16^th IHIW : Review of HLA typing by NGS

Abstract

Summary Human leukocyte antigens (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to; (i) template preparation including short and long range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

Comparative Analyses of Low, Medium and High-Resolution HLA Typing Technologies for Human Populations

“…HLA alleles information play a major role in solid organ and haematopoietic stem cell transplantation, autoimmune disease (e.g. diabetes, rheumatoid arthritis, coeliac), infectious diseases (HIV, Dengue, Hepatitis C), allergy, cancer, vaccine development and population structure [3,4,18]. The HLA loci sequencing have inherent limitations due to extreme polymorphism, ambiguities in haplotype phasing, unknown reference standards, technical problems like shorter read lengths etc [3,4].…”

“…The HLA region comprises over 200 genes, but six HLA genes (class I-A, B, C and class II-DR, DP, DQ) that are crucial for self and non-self recognition. Class I proteins expresses on the surface of all nucleated cells in the human body, but class II proteins can be found on the antigen-presenting phagocytes such as dendritic cells, mononuclear phagocytes [3,4]. The MHC class I complex consists of three peptide domains -α1, α2, and α3 and a non-MHC small petptide, β2-microglobulin.…”

“…The MHC class I complex consists of three peptide domains -α1, α2, and α3 and a non-MHC small petptide, β2-microglobulin. The central region of the α1/α2 heterodimer in the HLA complex forms antigen binding groove to presents the antigen(s) to CD8 T lymphocytes, thereby recognize the self and non-self fate of human cells [3,4]. Whereas class II complex consists of two chains, α and β peptides.…”

“…Whereas class II complex consists of two chains, α and β peptides. The class II antigen presenting peptide groove is formed through the interaction of heterodimer of α1 and β1 peptides, which presents the antigen to CD4 + lymphocytes [3,4].…”

“…Currently over 10,000 HLA class I and II alleles are available at IMGT(the international ImMunoGeneTics) database (http://www.ebi.ac.uk/imgt/hla/). The HLA proteins play a pivotal role in the immune response and are implicated in numerous human pathological conditions including autoimmune disease, infectious diseases, cancer and drug reactions [3,4,5]. Clinically, HLA gene sequence information is widely used in organ transplantion to identify matching donor and recipient HLA alleles.…”