HLA Typing Method Research Articles

132-P: DEVELOPMENT OF AN HLA TYPING METHOD FOR THE ION TORRENT PGM NEXT GENERATION SEQUENCING PLATFORM

The Ion Torrent PGM™ next generation sequencing platform allows for the simultaneous sequencing of up to 96 barcoded samples in a single two to four hour run, providing a method to process a large number of gene sequences in significantly less time than traditional sequencing. We are developing an HLA typing method based on the technology that enables read lengths of 400 bases on the PGM™ platform. Two different library preparation workflows have been evaluated. The first approach is based on long-range PCR (LR-PCR) followed by fragmentation and ligation of emulsion PCR adaptor sequences. This method allows coverage of an entire HLA gene with a single long-range PCR reaction for each sample. The basis for the second workflow is a tiled, multiplex, short-range PCR design. The amplicons in this design range in length from 350 - 400 base pairs and cover alternating sections of a gene in two primer pools. This approach offers a workflow that does not include the fragmentation and size selection steps required in the LR-PCR method, saving at least four hours of processing time while producing results equivalent to the LR-PCR workflow. Here we describe the development of a tiling assay that enables full coverage of the HLA-C gene. Approximately 100 primer sets were screened in designing the amplicons. Thirty samples have been sequenced using each method to compare coverage of region, read depth, accuracy, cis/trans ambiguity resolution, and ease of workflow. The current design is comprised of two PCR tubes, each with thirty-five primers, used to generate eighteen tiling amplicons. Each primer pool is designed to amplify every HLA-C gene with known genomic sequence. While the LR-PCR method has simplified primer designs, the short-range PCR approach enables a simplified workflow with equivalent genomic sequence results. The assay designs to date show the feasibility of the PGM™ platform for accurate, high throughput HLA gene sequencing. Bialozynski: Life Technologies: Employee. Dinauer: Life Technologies: Employee. Gifford: Life Technologies: Employee. Zhao: Life Technologies: Employee. Shi: Life Technologies: Employee. Radick: Life Technologies: Employee. Conradson: Life Technologies: Employee.

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Aim After transition to buccal swabs (SWs) for Be The Match Registry® recruitment activities in 2006, a study was initiated to evaluate the long-term stability of three sample types: frozen whole blood, whole blood spotted on filter paper (FP), and SW. Samples were analyzed for quality and quantity of DNA and accuracy of HLA typing. Data obtained from the first 5 years of the study are presented. Methods Fresh blood and SWs were collected from 30 volunteers with confirmed HLA typing. Fresh blood was stored as 1ml frozen aliquots and 75uL aliquots on FP. SWs and FP were stored at room temperature under controlled conditions. Samples were sent to 2 labs for testing at 6 time points (TP) immediately post collection and at one year storage intervals. Lab 1 performed high resolution HLA-A, B, C, DRB1 and DQB1 via SBT. Lab 2 performed intermediate resolution HLA-A, B, C and DRB1 via SSO and evaluated the quantity/quality of DNA within each sample. Results SSO and SBT HLA results were accurate at all loci through the 4 year TP. At 5 year TP, SSO results were 100% accurate, whereas SBT results were 99.4% accurate for FP and 97.3% for SWs. One FP and 4 SWs had amp failure, 5 SWs had allele drop-out and 1 SW had both. 19 FP and SW samples required repeat testing at multiple loci to obtain accurate HLA results. Sufficient DNA for HLA testing was extracted from all samples at all time points. DNA quality from frozen whole blood remained high thru year 5. DNA quality consistently decreased for the FP starting at the 3 year TP, 1 year TP for SWs. Conclusions Room temp storage of SWs and FP allows for DNA degradation, creating HLA testing inaccuracies and inefficiencies. Specifically, shorter amplicons were required to achieve accurate SBT results, though failures still persisted. DNA degradation did not affect SSO typing. Buccal swabs are a cost effective and efficient mechanism for registry DNA sample collection and storage, but shelf-life at room temperature is limited for Sanger SBT HLA typing method.

153-P: HIGH-RESOLUTION TYPING BY NEXT-GENERATION SEQUENCING IN A CLINICAL HLA LABORATORY

The aim of this study is to test the feasibility and compare the analytical performance of HLA typing of clinical and proficiency testing samples using next-generation sequencing against standard molecular HLA typing methods in a medium-sized clinical HLA laboratory without prior sequencing experience. We amplified HLA-A, -B, -C, -DRB1 and –DQB1 genes, from clinical samples, by long-range PCR using primers developed at our laboratory. Long-range PCR product identity was verified by Luminex SSO. HLA gene products were pooled and processed to prepare a sequencing library using the Nextera XT sample kit followed by sequencing on the Miseq instrument. Data was analyzed using the Omixon Target HLA software to assign HLA types. HLA assignment by next-generation sequencing was compared against results previously obtained by standard molecular typing methods. We report the concordance of HLA typing results obtained by next-generation sequencing and analysis compared with standard molecular typing methodologies. Importantly, the reagent cost of performing high-resolution typing by next-generation sequencing is similar to molecular methods currently used. Next generation sequencing can be a viable approach to high resolution HLA typing in medium-sized clinical HLA laboratories. Berces: Omixon: Employee; Stockholder. Major: Omixon: Employee. Hague: Omixon: Employee.

138-P: HLA TYPING AMBIGUITY OF SEQUENCE-SPECIFIC OLIGONUCLEOTIDE TYPING PROTOCOLS

Using Shannon’s entropy, we objectively measured the HLA typing ambiguity of various commercial Sequence-Specific Oligonucleotide (SSO) protocols (groups of SSO kits used in tandem). We previously compared the HLA typing ambiguity obtained by serology, allele family level DNA-based typing, SSO, and single-pass sequence based typing (SBT). However, commercial companies have made significant advances in SSO technology in recent years, and those results only evaluated early generic versions of SSO kits. For several US populations, and for 5 HLA loci relevant to transplantation (HLA-A, -B, -C, -DRB1, -DQB1), we identify a set of commonly used SSO protocols. We use population-level haplotype frequency data to generate a cohort of simulated subjects, followed by 5-locus genotype imputation to generate a list of genotypes that would result from SSO typing each simulated subject. The imputation step computes the relative likelihood of each genotype, which is then used for entropy calculation. Distribution of entropies across populations and for each locus is generally similar, while the magnitudes differ. For example, entropy for locus HLA-A in African American population reaches 0.19, while the maximum entropy in Caucasians is 0.055. We generally observed that protocols comprised of newer kits had lower ambiguity across all populations. In certain populations, some newer kits had higher entropy than older ones. This may indicate those kits were designed to detect alleles uncommonly found in those populations, but resolving ambiguity in other populations. Ambiguity in SSO typing kits has reduced substantially since their introduction in the mid-1990s. Results also indicate a European focus toward selection of additional probes, which is suboptimal for typing diverse populations. We hope to use Shannon’s entropy next to evaluate group-specific sequence primer strategies with SBT. These results will guide selection of HLA typing methods by researchers and laboratories.

Flow Cytometry Analysis with a New FITC-Conjugated Monoclonal Antibody-3E12 for HLA-B*57:01 Rapid Screening in Prevention of Abacavir Hypersensitivity in HIV-1–Infected Patients

Background: Rapid screening for the detection of HLA-B*57:01 in the prevention of abacavir hypersensitivity in HIV-1–infected patients is a hallmark for clinical services. Objective: The aim of this work was to analyze the utility of flow cytometry with a new FITC-conjugated B-17 monoclonal antibody (mAb3E12) for HLA-B*57:01 screening in a Spanish cohort of 577 HIV-1+ individuals. Methods: Cryopreserved peripheral blood mononuclear cell samples from HIV-1+ individuals were analyzed by flow cytometry with the mAb 3E12 that recognizes both HLA-B*57 and HLA-B*58 alleles (members of the group specificity, HLA-B17). Patients’ DNA samples had been previously typed for HLA-B*57:01 with PCR-SSO or PCR-SSP and additional DNA sequencing (EPI Study). The results obtained by flow cytometry were compared with the results obtained by the DNA-PCR techniques. Results: By flow cytometry, 46 samples (7.97%) were positive for HLA-B17, 530 (91.86%) were negative, and 1 (0.17%) was undetermined. All samples found negative by flow cytometry were negative for HLA-B*57:01 by DNA-PCR. Of the HLA-B17 positive samples, 31 (67.4%) were positive for HLA-B*57:01, 2 (3.25%) were positive for HLA-B*57:03, 11 (26.1%) were positive for HLA-B*58, and 2 (3.25%) were negative for both HLA-B*57 and HLA–B*58 antigens. The undetermined sample was negative for HLA-B*57 and HLA-B*58 alleles by DNA-PCR. Conclusions: This study shows that flow cytometry with mAb3E12 is a highly sensitive method (no false negatives) to implement prior to DNA-PCR analysis for rapid screening of HLA-B*57:01. Additional confirmation by molecular HLA typing method would be required in less than 10% of the cohort of HIV-1–infected individuals.

HLA Haplotyping from RNA-seq Data Using Hierarchical Read Weighting

Correctly matching the HLA haplotypes of donor and recipient is essential to the success of allogenic hematopoietic stem cell transplantation. Current HLA typing methods rely on targeted testing of recognized antigens or sequences. Despite advances in Next Generation Sequencing, general high throughput transcriptome sequencing is currently underutilized for HLA haplotyping due to the central difficulty in aligning sequences within this highly variable region. Here we present the method, HLAforest, that can accurately predict HLA haplotype by hierarchically weighting reads and using an iterative, greedy, top down pruning technique. HLAforest correctly predicts >99% of allele group level (2 digit) haplotypes and 93% of peptide-level (4 digit) haplotypes of the most diverse HLA genes in simulations with read lengths and error rates modeling currently available sequencing technology. The method is very robust to sequencing error and can predict 99% of allele-group level haplotypes with substitution rates as high as 8.8%. When applied to data generated from a trio of cell lines, HLAforest corroborated PCR-based HLA haplotyping methods and accurately predicted 16/18 (89%) major class I genes for a daughter–father-mother trio at the peptide level. Major class II genes were predicted with 100% concordance between the daughter–father-mother trio. In fifty HapMap samples with paired end reads just 37 nucleotides long, HLAforest predicted 96.5% of allele group level HLA haplotypes correctly and 83% of peptide level haplotypes correctly. In sixteen RNAseq samples with limited coverage across HLA genes, HLAforest predicted 97.7% of allele group level haplotypes and 85% of peptide level haplotypes correctly.

Definition of High-Risk Type 1 Diabetes HLA-DR and HLA-DQ Types Using Only Three Single Nucleotide Polymorphisms

Evaluating risk of developing type 1 diabetes (T1D) depends on determining an individual’s HLA type, especially of the HLA DRB1 and DQB1 alleles. Individuals positive for HLA-DRB1*03 (DR3) or HLA-DRB1*04 (DR4) with DQB1*03:02 (DQ8) have the highest risk of developing T1D. Currently, HLA typing methods are relatively expensive and time consuming. We sought to determine the minimum number of single nucleotide polymorphisms (SNPs) that could rapidly define the HLA-DR types relevant to T1D, namely, DR3/4, DR3/3, DR4/4, DR3/X, DR4/X, and DRX/X (where X is neither DR3 nor DR4), and could distinguish the highest-risk DR4 type (DR4-DQ8) as well as the non-T1D–associated DR4-DQB1*03:01 type. We analyzed 19,035 SNPs of 10,579 subjects (7,405 from a discovery set and 3,174 from a validation set) from the Type 1 Diabetes Genetics Consortium and developed a novel machine learning method to select as few as three SNPs that could define the HLA-DR and HLA-DQ types accurately. The overall accuracy was 99.3%, area under curve was 0.997, true-positive rates were >0.99, and false-positive rates were <0.001. We confirmed the reliability of these SNPs by 10-fold cross-validation. Our approach predicts HLA-DR/DQ types relevant to T1D more accurately than existing methods and is rapid and cost-effective.

Incorporation of a sequence-based HLA typing method into an immunology undergraduate laboratory course. (P4527)

Abstract Antibody-based laboratory techniques (e.g. Western blots and ELISA) are relatively easy to implement in an immunology course and widely applicable throughout the subfields of molecular biology. Incorporating lab activities with relevance to T cell immunology is more challenging because they often require proficiency in cell culture and sterile technique. HLA typing facilitates exploration of T cell immunology, including HLA presentation and the development of a T cell response. However, clinical HLA typing kits are costly. Numerous PCR amplifications require high yields of genomic DNA, entailing the challenges of working with blood as a source for DNA in the classroom. Also, typing requires precise gel loading of many samples, which can be challenging. We transformed a sequence-based HLA typing methodology recently developed for research use with plasma samples into a protocol for an immunology course lab. Since the methodology works with the low levels of DNA recoverable from plasma, the technique is well suited for DNA extraction from buccal swabs. The HLA typing process required the following labs to complete: (1) DNA extraction and quantitation (2) primary PCR set-up (3) optional verification of amplification success and secondary PCR set-up (4) verification of nested PCR amplification and sample preparation for sequencing. Additional time was required for editing sequence data and interpreting HLA types using dbMHC.

A new approach to HLA typing designed for solid organ transplantation: epityping and its application to the HLA-A locus

HLA-specific antibodies bind discrete clusters of amino acids called epitopes, but serological assignment of antibody specificities makes no reference to this. As HLA typing for solid organ transplantation is provided at only medium (serologically equivalent) resolution, this means that recipient HLA antibodies to donor HLA epitopes may not be identified. We have designed a novel and rapid HLA-A epitope typing method (epityping) using a two-stage PCR-SSP-based method to detect the HLA-A locus epitopes described by El Awar et al. 2007, Transplantation, 84, 532. The initial PCR step utilizes HLA-A locus-specific primers; the product is cleaned using the QIAquick Spin Purification procedure. The purified product is tested using our in-house epitope-specific primer panel, the results being visualized using gel electrophoresis. Twenty two UCLA DNA Exchange samples were epityped, blinded to the HLA type. Of the 75 primer pairs, the mean correlation coefficient was 0.95 with each sample giving 67 or more correct primer results. In all cases, it was possible to derive the first field classic HLA type from the epityping results. These results indicate that a method for identification of HLA epitopes which is comparable in time, cost and technical expertise to current HLA typing methods is achievable. Redesigning HLA typing to correlate with what the antibody binds should minimize inappropriate organ allocation. We suggest that epityping provides a more effective method than standard HLA typing for solid organ transplantation.

A glow of HLA typing in organ transplantation.

The transplant of organs and tissues is one of the greatest curative achievements of this century. In organ transplantation, the adaptive immunity is considered the main response exerted to the transplanted tissue, since the main goal of the immune response is the MHC (major histocompatibility complex) molecules expressed on the surface of donor cells. Cell surface molecules that induce an antigenic stimulus cause the rejection immune response to grafted tissue or organ. A wide variety of transplantation antigens have been described, including the major histocompatibility molecules, minor histocompatibility antigens, ABO blood group antigens and endothelial cell antigens. The sensitization to MHC antigens may be caused by transfusions, pregnancy, or failed previous grafts leading to development of anti-human leukocyte antigen (HLA) antibodies that are important factor responsible for graft rejection in solid organ transplantation and play a role in post-transfusion complication Anti-HLA Abs may be present in healthy individuals. Methods for HLA typing are described, including serological methods, molecular techniques of sequence-specific priming (SSP), sequence-specific oligonucleotide probing (SSOP), Sequence based typing (SBT) and reference strand-based conformation analysis (RSCA) method. Problems with organ transplantation are reservoir of organs and immune suppressive treatments that used to decrease rate of rejection with less side effect and complications.

Transplantation From 8/8-Matched or 7/8-Matched Unrelated Donors Compared with HLA-Matched Siblings in Acute Myeloid Leukemia in First Complete Remission

AbstractAbstract 2040▪▪This icon denotes a clinically relevant abstractComparable survival after 8/8-matched unrelated donor (URD) transplantation with HLA-matched siblings (MSD) has been reported in AML from a few multicenter studies. However, the role of 8/8-matched URD in acute myeloid leukemia (AML) with first complete remission (CR1) is uncertain because they applied various methods for HLA typing, such as low or intermediate resolution or partly high resolution (HR) typing, and/or included various disease status at transplantation. Additionally, there have been few studies comparing the clinical outcomes of 7/8-matched URD with current standard donors, such as MSD or 8/8-matched URD, particularly in AML, as a single disease.According to the risk-adapted treatment strategy, AML CR1 with intermediate or adverse cytogenetics received allogenetic transplantation as a post-remission treatment, if MSD or 8/8-matched URD was available. Patients with no available donor received 7/8-matched URD or autologous transplantation (only intermediate cytogenetics). Among 567 consecutive adult patients with AML who underwent transplantation between January 2002 and December 2009, in order to investigate the role of URD type in AML CR1, we assessed the transplantation outcomes of 8/8-matched URD (n=59) or 7/8-matched URD (n=36) classified by HR HLA typing, compared to MSD (n=165). Only intermediate or adverse cytogenetics was included, and we defined high-risk group as having one of the poor risk features, including adverse cytogenetics, hyperleukocytosis at diagnosis (over 100×109/L), older age over 60 years, or AML with myelodysplasia-related changes.In all, multivariate analyses showed that 8/8-matched URD had comparable 5-year disease-free survival (DFS; HR, 0.94; P=0.803), relapse (HR, 1.03; P=0.935), and non-relapse mortality (NRM; HR, 0.62; P=0.294) to MSD, while 7/8-matched URD had higher relapse (HR, 1.95; P=0.034) and a lower trend for DFS (HR, 1.47; P=0.154) than MSD without the difference in NRM (HR, 0.61; P=0.390). The equivalent outcomes of 8/8-matched URD were consistent irrespective of risk groups (high or standard). Particularly, only in standard-risk group, 7/8-matched URD (n=18) failed to show any benefit compared to concurrently performed autologous transplantation (n=67), contrasting to the superior survival of 8/8-matched URD (n=32) as well as MSD (n=108).In conclusion, the equivalent outcomes between MSD and 8/8-matched URD, irrespective of risk-group in AML CR1, suggest that 8/8-matched URD is useful for standard-risk as well as high-risk group of AML CR1, when lacking of MSD. On the other hand, the inferior outcome of 7/8-matched URD raise the necessity of trials comparing with other alternative graft sources, such as umbilical cord bloods or haploidentical family donors, particularly in AML with high-risk group. Disclosures:No relevant conflicts of interest to declare.

139-P

Aim The HLA-B ∗ 37:02 allele was first described in 1995 in a Syrian father and one of his sons during routine HLA typing. It has no assigned serologic equivalent and a mixed B27 and B37 serologic reactivity pattern has been suggested. In this report we characterize the serological reactivity and possible haplotypes association observed with this allele. Methods Of 73,355 subjects HLA typed in our laboratory, 644 were positive for B ∗ 37 with only 2 positive for B ∗ 37:02. One subject is an unrelated bone marrow donor and the other is a subject from the international cell exchange. The serological reactivity of both cells and probable haplotypes were analyzed. Results The serological reactivity were similar for both cells with BW4 positivity. Both cells reacted positively to polyspecific sera with B ∗ 37, B ∗ 27, and B ∗ 47 reactivity. Interestingly, neither of the cells reacted to operationally monospecific sera with B27, B37, or B47 reactivity or to polyspecific sera with B27 and B37 reactivity. No polyspecific sera with B37 and B47 reactivity were available for testing. A population genetics approach based on reported haplotypes frequencies suggest that the most probable haplotypes for both cells are A ∗ 24:02 B ∗ 18:01 C ∗ 05:01 DRB1 ∗ 03:01 & A ∗ 01:01 B ∗ 37:02 C ∗ 02:02 DRB1 ∗ 11:04 for the first cell and A ∗ 29:02 B ∗ 14:02 C ∗ 0202 DRB1 ∗ 07:01 & A ∗ 30 B ∗ 37:02 C ∗ 02:02 DRB1 ∗ 13 for the second cell. The former haplotype is similar to that of the first described B ∗ 37:02 allele. Conclusions This analysis emphasizes the value of serologic HLA typing methods in characterizing newly described and uncommon HLA alleles.

Measuring Ambiguity in HLA Typing Methods

In hematopoietic stem cell transplantation, donor selection is based primarily on matching donor and patient HLA genes. These genes are highly polymorphic and their typing can result in exact allele assignment at each gene (the resolution at which patients and donors are matched), but it can also result in a set of ambiguous assignments, depending on the typing methodology used. To facilitate rapid identification of matched donors, registries employ statistical algorithms to infer HLA alleles from ambiguous genotypes. Linkage disequilibrium information encapsulated in haplotype frequencies is used to facilitate prediction of the most likely haplotype assignment. An HLA typing with less ambiguity produces fewer high-probability haplotypes and a more reliable prediction. We estimated ambiguity for several HLA typing methods across four continental populations using an information theory-based measure, Shannon's entropy. We used allele and haplotype frequencies to calculate entropy for different sets of 1,000 subjects with simulated HLA typing. Using allele frequencies we calculated an average entropy in Caucasians of 1.65 for serology, 1.06 for allele family level, 0.49 for a 2002-era SSO kit, and 0.076 for single-pass SBT. When using haplotype frequencies in entropy calculations, we found average entropies of 0.72 for serology, 0.73 for allele family level, 0.05 for SSO, and 0.002 for single-pass SBT. Application of haplotype frequencies further reduces HLA typing ambiguity. We also estimated expected confirmatory typing mismatch rates for simulated subjects. In a hypothetical registry with all donors typed using the same method, the entropy values based on haplotype frequencies correspond to confirmatory typing mismatch rates of 1.31% for SSO versus only 0.08% for SBT. Intermediate-resolution single-pass SBT contains the least ambiguity of the methods we evaluated and therefore the most certainty in allele prediction. The presented measure objectively evaluates HLA typing methods and can help define acceptable HLA typing for donor recruitment.

Evaluation of DRB1 high resolution typing by a new SSO-based Luminex method

HLA testing is an essential part of the process to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow, peripheral blood or cord blood and the DNA typing in high resolution is now recommended as the Scientific Societies also describe in their standards. Recently the new PCR-Luminex HLA typing method, based on the reverse sequence specific oligonucleotide probes coupled with a microsphere beads in an array platform, has been well established. We report the data from 146 samples previously typed to a four digits level and used to evaluate the accuracy, sensitivity and performance of the new high definition DRB1 by PCR-Luminex kit. One hundred and forty-six samples from unrelated healthy donors, haematological patients or external proficiency tests were used in this study. The Luminex high definition DRB1 typing represents a versatile method and may be easily introduced in the routine, particularly when the technical team has already acquired experience on the technique. Only few HLA allelic combinations need an additional typing by PCR-SSP or SBT to solve the ambiguous results thus reducing the time necessary to produce a final report.

HLA*IMP—an integrated framework for imputing classical HLA alleles from SNP genotypes

Genetic variation at classical HLA alleles influences many phenotypes, including susceptibility to autoimmune disease, resistance to pathogens and the risk of adverse drug reactions. However, classical HLA typing methods are often prohibitively expensive for large-scale studies. We previously described a method for imputing classical alleles from linked SNP genotype data. Here, we present a modification of the original algorithm implemented in a freely available software suite that combines local data preparation and QC with probabilistic imputation through a remote server. We introduce two modifications to the original algorithm. First, we present a novel SNP selection function that leads to pronounced increases (up by 40% in some scenarios) in call rate. Second, we develop a parallelized model building algorithm that allows us to process a reference set of over 2500 individuals. In a validation experiment, we show that our framework produces highly accurate HLA type imputations at class I and class II loci for independent datasets: at call rates of 95-99%, imputation accuracy is between 92% and 98% at the four-digit level and over 97% at the two-digit level. We demonstrate utility of the method through analysis of a genome-wide association study for psoriasis where there is a known classical HLA risk allele (HLA-C*06:02). We show that the imputed allele shows stronger association with disease than any single SNP within the region. The imputation framework, HLA*IMP, provides a powerful tool for dissecting the architecture of genetic risk within the HLA. HLA*IMP, implemented in C++ and Perl, is available from http://oxfordhla.well.ox.ac.uk and is free for academic use.

A new PCR-SSP method for HLA DR-DQ risk assessment for celiac disease

Background Susceptibility to celiac disease is essentially restricted to carriers of specific HLA DQA1 and DQB1 alleles. We have developed a semi-automated sequence specific primer (SSP) PCR method for clinical HLA typing and compared the test results with those from a commercial method. Methods Primers for each DQA1 and DQB1 allele group were included in our PCR-SSP reaction to allow differentiation of homozygous from heterozygous carriers of risk alleles. Primers detecting the tightly linked DRB1*04, *03, *07 and *09 alleles were included to resolve potentially ambiguous results. Fluorescently labeled PCR products of 119 clinical samples were analyzed by capillary electrophoresis, and results were compared to those previously obtained from the DELFIA® Type 1 Diabetes Genetic Predisposition assay. Results The risk assessment derived from the two methods was 100% concordant. One previously unreported haplotype was detected and haplotype assignments in two of the 119 samples were improved from previous reports. Conclusions The use of three PCR reactions and a single electrophoretic step for DQA1, DQB1 and DRB1 typing provides distinction of celiac disease associated alleles and their homo- or heterozygous status. This multiplex analysis reduces reagent costs, personnel and instrument time, while enabling improved allelic assignment through HLA-DR-DQ haplotype association.

Shedding a new light on the HLA matching

Shedding a new light on the HLA matching

Is HLA Matching By SBT A Necessity For BMT?

Hematopoietic stem cell transplantation (HSCT) outcome has improved dramatically since the introduction of allele level HLA typing. Currently, HLA laboratories are using three different technologies for allele level matching, PCR amplification with sequence-specific primers (PCR-SSP), polymerase chain reaction-sequence-specific oligonucleotide probes (SSOP), and sequence-based typing (SBT). However, it is important to emphasize that SBT is the only HLA typing method so far that allows scrutiny of every nucleotide in the HLA gene sequence, therefore, it allows us to recognize new alleles and potential gene mutations that would ensure allele level typing to be as good as claimed.

Mismatch for the Minor Histocompatibility Antigen HA-2 and GVHD Occurrence in HLA-A*0201-positive Tunisian Recipients of HSCs

Graft-versus-Host disease (GVHD) has been widely linked to immunogenetic causes such as disparity between the recipient and its HLA geno-identical donor for some Non-HLA antigens called minor histocompatibility antigens (MiHAgs). HA-2 is one of potential human MiHAgs but its effect on the GVHD occurrence remains not clear. In order to examine such association in the Tunisian cohort of HSCs recipients, we performed a retrospective study on patients who received an HLA-identical HSCT between 2000 and 2009. The study was performed on 60 HLA-A2-positive patients who had received a haematopoietic stem cell transplant from an HLA-identical sibling. All patients received cyclosporine A and/or methotrexate for GVHD prophylaxis. HA-2 genotyping assay was performed with SSP-PCR method and HLA-A*0201 positive samples were identified mainly with Luminex HLA-Typing method. Luminex HLA-Typing assay showed that only 53 cases were positives for the HLA-A*0201 allele. Among these cases, only 3 pairs were mismatched for the MiHAg HA-2. Acute GVHD occurred in 01 HA-2-mismatched pair while chronic GVHD was detected in 02 disparate couples. Univariate and multivariate analyses showed that MiHAg HA-2 disparity does not have any significant effect on the occurrence of either acute or chronic GVHD. This last one appeared to be correlated only with the age of patient (adulthood) (p: 0.011, OR: 22.092). Our findings support the previously reported data denying the influence of the HA-2 disparity on the GVHD occurrence after HSCT.

Detection of a rare Caucasoid HLA-DRB1*0337 in a Taiwanese bone marrow donor using sequence-based typing method

We here describe the identification of HLA-DRB1*0337, using sequence-based typing (SBT) method, in a Taiwanese bone marrow donor intrigued by a casual curiosity on the donor's racial background. On high-resolution sequence-specific primer (SSP) typing we observed misleading reaction patterns due to similarity of DNA sequences in the exon 2 of DRB1*0301, *0317, *0337 and *1139. When encountering rare alleles in HLA typing, it is important to pay extra attentions to avoid pitfalls and shortcomings of SSP typing kits routinely used and bear in mind that constant up-dating of high-resolution SSP typing kits to be able to distinguish newly reported alleles. SBT may be considered as a back-up HLA typing method to confirm rare alleles.