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 40 patients and 23 related diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified 2 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, notably in order to identify potential candidates eligible to the new HIF-2a inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2a variants has been studied by using canonical and real time reporter luciferase assays. These functional assays used a newly edited vector containing an expanded region of the erythropoietin (EPO) promoter combined with distal regulatory elements which substantially enhanced the HIF-2a-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 15 patients and 23 related. 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 some patients with only erythrocytosis associated with germline mutations (A530S, Y532C) previously identified at somatic state in tumors, raising the complexity of the genotype/phenotype correlation. This study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2a inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.