ANALYSIS OF A SPLICING VARIANT IN DMD AND THE IMPACT AT mRNA LEVEL

MAZZANTI, CHIARA; CARCIONE, MICAELA; LEONELA LUCE; GILIBERTO FLORENCIA

Congreso; REUNIÓN ANUAL DE SOCIEDAD DE BIOCIENCIAS SAIC-SAI-AAFE-NANOMED.AR 2021 REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2021

TítuloANALYSIS OF A SPLICING VARIANT IN DMD AND THE IMPACT AT mRNA LEVELMazzanti, Chiara1,2; Luce, Leonela1,2; Carcione, Micaela1,2; Giliberto, Florencia1,2*.1 Laboratorio de Distrofinopatías, Cátedra de Genética, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.2 Instituto de Inmunología, Genética y Metabolismo (INIGEM), CONICET - Universidad de Buenos Aires, Buenos Aires, Argentina.Dystrophinopathies are rare progressive X-linked muscular diseases caused by mutations in DMD. Duchenne muscular dystrophy (DMD) is the most common and represents the most severe form. Although most of the alterations that occur in the DMD gene affect coding areas, ~ 7% are found in splicing regions. It is difficult to predict the effect that these variants will have on the splicing process.We present the case of an 8-year-old boy with dystrophinopathy, biopsy with preserved dystrophin and a NGS study where the probably pathogenic variant DMD:c.4519-2A> C (exon 33) was identified. Our aim was to analyze the consequence of the splicing acceptor site variant at the mRNA level.Muscle mRNA was extracted and cDNA was synthesized. Long-range and nested PCRs of exons flanking the variant were performed and sequenced by Sanger.Two isoforms of different molecular weight were observed. The most abundant with the highest molecular weight (A) had a 14bp deletion, while the less abundant with lower molecular weight (B) had a exon 33 complete deletion. The sequencing allowed the prediction that A would produce a frameshift (FS) with the appearance of a premature translation codon, leading to the absence of dystrophin protein. On the other hand, it is expected that FS will not be produced in B and a shorter dystrophin protein will be generated. This finding agrees with immunohistochemistry where DYS 1, 2 and 3 domains were preserved. The presence of isoform B, which product would generate a shorter but partially functional dystrophin, would explain the milder condition observed in the patient.In conclusion, the mRNA study allowed corroborating the deleterious impact of the variant at the splicing level, allowing the correlation between the splicing alteration, the dystrophin isoforms, the biopsy result, and the patient's clinical manifestations.