The folate pathway and variation in the genes encoding its enzymes play a central role in the etiology of birth defects [Bailey and Berry, 2005
; Boyles et al., 2009
; Eskes, 2006
; Patterson, 2008
; van der Linden et al., 2006
]. Maternal supplementation with folate in the periconceptional period protects against nonsyndromic CHDs [Pei et al., 2006
; van Beynum et al., 2006
]. Individuals with DS have abnormal folate metabolism; therefore, the potential role for altered DNA, or amino acid synthesis, or epigenetic effects in the etiology of DS-associated CHDs is intriguing [Chadefaux et al., 1985
; Pogribna et al., 2001
]. Also of interest, genes for two of the major components of the folate pathway, CBS
, are located on chromosome 21. CBS
plays an integral role in regulating folate metabolism by converting homocysteine into cystathionine, while SLC19A1
is the primary regulated transporter of 5-methyltetrahydrofolate into and out of the cytoplasm (). Overexpression of CBS
, which occurs with trisomy 21, creates a functional folate deficiency [Pogribna et al., 2001
]. Thus, cellular levels of many folate pathway components, such as homocysteine, methionine, SAM, and SAH, are altered in individuals with DS. Given the high risk for CHDs, particularly AVSDs, among individuals with DS, we tested SNPs in MTHFR
, and SLC19A1
for association with cases of DS and AVSD compared to controls with DS and no CHD.
At the gene level, cases affected with AVSD showed a significantly increased proportion of alleles shared across SLC19A1 than expected by chance (P = 0.01). Follow-up analysis of this association through individual SNP tests provided evidence consistent with association to a functional variant in or near SLC19A1. Based on the haplotype structure in CEPH pedigrees, these SNPs are in LD with rs1051266 (c.80A>G), a nonsynonymous coding variant in SLC19A1. We hypothesize that this variant may be the functional polymorphism, contributing to increased risk of AVSD in this population ().
Although the biochemical consequence of this SLC19A1
coding variant (c.80A>G, p.27H>R) has not been established, the c.80A>G has been studied in conjunction with birth defects frequently associated with dietary and metabolic folate deficiency, including neural tube defects, orofacial clefts, and heart defects. An association between c.80G and spina bifida was observed only in conceptions where the mother did not supplement with folic acid, while there was no genetic effect from rs1051266 on orofacial clefts [Shaw et al., 2002
]. Variants in SLC19A1
have been associated with conotruncal defects independent of maternal supplementation status, but the effects were further exacerbated if the mother did not supplement with folic acid during fetal heart development [Shaw et al., 2003
]. Similarly, Pei et al. 
observed that an offspring with a c.80G allele was at four times greater risk of any CHD, if the mother did not take a folate-containing supplement, an association further confirmed by family-based testing of the c.80G allele with CHD.
While the SNP and LD data are consistent with a functional role for SLC19A1 in AVSD susceptibility, this region of LD extends into the 3′ region of COL18A1 (). Fine mapping of the extended region, including genotyping of rs1051266, will help to determine whether the association with AVSD susceptibility is due to SLC19A1, COL18A1, or both.
The ancestral and fully enzymatically functional allele of MTHFR
, c.1298A [Marini et al., 2008
], also showed a unique pattern of association with DS-associated AVSD. The A allele was over-transmitted to cases (P
= 0.05, permuted P
= 0.272), under-transmitted to controls (P
= 0.02, permuted P
= 0.495; and ), and significantly associated with AVSD in FBAT analysis under both a dominant and an additive model (P
= 0.03 and P
= 0.01, respectively; ). While these associations do not withstand correction for multiple testing, the opposing pattern of transmission between cases and controls is compelling and warrants further study.
Previous studies have reached conflicting conclusions on the role of the c.1298A>C variant in nonsyndromic CHDs. Van Driel et al. 
observed a preponderance of c.1298AC and c.1298CC genotypes in cases affected with various CHDs and their fathers. Hobbs et al. 
, though, observed exactly the opposite—a significant under-transmission of the c.1298C allele to offspring affected with septal, conotruncal, or left/right obstructive defects. Most studies of nonsyndromic CHDs, though, have more commonly identified the c.677T allele or c.677TT genotype as a risk factor [Junker et al., 2001
; van Beynum et al., 2006
; Wenstrom et al., 2001
]. The T allele of c.677, similar to the C allele of c.1298, results in decreased enzymatic function due to increased thermolability [Frosst et al., 1995
]. For example, van Beynum et al. 
observed a three-fold increased risk of having a child with a variety of CHD for mothers with the c.677CT genotype and six-fold increase for mothers with the c.677TT genotype. Consistent with these results, Botto et al. 
observed that the c.677T allele is more prevalent in the Hispanic population, a group that is particularly susceptible to CHD. The data presented here suggest that variation in MTHFR
contributes less to the etiology of DS-associated cases of AVSD than nonsyndromic cases of CHD. These conclusions are complicated by the nonsyndromic studies investigating multiple and varied CHDs, while the present study looks specifically at cases of complete AVSD.
Given that our cases have both DS and AVSD and that folate polymorphisms have been associated with the occurrence of DS, we must be mindful that up to 80% of DS conceptuses are lost prior to birth [Hassold and Jacobs, 1984
]. The highly selected nature of the sample population could lead to identification of alleles or genotypes associated with survival of the offspring to term rather than those associated with abnormal heart development. A variant associated with survival of a fetus with trisomy 21, regardless of CHD status, should not be detected by an association in this DS-case/DS-control comparison, but would show over-transmission in both case and controls trios. In contrast, a variant associated with disease susceptibility, or survival of the fetus with that specific disease, would show a different pattern: a significant association in a case/control comparison and over-transmission in DS-case trios, but not in DS-control trios [Kerstann et al., 2004
]. The disproportionate over-transmission of the c.1298A allele to cases and the opposing under-transmission of c.1298A alleles to controls provide convincing evidence that c.1298A is associated with susceptibility to AVSD, not survival with trisomy 21. With diminished power in the trisomic TDT, we were unable to make as definitive an argument with respect to SLC19A1
variants, although the significant association among cases compared with controls suggests SLC19A1
variation contributes to AVSD susceptibility.
affect the level of 5,10-methylenetetrahydrofolate available in cells. SLC19A1
is a ubiquitously expressed transmembrane protein responsible for the regulated transport of 5-methyltetrahydrofolate, the physiologically active form of folate, into the cytoplasm [Chango et al., 2000
; Taparia et al., 2007
converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, the substrate for the conversion of homocysteine into methionine. Chango et al. 
suggest that the c.80G allele of SLC19A1
decreases the transport of folates into the cytoplasm, resulting in a functional folate deficiency. Conversely, the c.1298A allele of MTHFR
(p. 429E) is more enzymatically active than the c.1298C allele (p.429A); the associated variants of these two enzymes both function to limit the amount of available 5,10-methylenetetrahydrofolate. In dividing cells, 5,10-methylenetetrahydrofolate is a key substrate for DNA and RNA synthesis, whereas the product of MTHFR
, 5-methyltetrahydrofolate, is the methyl donor for generating methionine from homocysteine. Our data, suggesting diminished function of SLC19A1
and proper function of MTHFR
, support the hypothesis of Hobbs et al. 
, wherein these polymorphisms result in a functional cellular folate deficiency that decreases efficient and accurate DNA and RNA synthesis. Diminished DNA and RNA synthesis could, thereby, impede the proper proliferation of cells in the developing heart. In support of this hypothesis, mice-fed folate-deficient diets where shown to have heart malformations resulting from defects in proliferation [Li and Rozen, 2006
LIMITATIONS AND FUTURE STUDIES
Periconceptional folate supplementation has been recommended since associations with neural tube defects were confirmed in the mid-90s. A meta-analysis by Botto et al. 
, combining a diverse array of study designs and cardiac defects, observed a decrease in the rate of CHD by up to 50% with periconceptional folate supplementation. Combining genotype and maternal dietary folate supplementation data would be a powerful way to assess the role that the folate pathway plays in DS-associated CHDs. Because families were recruited over an extended period of time, we did not have folate supplementation data for the majority of mothers participating in this study.
Although SLC19A1 was significantly associated with AVSD at the gene level, after multiple test correction no individual SNP was significantly associated with AVSD. Thus, the significance of the gene-level test was a result of combined information from several of the tag SNPs across a large LD block. Other associations, such as the association of MTR c.2756G with AVSD in the TDT, are less convincing, and could be a signal of selection effects on survival to term. Since folate pathway gene variants have been associated with CHD in past studies, we felt it important to discuss all nominally significant associations. We acknowledge that these results may be false positives and require replication in a larger population.
This is one of the largest studies of AVSD in people with DS; however, our conclusions are hindered by the small sample size, particularly in trisomy-specific statistical analyses (i.e., trisomic TDT) where power is comparatively low. Continuing efforts, with the benefit of larger cohorts, will replicate current results, examine a greater number of genetic variants, incorporate environmental factors such as folate supplementation, and explore gene-environment interactions to further study the causes of DS-associated AVSD.