A common variant in MTHFD1L is associated with neural tube defects and mRNA splicing efficiency

Abstract

Polymorphisms in folate-related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer, and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs, we considered the more recently identified mitochondrial paralogue, MTHFD1L, as a candidate gene for NTD association. We identified a common deletion/insertion polymorphism, rs3832406, c.781-6823ATT(7-9), which influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying numbers of ATT repeats: Allele 1 consists of ATT(7), whereas Alleles 2 and 3 consist of ATT(8) and ATT(9), respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case-control logistic regression (P=0.002) and by transmission disequilibrium test (TDT) (P=0.001), whereas Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional single nucleotide polymorphism (SNP) genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts.

Figure 1

Analysis of the NTD associated rs3832406 and alternative splicing. A Sequence context of the intron 7 DIP within the polypyrimidine tract adjacent to alternative exon 8a. Allele 1 i.e., (ATT)₇ repeats are illustrated here. Exon 8a is illustrated as the boxed area as defined by Prassanan et al., (2003). B The potential protein products resulting from translation of the MTHFD1L long and short mRNAs. The protein products are identical up to amino acid residue 260. The longer transcript produces a monofunctional mitochondrial enzyme with 10-formyltetrahydrofolate synthetase activity localised to the C-terminal end as illustrated by the black bar. The shorter transcript differs at the extreme C-terminus due to inclusion of alternative exon 8a. The shorter protein lacks a synthetase domain. Based on a figure from Prassanan et al. (2003). C The ratio of MTHFD1L long and short transcripts differs by rs3832406 genotype in Coriell® lymphoblast cell lines. Homozygotes for Allele 1 have a significantly greater proportion (mean is 35% more) of the long transcript relative to the short transcript compared to homozygotes for Allele 2 as assessed by Mann Whitney U test (2-tailed; P= 0.006).

Figure 2

LD plot of pairwise values of D’ for 119 markers within and adjacent to MTHFD1L. The MTHFD1L gene (~250 kb) is depicted by an arrow indicating the direction of transcription. The LD plot was constructed using genotype data from 338 Irish controls. Brackets (a, b, c) mark three regions in which single markers were found to be associated with NTD risk. Region ‘a’ contains the rs3832406 DIP. Each individual polymorphism that showed an association with NTD are indicated by an asterisk (*). The single vertical line indicates the location of the SNP rs6922269:A>G (associated with coronary artery disease risk), which was not included in this LD plot.

Figure 3

Linkage disequilibrium (LD) Map of one associated region of the MTHFD1L Gene (marked region ‘a’ in Figure 2). LD plot of pairwise values of D’ for the 15 markers within the region spanning introns 7-10 of the MTHFD1L gene. Boxed markers were associated with NTD risk. The rs3832406 DIP is the third marker from the left.

Figure 4

A simplified schematic of folate metabolism in the mitochondria and cytoplasm. Based on a figure from Prassannan et al. (2003). THF= tetrahydrofolate. Enzyme abbreviations are underlined adjacent to the reaction(s) that they catalyse. Not all enzymes are included. MTHFD1L: monofunctional enzyme that catalyses the reversible synthesis of 10-formyltetrahydrofolate to formate and tetrahydrofolate. Reaction is shaded in gray. MTHFD1: trifunctional enzyme that catalyses the reversible synthesis of 10-formylTHF to formate and tetrahydrofolate in the cytoplasm and interconverts 5,10-methyenylTHF and 5,10-methyleneTHF via its cyclohydrolase and dehydrogenase activities. MTHFR: carries out the irreversible conversion of 5,10-methyleneTHF to 5-methylTHF that provides one-carbons for S-adenosylmethionine synthesis (SAM). SAM provides the methyl group required for cellular methylation reactions.