Identification and functional analysis of glycemic trait loci in the China Health and Nutrition Survey

Abstract

To identify genetic contributions to type 2 diabetes (T2D) and related glycemic traits (fasting glucose, fasting insulin, and HbA1c), we conducted genome-wide association analyses (GWAS) in up to 7,178 Chinese subjects from nine provinces in the China Health and Nutrition Survey (CHNS). We examined patterns of population structure within CHNS and found that allele frequencies differed across provinces, consistent with genetic drift and population substructure. We further validated 32 previously described T2D- and glycemic trait-loci, including G6PC2 and SIX3-SIX2 associated with fasting glucose. At G6PC2, we replicated a known fasting glucose-associated variant (rs34177044) and identified a second signal (rs2232326), a low-frequency (4%), probably damaging missense variant (S324P). A variant within the lead fasting glucose-associated signal at SIX3-SIX2 co-localized with pancreatic islet expression quantitative trait loci (eQTL) for SIX3, SIX2, and three noncoding transcripts. To identify variants functionally responsible for the fasting glucose association at SIX3-SIX2, we tested five candidate variants for allelic differences in regulatory function. The rs12712928-C allele, associated with higher fasting glucose and lower transcript expression level, showed lower transcriptional activity in reporter assays and increased binding to GABP compared to the rs12712928-G, suggesting that rs12712928-C contributes to elevated fasting glucose levels by disrupting an islet enhancer, resulting in reduced gene expression. Taken together, these analyses identified multiple loci associated with glycemic traits across China, and suggest a regulatory mechanism at the SIX3-SIX2 fasting glucose GWAS locus.

Fig 1. Principal components analysis of allele frequency for 8,403 subjects in the China Health and Nutrition Survey.

Dots representing each subject are colored by the province in which they reside.

Fig 2. Fasting glucose locus near G6PC2 exhibits two association signals in the CHNS.

The first association signal, rs34177044 (red diamond) shows the strongest association in the initial unconditioned analysis of fasting glucose. Coding variant rs2232326 (S324P; blue diamond), remained locus-wide significant after conditioning on rs34177044. The diamonds indicate the lead variants, which exhibited the strongest evidence of association at the locus among 1000 Genomes Project Phase 3-imputed variants. Variants are colored based on LD with the lead variants, rs34177044 (red) and rs2232326 (blue) within 8,403 CHNS subjects.

Fig 3. Pancreatic islet eQTL colocalizes with the fasting glucose GWAS locus.

(A) rs895636 (purple diamond) shows the strongest association with fasting glucose in the CHNS. Variants are colored based on East Asian LD from 1000 Genomes Project Phase 3. (B) rs12712929 (purple diamond) shows the strongest association with expression of SIX3 in pancreatic islets in European ancestry individuals. Variants are colored based on European LD from 1000 Genomes Project Phase 3. (C) Although the LD r² between rs895636 and rs12712929 is moderate in both European and East Asian populations (1000G Phase 3), one variant, rs12712928, exhibits high LD (r²>0.80) with rs895636 in East Asians and rs12712929 in Europeans (arrow). Red font indicates variants above r² of 0.80. Vertical bars indicate the genomic regions examined for allelic differences in regulatory function.

Fig 4. rs12712928 exhibits allelic differences in transcriptional activity and protein binding.

(A) A haplotype of four variant alleles associated with lower fasting glucose, GAGG, at rs10192373, rs10168523, rs12712928, and rs12712929 repeatedly showed greater transcriptional activity in both forward and reverse orientations with respect to SIX3 in MIN6 mouse insulinoma cells compared to the AGCT haplotype and an “Empty Vector” containing a minimal promoter. (B) Analysis of additional haplotypes created by site-directed mutagenesis of rs127129258 alleles show that haplotypes containing, rs12712928-C (GACG and AGCT), exhibited less transcriptional activity than haplotypes containing rs12712928-G (GAGG and AGGT) in the forward orientation. (C) EMSA with biotin-labeled probes containing the C or G allele of rs12712928 show an allele specific band (arrow; lane 7 versus 2) that is competed away more effectively by 45-fold excess of unlabeled probe containing the C allele (lane 8) than the G allele (lane 9). An arrow points to an allele-specific protein complex binding to the C allele. We observed a supershift with the addition of antibodies to the alpha subunit of GABP (denoted by *). (D) Model of rs12712928 as a functional regulatory variant at the SIX3-SIX2 locus. Alleles, including rs12712928-C, are associated with higher fasting plasma glucose levels and lower expression of SIX3 and other transcripts in human pancreatic islets. Arrows indicate the transcription start site (TSS) of the SIX3 and SIX2 genes. An oval represents GABP bound differentially to rs12712928-C, which exhibited lower transcriptional reporter activity compared to rs12712928-G.