Genetics of human neural tube defects

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Abstract
Neural tube defects (NTDs) are common, severe congenital malformations whose
causation involves multiple genes and environmental factors. Although more than
200 genes are known to cause NTDs in mice, there has been rather limited progress
in delineating the molecular basis underlying most human NTDs. Numerous genetic
studies have been carried out to investigate candidate genes in cohorts of patients,
with particular reference to those that participate in folate one-carbon metabolism.
Although the homocysteine remethylation gene MTHFR has emerged as a risk factor
in some human populations, few other consistent findings have resulted from this
approach. Similarly, attention focused on the human homologues of mouse NTD
genes has contributed only limited positive findings to date, although an emerging
association between genes of the non-canonical Wnt (planar cell polarity) pathway
and NTDs provides candidates for future studies. Priorities for the next phase of this
research include: (i) larger studies that are sufficiently powered to detect significant
associations with relatively minor risk factors; (ii) analysis of multiple candidate
genes in groups of well-genotyped individuals to detect possible genegene
interactions; (iii) use of high throughput genomic technology to evaluate the role of
copy number variants and to detect private and regulatory mutations, neither of
which have been studied to date; (iv) detailed analysis of patient samples stratified
by phenotype to enable, for example, hypothesis-driven testing of candidates genes
in groups of NTDs with specific defects of folate metabolism, or in groups of fetuses
with well-defined phenotypes such as craniorachischisis.
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INTRODUCTION
Congenital malformations are the leading cause of infant mortality in developed
countries and a major cause of health problems in surviving children. Neural tube
defects (NTDs) are a common group of central nervous system anomalies affecting
0.52 per 1000 pregnancies worldwide. NTDs arise when the neural tube, the
embryonic precursor of the brain and spinal cord, fails to close during neurulation.
The cranial region (anencephaly) or the low spine (open spina bifida;
myelomeningocele) are most commonly affected although, in the severe NTD
craniorachischisis, almost the entire neural tube remains open, from midbrain to low
spine.
Most individuals who survive with NTDs (particularly myelomeningocele) have a
multiple system handicap and a limited life expectancy. However, despite the high
prevalence and traumatic consequences for affected individuals and their families,
the causes of NTD are poorly understood. Identification of causative factors is
confounded by the fact that the majority of these malformations appear to result
from a combination of genetic and environmental factors. A strong genetic
component is indicated by the high recurrence risk for siblings of affected individuals
(1,2). Syndromic cases of NTD also exist, often associated with chromosomal
anomalies, but these represent <10% of all defects (1,35). The majority of NTDs are
sporadic, with recurrence fitting a multifactorial polygenic or oligogenic pattern,
rather than models on the basis of single gene dominant or recessives, with reduced
penetrance (2).
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GENETIC ANALYSIS OF HUMAN NTDS

Positional cloning strategies have been hampered by the paucity of large families
with multiple affected members. Nevertheless, genome-wide studies using
collections of smaller multiplex families have implicated chromosomes 2, 7 and 10 as
harbouring candidate risk loci for spina bifida (68). Although the causative genes
are yet to be identified, these studies may result in identification of candidate
sequences that can be evaluated in larger populations. An alternative approach
exploits the association of NTDs with chromosomal anomalies such as trisomies 13
and 18 (9), suggesting that gene-dosage can affect neural tube closure.
Rearrangements involving deletions, duplications or balanced translocations are
likely to be most informative, with fine mapping of chromosomal breakpoints
enabling identification of specific loci (10).
In some studies, direct mutation screening of candidate genes has been carried out
in cohorts of patients (11), but the vast majority involve statistical association
analysis of sequence variants in or near candidate genes. Most work has involved
casecontrol analysis, comparing the frequency of risk alleles in affected individuals
and/or mothers with a matched unaffected cohort. More sophisticated studies have
used the transmission disequilibrium test (TDT) in family trios (mother, father and
affected child), which is less dependent on population structure. In the remainder of
this article, we review the main candidate gene studies which have arisen primarily
from analysis of folate metabolic pathways and mouse models of NTDs. Boyles et al.
(11) published a comprehensive review of this field up to 2004, and an updated
candidate gene list is presented in Table 1.

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Table 1.

Candidate gene analysis in human NTDs

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CANDIDATE GENES FROM FOLATE METABOLISM

Epidemiological studies provide an opportunity to identify risk factors for NTDs, such
as dietary or teratogenic agents, to which susceptibility may be modified by genetic
predisposition (1214). Among environmental factors, folate status plays a key role
in determining NTD risk (15,16). Maternal supplementation with folic acid during
pregnancy reduces NTD frequency (17,18) whereas reduced serum folate and/or
elevated homocysteine (an inverse indicator of folate status) are observed in some
mothers of NTD fetuses, and are considered risk factors for NTDs (1921). However,
NTDs are not simply a condition of folate deficiency: maternal folate levels in most
human NTD-affected pregnancies are in the normal range (22), suggesting that low
folate status may increase susceptibility but is not directly causative. Similarly, in
mice dietary folate deficiency can cause significant embryonic growth retardation
but does not cause NTDs (16,23,24). Hence, sub-optimal folate status may predispose to NTDs in combination with additional factors, either environmental or
genetic.
The intricate interplay and cross-regulation between elements of one-carbon (folate)
metabolism (Fig. 1) complicates the teasing out of events that impinge on neural
tube closure. In mice, key cellular functions in the developing embryo include
methylation reactions and biosynthesis of nucleotides that support rapid cellular
proliferation (2,25). Cranial NTDs arise when the methylation cycle is inhibited
(26,27), and in null embryos for DNA methyltransferase 3B (28). In contrast,
exogenous homocysteine does not cause NTDs (2931), even in genetically
predisposed splotchembryos (24) and may be an indicator of impaired folate or
methylation cycle activity.

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Figure 1.

Summary of folate one-carbon metabolism showing the main pathways and

reactions that have been subject to analysis in the context of NTDs. Blue shading:
proteins involved in processing of folates in the digestive tract, transport and cellular
retention (by addition of glutamates). Yellow shading: the major part of the cycle
involving transfer of 1C groups between folate molecules, as required for purine and
pyrimidine biosynthesis. Pink shading: reactions of the methylation cycle. For clarity,
mitochondrial reactions that include generation of formate and cleavage of glycine
have been omitted. For explanation of abbreviations, see Table 1.
If folate status interacts with genetic factors in the causation of NTDs, this could
involve either folate-related or folate-independent genes. To date, most emphasis
has been placed on the evaluation of folate-related genes as NTD candidates (32,33)
(Table 1). Further support comes from analysis of primary cell lines obtained from
NTD fetuses, which indicates that a genetically-determined abnormality of folate
metabolism is present, in at least a proportion of cases (34). However, identifying
specific NTD-predisposing genetic lesions has proven far from straight forward.
Although a number of variants have been widely studied, inconsistent results
between different cohorts and populations (Table 1) indicate that very few, if any,
have a major causative effect. Below, we sub-divide the candidate folate-related
genes into three functional categories.
Methylation related genes

Among folate-related genes, 5,10-methylene tetrahydrofolate reductase (MTHFR) has

been the principal focus of attention, following reports that the 677C>T (A222V;
rs1801133) polymorphism is associated with increased risk of NTDs in Dutch and
Irish populations (3537). Other populations show no association (38,39) or even a
protective effect (40,41) (Table 1). A meta-analysis, including genotype data from 27
studies up to 2004, suggests that the 677TT genotype confers an overall 1.9 times
increase in NTD risk (Odds ratio: 1.9; 95% confidence interval: 1.62.2) (15). A more
recent meta analysis (42) found a positive association only in non-latin groups,
principally the Irish population.
The action of MTHFR generates 5-methylTHF for remethylation of homocysteine, at
the expense of other folates required for purine and pyrimidine biosynthesis (Fig. 1).
The A222V variant protein has reduced function and is associated with elevated
plasma homocysteine (36). Nullizygosity for MTHFR in mice also results in elevated
homocysteine and diminished DNA methylation (43), although NTDs are not
observed under either normal or folate-deficient conditions. Moreover, MTHFR
nullizygosity does not exacerbate the folate-responsive splotch mutation (4345).

These data suggest that in populations where MTHFR is a risk factor, additional
interacting factors are likely to be present.
The link between reduced methylation/elevated homocysteine and NTDs has
prompted analysis of variants in other genes that could influence the methylation
cycle through remethylation (MTR, MTRR, BHMT and BHMT2) or transsulfuration
(CBS) of homocysteine (11) (Table 1). In general, mildly elevated risks have been
identified in some studies but rarely replicated. MTRR (methionine synthase
reductase) functions to maintain activity of MTR (methionine synthase), and a
variant form (I22M, encoded by 66A>G) was reported as an NTD case and maternal
risk factor in some studies, but not others (Table 1). Mouse studies do not support a
role for these genes in NTDs: targeted deletion of Mtr is embryonic lethal prior to
neurulation stages and heterozygotes do not show NTDs (46). Similarly, reduced
activity of Mtrr and loss of cbs function do not cause NTDs, although elevated plasma
homocysteine is observed (47,48).
Folate cycle enzymes required for nucleotide biosynthesis

MTHFD1 encodes the cytoplasmic trifunctional C1THF synthase enzyme. A

polymorphism (1958G>A; rs2236225) which results in an R653Q substitution in the
10-formylTHF synthetase domain was found to be both a maternal and NTD case risk
factor, in the Irish and Italian populations (4951), although not in the Dutch (51,52)
or British (41). The R653Q polymorphism causes reduced C1THF synthase activity in
cell lines, resulting in diminished purine biosynthesis (53). A promoter polymorphism
(rs1076991C>T) in MTHFD1, that reduces transcriptional activity in vitro, was also
associated with NTD case and maternal risk, in combination with R653Q (54).
Folate transport

Another attractive group of candidate genes are those encoding proteins required for
transport, uptake and cellular retention of folates. This includes folate
receptors FR (Folr1 in mice), FR and FR, RFC1 (reduced folate
carrier), GCPII (folyl--glutamate carboxypeptidase) and FPGS(folylpolyglutamate
synthetase) (32,33). Increased risks associated with variants in RFC1 and GCPII are
not reproduced in all studies (Table 1), although the recently identified protoncoupled folate transporter PCFT(SLC46a1) is not required for embryonic survival or
neural tube closure in mice (55), but has not yet been investigated in humans. A
recent casecontrol study revealed a possible association with reduced risk of spina
bifida for a polymorphism in CUBN (Cubulin), which encodes a membrane-associated
multi-ligand endocytic receptor expressed in the neural folds and yolk sac (56).
Together, cubulin and its partner protein megalin are involved in binding and
endocytic uptake of a large number of different proteins, several of which could be
important for neural tube closure, including the intrinsic factor-cobalamin complex
(IF-B12) and folate binding protein (folate receptor) (57,58). Intriguingly, Cubn was
one of the most up-regulated genes in a microarray analysis of Rfc1 null mouse
embryos (59), which may reflect a compensatory mechanism to enhance endocytic
folate uptake via Folr1. Hence, CUBN merits further attention as a potential risk
factor, especially in conjunction with RFC1.
In view of the apparent resilience of mouse neurulation to specific genetic
disturbance of the methylation cycle, analysis of compound mutants with other
folate-related or NTD susceptibility alleles would be of considerable interest. In our
analysis of NTD cell lines, impaired folate cycle activity did not correlate with known
variants in MTHFR, MTHFD1, DHFR, GCPII, MTR,MTRR or RFC1 (34), encouraging the
view that currently unknown genetic influences on folate metabolism remain to be
identified in many NTD cases.
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CANDIDATE GENES FROM THE MOUSE

The potential complexity of NTD genetics is illustrated by the fact that 200 or more
different mouse genes result in NTD phenotypes either through naturally occurring,
induced or targeted mutations (2,25). Many of the NTD-causing mouse mutations
implicate specific signalling pathways such as non-canonical Wnt signalling (see
below), maintenance of the cell cycle, regulation of the actin cytoskeleton, chromatin
organization or epigenetic modifications including methylation and acetylation.
Recently, NTDs were observed in mice null for Mib2 (60), Smurf1/2 (61)
and Hectd1 (62), which all encode E3 ubiquitin ligases, suggesting a possible role in
neurulation for protein ubiquitination and targeted degradation. The human
homologues of some of these mouse NTD genes have been examined in casecontrol
association studies or directly sequenced in mutation screens, although with very
few significant findings to date (Table 1).
It is important to ask how appropriate are the mouse models as paradigms for
human NTDs? At the embryonic level, the events of neurulation appear extremely
similar between mice and humans. For example, the initial fusion event, Closure 1,
occurs at a closely similar stage and body axial level in both species, as does
initiation of closure in the forebrain (Closure 3) and completion of spinal closure at
the posterior neuropore. One point of variation concerns de novo initiation of closure
at the forebrain/midbrain boundary (Closure 2 in mice) which may be absent from
human neurulation (63). Hence, brain closure could be a rather simpler process in
humans than mice.
Another potential difference between mouse models and human NTDs is that many
gene-specific homozygous null mouse embryos exhibit phenotypes additional to
NTDs, such as prenatally lethal heart defects. Such syndromic examples do not
appear particularly close models for human NTDs which are primarily non-syndromic
(64). On the other hand, detailed analysis of a few of the mouse mutants suggests
that isolated NTDs can also result from the effect of hypomorphic alleles,
combinations of heterozygous mutations, genetic background effects and/or geneenvironmental interactions. This partial loss of function or multifactorial aetiologies
may more closely resemble human NTDs. For example, NTDs in splotch mice result
from homozygosity for mutations in Pax3 (23,65) but can also occur, or be
exacerbated, as a result of interaction with mutations in other genes
including neurofibromin1 (66) and grainyhead-like-3 (67). Environmental factors
including folate deficiency and arsenic can exacerbate NTDs in
homozygous splotch mutants, or induce NTDs in the usually unaffected
heterozygotes (24,68). Although association studies in humans have provided little
evidence to implicate PAX3 mutations in human NTDs (69,70), the possible
contribution of genegene and geneenvironment interactions indicates that larger
scale studies may be needed before a role for PAX3 in human NTDs can be
completely ruled out.
The curly tail mouse also exhibits features typical of the multifactorial aetiology of
human NTDs (71). Spinal NTDs are partially penetrant in homozygous ct/ct mutant
embryos, with the frequency of defects strongly affected by genetic modifiers (72).
The major ct gene is a hypomorphic allele of Grhl3, whose knockouts display
completely penetrant spina bifida (7375). The ct mutation appears to affect a
regulatory region, emphasising the need for consideration of possible non-coding
mutations in human NTDs. Moreover, there is a strong effect of environmental
factors, including a protective effect of supplemental inositol (76). A key role for
inositol in neural tube closure is supported by the finding that inositol deficiency in
vitro causes NTDs (77), inositol may prevent diabetes-associated NTDs (78) and the
recent finding of NTDs in embryos carrying a hypomorphic allele of inositol 1,3,4trisphosphate 5/6-kinase (Itpk1), a key enzyme in inositol phosphate metabolism
(79).
Planar cell polarity signalling and NTDs

A major advance in understanding the genetic basis of neurulation has been the
finding that initiation of closure at the hindbrain-cervical boundary (Closure 1)
requires non-canonical Wnt signalling: the so-called planar cell polarity (PCP)
pathway (Fig. 2). PCP signalling was defined originally in Drosophila, as a genetic
cascade involving the transmembrane receptor frizzled and the cytoplasmic protein
dishevelled, but without a requirement for -catenin (8083). This pathway is
required to specify planar polarity in epithelia including the wing and compound eye.
In vertebrates, non-canonical Wnt signalling is highly conserved, underpinning tissue
and cellular polarity during morphogenesis in systems as diverse as gastrulation and
the coordinated orientation of stereociliary bundles in inner ear hair cells (8490).

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Figure 2.

Diagrammatic representation of non-canonical Wnt signalling in a mammalian cell.

Black arrows indicate the signalling pathway necessary for establishment of planar
cell polarity (PCP). Known biochemical interactions are indicated by blue arrows and
genetic interactions are shown by red arrows. Ankdr6 is the mammalian homologue
of Diego which in Drosophila interacts with Fz, Vang and Pk, but has not been studied
in vertebrates.
A potential role for PCP in NTDs first came to light following positional cloning
of Vangl2 (the homologue of Drosophila strabismus/Van gogh) in the loop-tail mouse
mutant which exhibits the severe NTD, craniorachischisis (91,92). Subsequently, the
same NTD phenotype was found in other mouse mutants and targeted gene
knockouts (Table 2) almost all of which have been implicated biochemically in the
PCP pathway (e.g. Celsr1, Dvl) or which interact genetically with recognized PCP
genes (e.g. Scrb, Ptk7) (93,94). Interestingly, the double knockout
for Smurf1 andSmurf2 was recently found to display craniorachischisis and other
characteristic PCP defects. These genes encode ubiquitin ligases whose targets
include Prickle1 (Fig. 2), supporting the crucial nature of PCP signalling for initiation
of neural tube closure (61).

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Table 2.

Genes of the planar cell polarity pathwayinvolvement in mouse NTDs

In view of these findings in mice, PCP genes emerge as excellent candidates for
causation of craniorachischisis in humans. Nevertheless, sequence analysis has so
far failed to identify mutations in human VANGL2or its paralogue VANGL1 in a group
of patients with craniorachischisis (95,96). Reports of other PCP gene analysis in
similar patients are awaited. Although craniorachischisis is the obvious NTD
phenotype for study, theVANGL genes have also been analysed in patients with
anencephaly and open and closed spina bifida. No mutations were reported
in VANGL2(95,96) but several highly conserved and unique, heterozygous missense

variants were identified in VANGL1 in patients with either myelomeningocele or

closed spina bifida, as well as caudal regression syndrome (96,97). To date a
functional effect has been demonstrated for one of these putative mutations, where
V239I (identified in caudal regression syndrome) results in loss of interaction
between VANGL1 and DVL proteins (96,).
Interestingly, loss of Vangl1 function is insufficient to cause NTDs in mice, although
compound heterozygotes with Vangl2 (loop-tail) develop craniorachischisis (98).
Nevertheless, there is increasing evidence that PCP genes can contribute to NTDs
other than craniorachischisis (Table 2). For example, double heterozygotes carrying
both Vangl2 and Ptk7 develop spina bifida (94) although Vangl2 double mutants
with cordon-bleuC101 orCthrc1 develop exencephaly (99,100). In
contrast, Vangl2:Scrb andVangl2:Dvl3 double heterozygotes develop
craniorachischisis (101,102). It remains to be determined why Vangl2 displays this
variable phenotypic behaviour when combined with different PCP and other mutants.
Hence, although non-canonical Wnt signalling has been firmly linked with Closure 1
in mice, it is possible that genes in this pathway play more diverse roles in human
neural tube closure.
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CONCLUSIONS
The identification of genetic risk factors for human NTDs is complicated by the
multiplicity of genes participating in neurulation, and the importance of gene
environment interactions. Sequence analysis of candidate genes implicated from
their role in mouse models has revealed putative mutations in a few genes, but each
in only a small number of patients. Association studies of common polymorphic
variants, particularly related to folate one-carbon metabolism, indicate risk factors
such asMTHFR. However, no specific folate-related gene has yet been implicated as a
major determinant of risk for NTDs. Large-scale studies will be required to provide
sufficient statistical power to convincingly test whether such variants are truly NTD
susceptibility factors (56,103). It will also be essential, to evaluate multiple genes
(folate-related and others) in the same individuals in order to detect possible
compounding effects of combinations of risk alleles that, individually, might not be
statistically significant (11,39). To date, very few studies have been sufficiently large
to overcome issues of multiple testing bias in screening for genegene interactions
(39,56,104). Examination of specific hypotheses may be fruitful where fewer NTD
cases are available, particularly if combined with stratified sample sets in which
cases are sub-divided on the basis of phenotype. For example, NTDs with abnormal
folate metabolism have enabled a combined analysis of MTR and MTRR (105), and
fetuses with craniorachischisis provide a focus for determining the role of PCP genes.
Geneenvironment interactions appear likely to contribute to NTD predisposition,
with examples including interactions of MTHFR with multivitamin use (106), MTRR
with vitamin B12 (107) and PDGFRA with inositol and zinc (108).
One limitation of the association studies of multiple folate-related candidate genes in
NTDs is the predominant focus on known polymorphisms. In future, it will be
necessary also to consider the possible existence of private disease-causing
mutations. Moreover, the potential for deleterious gene expression changes resulting
from promoter mutations or copy number variation has been addressed in relatively
few studies (10,108110). Emerging technologies for high throughput sequencing
and analysis of genomic deletions and copy number variations (111) offer the
prospect, in the coming years, of progress in identification of candidate genes and
screening for novel mutations in human NTDs.