Program: Oral and Poster Abstracts
Session: 635. Myeloproliferative Syndromes: Basic Science: Poster III
SK and JJ : deserve equal credit ; SS and JTP : senior authors
Clonal
populations can be detected through indirect measures, such as the expression
of surrogate genes as in the case of X-chromosome inactivation. X-chromosome inactivation has been used to define the clonality of
malignant and premalignant disorders such as polycythemia vera (PV) and
essential thrombocythemia (ET). We developed a quantitative, transcriptional
clonality assay (qTCA) based on polymorphisms for five X-chromosome genes (MPP1,
FHL1, IDS, BTK, and G6PD) (Swierczek S, Blood, 112;p.3168,
2008). We found that the allelic usage ratio of these genes varies among normal
females and from tissue to tissue, but is the same in any given female in all
blood cell lineages and is stable in time. It was previously reported that
reactivation of inactivated X-chromosomes is seen in some human breast cancers
(Richardson A., Cancer cell, 9;p.121, 2006,). However, these
abnormalities did not lead to a global increase in X-chromosome transcription
but were associated with overexpression of a small subset of X-chromosomal
oncogenes and/or tumor suppressor genes (Thakur A., Mole Cancer Res, 2007). The
conditional deletion of Xist in hematopoietic stem cells in
female mice resulted in X-chromosome reactivation and led to complete
penetrance of a highly aggressive PV/ET-like syndrome that progressed to
invariably fatal lymphoma and/or acute leukemia (Yildirim E, Cell 152;p.
727, 2013).
In three PV/ET female patients, we observed expression of both alleles
of the IDS and G6PD genes in
clonal platelets and granulocytes, whereas in these three females only a single
other X-chromosome allele was expressed. We concluded that reactivation of
inactive IDS and G6PD, and perhaps other X-chromosome genes, occurred
in the PV/ET clones of these females (Kim SJ & Swierczek S, ASH abstract
#3225, 2014). We then investigated the epigenetic status of the IDS and G6PD
genes in these patients.
We performed chromatin immunoprecipitation of granulocyte DNA from
all three patients using histone H3 lysine 27 trimethylation (H3K27me3) antibody
to assess the level of polycomb silencing. The IDS and G6PD genes in these PV
females had decreased the H3K27me3 compared to normal controls.
We next performed DNA
methylation analysis focused on the X-chromosome CpG sites located within 1kb
from gene transcription start sites. We have analyzed 19 PV females 9 control
females and 22 control males. We observed discrete regions showing focal
hypomethylation in PV compared to control females (Fig. 1).
In
the PV females, there were large regions of X-chromosome, which were composed
of the same number of methylated and unmethylated regions as control females.
However, there were also segments of X-chromosome genes, which were less
methylated than in control females (Fig. 1). Some of these genes included variable
hypomethylation in promoter regions. We then isolated clonal platelets and
granulocytes, and transcripts of these genes were quantified by qT-PCR. Inexplicably,
the transcripts of these genes were decreased in most PV females (p<0.05) (Fig.
2). The molecular basis of this unexpected decrease in transcripts of these
hypomethylated X-chromosome genes in PV is currently being interrogated,
including possible activation of anti-sense transcripts.
In
conclusion, we demonstrate significant X-chromosome demethylation, which
appears to correlate with reactivation of some X-chromosome genes in PV and ET
females. Defining the molecular basis of these observations is important because
the conditional reactivation of inactivated X-chromosome genes in hematopoietic
stem cells leads to aggressive MPN-like disease in mice and expansion of their stem
cells. Further, PV females are more likely to have PV and have a different
clinical course.
Disclosures: No relevant conflicts of interest to declare.
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