The Finland-United States investigation of non-insulin-dependent diabetes mellitus genetics (FUSION) study. II. An autosomal genome scan for diabetes-related quantitative-trait loci

Type 2 diabetes mellitus is a complex disorder encompassing multiple metabolic defects. We report results from an autosomal genome scan for type 2 diabetes-related quantitative traits in 580 Finnish families ascertained for an affected sibling pair and analyzed by the variance components-based quantitative-trait locus (QTL) linkage approach. We analyzed diabetic and nondiabetic subjects separately, because of the possible impact of disease on the traits of interest. In diabetic individuals, our strongest results were observed on chromosomes 3 (fasting C-peptide/glucose: maximum LOD score [MLS] = 3.13 at 53.0 cM) and 13 (body-mass index: MLS = 3.28 at 5.0 cM). In nondiabetic individuals, the strongest results were observed on chromosomes 10 (acute insulin response: MLS = 3.11 at 21.0 cM), 13 (2-h insulin: MLS = 2.86 at 65.5 cM), and 17 (fasting insulin/glucose ratio: MLS = 3.20 at 9.0 cM). In several cases, there was evidence for overlapping signals between diabetic and nondiabetic individuals; therefore we performed joint analyses. In these joint analyses, we observed strong signals for chromosomes 3 (body-mass index: MLS = 3.43 at 59.5 cM), 17 (empirical insulin-resistance index: MLS = 3.61 at 0.0 cM), and 19 (empirical insulin-resistance index: MLS = 2.80 at 74.5 cM). Integrating genome-scan results from the companion article by Ghosh et al., we identify several regions that may harbor susceptibility genes for type 2 diabetes in the Finnish population.     

Familiality of quantitative metabolic traits in Finnish families with non-insulin-dependent diabetes mellitus. Finland-United States Investigation of NIDDM Genetics (FUSION) Study investigators.

Type 2 diabetes mellitus (NIDDM) is a complex disorder encompassing multiple metabolic defects. There exists strong evidence for a genetic component to NIDDM; however, to date there have been few reports of linkage between genetic markers along the genome and NIDDM or NIDDM-related quantitative traits. We sought to determine whether individual quantitative traits which determine glucose tolerance exhibit familiality in Finnish families with at least one NIDDM-affected sibling pair. Tolbutamide-modified frequently sampled intravenous glucose tolerance tests (FSIGT) were performed on unaffected offspring (n = 431) and spouses (n = 154) of affected sibling pairs sampled for the Finland-United States Investigation of NIDDM Genetics (FUSION) study. FSIGT data were analyzed using the Minimal Model to obtain quantitative measures of insulin sensitivity (SI), glucose effectiveness (SG), and insulin secretion assessed as the acute insulin response to glucose (AIR). The disposition index (DI), a measure of insulin resistance-corrected beta-cell function, was also derived as the product of SI and AIR. Variance components analysis was used to determine for each trait, the heritability (h2), the proportion of the total trait variance accounted for by additive genes. After adjustment for age, gender, and body mass index, h2 estimates were: SG: 18 +/- 9%, SI: 28 +/- 8%, AIR: 35 +/- 8%, and DI: 23 +/- 8%. We conclude that there is strong evidence for modest heritability of Minimal-Model-derived NIDDM-related quantitative traits in unaffected spouses and offspring of Finnish affected sibling pairs.     

An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians.

Genetic factors influence the development of type II diabetes mellitus, but genetic loci for the most common forms of diabetes have not been identified. A genomic scan was conducted to identify loci linked to diabetes and body-mass index (BMI) in Pima Indians, a Native American population with a high prevalence of type II diabetes. Among 264 nuclear families containing 966 siblings, 516 autosomal markers with a median distance between adjacent markers of 6.4 cM were genotyped. Variance-components methods were used to test for linkage with an age-adjusted diabetes score and with BMI. In multipoint analyses, the strongest evidence for linkage with age-adjusted diabetes (LOD = 1.7) was on chromosome 11q, in the region that was also linked most strongly with BMI (LOD = 3.6). Bivariate linkage analyses strongly rejected both the null hypothesis of no linkage with either trait and the null hypothesis of no contribution of the locus to the covariation among the two traits. Sib-pair analyses suggest additional potential diabetes-susceptibility loci on chromosomes 1q and 7q.     

Significance of platelet-activating factor acetylhydrolase in patients with non-insulin-dependent (type 2) diabetes mellitus

Background : Non-insulin dependent diabetes mellitus (NIDDM) represents an independent risk factor for cardiovascular diseases (CVD), being characterized by a continnous low-grade inflammation and endothelial activation state. Plasma platelet - activating factor - acetylhydrolases (PAF-AHs) are a subgroup of Ca²⁺ -independent phospholipase A₂ family (also known as lipoprotein-associated phospholipases A₂) that hydrolyze and inactivate the lipid mediator platelet-activating factor (PAF) and/or oxidized phospholipids. This enzyme is considered to play an important role in inflammatory diseases and atherosclerosis. The present study aims to investigate the relations between the levels of PAF-AH activity and LDL-cholesterol/HDL-cholesterol (LDL-ch/HDL-ch) ratio in NIDDM patients as compared to controls. Methods : serum PAF-AH activity was measured in 50 patients with dyslipidemia, in 50 NIDDM patients and in 50 controls (normal lipid and glucose levels). Total cholesterol, LDL-ch, HDL-ch, triglyceride and blood glucose were determined in all subjects. Results : All NIDDM patients display hiperlipidemia, with increased LDL-ch and triglyceride levels. There is a significant correlation between LDL-ch levels (especially LDL-ch / HDL-ch ratio) and PAF-AH activity in dyslipidemic and NIDDM patients. Conclusion : Diabetic and dyslipidemic patients have an increased plasma PAF-AH activity correlated with their LDL-ch levels and mainly with LDL-ch / HDL-ch ratio. Plasma PAF-AH high levels appear to be important as a risk marker for endothelial dysfunction in patients with NIDDM.     

Molecular pathology of insulin-dependent diabetes mellitus (type I)

Since sufficiently long time the average life expectancy in IDDM did not change. Only new data concerning the causes and pathogenesis of the disease may improve such set-back. The most promising field of creating the better insight into etiology of IDDM are molecular pathological studies. This review attempts to summarize the state of art in this area choosing the problems of clinical relevance. Many of them are controversial, disturbing the immunogenetic hypothesis of IDDM etiology, majority positively support this idea. 6 topics are critically discussed in respective sub-chapters: 1) genetic control of autoimmunization processes as pertinent to beta cells; 2) genesis, actions and clinical significance of various types of autoantibodies against autoantigens; 3) character of beta cell antigens; 4) suggested mechanism of beta cell destruction; 5) new idea of prediabetic state and 6) lessons from immunosuppressive therapeutical trials. Author brings the story of etiological mechanisms of IDDM up to date pointing that more direct and specific information is needed to be applicable in practice.     

Urinary excretion of glucagon-like peptide 1 (GLP-1) 7-36 amide in human type 2 (non-insulin-dependent) diabetes mellitus.

The urinary excretion of insulinotropic glucagon-like peptide 1 (GLP-1) was investigated as an indicator of renal tubular integrity in 10 healthy subjects and in 3 groups of type 2 diabetic patients with different degrees of urinary albumin excretion rate. No significant difference emerged between the groups with respect to age of the patients, known duration of diabetes, metabolic control, BMI, or residual beta-cell pancreatic function. Endogenous creatinine clearance was significantly reduced under conditions of overt diabetic nephropathy, compared with normo and microalbuminuric patients (p < 0.01). Urinary excretion of GLP-1 was significantly higher in normoalbuminuric patients compared to controls (490.4 +/- 211.5 vs. 275.5 +/- 132.1 pg/min; p < 0.05), with further increase under incipient diabetic nephropathy conditions (648.6 +/- 305 pg/min; p < 0.01). No significant difference resulted, in contrast, between macroproteinuric patients and non-diabetic subjects. Taking all patients examined into account, a significant positive relationship emerged between urinary GLP-1 and creatinine clearance (p = 0.004). In conclusion, an early tubular impairment in type 2 diabetes would occur before the onset of glomerular permeability alterations. The tubular dysfunction seems to evolve with the development of persistent microalbuminuria. Finally, the advanced tubular involvement, in terms of urinary GLP1 excretion, under overt diabetic nephropathy conditions would be masked by severe concomitant glomerular damage with the coexistence of both alterations resulting in a peptide excretion similar to control subjects.     

Serum fructosamine concentrations in patients with type II (non-insulin-dependent) diabetes mellitus during changes in management.

The serum fructosamine concentration was examined as a new means to monitor metabolic control in non-insulin-dependent diabetes during changes in management. Weekly fructosamine estimations were compared with glycosylated haemoglobin (HbA1c), 24 hour urinary glucose, and fasting plasma glucose concentrations in a 17 week study entailing withdrawal and reinstitution of oral treatment. The serum fructosamine concentration was more sensitive than the other measurements in detecting a deterioration in diabetic control after stopping oral hypoglycaemic drugs. The response to reinstitution of treatment was not significant in the first three weeks (p = 0.266), despite a highly significant reduction in fasting plasma glucose (p = 0.001) and 24 hour urinary glucose concentrations (p = 0.012). Compared with HbA1c, concentrations of fructosamine appeared more useful in monitoring short term (three to six weeks) changes after alterations in management of diabetes. Additional advantages were lower cost and technical simplicity of measurement.     

Utility of large consanguineous family-based model for investigating the genetics of type 2 diabetes mellitus

Objectives

This study examined the utility of a family-based model for replicating the results of genome-wide association studies (GWAS) of type 2 diabetes (T2D).

Methods and results

In a total of 232 members of a large consanguineous Omani Arab pedigree (age: 16–80 years), there were 27 diabetics and 50 prediabetics (17 with impaired fasting glucose and 33 with impaired glucose tolerance). All 232 individuals underwent anthropometric and biochemical investigations and genotyped for 14 known common gene variants of modest effect on T2D risk. Power analysis at a LOD score of 3, gave 80% power to locate a single specific locus that accounts for 52% of the total phenotypic variation. Measured genotype analysis (MGA) was used to determine heritability of various quantitative traits (QTs) which ranged 25–56%. Using MGA, some common gene variants were found to have little (< 5%) but significant impact on the heritability of T2D related QTs [KCNJ11 (rs5219), p = 0.004]; [IGF2BP2 (rs4402960), p = 0.02]; [SLC30A8 (rs13266634), p = 0.05]; [CAPN10 (rs2975760), p = 0.031]; [FTO (rs8050136), p = 0.023]; [FTO (rs9939609), p = 0.018] and [SLC30A8 (rs13266634), p = 0.05]. Sib-TDT analysis showed that some gene variants were significantly associated with T2D risk but didn't reach the level of significance after Bonferroni correction [KCNJ11 (rs5219), p = 0.047] and [CAPN10 (rs41266971), p = 0.035].

Conclusion

We have demonstrated that, in principle, a family-based model with minor limitations could be used to replicate some of the results of large GWAS case–control studies. This model could successfully be applied for the future discovery, by deep sequencing, of rare gene variants.     

Polymorphisms at the GLUT1 (HepG2) and GLUT4 (muscle/adipocyte) glucose transporter genes and non-insulin-dependent diabetes mellitus (NIDDM)

Summary In order to determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of non-insulin-dependent diabetes mellitus (NIDDM), two restriction fragment length polymorphisms (RFLPs) and the related haplotypes at this locus were studied in 48 Italian diabetic patients and 58 normal subjects. Genotype frequencies for the XbaI polymorphism were significantly different between patients and controls (XbaI: ² = 9.80, df= 2, P < 0.0079). A significant difference was also found in the allele frequencies between NIDDM patients and controls (² =9.39, df = 1, P < 0.0022), whereas no differences were found for the StuI RFLP. No linkage disequilibrium was detected between the XbaI and StuI RFLPs in this sample. The analysis of the four haplotype frequencies (X1S1, X1S2, X2S1, X2S2) revealed a significant difference between diabetic patients and controls (² = 14.26, df =3, P < 0.002). By comparing single haplotype frequencies, a significant difference between the two groups was found for the X1S1 and X2S2 haplotypes. A two-allele RFLP at the GLUT4 (muscle/adipocyte) glucose transporter gene, detected with the restriction enzyme KpnI, was also examined; no differences were found between patients and controls for this RFLP. The finding of an association between polymorphic markers at the GLUT1 transporter and NIDDM suggests that this locus may contribute to the inherited susceptibility to the disease in this Italian population.     

Genetic polymorphisms at the human liver/islet glucose transporter (GLUT2) gene locus in Caucasian and West Indian subjects with type 2 (non-insulin-dependent) diabetes mellitus.

The liver/islet glucose transporter (GLUT2) is mainly expressed in the hepatocytes of the liver and the beta-cells of the pancreatic islets and a defect in this transporter could lead to diabetic phenotypes, such as relative hypoinsulinaemia and reduced uptake and metabolism of glucose in the liver. DNA from unrelated individuals was digested with the restriction endonucleases Bgl-I and Taq-I followed by blotting and hybridisation with a 32P-labelled GLUT2 cDNA which revealed three restriction fragment length polymorphisms (RFLPs) (B1, T1 and T2) in both Caucasian and West Indian populations. Linkage analysis between these variant sites demonstrated that the alleles of these polymorphisms were in strong linkage disequilibrium. Disease association of genetic variants at the GLUT2 locus with type 2 diabetes was examined with these RFLPs in both Caucasian (n = 54) and West Indian (n = 46) populations with type 2 diabetes. There were no significant differences in the frequency of alleles, genotypes or haplotypes between diabetic patients and non-diabetic controls. However, there were significant differences in the allele frequencies of all these three polymorphisms between Caucasian and West Indian populations.     

Genetic heterogeneity of insulin-dependent (type I) diabetes mellitus: evidence from a study of extended haplotypes

Two hundred subjects with insulin-dependent (type I) diabetes mellitus (IDDM) were typed for HLA-B, HLA-DR, and properdin factor B (Bf). HLA and Bf antigen and haplotype frequencies in subjects were compared with control frequencies derived from the 8th HLA Workshop. Frequencies of extended haplotypes (defined by B-Bf-DR alleles on a chromosome) were also contrasted with control frequencies. Significant positive associations between IDDM and HLA-B8, DR3, DR4, BfS, and BfF1 were confirmed, as were significant negative associations between IDDM and HLA-B7, DR2, DR5, DR7, and BfF. One haplotype (B7-BfS-DR2) exhibited significant negative association, while five haplotypes (B8-BfS-DR3, B8-BfS-DR4, B15-BfS-DR4, B18-BfF1-DR3, and B40-BfS-DR4) exhibited significant positive associations with IDDM. In this sample, 64% of all probands carried at least one of the high-risk haplotypes. In conclusion, the occurrence of five "high-risk" haplotypes associated with IDDM provides evidence for previously undocumented genetic heterogeneity and suggests that possibly more than two HLA-region genes may be involved in IDDM susceptibility.     

Serum fructosamine concentration as measure of blood glucose control in type I (insulin dependent) diabetes mellitus.

Serum fructosamine activity was studied in 42 patients with type I (insulin dependent) diabetes mellitus and 30 non-diabetic volunteers as an index of blood glucose control. There was a significant correlation both between fructosamine and glycosylated haemoglobin values (r = 0.82) and between fructosamine and the fasting C peptide concentration (r = -0.81). Test results in 14 of the diabetics reflected the mean plasma glucose concentration calculated from 25 serial estimations in a single 24 hour period (r = 0.75; p less than 0.01) but not the mean amplitude of glycaemic excursion (r = 0.23; p greater than 0.05). Fructosamine concentrations measured in these multiple blood specimens did not change significantly throughout the day (mean coefficient of variation 4.1%) despite wide variability of the respective plasma glucose concentrations (mean coefficient of variation 36.2%). It is concluded that a single random serum sample analysed for fructosamine concentration provides a simple and reliable assessment of glucose homoeostasis in patients with type I diabetes mellitus.     

Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus.

The aim of the present study was to test the influence of obesity and the presence of type 2 diabetes mellitus (T2DM) on the expression of ten housekeeping genes and of the 18S rRNA in a group of human adipose tissue samples from the omental and subcutaneous depot. Adipose tissue biopsies were obtained by laparoscopic surgery from lean and obese patients. After the extraction, mRNA levels in adipose tissue samples were quantified by real-time PCR using the commercial HUMAN ENDOGENOUS CONTROL PLATES. From the genes analyzed, 18S rRNA exhibited the most stable expression levels in both depots regardless of the pathophysiological conditions of obesity and obesity-associated T2DM. Contrarily, GAPD was the gene with the highest variation in its expression levels, being upregulated (8.0-fold) in the obese group and downregulated (3.5-fold) in obesity-associated T2DM. Our results show that 18S rRNA may be the most suitable gene for normalization in expression studies performed in human adipose tissue samples obtained from patients suffering from obesity and/or obesity-associated T2DM, whereas GAPD is less appropriate for comparison purposes under these circumstances.     

Known data on the therapeutic use of Azadiracta indica (neem) for type 2 diabetes mellitus

There has been growing interest for the therapeutic use of traditional herbs in the management of diabetes mellitus (DM) and its complications. Data shows the hypoglycemic activity of Azadiracta indica in diabetes. Therefore, it is of interest to document known data on the therapeutic use of Azadiracta indica (neem) for type 2 diabetes mellitus (T2DM).     

Reproducibility of a glycemic response to mixed meals in type 2 diabetes mellitus.

OBJECTIVE: To evaluate the inter-patient and intra-patient reproducibility of the glycemic response to a mixed meal in individuals with type 2 diabetes mellitus (DM). SUBJECTS/SETTING: Six individuals with DM were admitted to the General Clinical Research Center for 6 days. INTERVENTION: Subjects consumed 3 different meal plans consisting of 4 meals daily (breakfast, lunch, dinner and snack) on 2 separate occasions. Serum insulin and glucose levels were sampled at 19 time points every day. The glycemic response (GR) to a meal was calculated as the area under the glucose response curve after consumption of a given meal. In addition, the incremental area under the curve (IGR) was calculated assuming a pre-prandial (baseline) glucose value before each meal as zero. RESULTS: Intra-patient correlation coefficients (R) of GR for meals in subjects with DM were quite good, ranging 0.69-0.94. The range of the inter-patient coefficients of variation (CV) for the same meals was 21.5-30.4%. For IGR, the R values ranged from 0.64 to 0.91 for 8 out of 12 meals, confirming good intra-patient reproducibility for these meals. CV for IGR ranged from 31% to 113%. CONCLUSIONS: For patients with DM, the GR of individual meals exhibits excellent intra-patient reproducibility, allowing prediction of the glycemic response to a given meal in an individual subject. However, significant inter-patient variability of the GR precludes its use for the prediction of post-prandial glucose concentrations in groups of patients with diabetes.     

Activity of Ca(+2)Mg(+2) --ATPase in erythrocyte membranes of women with diabetes mellitus type I]

The activity of Ca(++)-Mg++ ATP-ase present in erythrocyte membranes was determined in basal conditions and following stimulation with calmodulin in 8 women with insulin-dependent diabetes and in 9 healthy women. The isolation of erythrocyte membranes and the determination of activity of Ca(++)-Mg(++)-ATP-ase were carried out according to the method of Gietzen et al. A decrease in the activity of Ca(++)-Mg(++)-ATP-ase in basal conditions was found in fractions with the highest erythrocyte content obtained from diabetic patients. After stimulation with calmodulin the activity of Ca(++)-Mg(++)-ATP-ase in all the fractions was lower in diabetic patients than in the controls. Low activities of the enzyme were accompanied by high values of HbA1c. The results suggest that glycosylation of the ATP-ase or/and calmodulin may be the main cause of the observed fall in the enzyme activity in diabetes. Also the disturbances concerning the cumulation of intracellular calcium may be related to the changes caused by glycosylation of Ca(++)-Mg(++)-ATP-ase or/and calmodulin.     

A test for adequacy of chromium nutrition in humans--relation to type 2 diabetes mellitus

When Na(2)51CrO(4) is added to blood the 51CrO(4)(2-) ions enter the erythrocytes readily, and nearly exclusively, and are reduced to 51Cr(III) ions. We have observed that a fraction of these ions becomes bound to the cell membrane in a concentration which seemingly depends on that of the dietary derived intracellular Cr(III) ions. Thus, when constant amounts of 51CrO(4)(2-) ions enter constant amounts of erythrocytes, the resulting 51Cr(III) ions become bound to the cell membrane in a concentration that varies inversely as the initial, intracellular concentration of Cr(III) ions which, in turn, depends directly on the adequacy of chromium nutrition. Therefore, we have determined an arbitrary set of conditions under which the concentration of 51Cr(III) ions bound to the erythrocyte membrane becomes an indicator of the adequacy of chromium nutrition. The application of this test to 25 Type 2 diabetes mellitus subjects and 35 normal controls, both randomly selected, indicates that the concentrations of membrane bound 51Cr(III) ions in the two groups were not significantly different. Consequently, it is concluded that the level of chromium nutrition which is normally adequate in humans has only a minor role, if any, in the genesis of Type 2 diabetes mellitus.     

A search for association between the polymorphic markers of PON1 and PON2 genes and diabetic nephropathy in patients with type I diabetes mellitus

Alleles and genotypes of polymorphic markers of paraoxonase 1 and paraoxonase 2 genes (PON1 and PON2) encoding enzymes of the body antioxidative defense were compared in type 1 diabetes mellitus patients with or without diabetic nephropathy. The patients with nonoverlapping ("polar") phenotypes constituted different groups. The first group contained patients with diabetic nephropathy (DN+, n = 62), clinical proteinuria (albuminuria above 300 mg per day), and at least 15-year disease duration. In control group, the patients had no diabetic nephropathy (DN-, n = 68), their albuminuria was below 200 mg per day, and disease duration was at least 20 years. Comparative analysis with exact Fisher's test revealed no significant differences in frequencies of alleles and genotypes of the PON1 gene polymorphic marker Gln192Arg and of PON2 gene polymorphic markers Ala148Gly and Cys311Ser. Our results suggest that the polymorphic markers studied are not associated with diabetic nephropathy among Russian patients in Moscow.