Transcript NGS Beyond HLA Typing, Marcelo Fernandez-Vina

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
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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. 2015 Dec;76(12):928-38. doi:
10.1016/j.humimm.2015.05.001. PubMed PMID: 26027777.
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KIR typing
Selvaraj S, Schmitt AD, Dixon JR, Ren B.
Complete haplotype phasing of the MHC and KIR loci with
targeted HaploSeq.
BMC Genomics. 2015 Nov 5;16:900. doi: 10.1186/s12864015-1949-7. PubMed PMID: 26541200; PubMed Central
PMCID: PMC4636068.
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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. 2016 Aug 4;99(2):375-91. doi:
10.1016/j.ajhg.2016.06.023. PubMed PMID: 27486779;
PubMed Central PMCID: PMC4974113.
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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, 375–391
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
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, 375–391
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
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, 375–391
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
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, 375–391
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
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, 375–391
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
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. 2016 Jul 21. doi: 10.1111/vox.12432. [Epub
ahead of print] PubMed PMID: 27442304.
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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. 2016 May 20;17:374. doi: 10.1186/s12864016-2687-1. PubMed PMID: 27207383; PubMed Central
PMCID: PMC4874024.
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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.
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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. 2016 Oct;14(4):2967-74. doi:
10.3892/mmr.2016.5593. PubMed PMID: 27499173;
PubMed Central PMCID: PMC5042788.
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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.
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Expression of HLA alleles
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Nature Genetics 41, 591 - 595 (2009)
A genome-wide association study identifies
variants in the HLA-DP locus associated with
chronic hepatitis B in Asians
Yoichiro Kamatani, Sukanya Wattanapokayakit,
Hidenori Ochi, Takahisa Kawaguchi, Atsushi
Takahashi, Naoya Hosono, Michiaki Kubo,
Tatsuhiko Tsunoda, Naoyuki Kamatani,
Hiromitsu Kumada, Aekkachai Puseenam,
Thanyachai Sura, Yataro Daigo1, Kazuaki
Chayama, Wasun Chantratita, 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 rs9277534, which marks
HLA-DP cell-surface expression. The rs9277534G allele is associated
with high expression of HLA-DP, and the rs9277534A 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.
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;86:6979-6985
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;86:6979-6985
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; 373:599-609August 13, 2015DOI:
10.1056/NEJMoa1500140
DPB1 Genomic Structure, Associated DPB1 SingleNucleotide-Polymorphism Haplotypes, and Resulting HLA-DP
Expression.
Fleischhauer K. N Engl J Med 2015;373:669-672.
Hazard Ratios for Outcomes of HLA-DPB1 Mismatches in
Transplant Recipients, According to the rs9277534 Allele
Linked to the Mismatch.
Petersdorf EW et al. N Engl J Med 2015;373:599-609.
Probability of Grade II, III, or IV Acute Graft-versus-Host
Disease.
Petersdorf EW et al. N Engl J Med 2015;373:599-609.
DPB1 Genomic Structure, Associated DPB1 SingleNucleotide-Polymorphism Haplotypes, and Resulting HLA-DP
Expression.
Fleischhauer K. N Engl J Med 2015;373:669-672.
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
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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
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