Familial polycythemia vera with non-germ line JAK2V617F mutation sparing the abnormal and clonal granulopoiesis

Recently, Bellanné-Chantelot and co-workers have studied a series of 72 families with Philadelphia-chromosome-negative chronic myeloproliferative disorders (Ph CMPD) and found constant JAK2V617F mutation in 22 families.1 Although the distribution of Ph CMPD phenotypes within families suggested autosomal-dominant inheritance with incomplete penetrance, the absence of the JAK2V617F mutation both in purified B/T lymphocytes in 13 unrelated patients and variable ratios of the mutant allele in peripheral leukocytes indicated that JAK2V617F was acquired and not transmitted by germ line.1

We present a case of familial Ph CMPD with JAK2V617F mutation revealing different patterns of hematopoietic lineage involvement. The somatic nature of the mutation could be proven by comparison with non-hematopoietic tissues.

An 80-year-old woman suffered from polycythemia vera (PV) since 11 years, and recently PV was also diagnosed in her 49-year-old daughter. Diagnosis was confirmed by histological examination of bone marrow (BM) biopsies, detection of JAK2V617F in BM2 and elevated CD177 levels in peripheral blood (PB) granulocytes.3, 4 Cytogenetic and array-based comparative genomic hybridization analyses were performed as described.5 No aberrations were detected. Subsequently, JAK2V617F mutant gene dosage was quantified with the PCR-based pyrosequencer6 in separated hematopoietic lineages. Purification was performed either by laser microdissection of BM cells6 or by flow cytometric cell sorting of PB CD15+ granulocytes, CD19+ B and CD3+ T lymphocytes.6 As a germ line control, we used laser-microdissected gastric epithelial cells from the mother and buccal cells from the daughter. Analysis of JAK2V617F-mutated alleles in laser-microdissected BM cells of the mother revealed 78% for erythropoietic and 20% for megakaryocytic cells. Analysis of the daughter's cells rendered similar values (51 and 21%, respectively) (Figure 1a). Erythropoietic islands were identified by immunohistochemical labeling of hemoglobin (Figure 1b).

Figure 1

Molecular and hematological parameters in mother and daughter with polycythemia vera. (a) Several hematological lineages were analyzed: BM-derived hemoglobin-positive erythroid progenitors (HB+, 1:20, polyclonal rabbit, Quartett, Germany) and megakaryocytes separated by laser microdissection from BM biopsies and PB-derived CD15+, CD3+ and CD19+ cells sorted by flow cytometry. Non-hematological epithelial tissues served as germ line control: gastric epithelial cells from the mother (CD45+/CD68+ leukocytes-free, 1:200/1:100, monoclonal mouse, Dako, Denmark; diagnostically taken biopsy for exclusion of gastritis) and oral epithelial cells from the daughter (buccal cotton-swab sampling). Laser microdissection, DNA extraction and PCR amplification of 90 bp β-globin and 102 bp JAK2 fragments, and mutation analysis by pyrosequencing (Biotage, Uppsala, Sweden) was performed as described.6 Results were confirmed by direct sequencing (shown for CD15+ cells, JAK2 wild-type position 1849C/G or mutant A/T). In addition, density gradient-fractioned PB granulocytes/monocytes were analyzed and revealed a JAK2 wild-type status in the daughter (data not shown). HUMARA was performed as described3 and showed monoclonal (MO) CD15+ and polyclonal (PO) CD3+ and CD19+ cells. Relative CD 177 (polycythemia rubra vera 1 receptor, PRV1) mRNA-expression levels were determined as described3, 4 and were elevated in PB granulocytes of both mother (353.1) and daughter (91.1) compared to healthy controls (n=5, median 0.8, range: 0.4–1.7). Abbreviations: BM, bone marrow; HB, hemoglobin; HK, hematokrit; HUMARA, human androgen receptor gene assay; PB, peripheral blood; PLT, platelet counts; RBC, red blood cell counts; WBC, white blood cell counts. Elevated parameters were indicated (↑). For both, therapy was based on intermittent phlebotomy and hydroxyurea. Also, the mother received interferon-α, which resulted in normalization of initial erythrocytosis (7.0 × 109/l) and thrombocytosis (1000 × 109/l). Samples from the father were not available for analysis. (b) Laser microdissection of HB+ erythroid progenitors (upper row) and gastric glands (lower row, CD45+/CD68+ were indicated by asterisks, visualized by DAB-brown, counterstained with hematoxylin and methylene blue, respectively; original magnification × 200, PALM Laser-MicroBeam System, PALM, Wolfratshausen, Germany, with an Olympus IX70 microscope, Olympus, Hamburg, Germany). Sequence begins from the left with accurate isolation and subsequent catapultation into the lid of a PCR tube for further processing.

Interestingly, the daughter's CD15+ cells were clonal, as evidenced by human androgen receptor gene assay (HUMARA),3 but were devoid of the JAK2V617F mutation7 (Figure 1a). By contrast, the maternal CD15+ cells showed 81% JAK2V617F alleles. In addition, the mother's granulocytes exhibited a 4 times higher CD177 level than the daughter's, resembling sporadic PV.3 Epithelial cells and lymphoid cells revealed a germ line configuration of JAK2.

This case of familial PV sheds light on several aspects of the still incompletely understood biology of the JAK2V617F mutation in hematopoietic cells. Although familial, it is obviously not transmitted by germ line, as evidenced for the first time, by comparison with non-hematopoietic cells. The pattern of lineage involvement in familial JAK2V617F mutation may differ among diseased family members. Furthermore, the case illustrates that granulocytes in PV may be clonal, but they are not affected by the JAK2V617F mutation.7, 8 Despite the absence of the JAK2V617F mutation, the granulocytes of the daughter were enhanced in number and exhibited an exaggerated CD177 expression typically found in PV.3 Non-germ line transmission as well as abnormal and clonal but unmutated granulopoiesis may be considered as an indication that, at least in a subfraction of Ph CMPD, the JAK2V617F mutation constitutes a secondary event to still unknown primary genetic aberrations, which potentially convey an inherited predisposition for hematopoietic cells to acquire the JAK2V617F mutation.


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This study was supported by grants from Deutsche Krebshilfe, Dr Mildred Scheel Stiftung 10-2191 (OB, HK) and Deutsche Forschungsgemeinschaft –DFG BO 1954/1-1 (OB, HK).

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Correspondence to H Kreipe.

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Hussein, K., Bock, O., Ballmaier, M. et al. Familial polycythemia vera with non-germ line JAK2V617F mutation sparing the abnormal and clonal granulopoiesis. Leukemia 21, 2566–2568 (2007). https://doi.org/10.1038/sj.leu.2404846

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