Tuberous sclerosis complex (TSC) is due to inactivating TSC1/TSC2 mutationsleading to tumors, including facial angiofibroma (FAF) and cortical tubers, known to develop through two-hit mechanism. Here we explored frequency of second-hit mutation events.
We developed a Multiplex High-sensitivity PCR Assay (MHPA), enabling mutation detection at extremely low (<0.1%) variant allele frequencies (VAF).
TSC2-MHPA analysis of 81 samples, including 24 FAFs from TSC2 mosaic patients,revealed that UV-induced second-hit causing TSC2 inactivation was pervasive in TSC facial skin, with an average of 4.8 mutations per 2-mm FAF at median VAF=0.08%, generating >150,000 facial tumors (subclinical ‘micro-FAFs’) in the average TSC subject (Klonowska et al.,JCI,2022). TSC1-MHPA analysis of 6 FAFs from TSC1 mosaic patients showed that UV-mutations are less common in TSC1 (average 0.8 mutations) than in TSC2 (p=0.006). TSC2-MHPA of tubers identified nonsynonymous somatic SNV/indels in 6 of 13 (46%) tubers (median VAF=0.06%).
Our TSC2-MHPA analysis of FAF revealed that TSC facial skin can be viewed as harboring a patchwork of clonal fibroblast proliferations (micro-FAFs), a small proportion of which develop into clinically observable FAF. Such lesions are much less common in TSC1 than in TSC2. TSC2-MHPA of TSC tubers identified TSC2 second-hits in some lesions.