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MODY 5 and 6 have been shown to be low-penetrant MODYs. As the genetic background of unknown MODY is assumed to be similar, a new analytical strategy is applied here to elucidate genetic predispositions to unknown MODY. We examined to find whether there are major MODY gene loci remaining to be identified using SNP linkage analysis in Japanese. Whole-exome sequencing was performed with seven families with typical MODY. Candidates for novel MODY genes were examined combined with in silico network analysis. Some peaks were found only in either parametric or non-parametric analysis; however, none of these peaks showed a LOD score greater than 3.7, which is approved to be the significance threshold of evidence for linkage. Exome sequencing revealed that three mutated genes were common among 3 families and 42 mutated genes were common in two families. Only one of these genes, MYO5A, having rare amino acid mutations p.R849Q and p.V1601G, was involved in the biological network of known MODY genes through the intermediary of the INS. Although only one promising candidate gene, MYO5A, was identified, no novel, high penetrant MODY genes might remain to be found in Japanese MODY.

Aims: Only a few families with neuronal differentiation 1 (NEUROD1)-deficient diabetes, currently designated as maturity-onset diabetes of the young 6 (MODY6), have been reported, but mostly in Caucasian, and no mutation has been identified by family-based screening in Japanese. Accordingly, the phenotypic details of the disease remain to be elucidated., Methods: We examined a total of 275 subjects having diabetes suspected to be MODY who were negative for mutations in MODY1-5 referred from 155 medical institutions throughout Japan. So as not to miss low penetrant cases, we examined non-obese Japanese patients with early-onset diabetes regardless of the presence of family history by direct sequencing of all exons and flanking regions of NEUROD1 . Large genomic rearrangements also were examined., Results: Four patients with 3 frameshift mutations and 1 missense mutation, all of which were heterozygous and 3 of which were novel, were identified. Diabetic ketosis was found occasionally in these patients even under conditions of chronic hyperglycemia, for unknown reasons. Although the capacity of early-phase insulin secretion was low in these patients, the insulin secretory capacity was relatively preserved compared to that in hepatocyte nuclear factor (HNF)1A- and HNF1B-MODY. One of the patients and 2 of their diabetic mothers were found to have some mental or neuronal abnormality., Conclusions: This is the first report of NEUROD1 mutations in Japanese, who have a genetic background of intrinsically lower capacity of insulin secretion. NEUROD1-deficient diabetes appears to be low penetrant, and may occur in concert with other genetic factors., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A 57-year-old man was admitted for the treatment of steroid-induced diabetes mellitus (DM). He also had interstitial pneumonia and, to treat it, 20 mg prednisolone had been started in April 2014. Although glycated haemoglobin (HbA1c) level was 7.8% (62 mmol/mol), his glycated albumin (GA) level was normal (13.9%) and the ratio of GA to HbA1c (GA:HbA1c) was lower than that of normal participants and patients with type 2 DM. Plasma GA and GA:HbA1c levels became persistently lower. In September 2015, HbA1c levels measured by HPLC and immunoprecipitation methods were almost the same (6.8% (51 mmol/mol) and 6.7% (50 mmol/mol), respectively), but GA (10.2%) and GA:HbA1c (1.6) were much lower. We report the case of a patient with DM where steroid administration may have caused a decrease in plasma GA and GA:HbA1c levels via increased albumin turnover., (2016 BMJ Publishing Group Ltd.)

A 65-year-old woman had been visiting our department for the treatment of type-2 diabetes mellitus since December 2012. Her glycated haemoglobin levels were well controlled (≈5.8% (40 mmol/mol)) by metformin (500 mg). In July 2014, her white cell count increased suddenly to 33 530 cells/μL and she was diagnosed with Ph+ chronic myeloid leukaemia. She was started on dasatinib (100 mg), which immediately normalised plasma levels of WCC. Dasatinib improved the glycaemic index to <6.0% and also improved plasma levels of triglycerides (TGs) and high-density lipoprotein-cholesterol (HDL-c). Levels of low-density lipoprotein-cholesterol were increased but remained within the normal range. The TG:HDL-c ratio and Quantitative Insulin Sensitivity Check Index rapidly improved. Followed by an improvement in insulin sensitivity, plasma levels of adiponectin and leptin were increased. This case study suggests that dasatinib might have positive as well as negative effects on the metabolism of glucose and lipids., (2016 BMJ Publishing Group Ltd.)

Although lower extremity arterial disease is frequently accompanied by diabetes mellitus, the association of circulating biomarkers with flow components during the cardiac cycle in lower-leg arteries has yet to be fully elucidated. We enrolled 165 type 2 diabetic patients with normal ankle-brachial index (ABI 1.0-1.4), comprising 106 normoalbuminuric and 59 microalbuminuric patients, and 40 age-matched nondiabetic subjects consecutively admitted to our hospital. Serum high sensitivity C-reactive protein (hsCRP) level and plasma von Willebrand factor ristocetin cofactor activity (VWF) and vasoconstrictor serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) concentrations were measured. An automatic device was used to measure ABI and brachial-ankle pulse wave velocity (baPWV). Flow components during the cardiac cycle, total flow volume, and resistive index at popliteal artery were evaluated using gated magnetic resonance imaging. Although estimated glomerular filtration rate (eGFR), early diastolic flow reversal, heart rate, and ABI were similar between the groups, diabetic patients had higher log hsCRP (p<0.001), VWF (p<0.001), 5-HIAA (p=0.002), resistive index (p<0.001) and baPWV (p<0.001) and lower systolic (p=0.026) and late diastolic (p<0.001) forward flows and total flow volume (p<0.001) than nondiabetic subjects. Multivariate analyses demonstrated that 5-HIAA in microalbuminuric patients showed higher associations with systolic and late diastolic forward flows during the cardiac cycle, total flow volume and resistive index at popliteal artery, and eGFR compared to normoalbuminuric patients. In microalbuminuric patients, 5-HIAA was a significant independent determinant among these factors. Thus, increased plasma 5-HIAA levels are involved in the pathogenesis of impaired blood flow in lower extremities and renal insufficiency in diabetic patients with microalbuminuria.

Aim: Several studies have suggested that cigarette-smoking affects insulin sensitivity in Western populations. The present study evaluated glucose tolerance, pancreatic β-cell function and insulin sensitivity in relation to active and passive smoking among the Japanese., Methods: A total of 411 men and 586 women were recruited into a community-based cross-sectional study in Gifu, Japan. Diabetes, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) were screened for by a 75g oral glucose tolerance test. HOMA and insulinogenic (ΔI0-30/ΔG0-30) indexes were used to estimate insulin secretion and sensitivity. To assess the possible association of self-reported smoking status and parameters of glucose metabolism, logistic regression was applied after adjusting for potential confounders., Results: Currently smoking women were more likely to have diabetes, IGT or IFG compared with never-smoking women (OR: 2.26, 95% CI: 1.05-4.84). Heavy-smoking men (≥25 cigarettes/day) were likely to be in the lowest tertile group of ΔI0-30/ΔG0-30 compared with never-smoking men (OR: 2.64, 95% CI: 1.05-6.68, Ptrend=0.04). The number of cigarettes/day was borderline significantly associated with diabetes in men. Also with borderline significance, never-smoking women with smoking husbands were more likely to have diabetes, IGT or IFG (OR: 1.62, 95% CI: 1.00-2.62) and significantly more likely to have lower HOMA-β (OR: 2.17, 95% CI: 1.36-3.48) than those without smoking husbands., Conclusion: The greater the number of cigarettes smoked per day appears to be associated with diabetes among men whereas, among women, both active and passive smoking appear to be associated with diabetic states, including IGT and IFG. An association between smoking status and insulin secretion is also suggested, whereas no significant association was observed with HOMA-IR in this Japanese subjects, suggesting that the influence of smoking on glucose metabolism may differ among races., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Blood flow in lower extremity arteries is frequently impaired in diabetic patients even though they have a normal ankle-brachial index (ABI 1.0-1.4). Risk factors contributing to this lower extremity arterial disease have not been fully elucidated. We enrolled 52 type 2 diabetic patients with normal ABI and 30 age-matched nondiabetic subjects consecutively admitted to our hospital. Plasma B-type natriuretic peptide (BNP) concentrations were measured. Distensibility in ascending thoracic and abdominal aortas as well as total flow volume and resistive index at popliteal artery were evaluated by gated magnetic resonance imaging. An automatic device was used to measure ABI and brachial-ankle pulse-wave velocity (baPWV). Diabetic patients showed lower distensibility in ascending thoracic aorta (p<0.001) and total flow volume (p<0.001) and higher baPWV (p<0.001) and resistive index (p=0.005) and similar BNP and distensibility in abdominal aorta compared to nondiabetic subjects. Simple linear regression analyses revealed that distensibility in ascending thoracic (p=0.019) and abdominal (p=0.030) aortas positively as well as baPWV (p=0.020), resistive index (p<0.001) and BNP (p<0.001) negatively correlated with total flow volume. Stepwise multiple regression analysis demonstrated that increased BNP and resistive index were independent risk factors for total flow volume in diabetic patients (r(2)=0.639, p<0.001). These results indicate that increased plasma BNP levels and peripheral vascular resistance, but not decreased aortic distensibility, associate with impaired blood flow in lower extremity arteries in diabetic patients.

Background: Although there have been several reports that statins cause insulin resistance that leads to the occurrence of type 2 diabetes in Caucasians, there has been no Japanese prospective studies investigating the effects of statins on the glucose metabolism system., Materials and Methods: Our subjects were 86 Japanese patients with type 2 diabetes with hypercholesterolemia. Pitavastatin 2mg/day was administered for 12 months and the lipid-related values, glucose metabolism values, and the presence/absence of side effects were investigated., Results: None of these factors was found to differ between before and after administration of pitavastatin in overall analysis of all subjects. In subgroup analysis, fasting blood glucose showed a decrease in the BMI ≥ 25 group and there was a significant difference between the BMI<25 and BMI ≥2 5 groups (P-values: 0.021 and 0.0036). Although HbA1c showed an increase both in the group switched to pitavastatin and the BMI<25 group (P-values: 0.035 and 0.033) and HOMA-β showed a decrease in the BMI<25 group (P-values: 0.044), there were no significant differences in changes between each divided group and their counterparts., Conclusion: In the Japanese obese group with BMI ≥ 25, pitavastatin elicited a significant decrease in fasting blood glucose. It is not clear whether or not this is due to improved insulin resistance as a direct effect of pitavastatin, but in contrast to findings in Caucasians pitavastatin does not worsen insulin resistance in Japanese patients with type 2 diabetes complicated by hypercholesterolemia., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Aim: To compare the prevalence and clinical features of HNF1β-related MODY and HNF1α-related MODY in Japanese., Methods: We enrolled 230 Japanese patients with suspected MODY and examined them for HNF1α and HNF1β mutations. We characterized the clinical features of HNF1β-related MODY (HNF1β-MODY) and HNF1α-related MODY (HNF1α-MODY)., Results: Six patients had HNF1β mutations, four of which were large gene deletions and 24 patients had HNF1α mutations, which included one gene deletion. The mean fasting plasma glucose level at onset of HNF1β-MODY was considerably higher and the age of onset of HNF1β-MODY was considerably older than they were for HNF1α-MODY, while the mean BMI and C-peptide index at onset were similar. Three patients with HNF1β-MODY were found to have dorsal pancreatic agenesis and four of them had whole-gene deletion. Five of the patients with HNF1β-MODY had insulin secretion defects and were treated with insulin, and four of these did not have a parent with overt diabetes., Conclusion: HNF1β-MODY may present as β-cell dysfunction in Japanese rather than as hyperinsulinaemia, which it does among European/American. This dysfunction might result from an intrinsically lower capacity for insulin secretion in Japanese. HNF1β-MODY has an older age of onset than HNF1α-MODY, which may suggest lower penetrance of the disease. In addition, HNF1β-MODY has a broad spectrum of clinical manifestations, some of which are detectable by imaging. This may be helpful in some cases for selecting HNF1β-MODY candidates for genetic testing., (© 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.)

Type 2 11β-hydroxysteroid dehydrogenase encoded by the HSD11B2 gene converts cortisol to inactive cortisone, and alteration in this enzymatic activity might affect glucose homeostasis by affecting circulating levels or tissue availability of glucocorticoids. We investigated the association of HSD11B2 variant with glucose homeostasis. Subjects with normal glucose tolerance (n=585), impaired glucose tolerance (n=202) and type 2 diabetes (n=355) were genotyped for a highly polymorphic CA-repeat polymorphism in the first intron of HSD11B2. Allele and genotype frequencies differed between normal and impaired glucose tolerance (P = 0.0014 and 0.0407, respectively; 4 degree of freedom) or type 2 diabetes (P = 0.0053 and 0.0078), with significant linear trends between the repeat length and the phenotype fraction. In normal subjects, total CA-repeat length was negatively correlated with fasting insulin and HOMA-β. Thus, subjects having more CA repeats are susceptible to developing abnormal glucose tolerance, whereas normal subjects carrying more CA repeats appeared to have frugal characteristics in insulin secretion.

Aims: To evaluate the associations of circulating levels of proinflammatory molecules and endothelial factors with blood flow in lower-leg arteries in diabetic patients with normal ankle-brachial index (ABI>0.9)., Methods: We enrolled 123 type 2 diabetic patients with normal ABI and 30 age-matched nondiabetic subjects consecutively admitted to our hospital. Flow volume and resistive index, an index of peripheral vascular resistance, at the popliteal artery were evaluated using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. An automatic device was used to measure ABI and brachial-ankle pulse-wave velocity (baPWV) for evaluation of arterial stiffness. Plasma soluble intercellular adhesion molecule-1 (sICAM-1) and monocyte chemoattractant protein-1 (MCP-1) concentrations, serum high-sensitivity C-reactive protein (hsCRP) levels, plasma von Willebrand factor ristocetin cofactor activity (VWF), and plasma vasoconstrictor serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) concentrations were measured., Results: Diabetic patients had higher baPWV (P<.0001), resistive index (P<.0001), sICAM-1 (P<.0001), MCP-1 (P=.0224), log hsCRP (P<.0001), VWF (P<.0001), 5-HIAA (P=.0015), and lower blood flow (P<.0001) than nondiabetic subjects. VWF (P=.0019) or 5-HIAA (P=.0011), but not sICAM-1, MCP-1, and log hsCRP, was negatively correlated with blood flow in diabetic patients. A multivariate analysis revealed that the significant independent determinants of blood flow were hypertension, use of renin-angiotensin system inhibitors, VWF and 5-HIAA (r(2)=0.198, P<.0001) in diabetic patients., Conclusions: Plasma VWF and 5-HIAA concentrations are associated with blood flow and are involved in the pathogenesis of impaired peripheral circulation due to higher arterial stiffness and greater vascular resistance in lower-leg arteries in diabetic patients with normal ABI., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Metformin is a drug to improve glycemic control by reducing insulin resistance and is currently considered to be one of the first-choice drugs for type 2 diabetes mellitus (T2DM). However, during metformin use, adverse drug reactions (ADRs) including gastrointestinal adverse events were frequently observed. Thus, in the present study, we investigated the incidence of ADRs induced by metformin and further analyzed risk factors for ADRs in Japanese patients with type 2 diabetes mellitus who initially administered metformin (500-750 mg). One hundred and one hospitalized patients receiving metformin during September 1, 2009 and August 31, 2010 were studied. The incidence of ADRs and changes in laboratory data including hemoglobin A1c (HbA1c) were monitored retrospectively. The anti-glycemic effect of metformin was successfully observed as indicated by decreased HbA1c. Among ADRs, diarrhea was most frequently occurred during metformin use (26.7% of patients) although the symptom of diarrhea was mild in most cases and disappeared within 3 d after the initial use. A logistic regression analysis showed the existence of six risk factors, including initial dose (750 mg), female, age (≦65), body mass index (≧25), aspartate aminotransferase (≧30 IU/L) and alkaline phosphatase (≧270 IU/L). The incidence of diarrhea increased linearly as the number of risk factors increased. In conclusion, in order to avoid ADRs, especially diarrhea, subsequently improving the quality of life during metformin use, the optimization of the dose of metformin by considering risk factors would be beneficial for patients with T2DM.

TCF7L2 is a Wnt signaling-associated transcription factor and is ubiquitously expressed. Polymorphisms in the TCF7L2 gene exhibit the strongest association with type 2 diabetes among approximately twenty susceptibility gene variants identified to date. Although the mechanisms by which TCF7L2 affects susceptibility to type 2 diabetes remain to be elucidated, several studies have shown that decreased TCF7L2 protein inhibits the insulin secretory response to oral glucose through impaired incretin action(GLP-1, GIP). In this review, we discuss studies that investigate the association between polymorphisms of TCF7L2 and the diabetic phenotype, especially in vitro beta cell function with special reference to incretin action and the response to lifestyle intervention.

Objective: Early detection of nerve dysfunction is important to provide appropriate care for patients with diabetic polyneuropathy. The aim of this study was to assess the echo intensity of the peripheral nerve and to evaluate the relationship between nerve conduction study results and sonographic findings in patients with type 2 diabetes mellitus., Methods: Thirty patients with type 2 diabetes (mean +/- SD, 59.8 +/- 10.2 years) and 32 healthy volunteers (mean, 53.7 +/- 13.9 years) were enrolled in this study. The cross-sectional area (CSA) and echo intensity of the peripheral nerve were evaluated at the carpal tunnel and proximal to the wrist (wrist) of the median nerve and in the tibial nerve at the ankle., Results: There was a significant increase in the CSA and hypoechoic area of the nerve in diabetic patients compared with controls (wrist, 7.1 +/- 2.0 mm(2), 62.3% +/- 3.0%; ankle, 8.9 +/- 2.8 mm(2), 57.6% +/- 3.9%; and wrist, 9.8 +/- 3.7 mm(2), 72.3% +/- 6.6%; ankle, 15.0 +/- 6.1 mm(2), 61.4% +/- 5.3% in controls and diabetic patients, respectively; P < .05). Cross-sectional areas were negatively correlated with reduced motor nerve conduction velocity and delayed latency., Conclusions: These results suggest that sonographic examinations are useful for the diagnosis of diabetic neuropathy.

Vascular calcification is frequently accompanied by intima-media thickening, but the associations among these atherosclerotic features and bone-related peptides in diabetic patients are unclear. We enrolled 168 type 2 diabetic patients and 40 non-diabetic subjects consecutively admitted to our hospital. Mean intima-media thickness (mean-IMT) in common carotid arteries was measured by B-mode ultrasonography. Agatston coronary artery calcium score (CACS) was obtained using multidetector-row computed tomography (MDCT). Plasma bone-related peptides osteopontin and osteoprotegerin levels were measured. Diabetic patients had higher mean-IMT (p=0.0002) and log(CACS+1) (p<0.0001) and similar bone-related peptides compared to non-diabetic subjects. In diabetic patients classified into tertiles according to their CACS levels, those with the highest scores showed the highest mean-IMT (p=0.0004) and bone-related peptides (p<0.05) among the groups. log(CACS+1) and mean-IMT were associated (p<0.0001) and were positively correlated with osteopontin (p<0.01) and osteoprotegerin (p<0.01) in diabetic patients. Multivariate analyses revealed that the significant independent determinants of log(CACS+1) were age, duration of diabetes and osteopontin (p<0.0001) and those of mean-IMT were age, hypertension, osteopontin and osteoprotegerin (p<0.0001), respectively. We have demonstrated that vascular calcification in type 2 diabetic patients is frequently accompanied by intima-media thickening, and osteopontin may act as a vascular calcification inhibitor by increasing intima-media thickness.

Background: Higher arterial stiffness and greater peripheral vascular resistance reduce blood flow in lower-leg arteries and contribute to the development of ischaemic limb in diabetic patients even without peripheral artery occlusive disease. The aim of this study was to clarify whether these vascular parameters are associated with long-term hyperglycaemia in diabetic patients., Methods: We examined 45 type 2 diabetic patients and 38 age-matched nondiabetic subjects without peripheral artery occlusive disease assessed by ankle-brachial index consecutively admitted to our hospital, and followed them over a 3-year period (3.7 +/- 0.7 years) with no vasodilative medication. Blood flow and resistive index, a measure of peripheral vascular resistance, at the popliteal artery were evaluated using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. Brachial-ankle pulse wave velocity was measured to assess arterial stiffness., Results: At baseline, consistent with our previous report, diabetic patients showed higher brachial-ankle pulse wave velocity (p < 0.0001) and resistive index (p < 0.0001) and lower flow volume (p = 0.0044) than those of nondiabetic subjects. Stepwise multiple regression analysis revealed that duration of diabetes, mean HbA(1c) during the study, use of renin-angiotensin system inhibitors and change per year in resistive index were identified as significant independent variables predicting change per year in blood flow (r(2) = 0.733, p < 0.0001) in diabetic patients. Mean HbA(1c) during the study was positively correlated with changes per year in brachial-ankle pulse wave velocity (p = 0.00007) and resistive index (p = 0.0014) and was negatively correlated with that in blood flow (p < 0.0001) in diabetic patients., Conclusions: Long-term hyperglycaemia is a major cause of impaired peripheral circulation in lower-leg arteries in diabetic patients without peripheral artery occlusive disease.

Prediction of the disease status is one of the most important objectives of genetic studies. To select the genes with strong evidence of the association with type 2 diabetes mellitus, we validated the associations of the seven candidate loci extracted in our earlier study by genotyping the samples in two independent sample panels. However, except for KCNQ1, the association of none of the remaining seven loci was replicated. We then selected 11 genes, KCNQ1, TCF7L2, CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, HHEX, GCKR, HNF1B, KCNJ11 and PPARG, whose associations with diabetes have already been reported and replicated either in the literature or in this study in the Japanese population. As no evidence of the gene-gene interaction for any pair of the 11 loci was shown, we constructed a prediction model for the disease using the logistic regression analysis by incorporating the number of the risk alleles for the 11 genes, as well as age, sex and body mass index as independent variables. Cumulative risk assessment showed that the addition of one risk allele resulted in an average increase in the odds for the disease of 1.29 (95% CI=1.25-1.33, P=5.4 x 10(-53)). The area under the receiver operating characteristic curve, an estimate of the power of the prediction model, was 0.72, thereby indicating that our prediction model for type 2 diabetes may not be so useful but has some value. Incorporation of data from additional risk loci is most likely to increase the predictive power.

Genetic analysis of the KLF11 gene revealed two rare variants, A347S and T220M, segregating in families with early-onset type 2 diabetes, and one frequent polymorphic Q62R variant significantly associated with type 2 diabetes in Northern Europeans. Furthermore, it has been reported that over-expression of KLF11 has a deleterious effect on insulin promoter activity. Thus, an altered expression level of KLF11 may contribute to the occurrence of type 2 diabetes. To investigate the contribution of KLF11 to type 2 diabetes in Japanese, we surveyed the 5' flanking region of KLF11 by reporter assay and identified the minimal promoter region of the gene. The promoter region from -250 to +162 bp including five Sp1 binding sites showed basal promoter activity both in MIN6-m9 and HepG2 cells. We also examined the entire region of KLF11 to detect genetic variants. A total of 19 polymorphisms, six of which are novel, were identified, but none of them showed association with the occurrence of type 2 diabetes. Two of the identified polymorphisms, R29Q and S124F, are novel coding variants. Functional analyses of these variants were performed, and similarly reduced effects on transcriptional activities of insulin, catalase1, and the Smad7 gene were found. We conclude that variants of KLF11 are not a major factor in the occurrence of type 2 diabetes in Japanese. The promoter region of KLF11 identified in the present study should be useful in further elucidation of the transcriptional regulation mechanism of the gene and genetic analyses of type 2 diabetes.

Aims: Heterozygous mutations of the hepatocyte nuclear factor (HNF)-4alpha gene cause a particular form of maturity-onset diabetes of the young (MODY1). Recent genetic studies have shown that single nucleotide polymorphisms (SNPs) of the beta-cell type P2 promoter of the HNF-4alpha gene are associated with type 2 diabetes in some populations. In the Japanese population, a haplotype consisting of two SNPs (rs1884614 and rs2144908) in the P2 promoter region is reported to show a significant association with type 2 diabetes., Methods: Both rs1884614 and rs2144908 were genotyped in 349 type 2 diabetic patients and 203 non-diabetic controls. The relation of these SNPs to clinical characteristics was also examined in the diabetic subjects., Results: There were no differences in the genotype distribution of the two SNPs between the control and diabetic subjects, and the haplotype distribution was also similar in the two groups. However, the rs1884614 T/T genotype was significantly associated with a smaller area under the plasma insulin curve (AUC) during the OGTT in non-obese (BMI <25 kg/m(2)) patients (p=0.0272; adjusted for age and sex)., Conclusions: SNP rs1884614 in the P2 promoter region of the HNF-4alpha gene may influence insulin secretion in non-obese Japanese subjects with type 2 diabetes.

Mutations in the small heterodimer partner gene (NR0B2; alias SHP) are associated with high birth weight and mild obesity in Japanese children. SHP mutations may also be associated with later obesity and insulin resistance syndrome that induces diabetes. To investigate this possibility, the prevalence of SHP mutations in Japanese with and without type 2 diabetes mellitus and the functional properties of the mutant proteins were evaluated. Direct sequencing of two exons and flanking sequences of SHP in 805 diabetic patients and 752 non-diabetic controls identified 15 different mutations in 44 subjects, including 6 novel mutations. Functional analyses of the mutant proteins revealed significantly reduced activity of nine of the mutations. Mutations with reduced activity were found in 19 patients (2.4%) in the diabetic group and in 6 subjects (0.8%) in the control group. The frequency difference between DM and control subjects adjusted for sex and age was statistically significant (P=0.029, odds ratio 2.67, 95% CI 1.05-6.81, 1-beta=0.91). We conclude that SHP mutations associated with mild obesity in childhood increase susceptibility to type 2 diabetes in later life in Japanese., ((c) 2008 Wiley-Liss, Inc.)

Although the kidneys are the major source of proinflammatory cytokines, association of tumor necrosis factor-alpha (TNF-alpha) with severity of atherosclerosis or kidney function in diabetic patients is unclear. Two hundred type 2 diabetic patients and 30 age-matched nondiabetic subjects consecutively admitted to our hospital were enrolled. The Agatston coronary artery calcium score (CACS), a quantitative marker of coronary atherosclerosis, was obtained using multidetector-row computed tomography. Arterial stiffness was assessed by brachial-ankle pulse wave velocity (baPWV). Diabetic patients had higher log(CACS+1) (p=0.0089), baPWV (p=0.0293), frequency of elevated urinary albumin excretion (UAE) (p<0.0001) and TNF-alpha (p=0.0029) and similar estimated glomerular filtration rate (eGFR) compared to nondiabetic subjects. When diabetic patients were grouped into four subgroups with or without elevated UAE and renal insufficiency (UAE of >/=30 or <30mg/24h and eGFR of <60 or >/=60ml/min per 1.73m(2)), patients with micro- and macroalbuminuric renal insufficiency showed the highest log(CACS+1) (p<0.0001), baPWV (p=0.0068) and TNF-alpha (p<0.0001) of these groups. Log(CACS+1) (p=0.0008) and baPWV (p=0.0006) positively and eGFR (p<0.0001) negatively correlated with TNF-alpha in diabetic patients. We find that coronary artery calcification, arterial stiffness, and renal insufficiency associate with circulating levels of TNF-alpha in type 2 diabetic patients.

We carried out a multistage genome-wide association study of type 2 diabetes mellitus in Japanese individuals, with a total of 1,612 cases and 1,424 controls and 100,000 SNPs. The most significant association was obtained with SNPs in KCNQ1, and dense mapping within the gene revealed that rs2237892 in intron 15 showed the lowest Pvalue (6.7 x 10(-13), odds ratio (OR) = 1.49). The association of KCNQ1 with type 2 diabetes was replicated in populations of Korean, Chinese and European ancestry as well as in two independent Japanese populations, and meta-analysis with a total of 19,930 individuals (9,569 cases and 10,361 controls) yielded a P value of 1.7 x 10(-42) (OR = 1.40; 95% CI = 1.34-1.47) for rs2237892. Among control subjects, the risk allele of this polymorphism was associated with impairment of insulin secretion according to the homeostasis model assessment of beta-cell function or the corrected insulin response. Our data thus implicate KCNQ1 as a diabetes susceptibility gene in groups of different ancestries.

Aims: A missense mutation in the low density lipoprotein receptor-related protein 6 gene (LRP6) was recently shown to be responsible for a disorder characterized by early-onset coronary artery disease as well as diabetes mellitus (DM), hyperlipidemia, hypertension, and osteoporosis. Mice deficient in LRP5, a closely related paralog of LRP6, manifest a marked impairment in glucose tolerance. The aim of the present study was to examine whether common variants of LRP5 and LRP6 are associated with Type 2 DM or dyslipidemia in Japanese individuals., Methods: Thirteen single nucleotide polymorphisms (SNPs) of LRP6 and nine SNPs of LRP5 were genotyped in a total of 608 Type 2 DM patients and 366 nondiabetic control subjects (initial study). An association analysis was then performed for each SNP and for haplotypes. For some of the SNPs, we provided another sample panel of 576 cases and 576 controls for the replication study. The relation to clinical characteristics was also examined in diabetic subjects., Results: In the initial study, three SNPs of LRP6 were found to be associated with susceptibility to Type 2 DM. However, this association was not detected in the replication panel. None of SNPs in LRP5 were associated with Type 2 DM in the initial panel. Neither LRP6 nor LRP5 was associated with body mass index, HOMA-beta, HOMA-IR or serum lipid concentrations., Conclusions: We found no evidence for a substantial effect of LRP5 or LRP6 SNPs on susceptibility to type 2 diabetes or clinical characteristics of diabetic subjects in Japanese population.

Background: In Europeans and populations of European origin, several groups have recently identified novel type 2 diabetes susceptibility genes, including FTO, SLC30A8, HHEX, CDKAL1, CDKN2B, and IGF2BP2, none of which were in the list of functional candidates., Objective and Design: The aim of this study was to replicate in a Japanese population previously identified associations of single nucleotide polymorphisms (SNPs) within 10 candidate loci with type 2 diabetes using a relatively large sample size: 1921 subjects with type 2 diabetes and 1622 normal controls., Results: A total of 15 SNPs were genotyped. Eight SNPs in five loci were found to be associated with type 2 diabetes: rs3802177 [odds ratio (OR) = 1.16 (95% confidence interval (CI) 1.05-1.27); P = 4.5 x 10(-3)] in SLC30A8; rs1111875 [OR = 1.27 (95% CI 1.14-1.40); P = 1.4 x 10(-5)] and rs7923837 [OR = 1.27 (95% CI 1.13-1.43); P = 1.0 x 10(-4)] in HHEX; rs10811661 [OR = 1.27 (95% CI 1.15-1.40); P = 1.9 x 10(-6)] in CDKN2B; rs4402960 [OR = 1.23 (95% CI 1.11-1.36); P = 8.1 x 10(-5)] and rs1470579 [OR = 1.18 (95% CI 1.07-1.31); P = 8.3 x 10(-4)] in IGF2BP2; and rs7754840 [OR = 1.28 (95% CI 1.17-1.41); P = 4.5 x 10(-7)] and rs7756992 [OR = 1.27 (95% CI 1.15-1.40); P = 9.8 x 10(-7)] in CDKAL1. The first and second strongest associations were found at variants in CDKAL1 and CDKN2B, both of which are involved in the regenerative capacity of pancreatic beta-cells., Conclusion: Some of these variants represent common type 2 diabetes-susceptibility genes in both Japanese and Europeans.

Diabetic nephropathy is a major cause of lower-limb amputation. We enrolled 250 type 2 diabetic patients without apparent occlusive peripheral arterial disease (ankle-brachial indices >0.9) and 40 age-matched nondiabetic subjects consecutively admitted to our hospital. Flow volume and resistive index (RI), an index of vascular resistance, at the popliteal artery were evaluated using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. Brachial-ankle pulse wave velocity (baPWV) was measured as an index of arterial distensibility. Flow volume was negatively correlated with both baPWV (p=0.0009) and RI (p<0.0001) among the patients. When the patients were grouped into four subgroups with or without albuminuria and renal insufficiency according to the levels of urinary albumin excretion rate (>or=20 or <20microg/min) and estimated glomerular filtration rate (eGFR) (<60 or >or=60ml/min/1.73m(2)), albuminuric patients with renal insufficiency (n=30) showed the lowest flow volume (p=0.0078) and the highest baPWV (p=0.0006) and RI (p=0.0274) among the groups. Simple linear regression analyses demonstrated that eGFR correlated positively with flow volume (p=0.0020) and negatively with baPWV (p=0.0258) and RI (p=0.0029) in patients with albuminuria (n=92), but not with normoalbuminuria (n=158). Impaired peripheral circulation in lower-leg arteries associates with nephropathy in diabetic patients even though they have normal ankle-brachial indices.

The level of leptin increases with obesity, whereas that of adiponectin decreases with obesity. It is reported that the ratio of leptin to adiponectin (L/A) is associated with insulin resistance. It is difficult to evaluate insulin resistance in diabetic patients who have a dysfunction of insulin secretion. The aim of this study was to examine whether the L/A ratio is a useful marker for insulin resistance in diabetic patients. We examined L/A in the serum of a total of 139 Japanese patients with type 2 diabetes mellitus (66 women and 73 men) and 7 healthy individuals recruited in our hospital. Changes in the levels of leptin and adiponectin were observed using the oral glucose tolerance test and a hyper- and euglycemic clamp test. Twenty-one patients with type 2 diabetes mellitus were observed for more than 6 months after treatment with pioglitazone, and 31 patients with type 2 diabetes mellitus were observed for more than 6 months after the treatment with metformin. The mean value of L/A in 139 Japanese patients with type 2 diabetes mellitus was 1.22 +/- 1.41 (1.68 +/- 1.76 in women, 0.81 +/- 0.80 in men; P = .0002). In the clamp tests, L/A correlated with glucose infusion rate (GIR) (r(2) = 0.26, P = .0034). The correlation of L/A and GIR indicated a stronger correlation than either leptin (r(2) = 0.144, P = .03) or adiponectin alone (r(2) = 0.023, P = .41), or the homeostasis model assessment of insulin resistance (r(2) = 0.103, P = .08). The average hemoglobin A(1c) (HbA(1c)) improved from 10.2% +/- 1.2% to 9.2% +/- 1.6% (P = .0037) in 6 months after treatment with pioglitazone. Our results indicate pioglitazone to be effective for HbA(1c) improvement in subjects with high L/A and low L/A. The average HbA(1c) improved from 9.2% +/- 0.9% to 8.0% +/- 1.2% (P = .0002) in 6 months after treatment with metformin. Our results indicate metformin to be effective for HbA(1c) improvement in subjects with a low L/A. In conclusion, we demonstrate that L/A is different between male and female subjects. The correlation of L/A and GIR by the euglycemic hyperinsulinemic clamp test suggests that L/A is a useful indicator for the choice of drug to treat diabetes mellitus.

Transcription factor 7-like 2 (TCF7L2) has been shown to be associated with type 2 diabetes mellitus in multiple ethnic groups. Regarding the Asian population, Horikoshi et al. (Diabetologia 50:747-751, 2007) and Hayashi et al. (Diabetologia 50:980-984, 2007) reported that single nucleotide polymorphisms (SNPs) in TCF7L2 were associated with type 2 diabetes in the Japanese population, while contradictory results were reported for Han Chinese populations. The aim of this study was to investigate the associations of the TCF7L2 gene with type 2 diabetes using a relatively large sample size: 2,214 Japanese individuals with type 2 diabetes and 1,873 normal controls. The minor alleles of rs7903146, rs11196205, and rs12255372 showed significant associations with type 2 diabetes (OR=1.48, P=2.7 x 10(-4); OR=1.39, P=4.6 x 10(-4); OR=1.70, P=9.8 x 10(-5), respectively) in the combined sample sets. However, neither rs11196218 nor rs290487 showed a significant association. These results indicate that TCF7L2 is an important susceptibility gene for type 2 diabetes in the Japanese population.

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.

Because impaired insulin secretion is characteristic of type 2 diabetes in Asians, including Japanese, the genes involved in pancreatic beta-cell function are candidate susceptibility genes for type 2 diabetes. We examined the association of variants in genes encoding several transcription factors (TCF1, TCF2, HNF4A, ISL1, IPF1, NEUROG3, PAX6, NKX2-2, NKX6-1, and NEUROD1) and genes encoding the ATP-sensitive K(+) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) with type 2 diabetes in a Japanese cohort of 2,834 subjects. The exon 16 -3c/t variant rs1799854 in ABCC8 showed a significant association (P = 0.0073), and variants in several genes showed nominally significant associations (P < 0.05) with type 2 diabetes. Although the E23K variant rs5219 in KCNJ11 showed no association with diabetes in Japanese (for the K allele, odds ratio [OR] 1.08 [95% CI 0.97-1.21], P = 0.15), 95% CI around the OR overlaps in meta-analysis of European populations, suggesting that our results are not inconsistent with the previous studies. This is the largest association study so far conducted on these genes in Japanese and provides valuable information for comparison with other ethnic groups.

Context and Objective: Vascular endothelial growth factor plays a critical role both in neovascularization of proliferative diabetic retinopathy and in angiogenesis of islets in the pancreatic developmental stage in determining beta-cell mass and properties. Vascular endothelial growth factor mRNA levels increase as a result of increased transcriptional activation, mediated predominantly by hypoxia-inducible factor-1 alpha (HIF-1alpha) in response to hypoxia., Design and Patients: In this study, we examined all regions of the HIF-1alpha to detect single-nucleotide polymorphisms (SNPs), evaluated the pattern of linkage disequilibrium to analyze haplotypes, and performed association studies in Japanese type 2 diabetes patients with or without retinopathy., Results: A total of 35 SNPs were found in the gene, 27 of which were reported previously and eight of which were novel. Three of the 35 SNPs were located in coding regions, one in exon 2 (S28Y), and the others in exon 12 (P582S, A588T). The P582S HIF-1alpha mutation was associated with type 2 diabetes (P = 0.0028) by a consistently higher level of transcriptional activity than wild type, especially under hypoxic condition (P = 0.012), but no association with retinopathy was detected., Conclusion: This is the first report that HIF-1alpha is associated with the occurrence of type 2 diabetes and suggests that the P582S HIF-1alpha mutation should be assessed in larger studies as a risk factor for type 2 diabetes.

Diabetic and psychiatric symptoms often appear in patients with Wolfram syndrome, and obligate carriers of WFS1 have increased prevalence of type 2 diabetes and are more likely to require hospitalization for psychiatric illness including bipolar disorder. To identify the polymorphisms in Japanese, we examined a region of approximately 50 kb covering the entire WFS1 gene, and evaluated the patterns of linkage disequilibrium. We found a total of 42 variations including 8 novel coding single nucleotide polymorphisms (A6T, A134A, N159N, T170T, E237K, R383C, V412L, and V503G), 14 novel non-coding polymorphisms, and 2 linkage disequilibrium blocks. We also performed association studies in patients with type 2 diabetes mellitus and patients with bipolar disorder. The haplotype comprising R456 and H611 was most associated with type 2 diabetes (p = 0.013) and the haplotype comprising g. -15503C/T and g. 16226G/A was most associated with bipolar disorder (p = 0.006), but neither reached significant difference after multiple adjustment. These genetic variations and linkage disequilibrium patterns in WFS1 in Japanese should be useful in further investigation of genetic diversities of WFS1 and various related disorders.

Aims/hypothesis: Mutations in hepatocyte nuclear factor (HNF)-4alpha gene cause a form of maturity-onset diabetes of the young (MODY1). The T130I mutation is a rare missense mutation, which affects a conserved amino acid in a DNA binding domain. This mutation can be found in the general population, so this variant alone does not cause MODY. However, its significance in the development of late-onset Type 2 diabetes is not known., Methods: We screened 423 unrelated Japanese patients with late-onset Type 2 diabetes and 354 unrelated non-diabetic control subjects for the T130I mutation in the HNF-4alpha gene. The transactivation ability of T130I-HNF-4alpha was assessed using reporter gene assay., Results: The frequency of the T130I mutation was higher in Type 2 diabetic patients ( p=0.015, odds ratio 4.3, 95%CI 1.24-14.98) than control subjects. The serum HDL-cholesterol concentration was lower in Type 2 diabetic patients with the T130I mutation compared with those without this mutation ( p=0.006). Reporter gene analysis showed that T130I-HNF-4alpha transcriptional activity was not impaired compared with wild-type HNF-4alpha in Hela and MIN6 cells, but it was reduced in HepG2 and primary cultured mouse hepatocytes (27-78% of wild type, p<0.05)., Conclusion/interpretation: Our findings suggest that T130I-HNF-4alpha is a loss-of-function mutation in hepatocytes and that this mutation is associated with late-onset Type 2 diabetes in Japanese subjects. The T130I mutation in the HNF-4alpha gene might be involved in the development of Type 2 diabetes in the Japanese population.

The 112/121 haplotype combination defined by the UCSNP-43, -19, and -63 alleles in the calpain-10 gene is associated with type 2 diabetes in Mexican Americans. To determine whether this genetic variation constitutes risk of type 2 diabetes in Japanese, we investigated its frequency in 177 patients with type 2 diabetes and 172 controls. Though this variation occurs in Japanese more frequently than in Mexican Americans, there is no significant difference in frequency between diabetic (29.9%) and control (31.9%) subjects. We also screened all exons and the putative promoter of the calpain-10 gene for mutations in 96 of the genotyped patients, resulting in the identification of 7 coding variants, including 3 missense mutations and 5 nucleotide alterations in the promoter. However, their frequencies all are similar in patients and controls, suggesting that these genetic variations are not a major factor in the occurrence of type 2 diabetes in Japanese, although they could yet be associated with various phenotypes of the disease.

Aims/hypothesis: There is an emerging epidemic of Type II (non-insulin-dependent) diabetes mellitus of youth in Japan and in many other developed countries. The aim of this study was to determine the prevalence of mutations in the hepatocyte nuclear factor (HNF)-1alpha gene (TCF1) in a large group of Japanese patients with early-onset non-Type I (insulin-dependent) diabetes mellitus. Since approximately 20% of Caucasian patients with HNF-1alpha mutations have been shown to be obese or overweight, we also examined the association of genetic variations in TCF1 with body weight in Japanese subjects., Methods: We examined 203 patients with non-Type 1 diabetes who had been diagnosed before they reached 15 years of age. Ten exons and flanking introns of TCF1 of these patients were directly sequenced for mutations., Results: We found 14 different mutations in 18 patients (8.9%), including one that was found to be de novo. The patients with the mutations had lower BMI (20.1+/-3.0 kg/m(2)) at diagnosis than the patients without them (24.5+/-6.0 kg/m(2)) (p=0.0024). All of the patients with the mutations, except for one, Y120, had normal body weight (BMI<25 kg/m(2)); the frequency of HNF-1alpha mutations in the non-obese patients of this study was 17% (17/101). Patient Y120, who had atypical symptoms of mild obesity and insulin resistance at diagnosis, was found to have inherited an additional mutation in an obesity-related gene., Conclusion/interpretation: A considerable number of non-obese Japanese patients with non-Type 1 diabetes of youth have HNF-1alpha-deficient diabetes. Lack of obesity could well be a characteristic feature of this form of diabetes.

Heterozygous mutations in the genes encoding transcription factors in the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus. However, these genes are responsible for only approximately 20% of the cases of MODY in Japanese patients. Searching for a novel MODY gene in this population, we investigated a candidate for encoding the forkhead transcription factor HNF-3alpha, which also belongs to the HNF-transcription cascade. The human HNF-3alpha gene, which was assigned to the segment near microsatellites D14S75 and AFM200ZH4 on chromosome 14 by radiation hybrid mapping, spans approximately 5 kb and consists of two exons. Ninety-five Japanese subjects with MODY/early-onset non-ketotic diabetes were screened for mutations in this gene. Direct sequencing of the exons and flanking regions identified one missense mutation (Ala-83-Thr) in exon 2 and three nucleotide alterations in the non-coding regions. However, their frequencies were not significantly different between MODY and control subjects, indicating that mutations in the HNF-3alpha gene are not a major cause of MODY in Japanese patients.

Mutations in several genes encoding transcription factors of the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of early-onset diabetes mellitus. The ability of the orphan nuclear receptor small heterodimer partner (SHP, NR0B2) to modulate the transcriptional activity of MODY1 protein, the nuclear receptor HNF-4alpha, suggested SHP as a candidate MODY gene. We screened 173 unrelated Japanese subjects with early-onset diabetes for mutations in this gene and found five different mutations (H53fsdel10, L98fsdel9insAC, R34X, A195S, and R213C) in 6 subjects as well as one apparent polymorphism (R216H), all present in the heterozygous state. Interestingly, all of the subjects with the mutations were mildly or moderately obese at onset of diabetes, and analysis of the lineages of these individuals indicated that the SHP mutations were associated with obesity rather than with diabetes. Therefore, an additional group of 101 unrelated nondiabetic subjects with early-onset obesity was screened for mutations in the SHP gene. Two of the previously observed mutations (R34X and A195S) and two additional mutations (R57W and G189E) were identified in 6 subjects, whereas no mutations were identified in 116 young nondiabetic lean controls (P = 0.0094). Functional studies of the mutant proteins show that the mutations result in the loss of SHP activity. These results suggest that genetic variation in the SHP gene contributes to increased body weight and reveal a pathway leading to this common metabolic disorder in Japanese.

We investigated the presence and the function of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations in 26 Japanese subjects with type 2 diabetes. The subjects were between 20 and 39 years of age on diagnosis and had diabetic first-degree relatives. Two different frameshift mutations were found in 2 subjects (8 %). One novel mutation, T539fsdelC (deletion of C in codon 539 for Thr), is predicted to generate a protein of normal 539 residues at the N-terminus followed by an abnormal 119 amino acid protein. The mutation, P291fsinsC (insertion of C in codon 291 for Pro) should lead to production of a truncated protein of 315 amino acids. Transfection reporter assay using MIN6 and HepG2 cells revealed both mutations to have null function in the transactivation of reporter gene expression. When transfected with wild-type gene, these mutations behaved as dominant-negative regulators in both cells. An equimolar amount of T539fsdelC reduced wild-type activity by approximately 80% in MIN6 cells, while the same concentration of P291fsinsc reduced it by 30%. The sequences responsible for the transactivation activity of HNF-1alpha are confined largely to amino acids 547-628, so that the T539fsdelC mutation, which affects this entire region, replacing amino acids 540-631 with an abnormal 119 amino acid protein, may acquire a potent dominant-negative function.

Aims/hypothesis: Hepatocyte nuclear factor (HNF)-3beta, a transcription factor expressed in pancreatic beta cells, is an upstream regulator of HNF-1alpha/MODY3, HNF-4alpha/MODY1 and IPF1/MODY5 genes. Our previous screening of MODY subjects showed that mutations in the HNF-3beta gene are not a common cause of this form of diabetes in the Japanese. We tested the hypothesis that mutations in the HNF-3beta gene cause late-onset Type II (non-insulin-dependent) diabetes mellitus in this population., Methods: Genotyping of the polymorphic TCC repeat in the HNF-3beta gene was done in 112 Japanese subjects with Type II diabetes (age at diagnosis > 35 and family history of Type II diabetes among their second-degree relatives) and 96 Japanese control subjects. Furthermore, we screened 57 Type II diabetic patients for mutations of the HNF-3beta gene. Transactivation activity of variant HNF-3beta was investigated by transfection assay., Results: The distribution of alleles of the TCC repeat was similar between diabetic and control groups. Mutation screening identified two missense mutations, A86T and G114E. Neither mutation was observed in 225 control subjects. The transactivation activity of G114E-HNF-3beta was similar to that of wild typeHNF-3beta. In contrast, the activity of A86T-HNF-3beta was statistically significantly reduced to 83-86 % of that of wild type., Conclusions/interpretation: The A86T mutation in the HNF-3beta gene might be involved in the development of late-onset Type II diabetes in a small group of Japanese people.

Aims/hypothesis: Molecular defects of the genes for transcription factors, hepatocyte nuclear factor (HNF)-4 alpha, HNF-1 alpha, HNF-1 beta and insulin promoter factor-1 cause maturity-onset diabetes of the young (MODY1, 3, 5, and 4, respectively). This suggests the HNF-related transcription cascade is important in insulin secretion which is induced by glucose. These genes and the gene encoding glycolytic enzyme glucokinase (MODY2) are, however, responsible for only 15-20% of cases of MODY in the Japanese. Searching for a novel form of MODY in this population, we cloned a new candidate gene encoding human HNF-3 beta, a winged helix transcription factor, which also belongs to the same HNF-transcription cascade., Methods: The cDNA clone for human HNF-3 beta was isolated from a liver cDNA library. The gene was also cloned from a genomic library and its organization and chromosomal localization were determined. We screened 68 Japanese subjects with MODY/early-onset diabetes for mutations in this gene., Results: Human HNF-3 beta is composed of 457 amino acids. The human gene, which was mapped to the segment 30 cR from SHGC-37039 on chromosome 20p by radiation hybrid mapping, spans approximately 4.5 kb and consists of three exons. Direct sequencing of the exons and flanking regions identified one missense mutation A328 V and seven polymorphisms, although the functional significance of the mutation in the pathogenesis of diabetes is not known., Conclusion/interpretation: The characterization of the structure of the HNF-3 beta gene and its mapping in the framework of markers will be helpful in genetic studies of the various forms of diabetes mellitus.

Mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) cause maturity-onset diabetes of the young type 3 (MODY3), a form of diabetes mellitus characterized by autosomal dominant inheritance, early onset, and pancreatic beta-cell dysfunction. We have examined the effects of five diabetes-associated mutations (L12H, G191D, R263C, P379fsdelCT, and L584S585fsinsTC) on HNF-1alpha function including DNA binding ability, intracellular localization, and transactivation activity. L12H, P379fsdelCT, and L584S585fsinsTC mutations were found in patients with a clinical diagnosis of MODY, while G191D and R263C mutations were identified in patients diagnosed with type 2 diabetes. These mutations had diverse effects on the functional properties of HNF-1alpha. Comparison of the functional data with clinical information suggested that transactivation activity of mutant HNF-1alpha in beta cells like MIN6 may be the primary determinants of the phenotypic differences observed among diabetic patients with HNF-1alpha mutations., (Copyright 1999 Academic Press.)

Hepatocyte nuclear factor (HNF)-1beta, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1beta mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects. To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells. In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line. Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization. These results suggest that HNF-1beta mutations may impair pancreatic beta-cell function by loss-of-function and dominant-negative mechanisms.

Maturity-onset diabetes of the young (MODY) is a monogenic form of non-insulin-dependent diabetes mellitus (NIDDM) characterized by an early age of onset, often in childhood or adolescence and usually < 25 years of age, and autosomal dominant inheritance. Clinical characterization of patients with MODY indicates that impaired insulin secretion is the primary defect responsible for the hyperglycemia in these patients. Genetic studies have thus far identified five MODY susceptibility genes, four of which encode transcription factors; HNF (hepatocyte nuclear factor)-1 alpha, HNF-1 beta, HNF-4 alpha, and IPF1. The association of mutations in the genes for these transcription factors with early-onset familial diabetes indicates the importance of the HNF-regulatory network in determining pancreatic beta-cell function.

Maturity-onset diabetes of the young (MODY3), a monogenic subtype of non-insulin-dependent diabetes mellitus (NIDDM) with an early age of onset, is characterized by a primary defect in insulin secretion. Recently, it has been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1 alpha) cause MODY3. Since NIDDM in Japanese is characterized by insulin secretory defects due to primary beta-cell dysfunction, we screened 60 Japanese nonobese subjects with early-onset NIDDM for mutations in this gene, 45 of whom had a first-degree relative with NIDDM. Direct sequencing of the ten exons and flanking introns of the gene in these subjects identified eight nucleotide substitutions including two amino acid changes, Ile-27-Leu and Ser-487-Asn, the frequencies of which were not significantly different in subjects with early-onset NIDDM and nondiabetic subjects. These results suggest that mutations in the HNF-1 alpha gene are not a major cause of early-onset NIDDM in Japanese.