Abstract

First and second authors contributed equally.

During early female embryogenesis, the most of genes in either the maternal or paternal X-chromosomes are randomly inactivated; this remains remarkably constant in the progeny of these cells throughout life. The X-inactive specific transcript gene (XIST), which encodes long non-coding RNA, is expressed only from the inactive X-chromosome and plays a crucial role in this process. X-chromosome inactivation has been used to define clonality of malignant and premalignant disorders such polycythemia vera (PV) and essential thrombocythemia (ET). Using a quantitative, transcriptional clonality assay based on polymorphisms on 5 X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD), we found that the allelic usage ratio of these genes varies among normal females, and from tissue to tissue, but is the same in all blood cell lineages, including T-cells, and remained constant over 3 years of study (Prchal, J Exp Med, 1996). Further, in females heterozygous for more than 1 of these genes, the allelic usage ratio is the same for each marker. Over more than a decade, we analyzed over 150 informative PV and JAK2V617F-positive ET females and all were clonal. However, we recently encountered 4 exceptional cases. Two PV (P1 and P3) and 1 ET (P2) females appeared polyclonal using an IDS marker, and one female (P4), using a G6PD marker, in platelets and/or granulocytes, whereas all of these females appeared clonal using at least 1 other X-chromosome marker in these cells. Further, T-cell IDS allelic ratios were different from those in platelets and granulocytes; T-cell X-chromosome usage ratio is, in any female, a marker of normal hematopoiesis prior to a clonal event.

It was recently reported that the conditional deletion of Xist in hematopoietic stem cells in female mice leads to complete penetrance of a highly aggressive PV/ET-like syndrome that progresses to invariably fatal lymphoma and or acute leukemia. Further, reactivation of X-chromosome has been reported in ovarian and breast cancers. We propose that reactivation of inactive IDS and G6PD in these females, and perhaps other X-chromosome genes, contributes to the PV/ET phenotype.

In ongoing studies, we performed additional experiments on 2 of these females (P1 and P4). We analyzed their X-chromosome transcripts using clonogenic assays of early erythroid progenitors, which formed erythroid colonies (burst-forming units-erythroid, BFU-Es) in vitro when cultured in methylcellulose in the presence of EPO. In controls, each BFU-E expressed only a single X-chromosome transcript; however, in both of these females we observed at least 1 clone which expressed both X-chromosome genes. We then investigated the epigenetic status of the IDS gene in P1. We performed chromatin immunoprecipitation of granulocyte DNA from P1 using histone H3 lysine 27 trimethylation (H3K27m3) antibodies to assess the level of polycomb silencing in the immunoprecipitated granulocyte DNA fragments. In this analysis, the IDS gene was ~4 times less enriched for H3K27m3 compared to normal controls (see Figure). Ongoing analysis of the XIST gene revealed normal mRNA levels in P1 and P4 and inexplicably increased XIST gene transcript in P2 and P3. Analysis of XIST transcripts in platelets, erythroid progenitors and stem cells is ongoing, as well as sequencing of the XIST gene. Preliminary DNA methylation analysis of granulocytes and CD34+ cells from P1 revealed focal hypomethylation of the inactivated X-chromosome, including promoters of SRPL3, PIM2, PHKA1, PHKA2, and RPS6KA3 genes; in all four subjects, platelet and granulocyte transcripts of 2 of these genes, PIM2 and PHKA1, were overexpressed compared to normal controls.

These results suggest that reactivation of some X-chromosome genes in PV and ET clones present in some female patients plays an important role in the diversity of PV and ET phenotypes and may also play a role in the demonstrated differences in PV and ET phenotypes between males and females (Spivak JL, N Engl J Med, 2013).

Disclosures

Issa:Astex Pharmaceuticals, Inc.: Consultancy, Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.