Autosomal Dominant Erythrocytosis Caused by Non-Renal Erythropoietin (EPO) Due to EPO c.-136 G>A Germline Mutation.

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.