X-inactivation analysis in the 1990s: promise and potential problems

158

130

Clonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolution. Genetic abnormalities are found in ∼50% of patients with AA and, except for PIGA mutations and copy-neutral loss-of-heterozygosity, or uniparental disomy (UPD) in 6p (6pUPD), are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including DNMT3A, ASXL1, and BCOR/BCORL1. Mutations exhibit distinct chronological profiles and clinical impacts. BCOR/BCORL1 and PIGA mutations tend to disappear or show stable clone size and predict a better response to IST and a significantly better clinical outcome compared with mutations in DNMT3A, ASXL1, and other genes, which are likely to increase their clone size, are associated with a faster progression to MDS/AML, and predict an unfavorable survival. High frequency of 6pUPD and overrepresentation of PIGA and BCOR/BCORL1 mutations are unique to AA, suggesting the role of autoimmunity in clonal selection. By contrast, DNMT3A and ASXL1 mutations, also commonly seen in CH in the general population, indicate a close link to CH in the aged bone marrow, in terms of the mechanism for selection. Detection and close monitoring of somatic mutations/evolution may help with prediction and diagnosis of clonal evolution of MDS/AML and better management of patients with AA.

Section: Skewed X-chromosome Inactivationmentioning

confidence: 99%

Section: Skewed X-chromosome Inactivationmentioning

confidence: 99%

Clonal hematopoiesis in acquired aplastic anemia

Ogawa

2016

158

130

“…Polymerase chain reaction (PCR) amplification of the polymorphic CAG repeat at the HUMARA locus [12][13][14] was performed in tandem on undigested (6-FAM-labeled primer) and on HpaII-digested (HEX-labeled primer) DNA. PCR products were analyzed on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA) to determine the area under the curve (AUC) for each allele.…”

Section: Humara Clonality Assay and X-inactivation Ratio Determinationmentioning

confidence: 99%

“…Allelic skewing consistent with clonal granulopoiesis was defined as a 3:1 ratio between X-linked alleles, which is equivalent to DS at least 0.25. 12,15 Patients with "clonal granulopoiesis" have at least 50% clonally derived granulocytes but may have 50% or fewer admixed polyclonal cells; conversely, patients with "polyclonal granulopoiesis" may have 50% or fewer admixed clonal granulocytes. …”

mentioning

confidence: 99%

X-inactivation–based clonality analysis and quantitative JAK2V617F assessment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F-negative ET and MMM patients with clonal hematopoiesis

Levine

Bélisle

Wadleigh

et al. 2006

213

176

The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and in some patients with essential thrombocythemia (ET) and myeloid metaplasia/ myelofibrosis (MMM). We sought to investigate the relationship between granulocyte clonality and JAK2V617F allelic ratio. A total of 168 of 190 female patients were informative for a clonality assay at the HUMARA locus; 80% of MMM, 75% of PV, and 67% of ET patients demonstrated clonal granulopoiesis. The JAK2V617F allele was detected by quantitative real-time polymerase chain reaction (PCR) in 99% of PV, 72% of ET, and 39% of MMM. A correlation between clonality and JAK2V617F allelic ratio was demonstrated for PV (P < .001) but not for ET or MMM (both P > .6). These data suggest that acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary in ET and MMM. In addition, some ET and MMM patients with clonal granulopoiesis have somatic mutations other than JAK2V617F. IntroductionIn 1951, Dr William Dameshek classified polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia/myelofibrosis (MMM), and chronic myelogenous leukemia (CML) as phenotypically related myeloproliferative disorders (MPDs). 1 The molecular pathogenesis of BCR-ABL-negative MPDs was poorly understood until the recent identification of the JAK2V617F activating mutation. 2-8 JAK2V617F confers factor-independent growth and erythropoietin hypersensitivity to hematopoietic cells, 2,3 and expression of JAK2V617F in a murine bone marrow transplantation model results in erythrocytosis. 3 These data suggest that acquisition of JAK2V617F is an important pathogenetic event in JAK2V617F-positive MPDs. Nonetheless, some PV, ET, and MMM patients are JAK2V617F negative, as assessed by DNA resequencing. There are several potential explanations for this observation, including mutations in other genes that phenocopy JAK2V617F. Granulocytes from some patients with PV and ET are polyclonal, 9,10 and thus sequence analysis of granulocyte DNA may underestimate the true frequency of the JAK2V617F allele. Although granulocyte clonality and JAK2V617F mutational status were reported in ET, 11 no study has investigated the relationship between the acquisition of JAK2V617F and the emergence of clonal hematopoiesis in PV, ET, and MMM. We therefore investigated the relationship between clonality and JAK2V617F allelic ratio in MPDs. Study design Harvard Myeloproliferative Disorders StudyThe Harvard Myeloproliferative Disorders Study enrolled patients with PV, ET, and MMM to collect clinical information and biologic samples. 2 All subjects provided informed consent. Approval for the studies was obtained from the Dana-Farber Cancer Institutional Review Board. HUMARA clonality assay and X-inactivation ratio determinationPolymerase chain reaction (PCR) amplification of the polymorphic CAG repeat at the HUMARA locus 12-14 was performed in tandem on undigested (6-FAM-labeled primer) and on HpaII-digested (HEX-labeled primer) DNA. PCR produc...

“…31 It is likely that most cases of acquired skewing in apparently healthy individuals are due to a growth advantage conferred by parent-specific X-linked alleles (hemizygous selection), 33 although other causes, such as stem cell depletion and the expansion of premalignant clones, have been suggested. 29,34 Autologous HSCT experiments in female Safari cats have shown that low stem cell doses can result in skewed X-inactivation ratios, suggestive of oligoclonal hematopoiesis, and an extended period of clonal instability after transplantation. 29 Polyclonal hematopoiesis has been demonstrated in most human allogeneic HSCT recipients studied.…”

Section: Introductionmentioning

confidence: 99%

Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss

Thornley

Sutherland

Wynn

et al. 2002

Our inability to purify hematopoietic stem cells (HSCs) precludes direct study of many aspects of their behavior in the clinical hematopoietic stem cell transplantation (HSCT) setting. We indirectly assessed stem/progenitor cell behavior in the first year after HSCT by examining changes in neutrophil telomere length, X-inactivation ratios, and cycling of marrow progenitors in 25 fully engrafted allogeneic HSCT recipients. Donors were sampled once and recipients at engraftment and 2 to 6 months and 12 months after HSCT. Telomere length was measured by an in-gel hybridization technique, X-inactivation ratios were measured by the human androgen receptor assay, and cell cycle status was determined by flow cytometric analysis of pyronin Y-and Hoechst 33342-stained CD34 ؉ CD90 ؉ and CD34 ؉ CD90 ؊ marrow cells. Compared with their donors, recipients' telomeres were shortened at engraftment (؊424 base pairs [bp]; P < .0001), 6 months (؊495 bp; P ‫؍‬ .0001) after HSCT, and 12 months after HSCT (؊565 bp; P < .0001). There was no consistent pattern of change in telomere length from 1 to 12 months after HSCT; marked, seemingly random, fluctuations were common. In 11 of 11 informative recipients, donor X-inactivation ratios were faithfully reproduced and maintained. The proportion of CD34 ؉ CD90 ؉ progenitors in S/G 2 /M was 4.3% in donors, 15.7% at 2 to 6 months (P < .0001) after HSCT, and 11.5% at 12 months after HSCT (P < .0001, versus donors; P ‫؍‬ .04, versus 2-6 months). Cycling of CD34 ؉ CD90 ؊ progenitors was largely unchanged. We infer that (1) HSCT-induced accelerated telomere loss is temporary and unlikely to promote graft failure or clonal hematopoietic disorders and (2)