Analysis of 65 tuberous sclerosis complex (TSC) patients byTSC2DGGE,TSC1/TSC2MLPA, andTSC1long-range PCR sequencing, and report of 28 novel mutations

Abstract

Tuberous sclerosis complex (TSC) is a severe autosomal-dominant disorder characterized by the development of benign tumors (hamartomas) in many organs. It can lead to intellectual handicap, epilepsy, autism, and renal or heart failure. An inactivating mutation in either of two tumor-suppressor genes-TSC1 and TSC2-is the cause of this syndrome, with TSC2 mutations accounting for 80-90% of all mutations. Molecular diagnosis of TSC is challenging, since TSC1 and TSC2 consist of 21 and 41 coding exons, respectively, and the mutation spectrum is very heterogeneous. Here we report a new approach for detecting mutations in TSC: a denaturing gradient gel electrophoresis (DGGE) analysis for small TSC2 mutations, a multiplex ligation-dependent probe amplification (MLPA) analysis for large deletions and duplications in TSC1 or TSC2, and a long-range PCR/sequencing-based analysis for small TSC1 mutations. When applied in this order, the three methods provide a new sensitive and time- and cost-efficient strategy for the molecular diagnosis of TSC. We analyzed 65 Danish patients who had been clinically diagnosed with TSC, and identified pathogenic mutations in 51 patients (78%). These included 36 small TSC2 mutations, four large deletions involving TSC2, and 11 small TSC1 mutations. Twenty-eight of the small mutations are novel. For the missense mutations, we established a functional assay to demonstrate that the mutations impair TSC2 protein function. In conclusion, the strategy presented may greatly help small- and medium-sized laboratories in the pre- and postnatal molecular diagnosis of TSC.

Genetics of tuberous sclerosis complex: implications for clinical practice

“…In addition, mutations in the genes that encode other regulatory components of the mTOR pathway, eg, TBC1D7 and Rheb , have not been associated with TSC, ie, there is no known TSC3 gene. Mutations in TSC1 are often small insertions or deletions (indels) that result in shortened (truncated) protein, whereas TSC2 mutations include large deletions, indels, nonsense, and missense mutations 14,15. Splicing errors account for a small number of TSC1 and TSC2 mutations.…”

Giant angiofibromas in tuberous sclerosis complex: A possible role for localized lymphedema in their pathogenesis

“…This mutation has been reported in several patients. 17,18,[45][46][47][48] In conclusion, we describe two patients with TSC who presented with unusually large and disfiguring facial angiofibromas. We suggest that localized lymphedema contributes to the appearance of these lesions.…”

Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

“…A disease-associated mutation of the catalytic residue, N1643I [90,91], rendered TSC2 1525-1742 inactive, as expected, as did two other proximal mutations (H1640Y [92] and K1638N [91]). Among disease-associated mutations predicted to be located on the same face of the GAP domain as the catalytic residue, we found that the charge perturbing mutation E1558K [89,93] eliminated GAP activity and the conservative L1594M substitution [88] reduced GAP activity by approximately 80%, whereas the D1690Y mutation [94] did not impair GAP activity of TSC2 1525-1742 , despite its association with tuberous sclerosis disease. We were also able to show that a TSC2 polymorphic variant (D1636N) found in a TSC patient carrying an additional mutation [95] had no direct effect on GAP activity.…”