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

Abstract

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 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, to establish pathogenicity, and to identify potential candidates eligible for the new HIF-222inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-222variants 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 (EPO) promoter combined with distal regulatory elements which substantially enhanced the HIF-22-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-222inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

JAK2 unmutated erythrocytosis: 2023 Update on diagnosis and management

“…In the particular study, pulmonary arterial hypertension was described in two patients, and thrombosis was reported in six patients (five families). 94 On the other hand, in a prospective study of 8 patients harboring the HIF2A p.M535V variant, 5 experienced thrombotic events versus none of 17 HIF2A wild-type patients. 78 Furthermore, thrombotic events occurred despite ongoing phlebotomy with Hct <45% and in the absence of cardiovascular risks.…”

“…91 It is to be noted that interrogation of this pathway is an evolving area of investigation with recent identification of novel zinc finger domain PHD2 mutations, splicing mutations in VHL and HIF2A variants. [92][93][94] Germline EPAS1(HIF2A) variants 96 In a European collaborative study on 41 patients and 28 relatives with erythrocytosis associated with germline HIF2A variants, 11 mutations were classified as pathogenic with identification of four new mutations (D525G, L526F, G527K, A530S). 94 The study also unveiled two young cases with HIF2A variants in a mosaic state, suggesting that mosaicism may be present at a very low level that is not necessarily detectable by next-generation sequencing (NGS).…”

“…[92][93][94] Germline EPAS1(HIF2A) variants 96 In a European collaborative study on 41 patients and 28 relatives with erythrocytosis associated with germline HIF2A variants, 11 mutations were classified as pathogenic with identification of four new mutations (D525G, L526F, G527K, A530S). 94 The study also unveiled two young cases with HIF2A variants in a mosaic state, suggesting that mosaicism may be present at a very low level that is not necessarily detectable by next-generation sequencing (NGS). It is therefore necessary to lower the variant detection thresholds or to manually analyze the NGS results.…”

“…56,60,92,93,110 Furthermore, improved molecular characterization of "idiopathic erythrocytosis" cases through expanded and highly sensitive NGS platforms coupled with increased utilization of functional assays is anticipated in the near future. 94,153 In the current document we share a practical and comprehensive diagnostic approach to JAK2 unmutated erythrocytosis. Importantly, the prompt and accurate distinction between underlying physiological versus pathological causes of erythrocytosis is crucial, as management is directly impacted by etiology, and incorrect diagnosis may lead to under or over treatment.…”

“…Between 2012 and 2021, the Mayo Clinic laboratory tested 592 patients for HIF2A / PHD2 / VHL / EPOR alterations and identified a total of 12 pathogenic variants in HIF2A ( n = 6, 1%), PHD2 ( n = 3, 0.5%), and EPOR ( n = 2, 0.3%), while one of 446 (0.2%) patients harbored VHL variant 91 . It is to be noted that interrogation of this pathway is an evolving area of investigation with recent identification of novel zinc finger domain PHD2 mutations, splicing mutations in VHL and HIF2A variants 92–94 …”