El Habib Hani

Genomewide Search for Type 2 Diabetes–Susceptibility Genes in French Whites: Evidence for a Novel Susceptibility Locus for Early-Onset Diabetes on Chromosome 3q27-qter and Independent Replication of a Type 2–Diabetes Locus on Chromosome 1q21–q24

Despite recent advances in the molecular genetics of type 2 diabetes, the majority of susceptibility genes in humans remain to be identified. We therefore conducted a 10-cM genomewide search (401 microsatellite markers) for type 2 diabetes-related traits in 637 members of 143 French pedigrees ascertained through multiple diabetic siblings, to map such genes in the white population. Nonparametric two-point and multipoint linkage analyzes-using the MAPMAKER-SIBS (MLS) and MAXIMUM-BINOMIAL-LIKELIHOOD (MLB) programs for autosomal markers and the ASPEX program for chromosome X markers-were performed with six diabetic phenotypes: diabetes and diabetes or glucose intolerance (GI), as well as with each of the two phenotypes associated with normal body weight (body-mass index<27 kg/m(2)) or early age at diagnosis (<45 years). In a second step, high-resolution genetic mapping ( approximately 2 cM) was performed in regions on chromosomes 1 and 3 loci showing the strongest linkage to diabetic traits. We found evidence for linkage with diabetes or GI diagnosed at age <45 years in 92 affected sib pairs from 55 families at the D3S1580 locus on chromosome 3q27-qter using MAPMAKER-SIBS (MLS = 4.67, P=.000004), supported by the MLB statistic (MLB-LOD=3.43, P=.00003). We also found suggestive linkage between the lean diabetic status and markers APOA2-D1S484 (MLS = 3. 04, P=.00018; MLB-LOD=2.99, P=.00010) on chromosome 1q21-q24. Several other chromosomal regions showed indication of linkage with diabetic traits, including markers on chromosome 2p21-p16, 10q26, 20p, and 20q. These results (a) showed evidence for a novel susceptibility locus for type 2 diabetes in French whites on chromosome 3q27-qter and (b) confirmed the previously reported diabetes-susceptibility locus on chromosome 1q21-q24. Saturation on both chromosomes narrowed the regions of interest down to an interval of <7 cM.

Missense mutations in the pancreatic islet beta cell inwardly rectifying K+ channel gene (KIR6.2/BIR): a meta-analysis suggests a role in the polygenic basis of Type II diabetes mellitus in Caucasians.

Summary The K+ inwardly rectifier channel (KIR) is one of the two sub-units of the pancreatic islet ATP-sensitive potassium channel complex (IKATP), which has a key role in glucose-stimulated insulin secretion and thus is a potential candidate for a genetic defect in Type II (non-insulin-dependent) diabetes mellitus. We did a molecular screening of the KIR6.2 gene by single strand conformational polymorphism (SSCP) and direct sequencing in 72 French Caucasian Type II diabetic families. We identified three nucleotide substitutions resulting in three amino acid changes (E23K, L270V and I337V), that have also been identified in other Caucasian Type II diabetic subjects. These variants were genotyped in French cohorts of 191 unrelated Type II diabetic probands and 119 normoglycaemic control subjects and association studies were done. The genotype frequencies of the L270V and I337V variants were not very different between Type II diabetic subjects and control groups. In contrast, analysis of the E23K variant showed that the KK homozygocity was more frequent in Type II diabetic than in control subjects (27 vs 14 %, p = 0.015). Analyses in a recessive model (KK vs EK/EE) tended to show a stronger association of the K allele with diabetes (p = 0.0097, corrected p-value for multiple testing < 0.02). The data for the E23K variant obtained here and those obtained from three other Caucasian groups studied so far were combined and investigated by meta-analysis. Overall, the E23K variant was found to be significantly associated with Type II diabetes (0.001 ≤p≤ 0.0016, corrected p-values for multiple testing p≤ 0.01). This study shows that KIR6.2 polymorphisms are frequently associated with Type II diabetes in French Caucasians. Furthermore, a meta-analysis combining different Caucasian groups suggests an significant role of KIR6.2 in the polygenic context of Type II diabetes. [Diabetologia (1998) 41: 1511–1515]

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogenous disease. The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs ). We evaluated the role of IB1 in β-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma β-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IB1 protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on 149 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (S59N) that segregated with diabetes. In vitro , this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of β-cell function.

Cloning, Functional Expression, and Chromosomal Localization of the Human Pancreatic Islet Glucose-Dependent Insulinotropic Polypeptide Receptor

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27–amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 µmol/l). GIP binding was displaced by <20% by 1 µmol/l glucagon, glucagon-like peptide (GLP-I)(7–36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9–39) at 1 μumol/l displaced binding by ∼70 and ∼100% at 10 µmol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6–0.8 µmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9–39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 μmol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Mutation screening in 18 Caucasian families suggest the existence of other MODY genes

Summary Maturity-onset diabetes of the young (MODY) is a heterogeneous subtype of non-insulin-dependent diabetes mellitus characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. To date five MODY genes have been identified: hepatocyte nuclear factor-4 alpha (HNF-4α/MODY1/TCF14) on chromosome 20 q, glucokinase (GCK/MODY2) on chromosome 7 p, hepatocyte nuclear factor-1 alpha (HNF-1α/MODY3/TCF1) on chromosome 12 q, insulin promoter factor-1 (IPF1/MODY4) on chromosome 13 q and hepatocyte nuclear factor-1 beta (HNF-1β/MODY5/TCF2) on chromosome 17cen-q. We have screened the HNF-4α, HNF-1α and HNF-1β genes in members of 18 MODY kindreds who tested negative for glucokinase mutations. Five missense (G31D, R159W, A161T, R200W, R271W), one substitution at the splice donor site of intron 5 (IVS5nt + 2T→A) and one deletion mutation (P379fsdelT) were found in the HNF-1α gene, but no MODY-associated mutations were found in the HNF-4α and HNF-1β genes. Of 67 French MODY families that we have now studied, 42 (63 %) have mutations in the glucokinase gene, 14 (21 %) have mutations in the HNF-1α gene, and 11 (16 %) have no mutations in the HNF-4α, IPF1 and HNF-1β genes. Eleven families do not have mutations in the five known MODY genes suggesting that there is at least one additionnal locus that can cause MODY. [Diabetologia (1998) 41: 1017–1023]

Genetic studies of the sulfonylurea receptor gene locus in NIDDM and in morbid obesity among French caucasians

The sulfonylurea receptor (SUR) is a key component in glucose-stimulated insulin secretion. Obesity and NIDDM are frequently associated and share some metabolic abnormalities, suggesting that they might also share some susceptibility genes. Thus, the SUR encoding gene is a plausible candidate for a primary pancreatic β-cell defect and thus for hyperglycemia and weight gain. Through association and linkage studies, we have investigated the potential role of the SUR gene in families with NIDDM and in two independent sets of morbidly obese families. The exon 22 T-allele at codon 761 was more common in patients with NIDDM (7.7%) and morbid obesity (7.8%) than in control subjects (1.8%, P = 0.030 and P = 0.023, respectively). This variant was associated with morbid obesity (odds ratio 3.71, P = 0.017) and NIDDM (odds ratio 2.20, P = 0.04; association dependent on BMI). Although the frequencies for intron 24 variant were similar in all groups, morbidly obese patients homozygous for the c-allele had a more deleterious form of obesity. Sib-pair linkage studies with NIDDM in French Caucasian families gave no evidence for linkage to the SUR locus. However, in one set of the obese families, we found an indication for linkage with a SUR-linked microsatellite marker (D11S419, P = 0.0032). We conclude that in Caucasians, the SUR locus may contribute to the genetic susceptibility to NIDDM and obesity.

Genetics of NIDDM in France: Studies With 19 Candidate Genes in Affected Sib Pairs

As part of an ongoing search for susceptibility loci for NIDDM, we tested 19 genes whose products are implicated in insulin secretion or action for linkage with NIDDM. Loci included the G-protein–coupled inwardly rectifying potassium channels expressed in β-cells (KCNJ3 and KCNJ7), glucagon (GCG), glucokinase regulatory protein (GCKR), glucagon-like peptide I receptor (GLP1R), LIM/homeodomain islet-1 (ISL1), caudal-type homeodomain 3 (CDX3), proprotein convertase 2 (PCSK2), cholecystokinin B receptor (CCKBR}, hexokinase 1 (HK1), hexokinase 2 (HK2), mitochondrial FAD-glycerophosphate dehydrogenase (GPD2), liver and muscle forms of pyruvate kinase (PKL, PKM), fatty acid–binding protein 2 (FABP2), hepatic phosphofructokinase (PFKL), protein serine/threonine phosphatase 1 beta (PPP1CB), and lowdensity lipoprotein receptor (LDLR). Additionally, we tested the histidine-rich calcium locus (HRC) on chromosome 19q. All regions were tested for linkage with microsatellite markers in 751 individuals from 172 families with at least two patients with overt NIDDM (according to World Health Organization criteria) in the sibship, using nonparametric methods. These 172 families comprise 352 possible affected sib pairs with overt NIDDM or 621 possible affected sib pairs defined as having a fasting plasma glucose value of >6.1 mmol/1 or a glucose value of >7.8 mmol/1 2 h after oral glucose load. No evidence for linkage was found with any of the 19 candidate genes and NIDDM in our population by nonparametric methods, suggesting that those genes are not major contributors to the pathogenesis of NIDDM. However, some evidence for suggestive linkage was found between a more severe form of NIDDM, defined as overt NIDDM diagnosed before 45 years of age, and the CCKBR locus (11pl5.4; P = 0.004). Analyses of six additional markers spanning 27 cM on chromosome l ip confirmed the suggestive linkage in this region. Whether an NIDDM susceptibility gene lies on chromosome l ip in our population must be determined by further analyses.

Linkage Analyses of the MODY3 Locus on Chromosome 12q With Late-Onset NIDDM

Non-insulin-dependent diabetes mellitus (NIDDM) is a clinically and genetically heterogeneous disorder. Maturity-onset diabetes of the young (MODY), an autosomal dominant form of NIDDM, has been used as a model for genetic studies of NIDDM. We recently reported linkage between markers on chromosome 12q and diabetes in 25% of our French MODY families. To evaluate if this gene is also implicated in late-onset NIDDM, we performed linkage studies between two markers of the MODY region and diabetes in 172 families with late-onset NIDDM. Both parametric and nonparametric methods were used in a total of 600 affected sib-pairs. Linkage was rejected in this population by all methods, implying that the MODY gene on chromosome 12q is not a major gene for late-onset NIDDM in this population. However, we cannot exclude a modifying role in a polygenic disorder or an important role in some families.