Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

Abstract

Abstract Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disorder causing abnormal vessel formation and characterized by autosomal dominant transmission. About 80% of HHT cases are caused by pathogenic coding variants in ACVRL1 (also known as ALK1) and ENG, and 15% remain unexplained. We identified 2 variants, c.-79C > T and c.-68G > A, in the 5’UTR of ENG in 2 unrelated HHT patients. They are predicted to create upstream AUGs (uAUGs), which are in frame with a stop codon located within the CoDing Sequence (CDS), thus generating Overlapping upstream Open reading frames (uoORFs). Methods In order to assess the pathogenicity of these variants, we performed in vitro functional assays based on the expression of wild-type and mutant constructs in human cells and evaluated their effect on ALK1 activity in a BMP-response element (BRE) assay. This assay is a mandatory element before providing a definitive molecular diagnosis and has been so far applied only on coding ENG variants. Results We found that these 5’UTR variants were associated with a decrease of protein levels in HeLa and HUVEC cells and a decreased ability to activate BMP9-stimulated ALK1 receptor. We applied the same experimental workflow on 3 additional uoORF-creating variants (c.-142A > T, c.-127C > T and c.-10C > T) located in the 5’UTR of ENG and previously reported in HHT patients. We found that all the analyzed variants alter endoglin levels and function. Moreover, additional experiments relying on an artificial deletion in our mutated constructs show that identified uAUGs are able to initiate the translation indicating that the associated effect is likely caused by an alteration of the translation mechanism. Conclusion Overall, we here identified two 5’UTR ENG variations in HHT patients and shed new lights on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.