Molecular Diagnosis in Patients With Tuberous Sclerosis Complex: A Deep Sequencing Approach in Clinical Practice

Background Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous disorder caused by pathogenic variants in TSC1 or TSC2. Despite established clinical diagnostic criteria, a subset of patients remains molecularly unsolved after conventional genetic screening, often due to deep intronic variants, structural anomalies, or low-level somatic mosaicism. Methods A retrospective cohort study was performed. Patients without an identified pathogenic variant after conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) were selected for targeted high-depth next-generation sequencing (NGS), achieving a median sequencing depth of at least 800×. Clinical data were collected for phenotypic characterization. Results High-depth targeted sequencing identified pathogenic or likely pathogenic variants in the TSC2 gene in four out of seven previously unresolved patients, resulting in an incremental diagnostic yield of 57.1% (4/7). The identified variants were deep intronic structural alterations or splice-modifying changes (c.2838-122G>A, c.848+281C>T, and c.2640-16_2640-8del). Two unrelated patients carried the identical c.848+281C>T deep intronic variant. Patients with identified TSC2 variants exhibited a high prevalence of structural central nervous system anomalies, epilepsy, and multi-system tumor involvement compared to the molecularly unconfirmed subgroup. Discussion High-depth targeted sequencing using hybrid-capture enrichment provides a substantial diagnostic advantage for detecting deep intronic variants in previously unsolved TSC cases. These findings advocate for integrating deep sequencing into clinical practice. Although TSC remains mainly a clinical diagnosis, identifying pathogenic variants has an important role in diagnosing patients who do not meet complete clinical criteria, in providing genetic counseling, as well as access to potential targeted therapies.