Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH) identifies a new SP1 binding site

Objectives: To investigate the role of a novel intronic ENG variant found in a Patient with a PAH diagnosis followed by the identification of typical HHT clinical signs. The pathogenic role of this variant was demonstrated. Methods: We analysed all coding exons of ENG, ACVRL1 and BMPR2 by Sanger sequencing and mlPA. We expressed the ENG variant in vitro and evaluated protein levels by western blotting. Then, we confirmed the results by qRT-PCR on an RNA sample of the Patient. We used in silico tools to evaluate the presence of putative transcription factor binding-site, changed in the variant. EMSA analyses were performed to validate the involvement of the transcription factor Sp1. Results: We identified the ENG novel variant c.1852 + 42 C[T in a Patient with both PAH and HHT. No other disease-causing mutation was found. We proved by western blotting and qRT-PCR that the variant significantly reduced ENG expression. In silico analyses predicted that the variant changes a putative binding-site for the transcription factor Sp1, already demonstrated as involved in ENG expression. By EMSA, we observed that nuclear extract proteins of human fibroblasts bind with different affinity wild-type and mutated oligonucleotides. Conclusion: We identified a novel Sp1 binding-site in ENG intron14. We demonstrated the pathogenic role of ENG c.1852 + 42 C[T mutation, which impairs this Sp1 binding-site reducing the transcription level of the gene. These results stress the importance of joining genetic findings to functional studies, in order to understand the role of novel variants of uncertain significance in the disease pathogenesis.