Functional Investigations of HNF1A Identify Rare Variants as Risk Factors for Type 2 Diabetes in the General Population

Abstract

Variants in HNF1A encoding hepatocyte nuclear factor 1α (HNF-1A) are associated with maturity-onset diabetes of the young form 3 (MODY 3) and type 2 diabetes. We investigated whether functional classification of HNF1A rare coding variants can inform models of diabetes risk prediction in the general population by analyzing the effect of 27 HNF1A variants identified in well-phenotyped populations (n = 4,115). Bioinformatics tools classified 11 variants as likely pathogenic and showed no association with diabetes risk (combined minor allele frequency [MAF] 0.22%; odds ratio [OR] 2.02; 95% CI 0.73-5.60; P = 0.18). However, a different set of 11 variants that reduced HNF-1A transcriptional activity to <60% of normal (wild-type) activity was strongly associated with diabetes in the general population (combined MAF 0.22%; OR 5.04; 95% CI 1.99-12.80; P = 0.0007). Our functional investigations indicate that 0.44% of the population carry HNF1A variants that result in a substantially increased risk for developing diabetes. These results suggest that functional characterization of variants within MODY genes may overcome the limitations of bioinformatics tools for the purposes of presymptomatic diabetes risk prediction in the general population.

Figure 1

Position of HNF1A variants in the HNF-1A protein sequence identified in the study cohorts. Schematic illustration shows rare nonsynonymous HNF1A variants (orange), common variants (gray), and MODY3-associated variants (blue) identified in the cohorts studied (7). The total number of rare mutations reported in MODY families to date (25) is shown as the number for each functional domain.

Figure 2

Assessment of transcriptional activity of HNF-1A protein variants using a luciferase reporter assay. HeLa cells were transiently transfected with wild-type or variant HNF1A plasmids together with reporter plasmids pGL3-RA and pRL-SV40. Luciferase measurements are given in percentage activity compared with wild-type. Each point represents the mean (the range bars indicate the 95% CIs) of nine readings. Three parallel readings were conducted on each of 3 experimental days. Two MODY variants and three common variants were included in the study.

Figure 3

Analysis of nuclear localization of HNF-1A protein variants in HeLa cells. Cells were transiently transfected for 24 h and Xpress-epitope–tagged HNF-1A protein variants detected by immunofluorescence. A: Subcellular localization in a minimum of 200 cells was assessed for each HNF1A variant. The percentage of cells with nuclear accumulation alone is presented. B: Representative images of cells of the two most impaired nuclear localization variants. One MODY3 variant (p.Q466*) with abnormal subcellular localization was included as a control. HNF-1A was detected using tag-specific antibody and Alexa Fluor 488 (green). DNA staining (DAPI) is shown in blue. In more detail, the cytoplasmic signals of the cells expressing p.R131Q were more uniform, whereas the cells expressing p.H514R revealed a pattern resembling aggregated particles. For the purpose of clarity, the nuclei and cell membrane have been marked with a white line.