60 Years of clonal hematopoiesis research: From X-chromosome inactivation studies to the identification of driver mutations
2020; Elsevier BV; Volume: 83; Linguagem: Inglês
10.1016/j.exphem.2020.01.008
ISSN1873-2399
AutoresSami Ayachi, Manuel Buscarlet, Lambert Busque,
Tópico(s)Myeloproliferative Neoplasms: Diagnosis and Treatment
Resumo•X-chromosome inactivation analysis in females was the precursor of clonality determination and allowed to demonstrate the unicellular origin of human cancer.•Mutation in several genes such as DNMT3A, ET2 or ASXL1 occurs in blood cells of aging individuals, lead to clonal expansion, is compatible with normal blood counts but predispose to hematological cancers or cardiovascular disease.•Clonal Hematopoiesis has a modest genetic predisposition but is influenced by extrinsic factors such as inflammation, genotoxic stress or immune attack.•Clonal hematopoiesis may be a biomarker of unhealthy aging. The history of clonal hematopoiesis (CH) research is punctuated by several seminal discoveries that have forged our understanding of cancer development. The clever application of the principle of random X-chromosome inactivation (XCI) in females led to the development of the first test to identify clonal derivation of cells. Initially limited by a low level of informativeness, the applicability of these assays expanded with differential methylation-based assays at highly polymorphic genes such as the human androgen receptor (HUMARA). Twenty years ago, the observation that skewing of XCI ratios increases as women age was the first clue that led to the identification of mutations in the TET2 gene in hematologically normal aging individuals. In 2014, large-scale genomic approaches of three cohorts allowed definition of CH, which was reported to increase the risk of developing hematologic cancers and cardiovascular diseases. These observations created a fertile field of investigation aimed at investigating the etiology and consequences of CH. The most frequently mutated genes in CH are DNMT3A, TET2, and ASXL1, which have a role in hematopoietic stem cell (HSC) development and self-renewal. These mutations confer a competitive advantage to the CH clones. However, the penetrance of CH is age dependent but incomplete, suggesting the influence of extrinsic factors. Recent data attribute a modest role to genetic predisposition, but several observations point to the impact of a pro-inflammatory milieu that advantages the mutated clones. CH may be a barometer of nonhealthy aging, and interventions devised at curbing its initiation or progression should be a research priority. The history of clonal hematopoiesis (CH) research is punctuated by several seminal discoveries that have forged our understanding of cancer development. The clever application of the principle of random X-chromosome inactivation (XCI) in females led to the development of the first test to identify clonal derivation of cells. Initially limited by a low level of informativeness, the applicability of these assays expanded with differential methylation-based assays at highly polymorphic genes such as the human androgen receptor (HUMARA). Twenty years ago, the observation that skewing of XCI ratios increases as women age was the first clue that led to the identification of mutations in the TET2 gene in hematologically normal aging individuals. In 2014, large-scale genomic approaches of three cohorts allowed definition of CH, which was reported to increase the risk of developing hematologic cancers and cardiovascular diseases. These observations created a fertile field of investigation aimed at investigating the etiology and consequences of CH. The most frequently mutated genes in CH are DNMT3A, TET2, and ASXL1, which have a role in hematopoietic stem cell (HSC) development and self-renewal. These mutations confer a competitive advantage to the CH clones. However, the penetrance of CH is age dependent but incomplete, suggesting the influence of extrinsic factors. Recent data attribute a modest role to genetic predisposition, but several observations point to the impact of a pro-inflammatory milieu that advantages the mutated clones. CH may be a barometer of nonhealthy aging, and interventions devised at curbing its initiation or progression should be a research priority. "The Origin and Development of Human Tumors Studied with Cell Markers" is the title of a 1974 review article by the late Philip J. Fialkow [1Fialkow PJ The origin and development of human tumors studied with cell markers.N Engl J Med. 1974; 291: 26-35Crossref PubMed Scopus (152) Google Scholar]. It articulates both the necessity for and fascination with the study of clonality. The human body has to produce, over its life span, trillions of quality cells originating from a single fertilized egg. When this proliferation system derails, we are at risk of cancer. In this short review we provide a historical perspective of the study of clonal hematopoiesis and outline future opportunities and challenges. In 1962, Lyon [2Lyon MF Sex chromatin and gene action in the mammalian X-chromosome.Am J Hum Genet. 1962; 14: 135-148PubMed Google Scholar] proposed that each X-chromosome in excess of one is randomly inactivated in cells of the developing female embryo. Therefore, following X-chromosome inactivation (XCI), females present a mosaic pattern of two cell populations, one expressing maternal (Xm) and the other paternal (Xp) X-linked genes. Almost concurrently, Beutler et al. [3Beutler E Yeh M Fairbanks VF The normal human female as a mosaic of X-chromosome activity: studies using the gene for C-6-PD-deficiency as a marker.Proc Natl Acad Sci USA. 1962; 48: 9-16Crossref PubMed Scopus (250) Google Scholar] proved this theory in human females heterozygous for alleles of the glucose-6-phospate dehydrogenase (G6PD) gene [3Beutler E Yeh M Fairbanks VF The normal human female as a mosaic of X-chromosome activity: studies using the gene for C-6-PD-deficiency as a marker.Proc Natl Acad Sci USA. 1962; 48: 9-16Crossref PubMed Scopus (250) Google Scholar]. These observations paved the way to the first clonality studies. Linder and Gartler [4Linder D Gartler SM Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas.Science. 1965; 150: 67-69Crossref PubMed Scopus (263) Google Scholar] studied G6PD variants (A and B) by electrophoresis in five female heterozygotes and documented that 27 samples of leiomyomas had only one band, in contrast to 85 of 86 myometrium samples in which both alleles were present, indicating that these tumors had a single-cell origin [4Linder D Gartler SM Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas.Science. 1965; 150: 67-69Crossref PubMed Scopus (263) Google Scholar]. This study opened a fertile field of investigation that has allowed investigators to unravel some of the basic tenets of modern oncology [5Busque L Gilliland DG X-inactivation analysis in the 1990s: promise and potential problems.Leukemia. 1998; 12: 128-135Crossref PubMed Scopus (50) Google Scholar]. The applicability of XCI analysis with the G6PD assay was drastically limited by the low level of heterozygosity in the general population. This precluded analysis of rare disorders, analysis of large cohorts of females, and familial analysis. In the mid-1980s, Vogelstein et al. [6Vogelstein B Fearon ER Hamilton SR Feinberg AP Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.Science. 1985; 227: 642-645Crossref PubMed Scopus (331) Google Scholar] made a seminal contribution to the field by identifying DNA polymorphisms linked to residues differentially methylated between the active (Xa) and inactive (Xi) X-chromosomes. Differential methylation was validated as a surrogate for protein expression. Gilliland et al. [7Gilliland DG Blanchard KL Levy J Perrin S Bunn HF Clonality in myeloproliferative disorders: analysis by means of the polymerase chain reaction.Proc Natl Acad Sci USA. 1991; 88: 6848-6852Crossref PubMed Scopus (260) Google Scholar] developed a polymerase chain reaction (PCR)–based method allowing, for the first time, the study of small populations of cells. Another clever way of improving the informative value of XCI assays was to look for transcriptional polymorphisms of X-linked genes. Prchal et al. [8Prchal JT Guan YL A novel clonality assay based on transcriptional analysis of the active X chromosome.Stem Cells. 1993; 11: 62-65Crossref PubMed Scopus (35) Google Scholar,9Luhovy M Liu Y Belickova M Prchal JF Prchal JT A novel clonality assay based on transcriptional polymorphism of X chromosome gene p55.Biol Blood Marrow Transplant. 1995; 1: 81-87PubMed Google Scholar] were instrumental in developing such assays. These assays have the theoretical advantage of not relying on methylation patterns to assess the activity of the X-chromosome, as they rely directly on the exclusive transcriptional activity of Xa. In 1992, XCI studies expanded with the introduction of a novel assay of the human androgen-receptor gene (AR or HUMARA [GeneBank]) by Allen et al. [10Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar]. This assay benefitted from a multiallelic CAG short tandem repeat (STR) in the first exon of the gene that is closely linked to differentially methylated cytosine residues between Xa and Xi. The AR CAG repeat is polymorphic in 90% of females of all racial groups [11Edwards A Hammond HA Jin L Caskey CT Chakraborty R Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups.Genomics. 1992; 12: 241-253Crossref PubMed Scopus (1184) Google Scholar], rendering XCI analysis feasible in the majority of females with a single PCR-based assay. Interestingly, an RNA-based assay was developed and correlated with the HUMARA assay, supporting the reliability of the differential methylation patterns at this locus [12Busque L Zhu J DeHart D Griffith B Willman C Carroll R et al.An expression based clonality assay at the human androgen receptor locus (HUMARA) on chromosome X.Nucleic Acids Res. 1994; 22: 697-698Crossref PubMed Scopus (73) Google Scholar]. In fact, the concordance of transcriptional assays with HUMARA was challenged only once [13Swierczek SI Agarwal N Nussenzveig RH et al.Hematopoiesis is not clonal in healthy elderly women.Blood. 2008; 112: 3186-3193Crossref PubMed Scopus (51) Google Scholar] and ultimately further validated [14Busque L Paquette Y Provost S et al.Skewing of X-inactivation ratios in blood cells of aging women is confirmed by independent methodologies.Blood. 2009; 113: 3472-3474Crossref PubMed Scopus (69) Google Scholar,15Mossner M Nolte F Hutter G et al.Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay.J Med Genet. 2013; 50: 108-117Crossref PubMed Scopus (20) Google Scholar]. The HUMARA assay is currently the most frequently used method of XCI analysis (PubMed, January 2020: 323 publications using HUMARA key word). Significant skewing of the XCI ratio from the normal, theoretical 1:1 ratio has been arbitralily determined to be ≥3:1 (which corresponds to the expression of 75% of one allele) [16Allen RC Nachtman RG Rosenblatt HM Belmont JW Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.Am J Hum Genet. 1994; 54: 25-35PubMed Google Scholar, 17Busque L Mio R Mattioli J et al.Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age.Blood. 1996; 88: 59-65Crossref PubMed Google Scholar, 18Gale RE Wheadon H Linch DC X-chromosome inactivation patterns using HPRT and PGK polymorphisms in haematologically normal and post-chemotherapy females.Br J Haematol. 1991; 79: 193-197Crossref PubMed Scopus (97) Google Scholar, 19Gale RE Wheadon H Boulos P Linch DC Tissue specificity of X-chromosome inactivation patterns.Blood. 1994; 83: 2899-2905Crossref PubMed Google Scholar, 20Vogelstein B Fearon ER Hamilton SR et al.Clonal analysis using recombinant DNA probes from the X-chromosome.Cancer Res. 1987; 47: 4806-4813PubMed Google Scholar, 21Gale RE Wheadon H Linch DC Assessment of X-chromosome inactivation patterns using the hypervariable probe M27 beta in normal hemopoietic cells and acute myeloid leukemic blasts.Leukemia. 1992; 6: 649-655PubMed Google Scholar]. Determination of the prevalence of skewing should be straightforward because it can be simply estimated by using XCI assays in healthy females. However, initial estimation was contradictory. Using the phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl transferase (HPRT) X-inactivation probes, Vogelstein et al. [20Vogelstein B Fearon ER Hamilton SR et al.Clonal analysis using recombinant DNA probes from the X-chromosome.Cancer Res. 1987; 47: 4806-4813PubMed Google Scholar] found only 3 of 81 (3.7%) normal females to have skewing. Gale et al. found significant skewing in blood-derived cells in 23% of normal females using PGK and HPRT probes [18Gale RE Wheadon H Linch DC X-chromosome inactivation patterns using HPRT and PGK polymorphisms in haematologically normal and post-chemotherapy females.Br J Haematol. 1991; 79: 193-197Crossref PubMed Scopus (97) Google Scholar] and 22% of females using M27β [21Gale RE Wheadon H Linch DC Assessment of X-chromosome inactivation patterns using the hypervariable probe M27 beta in normal hemopoietic cells and acute myeloid leukemic blasts.Leukemia. 1992; 6: 649-655PubMed Google Scholar], which has been confirmed in other studies [20Vogelstein B Fearon ER Hamilton SR et al.Clonal analysis using recombinant DNA probes from the X-chromosome.Cancer Res. 1987; 47: 4806-4813PubMed Google Scholar,22Puck JM Stewart CC Nussbaum RL Maximum-likelihood analysis of human T-cell X chromosome inactivation patterns: normal women versus carriers of X-linked severe combined immunodeficiency.Am J Hum Genet. 1992; 50: 742-748PubMed Google Scholar]. Discordance in the incidence of skewing was initially explained by the diversity of assays used, the different criteria for skewing, and the small population sizes. However, a more plausible explanation came from studies that analyzed XCI patterns of different tissues in the same female. For example, Fey et al. [23Fey MF Peter HJ Hinds HL et al.Clonal analysis of human tumors with M27 beta, a highly informative polymorphic X chromosomal probe.J Clin Invest. 1992; 89: 1438-1444Crossref PubMed Scopus (91) Google Scholar] reported that the incidence of skewing was low in gastrointestinal mucosa and thyroid tissue, but was significantly higher in blood cells. Gale et al. [19Gale RE Wheadon H Boulos P Linch DC Tissue specificity of X-chromosome inactivation patterns.Blood. 1994; 83: 2899-2905Crossref PubMed Google Scholar] reported that 45% of females analyzed had different patterns of XCI when blood-derived cells were compared with muscle and skin samples. Interestingly, blood cells in both studies were more frequently skewed than other tissues. These studies suggested that XCI patterns were tissue specific. The second, and perhaps most important, clue to the discrepancy in incidence of skewing came with the analysis of XCI patterns in females of different age groups. An age difference in skewing was first suggested by Fey et al. [24Fey MF Liechti-Gallati S von Rohr A et al.Clonality and X-inactivation patterns in hematopoietic cell populations detected by the highly informative M27 beta DNA probe.Blood. 1994; 83: 931-938Crossref PubMed Google Scholar], who reported a higher incidence of skewing in older females compared with children using the M27β probe. We characterized the effect of age on skewing in a large cross-sectional study using the more robust and validated HUMARA assay in 295 normal females from three age groups: (1) neonates, (2) 28- to 32-year-olds, and (3) those >60 years old. We documented incidences of skewing (ratio ≥3:1) of 8.6% in neonates, 16.4% in the 28- to 32-year-olds, and 37.9% in women >60 years of age (p < 0.0001 vs. neonates, p = 0.064 vs. 28- to 32-year-olds) (17). These results have been confirmed by other investigators [15Mossner M Nolte F Hutter G et al.Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay.J Med Genet. 2013; 50: 108-117Crossref PubMed Scopus (20) Google Scholar,25Champion KM Gilbert JG Asimakopoulos FA Hinshelwood S Green AR Clonal haemopoiesis in normal elderly women: implications for the myeloproliferative disorders and myelodysplastic syndromes.Br J Haematol. 1997; 97: 920-926Crossref PubMed Scopus (138) Google Scholar, 26Gale RE Fielding AK Harrison CN Linch DC Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.Br J Haematol. 1997; 98: 512-519Crossref PubMed Scopus (195) Google Scholar, 27Tonon L Bergamaschi G Dellavecchia C et al.Unbalanced X-chromosome inactivation in haemopoietic cells from normal women.Br J Haematol. 1998; 102: 996-1003Crossref PubMed Scopus (74) Google Scholar, 28Racchi O Mangerini R Rapezzi D Rolfo M Gaetani GF Ferraris AM X chromosome inactivation patterns in normal females.Blood Cells Mol Dis. 1998; 24: 439-447Crossref PubMed Scopus (41) Google Scholar, 29El Kassar N Hetet G Briere J Grandchamp B X-chromosome inactivation in healthy females: incidence of excessive lyonization with age and comparison of assays involving DNA methylation and transcript polymorphisms.Clin Chem. 1998; 44: 61-67Crossref PubMed Scopus (44) Google Scholar, 30Hatakeyama C Anderson CL Beever CL Penaherrera MS Brown CJ Robinson WP The dynamics of X-inactivation skewing as women age.Clin Genet. 2004; 66: 327-332Crossref PubMed Scopus (110) Google Scholar, 31Sharp A Robinson D Jacobs P Age- and tissue-specific variation of X chromosome inactivation ratios in normal women.Hum Genet. 2000; 107: 343-349Crossref PubMed Scopus (271) Google Scholar]. A number of mechanisms have been put forward to explain the increased prevalence of skewing observed in hematopoietic cells including (1) acquired clonal hematopoiesis (mutation driven), (2) stochastic clonal dominance caused by hematopoietic stem cell (HSC) depletion, and (3) genetic predisposition [26Gale RE Fielding AK Harrison CN Linch DC Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.Br J Haematol. 1997; 98: 512-519Crossref PubMed Scopus (195) Google Scholar,32Sandovici I Naumova AK Leppert M Linares Y Sapienza C A longitudinal study of X-inactivation ratio in human females.Hum Genet. 2004; 115: 387-392Crossref PubMed Scopus (48) Google Scholar, 33Kristiansen M Knudsen GP Bathum L et al.Twin study of genetic and aging effects on X chromosome inactivation.Eur J Hum Genet. 2005; 13: 599-606Crossref PubMed Scopus (80) Google Scholar, 34Abkowitz JL Catlin SN Guttorp P Evidence that hematopoiesis may be a stochastic process in vivo.Nat Med. 1996; 2: 190-197Crossref PubMed Scopus (207) Google Scholar]. Several lines of evidence have supported a genetic contribution to the trait. Christensen et al. [35Christensen K Kristiansen M Hagen-Larsen H et al.X-linked genetic factors regulate hematopoietic stem-cell kinetics in females.Blood. 2000; 95: 2449-2451Crossref PubMed Google Scholar] analyzed peripheral blood cells from 71 elderly monozygotic (MZ) twin pairs and observed a strong correlation in the degree and direction of skewing between co-twins. Vickers et al. [36Vickers MA McLeod E Spector TD Wilson IJ Assessment of mechanism of acquired skewed X inactivation by analysis of twins.Blood. 2001; 97: 1274-1281Crossref PubMed Scopus (43) Google Scholar] studied the peripheral blood polymorphonuclear cells (PMNs) of 29 MZ and 18 dizygotic (DZ) twin pairs and observed a better intraclass correlation of XCI ratios between MZ twins than between DZ twins. They estimated heritability to account for 68% of the skewing observed in PMNs. Kristiansen et al. [33Kristiansen M Knudsen GP Bathum L et al.Twin study of genetic and aging effects on X chromosome inactivation.Eur J Hum Genet. 2005; 13: 599-606Crossref PubMed Scopus (80) Google Scholar] reported a comparable heritability estimate in a larger cohort of elderly MZ (n = 82) and DZ (n = 112) twin pairs. These studies collectively suggest that the skewing of XCI observed in hematopoietic cells is, at least in part, genetically determined. Evidence for an X-linked genetic component to skewing was provided by Abkowitz et al. [37Abkowitz JL Taboada M Shelton GH Catlin SN Guttorp P Kiklevich JV An X chromosome gene regulates hematopoietic stem cell kinetics.Proc Natl Acad Sci USA. 1998; 95: 3862-3866Crossref PubMed Scopus (82) Google Scholar], who analyzed the hematopoietic cells of aging female Safari cats (first-cross hybrids between Geoffroy and domestic cats), and found that XCI was random in young cats, but skewed in 67% of older cats. As recombination between the two parental X-chromosomes cannot occur in this animal model, the fact that skewing always favored the Geoffroy X-chromosome [37Abkowitz JL Taboada M Shelton GH Catlin SN Guttorp P Kiklevich JV An X chromosome gene regulates hematopoietic stem cell kinetics.Proc Natl Acad Sci USA. 1998; 95: 3862-3866Crossref PubMed Scopus (82) Google Scholar] suggested a hemizygous cell selection process and was not compatible with random processes. To investigate the genetic and nongenetic components of acquired skewing of XCI, we recruited 2,292 women of French-Canadian ancestry without any known hematologic disorders, ranging from 37 to 101 years of age. The cohort comprised 1,734 individuals belonging to 311 families and 558 unrelated individuals. We were intrigued by the observation that skewing was more prevalent in myeloid cells than in T cells or buccal cells and more age dependent. We hypothesized that skewing was probably multifactorial and that some subjects could have acquired true, mutation-driven clonal hematopoiesis. We first analyzed DNA from 3 elderly women with known skewing in their myeloid and polyclonal T cells using exome sequencing. We identified somatic mutations in TET2, DNMT3A, and SLC39A12 in one of them [38Busque L Patel JP Figueroa ME et al.Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis.Nat Genet. 2012; 44: 1179-1181Crossref PubMed Scopus (542) Google Scholar]. Extension of the analysis to various age groups led to the identification of missense, nonsense, and frameshift somatic TET2 mutations in the DNA from PMNs, but not from lymphocyte or epithelial cells, in 10 of 179 (5.6%) elderly subjects with XCI skewing, none of 105 elderly subjects without XCI skewing, and none of 96 younger subjects with XCI skewing. The hematologic parameters of TET2 mutant individuals did not differ from those of age-matched counterparts. This study was the first to report that an acquired mutation in a myeloid cancer-associated gene (TET2) is age dependent and compatible with normal hematopoiesis [38Busque L Patel JP Figueroa ME et al.Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis.Nat Genet. 2012; 44: 1179-1181Crossref PubMed Scopus (542) Google Scholar]. It also suggested that an acquired mutation in a driver gene is a small contributor to acquired XCI skewing in the general population (Figure 1). Simultaneously, Laurie et al. [40Laurie CC Laurie CA Rice K et al.Detectable clonal mosaicism from birth to old age and its relationship to cancer.Nat Genet. 2012; 44: 642-650Crossref PubMed Scopus (382) Google Scholar] reported an age-dependent clonal mosaicism for large chromosomal anomalies using single-nucleotide polymorphism (SNP) microarray data from more than 50,000 individuals recruited for genome-wide association studies. Clonal mosaicism was detected in 2%–3% of elderly individuals in contrast to 0.5% of individuals younger than 50 years. Furthermore, the presence of clonal mosaicism was associated with a 10-fold increased risk of developing a hematologic cancer. Jacobs et al. [41Jacobs KB Yeager M Zhou W et al.Detectable clonal mosaicism and its relationship to aging and cancer.Nat Genet. 2012; 44: 651-658Crossref PubMed Scopus (384) Google Scholar] and Forsberg et al. [42Forsberg LA Rasi C Razzaghian HR et al.Age-related somatic structural changes in the nuclear genome of human blood cells.Am J Hum Genet. 2012; 90: 217-228Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar] reported similar findings. In 2014, three groups reported analysis of DNA exome data sets from large cohorts of subjects and documented age-dependent mutations in driver genes [43Xie M Lu C Wang J et al.Age-related mutations associated with clonal hematopoietic expansion and malignancies.Nat Med. 2014; 20: 1472-1478Crossref PubMed Scopus (1068) Google Scholar, 44Genovese G Kahler AK Handsaker RE et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N Engl J Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1748) Google Scholar, 45Jaiswal S Fontanillas P Flannick J et al.Age-related clonal hematopoiesis associated with adverse outcomes.N Engl J Med. 2014; 371: 2488-2498Crossref PubMed Scopus (2204) Google Scholar]. These three cohorts had different inclusion criteria (nonhematologic cancer, psychiatric disorder, or cardiovascular disease), yet they identified a similar set of genes, suggesting a universal age-associated phenomenon. Although more than 70 different genes were identified, the most frequently mutated ones were the epigenetic modifiers DNMT3A, TET2, and ASXL1, nicknamed the "DAT" mutations. Importantly, the prospective data available for two of these studies revealed that subjects with clonal hematopoiesis had a roughly 10-fold heightened risk of developing a hematologic cancer [44Genovese G Kahler AK Handsaker RE et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N Engl J Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1748) Google Scholar,45Jaiswal S Fontanillas P Flannick J et al.Age-related clonal hematopoiesis associated with adverse outcomes.N Engl J Med. 2014; 371: 2488-2498Crossref PubMed Scopus (2204) Google Scholar]. The relative risk of mortality was modest at 1.4. However, clonal hematopoiesis was associated with an increased risk of cardiovascular events, which was further confirmed [46Jaiswal S Natarajan P Silver AJ et al.Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.N Engl J Med. 2017; 377: 111-121Crossref PubMed Scopus (920) Google Scholar]. This new entity has several designations, but it is generally accepted that age-related clonal hematopoiesis (ARCH) refers to clonal expansion irrelevant of the type of mutation [47Shlush LI Age-related clonal hematopoiesis.Blood. 2018; 131: 496-504Crossref PubMed Scopus (145) Google Scholar], and clonal hematopoiesis of indeterminate potential (CHIP), to clonal hematopoiesis caused by a mutation in a driver gene at a variant allele fraction (VAF) of >2% [48Steensma DP Bejar R Jaiswal S et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (964) Google Scholar]. Zink et al. [49Zink F Stacey SN Norddahl GL et al.Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly.Blood. 2017; 130: 742-752Crossref PubMed Scopus (334) Google Scholar], using a whole-genome sequencing (WGS) approach on a large cohort from Iceland, confirmed the high prevalence of mutations in driver genes, but an even higher rate of mutations in non-driver candidates. We recently reported the analysis of a large cohort of 2,530 normal individuals aged 55 to 101 years, using a more sensitive (relative to a genome-based approach) gene-targeted approach [50Buscarlet M Provost S Zada YF et al.DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions.Blood. 2017; 130: 753-762Crossref PubMed Scopus (180) Google Scholar]. We documented a high prevalence of mutations and even higher preponderance for mutations in DNMT3A or TET2—genes central to DNA methylation and demethylation. Clone size (VAF) was significant in a large proportion of affected individuals. The average VAF was 14.3%, which corresponds to 28.6% of cells originating from mutated stem cells. Almost half of the mutated cohort (48.7%) had a VAF ≥10%. There were no significant differences in blood cell parameters (cell numbers and indices) between mutant and aged-matched controls, except for a tendency toward reduced PMN in TET2 mutants. Interestingly, we have noted familial aggregation for TET2 mutations (Described in Etiology of ARCH/CHIP), as well as mutations in DNMT3A and TET2 originating at different levels of the hematopoietic hierarchy. DNMT3A lineage restriction patterns are compatible with a pluripotent stem cell origin, whereas TET2 mutations occur mainly in myeloid cells and sometimes in B cells [51Buscarlet M Provost S Zada YF et al.Lineage restriction analyses in CHIP indicate myeloid bias for TET2 and multipotent stem cell origin for DNMT3A.Blood. 2018; 132: 277-280Crossref PubMed Scopus (60) Google Scholar]. More disturbing to our current understanding of premalignant lesions, Young et al. [52Young AL Challen GA Birmann BM Druley TE Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults.Nat Commun. 2016; 7: 12484Crossref PubMed Scopus (334) Google Scholar] reported mutations in TET2 or DNMT3A at very low frequencies in 95% of individuals aged 50 to 60 years, suggesting that these mutations are almost ubiquitous after the age of 50. Aging HSCs face the potential of exhaustion. Several parameters differentiate old versus young HSCs (reviewed in [53de Haan G Lazare SS Aging of hematopoietic stem cells.Blood. 2018; 131: 479-487Crossref PubMed Scopus (134) G
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