Association between sarcoidosis and HLA polymorphisms in a Czech population from Central Europe: focus on a relationship with clinical outcome and treatment.

Background: Sarcoidosis is an immune-mediated systemic disease with unknown etiology affecting the lung predominantly. The clinical manifestation of sarcoidosis is rather diverse ranging from Löfgren's syndrome to fibrotic disease. Also, it differs among patients with distinct geographical and ethnic origins, consistent with environmental and genetic factors' role in its pathogenesis. Of those, the polymorphic genes of the HLA system have been previously implicated in sarcoidosis. Therefore, we have performed an association study in a well-defined cohort of Czech patients aiming to define how variation in HLA genes, may contribute to disease origin and development. Materials and methods: Total of the 301 Czech unrelated sarcoidosis patients were diagnosed according to international guidelines. In those, HLA typing was performed using next-generation sequencing. The allele frequencies at six HLA loci (HLA-A,-B,-C,-DRB1,-DQA1, and -DQB1) observed in the patients were compared with HLA allele distribution determined in 309 unrelated healthy Czech subjects; sub-analyses of relationships between HLA and distinct sarcoidosis clinical phenotypes were performed. Associations were assessed by two-tailed Fischer's exact test with correction for multiple comparisons. Results: We report two variants, HLA-DQB1*06:02, and HLA-DQB1*06:04, as risk factors for sarcoidosis, and three variants, HLA-DRB1*01:01, HLA-DQA1*03:01, and HLA-DQB1*03:02 as protective factors. HLA-B*08:01, HLA-C*07:01, HLA-DRB1*03:01, HLA-DQA1*05:01, and HLA-DQB1*02:01 variants associated with Löfgren's syndrome, a more benign phenotype. HLA- DRB1*03:01 and HLA-DQA1*05:01 alleles were connected with better prognosis-chest X-ray (CXR) stage 1, disease remission, and non-requirement of corticosteroid treatment. The alleles HLA-DRB1*11:01 and HLA-DQA1*05:05 are associated with more advanced disease represented by the CXR stages 2-4. HLA-DQB1*05:03 associated with sarcoidosis extrapulmonary manifestation. Conclusion: In our Czech cohort, we document some associations between sarcoidosis and HLA previously described in other populations. Further, we suggest novel susceptibility factors for sarcoidosis, such as HLA-DQB1*06:04, and characterize associations between HLA and sarcoidosis clinical phenotypes in Czech patients. Our study also extends the role of the 8.1 ancestral haplotype (HLA-A*01:01∼HLA-B*08:01∼HLA-C*07:01∼HLA-DRB1*03:01∼HLA-DQA1*05:01∼HLA-DQB1*02:01), already implicated in autoimmune diseases, as a possible predictor of better prognosis in sarcoidosis. The general translational application of our newly reported findings for personalized patient care should be validated by an independent study from another, international referral center.

Association of HLA alleles with Plasmodium falciparum severity in Malian children.

Pre-erythrocytic immunity to Plasmodium falciparum malaria is likely to be mediated by T-cell recognition of malaria epitopes presented on infected host cells via class I and II major histocompatibility complex (MHC) antigens. To test for associations of human leukocyte antigen (HLA) alleles with disease severity, we performed high-resolution typing of HLA class I and II loci and compared the distributions of alleles of HLA-A, -B, -C and -DRB1 loci in 359 Malian children of Dogon ethnicity with uncomplicated or severe malaria. We observed that alleles A*30:01 and A*33:01 had higher frequency in the group of patients with cerebral disease compared to patients with uncomplicated disease [A*30:01: gf = 0.2031 vs gf = 0.1064, odds ratio (OR) = 3.17, P = 0.004, confidence interval (CI) (1.94-5.19)] and [A*33:01: gf = 0.0781 vs gf = 0.0266, 4.21, P = 0.005, CI (1.89-9.84)], respectively. The A*30:01 and A*33:01 alleles share some sequence motifs and A*30:01 appears to have a unique peptide binding repertoire compared to other A*30 group alleles. Computer algorithms predicted malaria peptides with strong binding affinity for HLA-A*30:01 and HLA-A*33:01 but not to closely related alleles. In conclusion, we identified A*30:01 and A*33:01 as potential susceptibility factors for cerebral malaria, providing further evidence that polymorphism of MHC genes results in altered malaria susceptibility.

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q.

A gene involved in psoriasis susceptibility was localized to the distal region of human chromosome 17q as a result of a genome-wide linkage analysis with polymorphic microsatellites and eight multiply affected psoriasis kindreds. In the family which showed the strongest evidence for linkage, the recombination fraction between a psoriasis susceptibility locus and D17S784 was 0.04 with a maximum two-point lod score of 5.33. There was also evidence for genetic heterogeneity and although none of the linked families showed any association with HLA-Cw6, two unlinked families showed weak levels of association. This study demonstrates that in some families, psoriasis susceptibility is due to variation at a single major genetic locus other than the human lymphocyte antigen locus.

Exploring the ancestry and admixture of Mexican Oaxaca Mestizos from Southeast Mexico using next-generation sequencing of 11 HLA loci.

The Mestizos of Oaxaca resulted from the admixture of Zapotecan Natives with Spaniards and Africans. We selected 112 donors from Oaxaca and applied next-generation sequencing to characterize exon and intron variants in complete or extended HLA genes. Some alleles found, are unique to Mexican Natives and most likely will be absent in most major ethnicities, namely: Caucasians, Africans or Asians. Among these are HLA-A*68:03:01, HLA-A*68:05:01, HLA-C*03:04:01:02, HLA-C*15:09, HLA-C*3:05, HLA-C*03:06:01, HLA-B*39:05:01, HLA-B*35:14:01, HLA-B*35:12:01, HLA-B*35:43:01, HLA-B*40:05, HLA-B:40:08, HLA-B*51:02:01, HLA-B*35:24:01 and HLA-B*39:08. HLA-DQA1*05:05:01:05 and some HLA-DRB1 alleles were only present in Amerindians/Mestizos. Three haplotypes are unique to Mexican Natives, five to Middle-Eastern and Sephardi-Jews. We detected a novel HLA-DQA1*04:01:01 exon 4 variant. Any novel allele may have been positively selected to enlarge the peptide-binding repertoire, and some, like HLA-B*39:02:02 and HLA-B*39:05:01 were found with unique haplotype associations, suggesting convergent evolution events and/or allele lineage diversification. The allele frequencies were fairly evenly distributed in most HLA loci with the exception of HLA-DPB1. The application of NGS in Oaxaca is novel and will lead to better use in the clinical setting. It offers deep knowledge on the population structure, origins, migration, and discovery of new alleles and haplotypes that other techniques did not achieve.

Deciphering the fine nucleotide diversity of full HLA class I and class II genes in a well-documented population from sub-Saharan Africa.

With the aim to understand how next-generation sequencing (NGS) improves both our assessment of genetic variation within populations and our knowledge on HLA molecular evolution, we sequenced and analysed 8 HLA loci in a well-documented population from sub-Saharan Africa (Mandenka). The results of full-gene NGS-MiSeq sequencing compared with those obtained by traditional typing techniques or limited sequencing strategies showed that segregating sites located outside exon 2 are crucial to describe not only class I but also class II population diversity. A comprehensive analysis of exons 2, 3, 4 and 5 nucleotide diversity at the 8 HLA loci revealed remarkable differences among these gene regions, notably a greater variation concentrated in the antigen recognition sites of class I exons 3 and some class II exons 2, likely associated with their peptide-presentation function, a lower diversity of HLA-C exon 3, possibly related to its role as a KIR ligand, and a peculiar molecular diversity of HLA-A exon 2, revealing demographic signals. Based on full-length HLA sequences, we also propose that the most frequent DRB1 allele in the studied population, DRB1*13:04, emerged from an allelic conversion involving 3 potential alleles as donors and DRB1*11:02:01 as recipient. Finally, our analysis revealed a high occurrence of the DRB1*13:04-DQA1*05:05:01-DQB1*03:19 haplotype, possibly resulting from a selective sweep due to protection to Onchorcerca volvulus, a prevalent pathogen in West Africa. This study unveils highly relevant information on the molecular evolution of HLA genes in relation to their immune function, calling for similar analyses in other populations living in contrasting environments.

Development and characterization of desmoglein-3 specific T cells from patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is a cutaneous autoimmune disease characterized by blister formation in the suprabasilar layers of skin and mucosae and anti-desmoglein-3 (Dsg3) autoantibodies bound to the surface of lesional keratinocytes and circulating in the serum of patients. This disease can be reproduced in neonatal mice by passive transfer of patients' IgG, indicating that humoral immunity plays an important role in the pathogenesis of PV. Currently, the role of T lymphocytes in the development of PV is not clear. Here, we report that three immunoreactive segments of the ectodomain of Dsg3 specifically induced proliferation of T cells from PV patients. We found that T lymphocytes from 13 out of 14 patients responded to at least one of three Dsg3 peptides. T cells from controls and other patient groups did not respond to these Dsg3 peptides. The major T cell population stimulated by these Dsg3 peptides was CD4 positive. Dsg3-specific T cell lines and clones were developed and were shown to express a CD4 positive memory T cell phenotype. Upon stimulation, these cell lines and clones secreted a Th2-like cytokine profile. The Dsg3 responses of these T cells were restricted to HLA-DR, and not -DQ and -DP, of the major histocompatibility complex. This information will help to elucidate the cellular immune abnormalities leading to production of pathogenic IgG autoantibodies in patients with PV.

High-throughput next-generation sequencing to genotype six classical HLA loci from 96 donors in a single MiSeq run.

Next generation sequencing (NGS) methods have been established as an efficient approach for HLA typing because unlike traditional Sanger sequencing, they provide unambiguous results at a reasonable cost. We previously developed a multi-locus index method to genotype four HLA loci (A, B, C, and DRB1) on the Illumina MiSeq platform. We have now expanded this method to include two additional loci, HLA-DPB1 and DQB1. Contiguous full-length amplicons from 5'UTR through 3'UTR regions were generated using one long-range PCR reaction per locus for each of the six loci from 96 individuals of different ethnicities. The six amplicons from each donor were pooled, enzymatically fragmented and given a donor-specific index. This approach enabled sequencing of 576 loci from 96 individuals in a single MiSeq run. Donor-specific sequence reads were demultiplexed, and allele calls were generated from FASTQ files using commercially available software. Comparison to HLA genotypes generated from Sanger sequence-based typing (SBT) identified no discordances among any of the alleles analyzed in this study. Importantly, this method was able to resolve 22 DPB1 and 20 DQB1 alleles that were ambiguous with the SBT method. Furthermore, a novel allele in each of these two loci was identified, with the DQB1*05:01:24 allele having a frequency of greater than five percent. This method was subsequently validated against a blinded panel of 22 samples from the 17th International HLA and Immunogenetics Workshop. The flexibility of the method is further highlighted by successful genotyping of eight loci comprising all classical HLA loci for a subset of the samples. We now present a high-throughput, high-resolution, scalable NGS HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci.

Are changes in HLA Ags responsible for leukemia relapse after HLA-matched allogeneic hematopoietic SCT?

Loss of heterozygosity (LOH) has been shown to be associated with leukemia relapse after haploidentical transplantation. Whether such changes are an important cause of relapse after HLA-matched transplantation remains unclear. We retrospectively HLA-typed leukemic blasts for 71 patients with AML/myelodysplastic syndrome obtained from stored samples, and the results were compared with those obtained at diagnosis and/or before the transplant. No LOH or any other changes in HLA Ag were found in any of the samples tested post transplant as compared with pretransplant specimens. One patient had LOH in HLA class I Ag (HLA-A,-B and -C); however, these changes were present in the pretransplant sample indicating that they occurred before the transplant. We concluded that, in contrast with haploidentical transplantation, HLA loss does not have a major role as a mechanism of relapse after allogeneic transplantation with a closely HLA-matched donor.

Enzyme-linked DNA oligotyping. A practical method for clinical HLA-DNA typing.

The use of PCR and oligonucleotide hybridization has increased the accuracy and resolution of typing for HLA class II alleles, but current procedures, performed in batches, take too long and are not suited for testing single samples. We have developed a typing method using enzyme-linked oligonucleotides and PCR products immobilized in 96-well trays. Trays preloaded with typing probes, covalently linked with alkaline phosphatase, have been kept for weeks at 4 degrees C without loss of enzyme-probe activity. Bound alkaline phosphatase was detected using a color reaction with enzymatic amplification which produces readings in 30 minutes. Coupled with a quick DNA preparation method, results can be obtained in about 4 hours. This method can be easily performed in small laboratories. It is accurate, reproducible, and sensitive, and will make oligotyping for HLA alleles more convenient for testing clinical samples.

DNA typing for HLA class I alleles: I. Subsets of HLA-A2 and of -A28.

A group of HLA-A locus alleles known to be comprised of approximately 14 closely related variants are collectively called HLA-A2 and -A28. Variations among these alleles are given by differences in only a few codons, and in the case of A*6901, elements of A*6801 (exons 1 and 2) and of A*0201 are combined. The purpose of these experiments was to determine the possibility of designing oligonucleotide probes to identify and develop a typing method for all or most of the A2 and A28 variants. Because the regions of interest are also shared by alleles of other groups, allele-specific or group-specific primers were needed to amplify only the alleles under study. HLA-A2-specific amplification of exon 2 and selective amplification of portions of exon 3 of the A2-A28 group were accomplished with sequence-specific primers and after appropriate adjustments of the PCR conditions. Hybridization patterns using products of four PCR reactions with our set of probes distinguished 11 alleles. Two other alleles might be recognized with the reagents used, but were not found in the panels in this study. A*0201 and A*0209, which are different in exon 4, were not resolved because exon 4 was not tested. A new variant of Aw68, defined by a hybridization pattern obtained with our probes, was different from A*6801 only in that it was negative with probe A6. It was called A*68.3. Population studies were performed in North American whites, blacks, and Indians and in a sample of subjects from North China. HLA-A*0201 was the most frequent allele. A*0202 was found only in blacks, and A*0203 and A*0207 were found only in Chinese. Among the A28-positive subjects, Caucasoids were predominantly A*6801 or A*68.3; A*6802 was the most frequent subtype in American blacks; among American Indians the predominant type was A*68.3. The two A28-positive Chinese subjects studied had A*6901. The results obtained demonstrate that DNA typing is an efficient method for determining these alleles. The methodology should be applicable to other class I groups and should be useful for more extensive population studies, for matching for bone marrow transplantation, and for investigation of certain diseases associated with HLA class I alleles.

DQA1*03 subtypes have different associations with DRB1 and DQB1 alleles.

Polymorphisms outside the hypervariable regions of HLA class II alleles that do not affect the peptide-binding site are probably not under selective pressure and could therefore be useful as markers of the evolutionary pathways of the HLA class II haplotypes. We have analyzed such a polymorphism in the variants of DQA1*03, which differ at residue 160 encoded in exon 3. Our study included homozygous BCLs of the 10th IHWS and samples of a multiracial panel of 723 unrelated subjects which were also typed for allelic variations in exon 2 by hybridization with SSOP. BCLs having DQA1*03 and 131 selected DQA1*03-positive samples were typed for the dimorphism in exon 3 that distinguishes DQA1*0301 and DQA1*0302. DQA1*0301 was found to be exclusively associated with DQB1*0302, while samples carrying DQB1*0201, 0301, 0303, and 0401 always had DQA1*0302. A few haplotypes carrying DQB1*0302 had DQA1*0302. The fact that DQA1*0301 is completely included in DQB1*0302, and not vice versa, suggests that DQA1*0301 may have arisen from a mutation in a haplotype containing DQA1*0302-DQB1*0302. DQB1*0302 was found to be associated with all DR4 subtypes, suggesting possibly that the current variants of DRB1-DR4 may be of more recent origin. DRB1*0405 was the only subtype of DR4 which was not associated with DQA1*0301 and had multiple associations with the DQB1 alleles, therefore, perhaps representing the oldest allele of this group.

Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules.

The A*0201, A *0202, A*0203, A*0206, and A*6802 binding capacity of single amino acid substitution analogs of known A2-supertype binding peptides and of large nonredundant peptide libraries was measured. The results were utilized to rigorously define the peptide binding specificities of these A2-supertype molecules. Although each molecule was noted to have unique preferences, large overlaps in specificity were found. The presence of L, I, V, M, A, T, and Q residues in position 2, and L, I, V, M, A, and T residues at the C-terminus of peptide ligands were tolerated by all molecules. Likewise, whereas examination of secondary influences on peptide binding revealed allele specific preferences, shared features could also be identified. These shared features were utilized to define an A2-supermotif and were noted to correlate with crossreactivity. Over 70% of the peptides that bound A *0201 with high affinity were found to bind at least two other A2-supertype molecules. Because the A2-supertype molecules studied herein cover the variants most common in different major ethnicities, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.

Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations.

The HLA system is the most polymorphic of all human genetic systems. The frequency of HLA class I alleles and their linkage disequilibrium patterns differ significantly among human populations as shown in studies using serologic methods. Many DNA-defined alleles with identical serotypes may have variable frequencies in different populations. We typed HLA-A, B, and C loci at the allele level by PCR-based methods in 1,296 unrelated subjects from five major outbred groups living in the U.S.A (African, AFAM; Caucasians, CAU; Asian, ORI; Hispanic, HIS, and North American Natives, NAI). We detected 46, 100 and 32 HLA-A, B, and C alleles, respectively. ORI and HIS presented more alleles at each of these loci. There was lack of correlation between the levels of heterozygosity and the number of alleles detected in each population. In AFAM, heterozygosity (>90%) is maximized at all class I loci. HLA-A had the lowest heterozygosity in all populations but CAU. Tight LD was observed between HLA-B and C alleles. AFAM had weaker or nonexistent associations between alleles of HLA-A and B than other populations. Analysis of the genetic distances between these and other populations showed a close relationship between specific US populations and a population from their original continents. ORI exhibited the largest genetic distance with all the other U.S. groups and were closer to NAI. Evidence of admixture with CAU was observed for AFAM and HIS. HIS also had significant frequencies of AFAM and Mexican Indian alleles. Differences in both LD and heterozygosity levels suggest distinct evolutionary histories of the HLA loci in the geographical regions from where the U.S. populations originated.

Juvenile arthritis, HLA-A2 and binding of DEK oncogene-peptides.

Previous studies have shown that susceptibility to Pauciarticular Juvenile Arthritis is associated with HLA-A*0201. Recently, autoantibodies against the protein of the DEK oncogene have been found in sera of patients with this disease. If T cells are involved in the pathogenesis of joint lesions, it is possible that they target autoantigens presented by HLA-A*0201. Therefore, we investigated whether DEK-derived peptides can bind efficiently to HLA-A*0201. Nonameric peptides selected considering anchor positions 2 and 9, and preferred amino acids at other positions, were incubated either with the human TAP-deficient cell line 174CEM.T2 (T2) or with the homozygous B cell line JESTHOM (A*0201, B*2705, Cw1), previously depleted of endogenous peptides. Binding was measured as the increase of detection of fully assembled, HLA-A*0201 molecules by flow cytometry with the anti-HLA-A2 monoclonal antibody (mAb) BB7.2. Three out of ten selected DEK-derived peptides showed binding to HLA-A*0201, which was peptide concentration-dependent (1 microM to 100 microM). DEK155-163 (AMLKSICEV), which also has two preferred amino acid residues at positions 6 and 8, yielded the highest binding. DEK163-171 (VLDLERSGV) and DEK72-80 (SLQREPFTI), which also has one preferred amino acid residue at position 8, also were able to bind to HLA-A*0201. Furthermore, peptide-induced, fully assembled, HLA-A*0201 molecules were immunoprecipitated with the BB7.2 mAb from metabolically-labeled T2 cells incubated with DEK72-80, DEK155-163, and DEK163-171. A faint band was observed in the immunoprecipitates of cells incubated with DEK65-73 (it carries a preferred amino acid residue at position 6), suggesting that this peptide interacts weakly with HLA-A*0201. These results indicate that several nonameric peptides derived from the DEK protein can bind to HLA-A 0201 and suggest that the complexes formed may be able to stimulate CD8+ T cells in patients with Pauciarticular Juvenile Arthritis.

New alleles of the HLA-B15 family.

In a previous study of B locus alleles by sequence-specific oligonucleotide probe (SSOP) hybridization, we observed 18 novel patterns in a panel of 360 individuals. Four of these novel patterns were caused by alleles of the human leukocyte antigen (HLA)-B15 group, and three were available for this study. These alleles were found in Oriental, Latin American, African American, and Caucasian individuals. In addition, we analyzed a Caucasian subject who was found by serology to have an unusual B15 specificity. We sequenced these four samples by performing amplification from genomic DNA using polymerase chain reaction primers designed to obtain HLA class I products that included exon 2 and exon 3 as well as the intervening intron. The amplified segments were cloned and identified by colony hybridization with nonradioactive SSOP. Nucleotide sequences were obtained using an automated DNA sequencer. The allele B*1530 differs from B*1501 by a substitution of Asp for Asn in position 114 and Ser for Tyr in codon 116. The new allele B*1531 differs from B*1502 at amino acids 94, 95, and 152. The variant B*1524 was found to have N-77, I-80, A-81, L-82, R-83. A similar motif exists in B locus alleles that have the supertypic specificity Bw4 and in B*1513, B*1516, B*1517, and B*1523; it is likely to have been generated by gene conversion. Finally, the novel allele B*1527 is similar to B*1501 except for the presence of Phe instead of Tyr at position 99. Because this change exists also in B*1506, it is possible that B*1506 was derived from B*1501 through B*1527. It is of interest that a similar substitution (Cys for Tyr at position 99) distinguishes A*0201 from A*0207 and is known to determine an epitope recognized by T cells. Thus, B*1527 may also carry a change that is functionally relevant in cell-mediated immunity.

Novel HLA-B35 subtypes: putative gene conversion events with donor sequences from alleles common in native Americans (HLA-B*4002 or B*4801).

In a study of 523 normal subjects of differing ethnic groups, including 189 South American Indians, we have described novel hybridization pattern corresponding to 22 potentially new HLA-B locus alleles. Three of these alleles were subtypes of B35. The locally, assigned alleles, B-3504v, B-3505v, and B-3508v have been sequenced and were officially designated as B*3512, B*3517, and B*3518, respectively. In addition, we determined the nucleotide sequence of another new variant, locally designated B-3509.2. B*3517, was found in 3 individuals (2 Hispanic, 1 Caucasian), it differs from B*3505 by 3 nucleotide substitutions that lead to changes in residues 94, 95, and 103. B*3517 differs from B*3501 in residues 97 and 103. B*3518 was found in 7 South American Indian individuals (6 of 124 Toba Indians, 1 of 18 Pilaga Indians). It differs from B*3509 by 2 silent nucleotide substitutions and by one nonsynonymous substitution in codon 156 (Arg-->Leu). B*3512 differs from B*3504 by 3 nucleotides, one of them leading to a substitution in residue 103 (Val-->Leu). B*3509 was observed in 3 individuals from the Wichi tribe. The nucleotide sequence of one of these was determined and was found to differ from B*35091 by two synonymous nucleotide substitutions. The distinguishing amino acid substitutions in residues 95, 97, and 156 contribute to the structure of specificity pockets F, C, and E, and D and E respectively, therefore, it is possible that some of the new alleles may have different peptide binding profiles. It has been shown that differences at residue 156 may elicit different allorecognition and mediate graft-versus-host disease and rejection in bone marrow transplantation. The mechanisms for the generation of these novel alleles may involve gene conversion events in which short exon-3 segments from the common Native American alleles B*4002 or B*4801 were inserted in HLA-B35 backbone structures. The novel allele B*3518 is closely related to B*35092 and to B*3508. Two alternative hypotheses for its generation can be suggested, the most plausible one would involve B*35092, the putative progenitor of B*3518, since both alleles are prevalent in the same Indian tribes.

Evolution of HLA-class I compared to HLA-class II polymorphism in Terena, a South-American Indian tribe.

We have studied the HLA alleles of 60 unrelated healthy Terena and 10 Terena families. They are members of an isolated Brazilian tribe located in Mato Grosso do Sul (South Central Brazil). Six novel alleles were found in this population: HLA-A*0219 (gf = 0.02), A*0222 (gf = 0.15), HLA-B* 3520 (gf = 0.01), B*3521 (gf = 0.03), B*3912 (gf = 0.03) and B*4803 (gf = 0.16). Five of the six novel alleles differ from their putative progenitors by amino acid replacements in residues that contribute to the pockets of the peptide-binding site. Many of the variants defined by molecular methods were not identified correctly by serological typing. We calculated heterozygosity values (H) for HLA-A, -B, -C, DRB1, DQB1 and DPB . The highest values were observed at the HLA-B locus, followed by HLA-A, -DRB1 and DQB1. Residue positions 9, 24, 45, 62, 67, 95, 114, 116, 156, and 163 of HLA class I showed heterozygosity values greater than 0.50. Nine of them contribute to the peptide-binding specificity pockets and one to the T cell receptor binding site. If HLA antigens are useful for defense against pathogenic agents, heterozygosity would offer an advantage by allowing binding of a larger repertoire of peptides to the class I molecules. Individuals that are heterozygous at these positions would probably have a wider repertoire of peptide presentation to T cells. The observed results including the presence of novel alleles in the class I HLA loci suggest a functionally significant, more rapid evolution of class I compared to class II loci in this South American isolated population.

Strong linkage disequilibrium between HLA-B*3913 and DRB1*0807 in Brazilians.

The purpose of this research was to study the HLA-B39 distribution in 2560 healthy, unrelated, randomly selected individuals living in the southeastern region of Brazil (the states of Rio de Janeiro and São Paulo). Molecular methods were used to type HLA class I and II polymorphism: PCR-SSP, PCR-SSO, and PCR-SBT. HLA-B*39 was found in 7% (n = 182) of these individuals. HLA-B*3901, B*3906, and B*3913 were the most common alleles in this group (n = 57, 36, and 24, respectively). B*3913 was found associated with DRB1*0807 and DQB1*0402 in 16 of the 24 individuals and 13 of these were also associated with A*31012. This haplotype segregation was confirmed by family studies. Furthermore, in 5 of the 13 individuals carrying the A*31012, B*3913, DRB1*0807, and DQB1*0402 haplotype, HLA-DPB1*2701 was also present, suggesting that these alleles were found preferentially in cis association. DRB1-DPB1 linkage disequilibrium analysis was performed in 420 of the 2560 individuals and the association of DRB1*0807 with the uncommon DPB1*2701 was found to be highly significant (P <.0001). Because HLA-B*3913 and HLA-DRB1*0807 have been observed only in South American populations, it is possible that interlocus association has been selected to act on the same haplotype to collaborate in the class I and II restricted immune response to local pathogens and functional adaptation. Although numbers are small to predict which ethnic groups of the Brazilian population display this haplotype prevalently, it is possible to speculate that these data may have clinical application, such as in the selection of unrelated donors for bone marrow transplantation.

Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.