The following resources provide the latest information about the science and clinical aspects of histocompatibility and immunogenetics.
An international society for the advancement of the science and clinical application of histocompatibility and immunogenetics and setting the highest standards of laboratory testing.
A database of HLA sequences and part of the ImMunoGeneTics project (IMGT).
A repository of human KIR sequences.
An explanation of HLA nomenclature.
A database of allele frequencies from different polymorphic areas in the human genome.
A registery of stem cell donors and a dedicated cord blood bank.
This group supports the projects of the 13th international histocompatibility workshop, housed at Fred Hutchinson Cancer Research Center in Seattle, WA. They keep an inventory of B-LCL lines and HLA donors from diverse population groups.
The largest registry of marrow and umbilical blood donors in the world.
The World Marrow Donor Association (WMDA) is made up of organisations and individuals who promote global collaboration and best practices for the benefit of stem cell donors and transplant patients.
A group formed by histocompatibility and immunogenetics investigators to share immunogenomic data and to foster the consistent analysis of data by the immunogenetics community.
Smith AG, Pereira S, Jaramillo A, Stoll ST, Khan FM, Berka N, Mostafa AA, Pando MJ, Usenko CY, Bettinotti MP, Pyo CW, Nelson CW, Willis A, Askar M, Geraghty DE.
HLA, Sep 2019
Many clinical laboratories supporting solid organ transplant programs use mul- tiple 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 uti- lize 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), par- ticularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turn- around 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 mis- assignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS pro- vided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was com- pleted 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.
Nelson W, Pyo CW, Vogan D, Wang R, Pyon YS, Hennessey C, Smith A, Pereira S, Ishitani A, Geraghty DE.
Human Immunology, Dec 2015
Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1–7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1–4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.
Li S, Gilbert P, Tomaras G, Kijak G, Ferrari G, Thomas R, Pyo CW, Zolla-Pazner S, Montefiori D, Liao HX, Nabel G, Pinter A, Evans D, Gottardo R, Dai JY, Janes H, Morris D, Fong Y, Edelfsen PT, Li F, Frahm N, Alpert MD, Prentice H, Rerks-Ngarm S, Pitisuttihum P, Kaewkungwal J, Nitayaphan S, Robb ML, O’Connell RJ, Haynes BF, Michael NL, Kim JH, McElrath MJ, Geraghty DE.
Journal of Clinical Investigation, Sep 2014
The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.
Smith AG, Pyo CW, Nelson W, Gow E, Wang R, Shen S, Sprague M, Pereira SE, Geraghty DE, Hansen JA.
Human Immunology, Oct 2014
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.
Pyo CW, Wang R, Vu Q, Cereb N, Yang SY, Duh FM, Wolinsky S, Martin MP, Carrington M, Geraghty DE.
BMC Genomics, Feb 2013
The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization.
Shen S, Pyo CW, Vu Q, Wang R, Geraghty DE.
ILAR Journal, Jan 2013
Next-generation sequencing technologies have led to rapid progress in the fields of human and nonhuman primate (NHP) genomics. The less expensive and more efficient technologies have enabled the sequencing of human genomes from multiple populations and the sequencing of many NHP species. NHP genomes have been sequenced for two main reasons: (1) their importance as animal models in biomedical research and (2) their phylogenetic relationship to humans and use in derivative evolutionary studies. NHPs are valuable animal models for a variety of diseases, most notably for human immunodeficiency virus/acquired immunodeficiency syndrome research, and for vaccine development. Knowledge about the variation in primate immune response loci can provide essential insights into relevant immune function. However, perhaps ironically considering their central role in infectious disease, the accumulation of sequence detail from genomic regions harboring immune response loci, such as the major histocompatibility complex and killer immunoglobulin-like receptors, has been slow. This deficiency is, at least in part, due to the highly repetitive and polymorphic nature of these regions and is being addressed by the application of special approaches to targeted sequencing of the immune response genomic regions. We discuss one such targeting approach that has successfully yielded complete phased genomic sequences from complex genomic regions and is now being used to resequence macaque and other primate major histocompatibility complex regions. The essential detail contained within the genomics of the NHP immune response is now being assembled, and the realization of precise comparisons between NHP and human immune genomics is close at hand, further enhancing the NHP animal model in the search for effective treatments for human disease.
Pyo CW, Guethlein LA, Vu Q, Wang R, Abi-Rached L, Norman PJ, Marsh SG, Miller JS, Parham P, Geraghty DE.
PLoS One, Dec 29, 2010
The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ~6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ~1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region.
Pyo CW, Williams LM, Moore Y, Hyodo H, Li SS, Zhao LP, Sageshima N, Ishitani A, Geraghty DE.
Immunogenetics, Mar 2006
Despite several studies that defined the polymorphism of the nonclassical human leukocyte antigen-E (HLA-E), HLA-F, and HLA-G genes, most polymorphisms thus far examined in correlative studies were derived from the coding sequences of these genes. In addition, some discrepancies and ambiguities in the available data have persisted in current databases. To expand the data available and to resolve some of the discrepant data, we have defined protocols that allow for the amplification of 6 to 7 kb of contiguous genomic sequence for each gene, including all of the coding and intron sequences, approximately 2 kb of 5′ flanking promoter sequence, and 1 kb of 3′ flanking sequence. Using long-range polymerase chain reaction (PCR) protocols, generating either one or two PCR products depending on the locus, amplified genomic DNA was directly sequenced to completion using a set of about 30 primers over each locus to yield contiguous sequence data from both strands. Using this approach, we sequenced 33 genomic DNAs, from Asian, African American, and Caucasian samples. The results of this analysis confirmed several previously reported coding sequence variants, identified several new allelic variants, and also defined extensive variation in intron and flanking sequences. It was possible to construct haplotype maps and to identify tagging single nucleotide polymorphisms that can be used to detect the composite variation spanning all three genes.