Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 alpha (HIF-2 alpha) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2 beta variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2 alpha-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2 alpha inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Automated summary

Gain-of-function mutations in the EPAS1/HIF2A gene have been found in patients with hereditary erythrocytosis, which can lead to the development of paraganglioma, pheochromocytoma, and somatostatinoma. This study described a European collection of patients and relatives with erythrocytosis associated with a germline genetic variant in EPAS1. Functional assays were conducted to determine the pathogenicity of the genetic variants and to identify potential candidates for HIF-2 alpha inhibitor treatment. The study identified new mutations and highlighted the complexity of genotype/phenotype correlations in erythrocytosis.