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Aims/hypothesis: Psychological stress has long been considered a possible trigger of type 1 diabetes, although prospective studies examining the link between psychological stress or life events during pregnancy and the child's type 1 diabetes risk are rare. The objective of this study was to examine the association between life events during pregnancy and first-appearing islet autoantibodies (IA) in young children, conditioned by the child's type 1 diabetes-related genetic risk., Methods: The IA status of 7317 genetically at-risk The Environmental Determinants of Diabetes in the Young (TEDDY) participants was assessed every 3 months from 3 months to 4 years, and bi-annually thereafter. Reports of major life events during pregnancy were collected at study inception when the child was 3 months of age and placed into one of six categories. Life events during pregnancy were examined for association with first-appearing insulin (IAA) (N = 222) or GAD (GADA) (N = 209) autoantibodies in the child until 6 years of age using proportional hazard models. Relative excess risk due to interaction (RERI) by the child's HLA-DR and SNP profile was estimated., Results: Overall, 65% of mothers reported a life event during pregnancy; disease/injury (25%), serious interpersonal (28%) and job-related (25%) life events were most common. The association of life events during pregnancy differed between IAA and GADA as the first-appearing autoantibody. Serious interpersonal life events correlated with increased risk of GADA-first only in HLA-DR3 children with the BACH2-T allele (HR 2.28, p < 0.0001), an additive interaction (RERI 1.87, p = 0.0004). Job-related life events were also associated with increased risk of GADA-first among HLA-DR3/4 children (HR 1.53, p = 0.04) independent of serious interpersonal life events (HR 1.90, p = 0.002), an additive interaction (RERI 1.19, p = 0.004). Job-related life events correlated with reduced risk of IAA-first (HR 0.55, p = 0.004), particularly in children with the BTNL2-GG allele (HR 0.48; 95% CI 0.31, 0.76)., Conclusions/interpretation: Specific life events during pregnancy are differentially related to IAA vs GADA as first-appearing IA and interact with different HLA and non-HLA genetic factors, supporting the concept of different endotypes underlying type 1 diabetes. However, the mechanisms underlying these associations remain to be discovered. Life events may be markers for other yet-to-be-identified factors important to the development of first-appearing IA.

Chimpanzees have been used for some time as an animal model in research on immune-related diseases in humans. The major histocompatibility complex (MHC) region of the chimpanzee has also been the subject of studies in which the attention was mainly on the class I genes. Although full-length sequence information is available on the DRB region genes, such detailed information is lacking for the other class II genes and, if present, is based mainly on exon 2 sequences. In the present study, full-length sequencing was performed on DQ, DP, and DRA genes in a cohort of 67 pedigreed animals, thereby allowing a thorough analysis of the MHC class II repertoire. The results demonstrate that the number of MHC class II lineages and alleles is relatively low, whereas haplotype diversity (combination of genes/alleles on a chromosome) seems to have been maximised by crossing-over processes.

Aims: To assess the prevalence of autoantibodies (Aab) to insulin (IAA), glutamic acid decarboxylase 65 (GADA) and insulinoma antigen 2 (IA-2A), as well as human leukocyte antigen (HLA) class II alleles, in first degree relatives (FDR) of Mexican patients with type 1 diabetes (T1D), and to explore whether these parameters mirror the low incidence of T1D in the Mexican population., Methods: Aab titers were determined by ELISA in 425 FDR, 234 siblings, 40 offspring and 151 parents of 197 patients with T1D. Typing of HLA-DR and -DQ alleles was performed in 41 Aab-positive FDR using polymerase chain reaction with allele-specific oligotyping., Results: Seventy FDR (16.47%) tested positive for Aab. The siblings (19.2%) and the offspring (25%) had significantly higher prevalence of Aab than the parents (9.9%). GADA was the most frequent Aab. Almost half of the Aab-positive FDR had two different Aab (45.7%), and none tested positive for three Aab. The highest prevalence of Aab was found among women in the 15-29 years age group. Moreover, the positivity for two Aab was significantly more frequent among females. A considerable number of FDR (48.8%) carried the susceptible HLA-DR3, -DR4, -DQB1*0201 or -DQB1*0302 alleles, but almost none had the high risk genotype HLA-DR3/DR4., Conclusions: FDR of Mexican T1D patients have high prevalence of islet Aab, comparable to countries with the highest incidence of T1D. However, Aab positivity does not seem to be associated with HLA risk genotypes, which may have an impact on the low incidence of T1D in Mexico.

Rheumatoid arthritis (RA) is a chronic auto-immune disease primarily targeting the joints. Approximately 1% of the population is affected by RA, and despite the improvements in therapeutic interventions, elucidation of the disease pathogenesis is still in its infancy. RA patients can be subdivided on basis of the presence of autoantibodies, especially anti-citrullinated protein antibodies (ACPA). ACPA + and ACPA - disease most likely differ in aetiology, as different genetic and environmental risk factors are associated with these two disease entities. For ACPA + RA disease, the genetic factors associating with disease mainly comprised of human leukocyte antigen (HLA) class II molecules. The predisposing HLA-DR alleles have been depicted as the 'HLA Shared Epitope (SE) alleles', as these alleles encode a similar sequence, the shared epitope sequence, within the beta chain of the HLA-DR molecule. In addition to the involvement of the HLA-SE alleles in the development of ACPA + RA disease, other HLA-DR molecules have been shown to confer protection against this disease entity. The protective HLA molecules have, instead of the SE-motif, a different but shared sequence at the same location in the beta chain of HLA-DR molecules, consisting of the amino acid residues DERAA. The possible contributions of the predisposing and protective HLA molecules in association with ACPA-positive RA are discussed in this review.

The human leukocyte antigen (HLA) system is a major part of the human immune system and has an impact on tumor initiation, tumor progression, and immunosurveillance. Renal cell carcinoma tumors are considered to be immunogenic. Therefore, we studied the allele frequencies of four gene loci (HLA-A, -B, -C, and HLA-DR) in a cohort of German renal cell carcinoma (RCC) patients and in healthy controls. HLA-A-C were determined using serological methods, whereas HLA-C12, C14, C16, C18, and HLA-DR were characterized through the use of standard molecular biological methods. The occurrence of the HLA-C*12 allele was significantly increased in German RCC patients compared with healthy controls (P < 0.005; Fisher's exact test), whereas the occurrence of the HLA-DRB1*04 allele was significantly reduced in RCC patients compared with healthy controls (P < 0.05; Fisher's exact test). However, the presence of allele HLA-C*12 was not significantly associated with 10 year overall survival. We suggest that the frequency of HLA alleles can affect development of RCC and could add knowledge as predictive marker for future immunotherapies.

The polymorphism of major histocompatibility complex (MHC) class II DQ and DR genes in five common equine leukocyte antigen (ELA) haplotypes was determined through sequencing of mRNA transcripts isolated from lymphocytes of eight ELA homozygous horses. Ten expressed MHC class II genes were detected in horses of the ELA-A3 haplotype carried by the donor horses of the equine bacterial artificial chromosome (BAC) library and the reference genome sequence: four DR genes and six DQ genes. The other four ELA haplotypes contained at least eight expressed polymorphic MHC class II loci. Next generation sequencing (NGS) of genomic DNA of these four MHC haplotypes revealed stop codons in the DQA3 gene in the ELA-A2, ELA-A5, and ELA-A9 haplotypes. Few NGS reads were obtained for the other MHC class II genes that were not amplified in these horses. The amino acid sequences across haplotypes contained locus-specific residues, and the locus clusters produced by phylogenetic analysis were well supported. The MHC class II alleles within the five tested haplotypes were largely non-overlapping between haplotypes. The complement of equine MHC class II DQ and DR genes appears to be well conserved between haplotypes, in contrast to the recently described variation in class I gene loci between equine MHC haplotypes. The identification of allelic series of equine MHC class II loci will aid comparative studies of mammalian MHC conservation and evolution and may also help to interpret associations between the equine MHC class II region and diseases of the horse.

Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4(+) T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b(+)CD11c(+) cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b(+)CD11c(+) cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b(+)CD11c(+) cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4(+) T and CD8(+) T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy.

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide-MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0 .

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.

The DR region of particular primate species may display allelic polymorphism and gene copy number variation (region configuration polymorphism). The sum of these distinct types of polymorphism is defined as complexity. To date, however, the DR region of cynomolgus macaques (Macaca fascicularis) has been poorly defined. Transcriptome analysis of a pedigreed colony, comprising animals from Indonesia and Indochina, revealed a total of 15 Mafa-DRA and 57 DRB alleles, specifying 28 different region configurations. The DRA alleles can be divided into two distinct lineages. One lineage is polymorphic, but the majority of the amino acid replacements map to the leader peptide. The second lineage is at best oligomorphic, and segregates with one specific Mafa-DRB allele. The number of Mafa-DRB genes ranges from two to five per haplotype. Due to the presence of pseudogenes, however, each haplotype encodes only one to three bona fide DRB transcripts. Depending on the region configuration in which the Mafa-DRB gene is embedded, identical alleles may display differential transcription levels. Region configurations appear to have been generated by recombination-like events. When genes or gene segments are relocated, it seems plausible that they may be placed in the context of distinct transcription control elements. As such, DRB region-related transcription level differences may add an extra layer of polymorphism to this section of the adaptive immune system.

Aims/hypothesis: We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona., Methods: Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA-DRA, and with type 2 diabetes mellitus and prediabetic traits., Results: The A allele at rs9268852, which tags HLA-DRB1 02(1602), was associated both with higher HLA-DRB1 mRNA expression (n = 133, p = 4.27 × 10(-14)) and decreased risk of type 2 diabetes (n = 3,265, OR 0.723, p = 0.002). Among persons with normal glucose tolerance (n = 266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p = 0.005), higher mean 30 min insulin concentration during an oral glucose tolerance test (p = 0.017) and higher body fat percentage (p = 0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity., Conclusions/interpretation: HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.

Aims/hypothesis: We investigated the risk associated with HLA-B*39 alleles in the context of specific HLA-DR/DQ haplotypes., Methods: We studied a readily available dataset from the Type 1 Diabetes Genetics Consortium that consists of 2,300 affected sibling pair families genotyped for both HLA alleles and 2,837 single nucleotide polymorphisms across the major histocompatibility complex region., Results: The B*3906 allele significantly enhanced the risk of type 1 diabetes when present on specific HLA-DR/DQ haplotypes (DRB1 0801-DQB1 0402: p = 1.6 × 10(-6), OR 25.4; DRB1 0101-DQB1 0501: p = 4.9 × 10(-5), OR 10.3) but did not enhance the risk of DRB1 0401-DQB1 0302 haplotypes. In addition, the B 3901 allele enhanced risk on the DRB1 1601-DQB1 0502 haplotype (p = 3.7 × 10(-3), OR 7.2)., Conclusions/interpretation: These associations indicate that the B 39 alleles significantly increase risk when present on specific HLA-DR/DQ haplotypes, and HLA-B typing in concert with specific HLA-DR/DQ genotypes should facilitate genetic prediction of type 1 diabetes, particularly in a research setting.

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy, and a pathological manifestation of rapid eye movement during sleep. Narcoleptic pathogenesis is triggered by both genetic and environmental factors. Recently, development of genome-wide association studies (GWAS) has identified new genetic factors, with many more susceptibility genes yet to be elucidated. Using a new approach that consists of a combination of GWAS and an extensive database search for candidate genes, we picked up 202 candidate genes and performed a replication study in 222 narcoleptic patients and 380 controls. Statistical analysis indicated that six genes, NFATC2, SCP2, CACNA1C, TCRA, POLE, and FAM3D, were associated with narcolepsy (P<0.001). Some of these associations were further supported by gene expression analyses and an association study in essential hypersomnia (EHS), CNS hypersonia similar to narcolepsy. This novel approach will be applicable to other GWAS in the search of disease-related susceptibility genes.

Genes of the major histocompatibility complex (MHC) mainly code for proteins of the immune system of jawed vertebrates. In particular, MHC class I and II cell surface proteins are crucial for the self/non-self discrimination of the adaptive immune system and are the most polymorphic genes in vertebrates. Positive selection, gene duplications and pseudogenes shape the face of the MHC and reflect a highly dynamic evolution. Here, we present for the first time data of the highly polymorphic MHC class II DRB exon 2 of a representative of the mammalian order scandentia, the northern tree shrew Tupaia belangeri. We found up to eight different alleles per individual and determined haplotype constitution by intensively studying their inheritance. The alleles were assigned to four putative loci, all of which were polymorphic. Only the most polymorphic locus was subject to positive selection within the antigen binding sites and only alleles of this locus were transcribed.

Major histocompatibility complex class II plays a key role in the immune response, by presenting processed antigens to CD4+ lymphocytes. Major histocompatibility complex class II expression is controlled at the transcriptional level by at least four trans-acting genes: CIITA, RFXANK, RFX5 and RFXAP. Defects in these regulatory genes cause MHC class II immunodeficiency, which is frequent in North Africa. The aim of this study was to describe the immunological and molecular characteristics of ten unrelated Moroccan patients with MHC class II deficiency. Immunological examinations revealed a lack of expression of MHC class II molecules at the surface of peripheral blood mononuclear cells, low CD4+ T lymphocyte counts and variable serum immunoglobulin (IgG, IgM and IgA) levels. In addition, no MHC class II (HLA DR) expression was observed on lymphoblasts. The molecular analysis identified the same homozygous 752delG26 mutation in the RFXANK genes of all patients. This finding confirms the association between the high frequency of the combined immunodeficiency and the defect in MHC class II expression and provides strong evidence for a founder effect of the 752delG26 mutation in the North African population. These findings should facilitate the establishment of molecular diagnosis and improve genetic counselling for affected Moroccan families.

Poststreptococcal sequelae, especially acute rheumatic fever/rheumatic heart disease continues to occur in significant proportions in many parts of the world, especially in less developed countries. An important factor in the study of rheumatic heart disease is the human genetic susceptibility to the disease. The aim of the present study was to detect the most prevalent HLA class I and class II types associated with risk of rheumatic heart disease in Egyptian children. Our study was performed on 100 patients with rheumatic valvular heart diseases and 71 control subjects. Patients were recruited from the Heart Institute, Embaba, Egypt. HLA typing for HLA class I was performed by serotyping and HLDR allele genotyping was performed using INNO-LiPA kits. In the study of HLA class I, there was a statistically significant increase in the B5 allele (P = 0.03; odds ratio, 3.46 [1.12-10.72]) in patients compared to controls, while B49 and B52 alleles (P = 0.004 and P = 0.02) were found in controls only. There was a statistically significant increase in HLA DR* 04-02, 3.46 (1.12-10.72) and HLA DR *10-0101 5.75 (1.27-25.98) in patients. Meanwhile HLA DR*1309120 was found only in controls (P = 0.02). Our study provides further information on the genetic predisposition for rheumatic valvular disease and the protective genotypes in rheumatic heart disease. Further insight into the molecular mechanisms of the disease will be a useful tool for predicting clinical outcome in those patients and, thus, potentially offer new means and approaches to treatment and prophylaxis, including a potential vaccine.

The aetiology of anti-factor VIII (FVIII) autoantibody formation in acquired haemophilia remains unknown. We hypothesised that encounter of antigenically different, allogeneic FVIII may challenge inhibitor formation after presentation on MHC class II. Eighteen consecutive cases with acquired haemophilia were enrolled (nine females, nine males). A control group comprised 50 male and 50 female healthy blood donors. The coding region of the FVIII gene and the HLA-DRB1 genotype were studied. The presentation of foreign FVIII variants on the patient's MHC class II alleles was predicted using SYFPEITHI algorithm. A rare FVIII variant (E2004K) was found in one patient with acquired haemophilia after massive transfusion; the 2004 K allele was predicted to be presented on the patient's HLA-DRB1*0101. Moreover, distribution of a polymorphism (D1241E) was significantly skewed comparing patients and controls. Three of three patients with transfusion-associated disease carried 1241D in homozygous or hemizygous form and were predicted to present 1241E (foreign), but not 1241D (self), on their HLA-DRB1*0301. Therefore, encounter of 1241E may result in the presentation of a new T cell epitope in these patients. The same conditions were not found in any patient with acquired haemophilia of other causes. The expected frequency in the general Caucasoid population undergoing transfusion is 3% to 4%. In conclusion, encounter of variant allogeneic FVIII presented on a suitable MHC background could be a risk factor for inhibitor formation.

The DR region of primate species is generally complex and displays diversity concerning the number and combination of distinct types of DRB genes present per region configuration. A highly variable short tandem repeat (STR) present in intron 2 of nearly all primate DRB genes can be utilized as a quick and accurate high through-put typing procedure. This approach resulted previously in the description of unique and haplotype-specific DRB-STR length patterns in humans, chimpanzees, and rhesus macaques. For the present study, a cohort of 230 cynomolgus monkeys, including self-sustaining breeding groups, has been examined. MtDNA analysis showed that most animals originated from the Indonesian islands, but some are derived from the mainland, south and north of the Isthmus of Kra. Haplotyping and subsequent sequencing resulted in the detection of 118 alleles, including 28 unreported ones. A total of 49 Mafa-DRB region configurations were detected, of which 28 have not yet been described. Humans and chimpanzees possess a low number of different DRB region configurations in concert with a high degree of allelic variation. In contrast, however, allelic heterogeneity within a given Mafa-DRB configuration is even less frequently observed than in rhesus macaques. Several of these region configurations appear to have been generated by recombination-like events, most probably propagated by a retroviral element mapping within DRB6 pseudogenes, which are present on the majority of haplotypes. This undocumented high level of DRB region configuration-associated diversity most likely represents a species-specific strategy to cope with various pathogens.

To describe the polymorphism of the DRA gene in Macaca fascicularis, we have studied 141 animals either at cDNA level (78 animals from Mauritius, the Philippines, and Vietnam) or genomic level (63 animals from the Philippines, Indonesia, and Vietnam). In total, we characterized 22 cDNA DRA alleles, 13 of which had not been described until now. In the Mauritius population, we confirmed the presence of three DRA alleles. In the Philippine and Vietnam populations, we observed 11 and 14 DRA alleles, respectively. Only two alleles were present in all three populations. All DRA alleles but one differ from the consensus sequence by one to three mutations, most being synonymous; so, only seven DR alpha proteins were deduced from the 22 cDNA alleles. One DRA cDNA allele, Mafa-DRA*02010101, differs from all other alleles by 11 to 14 mutations of which only four are non-synonymous. The two amino acid changes inside the peptide groove of Mafa-DRA*02010101 are highly conservative. The very low proportion of non-synonymous/synonymous mutations is compatible with a purifying selection which is comparable to all previous observations concerning the evolution of the DRA gene in mammals. Homologues of the allele Mafa-DRA*02010101 are also found in two other Asian macaques (Macaca mulatta and Macaca nemestrina). The forces able to maintain this highly divergent allele in three different macaque species remain hypothetical.

Aim/hypothesis: We sought to clarify similarities and differences in the contribution of HLA to genetic susceptibility to three subtypes of type 1 diabetes: acute-onset, fulminant and slowly progressive., Methods: We genotyped 545 Japanese patients with type 1 diabetes (338 acute-onset, 80 fulminant, 127 slowly progressive) and 396 control participants at HLA-DRB1, -DQB1, -A, -B and -C, and at 101 candidate single nucleotide polymorphisms (SNPs) in an 8.5 Mb region of the extended HLA., Results: DRB1*0405-DQB1*0401, DRB1*0802-DQB1*0302 and DRB1*0901-DQB1*0303 were associated with acute-onset type 1 diabetes, with the DRB1*0405-DQB1*0401/DRB1*0802-DQB1*0302 genotype achieving the highest odds ratio of 42.7. DRB1*1501-DQB1*0602 and DRB1*1502-DQB1*0601 were negatively associated with acute-onset type 1 diabetes. A similar tendency was observed for slowly progressive type 1 diabetes. In contrast, only DRB1*0405-DQB1*0401 was associated with fulminant type 1 diabetes, with the DRB1*0405-DQB1*0401/DRB1*0405-DQB1*0401 genotype showing the highest odds ratio of 11.2. DRB1*0802-DQB1*0302, DRB1*0405-DQB1*0401/DRB1*0802-DQB1*0302 and DRB1*1501-DQB1*0602 were not associated with fulminant type 1 diabetes. The association of class I alleles and a panel of SNPs in an extended HLA region with fulminant type 1 diabetes was also different from that seen for the acute-onset and slowly progressive forms. The presence of both one and two susceptible haplotypes conferred susceptibility to slowly progressive type 1 diabetes, whereas the presence of two susceptible haplotypes was required to confer susceptibility to acute-onset and fulminant type 1 diabetes., Conclusions/interpretation: These data suggest that HLA associations with fulminant type 1 diabetes are qualitatively different from those with other subtypes of type 1 diabetes, whereas the HLA contribution to slowly progressive type 1 diabetes is qualitatively similar to, but quantitatively different from, that in acute-onset type 1 diabetes.

To confirm and refine associations of human leukocyte antigen (HLA) genotypes with variable antibody (Ab) responses to hepatitis B vaccination, we have analyzed 255 HIV-1 seropositive (HIV(+)) youth and 80 HIV-1 seronegatives (HIV(-)) enrolled into prospective studies. In univariate analyses that focused on HLA-DRB1, -DQA1, and -DQB1 alleles and haplotypes, the DRB1*03 allele group and DRB1*0701 were negatively associated with the responder phenotype (serum Ab concentration > or = 10 mIU/mL) (P = 0.026 and 0.043, respectively). Collectively, DRB1*03 and DRB1*0701 were found in 42 (53.8%) out of 78 non-responders (serum Ab <10 mIU/mL), 65 (40.6%) out of 160 medium responders (serum Ab 10-1,000 mIU/mL), and 27 (27.8%) out of 97 high responders (serum Ab >1,000 mIU/mL) (P < 0.001 for trend). Meanwhile, DRB1*08 was positively associated with the responder phenotype (P = 0.010), mostly due to DRB1*0804 (P = 0.008). These immunogenetic relationships were all independent of non-genetic factors, including HIV-1 infection status and immunodeficiency. Alternative analyses confined to HIV(+) youth or Hispanic youth led to similar findings. In contrast, analyses of more than 80 non-coding, single nucleotide polymorphisms within and beyond the three HLA class II genes revealed no clear associations. Overall, several HLA-DRB1 alleles were major predictors of differential Ab responses to hepatitis B vaccination in youth, suggesting that T-helper cell-dependent pathways mediated through HLA class II antigen presentation are critical to effective immune response to recombinant vaccines.

Aims/hypothesis: Larger childhood body size and rapid growth have been associated with increased type 1 diabetes risk. We analysed height, weight, BMI and velocities of growth in height, weight and BMI, for association with development of islet autoimmunity (IA) and type 1 diabetes., Methods: Since 1993, the Diabetes Autoimmunity Study in the Young (DAISY) has followed children at increased type 1 diabetes risk, based on HLA-DR, -DQ genotype or family history, for the development of IA and type 1 diabetes. IA was defined as the presence of autoantibodies to insulin, GAD or protein tyrosine phosphatase islet antigen 2 twice in succession, or autoantibody-positive on one visit and diabetic at the next consecutive visit within 1 year. Type 1 diabetes was diagnosed by a physician. Height and weight were collected starting at age 2 years. Of 1,714 DAISY children <11.5 years of age, 143 developed IA and 21 progressed to type 1 diabetes. We conducted Cox proportional hazards analysis to explore growth velocities and size measures for association with IA and type 1 diabetes development., Results: Greater height growth velocity was associated with IA development (HR 1.63, 95% CI 1.31-2.05) and type 1 diabetes development (HR 3.34, 95% CI 1.73-6.42) for a 1 SD difference in velocity., Conclusions/interpretation: Our study suggests that greater height growth velocity may be involved in the progression from genetic susceptibility to autoimmunity and then to type 1 diabetes in pre-pubertal children.

Purpose: We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III-IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III-IV malignant melanoma patients., Patients and Methods: A cohort of metastatic malignant melanoma patients (n = 91), in stage III (n = 26) or IV (n = 65) were analysed for HLA-A, -B, -Cw and -DRB1 types by PCR/sequence-specific primer method. The frequencies of HLA alleles in the patients were compared to that of healthy Swedish bone marrow donors. The effect of HLA types on prognosis was defined by Kaplan-Meier and Cox analysis., Results: The presence of the AHH 62.1 in clinical stage IV patients was significantly and independently associated with the worst survival rate recorded from the appearance of metastasis (HR = 2.14; CI = 1.02-4.4; P = 0.04). In contrast, the period from the primary diagnosis to metastasis was the longest in patients with this haplotype (HR = 0.40; CI = 0.17-0.90; P = 0.02)., Conclusions: Melanoma patients in our cohort with 62.1 AHH which is associated with autoimmune diseases have an initial strong anti-tumour control with longer metastasis-free period. These patients have rapid progression after the appearance of metastasis, responding poorly to chemo- or/and immunotherapy. This apparently paradoxical clinical process could be due to the interplay between tumour clones escape and immune surveillance ending up with a rapid disease progression.

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2(+) tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials.

Background: Human leukocyte antigen (HLA) molecules are known to play an important role in host-defense mechanisms. The aim of this study was to evaluate the association between HLA alleles and lung disease caused by nontuberculous mycobacteria (NTM) in Korean patients., Methods: Seventy-eight patients with NTM lung disease (48 patients with Mycobacterium avium-intracellulare complex [MAC] infection and 30 patients with Mycobacterium abscessus infection) were included in the study. HLA-A, -B, and -DRB1 genotyping was performed by polymerase chain reaction using sequence-specific primers. Data from 485 healthy Korean individuals were used as a control., Results: When compared to controls, patients with NTM lung disease showed an increased frequency of DRB1*11 (OR = 1.91, 95% confidence interval [CI] = 1.01-3.64, p = 0.045, corrected p [pC] > 0.05). In the subgroup analysis, patients with MAC lung disease had an increased frequency of B*46 (OR = 2.23, 95% CI = 1.05-4.73, p = 0.044, pC > 0.05)., Conclusions: Our data suggest that in a Korean population, patients with NTM lung disease and healthy subjects differ in the frequencies of some HLA alleles. However, when considering corrected p values, our findings are inconclusive.

Objective: To assess blood leucocytes gene profiling during recovery phase of septic shock; to test the relation between encoding gene expression and protein level., Study Design: Gene expression levels were studied at days 0, 1, 7 and 28 (D0, 1, 7 and 28) on a dedicated microarray of 340 genes involved in inflammatory processes., Settings: 16-bed intensive care unit, Lariboisière University hospital., Patients: Seventeen septic shock patients enrolled when at least one additional organ dysfunction occurred., Measurements and Results: Changes over time were compared with D0 via the ratio Dx/D0. The time-related gene expression study showed significant changes in ten genes. Among them, S100A8 and S100A12 had a reduced expression over time compared with D0, whereas CD74's expression increased. The microarray results were validated by RT-qPCR for four genes. The S100A8 plasma levels decrease along recovery in parallel with the gene expression decrease. The CD74 gene expression evolution significantly correlated with HLA-DR monocyte expression., Conclusions: These results are the first description of variations in expression of key inflammatory genes in the course of the septic shock recovery period.

Despite relatively frequent gene or segment duplications, the number of functional loci in the major histocompatibility complex (MHC) is relatively small. The dual function of MHC molecules (triggering the immune system and limiting T-cell receptor repertoires) is likely to balance the number of functional loci. The effect of this dual function on the number of functional MHC loci has been argued mainly in the theoretical and computer simulation studies, but the evidence from empirical data has not been fully examined. Here, we attempt to evaluate this effect based on the analysis of nucleotide sequence data. We hypothesized that due to the dual function, even becoming a pseudogene (pseudogenization) of MHC is advantageous for the organisms. To evaluate this hypothesis, we compared the distribution of the waiting time (T (W)) till pseudogenization for HLA (human MHC) with that of the human olfactory receptor (OR) and bitter taste receptor (T2R) genes. The result shows that T (W) in HLA has a tendency to be relatively shorter as the emergence time (T) of the gene becomes older, while in OR T (W) becomes proportionally longer as T becomes older and in T2R it is almost null irrespective of T. Furthermore, T (W) in HLA is strongly influenced by the extent of functional differentiation in the peptide-binding region. Taken together, these results show that MHC molecules have optimal numbers of functional loci, and these numbers are regulated by the advantageous pseudogenization of duplicated copies.

The discoveries in the 1970s of strong associations between various diseases and certain human leukocyte antigen (HLA) factors were a revolution within genetic epidemiology in the last century by demonstrating for the first time how genetic markers can help unravel the genetics of disorders with complex genetic backgrounds. HLA controls immune response genes and HLA associations indicate the involvement of autoimmunity. Multiple sclerosis (MS) was one of the first conditions proven to be HLA associated involving primarily HLA class II factors. We review how HLA studies give fundamental information on the genetics of the susceptibility to MS, on the importance of linkage disequilibrium in association studies, and on the pathogenesis of MS. The HLA-DRB1*1501 molecule may explain about 50% of MS cases and its role in the pathogenesis is supported by studies of transgenic mice. Studies of polymorphic non-HLA genetic markers are discussed based on linkage studies and candidate gene approaches including complete genome scans. No other markers have so far rivaled the importance of HLA in the genetic susceptibility to MS. Recently, large international collaborations provided strong evidence for the involvement of polymorphism of two cytokine receptor genes in the pathogenesis of MS: the interleukin 7 receptor alpha chain gene (IL7RA) on chromosome 5p13 and the interleukin 2 receptor alpha chain gene (IL2RA (=CD25)) on chromosome 10p15. It is estimated that the C allele of a single nucleotide polymorphism, rs6897932, within the alternative spliced exon 6 of IL7RA is involved in about 30% of MS cases.

Background/hypothesis: HLA, INS, PTPN22 and CTLA4 are considered to be confirmed type 1 diabetes susceptibility genes. HLA, PTPN22 and CTLA4 are known to be involved in immune regulation. Few studies have systematically investigated the joint effect of multiple genetic variants. We evaluated joint effects of the four established genes on the risk of childhood-onset type 1 diabetes., Methods: We genotyped 421 nuclear families, 1,331 patients and 1,625 controls for polymorphisms of HLA-DRB1, -DQA1 and -DQB1, the insulin gene (INS, -23 HphI), CTLA4 (JO27&#95;1) and PTPN22 (Arg620Trp)., Results: The joint effect of HLA and PTPN22 on type 1 diabetes risk was significantly less than multiplicative in the case-control data, but a multiplicative model could not be rejected in the trio data. All other two-way gene-gene interactions fitted multiplicative models. The high-risk HLA genotype conferred a very high risk of type 1 diabetes (OR 20.6, using the neutral-risk HLA genotype as reference). When including also intermediate-risk HLA genotypes together with risk genotypes at the three non-HLA loci, the joint odds ratio was 61 (using non-risk genotypes at all loci as reference)., Conclusion: Most established susceptibility genes seem to act approximately multiplicatively with other loci on the risk of disease except for the joint effect of HLA and PTPN22. The joint effect of multiple susceptibility loci conferred a very high risk of type 1 diabetes, but applies to a very small proportion of the general population. Using multiple susceptibility genotypes compared with HLA genotype alone seemed to influence the prediction of disease only marginally.

Background: Kawasaki disease is a leading cause of acquired heart disease in children. The prevalence rate varies in different ethnic groups. Recently, with the clinical application of molecular genetic technology, human leukocyte antigen (HLA) polymorphisms associated with several diseases have been identified by DNA analysis. This study aimed to assess the association of HLA alleles with susceptibility and complications of Kawasaki disease in Korean children., Methods: In this study, DNA was extracted from 74 children with a diagnosis of Kawasaki disease. The polymorphisms of the HLA-A, -B, -C, and -DRB1 alleles of patients with Kawasaki disease were determined by polymerase chain reaction (PCR)-amplification refractory mutation system (ARMS) and PCR-sequence-specific primer (SSP) analysis. The polymorphisms identified were compared with those of 159 normal healthy control subjects., Results: There was a significant increase in the frequencies of the HLA-B35, -B75, and -Cw09 alleles in patients with Kawasaki disease compared with the healthy control group. There was no increase in the frequency of HLA-DRB1 alleles among the Kawasaki disease patients compared with a healthy control group. When the patients with Kawasaki disease were divided into two subgroups, with or without coronary complications, the Kawasaki disease patients with coronary complications showed a significantly increased frequency of the HLA-DRB1*11 allele compared with the healthy control group and increased frequency of HLA-DRB1*09 in a comparison of the subgroups., Conclusions: This study suggests that polymorphisms in some alleles of B and C in HLA class I genes are associated with Kawasaki disease in Korean children.

In all but the most primitive vertebrates, multiple polymorphic genes associated with lymphocyte-mediated immunological surveillance are linked together within genomic regions termed the major histocompatibility complex (MHC). The extensive diversity at many MHC loci provides a valuable source of genetic markers for examining the complex relationships between host genotype and disease resistance or susceptibility. Such studies in domestic sheep (Ovis aries) have generally focused on exon 2 of the polymorphic class II MHC DRB1 gene and its adjacent sequences. We have determined the complete genomic sequences of two Ovar-DRB1 alleles, which has allowed an analysis of diversity at coding, non-coding and promoter regions of this gene. On the basis of these sequences, oligonucleotide primers were designed for amplification of full-length Ovar-DRB1 transcripts and used to evaluate diversity within an MHC-defined resource flock maintained at the Moredun Institute. We describe nine novel full-length Ovar-DRB1 sequences along with an improved direct-sequencing method for analysis of the entire exon 2 region of the Ovar-DRB1 gene based on previously unknown intronic sequences. We discuss how these data provide evidence on the evolution of MHC DRB diversity in domestic sheep.

Multiple sclerosis (MS) is a complex trait in which HLA-DRB1*15 bearing MHC haplotypes increase risk of MS in people of Northern European descent. In this investigation of 7,334 individuals from 1,515 MS families, the largest cohort used to study the HLA-DRB1 locus to date, we analysed the transmission of HLA-DRB1*15 haplotypes stratified by sex of transmitting parent. A significant over transmission of HLA-DRB1*15 from mothers was observed (chi (2) = 7.73, P = 0.0054), suggesting that parent of origin effects at the MHC determine susceptibility to MS.

Autoimmune diabetes mellitus, called type 1 diabetes mellitus (T1DM), is caused by autoimmune destruction of islet beta cells in the pancreas. T1DM susceptibility loci mapped by different genome screening are IDDM1-IDDM18. It has been estimated that HLA (IDDM1) provides up to 40-50 % of the familial clustering of T1DM (LOD score of 65.8). Many studies have verified that DQB1*0302 is a strong susceptibility gene and that the heterozygous combination of DQA1*0301-DQB1*0302 on the HLA-DR4 haplotype and DQA1*0501-DQB1*0201 on the HLA-DR3 haplotype results in a synergistically increased risk of T1DM. The presence of predisposing genes in autoimmune diabetes decreases with age, probably due to increasing influence of environmental factors. Autoimmune diabetes with manifestation in adults may have partly different immunogenetic etiopathogenesis than autoimmune diabetes with manifestation in childhood. Compared to fast progressing adult-onset T1DM, slowly progressing adult-onset type 1 diabetes (LADA) might involve genes leading to a slow progressive beta-cells destruction.

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.

Buerger disease (BD) is an occulusive vascular disease of unknown etiology. Although cigarette smoking is a well-known risk factor of BD, genetic factors may also play a role in the etiology. Because chronic bacterial infection such as oral periodontitis is suggested to be involved in the pathogenesis of BD, gene polymorphisms involved in the infectious immunity might be associated with BD as the genetic factor(s). We have previously reported that HLA-DRB1*1501 and B54 was associated with BD in Japanese. In this study, polymorphisms in HLA-DPB1, DRB1 and B were analyzed in 131 Japanese BD patients and 227 healthy controls. In addition, we investigated a functional promoter polymorphism, -260 C > T, of CD14 that is a main receptor of bacterial lipopolysaccharide. It was found that the frequencies of CD14 TT genotype [37.4 vs. 24.2%, P = 0.008 OR = 1.87, 95% confidence interval (CI); 1.18, 2.97], DRB1*1501 (34.4 vs. 13.2%, P (c) = 4.4 x 10(-5), OR = 3.44, 95%CI; 2.06, 5.73) and DPB1*0501 (79.4 vs. 55.1%, P (c) = 4.7 x 10(-5), OR = 3.14, 95%CI; 1.93, 5.11) were significantly higher in the patients than in the controls, demonstrating that at least three genetic markers were associated with BD. Stratification analyses of these associated markers suggested synergistic roles of the genetic factors. Odds ratios ranged from 4.72 to 12.57 in individuals carrying any two of these three markers. These findings suggested that the susceptibility to BD was in part controlled by genes involved in the innate and adaptive immunity.

The list of alleles in the HLA-DRB, HLA-DQA, and HLA-DQB gene loci has grown enormously since the last listing in this journal 8 years ago. Crystal structure determination of several human and mouse HLA class II alleles, representative of two gene loci in each species, enables a direct comparison of ortholog and paralog loci. A new numbering system is suggested, extending earlier suggestions by [Fremont et al. in Immunity 8:305-317, (1998)], which will bring in line all the structural features of various gene loci, regardless of animal species. This system allows for structural equivalence of residues from different gene loci. The listing also highlights all amino acid residues participating in the various functions of these molecules, from antigenic peptide binding to homodimer formation, CD4 binding, membrane anchoring, and cytoplasmic signal transduction, indicative of the variety of functions of these molecules. It is remarkable that despite the enormous number of unique alleles listed thus far (DQA = 22, DQB = 54, DRA = 2, and DRB = 409), there is invariance at many specific positions in man, but slightly less so in mouse or rat, despite their much lower number of alleles at each gene locus in the latter two species. Certain key polymorphisms (from substitutions to an eight-residue insertion in the cytoplasmic tail of certain DQB alleles) that have thus far gone unnoticed are highly suggestive of differences or diversities in function and thus call for further investigation into the properties of these specific alleles. This listing is amenable to supplementation by future additions of new alleles and the highlighting of new functions to be discovered, providing thus a unifying platform of reference in all animal species for the MHC class II allelic counterparts, aiding research in the field and furthering our understanding of the functions of these molecules.

We, in this paper, describe the identification of the novel human leukocyte antigen (HLA) blank allele DRB1*0710N that was detected in a male individual of Caucasian origin. The complete HLA class I typing was A*01, A*02, B*18, B*44, DRB1*0710N, DRB1*11, DQB1*02, and DQB1*03. Compared to DRB1*070101, the new variant is characterized by two consecutive nucleotide deletions at positions 175 and 176 of exon 2, leading to a frame shift that results in a stop codon (TAA), 6 bp further downstream. We identified three different possibilities as to which nucleotides were deleted to result in this non-expressed DRB1 allele. To verify the non-expression of the gene, an Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell line (B-LCL) of the individual was established and analyzed by flow cytometry for the cell surface expression of HLA-DRB1*07. Despite this non-expression of the cell surface protein, transcription of the DRB1*0710N sequence into messenger RNA (mRNA) was demonstrated.

Although the HLA class II alleles and immunological abnormalities are associated with type 1 diabetes mellitus (T1DM) in all racial groups, there are considerable variations in the genotypes and the prevalence of autoantibodies. In order to investigate the characteristics of the immunogenetic patterns and to use these as an early diagnostic tool and guideline for a therapeutic plan, we examined the clinical characteristics and the patterns of anti-GAD antibody (GADA), IA-2 antibody (IA-2A), HLA-DR and HLA-DQ in Korean adult-onset T1DM patients. Adult-onset patients had higher serum C-peptide levels than child-onset patients. In adult-onset patients, the prevalence of GADA and IA-2A were 59.5% and 15.3% respectively, and increased frequencies of HLADR4 and-DR9 were found. The frequencies of HLADQA1,-DQB1 and-DQ heterodimers were similar to those of the control, but child-onset patients had high frequencies of the HLA-DR3,-DR4,-DR9, DQA1*0301, DQA1*0501 and DQB1*0201 genotypes. In conclusion, Korean adult-onset T1DM patients had a lower prevalence of GADA, which was comparable to that found in Caucasian patients. The detection of GADA might help to predict the insulin dependency of adult-onset diabetes. Difference in the frequencies of diabetes associated with HLA type suggests that there might be a heterogeneity in the pathogenesis of diabetes according to the age of onset.

The aim of this study was to compare the frequency of the HLA-DRB1 and HLA-DQB1 alleles in Korean patients with idiopathic pulmonary arterial hypertension (IPAH) and in normal controls and to determine any association that may exist between clinical characteristics of IPAH and specific HLA alleles. IPAH patients seen between October 1998 and September 2001 were retrospectively assessed, and 19 patients and 193 controls were HLA typed at the HLA-DRB1 and DQB1 loci. Clinical characteristics and hemodynamic parameters were reviewed. The patients with IPAH had a significantly higher frequency of the HLA-DRB1*0406 allele (18% vs. 6%, p = 0.004) and the HLA-DQB1*0302 allele (24% vs. 12%, p = 0.034), as well as a significantly higher frequency of haplotype DRB1*0406-DQB1*0302 (p = 0.0006). All 6 patients with haplotype DRB1*0406-DQB1*0302 (H+ group) were women, compared with 8 of the 13 patients lacking the DRB1*0406-DQB1*0302 haplotype (H- group), but without statistical significance. Three of 19 patients showed a positive short-term hemodynamic response to NO inhalation, all 3 of whom had the DRB1*0406-DQB1*0302 haplotype. There were no other significant differences in clinical characteristics and hemodynamic parameters between the H+ and H- groups. We conclude from this study that the HLA-DRB1*0406-DQB1*0302 haplotype is associated with IPAH in Korean patients. These results suggest that certain clinical characteristics of IPAH may be controlled in part by patients' HLA alleles.

The generation and maintenance of allelic polymorphism in genes of the major histocompatibility complex (MHC) is a central issue in evolutionary genetics. Recently, the focus has changed from ex situ to in situ populations to understand the mechanisms that determine adaptive MHC polymorphism under natural selection. Birth-and-death evolution and gene conversion events are considered to generate sequence diversity in MHC genes, which subsequently is maintained by balancing selection through parasites. The ongoing arms race between the host and parasites leads to an adaptive selection pressure upon the MHC, evident in high rates of non-synonymous vs synonymous substitution rates. We characterised the MHC class II DRB exon 2 of free living bank voles, Clethrionomys glareolus by single-strand conformation polymorphism and direct sequencing. Unlike other arvicolid species, the DRB locus of the bank vole is at least quadruplicated. No evidence for gene conversion events in the Clgl-DRB sequences was observed. We found not only high allelic polymorphism with 26 alleles in 36 individuals but also high rates of silent polymorphism. Exceptional for MHC class II genes is a purifying selection pressure upon the majority of MHC-DRB sequences. Further, we analysed the association between certain DRB alleles and the parasite burden with gastrointestinal trichostrongyle nematodes Heligmosomum mixtum and Heligmosomoides glareoli and found significant quality differences between specific alleles with respect to infection intensity. Our findings suggest a snapshot in an evolutionary process of ongoing birth-and-death evolution. One allele cluster has lost its function and is already silenced, another is loosing its adaptive value in terms of gastrointestinal nematode resistance, while a third group of alleles indicates all signs of classical functional MHC alleles.

The HLA region harbors some of the most polymorphic loci in the human genome. Among them is the class II locus HLA-DRB1, with more than 400 known alleles. The age of the polymorphism and the rate at which new alleles are generated at HLA loci has caused much controversy over the years. Previous studies have mostly been restricted to the 270 base pairs that constitute the second exon and represent the most variable part of the gene. Here, we investigate the evolutionary history of the HLA-DRB1 locus on the basis of an analysis of 15 genomic full-length alleles (10-15 kb). In addition, the variation in 49 complete coding sequences and 322 exon 2 sequences were analyzed. When excluding exon 2 from the analysis, the diversity at the synonymous sites was found to be similar to the intron diversity. The overall diversity in noncoding region was also similar to the genome average. The DRB1*03 lineage has been found in human, chimpanzee, bonobo, gorilla, and orangutan. An ancestral "proto HLA-DRB1*03 lineage" appeared to have diverged in the last 5 million years into the human-specific lineages *08, *11, *13, and *14. With exception to exon 2, both the coding- and the noncoding diversity suggests a recent origin (<1 million years ago) for most of the alleles at the HLA-DRB1 locus. Sites encoding for amino acids involved in antigen binding [antigen recognizing sites (ARS)] appear to have a more ancient origin. Taken together, the recent origin of most alleles, the high diversity between allelic lineages, and the ancient origin of sequence motifs in exon 2, is consistent with a relatively rapid generation of novel alleles by gene conversion like events.

Human leucocyte antigen (HLA)-DRB1*15 is associated with predisposition to multiple sclerosis (MS), although conjecture surrounds the possible involvement of an alternate risk locus in the class I region of the HLA complex. We have shown previously that an alternate MS risk allele(s) may be encompassed by the telomerically extended DRB1*15 haplotype, and here, we have attempted to map the putative variant. Thirteen microsatellite markers encompassing a 6.79-megabase (D6S2236-G51152) region, and the DRB1 and DQB1 genes, were genotyped in 166 MS simplex families and 104 control families from the Australian State of Tasmania and 153 narcolepsy simplex families (trios) from the USA. Complementary approaches were used to investigate residual predisposing effects of microsatellite alleles comprising the extended DRB1*15 haplotype taking into account the strong predisposing effect of DRB1*15: (1) Disease association of the extended DRB1*15 haplotype was compared for MS and narcolepsy families--predisposing effects were observed for extended class I microsatellite marker alleles in MS families, but not narcolepsy families; (2) disease association of the extended DRB1*15 haplotype was investigated after conditioning MS and control haplotypes on the absence of DRB1*15--a significant predisposing effect was observed for a 627-kb haplotype (D6S258 allele 8-MOGCA allele 4; MOG, myelin oligodendrocyte glycoprotein) spanning the extended class I region. MOGCA allele 4 displayed the strongest predisposing effect in DRB1*15-conditioned haplotypes (p = 0.0006; OR 2.83 [1.54-5.19]). Together, these data confirm that an alternate MS risk locus exists in the extended class I region in Tasmanian MS patients independent of DRB1*15.

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.

We have previously reported that autoimmune pancreatitis (AIP) is a bioclinical entity characterized by high serum immunoglobulin G4 concentrations and association with the HLA-DRB1*0405-DQB1*0401 haplotype. However, the precise identity of gene(s) within this haplotype directly responsible for AIP pathogenesis is yet to be established. To dissect the genetic contribution of the incriminated haplotype, we have now performed an association analysis within the human leukocyte antigen (HLA) region using various types of polymorphic markers. Genomic DNAs from 43 AIP patients and 213 unrelated Japanese controls were used in this analysis. In each DNA sample, we established the genotype of 25 microsatellite markers distributed throughout the HLA region, that of single nucleotide polymorphism within the 5'-flanking regions of the TNFA and IkBLI (also known as NFKBIL1) as well as HLA class I and II genes. The HLA-linked susceptibility regions for AIP were localized to two segments: HLA-DRB1 (*0405; OR = 3.20, P = 0.00063, Pc = 0.0016) -DQB1 (*0401; OR = 3.29, P = 0.00046, Pc = 0.0069) in the HLA class II and C3-2-11 microsatellite (allele 219; OR = 2.96, P = 0.0076, Pc = 0.099) in the HLA class I regions. Upon stratification analysis in search for a synergistic effect given the extensive linkage disequilibrium within the major histocompatibility complex, it was established that each segment contributed to disease pathogenesis. The two critical HLA regions for susceptibility to AIP are limited to the HLA-DRB1*0405-DQB1*0401 in the class II and the ABCF1 proximal to C3-2-11, telomeric of HLA-E, in the class I regions.

Aims/hypothesis: Pathophysiological similarities between latent autoimmune diabetes in adults (LADA) and type 1 diabetes indicate an overlap in genetic susceptibility. HLA-DRB1 and HLA-DQB1 are major susceptibility genes for type 1 diabetes but studies of these genes in LADA have been limited. Our aim was to define patterns of HLA-encoded susceptibility/protection in a large, well characterised LADA cohort, and to establish association with disease and age at diagnosis., Materials and Methods: Patients with LADA (n = 387, including 211 patients from the UK Prospective Diabetes Study) and non-diabetic control subjects (n = 327) were of British/Irish European origin. The HLA-DRB1 and -DQB1 genes were genotyped by sequence-specific PCR., Results: As in type 1 diabetes mellitus, DRB1 0301&#95;DQB1 0201 (odds ratio [OR] = 3.08, 95% CI 2.32-4.12, p = 1.2 x 10(-16)) and DRB1 0401&#95;DQB1 0302 (OR = 2.57, 95% CI 1.80-3.73, p = 4.5 x 10(-8)) were the main susceptibility haplotypes in LADA, and DRB1 1501&#95;DQB1 0602 was protective (OR = 0.21, 95% CI 0.13-0.34, p = 4.2 x 10(-13)). Differential susceptibility was conferred by DR4 subtypes: DRB1 0401 was predisposing (OR = 1.79, 95% CI 1.35-2.38, p = 2.7 x 10(-5)) whereas DRB1 0403 was protective (OR = 0.37, 95% CI 0.13-0.97, p = 0.033). The highest-risk genotypes were DRB1 0301/DRB1 0401 and DQB1 0201/DQB1 0302 (OR = 5.14, 95% CI 2.68-10.69, p = 1.3 x 10(-8); and OR = 6.88, 95% CI 3.54-14.68, p = 1.2 x 10(-11), respectively). These genotypes and those containing DRB1 0401 and DQB1 0302 associated with a younger age at diagnosis in LADA, whereas genotypes containing DRB1 1501 and DQB1 0602 associated with an older age at diagnosis., Conclusions/interpretation: Patterns of susceptibility at the HLA-DRB1 and HLA-DQB1 loci in LADA are similar to those reported for type 1 diabetes, supporting the hypothesis that autoimmune diabetes occurring in adults is an age-related extension of the pathophysiological process presenting as childhood-onset type 1 diabetes.

HLA complex is composed of several closely linked loci, each containing several alleles, yielding a high expression of polymorphism. Vitiligo, a commonly acquired dermatological disorder, has been associated with different HLA antigens in different ethnic groups. In this study, HLA classes I (HLA-A, B, and C) and II (HLA-DR, DQ) antigens/alleles were analyzed in a group of 80 Saudi subjects consisting of vitiligo patients (40) and matched controls (40). The frequency of antigens of various HLA loci was tested using two-stage microcytotoxicity assays, while the frequency of alleles of HLA-DR was screened by polymerase chain reaction/sequence specific primers (PCR/SSP) method. The frequencies of HLA-B7, B15, Bw6, Cw6, Cw7, and DRB4*010101 were found to be significantly higher in vitiligo patients compared to controls [P = 0.029, 0.015, 0.033, 0.009, 0.043, and 0.015, respectively, with relative risk (RR) > or = 3, etiologic fraction (EF) > or = 0.4]. On the other hand, HLA-A9, B5, DQ1, and DRB3*010101 were significantly decreased in vitiligo patients compared to healthy Saudis [P = 0.008, 0.004, 0.028, and 0.04, respectively, with RR < 1 and preventive fraction (PF) < 0.5]. Among the patients, the highest allele frequency was noted for DRB4*010101(70%), while in controls it was for DRB3*010101 (72.5%). These results for antigens and allele frequency of various HLA Loci in vitiligo patients and control subjects suggested that HLA-B7, Bw6, Cw6, Cw7, and DRB4*010101 could be susceptible to vitiligo, while HLA-A9, B5, DQ1, and DRB3*010101 might be negatively associated with the development of vitiligo in Saudis.