Makiko Ogata

Organization and partial sequence of the hepatocyte nuclear factor-4 alpha/MODY1 gene and identification of a missense mutation, R127W, in a Japanese family with MODY

Hepatocyte nuclear factor-4α (HNF-4α) 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 exonintron organization and partial sequence of the human HNF-4α 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, Argl27 (CGG)→Trp (TGG) (designated R127W), located in the T-box, a region of the protein that may play a role in HNF-4α 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-4α gene may cause early-onset NIDDM/MODY in Japanese but they are less common than mutations in the HNF-1α/MODY3 gene. The information on the sequence of the HNF-4α 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 β-cell function.

Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with early- and late-onset NIDDM

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1α 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 >30 years of age). All 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, K205Q, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsinsTC). 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-1α 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.

Liver and Kidney Function in Japanese Patients With Maturity-Onset Diabetes of the Young

OBJECTIVE Heterozygous mutations in the transcription factors hepatocyte nuclear factor (HNF)-1α, HNF-1β, and HNF-4α are associated with maturity-onset diabetes of the young (MODY) and are believed to cause this form of diabetes by impairing pancreatic β-cell function. The HNFs also play a central role in the tissue-specific regulation of gene expression in liver and kidney, suggesting that patients with MODY due to a mutation in HNF-1α, HNF-1β, or HNF-4α may exhibit abnormal liver or kidney function. Here, we have examined liver and kidney function in a series of Japanese patients with HNF-4α/MODY1, HNF-1α/MODY3, and HNF-1β/MODY5 diabetes. RESEARCH DESIGN AND METHODS Clinical and biochemical data were obtained from Japanese subjects with HNF-1α, HNF-1β, and HNF-4α diabetes. The clinical data included information on BMI, age at diagnosis, current treatment, and the presence and nature of any complications. The biochemical studies examined liver and kidney function and included measures of alanine and aspartate aminotransferase, γ-glutamyl transpeptidase, blood urea nitrogen, creatinine, uric acid, total and HDL cholesterol, triglycerides, and 17 serum proteins. RESULTS The present age and duration of diabetes were similar in patients with HNF-1α, HNF-1β, or HNF-4α diabetes, as was the age at diagnosis of diabetes in the youngest generation. All subjects were lean. Of the subjects with HNF-1α and HNF-4α diabetes, 50% were treated with insulin, as were all three subjects with HNF-1β diabetes. Retinopathy was present in patients with each form of diabetes. None of the subjects with HNF-4α diabetes had evidence of nephropathy, whereas 36% of the patients with HNF-1α diabetes and 100% of those with HNF-1α diabetes showed diminished kidney function. The three subjects with HNF-1β diabetes also had abnormally high serum creatinine, uric acid, and blood urea nitrogen levels, which are consistent with impaired kidney function, and one of seven subjects with HNF-1α diabetes had a mild elevation in creatinine and blood urea nitrogen levels. These values were within the normal range in the three patients with HNF-4α diabetes. Although the HNFs play a role in regulating the expression of the genes for most, if not all, serum proteins, there was no decrease in the levels of any of the 17 serum proteins examined, and most were within or slightly above the normal range. Lipoprotein(a) [Lp(a)] levels were elevated in the three patients with HNF-4α diabetes and in one patient with HNF-1β diabetes, and in a second patient with HNF-1β diabetes, Lp(a) was at the upper limit of normal. CONCLUSIONS The results indicate that as in white patients, MODY resulting from mutations in the HNF-1α, HNF-1β, and HNF-4α genes in Japanese patients may be a severe disease similar to classic type 2 diabetes. In addition, they suggest that patients with HNF-1β diabetes may be characterized by diminished kidney function and perhaps abnormal liver function. Further studies are needed to determine whether tests of liver and kidney function will be useful in the diagnosis and subclassification of MODY.

Identification of KCNJ15 as a Susceptibility Gene in Asian Patients with Type 2 Diabetes Mellitus

Recent advances in genome research have enabled the identification of new genomic variations that are associated with type 2 diabetes mellitus (T2DM). Via fine mapping of SNPs in a candidate region of chromosome 21q, the current study identifies potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15) as a new T2DM susceptibility gene. KCNJ15 is expressed in the beta cell of the pancreas, and a synonymous SNP, rs3746876, in exon 4 (C566T) of this gene, with T allele frequency among control subjects of 3.1%, showed a significant association with T2DM affecting lean individuals in three independent Japanese sample sets (p = 2.5 x 10(-7), odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.76-3.67) and with unstratified T2DM (p = 6.7 x 10(-6), OR = 1.76, 95% CI = 1.37-2.25). The diabetes risk allele frequency was, however, very low among Europeans in whom no association between this variant and T2DM could be shown. Functional analysis in human embryonic kidney 293 cells demonstrated that the risk allele of the synonymous SNP in exon 4 increased KCNJ15 expression via increased mRNA stability, which resulted in the higher expression of protein as compared to that of the nonrisk allele. We also showed that KCNJ15 is expressed in human pancreatic beta cells. In conclusion, we demonstrated a significant association between a synonymous variant in KCNJ15 and T2DM in lean Japanese patients with T2DM, suggesting that KCNJ15 is a previously unreported susceptibility gene for T2DM among Asians.

Prevalence of A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA Leu(UUR) gene in Japanese patients with diabetes mellitus and end stage renal disease

AbstractThe A-to-G mutation at nucleotide 3243 of the mitochondrial tRNALeu(UUR) gene (mt.3243A>G) is associated with both diabetes mellitus and myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Recently, this mutation was found in three diabetic subjects with progressive kidney disease, suggesting that it may be a contributing factor in the development of kidney disease in patients with diabetes. The aim of this study was to evaluate the contribution of this mutation to the development of end stage renal disease (ESRD) in patients with diabetes. The study group consisted of 135 patients with diabetes and ESRD. The control group consisted of 92 non-diabetic subjects with ESRD who were receiving hemodialysis. The mt.3243A>G mutation was detected by polymerase chain reaction-restriction frag-ment length polymorphism (PCR-RFLP). We found the mt.3243A>G mutation in eight patients (8/135; 5.9%), all of whom were initially diagnosed with type II diabetes. Five of the eight patients were subsequently also diagnosed with MELAS. We did not find the mutation in any of the 92 non-diabetic subjects with ESRD. The prevalence of this mutation was 6.5-fold higher in patients with diabetes and ESRD than in those with diabetes alone (8/135 vs 5/550, respectively; χ2 = 13.704; P = 0.0002). The mt.3243A>G mutation may be a contributing genetic factor in the development of ESRD in Japanese patients with diabetes.

Genetic variants in the calpain-10 gene and the development of type 2 diabetes in the Japanese population.

AbstractVariation in the gene encoding the cysteine protease calpain-10 has been linked and associated with risk of type 2 diabetes. We have examined the effect of three polymorphisms in the calpain-10 gene (SNP-43, Indel-19, and SNP-63) on the development of type 2 diabetes in the Japanese population in a pooled analysis of 927 patients and 929 controls. We observed that SNP-43, Indel-19, and SNP-63 either individually or as a haplotype were not associated with altered risk of type 2 diabetes with the exception of the rare 111/221 haplogenotype (odds ratio (OR) =3.53, P=0.02). However, stratification based on the median age-at-diagnosis in the pooled study population (<50 and ≥50 years) revealed that allele 2 of Indel-19 and the 121 haplotype were associated with reduced risk in patients with later age-at-diagnosis (age-at-diagnosis ≥50 years OR=0.82 and 0.80, respectively; P=0.04 and 0.02). Thus, variation in the calpain-10 gene may affect risk of type 2 diabetes in Japanese, especially in older individuals.

GLP-1-related proteins attenuate the effects of mitochondrial membrane damage in pancreatic β cells

Glucagon-like peptide (GLP)-1 analog based therapies are used not only for their insulinotropic effects, but also for their pleiotropic effects that improve pancreatic β cell function. Liraglutide is a long acting derivative of human GLP-1(7-37), which is a cleavage product encompassing amino acids 7-37 of GLP-1. In this study, we examined whether Liraglutide treatment restore the glucose-stimulated mitochondrial response of β cells with chemically induced mitochondrial damage. We tested three GLP-1-related proteins: human GLP-1(1-37), GLP-1(7-37) and Liraglutide. To measure changes of the mitochondrial pH quantitatively in real-time, we have developed a bioengineered β cell line. We generated a mitochondrial damaged model by treating β cells with ethidium bromide (EtBr; 0.5 or 1 μg/mL for 48 h). EtBr treatment reduced the response to 25 mM glucose in mitochondrial pH in a dose- and time-dependent manner. GLP-1(7-37) (100 nM) enhanced the response of mitochondria to glucose stimulation in undamaged β cells. Preincubation with Liraglutide (1 nM) or GLP-1 (100 nM) for 3h recovered the mitochondrial response to glucose in damaged β cells, however, GLP-1(7-37) (100 nM) did not. When GLP-1(7-37) was administered in stepwise increments (i.e., starting with 20 nM to reach 100 nM in 3h), similar recovery of the mitochondrial function was observed. The results suggest that Liraglutide is effective to recover glucose-stimulated mitochondrial response in damaged β cells.

A new mitochondrial pH biosensor for quantitative assessment of pancreatic β-cell function

Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic β-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100μM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.

Pancreatic developmental defect evaluated by celiac artery angiography in a patient with MODY5

The hepatocyte nuclear factor 1β gene (HNF1B) is responsible for maturity-onset diabetes of the young type 5 (MODY5), which is characterized by early-onset diabetes mellitus and urogenital malformations. HNF1B is expressed during visceral endoderm formation. We identified a disruption of the great pancreatic artery in a patient with MODY5 with no pancreatic body or tail. Our finding supports the significance of HNF1B in the development of the pancreas.

Localization of hepatocyte nuclear factor‐4α in the nucleolus and nucleus is regulated by its C‐terminus

Aims/Introduction:  Mutations in hepatocyte nuclear factor‐4α (HNF4α) lead to various diseases, among which C‐terminal deletions of HNF4α are exclusively responsible for maturity onset diabetes of the young 1 (MODY1). MODY is an autosomal dominant disease characterized by a primary defect in insulin response to glucose, suggesting that the C‐terminus of HNF4α is important for pancreatic β‐cell function. To clarify the role of the C‐terminus of HNF4α, changes in cellular localization and the binding ability to its regulator were examined, specifically in the region containing Q268, which deletion causes MODY1.