A Novel homozygous missense mutation of melanocortin-4 receptor (MC4R) in a Japanese woman with severe obesity.

The melanocortin-4 receptor (MC4R) is a member of the seven membrane-spanning G protein-coupled receptor superfamily and signals through the activation of adenylyl cyclase. The MC4R mutations are the most common known monogenic cause of human obesity. However, no such mutations have been found in Japanese obese subjects. Here we report a novel homozygous missense mutation of MC4R (G98R) in a nondiabetic Japanese woman with severe early-onset obesity, which is located in its second transmembrane domain. Her birth weight was 3,360 g, and she gained weight progressively from 10 months of age. At 40 years of age, her weight reached 160 kg and a BMI of 62 kg/m(2). Her parents, who are heterozygous for the mutation, have BMIs of 26 and 27 kg/m(2). In vitro transient transfection assays revealed no discernable agonist ligand binding and cAMP production in HEK293 cells expressing the mutant receptor, indicating a severe loss-of-function mutation. This study represents the first demonstration of a pathogenic mutation of MC4R in Japan and will provide further insight into the pathophysiologic role of the hypothalamic melanocortin system in human obesity.     

Chemokine receptor genotype is associated with diabetic nephropathy in Japanese with type 2 diabetes.

Glomerular infiltration of monocytes/macrophages occurs in diabetic patients with nephropathy, and chemokine receptor signals are thought to play a key role in the development of nephropathy. Recently, polymorphism of the chemokine receptor (CCR)2 coding region V64I and the CCR5 promoter region 59029 (G/A) have been identified. Accordingly, we evaluated the effects of these genotypes on diabetic nephropathy. CCR2 V64I and CCR5 59029 (G/A) were detected by polymerase chain reaction-restriction fragment-length polymorphism in 401 patients with type 2 diabetes who had a serum creatinine <2.0 mg/dl. Although the CCR2 V64I genotype showed no association with nephropathy, the frequency of the CCR5 59029 A-positive genotype (G/A or A/A) was significantly higher in patients with microalbuminuria (urinary albumin-to-creatinine ratio [ACR] > or = 30 and <300 mg/gCre, 86%) and patients with macroalbuminuria (ACR > or = 300 mg/gCre, 87%) than in patients with normoalbuminuria (ACR <30 mg/gCre, 75%; P = 0.0095). Polytomic logistic regression analysis showed that the CCR5 59029 A-positive genotype was associated with nephropathy (odds ratio 2.243, P = 0.0074). These results suggest that the CCR5 promoter 59029 A genotype may be an independent risk factor for diabetic nephropathy in patients with type 2 diabetes.     

A polymorphism in the AMPKalpha2 subunit gene is associated with insulin resistance and type 2 diabetes in the Japanese population

AMP-activated protein kinase (AMPK) acts as a fuel gauge for glucose and lipid metabolism. The gene encoding the alpha2 isoform of the catalytic subunit of AMPK (PRKAA2) is located at one of the Japanese type 2 diabetes loci mapped by our previous genome scan (1p36-32). PRKAA2 is, therefore, a good candidate gene for insulin resistance and type 2 diabetes. We screened all nine exons, their exon-intron boundaries, and the 5' and 3' flanking regions of PRKAA2 to identify single nucleotide polymorphisms (SNPs), and we genotyped 192 type 2 diabetic patients and 272 nondiabetic subjects to assess possible associations between genotypes or haplotypes and type 2 diabetes. None of the 10 SNPs genotyped was associated with type 2 diabetes, but the haplotype analysis, consisting of six representative SNPs, revealed one haplotype, with the A (minor) allele for rs2051040 and a major allele for the other five SNPs, to be associated with type 2 diabetes (P = 0.009). This finding was confirmed in two larger replication samples (657 case and 360 control subjects, P = 0.021; and 356 case and 192 control subjects from the same area in Japan, P = 0.007) and a significant P value was obtained in the joint haplotype analysis of all samples (1,205 case and 824 control subjects, P = 0.0001). Furthermore, insulin resistance was associated with rs2051040 in nondiabetic subjects, and those with the A (minor) allele had a higher homeostasis model assessment of insulin resistance index than those who did not (initial control subjects [n = 272], P = 0.002; and joint replication control subjects [n = 552], P = 0.037). We speculate that the PRKAA2 gene influences insulin resistance and susceptibility to type 2 diabetes in the Japanese population.     

A functional variant in the human betacellulin gene promoter is associated with type 2 diabetes

Betacellulin (BTC) plays an important role in differentiation, growth, and antiapoptosis of pancreatic beta-cells. We characterized about 2.3 kb of the 5'-flanking region of human BTC gene and identified six polymorphisms (-2159A>G, -1449G>A, -1388C>T, -279C>A, -233G>C, and -226A>G). The G allele in the -226A>G polymorphism was more frequent in type 2 diabetic patients (n = 250) than in nondiabetic subjects (n = 254) (35.6% vs. 27.8%, P = 0.007), and the -2159G, -1449A, and -1388T alleles were in complete linkage disequilibrium with the -226G allele. The frequencies of the -279A and -233C alleles were low (7.0 and 2.0% in diabetic patients), and no significant differences were observed. In the diabetic group, insulin secretion ability, assessed by the serum C-peptide response to intravenous glucagon stimulation, was lower in patients with the -226G allele (G/G, 2.96 +/- 0.16 ng/ml; G/A, 3.65 +/- 0.18 ng/ml; A/A, 3.99 +/- 0.16 ng/ml at 5 min after stimulation; P = 0.008). Furthermore, in vitro functional analyses indicated that both the -226G and the -233C alleles caused an approximately 50% decrease in the promoter activity, but no effects of the -2159A>G, -1449G>A, -1388C>T, and -279C>A polymorphisms were observed. These results suggest that the -226A/G polymorphism of the BTC gene may contribute to the development of diabetes.     

Novel X-linked glomerulopathy is associated with a COL4A5 missense mutation in a non-collagenous interruption

A novel COL4A5 mutation causes rapid progression to end-stage renal disease in males, despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males have proteinuria, variable hematuria, and an early progression to end-stage renal disease. Renal biopsy findings include global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition. Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. The phenylalanine at position 222 is absolutely conserved among vertebrates. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. Immunostaining for α5(IV) collagen in renal biopsies from affected males was normal. This mutation, in a non-collagenous interruption associated with severe renal disease, provides evidence for the importance of this structural motif and suggests the range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. Hence, COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a presentation distinct from Alport syndrome.     

A missense mutation in pstpip2 is associated with the murine autoinflammatory disorder chronic multifocal osteomyelitis

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory disorder that primarily affects bone but is often accompanied by inflammation of the skin and/or gastrointestinal tract. The etiology is unknown but evidence suggests a genetic component to disease susceptibility. Although most cases of CRMO are sporadic, there is an autosomal recessive syndromic form of the disease, called Majeed syndrome, which is due to homozygous mutations in LPIN2. In addition, there is a phenotypically similar mouse, called cmo (chronic multifocal osteomyelitis) in which the disease is inherited as an autosomal recessive disorder. The cmo locus has been mapped to murine chromosome 18. In this report, we describe phenotypic abnormalities in the cmo mouse that include bone, cartilage and skin inflammation. Utilizing a backcross breeding strategy, we refined the cmo locus to a 1.3 Mb region on murine chromosome 18. Within the refined region was the gene pstpip2, which shares significant sequence homology to the PSTPIP1. Mutations in PSTPIP1 have been shown to cause the autoinflammatory disorder PAPA syndrome (pyogenic arthritis, pyoderma gangrenosum and acne). Mutation analysis, utilizing direct sequencing, revealed a single base pair change c.293T --> C in the pstpip2 gene resulting in a highly conserved leucine at amino acid 98 being replaced by a proline (L98P). No other mutations were found in the coding sequence of the remaining genes in the refined interval, although a 50 kb gap remains unexplored. These data suggest that mutations in pstpip2 may be the genetic explanation for the autoinflammatory phenotype seen in the cmo mouse.     

A new mouse model of type 2 diabetes, produced by N-ethyl-nitrosourea mutagenesis, is the result of a missense mutation in the glucokinase gene

Here we report the first cloned N-ethyl-nitrosourea (ENU)-derived mouse model of diabetes. GENA348 was identified through free-fed plasma glucose measurement, being more than 2 SDs above the population mean of a cohort of >1,201 male ENU mutant mice. The underlying gene was mapped to the maturity-onset diabetes of the young (MODY2) homology region of mouse chromosome 11 (logarithm of odds 6.0). Positional candidate gene analyses revealed an A to T transversion mutation in exon 9 of the glucokinase gene, resulting in an isoleucine to phenylalanine change at amino acid 366 (I366F). Heterozygous mutants have 67% of the enzyme activity of wild-type littermates (P < 0.0012). Homozygous mutants have less enzyme activity (14% of wild-type activity) and are even less glucose tolerant. The GENA348 allele is novel because no mouse or human diabetes studies have described a mutation in the corresponding amino acid position. It is also the first glucokinase missense mutation reported in mice and is homozygous viable, unlike the global knockout mutations. This work demonstrates that ENU mutagenesis screens can be used to generate models of complex phenotypes, such as type 2 diabetes, that are directly relevant to human disease.     

Impaired cathepsin L gene expression in skeletal muscle is associated with type 2 diabetes

To identify abnormally expressed genes associated with muscle insulin resistance or type 2 diabetes, we screened the mRNA populations using cDNA differential display combined with relative RT-PCR analysis from muscle biopsies of diabetes-prone C57BL/6J and diabetes-resistant NMRI mice fed with a high-fat or normal diet for 3 or 15 months. Six abnormally expressed genes were isolated from the mice after a 3-month fat feeding; one of them was cathepsin L. No significant difference in mRNA levels of these genes was observed between fat- and normal-diet conditions in either strains. However, cathepsin L mRNA levels in muscle were higher in normal diet-fed C57BL/6J mice compared with normal diet-fed NMRI mice at 3 months (0.72 +/- 0.04 vs. 0.51 +/- 0.04 relative units, P < 0.01, n = 8-10) and at 15 months (0.41 +/- 0.05 vs. 0.27 +/- 0.04 relative units, P = 0.01, n = 9-10). Further, cathepsin L mRNA levels in muscle correlated inversely with plasma glucose in both strains regardless of diets at 3 (r = -0.49, P < 0.01, n = 31) and 15 (r = -0.42, P = 0.007, n = 39) months. To study whether cathepsin L plays a role in human diabetes, we measured cathepsin L mRNA levels in muscle biopsies taken before and after an insulin clamp from 12 monozygotic twin pairs discordant for type 2 diabetes and from 12 control subjects. Basal cathepsin L mRNA levels were not significantly different between the study groups. Insulin infusion increased cathepsin L mRNA levels in control subjects from 1.03 +/- 0.30 to 1.90 +/- 0.32 relative units (P = 0.03). Postclamp cathepsin L mRNA levels were lower in diabetic twins but similar in nondiabetic twins compared with control subjects (0.66 +/- 0.22, 1.16 +/- 0.18 vs. 1.38 +/- 0.21 relative units, P < 0.02, NS, respectively). Further, postclamp cathepsin L mRNA levels were correlated with insulin-mediated glucose uptake (r = 0.37, P = 0.03), particularly, with glucose oxidation (r = 0.37, P = 0.03), and fasting glucose concentrations (r = -0.45, P < 0.01) across all three study groups. In conclusion, muscle cathepsin L gene expression is increased in diabetes-prone mice and related to glucose tolerance. In humans, insulin-stimulated cathepsin L expression in skeletal muscle is impaired in diabetic but not in nondiabetic monozygotic twins, suggesting that the changes may be secondary to impaired glucose metabolism.     

Insertion/deletion polymorphism of the ACE gene is associated with type 2 diabetes

In an attempt to examine the role of an ACE gene insertion/deletion (I/D) polymorphism in type 2 diabetes, we conducted a case-control association study among 132 couple-pairs from northern China. The genotype frequencies for II, ID, and DD were 39.8, 39.8, and 20.3%, respectively, in the case group and 44.8, 44.8, and 10.4% in the control group. The DD frequency was significantly higher in the case group than in the control group (chi(2)(1) = 4.77, P = 0.03), suggesting that the DD genotype is associated with an increased susceptibility to type 2 diabetes in our study population.     

The K121Q variant of the human PC-1 gene is not associated with insulin resistance or type 2 diabetes among Danish Caucasians

The human plasma-cell membrane differentiation antigen-1 (PC-1) has been shown to inhibit insulin receptor tyrosine kinase activity. Recently, a K121Q polymorphism in the human PC-1 gene was found in a Sicilian population and was shown to be strongly associated with insulin resistance. The objectives of the present investigation were to examine in the Danish Caucasian population whether the K121Q variant was associated with type 2 diabetes or, in glucose-tolerant subjects, with impaired whole-body insulin sensitivity. We genotyped 404 Danish type 2 diabetic patients and found that the allele frequency of the variant was 0.14 (95% CI 0.12-0.16), whereas the allele frequency was 0.16 (95% CI 0.13-0.19) among 237 matched glucose-tolerant control subjects (P = 0.6). In the control subjects, there were no significant differences among wild-type, heterozygous, or homozygous subjects in regard to 1) serum insulin and plasma glucose levels at fasting, 60 min, or 120 min during an oral glucose tolerance test (OGTT) or 2) the estimates of insulin resistance obtained from the homeostasis model assessment (HOMA). Furthermore, we investigated the impact of the variant in 2 other Danish population samples that comprised 356 young healthy subjects and 226 glucose-tolerant offspring of type 2 diabetic probands, respectively. In all of the study populations, the polymorphism was not associated with an altered insulin sensitivity index as estimated from an intravenous glucose tolerance test in combination with an intravenous injection of tolbutamide. In addition, among the 226 offspring, the variations in serum insulin and serum C-peptide responses measured during an OGTT were not related to the PC-1 genotype. In conclusion, the K121Q polymorphism of the human PC-1 gene is not associated with type 2 diabetes or insulin resistance among Danish Caucasians.     

A PC-1 amino acid variant (K121Q) is associated with faster progression of renal disease in patients with type 1 diabetes and albuminuria

Insulin resistance characterizes type 1 diabetes in patients with albuminuria. A PC-1 glycoprotein amino acid variant, K121Q, is associated with insulin resistance. We examined the impact of the PC-1 K121Q variant on the rate of decline of the glomerular filtration rate (GFR) by creatinine clearance derived from the Cockroft-Gault formula in 77 type 1 diabetic patients with albuminuria who were followed for an average of 6.5 years (range 2.5-15). Patients carrying the Q allele (n = 22; 20 with KQ and 2 with QQ genotypes) had a faster GFR decline than those patients with the KK genotype (n = 55) (median 7.2 vs. 3.7 ml x min(-1) x year(-1); range 0.16 to 16.6 vs. -3.8 to 16.0 ml x min(-1) x year(-1); P < 0.001). Significantly more patients carrying the Q allele belonged to the highest tertile of GFR decline (odds ratio = 5.7, 95% CI 4.1-7.2, P = 0.02). Levels of blood pressure, HbA1c, and albuminuria were comparable in the two genotype groups. Albuminuria (P = 0.001), mean blood pressure (P = 0.046), and PC-1 genotype (P = 0.036) independently correlated with GFR decline. Because all patients were receiving antihypertensive treatment, the faster GFR decline in the patients carrying the Q allele could be the result of reduced sensitivity to the renoprotective effect of antihypertensive therapy. PC-1 genotyping identifies type 1 diabetic patients with a faster progression of diabetic nephropathy.     

The alpha-adducin gene is associated with macrovascular complications and mortality in patients with type 2 diabetes

We examined the association between alpha-adducin 1 (ADD1) gene polymorphism (Gly460Trp) with macrovascular complications and mortality in type 2 diabetes in a Caucasian population aged >or=55 years. The study was part of the Rotterdam Study, a prospective population-based cohort study. ADD1 polymorphism was determined in 6,471 participants, including 599 patients with type 2 diabetes at baseline. The prevalence of hypertension in type 2 diabetic patients was 2.57 times higher in ADD1 TT carriers compared with GG carriers (95% CI 1.05-6.32, P = 0.03). Homozygous T carriers also had a higher mean common carotid intima media thickness (IMT) compared with GG carriers (mean difference 0.05 mm, P for trend = 0.03). In diabetic patients with hypertension, the risk of mortality was 1.83 times higher in homozygous T carriers compared with the GG genotype group (95% CI 1.07-3.16, P = 0.03). The increased risk was only present among TT carriers who did not use antidiabetes medication (hazard ratio 2.18 [95% CI 1.12-4.24], P = 0.02). The results of this population-based cohort study suggest that the ADD1 gene contributes to the risk of hypertension and increases mean common carotid IMT in patients with type 2 diabetes. Furthermore, the study indicates that the ADD1 polymorphism could be useful in identifying hypertensive type 2 diabetic patients with a high risk of mortality.     

A synonymous coding polymorphism in the alpha2-Heremans-schmid glycoprotein gene is associated with type 2 diabetes in French Caucasians

alpha2-Heremans-Schmid glycoprotein (AHSG) is an abundant plasma protein synthesized predominantly in the liver. The AHSG gene, consisting of seven exons and spanning 8.2 kb of genomic DNA, is located at chromosome 3q27, a susceptibility locus for type 2 diabetes and the metabolic syndrome. AHSG is a natural inhibitor of the insulin receptor tyrosine kinase, and AHSG-null mice exhibit significantly enhanced insulin sensitivity. These observations suggested that the AHSG gene is a strong positional and biological candidate for type 2 diabetes susceptibility. Direct sequencing of the AHSG promoter region and exons identified nine common single nucleotide polymorphisms (SNPs) with a minor allele frequency > or =5%. We carried out a detailed genetic association study of the contribution of these common AHSG SNPs to genetic susceptibility of type 2 diabetes in French Caucasians. The major allele of a synonymous coding SNP in exon 7 (rs1071592) presented significant evidence of association with type 2 diabetes (P = 0.008, odds ratio 1.27 [95% CI 1.06-1.52]). Two other SNPs (rs2248690 and rs4918) in strong linkage disequilibrium with rs1071592 showed evidence approaching significance. A haplotype carrying the minor allele of SNP rs1071592 was protective against type 2 diabetes (P = 0.014). However, our analyses indicated that rs1071592 is not associated with the evidence for linkage of type 2 diabetes to 3q27.     

SUMO4 M55V variant is associated with diabetic nephropathy in type 2 diabetes

OBJECTIVE-SUMO4 mRNA was recently found to be mainly expressed in the kidney, and the methionine-to-valine substitution at codon 55 (M55V) variant of SUMO4 may induce higher nuclear factor-kappaB (NF-kappaB) activity. Because NF-kappaB is known to mediate the development of diabetic nephropathy, we examined the association between the SUMO4 M55V variant and the severity of diabetic nephropathy. RESEARCH DESIGN AND METHODS-We recruited a total of 430 patients with type 2 diabetes. The M55V (rs237025, 163A-->G) polymorphism of SUMO4 was genotyped by real-time PCR, and urine albumin concentration was measured by radioimmunoassay. RESULTS-The frequencies of SUMO4 AA, GA, and GG were 52.6, 40.7, and 6.7%, respectively, in the normoalbuminuric group; 45.5, 47.3, and 7.1% in the microalbuminuric group; and 36.9, 46.2, and 16.9% in the macroalbuminuric group. We detected a significant linear trend for SUMO4 genotype between the macroalbuminuric and normoalbuminuric groups. The mean urine albumin-to-creatinine ratio (42.3 +/- 108.82 mg/mmol) in the GG group was significantly higher than in the AA (14.9 +/- 51.49 mg/mmol) and GA (17.0 +/- 43.74 mg/mmol) groups. Multivariate logistic regression analysis showed the SUMO4 M55V variant to be independently associated with the severity of diabetic nephropathy. CONCLUSIONS-This study indicates that the SUMO4 gene M55V variant is associated with severity of diabetic nephropathy in patients with type 2 diabetes.     

Susceptibility to type 1 diabetes is associated with ApoCIII gene haplotypes

Type 1 diabetes is a disease of beta-cell destruction leading to insulin deficiency. Genes for type 1 diabetes have been identified; however, much of the genetic risk remains unexplained. Genetic variation within the apolipoprotein CIII (apoCIII) gene alters apoCIII levels, which are increased in type 1 diabetes and induce beta-cell apoptosis. We therefore hypothesize haplotypes within the apoCIII gene are associated with type 1 diabetes. DNA from 584 type 1 diabetic patients and 591 control subjects were genotyped for six single nucleotide polymorphisms (SNPs) in the apoCIII gene (C-641A, C-482T, T-455C, C1100T, C3175G, and T3206G). Two alleles of a haplotype block (promoter SNPs + C3175G) were associated with type 1 diabetes. The A-T-C-C allele frequency was higher in type 1 diabetes (0.19 vs. 0.16, P = 0.05), and the C-C-T-C allele was reduced in type 1 diabetes (0.60 vs. 0.65, P = 0.04). The odds ratio (OR) for A-T-C-C allele increased with 0, 1, and 2 copies (OR of 1.00, 1.24, and 1.60, respectively; P = 0.05) and decreased for the C-C-T-C allele (1.00, 0.97, and 0.73, respectively; P = 0.03). This haplotype block contains an insulin response element. Screening for this haplotype may identify at-risk individuals, and this pathway may offer a target for prevention of type 1 diabetes.