Insights into the molecular mechanism for type 2 diabetes susceptibility at the KCNQ1 locus from temporal changes in imprinting status in human islets

Abstract

The molecular basis of type 2 diabetes predisposition at most established susceptibility loci remains poorly understood. KCNQ1 maps within the 11p15.5 imprinted domain, a region with an established role in congenital growth phenotypes. Variants intronic to KCNQ1 influence diabetes susceptibility when maternally inherited. By use of quantitative PCR and pyrosequencing of human adult islet and fetal pancreas samples, we investigated the imprinting status of regional transcripts and aimed to determine whether type 2 diabetes risk alleles influence regional DNA methylation and gene expression. The results demonstrate that gene expression patterns differ by developmental stage. CDKN1C showed monoallelic expression in both adult and fetal tissue, whereas PHLDA2, SLC22A18, and SLC22A18AS were biallelically expressed in both tissues. Temporal changes in imprinting were observed for KCNQ1 and KCNQ1OT1, with monoallelic expression in fetal tissues and biallelic expression in adult samples. Genotype at the type 2 diabetes risk variant rs2237895 influenced methylation levels of regulatory sequence in fetal pancreas but without demonstrable effects on gene expression. We demonstrate that CDKN1C, KCNQ1, and KCNQ1OT1 are most likely to mediate diabetes susceptibility at the KCNQ1 locus and identify temporal differences in imprinting status and methylation effects, suggesting that diabetes risk effects may be mediated in early development.

FIG. 1.

Schematic representation of imprinting control [as described in (17)] and type 2 diabetes-associated SNPs at 11p15.5. A: Chromosome 11:2,450,000–2,960,000. The closed circle at the DMR represents a high level of methylation; the open circle represents a low level of methylation. The transcribed sequence is shown in dark gray, and untranscribed is shown in light gray. Arrows indicate direction of transcription. B: Chromosome 11:2,650,000–2,880,000. The smaller region is distinguished by exonic (boxes) and intronic regions of KCNQ1 (dark gray), a region of KCNQ1OT1 transcription (light gray), and relative positions of top disease-associated SNPs (rs231362, chr11:2,691,471, and rs2237985, chr11:2,857,194). All genomic coordinates are b37/hg19 (graphics not to scale).

FIG. 2.

A: Imprinting status of genes in the 11p15.5 cluster CDKN1C. Fragment analysis traces demonstrate monoallelic (imprinted) expression of CDKN1C in adult islet (top) and fetal pancreas (bottom) samples. Left: genomic DNA, with the two size peaks characteristic of a heterozygote for del171APVA highlighted. Right: cDNA from the same heterozygous samples. In both cases, only one size peak is evident, indicating the presence of mRNA from only one chromosome. Every sample heterozygous for del171APVA at the gDNA level appeared homozygous at the cDNA level, indicating monoallelic expression. B: Imprinting status of genes in the 11p15.5 cluster KCNQ1, KCNQ1OT1, PHLDA2, and SLC22A18. Plots demonstrate flexibility of imprinting at the 11p15.5 cluster. The x-axis represents genotypes of reporter coding SNPs within the relevant gene (numbering arbitrary). The left- and right-hand clusters contain the two homozygote groups, whereas the center cluster contains heterozygous samples. The y-axis represents the ratio of mRNA expression level of a fluorescent probe specific to one allele of the coding SNP against the expression level of a differently labeled probe specific to the alternative allele. The two homozygous groups are therefore expected to have relatively high and low ratios, representing substantially more amplification of the probe specific for one allele. Under biallelic expression (all adult islet samples and PHLDA2 and SLC22A18 fetal pancreas samples), heterozygous samples form a central cluster with a ratio of ∼1, indicating equal expression from homologous chromosomes. Under monoallelic (imprinted) expression (KCNQ1 and KCNQ1OT1 fetal pancreas samples), heterozygote samples separate into two distinct groups, corresponding to the two homozygous clusters and indicating expression from only one chromosome.

FIG. 3.

Methylation according to rs2237895 type 2 diabetes risk genotype. Box plots show the effect of risk allele number (x-axis) on methylation level (y-axis) separated by tissue type. Boxes represent quartiles; whiskers encompass values within 1.5 times the interquartile range. Risk genotype is significantly associated with increased methylation levels at the CTCF binding site and Beckwith-Wiedemann diagnostic CpG island in fetal samples only and at the PLAGL1 binding site in adult islets only. T2D, type 2 diabetes.

FIG. 4.

Total expression according to rs2237895 type 2 diabetes risk genotype. Box plots show the effect of risk allele number (x-axis) on total mRNA expression level (y-axis), separated by tissue type. Boxes represent quartiles; whiskers encompass values within 1.5 times the interquartile range. There was no evidence for an effect of risk allele number on expression levels of any of the tested genes in either tissue type (P > 0.05 in all cases). T2D, type 2 diabetes.