Abstract
Aging hematopoietic stem cells acquire mutations that sometimes impart a selective advantage. Next-generation DNA sequencing (NGS) can be used to detect expanded peripheral blood progeny of a mutant clone, usually carrying just one cancer-driver mutation, most often in the epigenetic regulator genes, DNMT3A or TET2. This phenomenon is known as clonal hematopoiesis (CH), age-related CH (ARCH) when considering its association with age, and CH of indeterminate potential (CHIP) when the variant allele fraction (VAF) is at least 2% in peripheral leukocytes. CHIP is present in at least 10–15% of adults older than 65 years and is a risk factor for hematological neoplasms and diseases exacerbated by mutant, hyper-inflammatory, monocytes/macrophages, such as atherosclerotic cardiovascular disease. Therefore, the detection of CHIP has important clinical consequences. Herein, we present a protocol for the generation of targeted, amplicon-based, NGS libraries for ion semiconductor sequencing and CHIP detection, using Ion Torrent platforms.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jaiswal S, Fontanillas P, Flannick J et al (2014) Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 371:2488–2498
Genovese G, Kähler AK, Handsaker RE et al (2014) Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med 371:2477–2487
McKerrell T, Park N, Moreno T et al (2015) Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis. Cell Rep 10:1239–1245
Steensma DP, Bejar R, Jaiswal S et al (2015) Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood 126:9–16
Shlush LI (2018) Age-related clonal hematopoiesis. Blood 131:496–504
Abelson S, Collord G, Ng SWK et al (2018) Prediction of acute myeloid leukaemia risk in healthy individuals. Nature 559:400–404
Desai P, Mencia-Trinchant N, Savenkov O et al (2018) Somatic mutations precede acute myeloid leukemia years before diagnosis. Nat Med 24:1015–1023
Bowman RL, Busque L, Levine RL (2018) Clonal hematopoiesis and evolution to hematopoietic malignancies. Cell Stem Cell 22:157–170
Steensma DP (2018) Clinical consequences of clonal hematopoiesis of indeterminate potential. Blood Adv 2:3404–3410
Zhang Q, Zhao K, Shen Q et al (2015) Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6. Nature 525:389–393
Fuster JJ, MacLauchlan S, Zuriaga MA et al (2017) Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science 355:842–847
Jaiswal S, Natarajan P, Silver AJ et al (2017) Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease. N Engl J Med 377:111–121
Cull AH, Snetsinger B, Buckstein R et al (2017) Tet2 restrains inflammatory gene expression in macrophages. Exp Hematol 55:56–70.e13
Young AL, Challen GA, Birmann BM et al (2016) Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults. Nat Commun 7:12484
Rothberg JM, Hinz W, Rearick TM et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352
Sekeres MA, Othus M, List AF et al (2017) Randomized Phase II Study of azacitidine alone or in combination with lenalidomide or with vorinostat in higher-risk myelodysplastic syndromes and chronic myelomonocytic leukemia: North American Intergroup Study SWOG S1117. J Clin Oncol 35:2745–2753
Buscarlet M, Provost S, Zada YF et al (2017) DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions. Blood 130:753–762
Buscarlet M, Provost S, Zada YF et al (2018) Lineage restriction analyses in CHIP indicate myeloid bias for TET2 and multipotent stem cell origin for DNMT3A. Blood 132:277–280
Cook E, Izukawa T, Young S et al (2018) Feeding the fire: the comorbid and inflammatory backdrop of clonal hematopoiesis of indeterminate potential (CHIP) by mutation subtype. Blood 130:426
Coombs CC, Zehir A, Devlin SM et al (2017) Therapy-related clonal hematopoiesis in patients with non-hematologic cancers is common and associated with adverse clinical outcomes. Cell Stem Cell 21:374–382
Zink F, Stacey SN, Norddahl GL et al (2017) Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly. Blood 130:742–752
Acknowledgments
The authors thank Dr. Harriet Feilloter, Dr. Xudong Liu, Dr. Amy McNaughton, Dr. Xiao Zhang, and Dr. Paul Park for initial assistance with Ion Torrent Sequencing. Funding was provided by the Southeastern Ontario Academic Medical Organization (SEAMO) Innovation Fund, the University Hospitals Kingston Fund (UHKF)/Women’s Giving Circle, and the Ontario Institute for Cancer Research (OICR)/Ontario Molecular Pathology Research Network (OMPRN).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media New York
About this protocol
Cite this protocol
Snetsinger, B., Ferrone, C.K., Rauh, M.J. (2019). Targeted, Amplicon-Based, Next-Generation Sequencing to Detect Age-Related Clonal Hematopoiesis. In: Turksen, K. (eds) Stem Cells and Aging . Methods in Molecular Biology, vol 2045. Humana, New York, NY. https://doi.org/10.1007/7651_2019_216
Download citation
DOI: https://doi.org/10.1007/7651_2019_216
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9712-1
Online ISBN: 978-1-4939-9713-8
eBook Packages: Springer Protocols