Classic TSC brain pathology includes cortical tubers, subependymal nodules which can develop into giant cell astrocytomas, and white matter migration tracts (16
). More recent studies have identified a wider spectrum of abnormalities in the TSC patient brain, including cysts (35
), cerebellar pathology (17
), and MRI abnormalities of uncertain origin and nature (28
), as well as isolated giant and dysplastic cells in otherwise apparently normal regions of cortex (7
Despite this variety of cortical pathology in TSC, cortical tubers are the major neuropathologic feature in tuberous sclerosis. Their number and size have been shown to correlate, roughly, with both cognitive impairment and behavioral and social developmental issues (10
). In addition, tubers are often located within or adjacent to foci of treatment-refractory epilepsy, and thus they are resected on a regular basis to achieve seizure control in selected TSC patients.
The origin and pathogenesis of cortical tubers is poorly understood (7
). Their considerable size in many instances and apparent relative lack of change over time strongly suggests that they are neurodevelopmental lesions that occur during corticogenesis. However, it is also clear that there is ongoing inflammation in TSC tubers, which may play an important role in their clinical manifestations (3
), as well as some evidence of cell proliferation within them (20
We examined 46 cortical tuber samples from 34 TSC patients by deep sequencing. This approach enabled the identification of 25 small point mutations in TSC1 and TSC2 at heterozygote allele frequency in the majority of these individuals. Thirty-seven tubers were analyzed from these 25 patients with small germline point mutations, and none showed evidence for a shift in the allelic ratio of mutant to wild type allele. Thus, none of these 37 tubers showed this classic evidence of loss of the wild type allele. In addition, none of the 46 cortical tuber samples showed evidence of TSC1 or TSC2 copy number change by MLPA. These findings are consistent with previous reports, indicating that classic LOH is very rare in TSC cortical tubers (13
), even when laser capture microdissection is also used (performed in two cases (26
Our deep sequencing of brain tuber samples did yield identification of one low frequency second point mutation in TSC2 in a patient with a germline nonsense TSC2 mutation (see further discussion below). However, the other 40 cortical tuber samples that were analyzed had no small mutations identified that would be consistent with second hit events, strongly suggesting that this phenomenon is quite rare in cortical tubers.
Our study has some limitations. First it is possible that the samples studied did not consist solely of tuber. Indeed cortical tubers do not consist of giant and other abnormal cell types alone, but rather a mixture of cell types (). However, our deep sequencing approach is relatively tolerant to contamination by normal cells due to its sensitivity for low frequency mutations. In addition, all the samples were reviewed by expert neuropathologists (JC, HV).
A second potential limitation is that the depth of sequencing read coverage among the exons of TSC1 and TSC2 was somewhat uneven. However, it is notable that every germline mutation known to be present in these patients was detected in the deep sequencing analysis, at frequencies close to 50% expected except for a single variant (TSC2 334GGins). In addition, we studied two subependymal giant cell astrocytomas in this project, and the primary lab investigators were blinded to both the presence and the identification of those samples. Second hit events were detected in each of those lesions. Finally, based upon the distribution of read frequencies attained, we estimate that > 90% of small sequence changes (not genomic deletions) occurring at ≥ 5% frequency in these samples would have been detected. Since the mean fraction of pS6+ giant cells present in these tubers was 4.54%, not including other dysmorphic neurons and astrocytes, there was adequate power for detection of small mutations present in pS6+ cells in most cases. The approach used here is not designed, however, for detection of variants present at < 2% frequency.
Phosphorylation of TSC2 by extracellular regulated kinase (ERK) has been seen in some TSC giant cells, suggesting that the activation of MAP kinase pathway could co-operate with TSC1
haploinsufficiency to lead to tuber development (14
). However, how this process could occur so frequently (up to 50 cortical tubers per TSC patient) in TSC, and yet rarely affect non-TSC individuals is unexplained. In addition, MAPK phosphorylation is also seen in TSC SEGAs (14
) in which the two hit mechanism is clear, as shown here in two cases and previously (6
). Nonetheless, to explore the hypothesis that KRAS mutations (one of the most common mutational events occurring in cancer) might represent a second hit event complementing haploinsufficiency for TSC1 or TSC2 in these tubers, we performed KRAS deep sequencing analysis in 30 of the tuber samples. No significant sequence variants were identified, and these amplicons covered the amino acid residues that are the predominant site of mutation in this gene in cancer (amino acid residues 12, 13, and 61).
The single patient in whom we identified the same second point mutation, TSC2 1864C>T R622W, in multiple regions of cortex from one cerebral hemisphere in addition to a germline mutation in TSC2 (4375C>T, R1459X) appears to represent an extraordinary TSC patient, as this was not seen in any of the other TSC patient samples studied. We desired to perform more detailed correlations of the TSC2 1864C>T R622W mutation with individual brain cells in this individual, but the quality of the available tissue precluded such studies, as conventional (non-frozen) fixed paraffin-embedded pathologic material was not available for our use. Nonetheless, we speculate that in this individual an early second hit event occurred in the developing brain, which led to wide dissemination of this mutation throughout at least one hemisphere and likely both. Further, we expect that cells with both mutations, and consequent complete loss of functional TSC2 and activation of mTORC1, would demonstrate abnormal proliferation and development, leading to generation of giant cells and dysplastic cell types, and tuber formation in brain regions with high amounts of these cells.
Thus, in summary, we have identified a single TSC patient with a germline TSC2 nonsense mutation and widespread distribution of brain cells within the cortex with a secondary point mutation in TSC2 at frequencies as high as 10%. However, the majority of tuber samples analyzed (40 of 46) showed no evidence for LOH by analysis of mutations and intragenic SNPs, and MLPA assessment of TSC1 and TSC2 copy number, or for low frequency point mutations that might represent second hit events. In addition, 30 of these tubers showed no evidence of activating KRAS point mutation. Overall, this new data is consistent with previous reports, and suggests that both large genomic deletions, detectible as LOH, and small mutations in either TSC1 or TSC2 are rare in TSC cortical tubers. Thus, the molecular details of the pathogenesis of cortical tubers remain unclear for the majority of TSC patients, and requires additional investigation. Possibilities include second hit mutations in other TSC/mTOR pathway interacting genes, epigenetic silencing of the remaining TSC1 or TSC2 allele, or haploinsufficiency combined with some mechanism of activation of the MAPK pathway. In vitro studies and mouse models have shown that loss of one allele of TSC1 or TSC2 causes some changes in neuronal morphology, but alone does not cause giant cell formation or dysplasia. We did not find mutations in KRAS in these tubers, but there are many other genes interacting with either the MAPK or the PI3K–AKT pathway that are potential candidates.