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.

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.

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.

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 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.

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