other
family suggests that this
polymorphisms for in
gene can act as a risk factor
both cases and controls
for human NTD.
Excess of the Q allele in
Odds Ratio (p ¼ 0.003) and Mothers with the MTHFD1
mothers of cases and
traditional TDT
‘‘QQ’’ genotype have an
preferential transmission
(p ¼ 0.015) and TDT
increased risk (1.5to cases
based log linear (p¼0.007)
to2.0-fold) of having an
tests also significant
NTD-affected pregnancy.
Found polymorphisms,but
not significant
No polymorphisms found
Tested MLP1 polymorphisms Not significant, but the test
had low power
Screen for polymorphisms
TDT and Chi-square tests
(not sig)
Odds Ratio with 95% CI
(not sig)
Polymorphism tested was not Odds Ratio genotype test
significant
(not sig)
ETDT (not sig)
Polymorphisms found were
not significant allele-wise
or genotype-wise
Polymorphisms tested were
not significant
Polymorphisms found were
not significant
No polymorphisms within the Not significant, but the test
gene, tested markers
had low power
flanking the gene
96 cases, 113 mothers of cases, Spina bifida
Test for H475Y
97 fathers, and 101 controls
polymorphism
American Caucasians 459 patients and their parents
Isolated myelomeningocele Screen for polymorphisms
and Hispanics of
Mexican descent
Dutch Caucasians
Caucasian
Midwestern US
NTD families
Hol et al.[1998]
Stegmann et al.
[2001]
MTHFR is an enzyme Van der Put et al.
within the folate
[1995]
pathway, and the
C677T allele
decreases the
activity of
folate-dependent re
methylation of
homocysteine
Morrison et al.
[1998]
Genetic alterations of
MSX2 have been
shown to cause
failure of cranial
neural tube closure
in mice.
MTHFD encodes a
single protein with
three catalytic
properties
important in folate
metabolism
Members of all four
Volcik et al. [2002a]
clusters of the HOX
genes are implicated
in neural tube
closure in mice
MARCKS is important Stumpo et al. [1998]
to the development
of the central
nervous system;
mice lacking the
protein have a
higher frequency of
NTDs
MARCKS is important Stumpo et al. [1998]
to the development
of the central
nervous system;
mice lacking the
protein have a
higher frequency
of NTDs
Klootwijk et al.
[2003]
Primary folate receptor Trembath et al.
responsible for
[1999]
binding and
importing folate;
mutations may
reduce folate levels
H475Y polymorphism Vieira et al. [2002]
decreases enzyme
activity and is
associated with
decreased plasma
folate levels and
increased plasma
total homocysteine
Afman et al. [2003]
Human gene
Human locus
Type of candidate
Study rationale
Canadian (75%
English or
French)
Italian
84% Caucasian,
16% AfricanAmerican
Brazilian
Hispanic (Yucatan
area)
American Caucasian 175 cases and their families
and 195 controls
Christensen et al.
[1999]
Johanning et al.
[2002]
Cunha AL et al.
[2002]
Gonzalez-Herrera
et al. [2002]
Rampersaud et al.
[2003]
NTD
27 cases, 28 mothers of cases,
23 siblings of cases, and
159 controls
Revilla et al. [2003] Spanish
L-S Myelomeningocele
without folic acid
supplementation
Spina bifida
NTD (myelomeningocele,
anencephaly,
encephalocele)
104 cases, 106 mothers of cases, Non-syndromic, isolated
and 100 adult controls
spina bifida (separated
into lumbosacral and
thoracolumbar groups)
65 cases, 60 of their mothers
and 110 controls
25 cases with mothers,
75 controls
Pietrzyk et al. [2003] Polish
De Marco et al.
[2002]
Midwestern US
NTD families
Trembath et al.
[1999]
Screen for C677T and
A1298C
Screen for C677T
Screen for ‘‘thermolabile’’
variant (C677T)
Screen for C677T
C677T and A1298C
mutations in MTHFR
gene
NTD (myelomeningocele, ‘‘Thermolabile’’ (C677T)
56 cases, 62 mothers of cases,
mutation screen
97 control children,
anencephaly,
90 control mothers
encephalocele)
203 case, 98 of their mothers, Myelomeningocele,
Screen for A1298C
67 fathers, and 210 controls
meningocele, lipoma,
lipomyeloschisis, dermal
sinus, tight filum
terminalis
77 case and 77 controls
NTD (myelomeningocele, Thermolabile mutation:
anencephaly,
alanine to valine
encephalocele)
148 cases and 174 controls
Non-syndromic spina bifida Test for association with
(population-based test)
at any level (plus 8
C677T and A1298C
77 parent child triads and
anencephalics and
polymorphisms
110 mother-child pairs
2 encephalocele for
(family-based test)
molecular analysis)
128 families from Iowa 35 from NTD (myelomeningocele, Screen for polymorphisms
Minnesota, and 9 from
anencephaly,
(C677T mutation)
Nebraska (96% Caucasian)
encephalocele)
Type of study
Screen for ‘‘thermolabile’’
variant (C677T)
German
Diagnoses included
NTD (myelomeningocele,
anencephaly,
encephalocele)
Stegmann et al.
[1999]
Sample size
271 cases and 218 families
Population
studied
Shields et al. [1999] Irish
Reference
TABLE III. (Continued )
Summarized results
Test used (p-value)
Conclusion
The C677T variant frequency
is not different in cases
than controls
Evidence of association with
cases only, but no
evidence of unequal
transmission or a
previously reported
association with CBS
Statistically significant
differences in cases and
controls seen in
homozygous mothers and
children
There was no significant
difference in these
genotypes between cases
and controls. Therefore
we conclude these
polymorphisms have no
association with NTDs in
the Spanish population.
Evidence for an increased risk
in addition to other
candidate genes
Thermolabile mutation
may affect vitamin B12
and homocysteine
metabolism, which
possibly could contribute
to NTDs
No evidence for association
Increased risk for NTD
associated with the
C677T polymorphism
Evidence for in increased
risk when cases or their
mothers have a C allele
or fathers have a CC
genotype
Increases risk for
heterozygotes and valine
homozygotes
Evidence for in increased
risk in nonmyelomeningocele cases
Chi-square test (not sig)
No evidence for association
Odds Ratio significant in cases Increased risk associated with
(p ¼ 0.049) and mothers
the C677T polymorphism
(p ¼ 0.007)
in homozygous cases and
mothers
Odds Ratio (p < 0.05), PDT
(p > 0.1)
Odds Ratio (not sig)
Heterozygotes and
Odds Ratio with 95% CI
homozygotes had higher
(p < 0.05)
risk for NTD’s, with a
more significant difference
from 1998-1994 than
those born after 1994
Genotypes were associated
No difference in case and
with metabolite blood
control frequencies
levels
Not significant in
TDT (p ¼ 0.03 in nonmyelomeningocele cases,
myelomeningocele
however the mutation was
NTDs)
significantly different in
non myelomeningocele
(lipomyelomeningocele,
intradural lipoma and
sacral hypoplasia/agenesis)
Significant when mother and Odds Ratio (p < 0.05)
child are homozygous for
the variant allele
Cases, mothers, and fathers
Odds Ratio (all p < 0.05)
with the CC allele are
significantly different than
controls
The Tallele frequency is higher Odds Ratio (p ¼ 0.0005) and The T allele increases risk to
in cases than controls and
TDT based log linear
cases a modest additional
the TT genotype is
(p < 0.05)
risk is conferred by a
significant in cases and
maternal TT genotype
their mothers
No significant differences in Likelihood ratio test (not sig) No evidence for association
cases and controls; equal
and TDT (not sig)
transmission observed
Chick Model
11q23.1
NCAM1 (neural cell adhesion
molecule 1)
Folate Metabolism
Mouse Model
5p15.2-15.3
MTRR (methionine synthase
reductase)
Folate Metabolism
NAP1L2 (nucleosome assembly Xq12-q24
protein 1-like 2)
1q43
MTR or MS (Methionine
Synthase)
Italian
Southern Italian
De Marco et al.
[2002]
Gueant-Rodriguez
et al. [2003]
Spina bifida
TDT (not sig)
No differences between case
and control frequencies
Polymorphism increases risk
of NTD when cobalamin
status is low
Odds Ratio (p < 0.05 with
low cobalamin)
40 cases and 58 age and sex
matched controls
L-S Myelomeningocele
Screen for polymorphisms
5 polymorphisms screened,
one is significant
PDT (p ¼ 0.041), FBAT
(p ¼ 0.00045)
(Continued )
Significant evidence of
association in this
population
In the context of a
multifactorial origin, the
polymorphisms within the
50 CpG island of NAP1L2
may contribute to the
complex etiology of
NTDs
Increased risk associated with
the A66G polymorphism
in homozygous cases and
mothers
Evidence for risk after
accounting for the
MTHFR allele
Evidence for increase risk
from the polymorphism
The polymorphism was
associated with a reduced
risk for NTD
No evidence for association
No evidence for association
No evidence for association
Possible association in
conjunction with
MTHFR
Odds Ratio (p ¼ 0.046)
Odds Ratio (not sig)
TDT (not sig)
2 polymorphisms 900 and
154 kb away from MS
not associated
Found polymorphisms, but no Chi square with 95%
significant differences in
Confidence Interval
cases and controls
Polymorphism less common Odds Ratio (p < 0.05)
in cases
Equal transmission, but
approaches significance
with MTHFR
Evaluated the association of a MTR polymorphisms
MTR A2756G
significantly different in
polymorphisms alone and
cases than controls
with MTHFR C677T
66A > G polymorphism
Polymorphism only
check
significant when
cobalamin is low
Screen for A2756G variant
Screen for 2756A > G
polymorphism
Screen for polymorphisms
Screen for polymorphisms
Screen for C5049A and
A2756G
Myelomeningocele, all with Evaluated the association
MTRR polymorphisms were Odds Ratio (p ¼ 0.023)
failure of closure at or
of a MTRR A66G
associated with NTD risk
below position L-2
polymorphisms alone and
in cases having a MTHFR
with MTHFR C677T
677 CC wild genotype
104 cases, 106 mothers of cases, Non-syndromic, isolated
A66G polymorphism
Statistically significant
Odds Ratio significant in
and 100 adult controls
spina bifida (separated
differences in cases and
cases (0.034) and
into lumbosacral and
controls seen in
mothers (0.039)
thoracolumbar groups)
homozygous mothers
and children
114 cases
Spina bifida occulta to
Search for polymorphisms
Polymorphisms found were
n/a
craniorachischisis, but
not associated
were predominantly
spina bifida aperta (57)
and anencephaly (36)
56 cases, 58 mothers of cases,
97 control children,
89 control mothers
Mouse models exhibit Rogner et al. [2002] United Kingdom
NTDs closely
population of
resembling spina
mixed ethnic
bifida and
origins
anencephaly in
humans; plays a role
in the cell cycle
regulation of
developing neurons.
Controls cell migration Bastress et al. [2005] American Caucasian 477 sporadic and familial
in neural tissues; cell
cases and their families,
adhesion molecules
153 controls
are disturbed in
spontaneous NTDs
in chicks
Pietrzyk et al. [2003] Polish
Activates cobalaminWilson et al. [1999] Canadian
dependent
methionine
synthase as part of
the homocysteine re
methylation
pathway;
impairment of folate
and cobalamin
metabolism has
been observed in
families with NTDs.
Gueant-Rodriguez Southern Italian
et al. [2003]
Canadian
Christensen et al.
[1999]
Spina bifida
Spina bifida spina bifida
occulta, and
encephalocele
128 families from Iowa 35 from NTD (myelomeningocele,
Minnesota, and 9 from
anencephaly,
Nebraska (96% Caucasian)
encephalocele)
56 cases, 62 mothers of cases,
NTD (myelomeningocele,
97 control children,
anencephaly,
90 control mothers
encephalocele)
203 case, 98 of their mothers, Myelomeningocele,
67 fathers, and 210 controls
meningocele, lipoma,
lipomyeloschisis, dermal
sinus, tight filum
terminalis
40 cases and 58 age and sex
Myelomeningocele, all with
matched controls
failure of closure at or
below position L-2
85 case-parent triads
Irish
Midwestern US
NTD families
79 cases and their families
Dutch and British
Trembath et al.
[1999]
Converts intracellular
Morrison et al.
folate and
[1998]
homocysteine to
tetrahydrofolate and
methionine.
Tetrahydrofolate is a
crucial ingredient in
biosynthesis of
DNA and RNA,
while methionine
is important in
numerous
methylation
reactions
Brody et al. [1999]
17q22
Multiple sites
4q12
10q23-q24
21q22.3
PAX Gene Family
PDGFRA promotor
RALDH2 (retinaldehyde
dehydrogenase)
RFC-1 (reduced folate carrier
protein)
Human locus
Noggin
Human gene
Folate Metabolism
Retinoic Acid Pathway
Mouse Model
Mouse Models
Mouse Model
Type of candidate
38 familial cases, 79 sporadic
cases, and 300 controls
Dutch
Midwestern US
NTD families
A80G polymorphism
De Marco et al.
causes lower plasma
[2001]
folate levels
Exposure to high levels Dickerson et al.
or retinoic acid can
[2002]
cause NTDs,
Italian
Diagnoses included
Allelic association tests
NTD (myelomeningocele,
anencephaly,
encephalocele)
L-S Myelomeningocele
Summarized results
Check for A80 G
polymorphism, along
with MTHFR A1298 C
allele frequency
Screen for polymorphisms
Expression studies in human
cell line culture
n/a
Limited power for a linkage
study
Test used (p-value)
All polymorphisms found in
both cases and controls
The frequency of the
polymorphism in cases,
mothers, and fathers is
higher than in controls
Conclusion
Improper PDGFRA
expression may play a
role in the etiology
This promoter most likely
acts in combination with
other adverse factors
Associations may be with a
disease locus within the
same region as these
genes, therefore future
studies should focus on
this area
No evidence for association
No evidence for association
No evidence of linkage
No evidence for association
No evidence for association
Limited evidence for
association, but not likely
to be important in the
context of the whole
genome
Chi-square test, p < 0.05 for Both RFC-1 and MTHFR
cases, mothers, and fathers
polymorphisms may play
a role in NTD risk in
the Italian population
PDGFRA expression
n/a
enhanced by treatment
with retinoic acid or cyclic
AMP
Out of 9 polymorphisms, one FBAT (p ¼ 0.02)
was significant in one of
PDT (p > 0.05)
3 tests
TRANSMIT(p > 0.05)
Chi-square test (not sig)
n/a
Pax1 polymorphism found in n/a
one case and the maternal
grandmother, Pax3
polymorphism found in
cases and controls at equal
frequencies
Positive transmission of alleles TDT (Pax1 p ¼ 0.019, Pax7
for markers within PAX1,
p ¼ 0.011, Pax8
PAX7, and PAX8 were
p ¼ 0.013)
detected but not in phase
with disease
No evidence around PAX3
with a dominant or
recessive model
One variant found in a single n/a
case an unaffected family
members
One polymorphism present
in one SB patient, her
unaffected father, and in
one control individual
Tested for presence of specific Heterozygotes have an
PDGFRA promotor
increased risk for
haplotype combinations
malformations
Screen for polymorphisms
in Pax 3
Isolated myelomeningocele Screen for polymorphisms
All spina bifida except
1 encephalocele and
1 craniorachischisis
143 lumbosacral
Search for allelic variances
myelomeningocele
(143), thoracic
myelomeningocele
(12), thoracic
myelomeningocele (23),
lipomyelomeningocele
(12), and miscellaneous
(22)
NTD, including SBO
Linkage analysis in Pax 3
49 familial, 76 sporadic, and 77 Spina bifida
controls
203 unrelated nonsyndromic
cases, 98 mothers and
67 fathers
Type of study
Non-syndromic spina bifida Search for polymorphisms
aperta (meningocele and
myelomeningocele)
128 families from Iowa 35 from NTD (myelomeningocele,
Minnesota, and 9 from
anencephaly,
Nebraska (96% Caucasian)
encephalocele)
49 familial, 76 sporadic, and
Spina bifida
77 controls
American Caucasian 477 sporadic and familial
cases and their families,
153 controls
Pax-1 mouse models
Joosten et al. [2001] Dutch
have indicated
that deregulated
expression of the
gene encoding the
platelet-derived
growth factor alpha
receptor
(PDGFRA) causes
congenital NTDs.
Joosten et al. [2002] Dutch
Trembath et al.
[1999]
Volcik et al. [2002b] 59%Hispanic, 35%
459 patients and their parents
White, 6% other
17 informative multiplex
families
Sample size
American and
Dutch
American Caucasian 202 cases
Bauer et al. [2002]
179 cases
German
Population
studied
Felder et al. [2002]
Reference
PAX genes regulate
Chatkupt et al.
normal
[1995]
development, Pax 1
and Pax3 (splotch)
knock out mice
exhibit severe
NTDs
Hol et al. [1996]
Mouse models show
fully penetrant
skeletal
abnormalities and
defects in growth
and patterning of
the neural tube.
Study rationale
TABLE III. (Continued )
Mouse Model
T (human analogue of
Brachyury gene in mice)
6q27
Mouse, Chick and Frog
Models
SLUG (encodes a zinc finger
8q11
protein of the Snail family of
transcription factors)
Cytosolic form:
Folate Metabolism
17p11.2
mitochondrial
form: 12q13.2
SHMT (serine
hydroxymethyltransferase
Spina bifida
183 cases and 266 controls
(familial study)
Richter et al. [2002] German
13 informative sporadic cases
and their families
Midwestern US
NTD families
218 case-parent triads
79 cases and their families
150 cases (11 familial) and
136 controls
NTD (myelomeningocele,
anencephaly,
encephalocele)
NTD (myelomeningocele,
anencephaly,
encephalocele)
NTD (myelomeningocele,
anencephaly,
encephalocele)
Spina bifida spina bifida
occulta, and
encephalocele
Spina bifida aperta
109 cases, 120 mothers of cases, Spina bifida
and 420 controls
50 cases and 50 controls
(pilot study)
Dutch and British
German
Dutch
Midwestern US
Shields et al. [2000] Irish
SHMT catalyzes a
Heil et al. [2001]
reaction involved in
metabolism of folate
dependent
homocysteine
and elevated
homocysteine levels
and decreased
plasma folate levels
were observed in
mothers of children
with NTDs
SLUG is selectively
Stegmann et al.
expressed in the
[2001]
dorsal part of the
developing neural
tube; ablation
and antisense
experiments in
chicken suggest that
SLUG may be an
important factor
during neural tube
closure
Mouse knock outs
Morrison et al.
of its homologue,
[1998]
T (Brachyury),
have notochord
abnormalities
Trembath et al.
[1999]
Zhu et al. [2003a]
Check for T1VS7 allele
Screen for T1VS7-2
polymorphism in intron
7 of T gene
Screen for T1VS7-2
polymorphism
Screen for TIVS7C and
A530G
Screen for polymorphisms
Screen for mutations or
polymorphisms
Screen for mutations or
polymorphisms
25 embryos with axial structure Craniorachischisis and spina In situ hybridization for
abnormalities
bifida
expression
French
Screen for mutations
Check for A80G
polymorphism, along
with known MTHFR
polymorphisms
Check for A80G
polymorphism
78 families, 49 with NTD
(others have sacral agenesis
or polydactyly)
NTD
174 cases, 43 mothers of cases, Non-syndromic NTD
53 fathers of cases, and 156
controls
133 case infants and 188 control NTD (anencephaly, spina
infants
bifida cystica,
craniorachischisis,
or iniencephaly)
Irish
Italian
De Marco et al.
[2003]
Chick and Mouse Models SHH patterns the
Vargas [1998]
midline neuro-axis
and distal limb
structures and
abnormal
expression is seen in
mice with NTDs
Kirillova et al.
[2000]
7q36
SHH (Sonic Hedgehog)
California
Shaw et al. [2002]
n/a
No differences between case
and control frequencies
Allele markedly associated
with cases born before
1980, but not with more
recent cases.
Equal transmission seen
Preferential transmission of
the TIVS7C allele
One polymorphism found
in a case but also the
unaffected parent
(Continued )
This polymorphism is a
modest, but important
risk factor and works via
different mechanism than
MTHFR polymorphism
No evidence for association
TDT (p ¼ 0.01), MGRR
(p ¼ 0.02), and TDT
based log-linear
(p ¼ 0.006)
McNemar’s test (not sig)
No evidence for association
Significant evidence for
association
TDT (p ¼ 0.03)
TDT (not sig)
No evidence for association
No evidence for association
Abnormal expression
associated with neural
tube defects in embryos
No evidence for association
No evidence for association
Evidence for increased risk to
cases and mothers, but not
in combination with
MTHFR
No evidence for association
n/a
No known mutations
n/a
previously observed were
observed, one intronic
polymorphism identified
Two polymorphisms found,
Pearson Chi Square (not sig)
but neither significant
Abnormal patterns of
expression observed
However some evidence
Odds Ratio (not sig)
suggestive of an
interaction between infant
G80/G80 genotype and
maternal supplemental
vitamin use on the
occurrence of spina bifida
GG genotype more common Odds Ratio (p < 0.05 for
in the NTD cases and
homozygous cases and
mothers; no evidence for
mothers)
an association between
NTD phenotype and
combined MTHFR
C677T/RFC-a A80G
genotypes;
One sequence variation was n/a
found in two unrelated
individuals with NTD and
in none of the normal
control samples
Xq26.2
ZIC 3 (zinc finger protein of
cerebellum)
Mouse Model
Positional Candidate
Energy Metabolism
13q32
11q13
UCP2 (uncoupling protein 2)
Mouse Model
ZIC 2 (transcription factor)
6p24
TFAP2-alpha (transcription
factor activating
enhancer-binding protein
2 alpha)
Mouse Model
Positional Candidate
3q21-q28
TERC (telomerase RNA
component)
Vitamin B Pathway
Type of candidate
X-chromosome
11q11-q12
Human locus
TC (transcobalamin)
Human gene
Reference
40 cases and 58 controls
Southern Italian
Sample size
42 mothers of cases and
73 control mothers
Dutch
Population
studied
Execephaly and tail
defects in Bent tail
mice and sacral
anomalies and
NTDs in patients
with ZIC3
mutations
One large family with
multiple affected members
(43 sampled)
133 case infants and
188 control infants
38 multiplex families;
111 case samples
Australian, British,
Icelandic
Hispanic
Zhu et al. [2003b]
Hispanic
Diagnoses included
35 cases and controls
Type of study
Screen for variants in coding
region of the TC gene
Summarized results
Test used (p-value)
No effect on homocysteine
Odds Ratio with logistic
and NTD risk could be
regression
detected with any variant
discovered.
Linkage analysis
NTD (spina bifida or
anencephaly)
No evidence for association
No evidence for association
Evidence for TC playing a
role with MTHFR
Odds Ratio (p < 0.05)
Compelling evidence for
UCP2 as an NTD
risk factor
TDT based log linear (not sig) 1257C > T allele may confer
an increased risk
2-sided Fisher exact test
(not sig)
Conclusion
No evidence for association
with risk or homocysteine
levels
No differences between case
and control frequencies
No mutations or
polymorphisms were
identified
n/a
n/a
Odds Ratio (not sig)
No evidence for association
No evidence for association
No evidence for association
Apparent X-linked spina
Linkage and haplotype analysis No evidence for linkage in
bifida and anencephaly
this family
haplotypes were
extensively analyzed and
found to exclude linkage
to the X chromosome
Found polymorphisms but
Attempted TDT, but there
ZIC2 mutations are, at most,
were only 9 suitable triads
a very infrequent cause
in too few samples to
produce significant results
of NTDs
Found three polymorphisms
and the C1257T allele
approached significance
Combined homozygosity for
both UCP2 variants
resulted in a threefold or
more elevated risk of SB
(neither was significant
individually))
Found polymorphisms,
but not significant
Screen for mutations or SNPS No mutations or
polymorphisms were
identified in any of the
three exons studied
Screen for polymorphisms
Screen for 9H or 10H alleles
95% L-S myelomeningocele Screen for mutations
(5% encephalocele and
other NTDs)
Spina bifida and
anencephaly
Grouped into 3 groups: 1. Mutation screen
Anencephaly (including
craniorachischisis)
2. Encephalocele
3. Spina Bifida Aperta
NTD (myelomeningocele, Screen for polymorphisms
anencephaly,
of coding region and
encephalocele)
part of the introns
NTD (anencephaly,
Searched for insertion/
spina bifida cystica,
deletion at 30 UTR
craniorachischisis, or
iniencephaly)
Myelomeningocele, all with Evaluated the association
Polymorphism not significant Odds Ratio (p ¼ 0.028)
failure of closure at or
of a TC C777G
alone, but is in
below position L-2
polymorphisms alone and
combination with the
with MTHFR C677T
MTHFR CC genotype
L-S Myelomeningocele
Screen for polymorphisms
Two novel SNPs found, but PDT (not sig)
neither was significant
Non-syndromic NTD
NTD (spina bifida or
anencephaly)
3 multiplex families and 5 cases NTD (myelomeningocele,
anencephaly,
encephalocele)
69 controls and cases
Children’s Memorial 192 cases
Hospital Spina
Bifida Clinic
(Chicago)
Icelandic
California
Dutch
Carrel et al. [2001]
Polymophisms are
Volcik et al. [2003]
capable of affecting
energy metabolism,
body weight
regulation, and
possibly preventing
the buildup of
reactive oxygen
species, all factors
that could
contribute to neural
tube defect risk
through maternal
obesity and diabetes.
A large Icelandic
Newton R. et al.
pedigree displayed
[1994]
what appeared to
be X-linked
spina bifida and
anencephaly.
ZIC2 is in the
Brown et al. [2002]
chromosome 13q32
critical deletion
seen in the 13qsyndrome (included
encephalocele and
anencephaly);
targeted mutation
of ZIC2 in mouse
models result in
NTDs
Zhu et al. [2003b]
Klootwijk et al.
[2003]
Knockout mice have
Benz et al. [in press] American Caucasian 477 sporadic and familial cases
chromosomal
and their families, 153
instability which has
controls
been implicated in
failed neural tube
closure
AP-2 null and chimeric Stegmann et al.
German and Italian 204 cases (10 anencephaly,
mice exhibit
[1999]
8 encephalocele, and
exencephaly.
183 spina bifida aperta)
and 222 German controls
Low plasma TC levels Afman et al. [2002]
have previously
been associated with
an increased risk for
having a child with
an NTD
Gueant-Rodriguez
et al. [2003]
Study rationale
TABLE III. (Continued )
ARTICLE
mixed results as well [Christensen et al.,
1999; Wilson et al., 1999; Brody et al.,
2002].
Several candidate genes from mouse
models of NTDs have been studied in
human populations. The T locus is the
human homolog of the Brachyury gene in
mice, which is vital to axial development
and the formation of the posterior
mesoderm. No evidence has been found
for it being a major locus in human
NTDs [Trembath et al., 1999; Speer
et al., 2002]. An early study found
limited evidence in a small association
study, but it could only account for 6%–
18% of NTD incidence [Morrison et al.,
1998]. Splotch mice have a homozygous
mutation in the Pax3 gene and exhibit
NTDs, but no evidence has been found
for this gene to be a major NTD risk
factor in humans either [Trembath et al.,
1999; Speer et al., 2002]. As Table III
illustrates often compelling animal
model candidates fail to be associated in
human populations.
A few human NTD candidate gene
studies have recently been published that
look beyond the standard sources.
Uncoupling Protein 2 (UCP2) functions in energy metabolism and was
associated in a case-control study of a
Californian population [Volcik et al.,
2003]. A Vitamin B metabolizing
enzyme, transcobalomin (TC) was not
associated in a Dutch population, but did
have significant results in a Southern
Italian study [Afman et al., 2002;
Gueant-Rodriguez et al., 2003]. Utilizing evidence from a chick model of
NTDs involving cell adhesion molecules, there is recent evidence to support
a role for neural cell adhesion molecule 1
(NCAM1) in the etiology of NTDs.
While these findings may not hold up to
future investigation, they delve into new
sources of NTD candidate genes. Mouse
models and folate metabolism will always
provide new NTD candidates, but new
research investigating different pathways
and under utilized model organisms may
provide keys to NTD research.
CONCLUSIONS
Candidate gene testing in human neural
tubes has proved to have a burden of
AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.)
plenty—too many possible candidates
exist and few that are studied have
yielded positive results. Efficiently
prioritizing these possibilities based on
one source of data such as an animal
model, metabolic pathway, or positional
location is difficult, if not impossible.
Incorporating several types of data may
lead to a convergence of information,
highlighting crucial regions or pathways. Comparing several model organisms will clarify which genes play a
fundamental role in neural tube closure.
Detailed clarification of the phenotype is
of critical importance to these studies.
The most conservative approach is to
include only the narrowest group, but
this limits the samples available to most
studies. Increased sample numbers in
candidate gene studies will directly
increase their power to detect a significant association. While human NTD
samples are difficult to obtain, redoubling efforts to ascertain more families
from a variety of populations will enable
genomic screens and candidate gene
testing to be more effective in the future.
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