織田 直久

We have sequenced the insulin gene in 72 unrelated Japanese subjects (52 with type 2 diabetes mellitus and 20 with normal glucose tolerance). We identified 6 mutations and all were found at a low frequency (1% to 4%). Three mutations were new. These included a C-to-G substitution in the promoter region, a G-to-A substitution in codon-2 resulting in an Ala-to-Thr replacement in amino acid -2 of the signal peptide, and a G-to-A substitution in intron 2. We have no evidence that any of the mutations that we found are the cause of diabetes. Thus, mutations in the insulin gene do not appear to be an important genetic factor contributing to the development of diabetes in this population. Copyright (C) 2001 by W.B. Saunders Company.

Hepatocyte nuclear factor-4 alpha (HNF-4 alpha) is a member of the nuclear receptor superfamily, a class of ligand-activated transcription factors. A nonsense mutation in the gene encoding this transcription factor was recently found in a white family with one form of maturity-onset diabetes of the young, MODY1. Here, we report the exon-intron organization and partial sequence of the human HNF-4 alpha gene. In addition, we have screened the 12 exons, flanking introns and minimal promoter region for mutations in a group of 57 unrelated Japanese subjects with early-onset NIDDM/MODY of unknown cause. Eight nucleotide substitutions were noted, of which one resulted in the mutation of a conserved arginine residue, Arg127 (CGG)-->Trp (TGG) (designated R127W), located in the T-box, a region of the protein that may play a Pole in HNF-4 alpha dimerization and DNA binding, This mutation was not found in 214 unrelated nondiabetic subjects (53 Japanese, 53 Chinese, 51 white, and 57 African-American). The R127W mutation was only present in three of five diabetic members in this family, indicating that it is not the only cause of diabetes in this family. The remaining seven nucleotide substitutions were located in the proximal promoter region and introns. They are not predicted to affect the transcription of the gene or mRNA processing and represent polymorphisms and rare variants. The results suggest that mutations in the HNF-4 alpha gene may cause early-onset NIDDM/MODY in Japanese but they are less common than mutations in the HNF-1 alpha/MODY3 gene. The information on the sequence of the HNF-4 alpha gene and its promoter region will facilitate the search for mutations in other populations and studies of the role of this gene in determining normal pancreatic beta-cell function.

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene are the cause of maturity-onset diabetes of the young type 3 (MODY3). We have screened 193 unrelated Japanese subjects with NIDDM for mutations in this gene: 83 with early-onset NIDDM (diagnosis at <30 years of age) and 110 with late-onset NIDDM (diagnosis greater than or equal to 30 years of age). AU of the members of the latter group also had at least one sibling with NIDDM. The 10 exons, flanking introns, and promoter region were amplified using polymerase chain reaction and were sequenced directly. Mutations were found in 7 of the 83 (8%) unrelated subjects with early-onset NIDDM. The mutations were each different and included four missense mutations (L12H, R131Q, K205A, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsin-sTC). One of the 110 subjects with late-onset NIDDM was heterozygous for the missense mutation G191D. This subject, who was diagnosed with NIDDM at 64 years of age, also had a brother with NIDDM (age at diagnosis, 54 years) who carried the same mutation, suggesting that this mutation contributed to the development of NIDDM in these two siblings. None of these mutations were present in 50 unrelated subjects with normal glucose tolerance (100 normal chromosomes). Mutations in the HNF-1 alpha gene occur in Japanese subjects with NIDDM and appear to be an important cause of early-onset NIDDM in this population. In addition, they are present in about 1% of subjects with late-onset NIDDM.

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1 alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1 alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations mere identified in nine of these individuals, suggesting that mutations in the HNF-1 alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1 alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1 alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T-->G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1 alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell function.

Using three month-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a NIDDM model and Long-Evans Tokushima Otsuka (LETO) rats as the control, we examined the effect of ACEI on the onset of diabetes mellitus. Oral administration of captopril (ACEI) was started from the age of 13 to 32 weeks (A group) and from the age of 25 to 32 weeks (B group). Oral GTT (oGTT) was performed at 4-week intervals from 12 weeks to 32 weeks of the age in OLETF and LETO rats. Body weights did not change significantly in either the A or B groups, compared with that in the control group. In oGTT for OLETF rats at 28 and 32 weeks of age, blood glucose levels in the A group were lower at 120 min after glucose administration than those in the B and control groups. In LETO rats, there were no significant changes in blood glucose levels in oGTT in any group. GLUT4 protein concentrations in the skeletal muscle and adipocytes were not significantly changed in any group. These data suggest that ACEI improves the glucose intolerance in NIDDM model rats through some factor other than changes in GLUT4 concentrations.

Using three month-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a NIDDM model and Long-Evans Tokushima Otsuka (LETO) rats as the control, we examined the effect of ACEI on the onset of diabetes mellitus. Oral administration of captopril (ACEI) was started from the age of 13 to 32 weeks (A group) and from the age of 25 to 32 weeks (B group). Oral GTT (oGTT) was performed at 4-week intervals from 12 weeks to 32 weeks of the age in OLETF and LETO rats. Body weights did not change significantly in either the A or B groups, compared with that in the control group. In oGTT for OLETF rats at 28 and 32 weeks of age, blood glucose levels in the A group were lower at 120 min after glucose administration than those in the B and control groups. In LETO rats, there were no significant changes in blood glucose levels in oGTT in any group. GLUT4 protein concentrations in the skeletal muscle and adipocytes were not significantly changed in any group. These data suggest that ACEI improves the glucose intolerance in NIDDM model rats through some factor other than changes in GLUT4 concentrations.

THE disease maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM), characterized by early onset, usually before 25 years of age and often in adolescence or childhood, and by autosomal dominant inheritance(1). It has been estimated that 2-5% of patients with NIDDM may have this form of diabetes mellitus(2,3). Clinical studies have shown that prediabetic MODY subjects have normal insulin sensitivity but suffer from a defect in glucose-stimulated insulin secretion, suggesting that pancreatic beta-cell dysfunction rather than insulin resistance is the primary defect in this disorder(4,5). Linkage studies have localized the genes that are mutated in MODY on human chromosomes 20 (MODY1)(6), 7 (MODY2)(2) and 12 (MODY3)(7), with MODY2 and MODY3 being allelic with the genes encoding glucokinase(2), a key regulator of insulin secretion, and hepatocyte nuclear factor-1 alpha (HNF-1 alpha)(8), a transcription factor involved in tissue-specific regulation of liver genes but also expressed in pancreatic islets, insulinoma cells and other tissues. Here we show that MODY1 is the gene encoding HNF-4 alpha (gene symbol, TCF14), a member of the steroid/thyroid hormone receptor superfamily and an upstream regulator of HNF-1 alpha expressiong-(9,11).