Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus

Abstract

Type 2 diabetes mellitus is a common disabling disease with onset in middle-aged individuals, caused by an imbalance between insulin production and action. Genetic studies point to major genetic components, but, with the exception of maturity-onset diabetes of the young (MODY), specific diabetes susceptibility genes remain to be identified. Recent studies showed that a dominant negative mutation in the insulin promoter factor-1 (IPF-1), a pancreatic beta-cell specific transcription factor, causes pancreatic agenesis and MODY. Thus, we investigated 192 French, non-MODY type 2 diabetic families for mutations in IPF-1. We identified 3 novel IPF-1 mutations, including 2 substitutions (Q59L and D76N) and an in-frame proline insertion (InsCCG243). Functional transactivation assays of these IPF-1 mutant isoforms in a beta-pancreatic tumor cell line transfected with a transcriptional reporter and IPF-1 expression plasmids demonstrate a significant inhibition of basal insulin promoter activity (stronger with the InsCCG243 mutant). We find that the InsCCG243 mutation is linked, in 2 families, to an autosomal dominant-like late-onset form of type 2 diabetes, in which insulin secretion becomes progressively impaired. The lower penetrance D76N and Q59L mutations were more prevalent and were associated with a relative risk of 12.6 for diabetes and with decreased glucose-stimulated insulin-secretion in nondiabetic subjects. We propose that IPF-1 mutations can cause MODY or apparently monogenic late-onset diabetes and that they represent a significant risk factor for type 2 diabetes in humans.

Figure 1

Segregation of the IPF-1 InsCCG243 variation in pedigrees F-13 and F-498. Filled, half-filled, and open symbols represent subjects with overt type 2 diabetes mellitus, impaired glucose tolerance, and normal glucose tolerance, respectively. Hatched symbols represent untested subjects. The first line under the symbols corresponds to the identification number. The second line shows the genotype at codon 243 of IPF-1 (italicized): N, Normal allele; M, Mutant allele (InsCCG243). The third and fourth lines show, respectively, the current age of subjects and the age of diagnosis of diabetes (or glucose intolerance, italicized), when appropriate. The fifth line shows the BMI (kg/m²). §: Subject 10 presented with impaired glucose tolerance at 34 years of age.

Figure 2

Five-year evolution of insulin-secretion profiles in 4 middle-aged carriers of the IPF-1 InsCCG243 mutation. The plots show comparisons of plasma glucose levels (Figure 3a), plasma insulin levels (Figure 3b), and insulin/glucose ratios (Figure 3c), between 1992 (filled bars) and 1997 (hatched bars), with the paired Student’s t test. Basal (0 minutes) and 2 hours (120 minutes) mean values (± SEM) during oral glucose tolerance tests are presented. *P = 0.02; **P = 0.011.

Figure 3

Functional and Western immunoblot analyses of mutant IPF-1 proteins. (a) Transactivation assays were done in HIT-T15 insulinoma cells that express endogenous IPF-1. Cells were transiently transfected with a rat insulin-promoter-CAT reporter plasmid and 0.3–33 ng of expression plasmids for wild-type IPF-1 (WT) or the mutant IPF-1 isoforms. The number of independent experiments is 13–14, done on 2–3 different occasions. Basal CAT activities ranged from 6% to 15% conversion to acetylated forms of chloramphenicol. Mean values ± SEM are given compared to basal values (transfection with empty vector) normalized for the independent experiments. P values, shown above the bars (± SEM), were determined by the Wilcoxon rank sum test comparing the effect of each mutation to wild-type IPF-1 (WT). For the same comparisons, P values using the unpaired 2-sided Student’s t test were 0.052, 0.28, and < 0.001 for D76N, Q59L, and InsCCG243, respectively. (b) Determination of IPF-1 expression levels by Western immunoblot using antiserum α253 (14), specific for the detection of the COOH-terminal region of IPF-1: 500 ng of IPF-1 expression plasmids was transfected to the HIT-T15 cells. The relative expression levels of the wild-type (lane 2), Q59L (lane 4), and InsCCG243 (lane 5) IPF-1 forms were the same. The D76N form (lane 3) was expressed at a lower level than that of the others. After correction for expression efficiency by Western immunoblot analysis, the D76N mutant function was approximately 60% that of the wild-type IPF-1. Lane 1 corresponds to extracts from cells transfected with the empty vector.

Figure 4

Segregation of the IPF-1 D76N variation in the 9 type 2 diabetic families in which it was detected. These families belong to a large collection, ascertained in France for the presence of at least 2 sibs presenting with overt type 2 diabetes, and additional family members were recruited, when available (ref. 16). Filled, half-filled, and open symbols represent subjects with overt type 2 diabetes mellitus, impaired glucose tolerance, and normal glucose tolerance, respectively. Under each symbol, the first line corresponds the actual age; the second line shows the age at diagnosis of overt type 2 diabetes (when appropriate). The third line shows individual BMI values (kg/m²), and the fourth line shows the genotype at IPF-1 codon 76 (italicized): N, Normal allele; M, mutant allele.

Figure 5

Glucose levels and insulin-secretion profiles in young normoglycemic and glucose intolerant subjects from the 9 families carrying the IPF-1 D76N mutation. Only nonovertly diabetic subjects were included in the analysis. The maximal number glucose and insulin values for the studied subjects were included as possible, and the numbers of individual data used in the comparisons are shown above each line-point (N). Carriers of the IPF-1 D76N mutation (continuous line) compared with IPF-1 wild-type carriers (dotted line) for the mean (± SEM) of plasma glucose levels (Figure 5a), insulin levels (Figure 5b), and insulin/glucose ratios (Figure 5c) during standard oral glucose tolerance tests. For each OGTT time point (0, 30, 60, 90, and 120 minutes), the P values are shown.