Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare autosomal dominant disease (prevalence 1:5000) leading to vascular dysplasia. Genetic diagnosis is based on the presence of a pathogenic variant in one of the four HHT genes: ENG, ACVRL1, SMAD4, and GDF2. Here we present a large HHT family in which the Next-Generation Sequencing panel did not reveal any definite pathogenic variant. Previous linkage analysis suggested the involvement of ACVRL1 and one VUS in this gene co-segregates in the family: the c. 526-22 A>G, involving a putative branch point. This represents the first case of a branch point mutation in HHT.Purpose of the study: Demonstrate the pathogenicity of the variant by analyzing the ACVRL1 transcript.Materials and Methods: We performed exome (using a specific HHT virtual panel) and MLPA analysis on a clinically confirmed case from the family. Co-segregation of the variant in the other family members was confirmed by Sanger Sequencing. To understand the effects on splicing, we extracted RNA from a patient and a negative control’s peripheral blood. After retrotranscription and end-point PCR, the amplicons were run on a 2% agarose gel. The semi-quantitative analysis was carried out with the Image lab software (BIORAD). The sequence of each band was confirmed by Sanger Sequencing. Real-time PCR analysis of the transcripts is in progress. Results: The NGS revealed the presence of a rare ACVRL1 variant c. 526-22 A>G and co-segregation was confirmed. The patient’s RNA semi-quantitative analysis showed that the variant affects the splicing, leading to a change in the ratio between the wt coding RNA and the isoform characterized by exon 5 skipping. The presence of this alternative splicing, presenting a frameshift and a premature stop codon, was already reported both in patients and in unaffected subjects. Quantitative analyses are ongoing to confirm our data. Conclusion: We report the first case of a branch point pathogenic variant in HHT. This change affects the splicing process increasing, when compared with healthy individuals, the expression of the alternative spliced “non-coding” mRNA. This is, to the best of our knowledge, the first HHT-related variant modifying the quantity rather than the quality of the transcript.
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