Association of levels of fasting glucose and insulin with rare variants at the chromosome 11p11.2-MADD locus: Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study

Abstract

Background: Common variation at the 11p11.2 locus, encompassing MADD, ACP2, NR1H3, MYBPC3, and SPI1, has been associated in genome-wide association studies with fasting glucose and insulin (FI). In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study, we sequenced 5 gene regions at 11p11.2 to identify rare, potentially functional variants influencing fasting glucose or FI levels.

Methods and results: Sequencing (mean depth, 38×) across 16.1 kb in 3566 individuals without diabetes mellitus identified 653 variants, 79.9% of which were rare (minor allele frequency <1%) and novel. We analyzed rare variants in 5 gene regions with FI or fasting glucose using the sequence kernel association test. At NR1H3, 53 rare variants were jointly associated with FI (P=2.73×10(-3)); of these, 7 were predicted to have regulatory function and showed association with FI (P=1.28×10(-3)). Conditioning on 2 previously associated variants at MADD (rs7944584, rs10838687) did not attenuate this association, suggesting that there are >2 independent signals at 11p11.2. One predicted regulatory variant, chr11:47227430 (hg18; minor allele frequency=0.00068), contributed 20.6% to the overall sequence kernel association test score at NR1H3, lies in intron 2 of NR1H3, and is a predicted binding site for forkhead box A1 (FOXA1), a transcription factor associated with insulin regulation. In human HepG2 hepatoma cells, the rare chr11:47227430 A allele disrupted FOXA1 binding and reduced FOXA1-dependent transcriptional activity.

Conclusions: Sequencing at 11p11.2-NR1H3 identified rare variation associated with FI. One variant, chr11:47227430, seems to be functional, with the rare A allele reducing transcription factor FOXA1 binding and FOXA1-dependent transcriptional activity.

Keywords: genetic epidemiology; glucose; human genetics; insulin; molecular genetics.

Figure 1

Regional plots for association of fasting insulin with common and rare variants at the chromosome 11p11.2 locus. (A) Regional association at chromosome 11p11.2. The −log₁₀P value of variant associations with fasting insulin is plotted on the left y-axis versus chromosomal location on the x-axis (NCBI Genome Build 36). Regions targeted for sequencing are marked with black boxes along the x-axis. Common variant associations are shown as grey circles, and rare variant associations as green arrows that mark the −log₁₀ P from meta-analysis of SKAT and the genomic position of the gene region containing the aggregate rare variants tested. DNAse I hypersensitivity sites in pancreatic (blue dots) and hepatic cells (teal dots) are marked by their chromosomal position along the x-axis. The regional recombination rate is plotted in light blue with values on the right y-axis. The grey box indicates the NR1H3 region (meta-analysis P-value = 2.73 × 10⁻³) that is expanded and shown in Panel B. (B) Regional plot of 53 rare variants at NR1H3 and their individual percent contribution to the overall regional meta-analytic SKAT Q test statistic. Variants with functional prediction 1-3 are shown as purple circles and variants with other functional prediction or without annotation by grey circles. The x-axis shows the chromosomal position of the variants with the regions sequenced in black boxes. Additionally, DNAse I hypersensitivity sites in pancreatic (blue dots) and hepatic cells (teal dots) and an ideogram for the NR1H3 gene body (green line) are marked. The left y-axis shows the per-cent contribution of each variant to the overall meta-analysis SKAT Q statistic for the multi-variant test at NR1H3. The marked variant chr11:47227430 is predicted to be functional and has a large contribution to the SKAT Q statistic. The regional recombination rate is plotted in light blue with values on the right y-axis. The inset shows the consensus logo for the FOXA1 binding motif where the chr11:47227430 A-G substitution produces an adenine instead of a guanine in the fifth position, marked in the red box. The relative conservation of each nucleotide in the motif is displayed by its height.

Figure 2

The FI-associated A allele disrupts FOXA1 binding and transactivation potential at chr11:47227430. Panels A and B. Gel electrophoretic mobility shift assays (EMSA) show that FOXA1 binds to the chr11:47227430 site, with the reference G allele having higher affinity for FOXA1 compared to the FI-associated A allele. (A) A DIG-labeled oligonucleotide containing well-characterized FOXA1 binding site (FB) in the albumin gene enhancer was incubated with unprogrammed reticulocyte lysate (lane 1) or reticulocyte lysate programmed to express FOXA1 (Lanes 2-10). A retarded complex (upper arrowhead) is seen after nondenaturing acrylamide gel electrophoresis (lane 2), which is competed by 100-fold molar excess unlabeled oligonucleotide (lane 3), but not by an equivalent amount of nonspecific competitor oligonucleotide (NS, lane 4). While a synthetic oligonucleotide surrounding the reference G variant can efficiently compete with the DIG-labeled synthetic oligonucleotide carrying the FB site (lanes 5 to 7), the FI-associated A variant cannot displace the labeled probe even when used up to 100-fold molar excess (lanes 8 to 10), suggesting that G->A substitution at the chr11:47227430 site disrupts FOXA1 binding. (B) Binding and competition assays reciprocal to those in panel A confirm specific binding of FOXA1 to the reference G allele at chr11:47227430. Here, the synthetic oligonucleotide surrounding the reference G allele was labeled with DIG, and is shown to form a specific complex with FOXA1 (lane 2, upper arrowhead) that cannot be efficiently displaced by unlabeled NS oligonucleotide (lane 3). Increasing amounts of an unlabeled oligonucleotide containing the ref-G allele displaces the bound DIG-labeled ref-G oligonucleotide at lower molar fold excess than an unlabeled oligonucleotide carrying the FI-associated A variant (compare lanes 4-7 to lanes 8-10). Panels C-E. The reference G allele at chr11:47227430 has significantly transactivation potential compared to the FI-associated A allele in luciferase reporter assays. (C) Dual luciferase assays show that a reporter plasmid containing the 280bp region surrounding the chr11:47227430 site exhibits enhancer activity in HepG2 cells [Ref(G)] compared to the minimal HSV thymidine kinase (TK) promoter in the pGluc Mini-TK backbone (BB). (D) The 280bp region surrounding chr11:47227430 shows FOXA1-dependent enhancer activity in HepG2 cells. The FI-associated A variant [Ref(A)] is significantly less responsive to FOXA1 over-expression compared to the reference G allele [Ref(G)]. For each construct, luciferase measurements are normalized by the corresponding reporter activity measured in the absence of FOXA1 co-expression. (E) Luciferase activity was measured for reporter constructs containing 40-mer oligonucleotides centered on the chr11:47227430 reference [Ref(G)] or risk [FI(A)] alleles or a well characterized albumin enhancer FOXA1 binding site (FB). For each construct, luciferase measurements are normalized by the corresponding reporter activity measured in the absence of FOXA1 co-expression. All experiments were conducted using eight replicates.