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 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b 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-2α-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-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.
Hereditary erythrocytosis (HE) is a rare hematological disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2160 ...patients with erythrocytosis sequenced in 10 different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of the Hypoxia-Inducible Factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: in silico study of localization, conservation, and deleterious effects; analysis of hematological parameters of carriers identified in the UK Biobank; functional studies of the protein activity and stability; and comprehensive study of PHD2 splicing. Altogether, this study allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extented to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences with the variants of unknown significance regarding the severity of the developed disease (hematological parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare pathology to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematological diseases.
