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

## TL;DR

A new EPO gene mutation causes a type of inherited anemia by making red blood cells in non-kidney tissues.

## Contribution

The study identifies a novel germline EPO promoter mutation causing non-renal erythropoietin production in autosomal dominant erythrocytosis.

## Key findings

- The EPO c.-136 G>A mutation increases EPO expression in Hep3B cells under normoxic and hypoxic conditions.
- Patient samples show a more basic EPO isoform pattern, indicating reduced renal and increased non-renal EPO expression.
- The mutation creates a new hypoxia response element, leading to persistent non-renal EPO production.

## 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.

A novel erythropoietin (EPO) promoter mutation (c.‐136 G>A) causes autosomal dominant erythrocytosis via non‐renal expression of EPO.