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: email@example.com 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.  Trembath et al.  Speer et al.  Dickerson et al.  Klootwijk et al.  Dickerson et al.  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.  Dickerson et al.  Irish Dutch and British German Canadian Population studied Ramsbottom et al.  Morrison et al.  Felder et al.  Morin et al.  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.  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. Stegmann et al.  MTHFR is an enzyme Van der Put et al. within the folate  pathway, and the C677T allele decreases the activity of folate-dependent re methylation of homocysteine Morrison et al.  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.  to the development of the central nervous system; mice lacking the protein have a higher frequency of NTDs MARCKS is important Stumpo et al.  to the development of the central nervous system; mice lacking the protein have a higher frequency of NTDs Klootwijk et al.  Primary folate receptor Trembath et al. responsible for  binding and importing folate; mutations may reduce folate levels H475Y polymorphism Vieira et al.  decreases enzyme activity and is associated with decreased plasma folate levels and increased plasma total homocysteine Afman et al.  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.  Johanning et al.  Cunha AL et al.  Gonzalez-Herrera et al.  Rampersaud et al.  NTD 27 cases, 28 mothers of cases, 23 siblings of cases, and 159 controls Revilla et al.  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.  Polish De Marco et al.  Midwestern US NTD families Trembath et al.  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.  Sample size 271 cases and 218 families Population studied Shields et al.  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.  Gueant-Rodriguez et al.  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.  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.  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.  Polish Activates cobalaminWilson et al.  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.  Canadian Christensen et al.  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.  Converts intracellular Morrison et al. folate and  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.  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  folate levels Exposure to high levels Dickerson et al. or retinoic acid can  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.  Dutch have indicated that deregulated expression of the gene encoding the platelet-derived growth factor alpha receptor (PDGFRA) causes congenital NTDs. Joosten et al.  Dutch Trembath et al.  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.  179 cases German Population studied Felder et al.  Reference PAX genes regulate Chatkupt et al. normal  development, Pax 1 and Pax3 (splotch) knock out mice exhibit severe NTDs Hol et al.  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.  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.  Irish SHMT catalyzes a Heil et al.  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  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,  T (Brachyury), have notochord abnormalities Trembath et al.  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.  Chick and Mouse Models SHH patterns the Vargas  midline neuro-axis and distal limb structures and abnormal expression is seen in mice with NTDs Kirillova et al.  7q36 SHH (Sonic Hedgehog) California Shaw et al.  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.  Polymophisms are Volcik et al.  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  what appeared to be X-linked spina bifida and anencephaly. ZIC2 is in the Brown et al.  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.  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  8 encephalocele, and exencephaly. 183 spina bifida aperta) and 222 German controls Low plasma TC levels Afman et al.  have previously been associated with an increased risk for having a child with an NTD Gueant-Rodriguez et al.  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. 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