Genome-wide minor histocompatibility matching as related to the risk of graft-versus-host disease.

The risk of acute graft-versus-host disease (GVHD) is higher after allogeneic hematopoietic cell transplantation (HCT) from unrelated donors as compared with related donors. This difference has been explained by increased recipient mismatching for major histocompatibility antigens or minor histocompatibility antigens. In the current study, we used genome-wide arrays to enumerate single nucleotide polymorphisms (SNPs) that produce graft-versus-host (GVH) amino acid coding differences between recipients and donors. We then tested the hypothesis that higher degrees of genome-wide recipient GVH mismatching correlate with higher risks of GVHD after allogeneic HCT. In HLA-genotypically matched sibling recipients, the average recipient mismatching of coding SNPs was 9.35%. Each 1% increase in genome-wide recipient mismatching was associated with an estimated 20% increase in the hazard of grades III-IV GVHD (hazard ratio [HR], 1.20; 95% confidence interval [CI], 1.05-1.37; P = .007) and an estimated 22% increase in the hazard of stage 2-4 acute gut GVHD (HR, 1.22; 95% CI, 1.02-1.45; P = .03). In HLA-A, B, C, DRB1, DQA1, DQB1, DPA1, DPB1-phenotypically matched unrelated recipients, the average recipient mismatching of coding SNPs was 17.3%. The estimated risks of GVHD-related outcomes in HLA-phenotypically matched unrelated recipients were low, relative to the large difference in genome-wide mismatching between the 2 groups. In contrast, the risks of GVHD-related outcomes were higher in HLA-DP GVH-mismatched unrelated recipients than in HLA-matched sibling recipients. Taken together, these results suggest that the increased GVHD risk after unrelated HCT is predominantly an effect of HLA-mismatching.

Predicting multiallelic genes using unphased and flanking single nucleotide polymorphisms.

Recent advances in genotyping technologies have enabled genomewide association studies (GWAS) of many complex traits including autoimmune disease, infectious disease, cancer and heart disease. To facilitate interpretations and establish biological basis, it could be advantageous to identify alleles of functional genes, beyond just single nucleotide polymorphisms (SNPs) within or nearby genes. Leslie et al. ([2008] Am J Hum Genet 82:48­56) have proposed an Identity-by-Decent method (IBD-based) for predicting human leukocyte antigen (HLA) alleles (multiallelic and highly polymorphic) with SNP data, and predictions have achieved a satisfactory accuracy on the order of 97%. Building upon their success, we introduce a complementary method for predicting highly polymorphic alleles using unphased SNP data as the training data set. Due to its generality and flexibility, the new method is readily applicable to large population studies. Applying it to HLA genes in a cohort of 630 healthy individuals as a training set, we constructed predictive models for HLA-A, B, C, DRB1 and DQB1. Then, we performed a validation study with another cohort of 630 healthy individuals, and the predictive models achieved predictive accuracies for HLA alleles defined at intermediate or high resolution ranging as high as (100%, 97%) for HLA-A, (98%, 96%) for B, (98%, 98%) for C, (97%, 96%) for DRB1 and (98%, 95%) for DQB1, respectively. These preliminary results suggest the feasibility of predicting other polymorphic genetic alleles, since HLA loci are almost certainly among most polymorphic genes.

Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

Human T cell receptor occurrence patterns encode immune history, genetic background, and receptor specificity.

The T cell receptor (TCR) repertoire encodes immune exposure history through the dynamic formation of immunological memory. Statistical analysis of repertoire sequencing data has the potential to decode disease associations from large cohorts with measured phenotypes. However, the repertoire perturbation induced by a given immunological challenge is conditioned on genetic background via major histocompatibility complex (MHC) polymorphism. We explore associations between MHC alleles, immune exposures, and shared TCRs in a large human cohort. Using a previously published repertoire sequencing dataset augmented with high-resolution MHC genotyping, our analysis reveals rich structure: striking imprints of common pathogens, clusters of co-occurring TCRs that may represent markers of shared immune exposures, and substantial variations in TCR-MHC association strength across MHC loci. Guided by atomic contacts in solved TCR:peptide-MHC structures, we identify sequence covariation between TCR and MHC. These insights and our analysis framework lay the groundwork for further explorations into TCR diversity.

Common and well-documented HLA alleles: report of the Ad-Hoc committee of the american society for histocompatiblity and immunogenetics.

In histocompatibility testing some genotype ambiguities are almost always resolved into the genotype with the most common alleles. To achieve unambiguous assignments additional unwieldy tests are performed. The American Society for Histocompatibility and Immunogenetics formed a committee to define what human leukocyte antigen (HLA) genotypes do not need to be resolved in external proficiency testing. The tasks included detailed analysis of large datasets of high-resolution typing and thorough review of the pertinent scientific literature. Strict criteria were used to create a catalogue of common and well-documented (CWD) alleles. In total, 130, 245, 81, and 143 of the highly polymorphic HLA-A, -B, -C, and DRB1 loci fell into the CWD category; these represent 27%-30% of all alleles recognized. For the loci DRB3/4/5, DQA1, DQB1, and DPB1, a total of 29, 16, 26, and 52 CWD alleles were identified. A recommendation indicated that an acceptable report should only include one possible genotype; multiple genotypes can only be reported if only one of these includes two alleles of the CWD group. Exceptions in which resolution is not necessary are ambiguities involving functional alleles with identical sequences in the antigen recognition site. The criteria were established for proficiency testing, which could be a valuable tool when making clinical histocompatibility decisions.

DQB1*0301 and DQB1*0601 modulate narcolepsy susceptibility in Koreans.

The association of narcolepsy with HLA-DQB1*0602 is established in Japanese, African-Americans, European, and North American Caucasians. We examined DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 in 163 patients with centrally mediated daytime sleepiness (100 with narcolepsy) and 211 Korean controls. In this population, the DQB1*0602 association was always evident in the context of the DRB1*1501-DQA1*0102-DQB1*0602 haplotype. The DQB1*0602 association was highest in cases with hypocretin deficiency (100% vs 13% in controls), most of which had narcolepsy-cataplexy (81%). A weaker DQB1*0602 (45%) association was present in cases without cataplexy. No human leukocyte antigen (HLA) association was present in idiopathic hypersomnia or in cases with normal cerebrospinal fluid (CSF) hypocretin-1. As in other populations, DQB1*0602 homozygosity increased risk in cases with cataplexy and/or hypocretin deficiency (odds ratio = 2.0 vs heterozygotes). Non-DQB1*0602 allelic effects were also observed but could not be interpreted in the context of DQB1*0602 overabundance and linkage disequilibrium. We therefore next analyzed compound heterozygote effects in 77 subjects with either hypocretin deficiency or cataplexy and one copy of DRB1*1501-DQA1*0102-DQB1*0602, a sample constructed to maximize etiologic homogeneity. In this analysis, we found additional predisposing effects of DQB1*0301 and protective effects for DQA1*0103-DQB1*0601. Unexpectedly, the predisposing effects of DQB1*0301 were present in the context of various DQA1-bearing haplotypes. A predisposing effect of DQA1*0303 was also suggested. These results indicate a remarkable consistency in the complex HLA association present in narcolepsy across multiple ethnic groups.

An integrated genotyping approach for HLA and other complex genetic systems.

Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1-7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1-4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.

Molecular characterization of the HLA-Cw*0409N allele.

Various molecular methods in conjunction with serologic assays are used for clinical human leukocyte antigen (HLA) typing. Although serologic reagents detect most HLA-A and -B allospecificities, serologic HLA-C typing is hampered by the lack of informative antisera for many of the known HLA-C gene products. HLA antigens not detected by serology, but detected by molecular methods, are often referred to as "blank" antigens. Their lack of reactivity with antibodies in serological assays often reflects the presence of null alleles. The present study has characterized an HLA-Cw*04 allele (Cw*0409N) detected by DNA typing but not by serology. In cultured B-lymphoid 13W09501 cells carrying this Cw*04 null allele, isoelectric focusing analysis could not detect any component with a pattern compatible with that of the product of the HLA-Cw*0401 allele, but detected components reacting with an anti-HLA-Cw4 and Cw6 monoclonal antibody. Sequencing analysis of the full length HLA-Cw4 cDNA amplified from the cell line 13W095-01 revealed a base deletion at codon 365 in exon 7, resulting in a reading frame shift that added 32 amino acids at the C-terminal of the HLA-Cw4 heavy chain. These results indicate that the HLA-Cw*0409N allele may produce a putative long HLA-Cw4 heavy chain that is not expressed on the cell surface.

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors.

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.

Prospective monitoring for alloimmunization in cord blood transplantation: "virtual crossmatch" can be used to demonstrate donor-directed antibodies.

Preformed host antibodies may contribute to graft rejection after hematopoietic stem-cell transplantation. In cord blood transplantation (CBT), donor-directed host antibodies may be particularly relevant because patients are often markedly mismatched to donors, and limited donor cells preclude cross-matching. The recent development of single human leukocyte antigen (HLA) microbead array assays allows characterization of host alloreactivity to individual HLA antigens with sufficient sensitivity and specificity to allow consideration of "virtual crossmatch" testing as a surrogate for conventional crossmatch testing in the CBT setting. We report results of prospective monitoring for alloimmunization in our recent CBT experience. Among 46 consecutive patients, four patients (9%) (5 of 88 units [6%]) had evidence of at least moderate antibodies to HLA antigens on cord units originally selected for transplantation. Virtual crossmatch can be used to screen for donor-directed antibodies in CBT. As possible, units should be changed to avoid sensitized mismatches.

Comprehensive analysis of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci and squamous cell cervical cancer risk.

Variation in human major histocompatibility genes may influence the risk of squamous cell cervical cancer (SCC) by altering the efficiency of the T-cell-mediated immune response to human papillomavirus (HPV) antigens. We used high-resolution methods to genotype human leukocyte antigen (HLA) class I (A, B, and Cw) and class II (DRB1 and DQB1) loci in 544 women with SCC and 542 controls. Recognizing that HLA molecules are codominantly expressed, we focused on co-occurring alleles. Among 137 allele combinations present at >5% in the case or control groups, 36 were significantly associated with SCC risk. All but one of the 30 combinations that increased risk included DQB1*0301, and 23 included subsets of A*0201-B*4402-Cw*0501-DRB1*0401-DQB1*0301. Another combination, B*4402-DRB1*1101-DQB1*0301, conferred a strong risk of SCC (odds ratio, 10.0; 95% confidence interval, 3.0-33.3). Among the six combinations that conferred a decreased risk of SCC, four included Cw*0701 or DQB1*02. Most multilocus results were similar for SCC that contained HPV16; a notable exception was A*0101-B*0801-Cw*0701-DRB1*0301-DQB1*0201 and its subsets, which were associated with HPV16-positive SCC (odds ratio, 0.5; 95% confidence interval, 0.3-0.9). The main multilocus associations were replicated in studies of cervical adenocarcinoma and vulvar cancer. These data confirm that T helper and cytotoxic T-cell responses are both important cofactors with HPV in cervical cancer etiology and indicate that co-occurring HLA alleles across loci seem to be more important than individual alleles. Thus, certain co-occurring alleles may be markers of disease risk that have clinical value as biomarkers for targeted screening or development of new therapies.

Comparison of variable number tandem repeat and short tandem repeat genetic markers for qualitative and quantitative chimerism analysis post allogeneic stem cell transplantation.

BACKGROUND: Analysis of donor chimerism has become a routine procedure for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation. Quantitative analysis of chimerism kinetics has been shown to predict graft failure or relapse. In this study, we compared the use of variable number tandem repeats (VNTR) and short tandem repeats (STR) as polymorphic genetic markers in chimerism analysis. This study included qualitative and quantitative assessment of both techniques to assess informative yield and sensitivity. PATIENTS AND METHODS: We analyzed 206 samples representing 40 transplant recipients and their HLA-identical sibling donors. A panel of six VNTR loci, 15 STR loci and 1 sex chromosome locus was used. Amplified VNTR products were visualized in an ethidium bromide-stained gel. STR loci were amplified using fluorescent primers, and the products were analyzed by capillary electrophoresis. RESULTS: VNTR and STR analysis gave comparable qualitative results in the majority of cases. The incidence of mixed chimerism (MC) by STR analysis was 45% compared to 32% in cases evaluated by VNTR analysis. STR markers were more informative; several informative loci could be identified in all patients. Unique alleles for both patient and donor could be identified in all patients by STR versus 32/40 by VNTR analysis. The STR markers were also more sensitive in the detection of chimerism. The size of VNTR alleles and differences between the size of donor and recipient VNTR alleles affected the sensitivity of detection. With both techniques, quantitative assessment of chimerism showed some discrepancies between the estimated and the calculated percentage of donor DNA. Discordance between the two estimates was observed in 8/19 patients with MC. However, sequential monitoring of the relative band intensity of VNTR alleles offered some insight into the direction of change in engraftment over time. CONCLUSION: The higher yield of informative loci with STR and the automated measurement of amplified STR 103 products offered the advantages of more rapid and accurate quantitative assessment of chimerism. The choice between these two techniques depends on the need for quantitative or qualitative information, the availability of equipment, and the cost.

Limits of HLA mismatching in unrelated hematopoietic cell transplantation.

HLA matching between the donor and recipient improves the success of unrelated hematopoietic cell transplantation (HCT). Matched donors are available for only a minority of patients. Further information is needed to evaluate the limits of HLA mismatching. We examined the association of mortality with HLA-A, -B, -C, -DRB1, and -DQB1 mismatching in 948 patients who received a T-replete unrelated HCT for treatment of a marrow disorder. A single HLA allele or antigen mismatch was associated with increased mortality among patients with chronic myeloid leukemia (CML) within 2 years after diagnosis compared to patients with no HLA mismatch, but not among those with more advanced malignancy. In particular, a single HLA-C mismatch conferred increased risk of mortality compared to matches. There was a suggestion for increased mortality with multiple mismatches involving HLA-DQB1 compared to multiple mismatches not involving HLA-DQB1. Donors with a single HLA allele or antigen mismatch may be used for HCT when a fully matched donor is not available for patients with diseases that do not permit time for a lengthy search. Whenever possible, HLA-C mismatches should be avoided for patients with early stage CML, and HLA-DQB1 mismatches should be avoided for patients with multiple mismatches.

Human leukocyte antigen class II and cervical cancer risk: a population-based study.

The critical role of the human leukocyte antigen (HLA) system in presenting peptides to antigen-specific T cell receptors may explain why only some human papillomavirus (HPV)-infected women progress to cervical cancer. HLA class II DRB1 and DQB1 genes were examined in 315 women with invasive squamous cell cervical cancer (SCC) and 381 control subjects. Increased risks of SCC were associated with DRB1*1001, DRB1*1101, and DQB1*0301, and decreased risks were associated with DRB1*0301 and DRB1*13. Of squamous cell tumors, those containing HPV-16 were different from those not containing HPV-16 for 3 alleles: DRB1*0401, DRB1*07, and DQB1*06. Increased risks of SCC were associated with DRB1*0401-DQB1*0301 (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.1-2.7) and DRB1*1101-DQB1*0301 (OR, 2.5; 95% CI, 1.4-4.5), and decreased risks were associated with DRB1*0301-DQB1*02 (OR, 0.7; 95% CI, 0.5-1.0) and DRB1*13-DQB1*06 (OR, 0.6; 95% CI, 0.4-0.9) haplotypes. These results add to the evidence that certain HLA class II alleles or allele combinations, or genes linked to them, make some women more susceptible to SCC.

Molecular analysis of HLA antigens and alleles in the unusual association of idiopathic myastenia gravis, seropositive rheumatoid arthritis, d-penicillamine induced myastenia gravis anf Graves' disease / Análise molecular dos antígenos e alelos HLA na rara associaçäo de miastenia gravis idiopática, artrite reumatóide soropositiva, miastenia gravis induzida por d-penicilamina e doença de Graves

Um paciente apresentando complexa associaçäo de miastenia gravis idiopática, artrite reumatóide, miastenia gravis induzida por penicilamina e doença de Graves foi analisado com relaçäo aos alelos de histocompatibilidade. Alelos HLA de classe I e II foram determinados por microlinfocitotoxicidade. Os alelos DRB1, DRB5, DOB1 e DOA1 foram determinados por análise de DNA amplicado por reaçäo da polimerase em cadeia por meio de soondas de nucleotídeos sequência-específicas. O paciente apresentou os seguintes alelos: HLA-A1/24, B8/35, Cw4/w-, DR2/3, DQ1/2, DRB1*1601/*0301, DRB5*02, DQB1*0502/*0201, and DQA1*0102/0501. Interessantememtne, este paciente apresentava um alelo associado à doença de Graves e miastenia gravis (DQB1*0201), um alelo associado à miastenia gravis idiopática (DR3) e um alelo associado à miastenia gravis induzida por droga (B35), mas nenhum alelo associado a artrite reumatóide. Entretanto, encontramos que o alelo DQB1*0201 codifica uma molécula contendo o motivo RKRAA em regiäo similar à do epítopo compartilhado da artrite reumatóide QKRAA e QRRAA. Este achado reforça a importância do epítopo compartilhado e enfatiza o papel das moléculas de HLA-DQ na suscetibilidade e patogênese da artrite reumatóide