High‐resolution, high‐throughput HLA genotyping by next‐generation sequencing

Bentley, Gordon; Higuchi, Russell; Höglund, Bryan; Goodridge, Damian; Sayer, D.; Trachtenberg, Elizabeth; Erlich, Henry A.

doi:10.1111/j.1399-0039.2009.01345.x

Cited by 177 publications

(133 citation statements)

References 22 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…Recently, several laboratories (16)(17)(18)(19)(20) have developed highthroughput HLA genotyping methodologies using massively parallel sequencing strategies such as Roche/454 sequencing (21). In all these high-throughput HLA-genotyping studies, with the exception of the study by Lind et al (19), a few polymorphic exons were amplified separately and sequenced in a multiplexed manner.…”

Section: Discussionmentioning

confidence: 99%

High-throughput, high-fidelity HLA genotyping with deep sequencing

Wang

Krishnakumar

Wilhelmy

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

177

144

View full text Add to dashboard Cite

Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.hematopoietic stem cell transplantation | sequence-based typing H uman leukocyte antigen (HLA) genes encode cell-surface proteins that bind and display fragments of antigens to T lymphocytes. This helps to initiate the adaptive immune response in higher vertebrates and thus is critical to the detection and identification of invading microorganisms (1). Six of the HLA genes (HLA-A, -B, -C, -DQA1, -DQB1, and -DRB1) are extremely polymorphic and constitute the most important set of markers for matching patients and donors for bone marrow transplantation (2, 3). Specific HLA alleles have been found to be associated with a number of autoimmune diseases, such as multiple sclerosis (4), narcolepsy (5), celiac disease (6), rheumatoid arthritis (7), and type I diabetes (3,8). Alleles have also been noted to be protective in infectious diseases such as HIV (9, 10), and numerous animal studies have shown that these genes are often the major contributors to disease susceptibility or resistance (11-13).HLA genes are among the most polymorphic in the human genome, and the changes in sequence affect the specificity of antigen presentation and histocompatibility in transplantation. A variety of methodologies have been developed for HLA typing at the protein and nucleic acid level. Whereas earlier HLA typing methods distinguished HLA antigens, modern methods such as sequence-based typing (SBT) determine the nucleotide sequences of HLA genes for higher resolution. However, due to cost and time constraints, HLA sequencing technologies have traditionally focused on the most polymorphic regions encoding the peptide-...

show abstract

Section: Discussionmentioning

confidence: 99%

High-throughput, high-fidelity HLA genotyping with deep sequencing

Wang

Krishnakumar

Wilhelmy

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

177

144

View full text Add to dashboard Cite

show abstract

“…We performed amplicon sequencing of exons 2 and 3 of HLA-DQB1 and exon 2 of HLA-DRB3/4/5, HLA-DRB1, and HLA-DQA1 in 20 unrelated EUR vitiligo patients, on the 454 Life Science GS FLX, as previously described (40,41), except that the GS GType HLA HR primers (Roche Applied Science) were used. The manufacturer's protocol for GS Titanium Sequencing was followed, except that 0.5 copies of DNA per bead were used and 70% of the recommended amount of DNA beads was loaded on the PicoTiter PlateT.…”

Section: Methodsmentioning

confidence: 99%

MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo

Cavalli

Hayashi

Jin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genomewide association study of 2,853 Caucasian vitiligo patients. The superenhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5-and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.A utoimmune diseases are a group of over 80 disorders that together affect 3-5% of the United States population (1, 2). Many autoimmune diseases are associated with genetic variation in the HLA class I and class II gene regions of the major histocompatibility complex (MHC) on chromosome 6p21.3. HLA class I molecules present peptide antigens on the surface of almost all cells, whereas HLA class II molecules present antigens on the surface of antigen-presenting cells, such as dendritic cells, mononuclear phagocytes, and B cells. Contributions of HLA molecules to autoimmunity have almost exclusively focused on antigenic diversity and specificity. Although polymorphisms in intergenic regions of the MHC might additionally affect the complex transcriptional regulation of HLA genes, the potential role of these noncoding regions in the pathogenesis of autoimmune diseases has received much less attention.Vitiligo is an autoimmune disease in which white spots of skin and overlying hair result from progressive destruction of melanocytes by autoreactive T cells (3). In previous genome-wide association studies of autoimmune vitiligo in European-derived Caucasian (EUR) populations, we have identified association with 27 different loci (4-6), most strongly with MHC class II r...

show abstract

“…1,2 It has become apparent that NGS platforms will have practical applications in clinical diagnostics and applications, such as detection of EGFR mutations in lung adenocarcinoma, 3 characterizing RAS and methylation pathway alterations in myeloproliferative diseases and myeloid leukemias, [4][5][6][7] or high-resolution, high-throughput human leukocyte antigen genotyping have been developed. 8,9 Thus far, limited data are available on the technical performance of amplicon deep sequencing in a clinical diagnostic setting. Here, we reportFas an international consortiumFthe results of our investigation concerning the robustness, precision and reproducibility of amplicon NGS across 10 laboratories from eight countries.…”

Section: Introductionmentioning

confidence: 99%

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

et al. 2011

View full text Add to dashboard Cite

Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon nextgeneration deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.

show abstract

High‐resolution, high‐throughput HLA genotyping by next‐generation sequencing

Cited by 177 publications

References 22 publications

High-throughput, high-fidelity HLA genotyping with deep sequencing

High-throughput, high-fidelity HLA genotyping with deep sequencing

MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

Contact Info

Product

Resources

About