Next-generation Sequencing (NGS)-based Genetic Testing for Tuberous Sclerosis Complex (TSC) - Highlighting on Focal Lesion Sequencing & Clinical Research Database

Tuberous sclerosis complex (TSC) is an autosomal dominant disease due to causative variants at either TSC1 or TSC2. The disease-causating mutations are highly variable and 10–20% of TSC individuals have no causative variants identified after thorough conventional molecular diagnostic assessment. In this study, 110 definite TSC probands were studied using next-generation sequencing (NGS) to search for pathogenic variants at TSC1 and TSC2 genes. “Pathogenic” or “likely pathogenic” variants were identified in 96 (87%) probands; however, 14 (13%) probands could not be assigned causative variants. Among the 96 identified causative variants, 58 (60%) were single nucleotide substitutions, 28 (29%) were small indels, and 10 (10%) were large deletions/duplications. Fourteen (15%) probands were found to have variants at TSC1 and 82 (85%) were found to have variants at TSC2. Seven individuals were mosaic regarding to their causative variants, including 5 probands and parents of other two non-mosaic probands. A splicing site variant “TSC2, c.138+5G>T” was identified in one TSC family co-segregated in 2 affected family members. With the intent to investigate the genetic etiology in those TSC patients without identifiable causative variants at TSC1 or TSC2, we conducted sequencing using focal lesion DNA. A total of 10 focal lesions obtained from 3 no-mutation-identified probands and 2 probands with one germline mutation (positive control) were studied. One pathogenic mutation at TSC2 with 0.6% of allele percentage was detected in the focal lesion DNA of one no-mutation-identified proband. This variant was considered as the second-hit mutation and indicated that the germline mutation in this patient is located in TSC2 gene as well. However, no potential second-hit mutation was identified in the positive control samples. These findings concluded and suggested that there were several possible reasons for no-mutation-identified status in TSC patients: 1) mosaicism with very low allele percentage for variants at TSC1 or TSC2; 2) variants in introns that affect splicing, or in promoter and enhancer regions of TSC1 and TSC2; 3) variants detection failure due to technical issues; 4) variants at a yet-unknown third TSC gene. Furthermore, to manage the clinical research data collected from TSC Genetic Research, a user-friendly online clinical research database - Clinical Study Information System (CSIS) was established. The study-specified data entry forms were designed for TSC Genetic Research and the system was under security protection by personal ID and password for researchers who were delegated to this study. All the available data including the clinical information from subjects and genetic test results were entered and managed by our study team accordingly.