Non-canonical regulation of Endoglin by rare and common variants: new molecular and clinical perspectives for Hereditary Hemorrhagic Telangiectasia and beyond

Endoglin, encoded by the ENG gene, is a transmembrane glycoprotein with a major implication in angiogenesis. Loss-of function ENG variants are responsible for Hereditary Hemorrhagic Telangiectasia (HHT), a rare vascular disease, characterized with a large inter-individual clinical heterogeneity. But, Endoglin and its soluble form have also been reported to be involved in other pathologic conditions including cancer and thrombosis. Thus, dissecting the genetic regulation of Endoglin holds the potential to deepen our understanding of the pathophysiology underlying HHT and other human diseases. To follow-up our latest study in which we characterized 5 rare HHT-causing variations in the 5’UTR of ENG, all creating overlapping upstream Open Reading Frames (upORFs) initiated with upstream AUG, we here performed an exhaustive in silico analysis of all possible single nucleotide variants (n=328) predicted to create or modify any type of upORF in the 5’UTR of ENG. We demonstrated that 85% (11/13) of variants creating uAUGs in frame with the same stop codon located at position c.125, decrease the Endoglin levels in vitro. We identified the moderate effect on ENG of a rare uCUG-creating variant found in HHT patients. Our obtained experimental results were in partial correlation with bioinformatics predictions based on Kozak sequence and PreTIS scores. In parallel, we leveraged results from large scale plasma proteogenomics resources and identified 8 loci ( ABO, ASGR1, B3GNT8, ENG, HBS1L, NCOA6, PLAUR, and TIRAP ), presenting common polymorphisms, significantly associated with Endoglin levels. The ABO locus, coding for the ABO blood groups, explain ∼5% of the inter-individual variability of ENG plasma levels. Overall, these loci candidates could contribute to explain the incomplete penetrance of known pathogenic mutations and/or the clinical heterogeneity of HHT patients. Of note, 4 of these loci are also associated with venous thrombosis in the latest INVENT Genome Wide Association Study initiative. This project brings new insights on the interpretation of ENG non-coding variants and on molecular mechanisms participating to the regulation of Endoglin. It also exemplifies how the incorporation of genotype data on common polymorphisms could enhance the management of rare diseases. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was carried out in the framework of the French National Research Agency (ANR) ANR-23-CE17-0042-01 program as part of the ENDOMORF project and of the INSERM GOLD Cross-Cutting program (D-A.T.). O.S was financially supported by a grant of the Lefoulon-Delalande Foundation. The 3C proteomics project was supported by a grant overseen by the French National Research Agency (ANR) as part of the -Investment for the Future Program-ANR-18-RHUS-0002 and by the Precision and Global Vascular Brain Health Institute (VBHI) funded by the France 2030 IHU3 initiative. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: These resources include 10,708 participants from the Fenland study, 35,559 Icelander participants of the Decode project, and 1,056 population-based participants of the 3C-Dijon study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes ENG constructs generated during this study are available upon request by email from the corresponding author (omar.soukarieh{at}inserm.fr).