Replication and exploratory analysis of 24 candidate risk polymorphisms for neural tube defects

Neural tube defects (NTDs) are common, severe birth defects that affect the development of the spinal cord and/or brain. NTDs mainly present as spina bifida, anencephaly and encephalocele. Although the NTD rate has been reduced in areas with folic acid fortification [1]-[4], reported NTD rates include ~1 in 1000 births in the US [5],[6], 0.1-1.1 in 1000 births in Europe [7], 1–4.8 in 1000 pregnancies in China [3], 3.9-8.8 in 1000 births in India [8], and 0.2-9.6 in 1000 births in Latin America [9]. Both environmental and genetic factors are involved in the development of NTDs. In western countries, dietary folate is the strongest known environmental factor. Mothers with an NTD pregnancy have significantly lower levels of folate [10], and periconceptional folate supplementation can lower NTD risk by 50-70% [3],[11],[12]. The most firmly established genetic factor is a common polymorphism (rs1801133, c.677C > T) in the 5, 10-methylene-tetrahydrofolate reductase (MTHFR) gene. The alternate nucleotide in this single nucleotide polymorphism (SNP) changes an alanine to a valine (p.A222V), resulting in a thermolabile form of the protein [13]. The MTHFR c.677 TT genotype has been significantly associated with NTD cases in many populations (reviewed in [14],[15]). Other NTD risk SNPs have been identified in a number of populations (e.g., rs2236225 (c.1958G > A, p.R653Q) in methylenetetrahydrofolate dehydrogenase (NADP + dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase (MTHFD1) [16]-[21], rs1805087 (c.2756A > G, p.D919G) in methionine synthase (MTR) [15],[16],[22]-[29], rs1051266 (c.80A > G, p.H27R) in the reduced folate carrier (RFC1/SLC19A1) [15],[21],[30]-[33]), and rs70991108 (19 bp indel; c.86 + 59_86 + 60insACCTGGGCGGGACGCGCCA) in dihydrofolate reductase (DHFR) [34]-[36]). The majority of associations have not been subjected to robust replication.

Rigorously establishing genetic risk for any multifactorial disorder is important but inherently difficult. Genetic risk is optimally detected with a large number of racially/ethnically homogenous samples that include cases with a well-defined, highly penetrant phenotype. These are difficult criteria to meet for rare, complex disorders, meaning that statistical power is often compromised to some extent. This, in combination with the bias that significant associations are more likely to be published [37]-[40], contributes to many associations in the literature that would not survive correction for multiple tests.

Specifically in the field of genetic risk for NTDs, studies including large numbers of candidate polymorphisms and/or genes have implicated many candidate SNPs exhibiting nominal NTD associations [21],[41]-[45]. Each of the cited studies examined a minimum of 37 SNPs for NTD risk, yet only the association of rs1907362 in cubilin (CUBN) with NTDs survived correction in the original study [21]. The most likely explanation is that the reported nominally significant associations were due to chance. However, the large number of tests performed, reduced power due to limited availability of samples, and/or the population-specific effect of a risk allele can all contribute to Type II errors. Therefore replication studies are essential for the validation of any genetic risk factor for NTDs.

In one of the largest studies to date we previously reported evaluation of the common genetic variation in 82 candidate genes for NTD risk in an Irish population [45]. The inclusion of 570 NTD cases (~95% spina bifida cases), their parents and 999 controls made it possible to use both family based methods (the transmission disequilibrium test and log-linear analysis) and case–control based methods (logistic regression) to evaluate genetic risk for NTDs in cases (case effect) as well as genetic risk for an NTD pregnancy in mothers (maternal effect). In the current study, we test 24 of the resulting nominally significant association signals using 9 case–control tests and 14 family-based tests in racially/ethnically similar populations. The 9 SNPs that showed nominal significance by case–control analysis in the previously studied Irish NTD cohort were additionally evaluated for NTD risk using varying models in racially/ethnically similar and distinct populations.

The primary aim of this study was to perform replication analyses of the nominally significant NTD associations identified in a recent study in an Irish population [45]. The secondary aim was to test a subset of these candidate SNPs for association using alternate risk models and populations.

Our study addressed two questions. First, are SNPs that were nominally significant upon testing for NTD association in an Irish population also associated with NTDs in a similar, independent population? Second, are the SNPs that were nominally significant upon testing for NTD association in an Irish population by logistic regression also associated with NTDs in different racial/ethnic populations when using a broader range of association tests, genetic models and NTD case groupings?

Our criteria for replication were stringent, and only performed for a SNP when the same association test and genetic model could be applied. Because we did not have samples from mothers of NYS NTD cases, we were unable to test for a maternal risk for nine independent mother-control signals in seven genes. Nine SNPs were re-tested for a case effect by logistic regression in a white NTD sample from NYS, and log-linear analyses were used to re-test ten SNPs for a case effect and five SNPs for a maternal effect in a UK NTD sample. Of 14 SNPs previously observed to be associated with NTDs by family-based analysis in an Irish population, two showed nominally significant NTD association in trios from the UK (Table 2). ADA rs452159 falls in the first intron, and is at the border of a D’ block encompassing the first exon and intron of the gene. MTR rs10925260 is in intron 23 of the gene, and is part of a large block of D’ LD encompassing the entire gene. Due to their strong D’ linkage, these nominally significant associations may reflect a direct signal or that of a causative SNP linked to the tested SNP(s).

Although none of the 9 SNPs found to be nominally significant in Irish NTDs were replicated in isolated NYS white NTD cases, two were nominally associated with NTDs under different conditions. ARID1A rs11247593 did not replicate by logistic regression using a continuous model in isolated NTDs (Table 1), but was significant when all white NTD cases and controls were tested using a dominant model (Table 5). ARID1A rs11247593 is intronic and is part of a large D’ block extending over the entire gene, so the signal may be due to the tested SNP or a linked causative SNP. In addition, ALDH1A2 rs7169289 was significantly associated with isolated NTDs and isolated spina bifida cases in the African American population (Table 5). These results most likely represent a single association signal as the genetic models (continuous, dominant) and NTD sets (isolated NTDs, isolated SB) for these analyses overlap. The estimated effects observed in African American NTD cases from NYS (Table 5; OR = 0.46-0.57, p = 0.017-0.041) are similar to the original effect observed in an Irish population (Table 1; OR = 0.67 [0.52-0.86], p = 0.0016). ALDH1A2 rs7169289 is just downstream of the gene. It falls between two blocks of D’ LD, and the association signal may be due to this SNP or a linked SNP in either block.

As was found in the original study, none of the observed associations in the NYS and UK sample sets would have survived correction for multiple tests. One interpretation is that these associations are indeed due to chance and that the tested SNPs do not contribute to NTD risk. It is not clear, however, that Bonferroni correction is appropriate for replication studies. All the tests performed in this study were clearly based on individual, a priori hypotheses generated for our initial study and supported by previous data. Additionally, factors that can contribute to Type II error should be considered, such as population stratification and genetic heterogeneity.

The most important factor may be limited sample size, which compromises the power to detect true associations. Compared to the number of NTD cases (n ~ 570) in the original Irish cohort, a limited number of white NYS isolated NTD cases (n ~ 190) were available in the current study. With ~530 NTD trios, however, the UK NTD cohort appears comparable, but this is before taking into consideration the power required to replicate an association. Due to the “winner’s curse,” or the tendency of an initial observation to overestimate the effect size or significance of a true association, replication studies generally require larger study sample numbers to detect the original effect [51],[52]. This may explain why the well known NTD risk allele of MTHFR rs1801133 (T) is not associated with NTDs by TDT (p = 0.742) in our large UK NTD cohort. In fact, it is nominally significant as a protective factor in a recessive model by log-linear analysis (GRR = 0.663 [0.441-0.997], p = 0.049). Considering that this association has been replicated in many studies in other populations supporting its role in NTD risk, it would be surprising if the MTHFR rs1801133 TT genotype does not contribute to NTDs in the UK population. A lack of replication power and chance seem the most likely explanations for this failure to replicate, which must be taken into consideration for the other SNPs tested for replication in this study. Authentic validation of any genetic association requires accumulation of evidence over time, involving multiple studies in independent populations.

Lack of adequate power is a pervasive problem in the field of NTD genetics. One recent review estimated that ~1000 cases would be required to attain 80% power to detect an odds ratio of 2 or under, yet approximately one quarter of published NTD association studies used fewer than 100 cases [53]. This problem is compounded when attempting to evaluate candidate risk SNPs in less studied populations. While we were able to examine candidate risk SNPs of interest in African American and Hispanic populations from NYS, far fewer cases were available compared to whites (Table 4). Although some population-based studies have included African American cases in aggregate analyses of genetic risk for NTDs [54]-[56], and there are studies of NTD risk in African cohorts [57], this is the first report of candidate NTD SNPs evaluated in an African American case–control cohort. The nominal association observed for ALDH1A2 rs7169289 may be real, but needs confirmation.

Identifying potential NTD risk SNPs has proven much easier than validating them. A single replication study is not definitive, and the limited numbers in existing NTD cohorts may contribute to subsequent underpowered studies failing to confirm reported associations. By combining data from multiple published studies, meta-analyses increase power and confidence in whether a SNP truly contributes to NTD risk. Meta-analyses involving aggregate data from several hundred to several thousand NTD cases and controls have confirmed MTHFR rs1801133 (c.677C > T) as a maternal [58] and case risk factor for NTDs [14],[15],[58],[59]. Although comparatively fewer data were available, meta-analyses show 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) rs1801394 (c/66A > G) is a maternal risk factor for NTDs [60],[61], while MTR rs1805087 (c/2756A > G) does not contribute to maternal or case risk [15],[22],[60],[62]. However, meta-analysis requires a large amount of data including full genotype information. These data are absent in publications for the majority of NTD risk SNPs that show nominal significance. It is therefore essential to publish all association study data in a format that allows future meta-analyses to be performed (i.e., genotype counts or a way to unambiguously determine them). As such, we have reported the genotype data for all 24 SNPs tested in our study (Additional file 1: Table S1 and Additional file 2: Table S2).

In summary, this study represents both the effort to validate nominally significant NTD associations via replication, and to test a subset of these SNPs in alternate NTD groupings and racial/ethnic populations. The NTD associations we observed with four SNPs would not have survived correction for multiple tests. However, we believe it would be overly severe to apply Bonferroni correction to ADA rs452159 and MTR rs10925260, which were nominally significant by strict replication analysis, as well as ARID1A rs11247593, which was found to be associated by a different model and expanded definition of NTDs. Additionally, to our knowledge, this is the first report of a genetic association with NTDs in African Americans and so the nominal significance of ALDH1A2 rs7169289 in the small NYS NTD cohort warrants follow-up. Further testing in independent populations and meta-analyses are needed to clarify the role of these and a multitude of nominally significant SNPs associated with NTDs.