Analysis of TSC Cortical Tubers by Deep Sequencing of TSC1, TSC2 and KRAS Demonstrates that Small Second‐Hit Mutations in these Genes are Rare Events

Abstract

Tuberous sclerosis complex (TSC) is an often severe neurocutaneous syndrome. Cortical tubers are the predominant neuropathological finding in TSC, and their number and location has been shown to correlate roughly with the severity of neurologic features in TSC. Past studies have shown that genomic deletion events in TSC1 or TSC2 are very rare in tubers, and suggested the potential involvement of the MAPK pathway in their pathogenesis. We used deep sequencing to assess all coding exons of TSC1 and TSC2, and the activating mutation hot spots within KRAS in 46 tubers from TSC patients. Germline heterozygous mutations were identified in 81% of tubers. The same secondary mutation in TSC2 was identified in 6 tuber samples from one individual. Further study showed that this second hit mutation was widely distributed in the cortex from one cerebral hemisphere of this individual at frequencies up to 10%. No other secondary mutations were found in the other 40 tubers analyzed. These data indicate that small second hit mutations in any of these three genes are very rare in TSC tubers. However, in one TSC individual, a second hit TSC2 point mutation occurred early during brain development, and likely contributed to tuber formation.

Genotype and cognitive phenotype of patients with tuberous sclerosis complex

“…Our explorations confirm that cognition and epilepsy are interrelated in TSC, showing greater frequencies of epilepsy and IS in mutation subgroups with a higher prevalence of MR and low mean IQ/DQs, which may contribute to the large ranges of IQ/DQ per mutation subgroup. In addition, it is still unclear if second hits are absolutely necessary for the formation of tubers, 17,18 which are also associated with intellectual outcomes. 9,43 A drawback for this type of study is the use of multiple cognitive measures, which is inherent to the inclusion of different age groups.…”

“…10 TSC patients with germline TSC1 and TSC2 mutations have only one fully functional TSC2 allele in all their cells, and this condition could lead to neurocognitive dysfunction through the mechanism of haplo-insufficiency, [14][15][16] similar to Fragile-X syndrome and Neurofibromatosis type 1. [14][15][16] However, in TSC there are additional factors which may contribute to cognitive impairment, including loss of heterozygosity, which may contribute to tuber development, 17,18 and effects of early onset and refractory epilepsy. Thus far, no associations have been found between specific TSC mutation types and cognitive outcomes, 10,19 although there are reports on associations with epilepsy and psychiatric features.…”

mTOR Signaling in Epilepsy: Insights from Malformations of Cortical Development

“…Renal and pulmonary lesions in TSC follow a "twohit" mutational model in which a somatic inactivating mutation including loss of heterozygosity or a point mutation, in the unaffected allele, is superimposed on the existing germline mutation. Two recent reports suggest that tubers contain both germline and somatic mutations, suggesting a mechanism of biallelic gene inactivation Qin et al 2010). Mouse models that show abnormal cortical structure have required full Tsc1 or Tsc2 knockout (e.g., Feliciano et al 2011); heterozygous mice do not show neuropathological changes.…”

Epilepsy Related to Developmental Tumors and Malformations of Cortical Development

“…By sequencing TSC1 and TSC2 in microdissected pS6-immunolabeled giant cells, a recent study demonstrated that somatic inactivating mutations may be detected in tubers, thus supporting a "twohit" mechanism for tuber formation [213]. However, another study implementing deep sequencing technologies of genomic DNA extracted from whole tubers reported a somatic mutation in TSC1 in only 1 out of 46 tuber samples [214]. The differences in results between these two studies may reflect differences in sequencing approaches, for example DNA extracted from single cells versus whole tubers.…”