ABSTRACT We previously reported a five‐generation kindred with autosomal dominant erythrocytosis associated with a novel germline promoter variant in the erythropoietin (EPO) gene ( EPO c.‐136 G>A). This mutation creates a new hypoxia response element (HRE) consensus sequence on the reverse strand suggesting a gain of function mutation. CRISPR/Cas9‐edited Hep3B cells harboring the c.‐136 G>A variant had increased EPO mRNA and protein expression under both normoxic and hypoxic conditions compared to wild‐type cells; functional assays confirmed the activity of the c.‐136 G>A variant‐induced EPO. Isoelectric focusing analyses of patient urine and plasma showed a more basic EPO isoform pattern, consistent with the reduced sulfated N‐glycan contribution, suggesting decreased renal and increased non‐renal expression. Luciferase reporter assays confirmed increased transcriptional activation of the mutant promoter. However, chromatin immunoprecipitation did not verify direct hypoxia‐inducible factor (HIF)‐1/2 binding, suggesting the possible involvement of alternative regulatory elements. These findings support a model in which the EPO c.‐136 G>A promoter variant introduces a new HRE that overrides the normal kidney expression resulting in persistent or ectopic non‐renal EPO production postnatally. This study expands the spectrum of molecular mechanisms underlying hereditary erythrocytosis and provides novel mechanistic insights into EPO regulation, including its tissue‐specific expression.
Lanikova et al. (Fri,) studied this question.