Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10-15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI.

Fig 1. Pie charts displaying the mutation types and frequencies in 53 TSC NMI subjects.

(A) Proportion of subjects with mutations identified vs. remaining as persistent NMI. (B) Proportion of mutations in TSC1 vs. TSC2. (C) Proportion of heterozygous vs. mosaic mutations. (D) Different types of identified mutations.

Fig 2. Intronic mutations in 18 TSC NMI subjects.

The locations of 18 splice site mutations identified are shown relative to the canonical consensus sequences present at the 3’ exon region, the branch site, and the 5’ exon region.

Fig 3. Correlation between clinical features and mutation status in 53 NMI subjects.

(A) The proportion of subjects with 2, 3, or 4 organs affected; with heterozygous or mosaic mutations, or persistent NMI status. P = 0.003. (B) The number of major symptoms seen for each subject sorted according to mutation status. Note that differing levels of mosaicism have different symbols according to allele frequency (AF). (C) Age at the time of clinical evaluation, sorted according to mutation status. Het, heterozygous; Mos, mosaic. P values determined by chi square test (A) and Mann-Whitney unpaired test (B and C). Results with p < 0.05 are considered statistically significant.