GWAS identifies population-specific new regulatory variants in FUT6 associated with plasma B12 concentrations in Indians

Abstract

Vitamin B12 is an important cofactor in one-carbon metabolism whose dysregulation is associated with various clinical conditions. Indians have a high prevalence of B12 deficiency but little is known about the genetic determinants of circulating B12 concentrations in Indians. We performed a genome-wide association study in 1001 healthy participants in the Pune Maternal Nutrition Study (PMNS), replication studies in 3418 individuals from other Indian cohorts and by meta-analysis identified new variants, rs3760775 (P = 1.2 × 10-23) and rs78060698 (P = 8.3 × 10-17) in FUT6 to be associated with circulating B12 concentrations. Although in-silico analysis replicated both variants in Europeans, differences in the effect allele frequency, effect size and the linkage disequilibrium structure of credible set variants with the reported variants suggest population-specific characteristics in this region. We replicated previously reported variants rs602662, rs601338 in FUT2, rs3760776, rs708686 in FUT6, rs34324219 in TCN1 (all P < 5 × 10-8), rs1131603 in TCN2 (P = 3.4 × 10-5), rs12780845 in CUBN (P = 3.0 × 10-3) and rs2270655 in MMAA (P = 2.0 × 10-3). Circulating B12 concentrations in the PMNS and Parthenon study showed a significant decline with increasing age (P < 0.001), however, the genetic contribution to B12 concentrations remained constant. Luciferase reporter and electrophoretic-mobility shift assay for the FUT6 variant rs78060698 using HepG2 cell line demonstrated strong allele-specific promoter and enhancer activity and differential binding of HNF4α, a key regulator of expression of various fucosyltransferases. Hence, the rs78060698 variant, through regulation of fucosylation may control intestinal host-microbial interaction which could influence B12 concentrations. Our results suggest that in addition to established genetic variants, population-specific variants are important in determining plasma B12 concentrations.

Figure 1

Meta-analysis forest plots of association of plasma B12 concentrations with (A) rs3760775 in FUT6, (B) rs78060698 in FUT6, (C) rs281379 in FUT2 and (D) genetic score (GS) derived from eight independently associated SNPs (rs12780845 in CUBN, rs602662 in FUT2, rs708686 and rs3760775 in FUT6, rs2270655 in MMAA, rs34324219 and rs34528912 in TCN1 & rs1131603 in TCN2) identified from conditional analysis. GWAS, genome-wide association study; PMNS, Pune Maternal Nutrition Study; PS, Parthenon Study; MMNP, Mumbai Maternal Nutrition Project; ES, effect size.

Figure 2

Trend of plasma B12 concentrations with increasing age in four quantiles derived from eight SNPs genetic score (GS) in (A) Pune Maternal Nutrition Study (PMNS) children and (B) Parthenon Study (PS) children. The horizontal midlines in each group indicate the median point.

Figure 3

Regional plot of FUT6 and functional regulatory marks. Genomic region of FUT6 after meta-analysis showing two new SNPs rs78060698 and rs3760775, and previously reported variants rs3760776 and rs708686 with functional regulatory mark of DNase I hypersensitive sites, transcription factor binding sites and active histone modification marks in UCSC genome browser. The r² values in the regional plot were derived using hg19/1000 Genome Nov 2014 SAN Genome build and LD population in LocusZoom.

Figure 4

Functional analysis of SNPs, rs78060698, rs3760775 and rs3760776 in the FUT6 region by luciferase assay for promoter and enhancer activity. The Y-axis shows different constructs carrying the specific allele of each variant, and the X-axis represents normalized relative luciferase activity to the basal promoter pGL4.10 and enhancer pGL4.23 constructs. Except rs3760775 promoter constructs, each construct carrying the specific allele showed significant promoter or enhancer activity compared to respective basal constructs (P < 0.01).

Figure 5

Consensus binding motif of HNF4α and HNF4γ and electrophoretic-mobility shift assay of rs78060698 alternate alleles. (A) Change in binding motif of HNF4α and HNF4γ due to A and G alleles at rs78060698. (B) Gel shift and super-shift of HNF4α consensus sequence, oligonucleiotide probes of A and G alleles of rs78060698. Arrows indicate the shift and supershift bands. NE, nuclear extract from HepG2 cells; HNF4α con; HNF4α consensus sequence; Ab, Antibody.