OBJECTIVE -The aim of the present study was to define heterogeneity of adult-onset autoimmune diabetes based on characterization of GAD antibodies (GADAs). RESEARCH DESIGN AND METHODS-Patients enrolled in a nationwide survey, the Non Insulin Requiring Autoimmune Diabetes (NIRAD) Study, have been screened for GADAs and IA-2 antibodies (IA-2As) and further characterized for GADA titer, antibodies to thyroid peroxidase (TPO), and HLA DRB1-DQB1 polymorphisms.RESULTS -Of 4,250 consecutive type 2 diabetic patients, 4.5% had either GADAs and/or IA-2As. Patients with autoimmune diabetes showed a clinical phenotype significantly different from that of type 2 diabetes, including higher fasting glucose and A1C, lower BMI and uric acid, lower prevalence of metabolic syndrome and its components, and higher frequency of TPO antibodies. More interestingly, analysis of GADA titers showed a bimodal distribution that identified two subgroups of patients with high (Ͼ32 GADA arbitrary units) and low (Յ32 GADA arbitrary units) GADA titers. Compared with those with low GADA titers, patients with high GADA titers had more prominent traits of insulin deficiency and a profile of more severe autoimmunity resulting in higher A1C, lower BMI, a lower prevalence of metabolic syndrome and its components (P Ͻ 0.02 for all), a higher prevalence of IA-2As, TPO antibodies (P Ͻ 0.003 for both), and DRB1*03-DQB1*0201 (50 vs. 26.8%, P ϭ 0.001), and a decreasing frequency of DQB1*0602 and DRB1*0403 (from type 2 to low and to high GADA titer autoimmune diabetes; P Ͻ 0.001 for trend for both comparisons).CONCLUSIONS -GADA titers identify two subgroups of patients with adult-onset autoimmune diabetes having distinct clinical, autoimmune, and genetic features. Diabetes Care 30:932-938, 2007

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 ¼ *0401 ¼ *0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that metaanalyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

OBJECTIVEZinc transporter 8 (ZnT8) is an islet β-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes.RESEARCH DESIGN AND METHODSZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH2-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study.RESULTSZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH2 were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all Ptrend < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001).CONCLUSIONSZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.

Background-Excess fat is one of the main determinants of insulin resistance, representing the metabolic basis for developing future cardiovascular disease. The aim of the current study was to find an easy-to-detect clinical marker of insulin resistance which can be used to identify young subjects at increased risk of cardiovascular disease. Methods and Results-Four-hundred and seventy-seven overweight/obese children and adolescents (mean age 10.31Ϯ2.80 years) were consecutively enrolled. Standard deviation score body mass index, fasting biochemical parameters, and homeostasis model assessment of insulin resistance were evaluated. Statistical differences were investigated using multiple linear regression analysis. Manual measure of wrist circumference was evaluated in all children and adolescents. Fifty-one subjects, randomly selected, underwent nuclear magnetic resonance imaging of the wrist to evaluate transversal wrist area at the Lister tubercle level. A statistically significant association was found between manual measure of wrist circumference and insulin levels or homeostasis model assessment of insulin resistance (␤ϭ0.34 and 0.35, respectively; PϽ10 Ϫ5 for both comparisons). These associations were more significant than those between SD score body mass index and insulin levels or homeostasis model assessment of insulin resistance (␤ϭ0.12 and 0.10, respectively; PՅ0.02 for both comparisons). Nuclear magnetic resonance imaging acquisition clarified that the association between wrist circumference and insulin levels or homeostasis model assessment of insulin resistance reflected the association with bone tissue-related areas (PՅ0.01 for both) but not with the adipose tissue ones (PϾ0.05), explaining 20% and 17% of the variances of the 2 parameters. Conclusions-Our findings suggest a close relationship among wrist circumference, its bone component, and insulin resistance in overweight/obese children and adolescents, opening new perspectives in the prediction of cardiovascular disease.

We investigated the association of the Ϫ11,391GϾA, Ϫ11,377GϾC, ϩ45TϾG, and ϩ276GϾT adiponectin single-nucleotide polymorphisms (SNPs) and expected haplotypes with the insulin resistance (IR) state in overweight/ obese children; by using the haplotype background analysis, we also assessed the effect of each SNP independently. GG genotype at the Ϫ11,391 locus was associated with higher fasting insulin levels and homeostasis model assessment-IR index and lower adiponectin levels compared with GA ϩ AA genotypes (p ϭ 0.01, 0.002, and 0.03, respectively). Those heterozygous and homozygous for G allele at the Ϫ11,377 locus showed higher fasting glucose (p ϭ 0.001 for both), fasting insulin (p ϭ 0.001 for both), homeostasis model assessment-IR index (p Ͻ 0.001 for both), and triglyceride levels (p ϭ 0.02 and 0.03, respectively) and lower adiponectin levels (p ϭ 0.002 and 0.02, respectively) compared with C homozygotes. The ϩ45G carriers showed higher fasting and 2-hour glucose levels (p ϭ 0.01 for both) and lower adiponectin levels (p ϭ 0.02) compared with non-carriers. Haplotype analysis suggested that, considering the same haplotypic background, each of the three polymorphisms exerted an independent effect on investigated parameters. The Ϫ11,391GϾA, Ϫ11,377CϾG, and ϩ45TϾG SNPs are associated with IR syndrome in overweight/obese children; they independently influence the investigated variables. The effect of ϩ45TϾG SNP seems to be marginal compared with the promoter SNPs. The GGT haplotype is associated with the highest degree of IR.

The genetic predisposition to type 1 diabetes among Filipinos was examined by PCR/SSOP HLA class I and II typing of 90 patients and 94 general population controls. The HLA-DRB1, DQB1, and the A, B, and C loci were typed using the reverse SSO probe line-blot method while the DPB1 and DPA1 loci were typed using the SSO probe dot blot method. The Filipino population has a distinctive frequency distribution of HLA class II alleles as well as linkage disequilibrium patterns: a DR-DQ haplotype, unique to Filipinos, contains a DRB1 allele (*0405) positively associated with type 1 diabetes in other populations and DQA1 and DQB1 alleles (*0101-*0503) that are negatively associated in other populations. Specific DR-DQ haplotypes or alleles could be identified as susceptible, neutral or protective based on the distribution among Filipino patients and controls. The DR9 and DR3 haplotypes showed the most dramatic increase among patients (0.156 vs 0.063) and (0.172 vs 0.042), respectively. Among Filipinos, the DR3/9 genotype confers approximately the same risk as the well-known high-risk DR3/4 genotype, similar to that for DR3/3 and DR9/9. The common DR2 haplotype in the Philippines (DRB1*1502-DQB1*0502) was only slightly decreased in type 1 diabetic patients (0.200 in patients vs 0.270 in controls). Another DR2 haplotype, DRB1*1502-DQB1*0501, was significantly decreased among patients. In addition, haplotypes containing DQB1*06 alleles, such as the DRB1*0803-DQB1*0601 (OR = 0.1), are strongly protective. The DR4 allele group was also increased in Filipino patients compared to controls. In this population there is, as in other populations, a hierarchy of type 1 diabetes associations among the many different DR4 haplotypes (n = 15). The high-risk haplotypes in this population are the very rare DRB1*0405-DQB1*0302 and DQB1*0405-DQB1*0201, followed by the more common DRB1*0405-DQB1*0401 and DRB1*0405-DQB1*0402. The DRB1*0403-DQB1*0302 is protective. The DRB1*0405-DQB1*05031 haplotype, which is unique to Filipinos, appears to be "neutral". HLA-DPB1*0202 was significantly increased among patients (0.056 vs 0.011; with OR = 5.3); this increase does not appear to simply reflect linkage disequilibrium with high risk DR-DQ haplotypes. The observed distribution of HLA class II alleles among Filipino patients and controls strongly supports the notion that specific combinations of alleles at the DRB1, DQB1, DQA1, and DPB1 loci are critical in determining the risk for type 1 diabetes. Specific HLA class I alleles also show significant associations with type 1 diabetes in this population. HLA-A*2402 and *2403 were increased among patients; however, 2407 was decreased. Inaddition, A *1101 was significantly decreased among patients (OR = 0.51). Moreover, these HLA-A associations do not appear attributable to linkage disequilibrium with the DR-DQ region. The allele B*5801 was increased in patients while B*1301 was decreased; both of these associations, however, reflected linkage disequilibrium with high-risk and with protective DR-DQ haplotype...

HLA class II is the primary susceptibility gene to type 1 diabetes and the analysis of HLA class II association could help to clarify the relative weight of genetic contribution to the incidence of the disease. Here we present an extensive typing for HLA class II alleles and their haplotypes in a homogenous population of type 1 diabetic patients (n=134) and controls (n=128) and in simplex (n=100) and multiplex families (n=50) from continental Italy (Lazio region). Among the various haplotypes tested, the DRB1*0301-DQA1*0501-DQB1*0201 was the most frequent found in type 1 diabetic patients and was transmitted in 82% of affected siblings, whereas DRB1*0402-DQA1*0301-DQB1*0302 appeared to have the highest odds ratio (10.4), this haplotype was transmitted in 96.3% of affected siblings, followed by DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0405-DQA1*0301-DQB1*0201, DRB1*0401-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302. The following haplotypes showed a significant decreased transmission to diabetic siblings: DRB1*0701-DQA1*0201-DQB1*0303, DR2-DQA1*01-DQB1*0602, DR5-DQA1*0501-DQB1*0301. We suggest that the HLA DR/DQ haplotype/genotype frequencies observed could in part explain the low incidence of type 1 diabetes registered in Lazio region (8.1/100.000/year), for a number of reasons: i) the low frequency, in the general control population, of the most susceptible haplotypes and genotype for type 1 diabetes DRB1*0301-DQA1*0501-DQB1*0201 (14%), and DR4-DQA1*0301-DQB1*0302 (9%) and DRB1*0301-DQA1*0501-DQB1*0201/DR4-DQA1*0301-DQB1*0302 (0.8%) compared to other countries characterised by high incidence rate of the disease, Sardinia and Finland, respectively; ii) a significant lower ratio, in the control population, between the susceptible DRB1*0301-DQA1*0501-DQB1*0201 and the neutral DRB1*0701-DQA1*0501-DQB1*0201 haplotypes compared to the Sardinian population; iii) the high frequency of protection haplotypes/genotypes as the DR5-DQA1*0501-DQB1*0301, and DR5-DQA1*0501-DQB1*0301/DR5-DQA1*0501-DQB1*0301 very common in the control population of Lazio region and the DRB1*1401-DQA1*0101-DQB1*0503 haplotype.

Hashimoto's thyroiditis (HT) is an autoimmune disease resulting from a complex interaction between genetic and environmental factors. The genetic loci conferring susceptibility need to be still defined. The aim of the present study was to determine whether Cytotoxic T-Lymphocyte-Associated Antigen-4 (CTLA-4), HLA DRB1, and DQB1 genes were associated to HT in an Italian population. We evaluated the allele distribution of the following loci: CTLA-4 exon 1 A49G dimorphism, which resulted in an amino acidic exchange (Thr/Ala) in the leader peptide, CTLA-4 3' microsatellite, HLA DRB1 and DQB1 in 126 patients with HT and in 301 control subjects from an Italian population (Lazio region). CTLA-4 exon 1 A49G dimorphism was typed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP); CTLA-4 3' microsatellite alleles were defined using a fluorescence-based method. HLA DRB1 and DQB1 alleles were typed using a SSO reverse line blot method and a probeless procedure based on allele group-specific amplification followed by DNA heteroduplex analysis, respectively. Data were initially analyzed by chi2 test or Fisher's exact test. Multiple logistic regression analysis was then applied on factors with significant crude odds ratios and on CTLA-4 exon 1 A49G dimorphism to investigate their independent effects. The two polymorphic sites at CTLA-4 gene did not increase the risk for HT. The distribution of HLA DRB1 and DQB1 alleles did not show any significant difference between patients and controls, however, the DRB1*04-DQB1*0301 haplotype was significantly increased in patients. Other factors that increase the risk of disease were gender and age. Females showed approximately 18 times more risk than males; subjects older than 50 years had an odds ratio of 6.6. These data suggest that these two polymorphic sites at CTLA-4 do not play a major role in the susceptibility of the disease in an Italian population while female gender, age over 50 years, HLA DRB1*04-DQB1*0301 haplotype increase the risk of developing HT.