Sequence variation in folate pathway genes and risks of human cleft lip with or without cleft palate

Abstract

In an effort to comprehensively interrogate genetic variation in the folate pathway for risk of cleft lip with or without cleft palate(CLP), we evaluated 504 common and rare variants in 35 folate-related genes in a panel of 330 infants with CLP and 367 non-malformed controls. Odds ratios (OR) with 95% confidence intervals were computed for common genotypes. A Case-Control Difference metric was calculated for rare variants to highlight differentially occurring alleles. Interactions between variants and a maternal folate intake variable were also evaluated. In gene-only results, significant odds ratios were observed for multiple variants in the BHMT/BHMT2/DMGDH gene cluster, particularly in Hispanic infants. Also in this cluster, rare variant analysis highlighted a substantial case-control difference in BHMT rs60340837 (synonymous Y284Y). In Hispanics, the ALDH1L1 I812V variant (rs4646750) was the most significant risk allele: OR = 3.8 (95% CI = 1.6–9.2) when heterozygous. In non-Hispanic white infants, we observed significant risk for AHCYL2 rs1095423 (homozygous OR = 3.0, 95%CI 1.1–7.8) and the 68 bp CBS insertion (c.844ins68; heterozygous OR = 2.4, 95%CI = 1.1–5.3). Rare variant analysis in this group revealed case-control differences in MTRR and several other methionine cycle genes, a process implicated previously in clefting risk. In women with low folate intake specifically, increased risks were observed for CBS rs2851391 (OR 3.6, 95%CI = 1.3–9.6) and the R259P nonsynonymous variant of TCN2 (rs1801198; OR = 2.8, 95%CI 1.2–6.3). This comprehensive study provides further direction on candidate loci to help disentangle the folate-related developmental phenomena in human clefting risk.

Accumulation of Rare Coding Variants in Genes Implicated in Risk of Human Cleft Lip with or without Cleft Palate

“…We observed a small number of nominally significant associations that are in agreement with previous studies. For example, association of nonsynonymous SNPs in DMGDH (S279P, homozygous OR = 0.5 (0.3-0.9), p = 0.022) and ALDH1L1 (I812V, heterozygous OR = 2.7 (1.3-5.5), p = 0.008) have been reported previously [Boyles et al, 2009;Marini et al, 2016].…”

“…However, many of the known common pathway SNPs have been evaluated, yet the results are still somewhat unconvincing. The exception to this seems to be the fairly consistent association of the BHMT/BHMT2/DMGDH locus on Chromosome 5 with CLP observed in a number of studies [Marini et al, 2016]. Interestingly, while the locus often shows association, the local SNPs driving the association can vary between studies.…”

“…Variant Calling Accuracy. The accuracy of the variant calls in the filtered dataset was evaluated in two ways: 1) the concordance of genotype calls by Illumina sequencing and by TaqMan allelic discrimination assays (described in Marini et al, 2016) was determined for 8,537 variant calls (mostly common alleles) in 476 samples, and 2) confirmation by Sanger sequencing for the subset of rare variants in genes with striking case-related trends (~15% of all protein-altering variants identified in the study).…”

“…In the first, we established the concordance between the genotypes called from Illumina sequencing with those obtained from TaqMan allelic discrimination assays for many multiply-occurring loci (i.e. not singletons) using TaqMan assays previously described [Marini et al, 2016]. 91 variant positions in 24 genes were compared in 476 samples.…”

“…Although some reports do not observe a reduction in risk associated with maternal folate supplementation [see Little et al, 2008], a recent meta-analysis indicated a significant reduction in risk of CLP with maternal folic acid use [Jahanbin et al, 2018]. Because of this folic acid link, folate/one-carbon pathway genes have been a logical place to look for risk variants [Boyles et al, 2009;Blanton et al, 2011;Marini et al, 2016]. However, many of the known common pathway SNPs have been evaluated, yet the results are still somewhat unconvincing.…”