Maria Clara de Freitas Pinho

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by the presence of pathogenic germline variants in TSC1 or TSC2 genes. However, most TSC1 and TSC2 genetic testing strategies are limited and about 20% of patients have inconclusive or negative results. In this study, we standardized a low-cost technique based on RT-PCR followed by Sanger sequencing, aimed to complement genetic testing strategies. Patients with clinical suspicion of TSC and TSC1 and TSC2 inconclusive (n=3) or negative (n=1) genetic testing results were recruited. Exon 5 skipping in TSC1 gene was identified in one patient with a previous negative genetic testing result. In addition, a 9-base-pair intronic inclusion was found in the TSC1 of an individual with a previously identified variant of uncertain significance, c.664-10A>C. No alterations were found in the other two patients. The present study was successful in identifying errors in mRNA processing in two TSC patients with previously negative or inconclusive genetic testing results. Furthermore, this strategy was useful to reclassify a VUS to pathogenic. Finally, the validated technique could be used to complement coding region genetic tests and to investigate causality of VUS in TSC and other monogenic diseases. Highlights: - A novel cDNA analysis method revealed the presence of exon 5 skipping in the - TSC1 gene - A nine-base-pair retention in TSC1 mRNA is caused by the intronic variant c.664-10A>C - The variant of uncertain significance c.664-10A>C in TSC1 was reclassified as pathogenic - A strategy using cDNA could be a good complement to conventional molecular diagnosis protocols