HLA Allele Sequences Research Articles

Confirmation and extension of 18 HLA alleles identified in donors from the Russian bone marrow registry.

Eighteen HLA allele sequences were confirmed and extended: 3 HLA-A, 6 HLA-B, 3 HLA-C, 2 HLA-DRB1, and 4 HLA-DQB1 alleles.

Different eplet software programs give discordant and incorrect results: An analysis of HLAMatchmaker vs Fusion Matchmaker Eplet calling software.

HLA eplet matching is a novel approach to define acceptable HLA mismatches for transplant recipients. We performed an eplet analysis of three different transplant case-series to determine if the available software programs gave accurate results. Eplet analysis was performed for three different transplant case-series typed by NGS for all HLA class I and II loci. The three different HLA datasets were entered into both the HLAMatchmaker program (v2.1) and OLI Fusion MatchMaker (v4.2) software tools. Eplet results which were discordant were cross referenced against eplet registry and published HLA allele sequence data to determine the correct assignments. The comparison reveals that there was poor concordance between the two eplet programs. Analysis of the same donor/recipient pair often gave rise to different total eplet scores, incorrect eplet mismatches and antibody verification status, and both programs have eplets assigned to incorrect HLA alleles. Overall, the OLI Fusion MatchMaker eplet tool gave more accurate and useful eplet results. Eplet matching is still primarily a research tool. Before eplet matching can enter routine clinical practice further work is required to validate the accuracy of available eplet software programs. Incorrect eplet assignment could have serious adverse consequences in the clinical transplant setting.

TypeLoader2: Automated submission of novel HLA and killer-cell immunoglobulin-like receptor alleles in full length.

The Immuno Polymorphism Database (IPD) databases provide global, curated repositories for information regarding polymorphisms of genes of the immune system, thereby generating immense value for the research and clinical communities. The advent of high‐throughput genotyping in immunogenetics has led to dramatically growing numbers of heretofore unknown HLA and lately also killer‐cell immunoglobulin‐like receptor (KIR) alleles, which are to be curated and deposited in the IPD‐IMGT/HLA and IPD‐KIR databases, respectively. It is highly desirable that these novel alleles are characterised and submitted in full length, and that known alleles are extended to cover the complete gene sequence.However, the manual annotation and submission of sequences to European Molecular Biology Laboratory's European Nucleotide Archive and the IPD‐IMGT/HLA and IPD‐KIR databases is time‐consuming and error‐prone. Here, we report the substantial extension of the HLA allele submission tool TypeLoader, which now also supports the annotation and submission of KIR alleles. To enable a more widespread use of this tool, we have made it available as a stand‐alone application that can easily be installed on standard Windows or Linux computers. Furthermore, an internal SQLite database was added to store a wide range of metadata about each allele. This allows TypeLoader2 to be used as a lab's central information platform for the annotation, curation and submission of full‐length HLA and KIR allele sequences. The software is freely available from GitHub (https://github.com/DKMS-LSL/typeloader).We hope that the increased convenience and scope of TypeLoader2 will foster the submission of more full‐length sequences to the IPD‐IMGT/HLA and IPD‐KIR databases, ultimately promoting the use of full‐length sequencing for genotyping both HLA and KIR.

GRIMM: GRaph IMputation and matching for HLA genotypes.

For over 10 years allele-level HLA matching for bone marrow registries has been performed in a probabilistic context. HLA typing technologies provide ambiguous results in that they could not distinguish among all known HLA alleles equences; therefore registries have implemented matching algorithms that provide lists of donor and cord blood units ordered in terms of the likelihood of allele-level matching at specific HLA loci. With the growth of registry sizes, current match algorithm implementations are unable to provide match results in real time. We present here a novel computationally-efficient open source implementation of an HLA imputation and match algorithm using a graph database platform. Using graph traversal, the matching algorithm runtime is practically not affected by registry size. This implementation generates results that agree with consensus output on a publicly-available match algorithm cross-validation dataset. The Python, Perl and Neo4j code is available at https://github.com/nmdp-bioinformatics/grimm. Supplementary data are available at Bioinformatics online.

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

P071 Limited exon sequence mismatches outside the antigen recognition domain in a cohort of 4646 high resolution 10/10 HLA-matched donor and recipient

Aim In hematopoietic stem cell transplantation (HCT), HLA allele matching between donor and recipient has traditionally focused on the polymorphic antigen recognition domain (ARD). Mismatching at the ARD is known to influence outcomes. This study investigated the genetic differences in the non-ARD regions among ARD-matched donor-recipient pairs to determine possible association with the transplant outcomes. Methods We compared the full-length HLA Class I allele sequences (HLA-A, -B, -C) and partial-length (partial intron 1 through partial intron 3) Class II allele sequences (HLA-DRB1, DQB1) for 4646 high-resolution 10/10 HLA-matched HCT donor-recipient pairs. We developed a comprehensive HLA allele sequence comparison pipeline, which identifies and annotates the mismatched positions between two alleles by their functional region and their protein sequence differences using IMGT/HLA Database (v3.31.0). Results For HLA Class I alleles, 95.4% of the ARD matched alleles have identical sequences outside the ARD, including introns and non-ARD exons. 0.3% of the mismatches were synonymous variants from the ARD region while 0.2% and 0.1% of mismatches found from non-ARD exons were synonymous and nonsynonymous variants, respectively. The intronic variation accounted for 4.2% of the mismatches. Similarly, for HLA Class II alleles, 0.3% of mismatches were synonymous ARD variants, and the mismatches in the non-ARD exons were also very rare (synonymous: 0.3%; nonsynonymous: 0.2%). However, a high degree of polymorphism was observed in the intronic regions of the Class II genes with only 77.3% of the allele pairs having identical sequences. 0.2% and 4.6% of Class I and Class II allele pairs, respectively, showed both exonic and intronic mismatches. Conclusions HCT donor/recipient pairs matched at high resolution for HLA-A, B, C, DRB1 and DQB1 have limited coding variation outside of the ARD. Intronic variation was observed at a higher rate for HLA Class II in our study compared to prior publications (17.3% vs.

Saddlebags: A software interface for submitting full-length HLA allele sequences to the EMBL-ENA nucleotide database.

Submission of full-length HLA allele sequences presents a unique challenge, both for high-throughput sequencing laboratories and smaller diagnostic laboratories. HLA's extensive polymorphism means that accurate representation and annotation of allele sequence is of critical importance, and curators of nucleotide databases must establish submission formats to ensure high-quality data and prevent ambiguities. The IPD-IMGT/HLA database is established as the standard repository for HLA sequences, and it is a major goal of the 17th International HLA and Immunogenetics Workshop to fill the IPD-IMGT/HLA database with full-length HLA sequences. The process of preparing sequence annotation and metadata is cumbersome and error prone, and it is desirable to create a straightforward and concise method of preparing sequence submissions. We introduce Saddlebags, a software tool for rapid generation of HLA (novel) full-length allele sequence submissions. HLA allele sequences are submitted first to EMBL European Nucleotide Archive (EMBL-ENA), and metadata is gathered for subsequent preparation of an IPD-IMGT/HLA formatted submission. Combining these steps into a pipeline reduces effort and minimizes errors for submitting laboratories. This software has been used by Maastricht University Medical Center Transplantation Immunology Laboratory to submit 79 novel alleles to EMBL-ENA, and the tool is freely available for the HLA community.

P019 Collection of major HLA allele sequences in japanese population towards precise NGS based HLA DNA typing at the field 4 level

Aim We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method. However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT®) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. Here, we report on sequence-based genotyping from the promoter-enhancer region to 3 ′ UTR of the major HLA genes in the Japanese using the PacBio RS II and Ion PGM NGS systems. Method Forty-six DNA samples were obtained from a reference set used previously to establish the HLA allele frequency data in the Japanese population. The reference samples represented more than 99.5% of the HLA alleles at each of the nine HLA loci. The genomic DNA samples were amplified by long ranged PCR using eleven HLA loci specific primer pairs (A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1). After NGS, consensus sequences were obtained via Long Amplicon analysis such as overlapping, clustering of filtered sub-reads, phasing and removing PCR artifacts. The HLA allele sequences for generating a library of consensus sequences were determined by mapping of sequence reads obtained from Ion PGM sequencer. Results A total of 219 HLA allele sequences (20 A, 40 B, 23 C, 31 DRB1, 36 DQA1, 14 DQB1, 15 DPA1 and 40 DPB1) that covered the promoter-enhancer region to 3’UTR were determined for the 46 samples. The classification of the newly identified SNPs and/or indels revealed at least three non-synonymous substitutions for one B and two DPA1 alleles, although most of the substitutions were observed in intron regions. Conclusion We determined at least 219 Japanese major HLA alleles at the field 4 level by NGS and we expect that this HLA genotyping method of entire HLA gene regions by NGS will help to precisely detect rare, novel and null alleles in population genetic and disease studies.

P066 Next-generation sequencing (NGS) HLA typing: Beyond allele assignment

Aim To expand the analytical potential of NGS in HLA typing. Studies based on SNP analysis have revealed the DNA sequence of HLA genes to carry important biological information typically overlooked in the simple exercise of allele assignment. NGS software is optimised to match sample sequences against databases of HLA allele sequence references. The goal here, however, is to create a method to open the data collected by NGS to any kind of query. Methods BLAST is still one of the most robust and efficient sequence-matching and sequence-alignment methods. Typically BAST databases are built with reference sequences against which a sample sequence is queried for matches. Here we reverse the approach and build a database of sample sequences against which we query for matches for particular sequences of interest following these steps: 1. Compile a database of sample sequences included in a given study. 2. Convert sample sequences to FASTA format. 3. Create a BLAST database with the study sample sequences. 4. Run any BLAST query against the BLAST database of the study sample sequences. Results This method was implemented using a dataset collected for typing purposes. The HLA-C 3’UTR polymorphisms which bind to miRNAs and regulate the level of expression (Nature 2011; 472:7344) were used. The BLAST output provided accurate information about which sequences carried the query polymorphism, which matched what is known about the association of these SNPs with HLA-C alleles. This method has also been successfully used to look for mutations that render HLA alleles null, and other SNP analysis queries. Conclusions NGS provides data that goes beyond the need for simple allele assignment. The method presented here provides (1) a robust and reliable way to store this accumulated information, (2) a quick and simple way to query this database of sequence data; and (3) an open method to ask any sequence question. M. Li: Employee; Company/Organization; Immucor.

Genetic Basis of Chronic Hepatitis C Virus and Autoimmune Hepatitis: A Comparative Study

Background: It is now well established that HCV is of global importance affecting all countries, leading to a major global health problem that requires widespread active interventions for its prevention and control. Chronic hepatitis C was linked to the development of cirrhosis and Hepatocellular Carcinoma (HCC) in many areas of the world. WHO reported that Egypt has the highest prevalence (22%) in the world? Objectives: Susceptibility to infection has been related to immunological disturbances.HCV and autoimmune hepatitis have been associated with HLA A1, A6, A8,-DRB∗01-∗15 alleles. Methods: HLA alleles were detected by Sequence Specific Oligoneucleotide Probe (SSOP) using INNO-LiPA based on reverse hybridization after amplification of target HLA allele sequence by Real Time Polymerase Chain Reaction (RT-PCR). The non-organ-specific Antibodies Include Antinuclear (ANA), Smooth Muscle (ASMA), Anti- Mitochondrial Antibody (AMA), Perinuclear Antineutrophil Cytoplasmic(pANCA), Liver-Kidney Microsomal Antibodies (LK/MA) and Anticardiolipn Antibodies (ACA) were all measured by commercial Indirect ImmunoFluorescence (IF). Clinical significance of these autoantibodies was analyzed in comparison to HCV load by Real Time Polymerase Chain Reaction (RT-PCR) and genotype by 4 byline probe assay INNO-LiPA. Conclusion: Current understanding of genetic basis (HLA alleles) of chronic HCV genotype4 cases and autoimmune hepatitis cases free from HCV will aid a lot in treating both types of hepatitis using successful line of therapy as early as possible to save money and side effects of wrong medications.

Filling the gaps – The generation of full genomic sequences for 15 common and well-documented HLA class I alleles using next-generation sequencing technology

Many common and well-documented (CWD) HLA alleles have only been partially characterized. The DNA sequence of these incomplete alleles, as published in the IMGT/HLA database, is most often limited to exons that code for the extracellular domains of the mature protein. Here we describe the application of next-generation sequencing technology to obtain full length genomic sequence from a single long-range PCR amplicon for 15 common and well-documented HLA Class I alleles. This technology is well suited to fill in the gaps of the current HLA allele sequence database which is largely incomplete. A more comprehensive catalog of HLA allele sequences would be beneficial in the evaluation of mismatches in transplantation, studies of population genetics, the evolution of HLAs, regulatory mechanisms and HLA expression, and issues related to the genomic organization of the MHC.

Bioinformatic Databases and Resources in the public domain to aid HLA research

Research in HLA as in any other field depends on information. Different groups have generated generic and specific resources and tools to support this research. The present review describes a qualified subset of these resources, which should cover the most important starting points for research in the HLA field. It discusses access to HLA allele sequences, allele frequencies, continues with general support to access to literature, DNA and protein sequence information, structural models, teaching books, databases with phenotypic datasets, alignment tools, peptide binding, statistical tools, guidelines and ambiguity coding. Following functionalities and databases have been included: IMGT/HLA, Immuno Polymorphism Database (IPD), allele frequencies*.net, a detailed look into NCBI (National Center of Biotechnology Information) with a subset of databases and tools, focusing on literature research, sequences, user-specific support tools and applications (PubMed, GenBank, MyNCBI, blast, Gene, MapViewer, Structure, CN3D, WorkBench, and dbMHC). This is followed by a brief survey of EBI-EMBL/Ensemble, the sequence alignment tool Clustal, the peptide and ligand databases SYFPEITHI and Immune Epitope Database, and last but not least statistical packages and HLA allele coding resources PyPop, the Immuno-genomics Data Analysis Working Group and the NMDP informatics section. All databases and tools can be freely accessed. Data linked to individuals, however, might require authorization by a data access committee.

Nomenclature for factors of the HLA system, update February 2010

The following sequences have been submitted to the Nomenclature Committee since the January 2010 nomenclature update and, following agreed policy, have been assigned official allele designations. Full details of all sequences will be published in a forthcoming report. Below are listed the newly assigned sequences (Table 1) and confirmations of previously reported sequences (Table 2). Together with the new format HLA allele sequence name, the designation prior to April 2010 is also listed. Full details of the change to the HLA nomenclature format is given in the recent nomenclature report (1). The accession number of each sequence is given and these can be used to retrieve the sequence files from the EMBL, GenBank or DDBJ data libraries. Although accession numbers have been assigned by the data-libraries and most sequences are already available, there is still the possibility that an author may not yet have allowed the sequence to be released; in such a case you will have to contact the submitting author directly. Additional information pertaining to new sequences is often included in the publications describing these alleles; a listing of recent publications that describe new HLA sequences is given in Table 3. All new and confirmatory sequences should now be submitted directly to the WHO Nomenclature Committee for Factors of the HLA System via the IMGT/HLA Database using the sequence submission tool provided. The IMGT/HLA Database may be accessed via the World Wide Web at: www.ebi.ac.uk/imgt/hla

Nomenclature for factors of the HLA system, 2004

Nomenclature for factors of the HLA system, 2004

Nomenclature for factors of the HLA system, 2002.

This chapter provides the nomenclature for factors of the HLA system, 2002. A number of previously described class I and II gene fragments within the HLA region are named in this system. Official designations are given to these gene fragments. The names LMP2 and LMP7 used previously for the two proteasome genes in the HLA class II region have been renamed by the Human Genome Nomenclature committee (HGNC) as PSMB9 and PSMB8, respectively. This system introduces the additional digit for synonymous variation and all allele names that are currently five digits or above are renamed accordingly. The use of an optional “N’” or “L” suffix to an allele name to indicate either “Null” or “Low” expression was introduced in previous Nomenclature Reports. Three new suffixes are introduced in this system. An “S” to denote an allele specifying a protein that is expressed as a soluble “Secreted” molecule but is not present on the cell surface; a “C” to indicate an allele product that is present in the “Cytoplasm” but not at the cell surface; an “A” to indicate “Aberrant” expression where there is some doubt as to whether a protein is expressed or not. There is evidence of differential splicing of HLA-G that leads to the production of both membrane-bound and soluble forms of the same allele. The IMGT/HLA Sequence Database contains sequences for all HLA alleles officially recognized by the WHO Nomenclature Committee for Factors of the HLA System and, provides users with online tools and facilities for their retrieval and analysis.

HLA allele detection using molecular techniques.

There are now many molecular biological techniques available to define HLA class I and class II alleles. Some of these are also applicable to other human polymorphic genes, in particular to those non-HLA genes encoded within the Mhc. The range of techniques available allows laboratories to choose those most suited to their purpose. The routine laboratory supporting solid organ transplants will need to type large numbers of potential recipients over a period of time, probably using PCR-SSOP while donors will be typed singly and rapidly using PCR-SSP with HLA allele compatibility determined by heteroduplex analysis. Laboratories supporting bone marrow transplantation, where time is less pressing, can choose from the whole range of techniques to determine accurately donor recipient Mhc compatibility. For disease studies, techniques defining precise HLA allele sequence polymorphisms are needed and high sample numbers have to be accommodated. When an association is established allele sequencing has to be used. In the near future, the precise role of HLA alleles in transplantation and disease susceptibility is likely to be established unambiguously.