Marcelo Fernández-Viña

Transcription

1 Marcelo Fernández-Viña Histocompatibility, Immunogenetics and Disease Profiling Laboratory Stanford Blood Center Applications of NGS in Immunogenetics HLA Typing KIR Typing Blood Group Typing Engraftment tests HLA expression 2 KIR typing Nelson WC, Pyo CW, Vogan D, Wang R, Pyon YS, Hennessey C, Smith A, Pereira S, Ishitani A, Geraghty DE. An integrated genotyping approach for HLA and other complex genetic systems. Hum Immunol Dec;76(12): doi: /j.humimm PubMed PMID:

2 KIR typing Selvaraj S, Schmitt AD, Dixon JR, Ren B. Complete haplotype phasing of the MHC and KIR loci with targeted HaploSeq. BMC Genomics Nov 5;16:900. doi: /s PubMed PMID: ; PubMed Central PMCID: PMC KIR Norman PJ, Hollenbach JA, Nemat-Gorgani N, Marin WM, Norberg SJ, Ashouri E, Jayaraman J, Wroblewski EE, Trowsdale J, Rajalingam R, Oksenberg JR, Chiaroni J, Guethlein LA, Traherne JA, Ronaghi M, Parham P. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing. Am J Hum Genet Aug 4;99(2): doi: /j.ajhg PubMed PMID: ; PubMed Central PMCID: PMC

3 Figure 1. Pipeline for Analyzing Sequence Data from Highly Polymorphic and Structurally Divergent KIR Haplotypes(A) The PING (Pushing Immunogenetics to the Next Generation) pipeline has two broad arms and two modules. The first module (PING_gc) determines KIR... Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing null, Volume 99, Issue 2, 2016, Figure 2. KIR Gene Copy-Number Genotype Determined by Read DepthThe ratio of reads mapping to a specific KIR gene to those mapping to KIR3DL3 can be used for calculating KIR copy number. The results from 97 samples are shown and sorted by ratio. KIR2DL4 (left)... Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing null, Volume 99, Issue 2, 2016, Figure 3. The KIR Region Is >99.99% Covered by Sequence Data(A) Target KIR region on chromosome 19: the gene locations are shown in orange, and pseudogenes are shown in gray. The KIR region varies in gene content, and shown are examples of two frequent A an... Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing null, Volume 99, Issue 2, 2016,

4 Figure 6. High-Resolution KIR Allele and Copy-Number Genotypes of 97 IHWG CellsFour examples of high-resolution allele and copy-number genotypes of KIR. Individual 1 (SP0010) is homozygous for the KIR A haplotype. Individual 2 (CB6B) has two different B haplot... Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing null, Volume 99, Issue 2, 2016, Figure 7. Capture of HLA Class I Genes for High-Resolution Allele Genotyping(A) Shown is the read depth across each of the HLA class I genes from a representative sample (chosen by virtue of having a read number closest to the mean number of HLA-specific reads... Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing null, Volume 99, Issue 2, 2016, Blood Groups Fichou Y, Mariez M, Le Maréchal C, Férec C. The experience of extended blood group genotyping by nextgeneration sequencing (NGS): investigation of patients with sickle-cell disease. Vox Sang Jul 21. doi: /vox [Epub ahead of print] PubMed PMID:

5 13 14 ABO Lang K, Wagner I, Schöne B, Schöfl G, Birkner K, Hofmann JA, Sauter J, Pingel J, Böhme I, Schmidt AH, Lange V. ABO allele-level frequency estimation based on populationscale genotyping by next generation sequencing. BMC Genomics May 20;17:374. doi: /s PubMed PMID: ; PubMed Central PMCID: PMC

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7 ABO Typing Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples. 19 Aloisio M, Licastro D, Caenazzo L, Torboli V, D'Eustacchio A, Severini GM, Athanasakis E. A technical application of quantitative next generation sequencingfor chimerism evaluation. Mol Med Rep Oct;14(4): doi: /mmr PubMed PMID: ; PubMed Central PMCID: PMC NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44-amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standardion AmpliSeq library preparation and I on Torrent Personal Genome Machineguidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification. 21 7

8 Expression of HLA alleles 22 Nature Genetics 41, (2009) A genome-wide association study identifies variants in the HLA-DPlocus associated with chronic hepatitis B in Asians YoichiroKamatani, SukanyaWattanapokayakit, HidenoriOchi, TakahisaKawaguchi, Atsushi Takahashi, NaoyaHosono, Michiaki Kubo, Tatsuhiko Tsunoda, NaoyukiKamatani, HiromitsuKumada, Aekkachai Puseenam, Thanyachai Sura, YataroDaigo 1, Kazuaki Chayama, WasunChantratita, Yusuke Nakamura & Koichi Matsuda A Novel Variant Marking HLA-DP Expression Levels Predicts Recovery from Hepatitis B Virus Infection Rasmi Thomasa, Chloe L. Thio, Richard Apps,Ying Qa, Xiaojiang Gao, Darlene Marti, Judy L. Stein, Kelly A. Soderberg, M. Anthony Moody, James J. Goedert, Gregory D. Kirk, W. Keith Hoots, Steven Wolinsky and Mary Carrington Variation in the 3 untranslated region of HLA-DPB1 is associated with spontaneous clearance of hepatitis B virus in both Japanese and U.S. populations. The mechanism facilitating viral clearance may be related to the A G single-nucleotide polymorphism rs , which marks HLA-DP cell-surface expression. The rs g allele is associated with high expression of HLA-DP, and the rs a allele is associated with low expression. The 496GG genotype, which confers recessive susceptibility to HBV persistence, also associates in a recessive manner with significantly higher levels of HLA-DP surface protein and transcript level expression in healthy donors, suggesting that differences in expression of HLA-DP may increase the risk of persistent HBV infection. 8

9 HLA-DP surface protein levels correlate with the 496A/G genotype in the 3 UTR region of HLA-DPB1. Rasmi Thomas et al. J. Virol. 2012;86: HLA-DPB1 mrna levels correlate significantly with the 496A/G variant in the 3 UTR region of HLA-DPB1. Rasmi Thomas et al. J. Virol. 2012;86: High HLA-DP Expression and Graft-versus-Host Disease Effie W. Petersdorf, M.D., Mari Malkki, Ph.D., Colm O huigin, Ph.D., Mary Carrington, Ph.D., Ted Gooley, Ph.D., Michael D. Haagenson, M.S., Mary M. Horowitz, M.D., Stephen R. Spellman, M.B.S., Tao Wang, Ph.D., and Philip Stevenson, M.S. N Engl J Med 2015; 373: August 13, 2015DOI: /NEJMoa

10 DPB1 Genomic Structure, Associated DPB1 Single- Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression. Fleischhauer K. N Engl J Med 2015;373: Hazard Ratios for Outcomes of HLA-DPB1 Mismatches in Transplant Recipients, According to the rs Allele Linked to the Mismatch. Petersdorf EW et al. N Engl J Med 2015;373: Probability of Grade II, III, or IV Acute Graft-versus-Host Disease. Petersdorf EW et al. N Engl J Med 2015;373:

11 DPB1 Genomic Structure, Associated DPB1 Single- Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression. Fleischhauer K. N Engl J Med 2015;373: Expression of HLA alleles Define expression variants Determine variation at the RNA level NGS may provide additional specificity for further characerization of Genotypes Possible application for evaluation in tissue expression 32 Summary KIR and Blood Group Typing has been developed Test Formats and combinations with HLA typing Quantitation and Expression studies could be applied in the clinical routine setting 33 11