Risk HLA Genotypes Research Articles

Genetic variation in environmental enteropathy and stunting in Zambian children: A pilot genome wide association study using the H3Africa chip.

Stunting is known to be heavily influenced by environmental factors, so the genetic contribution has received little attention. Here we report an exploration of genetic influences in stunted Zambian children with environmental enteropathy. Children with stunting (LAZ < -2) were enrolled and given nutritional therapy. Those that were non-responsive to therapy were designated as cases, and children with good growth (LAZ > -1) from the same community as controls. Blood and stool samples were taken to measure biomarkers of intestinal inflammation, epithelial damage, and microbial translocation. Single nucleotide polymorphism array genotyping was carried out on saliva samples using the H3Africa consortium array. Genome wide associations were analysed in 117 cases and 41 controls. While no significant associations with stunting were observed at P<5x10-8, likely due to the small sample size, interesting associations were observed at lower thresholds. SNPs associated with stunting were in genomic regions known to modulate neuronal differentiation and fatty acid biosynthesis. SNPs associated with increased microbial translocation were associated with non-integrin membrane ECM interactions, tight junctions, hemostasis, and G-alpha signalling events. SNPs associated with increased inflammation were associated with, ECM interactions, purine metabolism, axon guidance, and cell motility. SNPs negatively associated with inflammation overlapped genes involved in semaphoring interactions. We explored the existing coeliac disease risk HLA genotypes and found present: DQ2.5 (7.5%), DQ8 (3.5%) and DQ2.2 (3.8%); however, no children were positive for coeliac antibodies. We detected HLA-DRB:1301 and HLA-C:1802 with high odds ratios and P<0.05 in stunted children compared to controls. Genetic variations associated with stunting and the enteropathy underlying it, include variants associated with multiple pathways relating to gene expression, glycosylation, nerve signalling, and sensing of the nutritional and microbiological milieu.

The oxylipin profile is associated with development of type 1 diabetes: the Diabetes Autoimmunity Study in the Young (DAISY)

Oxylipins are lipid mediators derived from polyunsaturated fatty acids. Some oxylipins are proinflammatory (e.g. those derived from arachidonic acid [ARA]), others are pro-resolving of inflammation (e.g. those derived from α-linolenic acid [ALA], docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) and others may be both (e.g. those derived from linoleic acid [LA]). The goal of this study was to examine whether oxylipins are associated with incident type 1 diabetes. We conducted a nested case-control analysis in the Diabetes Autoimmunity Study in the Young (DAISY), a prospective cohort study of children at risk of type 1 diabetes. Plasma levels of 14 ARA-derived oxylipins, ten LA-derived oxylipins, six ALA-derived oxylipins, four DHA-derived oxylipins and two EPA-related oxylipins were measured by ultra-HPLC-MS/MS at multiple timepoints related to autoantibody seroconversion in 72 type 1 diabetes cases and 71 control participants, which were frequency matched on age at autoantibody seroconversion (of the case), ethnicity and sample availability. Linear mixed models were used to obtain an age-adjusted mean of each oxylipin prior to type 1 diabetes. Age-adjusted mean oxylipins were tested for association with type 1 diabetes using logistic regression, adjusting for the high risk HLA genotype HLA-DR3/4,DQB1*0302. We also performed principal component analysis of the oxylipins and tested principal components (PCs) for association with type 1 diabetes. Finally, to investigate potential critical timepoints, we examined the association of oxylipins measured before and after autoantibody seroconversion (of the cases) using PCs of the oxylipins at those visits. The ARA-related oxylipin 5-HETE was associated with increased type 1 diabetes risk. Five LA-related oxylipins, two ALA-related oxylipins and one DHA-related oxylipin were associated with decreased type 1 diabetes risk. A profile of elevated LA- and ALA-related oxylipins (PC1) was associated with decreased type 1 diabetes risk (OR 0.61; 95% CI 0.40, 0.94). A profile of elevated ARA-related oxylipins (PC2) was associated with increased diabetes risk (OR 1.53; 95% CI 1.03, 2.29). A critical timepoint analysis showed type 1 diabetes was associated with a high ARA-related oxylipin profile at post-autoantibody-seroconversion but not pre-seroconversion. The protective association of higher LA- and ALA-related oxylipins demonstrates the importance of both inflammation promotion and resolution in type 1 diabetes. Proinflammatory ARA-related oxylipins may play an important role once the autoimmune process has begun.

Factors associated with acquired Anti IFN- γ autoantibody in patients with nontuberculous mycobacterial infection.

BackgroundThe clinical syndrome of disseminated nontuberculous mycobacterial (NTM) infection in patients who were previously healthy is now well recognized to be associated with an acquired autoantibody to Interferon gamma (Anti IFN- γ autoantibody). However, the risk factors of this syndrome remain unknown.MethodWe performed an unmatched case control study among patients with NTM diseases who were diagnosed and treated at Siriraj Hospital, Bangkok, Thailand. Anti-IFN autoantibody was detected by enzyme-linked immunosorbent assay (ELISA) method. Cases were patients with NTM diseases and detectable anti IFN- γ autoantibody. Controls were randomly selected from those with undetectable anti IFN- γ autoantibody. Data from both groups including demographic data, clinical presentation, laboratory results, other risk factors and HLA genotypes were collected. Univariate and multivariate analyses were performed to identify independent risk factors for this syndrome.Results70 cases (mean age 50 ± 11 years) and 70 controls (mean age 58 ± 18 years) were enrolled into the study. Mycobacterial abscessus was the most common NTM pathogen found in both groups (72.9% in cases and 41.4% in controls respectively). However, disseminated NTM disease was significantly more common in cases (92.9%) than in the controls (14.3%, p<0.001). Binary logistic regression analysis showed that previous OIs (adjusted OR14.87, 95% CI 2.36–93.86), birthplace outside Central region (adjusted OR 19.19, 95% CI 3.86–95.35), lack of comorbidities lead to immunosuppression, such as HIV infection or diabetes mellitus (adjusted OR 23.68, 95% CI 4.01–139.94), and presence of HLA DRB1*15/16 (adjusted OR 153.28, 95% CI 16.87–139.88) were independent factors associated with this syndrome.ConclusionPatients with NTM disease associated with anti IFN- γ autoantibody are almost always previously healthy and HIV negative. Most of these patients presented with disseminated NTM disease with generalized lymphadenitis and often with reactive skin lesions. Factors associated with detectable anti IFN- γ autoantibody are HLA-DRB1 and DQB1 alleles, and history of previous OIs in patients without comorbidity that leads to immunosuppression. Further studies are needed to better understand these associations and to improve the treatment outcome.

Feeding the Infant at High-Risk of Celiac Disease - An Update.

Celiac disease is a life-long condition with significant morbidity and health-care costs (1). Conventional understanding of etiopathological factors, specifically the roles played by the HLA genes (DQ2 and DQ8), has greatly advanced in recent years. Furthermore, it is now well accepted that these HLA genes contribute to an increased risk of celiac disease for first-degree relatives of those affected by the condition. One such group is infants born to a mother with known celiac disease. Current guidelines for such high-risk infants include the recommendation to introduce gluten between 4 and 6 months of age and ideally, while still breastfeeding. Emerging data from a number of large studies refute the basis for these recommendations, thereby necessitating a new approach. Existing guidelines pertaining to gluten introduction are largely based on observational, retrospective data. In 1980s, the Swedish epidemic of celiac disease occurred following a general recommendation to delay the introduction of gluten to beyond 6 months of age (2). The dramatic increase in celiac diagnoses was halted in Sweden when this recommendation was revised so that gluten was instead introduced at 4 months of age. Subsequent evidence emerged showing that the introduction of gluten, either prior to 3 months or after 7 months of age, increased the risk of celiac disease (3). Similarly, the advice to introduce gluten while infants were still being breastfed was in keeping with the best available evidence, which suggested a negative association between duration of breastfeeding and the development of celiac disease. A series of large prospective studies conducted in recent years has now provided new evidence on which to base our management of these infants at greater risk of celiac disease. In a well-designed study conducted across multiple locations in Italy, Lionetti et al. (4) confirmed that children with a high-risk HLA genotype (DQ2 or DQ8 positive) were more likely than those with standard risk HLA genotype (DQ2 or DQ8 negative) to develop celiac disease autoimmunity. Interestingly, following randomization to having gluten introduced at either 6 or 12 months of age, there was no difference in the rates of celiac disease autoimmunity or biopsy-proven celiac disease between the groups. Results from this study did, however, demonstrate that delayed introduction of gluten was associated with a later onset of disease. In this study, breastfeeding had no modifying effect on subsequent development of celiac disease. A further multicentre, double-blinded randomized controlled trial included children who were either HLA-DQ2 or HLA-DQ8 positive and who had a first-degree relative with celiac disease (5). Each arm was randomized to receive either a small amount of gluten or placebo at between 4 and 6 months of age. This study reported no difference in the incidence of celiac disease between the groups at 3 years of age. The authors’ also conclude that exclusivity or duration of breastfeeding appeared to have no impact on celiac disease development in this cohort. From the Netherlands, a nested subset of children who were either HLA-DQ2 or HLA-DQ8 positive within a population-based cohort study were retrospectively assessed with regards to gluten introduction, breastfeeding, and the development of celiac disease (6). The findings arising from this study demonstrated that neither the delayed introduction of gluten to beyond 6 months of age nor duration of breastfeeding of at least 6 months had any effect on developing celiac disease autoimmunity. Similar to the Dutch study, a genetically high-risk sub-group within a multi-national birth cohort was followed up with regards to risk factors for subsequent development of celiac disease (7). In the latter study, gluten introduction either before 4 months or after 6 months of age appeared to have no effect on celiac disease risk. By contrast, evidence from a Norwegian cohort study suggested that introduction of gluten after 6 months of age and breastfeeding beyond 12 months of age conferred additional risk for developing celiac disease (8). It is important to note, however, that the cohort in this Norwegian study was not stratified according to genetic risk (unlike the previously referenced reports). While each of the recent studies has provided useful evidence, it is important to note that these reports have done so by asking slightly different questions. The consensus, however, is clear – the approach to a first-degree infant of someone with celiac disease must change. Although it still appears appropriate and important to introduce gluten-containing foods in mid-infancy, the previous recommendation of coordinating this with ongoing breastfeeding is no longer valid. That said, the myriad benefits of breastfeeding still hold true: none of the presented evidence warrants changing advice in this regard. While several aspects of the optimal management of genetically susceptible infants are not known, these recent studies at least necessitate revision of the current recommendations.

Temporal Trends of HLA, CTLA-4 and PTPN22 Genotype Frequencies among Type 1 Diabetes in Continental Italy

The incidence of type 1 diabetes has, progressively, increased worldwide over the last decades and also in Continental Italian population. Previous studies performed in northern European countries, showed, alongside a general increase in the disease incidence, a decreasing frequency of the highest risk HLA genotype in type 1 diabetes populations, thus emphasizing the role of environmental factors. The aim of the study was to evaluate whether a decreasing trend of high risk HLA, CTLA-4 and PTPN22 genotypes would be present in type 1 diabetes subjects of Continental Italy, a country considered at low incidence of the disease compared to northern European populations. N = 765 type 1 diabetes patients diagnosed from 1980 to 2012 in Lazio region were included. For HLA, CTLA4 and PTPN22 temporal trend evaluation, subjects were subdivided into groups of years according to age at diagnosis. All subjects were typed for HLA-DRB1 and DQB1 by a reverse line blot. The CT60 polymorphism of the CTLA4 and C1858T of the PTPN22 gene were genotyped using ABI PRISM 7900HT (n = 419 and n = 364 respectively). HLA genotypes were divided in high, moderate and low risk categories. The proportion of the HLA risk categories was not statistically different over the three decades in subjects with age of onset <15 years and ≥15 years. The genotype distribution of CT60 polymorphism of CTLA4 gene did not show any change in the frequencies during time. The analysis of the PTPN22 C1858T variant revealed, instead, that the frequency of CT+TT susceptibility genotypes decreased during time (23.9% vs 13.6%, p = 0.017). We can hypothesize that the pressure of the diabetogenic environment could be milder and therefore not sufficient to reduce the need of a strong genetic background (HLA) “to precipitate” diabetes; the increased pressure of the environment could have, instead, some effects on minor susceptibility genes in our population.

Family history of diabetes and distribution of class II HLA genotypes in children with newly diagnosed type 1 diabetes: effect on diabetic ketoacidosis

Our purpose was to assess whether family history of diabetes or the HLA-DR-DQ genotype of the index case was associated with the frequency of diabetic ketoacidosis (DKA) at diagnosis of childhood type 1 diabetes. The study cohort comprised 1518 children aged <15 years and diagnosed with type 1 diabetes in Finland in 2002-2005. Family history of type 1 and type 2 diabetes among first-degree relatives (FDRs) and grandparents was assessed at diagnosis. HLA-DR-DQ genotypes were analysed using time-resolved fluorometry. In total, 12.6 and 1.7% of children had at least one FDR affected with type 1 or type 2 diabetes, respectively, and 6.6 and 34.8% had at least one grandparent with type 1 or type 2 diabetes. DKA (pH <7.30) occurred less frequently in children having a type 1 diabetes affected FDR (7.4 vs 20.5%, P<0.001). Type 2 diabetes among the parents or grandparents had no such effect. Lower risk HLA genotypes were observed to predispose to DKA (P<0.024). In a logistic regression analysis, the risk of DKA was independently associated with the absence of a family member affected by type 1 diabetes, the presence of a low-risk HLA genotype and older age at diagnosis (odds ratio 3.23, 1.45 and 1.07 respectively). The presence of type 1 diabetes in an FDR is associated with an decreased risk of DKA at diagnosis. The rate of DKA seems to be higher in children with lower HLA-conferred risk for type 1 diabetes.

Analysis of pathogenesis of juvenile new-onset diabetes

Measurement of anti-islet autoantibodies at the time of disease onset contributes greatly to the differentiation of Type 1A diabetes with HLA Class II subtyping also contributing. Blood samples were obtained from 900 patients with age from 1 month to 25 years (median age 11.1 years) within 2 weeks of diabetes onset to test anti-islet autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma antigen (IA-2AA), the zinc transporter-8 (ZnT8AA), and islet-cell antibodies (ICA). Polymorphisms of the HLA Class II gene were typed in 547 randomly selected patients. Of the 900 subjects analyzed, 145 (16.1%) were negative for all five anti-islet autoantibodies, and autoantibody negativity significantly increased with age: 10.2% (38/372) among children <10 years of age, 14.2% (46/325) in those 10-14 years of age, and 30.1% (61/203) in those >14 years of age (P < 0.001). The prevalence of IA-2AA was the highest among young children. The prevalence of GADA increased with age while the prevalence of IAA was inversely correlated with age. At diagnosis, the subjects with negative antibodies had a higher body mass index (P < 0.001) and less high risk HLA genotype DR3-DQ2/DR4-DQ8 (P < 0.01). A large percentage of children and youths negative for all anti-islet autoantibodies at the onset of diabetes are likely to have the non-immune form, especially those without DR3/DR4 and obese patients. Among autoantibody-positive Type 1A patients, IAA and GADA showed a reciprocal prevalence, suggesting differential disease pathogenesis.

Primary Dietary Intervention Study to Reduce the Risk of Islet Autoimmunity in Children at Increased Risk for Type 1 Diabetes

OBJECTIVETo determine whether delaying the introduction of gluten in infants with a genetic risk of islet autoimmunity is feasible, safe, and may reduce the risk of type 1 diabetes–associated islet autoimmunity.RESEARCH DESIGN AND METHODSA total of 150 infants with a first-degree family history of type 1 diabetes and a risk HLA genotype were randomly assigned to a first gluten exposure at age 6 months (control group) or 12 months (late-exposure group) and were followed 3 monthly until the age of 3 years and yearly thereafter for safety (for growth and autoantibodies to transglutaminase C [TGCAs]), islet autoantibodies to insulin, GAD, insulinoma-associated protein 2, and type 1 diabetes.RESULTSAdherence to the dietary-intervention protocol was reported from 70% of families. During the first 3 years, weight and height were similar in children in the control and late-exposure groups, as was the probability of developing TGCAs (14 vs. 4%; P = 0.1). Eleven children in the control group and 13 children in the late-exposure group developed islet autoantibodies (3-year risk: 12 vs. 13%; P = 0.6). Seven children developed diabetes, including four in the late-exposure group. No significant differences were observed when children were analyzed as per protocol on the basis of the reported first gluten exposure of the children.CONCLUSIONSDelaying gluten exposure until the age of 12 months is safe but does not substantially reduce the risk for islet autoimmunity in genetically at-risk children.

Receptor for advanced glycation end-products (RAGE) provides a link between genetic susceptibility and environmental factors in type 1 diabetes

This group of studies examines human genetic susceptibility conferred by the receptor for advanced glycation end-products (RAGE) in type 1 diabetes and investigates how this may interact with a western environment. We analysed the AGER gene, using 13 tag SNPs, in 3,624 Finnish individuals from the FinnDiane study, followed by AGER associations with a high risk HLA genotype (DR3)-DQA1*05-DQB1*02/DRB1*0401-DQB1*0302 (n = 546; HLA-DR3/DR4), matched in healthy newborn infants from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study (n = 373) using allelic analysis. We also studied islets and circulating RAGE in NODLt mice. The rs2070600 and rs17493811 polymorphisms predicted increased risk of type 1 diabetes, whereas the rs9469089 SNP was related to decreased risk, on a high risk HLA background. Children from the DIPP study also showed a decline in circulating soluble RAGE levels, at seroconversion to positivity for type 1 diabetes-associated autoantibodies. Islet RAGE and circulating soluble RAGE levels in prediabetic NODLt mice decreased over time and were prevented by the AGE lowering therapy alagebrium chloride. Alagebrium chloride also decreased the incidence of autoimmune diabetes and restored islet RAGE levels. These studies suggest that inherited AGER gene polymorphisms may confer susceptibility to environmental insults. Declining circulating levels of soluble RAGE, before the development of overt diabetes, may also be predictive of clinical disease in children with high to medium risk HLA II backgrounds and this possibility warrants further investigation in a larger cohort.

Human Enterovirus RNA in Monthly Fecal Samples and Islet Autoimmunity in Norwegian Children With High Genetic Risk for Type 1 Diabetes

OBJECTIVETo test whether the frequency of human enterovirus RNA in fecal samples collected monthly from early infancy was associated with development of multiple islet autoantibodies in children with the highest risk HLA genotype.RESEARCH DESIGN AND METHODSIndividuals carrying the HLA DRB1*0401-DQA1*03-DQB1*0302/DRB1*03-DQA1*05-DQB1*02 genotype were identified at birth and followed with monthly stool samples from age 3 to 35 months. Blood samples taken at age 3, 6, 9, and 12 months and then annually were tested for autoantibodies to insulin, GAD 65 and IA-2. Among 911 children, 27 developed positivity for two or more islet autoantibodies in two or more consecutive samples (case subjects). Two control subjects per case subject were matched by follow-up time, date of birth, and county of residence. Stool samples were analyzed for enterovirus with a semiquantitative real-time RT-PCR.RESULTSThe frequency of human enterovirus RNA in stool samples from case subjects before seroconversion (43 of 339, 12.7%) did not differ from the frequency in control subjects (94 of 692, 13.6%) (P = 0.97). Results remained essentially unchanged after adjustment for potential confounders, restriction to various time windows before seroconversion, or infections in the 1st year of life or after inclusion of samples collected after seroconversion. There was no difference in the average quantity of enterovirus RNA or in the frequency of repeatedly positive samples. The estimated relative risk for islet autoimmunity per enterovirus RNA–positive sample during follow-up (nested case-control analysis) was 1.12 (95% CI 0.66–1.91).CONCLUSIONSThere was no support for the hypothesis that fecal shedding of enteroviral RNA is a major predictor of advanced islet autoimmunity.

Definition, epidemiology and classification of diabetes in children and adolescents

Diabetes mellitus is a group of metabolic diseases characterised by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The abnormalities in carbohydrate, fat, and protein metabolism that are found in diabetes are due to deficient action of insulin on target tissues. If ketones are present in blood or urine, treatment is urgent, because ketoacidosis can evolve rapidly.

Omega-3 Polyunsaturated Fatty Acid Intake and Islet Autoimmunity in Children at Increased Risk for Type 1 Diabetes

Cod liver oil supplements in infancy have been associated with a decreased risk of type 1 diabetes mellitus in a retrospective study. To examine whether intakes of omega-3 and omega-6 fatty acids are associated with the development of islet autoimmunity (IA) in children. A longitudinal, observational study, the Diabetes Autoimmunity Study in the Young (DAISY), conducted in Denver, Colorado, between January 1994 and November 2006, of 1770 children at increased risk for type 1 diabetes, defined as either possession of a high diabetes risk HLA genotype or having a sibling or parent with type 1 diabetes. The mean age at follow-up was 6.2 years. Islet autoimmunity was assessed in association with reported dietary intake of polyunsaturated fatty acids starting at age 1 year. A case-cohort study (N = 244) was also conducted in which risk of IA by polyunsaturated fatty acid content of erythrocyte membranes (as a percentage of total lipids) was examined. Risk of IA, defined as being positive for insulin, glutamic acid decarboxylase, or insulinoma-associated antigen-2 autoantibodies on 2 consecutive visits and still autoantibody positive or having diabetes at last follow-up visit. Fifty-eight children developed IA. Adjusting for HLA genotype, family history of type 1 diabetes, caloric intake, and omega-6 fatty acid intake, omega-3 fatty acid intake was inversely associated with risk of IA (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.21-0.96; P = .04). The association was strengthened when the definition of the outcome was limited to those positive for 2 or more autoantibodies (HR, 0.23; 95% CI, 0.09-0.58; P = .002). In the case-cohort study, omega-3 fatty acid content of erythrocyte membranes was also inversely associated with IA risk (HR, 0.63; 95% CI, 0.41-0.96; P = .03). Dietary intake of omega-3 fatty acids is associated with reduced risk of IA in children at increased genetic risk for type 1 diabetes.

Progress in the characterization of slowly progressive autoimmune diabetes in adult patients (LADA or type 1.5 diabetes).

LADA or type 1.5 diabetes is a slowly progressive form of autoimmune diabetes of adults and represents a considerable proportion (about 5-10%) of all diabetic patients. Associations with high risk HLA genotypes and autoimmune phenomena (GAD, IA2, ICA) show similarities with type 1 diabetes, but phenotypical characteristics of these patients do not allow the correct identification without screening of GAD antibodies. The relatively low antibody titers against islet-cell antigens in LADA patients may be sign of a less aggressive form of autoimmune diabetes and could be responsible for the long non-insulin requirement phase of this diabetes type. Similar as in prediabetic relatives of type 1 diabetic patients the risk for beta cell failure in adult "type 2 diabetic" patients increases with the number of antibodies positive. Consequently, low titers of GAD--in particular in elderly patients--do not predict a progressive and rapid loss of beta-cell failure, when associations with high risk genotypes or other islet-cell antibodies are lacking. Patients with LADA share insulin resistance with type 2 diabetic patients, but display a more severe defect in stimulated beta-cell capacity than patients with classical type 2 diabetes. With respect to features of the metabolic syndrome, patients with LADA have lower BMI, blood pressure and triglyceride levels compared with classical type 2 diabetes patients. Early identification of LADA patients will be mandatory, when effective immune interventions are available for prevention of the beta-cell destructive process and insulin requirement of these patients.

HLA-DR, DQ and anti-GAD antibodies in first degree relatives of type I diabetes mellitus

The differential antibody response to glutamic acid decarboxylase (anti-GAD) and to islet cell cytoplasm (ICA) according to HLA-DR and DQ genotypes were examined in 28 Spanish patients with Type I diabetes mellitus (11.1 ± 10.4 year diabetes duration) and their 41 first degree non-diabetic relative. Anti-GAD was detected by radioimmunoprecipitation and ICA by indirect immunofluorescence and HLA-DR/DQ alleles were assigned by PCR and sequence specific oligonucleotide probes. The frequency in patients of positivity for ICA was 7.1% and of anti-GAD − 64.3%, and in relatives, the frequency of ICA − was 4.9%, and anti-GAD + 9.8%. Concurrent positivity for ICA and anti-GAD existed in only one patient, and in none of the relatives. We confirm for a Spanish population the high frequency of risk genotypes for Type I, involving DR3, DR4 and DQB1*0302 (DQ8), which were present in 26 of 28 (93%) patients and 32 of 41 (78%) relatives. The most frequent genotypes were DR3/DQB1*0201/DQA1*0501-DR4/DQB1*0302/DQA1*0301(9 patients, 32%; 6 relatives, 15%) DR3/DQB1*0201/ DQA1*0501-DR3/DQB1*0201/DQA1*0501 (5 patients, 18% 7 relatives, 17% and DR3/DQB1*0201/DQA1*0501-DR1/DQB1*0501/DQA1*0101 (5 patients, 18%; 1 relative, 2%). Positivity for anti-GAD or for ICA did not correlate with gender, or age at onset or duration of DM. The distribution of high risk HLA genotypes were similar regardless the anti-GAD or anti-ICA status either in patients or in their relatives.