Background: A pivotal developmental question is whether tubers in tuberous sclerosis complex (TSC) form by germline and somatic TSC1 or TSC2 gene mutations. Loss of TSC1 or TSC2 in vitro and in vivo leads to mTORC1 cascade activation and ribosomal protein S6 phosphorylation (P-S6). Giant cells (GCs) in tubers exhibit S6 phosphorylation, suggesting cell-specific loss of TSC gene function.

Methods: TSC1 and TSC2 gene mutations were investigated in DNA extracted from tuber sections (n = 6) and microdissected P-S6–labeled GCs by sequencing and loss of heterozygosity (LOH) analysis to define germline and somatic mutations.

Results: A germline TSC1 mutation was defined in 1 case and TSC2 mutations were defined in 5 cases. LOH was not detected in whole tuber sections or microdissected P-S6–labeled GCs. TSC1 and TSC2 were sequenced in microdissected P-S6–immunolabeled GCs. In 5 specimens, a somatic mutation was identified in single GCs that was not detected in whole tuber sections or leukocyte DNA. Four somatic mutations were novel variants (1 nonsense and 3 missense mutations) and 1 additional nonsense somatic mutation was previously reported as a germline mutation. In 1 case, no somatic mutation was identified. There was reduced expression of TSC1 or TSC2 transcripts in the TSC1 or TSC2 associated specimens. In the cases containing a nonsense mutation, no transcript mRNA was detected, suggesting nonsense-mediated degradation.

Conclusions: We provide evidence to support the hypothesis that tubers form by biallelic TSC1 or TSC2 gene inactivation reflecting a “2-hit” mechanism of germline and somatic mutational events.

AML = angiomyolipoma; DN = dysplastic neuron; FFPE = formalin fixed, paraffin embedded; GC = giant cell; H-E = hematoxylin and eosin; LOH = loss of heterozygosity; TSC = tuberous sclerosis complex.