Routine HLA Typing Research Articles

Characterisation of the novel HLA-B*15:699 and HLA-C*03:677 alleles identified in Danish individuals.

Two novel HLA alleles HLA-B*15:699 and HLA-C*03:677 were detected during routine HLA typing by next generation sequencing.

Identification of the novel HLA-C*14:159 allele by next-generation sequencing.

The novel HLA-C*14:159 allele was detected during the routine HLA typing for kidney transplantation.

Identification of the novel HLA-C*01:65:02 allele by next-generation sequencing.

The novel HLA-C*01:65:02 allele was detected during routine HLA typing.

Identification of the novel HLA-C*06:02:01:94 and -C*15:02:01:58 alleles in individuals from Madhya Pradesh.

Novel HLA-C*06:02:01:94 and -C*15:02:01:58 alleles were detected during the routine HLA typing process.

Identification of the HLA-B*15:665 and HLA-C*12:02:02:23 alleles in individuals from the South Asia.

The novel HLA-B*15:665 and HLA-C*12:02:02:23 alleles were detected during the routine HLA typing process.

Detection and characterization of the novel HLA-DPA1*02:66:02N allele, with a premature stop codon in exon 2.

The failure to identify HLA null alleles in bone marrow transplantation could be life-threatening because this could result in an HLA mismatch with the ability to trigger the graft-vs-host disease (GVHD) and to reduce patient's survival. In this report we describe the identification and characterization of the novel HLA-DPA1*02:66:02N allele with a non-sense codon in exon 2. This new allele was discovered in two unrelated bone marrow donors during routine HLA-typing using next-generation sequencing (NGS). DPA1*02:66:02N is homologous to DPA1*02:01:01:03 with a single nucleotide difference in exon 2, codon 50, where the replacement of C located at genomic position 3825 by T, causes the formation of a premature stop codon (TGA), resulting in a null allele. This description illustrates the benefits of HLA typing by NGS since it permits to reduce ambiguities, identify new alleles, analyze multiple HLA loci and improve transplantation outcome.

Identification of the HLA-B*40:01:02:59 and -C*05:01:73 alleles in individuals from the northern regions of India.

The novel HLA-B*40:01:02:59 and HLA-C*05:01:73 alleles were detected during the routine HLA typing process.

Identification of the novel HLA-A*01:01:01:111 allele in two individuals from Uttar Pradesh, India.

The novel HLA-A*01:01:01:111 allele was detected during routine HLA typing.

Two novel HLA class I alleles, HLA-C*04:489 and -C*07:01:115, were identified by next-generation sequencing.

The novel HLA-C*04:489 and -C*07:01:115 alleles were detected during routine HLA typing by next-generation sequencing.

Identification of the novel HLA-DQA1*01:01:01:11 and -DQA1*01:03:01:13 alleles in individuals from India.

The novel alleles HLA-DQA1*01:01:01:11 and -DQA1*01:03:01:13 were detected during the routine HLA typing process.

Identification of the novel HLA-DPA1*01:03:01:64 allele in an individual from Uttar Pradesh, India.

The novel HLA-DPA1*01:03:01:64 allele was detected during routine HLA typing.

Identification of the novel HLA-C*12:02:47 allele in an individual from Maharashtra, India.

The novel HLA-C*12:02:47 allele was detected during routine HLA typing.

Identification of the novel HLA-DQB1*06:01:01:04 allele in an individual from Western India.

The novel HLA-DQB1*06:01:01:04 allele was detected during routine HLA typing.

Identification of the novel HLA-B*51:01:01:108 allele and confirmation of HLA-B*40:387 in two Indian individuals.

The HLA-B*51:01:01:108 and HLA-B*40:387 alleles were detected during routine HLA typing.

The novel HLA-B*07:461 allele and confirmation of the HLA-C*15:193 allele in individuals from Eastern India.

A novel HLA-B*07:461 and an extended HLA-C*15:193 allele were detected during routine HLA typing process.

Four novel HLA class II alleles identified using next-generation sequencing.

We describe here four novel HLA class II alleles, DPA1*01:33, DPB1*1082:01, DPB1*1144:01, and DQA1*01:45, identified in our laboratory following the implementation of next generation sequencing (NGS) for routine high resolution HLA typing of donors and recipients for solid organ transplant and hematopoetic stem cell transplant. The NGS technology has improved our ability to match at all clinically HLA loci and to facilitate the interpretation of allele specific antibody and eplet matching.

Immunogenetic characteristics of potential donors of hemapoietic stem cells recruited in the North Caucasus

Aim of the study was to investigate the immunogenetic characteristics of the potential donors of hematopoietic stem cells recruited in the North Caucasus and the distribution features of HLA alleles and multilocus haplotypes. Material and methods . Next Generation Sequencing technology was used to identify HLA-A, -B, -C, -DRB1 and -DQB1 alleles from 2663 unrelated bone marrow volunteers living in the Chechen Republic, Stavropol region, Republic of Dagestan. Mass parallel sequencing was performed using the MiSeq™ System («Illumina Inc.», USA). HLA allele and haplotype frequencies were estimated via maximum-likelihood analysis from genotypic data through an expectationmaximization (EM) algorithm for unknown gametic phase using Arlequin v. 3.5.2.2. Results and discussion . In studied population, 47 HLA-A, 77 HLA-B, 39 HLA-C, 54 HLA-DRB1, 22 HLA-DQB1 alleles were selected. Thirteenth alleles, HLA-A*02:01, HLA-A*01:01, HLA-B*13:02, HLA-B*51:01, HLA-C*06:02, HLA-C*04:01, HLA-C*07:02, HLADRB1*07:01, HLA-DRB1*13:01, HLA-DQB1*03:01, HLA-DQB1*02:02, HLA-DQB1*06:03, DQB1*03:02 exhibit frequencies over 10 %. The highest frequency extended haplotype HLA-A*02:01-B*13:02-C*06:02-DRB1*07:01DQB1*02:02, was observed frequencies of 4,5 %. Routine HLA typing allowed us to define 13 new HLA alleles.

Detection of novel and confirmation of very rare and rare HLA alleles by next generation sequencing in Croatia.

Routine HLA typing in clinical practice encompassing solid organ and hematopoietic stem cells transplantation programs, disease association typing, volunteer marrow donor typing and population studies, provided a large dataset for studying HLA allele polymorphism in the Croatian population which led to the identification of new, very rare and rare HLA alleles. Over the last 4 years we have identified six new HLA alleles (HLA-A*01:200, A*02:836, A*11:01:01:44, B*08:251, B*18:169 and C*05:46:01:02) and a number of very rare (HLA-B*08:78, DRB1*12:39, DRB1*13:23:02 and DQB1*06:09:04) or rare (HLA-A*24:41, B*39:40:01N, B*51:78:01, DRB1*01:31 and DRB1*14:111) alleles using sequence-based typing methods. The reported data enhance the knowledge about HLA polymorphisms in the Croatian population and provide a foundation for further studies in population genetics.

P097 An HLA typing assay using the illumina NGS system allows for the detection of poorly amplified HLA alleles, virtually eliminating allele dropout

Aim DNA sequencing has long been the gold standard for HLA typing, offering the ability to interrogate each base pair of a targeted region. Sanger sequencing-based typing assays are known to suffer from allele dropout, whereby one of two encoded alleles is less represented in the results, due to poor amplification. Next-generation sequencing (NGS) includes several distinct technologies, defined by their ability to sequence clonal DNA fragments in a high-throughput manner. One such technology, provided by Illumina, sequences thousands of short DNA fragments, with read depth orders of magnitude higher than Sanger sequencing, which may allow for the identification of poorly-amplified alleles. Here we sought to determine the rate of allele dropout in an Illumina-based HLA typing assay. Methods NGSgo (GenDx) was used to produce libraries for the HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 loci for sequencing on a MiSeq (Illumina). NGSengine (GenDx) was used to analyze the data and type the loci. 85 unique samples were run in parallel by a combination of previously-validated assays (SBT, SSP, rSSOP) and by NGS. Results The NGS assay resulted in very high read depths (>100 for our clinical results) and a very low error rate. Use of NGSengine software allowed detection of both alleles at heterozygous loci when the alleles were unevenly represented (imbalanced). In cases of extreme allele imbalance, data visualization by NGSengine allowed detection of the minor allele during manual review. Of the 811 patient loci tested by the previously-validated assays, 584 were heterozygous, 108 were homozygous and 119 were absent; the NGS results were 100% concordant. Conclusions In our experience with a clinical NGS typing assay, we found the Illumina sequencing technology was able to successfully identify all alleles present in tested samples, including poorly-amplified alleles. While the possibility of rare allele dropout still exists with this technology, due to alleles with novel polymorphisms at a primer-binding site, we found that with an appropriate combination of NGS library construction, sequencing technology, and analysis software, allele dropout is a low frequency, to non-existent, event. This reduces the need for extra testing to confirm homozygous loci during routine clinical HLA typing.

P166 Validation of a next generation sequencing based HLA typing system

Aim Very high resolution typing of HLA genes by Next Generation Sequencing (NGS) methods is becoming mainstream in histocompatibility laboratories for the workup of haematopoietic stem cell and solid organ transplant patients and donors. Determining the full sequence for class I and extended exon coverage for class II genes produces HLA types with far fewer ambiguities than traditional Sanger sequencing based typing (SBT) and other lower resolution typing methods. In addition NGS brings benefits of higher throughput and lower cost. Methods We have validated the Illumina TruSight HLAv2 typing kit for HLA-A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1 and DPB1. A total of 151 samples were tested following the method described by the vendor. All method steps were carried out manually. Testing comprised 6 batches of 24 samples and 1 batch of 12 samples, which were made up of 73 local DNA samples typed previously by a combination of SBT, SSP and SSO, 54 DNA samples from the UCLA reference panel and 24 samples from the 17th International HLA Workshop Study blinded proficiency panel. Reference types for all eleven loci were not available for all local samples, four digit types were available for all eleven loci for the UCLA samples. Sequence analysis and genotype calling was performed using Illumina Assign for Trusight HLA v2.1 and GenDX NGSEngine. HLA type concordance rate, ambiguity rate and accuracy were examined. Results Total percent concordance between the reference type (n = 130) and genotype determined by Assign was as follows: A: 100; B: 99.2; C: 99.2; DRB1: 97.6.5; DRB345: 98.4; DQA1: 99.2; DQB1: 100; DPA1: 100; and DPB1: 97.6. No allele (1st, 2nd and 3rd field) or genotype ambiguity was found for HLA-A, B, C, DRB345 DQB1 or DPA1. An ambiguous type was determined in 4 cases for DRB1, 3 cases for DQA1 and 29 cases for DPB1. In the case of DRB1 and DPB1 this was due to lack of part of the gene sequence and for DQA1 it was due to poor read depth in those samples. Average read depth across all loci was 223. Repeat rates varied between 1.3% (A & B) and 10.9% (DPA1). Conclusions The Illumina TruSight HLAv2 typing kit provides a suitable first pass high resolution typing system for stem cell and solid organ transplant patients and donors. Accuracy is excellent and the ambiguity levels are very low. We intend to introduce this method for routine HLA typing.