Validation of a Novel Clonality Assay in Patients with Clonal Hematologic Disorders

Abstract Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. They are useful in the diagnosis and assessment of therapeutic response in clonal hematologic disorders, especially in absence of an identifiable somatic mutations. Skewing of X-chromosome allelic-usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in ~30% elderly women, precluding the use of this assay in elderly patient (>65 years of age). Using a quantitative, transcriptionally-based clonality assay, we did not detect clonal hematopoiesis in >200 healthy non-elderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in forty elderly women (age 65–92, mean 81.3 years), using a novel, quantitative qPCR transcriptional clonality assay. In this assay, mRNA transcribed from five X-chromosome polymorphic genes expressed in peripheral blood neutrophils is quantified by real time, allele specific RT-PCR. We did not detect clonal hematopoiesis in any of the elderly women. However, using HUMARA assay, 30% of these elderly women were detected to have monoallelic methylation of the HUMARA gene locus, consistent with previously reported literature. We concluded that our novel transcriptional clonality assay is suitable for evaluation of clonal hematopoiesis in all women including elderly women (Swierczek S, Agarwal N et al. Blood 2008, July 18 epub). Using this novel assay, we detected clonal hematopoiesis in 31 out of 32 well characterized patients with myeloproliferative disorders: polycythemia vera (all fourteen patients were clonal by our assay and all were JAK2V617F positive), essential thrombocytosis (nine out of ten patients were clonal by our assay, one out of ten patients was cMPLW515L positive, seven out of ten were JAK2 V617F positive; however one subject with low JAK2 V617F allelic burden was polyclonal by our assay), and primary myelofibrosis (all eight patients were clonal by our assay and two of them were positive for JAK2V617F). In addition, we detected clonal hematopoiesis in 4 patients with unexplained anemia (two eventually evolved in to myelodysplastic syndrome), and in one patient with persistent leukocytosis (eventually found to be cMPLW515L positive). Using our assay we did not detect clonal hematopoiesis in 10 patients with reactive or secondary erythrocytosis, thrombocytosis or leukocytosis. We conclude that our novel transcriptional clonality assay is suitable for detection of clonal hematopoiesis in patients with clonal hematologic disorders, especially in patients lacking known somatic mutation. Studies to detect an emerging clone in milieu of polyclonal hematopoiesis (such as seen in PNH or early stages of clonal hematological disorders) by comparison of X-chromosome allelic usage ratio in myeloid cells and in T lymphocytes are underway.

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Hematopoiesis is not clonal in healthy elderly women

Blood ◽

10.1182/blood-2008-03-143925 ◽

2008 ◽

Vol 112 (8) ◽

pp. 3186-3193 ◽

Cited By ~ 40

Author(s):

Sabina I. Swierczek ◽

Neeraj Agarwal ◽

Roberto H. Nussenzveig ◽

Gerald Rothstein ◽

Andrew Wilson ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

X Chromosome ◽

Elderly Women ◽

Quantitative Polymerase Chain Reaction ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Healthy Women ◽

Healthy Elderly ◽

Age Related

Abstract Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. Skewing of X-chromosome allelic usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in approximately 30% of elderly women. Using a quantitative, transcriptionally based clonality assay, we reported X-chromosome–transcribed allelic ratio in blood cells of healthy women consistent with random X-inactivation of 8 embryonic hematopoietic stem cells. Furthermore, we did not detect clonal hematopoiesis in more than 200 healthy nonelderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in elderly women. Forty healthy women (ages 65-92 years; mean, 81.3 years) were tested by a novel, quantitative polymerase chain reaction (qPCR) transcriptional clonality assay. We did not detect clonal hematopoiesis in any of the tested subjects. We also tested DNA from the same granulocyte samples using the methylation-based HUMARA assay, and confirmed previous reports of approximately 30% extensively skewed or monoallelic methylation, in agreement with likely age-related deregulated methylation of the HUMARA gene locus. We conclude that the transcriptionally based X-chromosome clonality assays are suitable for evaluation of clonal hematopoiesis in elderly women.

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Abstract WP325: X, but Not Y, Sex Chromosome Contributes to Stroke Sensitivity in Aged Mice

Stroke ◽

10.1161/str.51.suppl_1.wp325 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Shaohua Qi ◽

Abdullah Al Mamun ◽

Romana Sharmeen ◽

Conelius Ngwa ◽

Louise D. McCullough ◽

...

Keyword(s):

X Chromosome ◽

Sex Chromosome ◽

Elderly Women ◽

The Elderly ◽

Artery Occlusion ◽

Natural Menopause ◽

Aged Mice ◽

Tnf Α ◽

Significant Difference ◽

Four Core Genotype

Introduction: Stroke is a sexually dimorphic disease. Women are protected against ischemia compared to men before menopause due to estrogen’s neuroprotection; after menopause the elderly women become vulnerable to stroke attack. Our previous studies with four core genotype mice found a chromosomal effect (either X or Y) in stroke sensitivity. Recently, we found two X-linked genes ( Kdm6a and Kdm5c ) that escape from X chromosome inactivation (XCI) are higher expressed in aged female vs. male microglia after stroke. KDM6A and KDM5C are histone demethylases that modify gene expression of inflammatory mediators. By these early studies, we hypothesized that the second X chromosome contributes to stroke sensitivity in aged mice through immune responses mediated by KDM6A and KDM5C. Methods: XY* aged (18-22 months) mice (natural menopause cohort) that have four genotypes (XO, XX, XY, XXY) were subjected to middle cerebral artery occlusion (MCAO). Another cohort of gonadectomized XY* mice were also used as the “surgical menopause” cohort. Infarct volumes and behavior deficits were quantified 3 days after MCAO. KDM6A and KDM5C localization with microglial marker TMEM119 was examined by IHC. Plasma inflammatory cytokine (IL-1β, TNF-α, IL-6, IL-4, TL-10, etc.) levels were analyzed with MultiPlex. The contribution of the second X-chromosome to stroke sensitivity was determined by comparing XX vs. XO or XXY vs. XY mice, and the effect of the Y-chromosome was evaluated by a comparison between XY vs. XO and XXY vs. XX mice. Results: In both surgical and natural menopause cohorts, XX and XXY mice showed worse stroke outcomes compared to XO or XY mice respectively; however, no significant difference was found between XX vs. XXY or XO vs. XY mice. IHC results showed higher expression of KDM6A and KDM5C in TMEM119 positive cells in mice with two vs. one copy of X chromosome. XXY mice had significantly higher levels of circulating TNF-α and IL-6 than XY mice. Conclusion: The second X chromosome contributes to stroke sensitivity in mice. Kdm6a and Kdm5c may play important roles in mediating post-stroke inflammation. Future work will genetically manipulate the expression of Kdm6a and Kdm5c in microglia to examine the roles of the two XCI escapee gene in stroke.

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Clonal Hematopoiesis, Cardiovascular Diseases and Hematopoietic Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21217902 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7902

Author(s):

Oleg Kandarakov ◽

Alexander Belyavsky

Keyword(s):

Cardiovascular Diseases ◽

Cell Biology ◽

Driving Force ◽

The Elderly ◽

Stem Cell Biology ◽

Driver Genes ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Somatic Mutagenesis ◽

Progression Of Atherosclerosis

Cardiovascular diseases and cancer, the leading causes of morbidity and mortality in the elderly, share some common mechanisms, in particular inflammation, contributing to their progression and pathogenesis. However, somatic mutagenesis, a driving force in cancer development, has not been generally considered as an important factor in cardiovascular disease pathology. Recent studies demonstrated that during normal aging, somatic mutagenesis occurs in blood cells, often resulting in expansion of mutant clones that dominate hematopoiesis at advanced age. This clonal hematopoiesis is primarily associated with mutations in certain leukemia-related driver genes and, being by itself relatively benign, not only increases the risks of subsequent malignant hematopoietic transformation, but, unexpectedly, has a significant impact on progression of atherosclerosis and cardiovascular diseases. In this review, we discuss the phenomenon of clonal hematopoiesis, the most important genes involved in it, its impact on cardiovascular diseases, and relevant aspects of hematopoietic stem cell biology.

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Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0054 ◽

2019 ◽

Vol 44 (4) ◽

pp. 492-498

Author(s):

Gonca Gulbay ◽

Elif Yesilada ◽

Mehmet Ali Erkurt ◽

Harika Gozukara Bag ◽

Irfan Kuku ◽

...

Keyword(s):

Blood Cell ◽

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Hematological Parameters ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

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Blast Transformation in a Patient with Primary Myelofibrosis Initiated from JAK2 V617F Progenitor.

Blood ◽

10.1182/blood.v110.11.4665.4665 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4665-4665 ◽

Cited By ~ 1

Author(s):

Sabina I. Swierczek ◽

Donghoon Yoon ◽

Josef T. Prchal

Keyword(s):

X Chromosome ◽

Real Time Pcr ◽

De Novo ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Quantitative Real Time Pcr ◽

Leukemic Transformation ◽

Cd34 Cells ◽

V617f Mutation

Abstract Myeloproliferative disorders (MPDs) are caused by clonal proliferation arising from a single multi-lineage stem cell. The JAK2 V617F mutation has been reported in greater than 90% of patients with polycythemia vera (PV), and ∼50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). However, several studies have indicated that the JAK2 V617F mutation is not an MPD initiating mutation but rather represents clonal evolution of these MPDs. Jelinek and colleagues first reported that most PV transformed acute leukemias are JAK2 V617F negative (Jelinek, Blood, 2005 106:3370). More recently, the role of the JAK2 V617F mutation in leukemic transformation in 27 patients with MPDs revealed that most JAK2 V617F -positive MPD patients transformed to a JAK2 V617F -negative AML (Theocharides, Blood, 2007 110:375); however, in the 4 patients with an apparent JAK2 V617F -positive leukemia clonality of leukemic blasts and mature granulocytes was not determined. Two models proposed by Theocharides et al may explain these findings. First, MPD and AML represent 2 independent clones that arose de novo from different progenitors. Second, MPD and AML are 2 subclones derived from a common progenitor. Here, we describe a woman with PMF with transformation to AML. We determined her JAK2 V617F mutation status by sensitive and quantitative real-time PCR (Nussenzveig, Exp Hematol, 2007 3:32). At the time of her transformation to AML, her normal appearing peripheral blood granulocytes were purified and the frequency of mutant JAK2 allele T was 6%. However, all FACS-sorter isolated CD34+ cells (enriched to 95% purity) were heterozygous for the JAK2 V617F mutation. To determine if MPD and AML clones arose de novo or from the same progenitor, we performed clonality studies using a newly developed sensitive and quantitative real-time PCR based on the X-chromosome inactivation principle using transcriptional clonality assays in granulocytes and CD34+ purified cells from peripheral blood at both stages of disease (see Swierczek et al, abstract, this meeting). When this woman’s PMF was first discovered, hematopoiesis was clonal, based on heterozygosity for three X-chromosome genes, FHL1, G6PD and IDS (Liu, Blood, 2003 101:3294) and their single allelic expression in granulocytes and platelets. At the time of leukemic transformation, both her granulocytes and leukemic CD34+ cells expressed all three identical isoforms from the same parental X chromosome. Our findings indicate that leukemic transformation does not invariably arise from a JAK2 V617F negative progenitor. This has important implication for therapy of MPDs with JAK2 V617F inhibitors, as these would not prevent leukemic transformation. It remains to be determined if the JAK2 background of leukemic progenitors is variable, and if there are differences between PV and PMF.

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Combination approach to diagnosis and treatment of an elderly patient with chronic Ph-negative myeloproliferative neoplasm and concomitant surgical pathology. Clinical observation

MD-Onco ◽

10.17650/2782-3202-2021-1-1-61-65 ◽

2021 ◽

Vol 1 (1) ◽

pp. 61-65

Author(s):

Yu. E. Ryabukhina ◽

P. A. Zeynalova ◽

O. I. Timofeeva ◽

F. M. Abbasbeyli ◽

T. V. Ponomarev ◽

...

Keyword(s):

Elderly Patient ◽

Essential Thrombocythemia ◽

Early Stage ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Left Femur ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms

Chronic myeloproliferative neoplasms (CMPN), Ph-negative, are of clonal nature, develop on the level of hematopoietic stem cell and are characterized by proliferation of one or more hematopoietic pathways. Currently, the group of Ph-negative CMPN includes essential thrombocythemia, primary myelofibrosis, polycythemia vera, myeloproliferative neoplasm unclassifiable.Identification of mutations in the Jak2 (V617F), CALR, and MPL genes extended understanding of biological features of Ph-negative CMPN and improved differential diagnosis of myeloid neoplasms. Nonetheless, clinical practice still encounters difficulties in clear separation between such disorders as primary myelofibrosis, early-stage and transformation of essential thrombocythemia into myelofibrosis with high thrombocytosis. Thrombocytosis is one of the main risk factors for thromboembolic complications, especially in elderly people.A clinical case of an elderly patient with fracture of the left femur developed in the context of Ph-negative CMPN (myelofibrosis) with high level of thrombocytosis is presented which in combination with enforced long-term immobilization and presence of additional risk created danger of thrombosis and hemorrhage during surgery and in the postoperative period.

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Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.482.482 ◽

2015 ◽

Vol 126 (23) ◽

pp. 482-482

Author(s):

Koki Ueda ◽

Kazuhiko Ikeda ◽

Kazuei Ogawa ◽

Akiko Shichishima-Nakamura ◽

Kotaro Shide ◽

...

Keyword(s):

Disease Progression ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Extramedullary Hematopoiesis ◽

Hematopoietic Stem ◽

Cell Counts ◽

Epigenetic Modifiers ◽

One Year

Abstract Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression. Disclosures No relevant conflicts of interest to declare.

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Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Blood ◽

10.1182/blood.v124.21.4590.4590 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4590-4590

Author(s):

Xylina Gregg ◽

Sabina Swierczek ◽

Soo Jin Kim ◽

Josef T. Prchal

Keyword(s):

T Cells ◽

X Chromosome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Calr Mutations ◽

Calr Mutation

Abstract First and second authors contributed equally During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio. JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent. We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes. Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2V617F-positive ET or PMF females all had clonal hematopoiesis. We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described. Disclosures No relevant conflicts of interest to declare.

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Methylation of AR locus does not always reflect X chromosome inactivation state

Blood ◽

10.1182/blood-2011-11-390351 ◽

2012 ◽

Vol 119 (13) ◽

pp. e100-e109 ◽

Cited By ~ 30

Author(s):

Sabina I. Swierczek ◽

Lucie Piterkova ◽

Jaroslav Jelinek ◽

Neeraj Agarwal ◽

Sue Hammoud ◽

...

Keyword(s):

Dna Methylation ◽

X Chromosome ◽

Elderly Women ◽

Methylation Status ◽

Methylation Pattern ◽

Elderly Persons ◽

Quantitative Expression ◽

Clonal Hematopoiesis ◽

Healthy Elderly ◽

Gene Assay

Abstract Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

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JAK2 V617F Mutation in Essential Thrombocythemia.

Blood ◽

10.1182/blood.v108.11.4925.4925 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4925-4925

Author(s):

Jeong Yeal Ahn ◽

Pil Whan Park ◽

Yiel Hea Seo ◽

Dong-Bok Shin ◽

Jae-Hoon Lee ◽

...

Keyword(s):

Bone Marrow ◽

Essential Thrombocythemia ◽

Statistical Significance ◽

Vascular Complications ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Janus Kinase ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Specific Association

Abstract Background: Essential thrombocythemia (ET) is thought to reflect transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis has remained obscure. But tyrosine kinase, especially Janus kinase 2 (JAK2) has been implicated in myeloproliferative disorders other than chronic myeloid leukemia. We investigated the incidence and its correlation with other clinicopathologic variables of JAK2 mutation in patients with ET and reactive thrombocytosis (RT). Method: JAK2 mutation analysis, using allele-specific polymerase chain reaction, was undertaken on genomic DNA from bone marrow aspirates of 24 patients with ET and peripheral blood in 36 patients with RT. Results: JAK2 mutation was detected in 11 patients (46%) among the 24 patients with ET and was not found in 36 patients with RT. In patients with ET, older age and leukocytosis were related with JAK2 mutation without statistical significance (p=0.172 and 0.094, respectively). But this mutation was not correlated with sex, hemoglobin, platelet count, splenomegaly, increased cellularity of bone marrow, bone marrow fibrosis and vascular complications. Conclusions: The current observation strengthens the specific association between JAK2 mutation and ET. At the diagnosis of ET, identification of JAK2 mutation should be incorporated in foundation for new approaches.

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