Abstracts

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder resulting from mutations in the TSC1 or TSC2 genes encoding the proteins hamartin or tuberin. Tubers are developmental malformations of the cerebral cortex associated with epilepsy characterized histologically by dysmorphic giant cells (GCs) that exhibit cytomegaly. A pivotal unanswered question is whether tubers arise as a consequence of a second-hit somatic mutation within the TSC1 or TSC2 genes. We identify a marker for GCs in human tubers, phosphorylated ribosomal S6 protein (phospho-RS6) and show that GCs are formed as a direct consequence of an inactivating somatic second-hit TSC gene mutation.
Tubers were obtained from 13 patients with clinically diagnosed TSC (mean age 7.25 years). One tuber specimen, frontal cortex and cerebellum were obtained post-mortem from 1 TSC patient (age 3 years).
The Eker rat model results from a truncating Tsc2 mutation and exhibits single subependymal nodules in the lateral ventricles. Tsc1 astrocyte-specific conditional knockout (Tsc1 cKO) mice result in loss of hamartin expression in astrocytes.
Brain sections from human specimens, Eker rats, and Tsc1 cKO mice were probed with phospho-RS6 (1:500 dilution) or tuberin antibodies (C-20, 1:200).
Genomic DNA was extracted from the tuber, frontal cortex, and cerebellum obtained post-mortem. Exon specific flanking PCR primers were generated and amplicons from all 41 exons of TSC2 were sequenced. Single phospho-RS6 immunoreactive GCs as well as non-immunoreactive pyramidal neurons in cortex and Purkinje cells in cerebellum were microdissected and TSC2 mRNA was amplified by RT-PCR with overlapping cDNA primers and sequenced. Studies were approved by the University of Pennsylvania Institutional Review Board.
Phospho-RS6 is a marker for cells such as astrocytes lacking hamartin in the Tsc1 cKO mouse and GCs lacking tuberin in the Eker rat model Phospho-RS6 is highly expressed in human GCs but absent in control neurons. DNA sequencing of TSC2 in a tuber, normal cortex, and the cerebellum from one patient revealed a nonsense mutation (1371 C[gt]T, 458R[gt]X) in exon 13. RT-PCR amplification and sequencing of TSC2 mRNA from single microdissected, phospho-RS6 immunoreactive GCs revealed a multi-exon inactivating deletion resulting in loss of tuberin immunoreactivity in the GCs. This mutation was not identified in adjacent, phospho-RS6 non-immunoreactive neurons.
Expression of phospho-RS6 and single cell mutational analysis provides direct evidence that GCs are generated as a consequence of loss of hamartin-tuberin pathway resulting from a somatic inactivating TSC gene mutation.
[Supported by: (PBC) by the Tuberous Sclerosis Alliance, NINDS NS045021, and the Esther A. and Joseph Klingenstein Fund. ]