| Abstrakt: |
Introduction: Next generation sequencing (NGS) is a method which is becoming increasingly available in clinical setting, especially in genetically heterogenous diseases as hereditary hearing loss. For each patient there are thousands of variants, including benign polymorphisms, pathogenic variants and variants of unknown clinical significance. The last ones are difficult to interpret, especially in case of silent variants and variants in non-coding parts of the gene - their mode of pathogenicity might be more elusive, i.e. alteration of splicing. Such variants require functional studies to properly assess their impact and pathogenic potential. Aim: To assess the pathogenicity of 11 novel variants with possible effect on splicing using minigene assay. Material and methods: Selected variants were: ATP2B2 c.941-7C>G, EYA1 c.1475+1G>T, EYA4 c.1282-12T>A, GSDME c.991delT, GSDME c.1127A>G, MYO6 c.816+1G>A, MYO6 c.1984-1G>A, MYO6 c.3281-13A>G, MYO7A c.2829G>A, MYO15A c.9230-4 A>T, SLC26A4 c.1001+1G>A. Each variant was detected in the custom HL gene panel performed for patient of Department of Genetics, Institute of Physiology and Pathology of Hearing. Fragments of genes of interest encompassing closest introns and exons were introduced into expression vector pDEST pCI-Neo RHO using Gateway cloning system. Cell cultures of HEK293T line were transfected with expression vectors for each gene, containing either wild type sequence or sequence with studied variant. After 48 h of incubation cell lysis and RNA isolation were performed. Transcripts were analyzed by subsequent RT-PCR, gel elec-trophoresis and Sanger sequencing. Results: The majority of studied variants displayed their effect on splicing (9 out of11, 82%). The most common aberrations were exon skipping and incorporation of intron fragment to the transcript, which usually resulted in frameshift and introduction of premature stop codon. In case of variants ATP2B2 c.941-7C>G and MYO15A c.9230-4 A>T there were no observable signs of splicing alteration. Conclusions: Genetic variants affecting splicing emerge as an important contributor to HL. The performed minigene assays allowed for better variant interpretation, which in turn allowed for the correct genetic diagnosis. The study demonstrates the significance of functional testing especially when it comes to the silent variants and intronic variants. [ABSTRACT FROM AUTHOR] |
