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. Author manuscript; available in PMC: 2012 Nov 21.
Published in final edited form as: Am J Med Genet A. 2010 Dec 10;155A(1):14–21. doi: 10.1002/ajmg.a.33755

Evaluation of 64 Candidate Single Nucleotide Polymorphisms as Risk Factors for Neural Tube Defects in a Large Irish Study Population

Tonia C Carter 1,*, Faith Pangilinan 2,*, James F Troendle 1, Anne M Molloy 3, Julia VanderMeer 2, Adam Mitchell 2, Peadar N Kirke 4, Mary R Conley 1, Barry Shane 5, John M Scott 3, Lawrence C Brody 2, James L Mills 1
PMCID: PMC3503244  NIHMSID: NIHMS418549  PMID: 21204206

Abstract

Individual studies of the genetics of neural tube defects (NTDs) contain results on a small number of genes in each report. To identify genetic risk factors for NTDs, we evaluated potentially functional single nucleotide polymorphisms (SNPs) that are biologically plausible risk factors for NTDs but that have never been investigated for an association with NTDs, examined SNPs that previously showed no association with NTDs in published studies, and tried to confirm previously reported associations in folate-related and non-folate-related genes. We investigated 64 SNPs in 34 genes for association with spina bifida in up to 558 case-families (520 cases, 507 mothers, 457 fathers) and 994 controls in Ireland. Case-control and mother-control comparisons of genotype frequencies, tests of transmission disequilibrium, and log-linear regression models were used to calculate effect estimates. Spina bifida was associated with over-transmission of the LEPR (leptin receptor) rs1805134 minor C allele (genotype relative risk (GRR): 1.5; 95% confidence interval (CI): 1.0, 2.1; P = 0.0264) and the COMT (catechol-O-methyltransferase) rs737865 major T allele (GRR: 1.4; 95% CI: 1.1, 2.0; P = 0.0206). After correcting for multiple comparisons, these individual test P-values exceeded 0.05. Consistent with previous reports, spina bifida was associated with MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA promoter haplotype combinations. The associations between LEPR SNPs and spina bifida suggest a possible mechanism for the finding that obesity is a NTD risk factor. The association between a variant in COMT and spina bifida implicates methylation and epigenetics in NTDs.

Keywords: congenital abnormalities, folic acid, neural tube defects, single nucleotide polymorphism, spina bifida

INTRODUCTION

Neural tube defect (NTD) prevalence ranges between 0.5 and 5.0 per 1,000 pregnancies [Feuchtbaum et al., 1999; McDonnell et al., 1999]. The findings of increased prevalence (2–5%) in siblings of affected individuals [Sebold et al., 2005] and in same-sex twins (including monozygotic pairs) [Windham and Sever, 1982], compared with the general population, suggest that genetic factors contribute to NTD etiology. The observation that maternal periconceptional folic acid supplementation reduces the risk for NTDs by 50–70% has identified folate as an important environmental factor [MRC Vitamin Study Research Group, 1991; Czeizel and Dudas, 1992]. Homozygosity for a common variant in the folate gene 5,10-methylenetetrahydrofolate reductase, MTHFR 677C>T, has been associated with NTDs but only 11–26% of cases can be attributed to this variant [Kirke et al., 2004]. Animal, and some human, studies suggest that both folate-related and non-folate-related genes play a role in the etiology of NTDs.

We used several strategies to look for genetic risk factors for NTDs. First, we chose single nucleotide polymorphisms (SNPs) that have not been evaluated previously for NTD risk but that have potential functional consequences and are located in genes that could be biologically related to neural tube development. Second, we examined a group of biologically plausible candidate SNPs previously tested for association with NTDs but found to be unassociated. In these mostly small studies, this lack of association could have resulted from inadequate power. Third, we sought to confirm associations between NTDs and SNPs in folate-related and non-folate-related genes reported by others, often using smaller sample sizes than were available in this study. Fourth, now using a larger study population, we re-examined some SNPs that have been investigated by our research group.

MATERIALS AND METHODS

Cases and controls

Details of case and control recruitment have been described previously [Pangilinan et al., 2010; Kirke et al., 1993]. NTD-affected families (583 full and partial trios that included 544 cases, 531 mothers, and 478 fathers) were recruited in the Republic of Ireland from 1993 to 2004 with assistance from the Irish Association for Spina Bifida and Hydrocephalus and the Public Health Nurses. Controls (N = 999) were randomly selected from a bank of blood samples obtained between 1986 and 1990 from 56,049 pregnant women receiving prenatal care at the three main maternity hospitals in Dublin, excluding women whose past or current pregnancies were affected with an NTD. Informed consent was obtained for the collection of all blood and buccal samples. Buccal cells were collected onto cotton swabs and placed in stabilizing buffer before being processed. Ethical approval for the study was provided by the Research Ethics Committee of the Health Research Board of Ireland and the Institutional Review Board of the National Human Genome Research Institute.

SNP selection

We examined a total of 64 SNPs in 34 genes (for gene names and functions see supporting information Table I which may be found in the online version of this article). Six SNPs (MTHFR 677C>T and 1298A>C, MTR D919G, T (Brachyury) rs3127334, UCP2 -866G>A and A55V) were previously evaluated by this research group, albeit with a smaller number of study subjects (<400 cases or case-families) [Brody et al., 1999; Shields et al., 1999; Shields et al., 2000; Parle-McDermott et al., 2003; Kirke et al., 2004; Mitchell et al., 2009]. Another group of 12 SNPs (ALDH1A2 rs3784260, rs3784259, and rs16939660, BHMT rs3733890, BRCA1 rs1799966, CCL2 rs1024611, ERCC2 rs13181, LEPR rs1137100, NAT1 rs15561, PDGFRA rs6554162, rs1800812, and rs1135534) were previously reported by other groups to be associated with NTDs [Joosten et al., 2001; Zhu et al., 2004; Deak et al., 2005; Jensen et al., 2005; Olshan et al., 2005; Jensen et al., 2006; King et al., 2007; Davidson et al., 2008; Shaw et al., 2009].

The remaining 46 SNPs (Table I) either were previously evaluated for NTD risk and no association was observed (5 SNPs: BHMT2 rs626105, CITED2 rs1131431, LEPR rs1137101 and rs8179183, SHH rs1233555), were examined as components of haplotypes associated with NTDs (4 SNPs: NAT1 rs4986782, rs4986783, and rs56172717, PEMT rs7946), or had not been tested previously for an association with NTDs (37 SNPs). The 46 SNPs were selected from genes related to folate/homocysteine metabolism (BHMT2, CBS, CTH, GGH, GNMT, PEMT, TK1), craniofacial morphogenesis (IRF6, MSX1, SHH), obesity which is a risk factor for NTDs (LEP, LEPR), and genes that cause NTDs in animals (BRCA1, CITED2, DNMT3B, LRP6). We also included SNPs in genes that have been associated with other disease phenotypes (ABCG2, CDKN2A, COMT, HSPA8, NAT1, NDOR1, NOS3, PADI4, RAC2); these associations suggest that these SNPs could have functional consequences. We included SNPs from this last group of genes in our study because these genes regulate diverse cellular processes that could be involved in neural tube development and because one of our goals was to investigate novel candidate SNPs for NTDs.

Table I.

Single nucleotide polymorphisms investigated for associations with neural tube defects.

Gene SNP Description SNP association with protein function or disease phenotype Referencea
ABCG2 rs2231142 Q141K reduced ABCG2 activity; associated with nonpapillary renal cell carcinoma Mizuarai et al., 2004; Korenaga et al., 2005
BHMT2 rs626105 intron NTD study examined rs626105 as a BHMT2 marker Zhu et al., 2005; Shaw et al., 2009
BRCA1 rs3737559 intron breast cancer Cox et al., 2005; Tommasi et al., 2005
BRCA1 rs8176166 intron
BRCA1 rs16942 K1183R
BRCA1 rs799917 P871L
CBS rs234706 Y233Y reduced risk of coronary artery disease Kruger et al., 2000
CDKN2A rs3731249 A148T malignant melanoma Debniak et al., 2005
CITED2 rs1131431 3′UTR present in some patients with congenital heart defects Sperling et al., 2005
COMT rs737865 intron reduced COMT activity, plasma homocysteine concentration, schizophrenia Lavigne et al., 1997; Goodman et al., 2001; Shifman et al., 2002; Chen et al., 2004; Tunbridge et al., 2008
COMT rs4680 V158M
CTH rs1021737 S403I plasma homocysteine concentration Wang et al., 2004
DNMT3B rs6087990 5′ near gene evaluated for risk of primary lung cancer Lee et al., 2005
GGH rs11545078 T151I evaluated because amino acid change might affect protein function Cheng et al., 2004
GGH rs719235 5′ near gene SNPs in promoter region could affect GGH expression Chave et al., 2003
GGH rs3758149 5′ near gene
GGH rs719236 5′ near gene
GNMT rs10948059 5′ near gene plasma homocysteine concentrations Beagle et al., 2005
HSPA8 rs1136141 5′UTR mild mental impairment Butcher et al., 2005
IRF6 rs2235371 V274I oral clefts Zucchero et al., 2004
IRF6 rs2013162 S153S
LEP rs7799039 -2548G>A overweight/obesity, glucose homeostasis Mammes et al., 2000; Mammes et al., 2001; Yiannakouris et al., 2001; Lakka et al., 2004
LEP rs2167270 -39G>A
LEP rs2060713 intron
LEPR rs3790435 5′UTR
LEPR rs1137101 Q223R
LEPR rs1805134 S343S
LEPR rs8179183 K656N
LRP6 rs2302685 V1062I bone fractures in elderly men van Meurs et al., 2006
MSX1 rs33946149 intron evaluated for association with oral clefts Beaty et al., 2001; Fallin et al., 2003
MSX1 rs1042484 intron
MSX1 rs34165410 G116G
NAT1 rs4986782 R187Q association with spina bifida when SNPs were tested as components of a haplotype Jensen et al., 2006
NAT1 rs4986783 S214A
NAT1 rs56172717 D251V
NDOR1 rs62587579 V522I reduced NDOR1 activity Finn et al., 2005
NOS3 rs2070744 intron vascular invasion in breast cancer Ghilardi et al., 2003
PADI4 rs11203366 S55G rheumatoid arthritis Suzuki et al., 2003; Harney et al., 2005
PADI4 rs11203367 A82V
PADI4 rs874881 A112G
PADI4 rs1748033 L117L
PADI4 rs2240339 intron
PEMT rs7946 V175M reduced PEMT activity; possible association with nonalcoholic fatty liver disease Song et al., 2005
RAC2 rs13058338 intron anthracycline-induced cardiotoxicity Wojnowski et al., 2005
SHH rs1233555 intron associated with dideoxy DNA fingerprinting pattern of gene fragment containing exon 2 of SHH Zhu et al., 2003
TK1 rs1132543 V106M reduced enzyme stability Berenstein et al., 2000
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a

References are listed in supplemental material (see supporting information Supplement I which may be found in the online version of this article)

Abbreviation: SNP – single nucleotide polymorphism

Genotyping

Genomic DNA was extracted from blood or buccal samples using the QIAamp DNA Blood Mini Kit (Qiagen, Sussex, UK). Genotyping was performed by either of two methods: the Illumina GoldenGate assay (Illumina, San Diego, CA) or Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (Sequenom, San Diego, CA) of allele-specific extension products. Genotyping quality was assessed by repeat genotyping 2.5% of samples on the Illumina GoldenGate platform and 10% of samples using MALDI-TOF mass spectrometry; reproducibility exceeded 99.5% for each platform. For 10 SNPs, genotype concordance was assessed between the two genotyping methods. Concordance was observed among >98.5% of samples for each of the 10 SNPs. Of the 33 SNPs genotyped using the Illumina GoldenGate assay, the proportion of successful genotype calls was ≥95% for 25 SNPs, 90.0–94.9% for 7 SNPs (COMT rs737865, IRF6 rs2235371, MSX1 rs1042484, PADI4 rs874881 and rs1748033, PDGFRA rs6554162, UCP2 -866G>A), and 86% for 1 SNP (MSX1 rs33946149). Of the 31 SNPs genotyped using MALDI-TOF mass spectrometry, ≥95% of genotypes were called successfully for 30 SNPs; 93% of genotypes were called successfully for one SNP (LEP rs2167270).

All SNPs were tested for Hardy-Weinberg equilibrium separately among controls, cases, case-mothers, and case-fathers. Using a cutoff of P = 0.01, one SNP was not in Hardy-Weinberg equilibrium among controls (GGH rs719235, P = 0.00017). There was no deviation from Hardy-Weinberg equilibrium among cases, case-mothers, and case-fathers for any of the SNPs tested (P > 0.01).

Exclusions

Individuals with discordant genotypes, or trios displaying non-Mendelian inheritance for one or two SNPs were excluded from analyses for those SNPs. Trios that showed non-Mendelian inheritance for >2 SNPs (20 cases and their parents) and individuals that had discordant genotypes for >2 SNPs (5 controls, 4 cases, 4 case-mothers, 1 case-father) were excluded from all analyses. The remaining 558 case families and 994 controls were included in the analysis.

Linkage disequilibrium

Linkage disequilibrium (LD) measures were estimated using Haploview (http://www.broadinstitute.org/haploview/haploview) [Barrett et al., 2005] based on the genotypes of control samples.

Statistical methods

Most of the case families had a child affected with spina bifida; therefore, the main analysis was restricted to this defect group. Analyses were repeated adding case families affected by other NTDs. Logistic regression was used to model dominant and recessive effects of genetic risk. These tests were applied to case-control and mother-control analyses to generate odds ratios (ORs) and 95% confidence intervals (CIs). Family-based analyses, which included the transmission disequilibrium test (TDT) and log-linear analysis, produced estimates of genotype relative risk (GRR) and 95% CIs. The log-linear models were fitted to test dominant and recessive effects of genetic risk; they assessed case effects as well as direct maternal effects. We adjusted for multiple comparisons using permutation (N = 9,999). Multivariate permuting of trios for the TDT and log-linear analysis was performed by treating the test as a one-sample test, and permuting the hypothetical risk allele. Permutations of trios were independent of those for cases and controls, and the results were combined by Bonferroni adjustment so that the resulting adjusted P-values accounted for all comparisons while controlling the probability of any false-positives at 5%.

The six SNPs previously examined by this group were tested using case-control, mother-control, and log-linear analyses. The 12 SNPs previously reported by other groups to be associated with NTDs were tested using analyses similar to those in the original reports [Joosten et al., 2001; Zhu et al., 2004; Deak et al., 2005; Jensen et al., 2005; Olshan et al., 2005; Jensen et al., 2006; King et al., 2007; Davidson et al., 2008; Shaw et al., 2009]. We performed case-control and mother-control analyses to test for the recessive effect of PDGFRA promoter haplotype combinations associated with low or high transcriptional activity. Low transcriptional activity was signified by either the joint presence of the A and T alleles for PDGFRA rs6554162 (-1467G>A) and rs1800812 (-794G>T), respectively, or the presence of the A allele for PDGFRA rs1135534 (-909C>A); other haplotype combinations indicated high transcriptional activity. Because we tested a priori hypotheses, the analyses for these 18 SNPs were not adjusted for multiple comparisons.

Of the other 46 SNPs (Table I), 37 were novel, unpublished candidates for NTD risk and nine were previously tested in mainly small study populations. We evaluated these SNPs by case-control, mother-control, and log-linear analyses, and the TDT. These analyses were adjusted for multiple comparisons by the permutation method described above.

RESULTS

The 558 case families included 449 full trios, 21 case-mother pairs, 5 case-father pairs, 45 cases only, and 38 families with parents only. A family history of NTDs was reported by 31.7% (177/558) of families. For the 31 SNPs genotyped by MALDI-TOF mass spectrometry, all samples were tested (558 case families and 994 controls). For the remaining 33 SNPs genotyped on the Illumina GoldenGate platform, 314 case families and 338 controls were tested (Table II). The genotype distribution for each SNP was obtained for controls, cases, case-mothers, and case-fathers (see supporting information Table II which may be found in the online version of this article).

Table II.

Irish case families that had a child affected with a neural tube defect.

Study subjects Total Subset genotyped on Illumina platform
Case families 558 314
 Anencephaly 7 5
 Spina bifida 528 298
 Encephalocele 23 11
All neural tube defects
 Cases 520 295
 Case mothers 507 284
 Case fathers 457 270
Spina bifida
 Cases 498 284
 Case mothers 477 268
 Case fathers 436 259
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For the six SNPs that we previously investigated, we increased the size of the case group by 100 or more subjects, compared with our earlier reports. In case-control analysis, spina bifida was associated with homozygosity for the MTHFR 677C>T (rs1801133, A222V) minor T allele (OR: 1.3; 95% CI: 1.0, 1.6; P = 0.0451). The T (Brachyury) rs3127334 major A allele was also associated with spina bifida in case-control analysis (OR: 2.4; 95% CI: 1.1, 5.7; P = 0.0288; dominant model). There was no association with MTHFR 1298A>C (rs1801131, E429A), MTR D919G (rs1805087), UCP2 -866G>A (rs659366), and UCP2 A55V (rs660339), similar to our previous reports.

Our results for variants reported to be NTD risk factors by other groups (Table III) show that spina bifida was associated with transmission of the LEPR (leptin receptor) rs1137100 (K109R) major A allele (GRR: 1.4; 95% CI: 1.0, 1.8; P = 0.0236) and with PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) haplotype combinations with high transcriptional activity (OR: 1.5; 95% CI: 1.0, 2.0); P = 0.0252; recessive effect). No association with spina bifida was observed for the other SNPs reported in previous studies.

Table III.

Effect estimates for associations between spina bifida and specific SNPs or haplotypes previously reported to be associated with neural tube defects.

Gene SNP/Haplotype Description Allele Analysis Effect estimate (95% CI)
ALDH1A2 rs3784259 intron A (major allele) TDT GRR: 1.0 (0.8, 1.4)
ALDH1A2 rs3784260 intron C (minor allele) TDT GRR: 0.9 (0.7, 1.2)
ALDH1A2 rs16939660 A151A C (minor allele) TDT GRR: 1.3 (0.3, 6.0)
BHMT rs3733890 R239Q A (minor allele) Case-control, recessive model OR: 1.4 (0.9, 2.1)
BRCA1 rs1799966 S1613G G (minor allele) TDT GRR: 1.2 (0.9, 1.6)
CCL2 rs1024611 5′ near gene C (minor allele) Mother-control, dominant model GRR: 0.9 (0.7, 1.2)
ERCC2 rs13181 K751Q G (minor allele) Case-control, dominant model OR: 1.1 (0.9, 1.4)
LEPR rs1137100 K109R A (major allele) TDT GRR: 1.4 (1.0, 1.8)
NAT1 rs15561 1095C>A A (minor allele) Log linear, case effect, dominant model GRR: 1.0 (0.7, 1.3)
Log linear, maternal effect, dominant model GRR: 1.1 (0.8, 1.5)
PDGFRA Promoter haplotype combinations that affect transcriptional activity Low activity: (rs6554162 = A and rs1800812 = T) or rs1135534 = A; Other = high activity - Case-control analysis, recessive for low transcriptional activity OR: 1.0 (0.5, 1.9)
Mother-control analysis, recessive for low transcriptional activity OR: 1.1 (0.6, 2.2)
Case-control analysis, recessive for high transcriptional activity OR: 1.5 (1.0, 2.0)
Mother-control analysis, recessive for high transcriptional activity OR: 0.7 (0.5, 1.0)
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Abbreviations: SNP – single nucleotide polymorphism, TDT – transmission disequilibrium test, GRR – genotype relative risk, OR – odds ratio, CI – confidence interval

We examined associations for the other 46 SNPs that either showed no association with NTDs in previous reports or were not tested previously for an association with NTDs (see supporting information Tables III and IV which may be found in the online version of this article); eight were associated with spina bifida (Table IV). Two of these SNPs were associated with spina bifida based on the results of two different statistical tests. Increased spina bifida risk was associated with the LEPR rs1805134 minor C allele when tested using the TDT (GRR: 1.5; 95% CI: 1.0, 2.1; P = 0.0264) and log-linear analysis (GRR: 1.7; 95% CI: 1.1, 2.6; P = 0.0179), and with the COMT (catechol-O-methyltransferase) rs737865 major T allele when tested using the TDT (GRR: 1.4; 95% CI: 1.1, 2.0; P = 0.0206) and log-linear analysis (GRR: 2.1; 95% CI: 1.1, 4.3; P = 0.0364). Correction for multiple comparisons performed for the 46 SNPs rendered these P values > 0.05. Adding case families with other NTDs produced few changes in the results obtained.

Table IV.

Results of statistical analyses of associations between spina bifida and candidate single nucleotide polymorphisms.

Gene SNP Description Allele Analysis method Effect estimate (95% CI) Uncorrected P-valuea
ABCG2 rs2231142 Q141K C Case-control, recessive model OR: 1.5 (1.0, 2.1) 0.0449
ABCG2 rs2231142 Q141K C Mother-control, recessive model OR: 1.5 (1.0, 2.3) 0.0312
BRCA1 rs3737559 intron G Case-control, recessive model OR: 1.9 (1.2, 2.9) 0.0075
BRCA1 rs3737559 intron G Mother-control, recessive model OR: 2.1 (1.3, 3.4) 0.0026
BRCA1 rs799917 P871L C Mother-control, dominant model OR: 1.8 (1.1, 3.1) 0.0282
COMT rs737865 intron T Mother-control, dominant model OR: 0.4 (0.2, 0.8) 0.0105
COMT rs737865 intron T Transmission disequilibrium test GRR: 1.4 (1.1, 2.0) 0.0206
COMT rs737865 intron T Log-linear, case effect, dominant model GRR: 2.1 (1.1, 4.3) 0.0364
DNMT3B rs6087990 5′ near gene T Log-linear, mother effect, recessive model GRR: 1.4 (1.0, 1.9) 0.0356
LEPR rs1805134 S343S C Transmission disequilibrium test GRR: 1.5 (1.0, 2.1) 0.0264
LEPR rs1805134 S343S C Log-linear, case effect, dominant model GRR: 1.7 (1.1, 2.6) 0.0179
LEPR rs8179183 K656N C Log-linear, case effect, recessive model GRR: 2.4 (1.1, 5.5) 0.0439
SHH rs1233555 intron C Log-linear, case effect, recessive model GRR: 1.7 (1.2, 2.5) 0.0023
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After adjustment for multiple comparisons using permutation (N = 9,999), these P-values exceeded 0.05.

Abbreviations: SNP – single nucleotide polymorphism, OR – odds ratio, GRR – genotype relative risk, CI – confidence interval

Multiple SNPs in BRCA1 and LEPR that showed an association with spina bifida did not exhibit intragenic LD. BRCA1 rs1799966 and rs799917 were in strong LD (r2 = 0.97); neither was in strong LD with BRCA1 rs3737559 (r2 < 0.22). LEPR rs1137100, rs1805134, and rs8179183 were not in strong LD (r2 < 0.20).

DISCUSSION

We used several strategies to select SNPs to assess as genetic risk factors for NTDs. We investigated potentially functional variants that have not previously been evaluated for NTD risk, examined candidates that previously showed no association but were deserving of further study, and attempted to confirm previously reported associations in folate-related and non-folate-related genes. We observed statistically significant associations between spina bifida and MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA haplotype combinations with high transcriptional activity. Although not statistically significant after correction for multiple comparisons, spina bifida was associated with COMT rs737865 and LEPR S343S using both the TDT and log-linear analysis.

Among the candidate SNPs that have never been investigated as NTD risk factors or that previously showed no association with NTDs, most were not associated with spina bifida in our study population, but there were some notable exceptions. SNPs in LEPR and COMT showed associations with spina bifida using two different statistical tests. There are conflicting reports on the importance of LEPR as a risk factor for spina bifida [Shaw et al., 2000; Davidson et al., 2008]. We observed that the LEPR K109R major K allele was associated with increased spina bifida risk, confirming a previous report of over-transmission of the major K allele to spina bifida cases [Davidson et al., 2008]. Our finding of associations between spina bifida and three LEPR variants that are not in strong LD with each other, and the higher NTD risk in the offspring of obese women [Waller et al., 1994], suggest that LEPR is a strong candidate for additional study. COMT encodes catechol-O-methyltransferase, an enzyme that transfers a methyl group from S-adenosylmethionine to catecholamines, and thus generates homocysteine. The V158M polymorphism (rs4680) occurs in up to 75% of Caucasians and gives rise to enzymes of substantially different activity such that MM homozygotes have between two and three times lower activity than VV homozygotes [Lavigne et al., 1997; Goodman et al., 2001]. The high activity enzyme (VV) has been linked to elevated plasma homocysteine, especially in the presence of the MTHFR TT genotype [Tunbridge et al., 2008]. SNPs in this gene have not been tested previously for NTD risk, but the finding that mothers carrying NTD-affected fetuses have higher homocysteine concentrations [Mills et al., 1995] makes COMT a good candidate gene for NTD risk assessment. We found no effect of the V158M polymorphism on spina bifida risk, but our observation of increased risk with the intronic SNP, rs737865, suggests that a role for this gene in NTD risk should be investigated further.

We also observed associations between spina bifida and other variants that have not been previously reported as NTD risk factors (ABCG2 Q141K, BRCA1 rs3737559 and P871L, DNMT3B rs6087990, and SHH rs1233555), although correction for multiple comparisons made these results not statistically significant. Nevertheless, it is biologically plausible that these variants could be related to NTDs. ABCG2 is a protein involved in xenobiotic transport; and it can transport methotrexate, an antifolate, and mono- and poly-glutamyl folates across cell membranes [Chen et al., 2003]. Brca1-mutant mice have disorganized neuroepithelia and NTDs [Gowen et al., 1996]. DNMT3B encodes a DNA methyltransferase required for de novo DNA methylation during embryogenesis and Dnmt3b-deficient mice have NTDs [Okano et al., 1999]. SHH signaling is involved in patterning of the brain and spinal cord [Chiang et al., 1996] and SHH mutations are associated with craniofacial malformations in humans [Roessler et al., 2009]. While our results did not withstand correction, in combination with these reports, they suggest that variants in ABCG2, BRCA1, DNMT3B, and SHH could be involved in neural tube development.

Other studies have identified potential genetic risk factors for NTDs that require confirmation. We attempted to confirm some of these associations. In addition to our finding for LEPR K109R, we also observed an association with promoter haplotypes of PDGFRA, a gene associated with NTDs in animals [Soriano, 1997]. Haplotypes with low transcriptional activity have been associated with increased NTD risk in some human studies [Joosten et al., 2001; Zhu et al., 2004; Toepoel et al., 2009] but not all [Au et al., 2005]. In our study, being homozygous for PDGFRA haplotypes with high transcriptional activity was associated with spina bifida. We did not, however, confirm associations between spina bifida and a variant (R239Q) in BHMT, a folate/homocysteine enzyme, or other biologically plausible candidate SNPs (ALDH1A2 rs3784260, rs3784259, and rs16939660, BRCA1 rs1799966, CCL2 rs1024611, ERCC2 rs13181).

Using a larger study population, we evaluated variants in folate-related and non-folate-related genes that had been previously examined by our research group. Maternal folate deficiency has been identified as the most significant environmental factor influencing NTD risk; however, the mechanism through which this deficiency leads to NTDs is unknown. Our results bolster the evidence that MTHFR 677C>T, a variant in a folate enzyme gene, is associated with NTDs. There have been conflicting reports [Morrison et al., 1996; Richter et al., 2002; Speer et al., 2002] regarding an association with the rs3127334 variant of T (Brachyury) which encodes a transcription factor expressed in the notochord during neurulation [Herrmann, 1991]. In this study, we observed an association between this SNP and spina bifida, similar to our previous report [Shields et al., 2000].

A major strength of our study was the large number of affected families for which genotype data were available; this was valuable in our examination of previously reported associations, some of which were from studies with small sample sizes. Other strengths included the use of an ethnically homogenous study population and the ability to examine maternal effects as well as case effects. However, we lacked data on environmental exposures and the small number of families affected by anencephaly and encephalocele did not permit us to examine associations separately for these sub-groups. There were nine SNPs for which a genotype call rate of <95% was obtained, which could cause concern about the reliability of these data. Low call rates can be associated with poorly performing genotype assays. Such assays can introduce bias by over- or under-calling one allele. For these nine SNPs, additional data suggest that the genotypes are being accurately called. These SNP assays were highly reproducible (>99.5%) upon repeat genotyping, the SNPs were all in Hardy-Weinberg equilibrium, and the genotype and allele frequencies we obtained were consistent with those reported in reference populations (that is, HapMap). Although limited resources did not permit us to perform genotyping of all subjects on the Illumina GoldenGate platform, there was 80% power to detect an OR of at least 1.5 in case-control analyses for an allele frequency of 10%. The larger sample size genotyped by MALDI-TOF mass spectrometry further improved the power of the study: in case-control analyses, there was 80% power to detect an OR of at least 1.4 for an allele frequency of 10%, and an OR of at least 1.6 for an allele frequency of 5%.

In contrast to the NTD cases, which were recruited from the entirety of the Republic of Ireland, our controls were randomly selected from a large group of women attending their first prenatal care visit at the three main maternity hospitals in Dublin. Approximately one-third of all births in the Republic of Ireland, and 90% of all births in and around the Dublin area, occur in these hospitals. We investigated SNPs in genes located on autosomes, and previous reports indicate that, among this group of subjects, the allele frequencies of the genes on autosomes reflect those of the underlying Irish population [Parle-McDermott et al., 2003; Kirke et al., 2004]. Therefore, our results are unlikely to be biased by the use of this control group.

Our results add to our knowledge of NTD genetics. We confirmed associations in folate-related and other genes that were previously reported by others. In addition, we tested a number of variants that have not been previously examined for an association with NTDs. Among these novel candidate variants, SNPs in LEPR and COMT showed associations with spina bifida using two different statistical tests. Our results for the leptin receptor gene, LEPR, suggest that the increased risk of NTDs associated with obesity could involve leptin signaling. In a similar fashion, our findings for a variant in COMT implicate a specific methylation reaction that may interact with folate and should be investigated further in relation to NTDs.

Supplementary Material

Supplementary Data

Acknowledgments

This work was supported by the Intramural Research Programs of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Human Genome Research Institute. We are grateful to the families who participated in this study and for their recruitment by the Irish Association for Spina Bifida and Hydrocephalus and the Public Health Nurses. We thank Tracey Claxton for critical technical assistance. We also thank the staff of the Center for Inherited Disease Research at Johns Hopkins University for carrying out the Illumina-based genotyping.

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