Genetic and Functional Assessment of the Role of the rs13431652-A and rs573225-A Alleles in the G6PC2 Promoter That Are Strongly Associated With Elevated Fasting Glucose Levels

Abstract

OBJECTIVEGenome-wide association studies have identified a single nucleotide polymorphism (SNP), rs560887, located in a G6PC2 intron that is highly correlated with variations in fasting plasma glucose (FPG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. This study examines the contribution of two G6PC2 promoter SNPs, rs13431652 and rs573225, to the association signal.RESEARCH DESIGN AND METHODSWe genotyped 9,532 normal FPG participants (FPG <6.1 mmol/l) for three G6PC2 SNPs, rs13431652 (distal promoter), rs573225 (proximal promoter), rs560887 (3rd intron). We used regression analyses adjusted for age, sex, and BMI to assess the association with FPG and haplotype analyses to assess comparative SNP contributions. Fusion gene and gel retardation analyses characterized the effect of rs13431652 and rs573225 on G6PC2 promoter activity and transcription factor binding.RESULTSGenetic analyses provide evidence for a strong contribution of the promoter SNPs to FPG variability at the G6PC2 locus (rs13431652: β = 0.075, P = 3.6 × 10−35; rs573225 β = 0.073 P = 3.6 × 10−34), in addition to rs560887 (β = 0.071, P = 1.2 × 10−31). The rs13431652-A and rs573225-A alleles promote increased NF-Y and Foxa2 binding, respectively. The rs13431652-A allele is associated with increased FPG and elevated promoter activity, consistent with the function of G6PC2 in pancreatic islets. In contrast, the rs573225-A allele is associated with elevated FPG but reduced promoter activity.CONCLUSIONSGenetic and in situ functional data support a potential role for rs13431652, but not rs573225, as a causative SNP linking G6PC2 to variations in FPG, though a causative role for rs573225 in vivo cannot be ruled out.

Moving on from GWAS: Functional Studies on the G6PC2 Gene Implicated in the Regulation of Fasting Blood Glucose

“…Two SNPs in the G6PC2 promoter, rs13431652 and rs2232316, were shown to affect G6PC2 fusion gene expression by modulating NF-Y and Foxa2 binding, respectively [90, 50]. In addition, two SNPs in the third G6PC2 intron, rs560887 and rs2232321, were shown to affect G6PC2 RNA splicing [50], likely by modulating the strength of a branch point sequence, a key element in RNA splicing [91, 92].…”

“…In addition, two SNPs in the third G6PC2 intron, rs560887 and rs2232321, were shown to affect G6PC2 RNA splicing [50], likely by modulating the strength of a branch point sequence, a key element in RNA splicing [91, 92]. The in vitro and in situ molecular data suggest that all four SNPs are potentially causative since the allele that results in elevated G6PC2 expression is associated with elevated FBG [90, 50]. In contrast, for another G6PC2 promoter SNP, rs573225, that also affects G6PC2 fusion gene expression by modulating Foxa2 binding, the allele that results in elevated G6PC2 expression is associated with reduced FBG [90, 50], suggesting that rs573225 is a functional SNP that opposes the action of causative SNPs on G6PC2 expression [90, 50], a conclusion that contrasts with an earlier study [93].…”

“…First, because these SNPs are in high linkage disequilibrium, it is difficult to definitely determine whether one or all of these SNPs are truly causative [90, 50]. Second, because the G6PC2 gene is only expressed in pancreatic islet beta cells and because the effects of these SNPs are subtle, the lack of sufficient human samples has limited the ability to correlate genotypes with endogenous G6PC2 expression.…”

“…Second, because the G6PC2 gene is only expressed in pancreatic islet beta cells and because the effects of these SNPs are subtle, the lack of sufficient human samples has limited the ability to correlate genotypes with endogenous G6PC2 expression. Finally, multiple caveats are associated with analyzing G6PC2 promoter SNPs using fusion genes in islet-derived cell lines [90, 50]. Furthermore, most of these cell lines are derived from rodent islets and recent studies suggest the existence of significant differences between rodent and human islets [94].…”

Genetic Variants in CCNB1 Associated With Differential Gene Transcription and Risk of Coronary In-Stent Restenosis

“…21 In summary, our findings suggest that allele-dependent transcriptional regulation of CCNB1 associated with the SNPs rs350099, rs350104, and rs164390 affects ISR risk through differential recruitment of NF-Y, AP-1, and SP1 (Figure 8). Interestingly, Bouatia-Naji et al 42 allele of rs13431652 in the G6PC2 promoter generates a functional NF-Y-binding CCAAT box and is strongly associated with elevated fasting plasma glucose in humans. Moreover, a mutation in the CCAAT box of the TERC promoter that abrogates NF-Y binding has been associated with human telomere disease, thus providing further evidence that allelespecific differences in the recruitment of NF-Y can contribute to human disorders.…”