CTLA4+49G allele associates with early onset of type 1 diabetes in North Indians.

Type 1 diabetes (T1D) is a complex autoimmune disease with strong genetic influence. In this study, we investigated +49A/G SNP (rs 231775) in exon 1 of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) by PCR-RFLP and its influence as a risk factor for the disease in the North Indian population. This polymorphism at codon 17 results in an amino acid substitution (Thr/Ala) in the leader peptide of the molecule. The study included 232 patients with T1D (age at onset of disease (AOD): 0.5-37 years) and 305 ethnically matched healthy controls. The DNA obtained from these 537 individuals was amplified using a set of specific primers followed by restriction enzyme digestion with Fnu4HI. The +49G allele as well as its homozygous genotype G/G was observed to be significantly higher in patients as compared to the healthy controls {(37.3% vs. 25.6%, P = 4.96E(-05) , OR = 1.73; 95%CI = 1.33-2.25) (15.52% vs. 6.6%, P = 0.001, OR = 2.62; 95% CI = 1.48-4.63) respectively}. The frequency of G/G genotype was significantly higher in patients with early age at onset of disease (AOD:<12 years) as compared to that in the late-onset patients with AOD: ≥12 years (21.1% vs. 10.6%, P = 0.042, OR = 2.26; 95% CI = 1.09-4.67) as well as to that in the healthy controls (21.1% vs. 6.6%, P = 0.00004, OR = 3.8; 95% CI = 2.01-7.2). Further analysis revealed that the median AOD significantly reduced (P = 0.049) from 14 years in patients with A/A genotype to 11 and 10 years in those with A/G and G/G genotypes, respectively. These results suggest that CTLA4+49G allele, particularly in homozygous G/G condition, associates with early onset of T1D.

Association of PTPN22+1858C/T polymorphism with Type 1 diabetes in the North Indian population.

A nonsynonymous SNP +1858C/T (rs2476601) in the protein tyrosine phosphatase, nonreceptor type 22(PTPN22) gene leading to Arg 620 Trp substitution is known to be associated with susceptibility to type 1 diabetes (T1D) and several other autoimmune diseases. We studied this polymorphism in 145 T1D patients and 210 healthy controls from North India. The minor allele +1858T was observed to be significantly increased among patients as compared to healthy controls (2.76% vs 0.5%, P = 0.027, OR = 5.93; 95% CI = 1.4-24.8). The association was also observed at the level of heterozygous C/T genotype (5.5% vs 0.95%, P = 0.026, OR = 6.07; 95% CI = 1.43-25.6). The T allele and C/T genotype were predominantly found among patients who were positive for both glutamic acid decarboxylase 65 (GAD65) and insulin antigen 2 (IA2) autoantibodies and showed significantly increased frequencies (10%, P = 0.034, OR = 11.67; 95% CI = 1.58-84.1 and 20%, P = 0.031, OR = 13.0; 95% CI = 1.66-97.5, respectively) as compared to patients negative for these autoantibodies (0.95% and 1.9%, respectively). The results suggest that the PTPN22+1858T allele is positively associated with T1D in the North Indian population.

16(th) IHIW: analysis of HLA population data, with updated results for 1996 to 2012 workshop data (AHPD project report).

We present here the results of the Analysis of HLA Population Data (AHPD) project of the 16th International HLA and Immunogenetics Workshop (16IHIW) held in Liverpool in May-June 2012. Thanks to the collaboration of 25 laboratories from 18 different countries, HLA genotypic data for 59 new population samples (either well-defined populations or donor registry samples) were gathered and 55 were analysed statistically following HLA-NET recommendations. The new data included, among others, large sets of well-defined populations from north-east Europe and West Asia, as well as many donor registry data from European countries. The Gene[rate] computer tools were combined to create a Gene[rate] computer pipeline to automatically (i) estimate allele frequencies by an expectation-maximization algorithm accommodating ambiguities, (ii) estimate heterozygosity, (iii) test for Hardy-Weinberg equilibrium (HWE), (iv) test for selective neutrality, (v) generate frequency graphs and summary statistics for each sample at each locus and (vi) plot multidimensional scaling (MDS) analyses comparing the new samples with previous IHIW data. Intrapopulation analyses show that HWE is rarely rejected, while neutrality tests often indicate a significant excess of heterozygotes compared with neutral expectations. The comparison of the 16IHIW AHPD data with data collected during previous workshops (12th-15th) shows that geography is an excellent predictor of HLA genetic differentiations for HLA-A, -B and -DRB1 loci but not for HLA-DQ, whose patterns are probably more influenced by natural selection. In Europe, HLA genetic variation clearly follows a north to south-east axis despite a low level of differentiation between European, North African and West Asian populations. Pacific populations are genetically close to Austronesian-speaking South-East Asian and Taiwanese populations, in agreement with current theories on the peopling of Oceania. Thanks to this project, HLA genetic variation is more clearly defined worldwide and better interpreted in relation to human peopling history and HLA molecular evolution.

Role of killer immunoglobulin-like receptor-ligand interactions in human leukocyte antigen-matched sibling hematopoietic stem cell transplantation.

INTRODUCTION: Killer immunoglobulin-like receptor (KIR)-ligand mismatches lead to natural killer cell alloreactivity after hematopoietic stem cell transplantation (HSCT). However, their clinical impact on HSCT outcomes is controversial due to complexity of KIR haplotypes, genotypes, and phenotypes as well as their diversity among patient populations. The present study investigated the role of KIR-ligand interactions in human leukocyte antigen (HLA)-matched sibling transplants. METHODS: The recipient cohort, which included patients diagnosed with aplastic anemia, acute leukemia, and myelodysplastic syndrome, received granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells. HLA typing was performed using polymerase chain reaction - sequence specific oligo probes (PCR-SSO). The KIR genotype of the donors and the ligands C1 (Asparagine 80), C2 (Lysine 80), and Bw4 recipient typings were performed using polymerase chain reaction - sequence specific primers (PCR-SSP). We assessed acute and chronic graft-versus-host disease (GVHD), relapse, and overall survival. RESULTS: While 84.5% of donors carried a Bx KIR, 15.5% carried the AA haplotype. The effect of a recipient's lack of ligands among 88.5% of cases was associated with 39% of subjects developing GvHD. Lack of C1 may lead to manifestations of acute GvHD and lack of C2 to manifestation of chronic GvHD. The presence of both C1 and C2 seemed to be protective against both forms of GvHD. The role of two Bw4 alleles, threonine (T) or isoleucine (I) at position 80, was evaluated. 73% of recipients who carried Bw4 80(I) versus 27% with the Bw4 80(T) allele. The presence of Bw4-80(T) allele appeared to reduce the risk of GvHD, indicating its stronger inhibitory effect than its 80(I) counterpart. CONCLUSION: KIR-ligand interactions influenced HSCT outcomes.

Audit of peripheral stem cell transplantation for aplastic anemia in multitransfused infected patients.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation is a curative modality for aplastic anemia; the preferred stem cell source is bone marrow. However, allogeneic peripheral blood stem cell transplantation (PBSCT) used in high-risk patients is associated with higher risk of chronic graft-versus-host disease (GVHD). Our center receives multitransfused, alloimmunized, infected, late referrals for transplant. METHODS: Forty-one patients of median age 22 years (range 8-37) received allogeneic-PBSCT from human leukocyte antigen (HLA)-matched sibling donors. The median time since diagnosis was 12 months (range 4-65) and median pretransplant transfusions were 37 (range 6-160). Six patients were platelet refractory and one alloimmunized for pan-red blood cell (RBC) antigens. Several patients had pretransplant icterus or renal dysfunction and 26 (63.4%) had unresponsive bacterial/fungal infections. Our conditioning regimen included fludarabine 30 mg/m(2) for 6 days (days -10 to -5), cyclophosphamide 60 mg/kg/d for 2 days (days -6 to -5), and antithymocyte globulin (ATGAM) 30 mg/kg/d for 4 days (day -4 to -1), which was reduced to 2 days in 2 patients. We used standard GVHD prophylaxis with cyclosporine and methotrexate on days 1, 3, 6, 11. RESULTS: The median follow-up period was 29 months (range 6-78) and median engraftment time 10 days (range 8-17). Thirty-one patients (75.6%) were treated for infections, with 20 of these on antifungals for preexisting infections. There were two graft rejections and 10 (24.4%) deaths, with three intracranial hemorrhages, two rejections with infection, three cases of refractory GVHD (acute/overlap syndrome) with cytomegalovirus reactivation, and two invasive fungal infections. Overall incidence of acute GVHD was 39% with 2 grade IV cases. Ten (25%) cases developed chronic GVHD, with extensive GVHD in four. CONCLUSION: With more experience using shortened course of ATGAM, HLA-matched donor transfusions, and availability of newer antifungals, we have been able to decrease PBSCT-related mortality. Further improvement will be possible with early referrals.

A novel HLA-DPB1 allele, DPB1*125:01, identified by sequence-based typing in an Indian individual.

A novel DPB1*125:01 allele differs from DPB1*26:01:02 at two positions in exon 2, leading to changes at codons 9 and 35.

Asian Indian donor marrow registry: All India Institute of Medical Sciences experience.

A major limitation in hematopoietic stem cell transplantation (HSCT) is the availability of a genetically matched donor, particularly with respect to the human leukocyte antigens (HLA)-linked immune response genes located on chromosome 6 in humans. During the last 5 years, a total of 688 patients requiring HSCT underwent HLA testing in our department to identify a matched donor from their families. The sibship size ranged from 1 to > or =5 in all disease categories, except thalassemia major where the majority of patients had only 1 sibling. Family genotype analysis revealed that 39.3% of the total number of patients had an HLA-matched sibling and that families with sibship size of > or =4 had a higher probability (68.8%) compared with those with sibship size of < or =3 (29.7%). Because the Indian population is characterized by the presence of novel HLA alleles and unique haplotypes (HLA-A*0211, B*2707, A*26-B*08-DRB1*03), patients with rare HLA alleles have much less probability of finding an unrelated optimally matched donor than those with common HLA phenotypes. Smaller family size and unique HLA profile are limitations that can be overcome by developing unrelated volunteer marrow donor registries. The Asian Indian Donor Marrow Registry at our institute is regularly providing services to such patients.

Immunogenetic association and thyroid autoantibodies in juvenile autoimmune thyroiditis in North India.

OBJECTIVES: Autoimmune thyroid diseases (AITD) encompass a number of conditions that have in common cellular and humoral responses targeting the thyroid gland. Interactions between susceptibility genes and environmental triggers are thought to initiate an autoimmune response to thyroid antigens leading to disease manifestation. Commencement of the disease in childhood leads to the presumption that genetics may have an important role in the causation of the disease. DESIGN: The present study was aimed at evaluating the human leucocyte antigen (HLA) encoded susceptibility to develop juvenile autoimmune thyroiditis (JAT) in patients from North India. PATIENTS: We studied 48 consecutive patients of JAT along with 176 first-degree relatives for their thyroid function (FT4, TSH) and anti-thyroid peroxidase antibody status (AbTPO). MEASUREMENTS: HLA studies were carried out using serology for HLA-class I antigens and DNA analysis of HLA-class II alleles. The data were compared with a cohort of 308 ethnically matched healthy individuals. RESULTS: We observed overt hypothyroidism in 50% and AbTPO positivity in 70.8% of the index cases. Among the first-degree relatives, goitre was observed in 51.7%, thyroid dysfunction in 28.4% and AbTPO in 29.5% of individuals. Of the 37 relatives who underwent fine-needle aspiration cytology (FNAC), 60% had evidence of chronic lymphocytic thyroiditis (CLT). A strong positive association of HLA-DRB1*1404 was observed with the JAT (35.4%vs. 10.4%, chi2 = 19.8, Pc = 0.0001). We also observed a higher (72%, P = 0.03) paternal transmission of HLA-DRB1*1404 to affected offspring in comparison to unaffected offspring. HLA-DRB1*03 was also increased among JAT patients but did not reach statistical significance. CONCLUSION: These studies point towards an important role of immune modifying genes, such as HLA, in influencing susceptibility to juvenile-onset AITD.

Molecular diversity of the HLA-A*19 group of alleles in North Indians: possible oriental influence.

The present study aims to determine the genetic diversity of the HLA-A19 allelic family in the North Indian and Japanese populations. The HLA-A*19 group of alleles occurred at similar frequencies in North Indians and Japanese as in Caucasians. All the known serological splits of HLA-A19 were observed among the North Indians, i.e. A*33 (15.6%), A*32 (8.6%), A*31 (3.5%), A*30 (3%), A*29 (1.2%) and A*74 (0.77%), while only A*30 (0.7%), A*31 (17.6%) and A*33 (11.7%) were observed in the Japanese. High resolution analysis indicated that the A*29, A*30, A*31 and A*32 alleles were represented by only single subtypes among the North Indians while the HLA-A*33 group comprised two alleles, A*3301 (4.3%) and A*3303 (43.7%). All 15 of the HLA-A*33 positive samples from the Tamil population of South India were found to be A*3303. One novel subtype of A*33, A*3306 was also observed in the North Indian sample. Conversely, only one subtype each of A*30, A*31 and A*33 was encountered in the Japanese population, of which A*3101 and A*3303 were the most frequent (58.5% and 39%, respectively, among the HLA-A*19 group of alleles). All other subtypes of A19 were not found in the Japanese in the present study. The study suggests a significant amount of genetic admixture in the North Indian gene pool from other racial groups, with profound oriental influence.

Influence of HLA compatibility on renal graft survival using live unrelated & cadaver donors in India.

BACKGROUND & OBJECTIVES: Living unrelated donor (LURD) renal transplantation has shown a rising trend over the last 5 yr at our center following the passing of The Transplantation of Human Organs Act by the Government of India in 1994. In this paper, the results of LURD and cadaver (CAD) donor renal transplantation are compared. We have also looked into factors that have a bearing on graft survival such as the extent of HLA mismatch (MM), infections, acute rejections (AR), donor age and sex. METHODS: A total of 42 LURD and 25 CAD renal transplants performed between March 1994 and February 1999 has been included in the study. HLA typing, panel reactive antibody (PRA) screening and T and B cell cross match assay were performed by the complement dependent cytotoxicity (CDC) method for all patients. RESULTS: The graft survival rates were generally higher in the LURD category as compared to the CAD group and were significant at 6 month period (90 vs 56%, P = 0.002). A follow up of the patients up to 60 months revealed a matching effect since the 3, 4 allele MM group had better survival rates as compared to the 5, 6 MM group. Twenty six of the 67 recipients (39%) experienced episodes of acute rejection (AR). Patients with 3, 4 MM had fewer such episodes than those with 5, 6 allele MM (P < 0.05). Of the 32 deaths, 20 were those with a functional kidney, of which 15 were caused by severe infections. INTERPRETATION & CONCLUSION: Better HLA matching ensures fewer episodes of rejection and better long term graft survival in comparison to the poorly matched grafts. The graft survival for LURD recipients was appreciably higher than that of CAD recipients.