We studied 10 affected and 11 non affected relatives of a five generation kindred with autosomal dominant familial erythrocytosis. We have excluded other known inherited forms of erythrocytosis. i.e., mutations of globin, the 2,3 DPG generating PBGM gene causing increased Hb/O2 affinity (low p50), gain-of-function mutations of erythropoietin receptor (EPOR), germ-line JAK2 mutations, and hypoxia inducible factor 2A (HIF2-A(EPAS1)),PHD2(EGLN1), and VHL mutations associated with augmented oxygen-sensing pathway. Those affected family members had moderately increased erythropoietin (EPO) levels, no splenomegaly, normal leukocyte and platelet numbers and normal p50 (presented at this mtg, Blood. 2003;102,162b). We sequenced whole exomes and adjacent portions of introns of two affected individuals and found a novel heterozygous 5`UTR EPO variant with change -136 nt upstream from the ATG EPO initiation site (NG_021471 -136 G>A). This variant segregated with the erythrocytosis phenotype in 15 relatives examined: the 7 affected subjects were heterozygous for this variant and the 8 unaffected were negative, suggesting its causative role in erythrocytosis (presented at this mtg, Blood. 2013;122,950).

Other authors (NEJM 2018; 378:924) reported a variant of autosomal dominant familial erythrocytosis with a different EPO mutation: a single-nucleotide deletion (c.32delG) in exon 2 of the EPO gene causing a frameshift and alternative EPO mRNA transcripts, leading to increased production of functional EPO protein with shortened signal peptide and a novel N-terminus as cause of their familial erythrocytosis.

In order to characterize function of our 5`UTR EPO variant, we introduced it into the EPO producing human hepatoma cell line Hep3B using CRISPR/Cas9 editing system by homologous recombination with single-stranded donor oligonucleotides. The targeted cells were sorted in 96 well plates (20 cells per well) and then each well tested for presence of -136 G>A variant by allele-specific PCR. We identified 3 heterozygous Hep3B for EPO-136 G>A; the second round of targeting generated homozygous Hep3B clones. The EPO mRNA of homozygous recombinants was greatly increased and detected even in normoxia, unlike non-edited Hep3B cells. No alternative EPO mRNA transcripts were detected in the engineered and non-edited Hep3B cells. To emulate human phenotype, the supernatants of cultured three heterozygousEPO-136 G>A Hep3B single-cell derived clones and controls in normoxic and hypoxic conditions were used to detect production of EPO. The hypoxic treatment increased ~2x the production of EPO from edited clones compared to non-edited Hep3B cells. The secreted EPO from heterozygousEPO-136 G>A Hep3B clones supported growth of EPO-dependent BaF3-EPOR cells more than supernatants from non-edited Hep3B cells. We then measured EPO transcript levels in Hep3B with EPO-136 G>A and parental Hep3B cell lines in normoxia and hypoxia. The hypoxia increased the relative expression of EPO-136 G>A allele in all three targeted heterozygousEPO-136 G>A Hep3B cell lines. The EPO gene promoter was largely unmethylated in both wild and mutated clones. To evaluate activity of this mutant EPO promoter, we sub-cloned wild and mutated 5`UTR EPO sequence upstream of luciferase reporter gene and transfected them into two EPO producing cell lines - Hep3B, Hep2G. The mutant significantly increased activity of the reporter. To study the interaction of EPO-136 G>A mutant promoter with HIF2 (principal transcription factor regulating EPO), we co-transfected EPO-luc reporter with HIF2-A expression plasmid. The activity of reporter with mutated EPO-136 G>A was further increased in these cells with augmented HIF2 levels. Indeed, alignment tools predicted the EPO-136 G>A genomic region as putative HIF2 binding site. This suggests that mutated 5`UTR of EPO augments interaction with HIF2, leading to increase production of EPO. Chromatin immunoprecipitation experiments are ongoing to model the transcriptional regulatory network accounting for augmented transcriptional regulation of this 5`UTR EPO gene variant.

Here we report a novel mechanism of inherited erythrocytosis caused by increased transcription of mutated 5`UTR of EPO.

Supported by Czech HRC, grant NV19-07-00412 and Ministry of Education, grant LTAUSA17142 and University of Utah.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.