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.

The authors have declared no competing interest.

This work was supported by Fundo de Incentivo a Pesquisa e Eventos (FIPE) of Hospital de Clinicas de Porto Alegre, grant number 2015-0049. Patricia Ashton-Prolla is a researcher of CNPq (category 1B, process 313806-2021-7). Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS)(Edital Fapergs number 09-2023) for Financial support.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was approved by the Research Ethics Committee of Hospital de Clinicas de Porto Alegre under the protocol number 44739715.7.0000.5327 (Plataforma Brasil).

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The clinical data of individuals that support the findings of this study are openly available in ClinVar at SUB15042297. The primer sequences used to analyze the specific regions and raw data produced by Sanger sequencing of all analyzed regions are available as supplementary data. All the data were reported in accordance with STROBE (STrengthening the Reporting of OBservational studies in Epidemiology) statements. The manuscript was published in a preprint version in MedRxiv.