Abstract
Hereditary Hemorrhagic Telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder. About 80% of cases are caused by pathogenic variants in ACVRL1 (also known as ALK1) and ENG, with the remaining cases being unexplained. We identified two variants, c.-79C>T and c.-68G>A, in the 5'UTR of ENG in two unrelated patients. They create upstream AUGs at the origin of upstream overlapping open reading frames (uoORFs) ending at the same stop codon. To assess the pathogenicity of these variants, we performed 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 assay. This assay is mandatory for molecular diagnosis and has been so far only applied to coding ENG variants. These variants were associated with a decrease of protein levels in HeLa and HUVEC cells and a decreased ability to activate ALK1. We applied the same experiments on three 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 protein levels and function. Additional experiments relying on an artificial deletion in our mutated constructs show that identified uAUGs could initiate the translation indicating that the associated effect is translation-dependent. Overall, we have identified two 5'UTR ENG variations in HHT patients and shed new light on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.