Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex

Abstract

Tuberous sclerosis complex (TSC) is characterized by the formation of tumors in multiple organs and is caused by germline mutation in one of two tumor suppressor genes, TSC1 and TSC2. As for other tumor suppressor gene syndromes, the mechanism of somatic second-hit events in TSC tumors is unknown. We grew fibroblast-like cells from 29 TSC skin tumors from 22 TSC subjects and identified germline and second-hit mutations in TSC1/TSC2 using next-generation sequencing. Eighteen of 22 (82%) subjects had a mutation identified, and 8 of the 18 (44%) subjects were mosaic with mutant allele frequencies of 0 to 19% in normal tissue DNA. Multiple tumors were available from four patients, and in each case, second-hit mutations in TSC2 were distinct indicating they arose independently. Most remarkably, 7 (50%) of the 14 somatic point mutations were CC>TT ultraviolet 'signature' mutations, never seen as a TSC germline mutation. These occurred exclusively in facial angiofibroma tumors from sun-exposed sites. These results implicate UV-induced DNA damage as a cause of second-hit mutations and development of TSC facial angiofibromas and suggest that measures to limit UV exposure in TSC children and adults should reduce the frequency and severity of these lesions.

Figure 1.

Immunoblot analysis of skin tumor and control fibroblasts from TSC subjects. Subjects are indicated as P#. C, control fibroblasts; T, skin tumor fibroblasts and multiple skin tumors from one subject are indicated as T#. Immunoblotting is shown for TSC1, TSC2, ribosomal protein S6, phospho-S6 (Ser-235/236) (pS6) and β-actin.

Figure 2.

IGV screenshot demonstrating that TSC2 c.1803C>G (splice) and TSC2 c.1830_1831CC>TT, p.R611W mutations do not occur in the same amplified DNA molecules generated from cultured skin tumor P7T1 and thus arose on opposite alleles of TSC2.

Figure 3.

Distribution of types of point mutations seen in the germline in TSC2 (2, http://chromium.liacs.nl/LOVD2/TSC/home.php), left; and seen in this study as somatic mutations in TSC2, right.