Our functional analysis and association study of the MTHFD1L
gene has identified rs3832406 as a risk factor for NTDs by impacting on alternative splicing efficiency. This interesting polymorphism resides within the PPT of intron 7; an important element that is recognised by the splicing machinery. The consensus ‘UCUU’ within a pyrimidine rich sequence provides the optimal binding site for the polypyrimidine tract binding protein (PTB) (Perez et al., 1997
). PTB, also known as hnRNP-I, acts as a splicing repressor through interference with a necessary component of the spliceosome, U2AF (Sharma et al., 2005
; Izquierdo et al., 2005
). Splicing repression via
PTB involves binding at sites both upstream and downstream of the exon (Amir-Ahmady et al., 2005
). The upstream site adjacent to the 3′ splice site appears to bind PTB with high affinity i.e., equivalent to the PTB site adjacent to rs3832406 in MTHFD1L
. Cooperative binding with weaker PTB binding site(s) is necessary for splicing repression. MTHFD1L
contains at least four consensus ‘UCUU’ sites located both upstream and downstream of alternate exon 8a. Our data indicates that a change in the length of the polypyrimidine tract at the high affinity PTB binding site interferes with the efficiency of PTB-mediated repression, possibly by weakening cooperative binding of multiple PTBs. A shortened polypyrimidine tract as in Allele 1 (ATT7
) appears to result in less efficient splicing of exon 8a, while extending the tract by 3 bases as in Allele 2 (ATT8
) results in more efficient splicing of exon 8a. The functional consequences of this are that Allele 1 carriers, particularly in the homozygous state, have a higher proportion of functional MTHFD1L mRNA compared to Allele 2 carriers (the shorter alternatively spliced mRNA lacks enzyme activity). Direct assessment of endogenous MTHFD1L protein level and activity is complicated by the difficulty of separating the mitochondrial form of this enzyme from the more abundant cytoplasmic form as illustrated by the controversy that surrounded the existence of MTHFD1L initially (Prasannan et al., 2003).
The association analysis of rs3832406 in our NTD study cohort provides strong evidence that this common variant increases the case risk of an NTD. The case-control analysis association was strongly supported by the TDT analysis which identified opposite effects on risk for two of the alleles. Allele 1 was associated with an increase of NTD risk while Allele 2 was associated with a decreased risk (). Allele 3 appeared to have no impact on NTD risk, possibly by not significantly influencing the ratio of the long to short mRNA form of MTHFD1L; but this requires further investigation. The association of rs3832406 prompted an assessment of other variants within the gene region. Our screen of an additional 118 SNP markers in a subset of our NTD study cohort confirmed the region surrounding and including intron 7 as harbouring variant(s) that influence the risk of NTDs in the Irish population (Figures and ). Haplotype analysis did not detect a significant association with any of the haplotypes and risk of NTDs but haplotype ‘GG1AGAG’ showed a frequency of 13% in controls compared to 19% in cases; while haplotype ‘GA2GGAA’ showed a frequency of 12% in controls compared to 8% in cases. The association of the MTHFD1L gene region incorporating introns 7 to 10 with NTD risk may be due to rs3832406 itself, the DIP in the context of other variants in the haplotype or there may be an unidentified disease causing polymorphism(s) that remain to be identified. However, the location of the rs3832406 within the PPT adjacent to exon 8a, the impact on alternative splicing efficiency and NTD disease association points toward this triallelic polymorphism as the strongest candidate for directly contributing to disease causation. Our analysis also identified two other regions of the MTHFD1L gene as harbouring additional variants that are associated with NTDs.
Evidence is accumulating that the MTHFD1L
gene is not just important for risk of NTDs. A recent genome wide association study also identified MTHFD1L
as a risk factor of coronary artery disease (CAD) in both UK and German populations (Samani et al., 2007
). A lead positive SNP in their analysis, rs6922269:A>G, resides in intron 11 with a recombination hotspot occurring between it and rs3832406. Although not genotyped in the current study, its physical location excludes it from the three SNP clusters showing NTD association (). Thus, it appears that rs6922269:A>G and rs3832406 represent separate risks in their respective disease associations.
Approximately 41% of populations of North European descent are homozygous for Allele 1. Our Q RT-PCR data predict that these individuals could have up to 70% more functional MTHFD1L and this somehow alters their risk of having an NTD. How does having more MTHFD1L increase ones risk of having an NTD? Studies have shown that folate metabolism is compartmentalized between the cytoplasm, mitochondrion (Appling, 1991
) and more recently the nucleus (Anderson et al., 2007
). This compartmentalisation is thought to facilitate the different metabolic roles within the cell (Anderson et al., 2007
). The mitochondrial folate pathway is believed to play an important role during embryogenesis by ensuring an adequate supply of formate and glycine (Christensen and MacKenzie, 2006
). Formate is the preferred one-carbon donor for purine synthesis. Mitochondrial C1
-Tetrahydrofolate Synthase encoded by MTHFD1L
supplies this formate by catalyzing the reversible synthesis of 10-formyltetrahydrofolate to formate and tetrahydrofolate (). Increased production of formate in Allele 1 homozygotes may disrupt the one-carbon flux through the mitochondria and thus, interfere with cellular proliferation. Alternatively, a higher level of formate itself may be toxic to cells which also results in disrupted cellular production during embryogenesis.
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 (more ...)
In conclusion, we have identified an MTHFD1L functional polymorphism that appears to influence NTD disease risk by affecting splicing efficiency. We acknowledge that replication of this association in another population is required to demonstrate whether this gene has relevance for NTDs outside of Ireland. The MTHFD1L gene is now implicated in two distinct common diseases i.e., NTDs and CAD. The role of MTHFD1L in disease risk highlights the importance of folate metabolism in maintaining health.