Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Abstract

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

“…On the other hand, germline mutation was detected in one (4.5%) of 22 patients with sporadic LAM, whereas the majority of patients with sporadic LAM had neither of TSC1 nor TSC2 germline mutations. Our detection rate of germline mutations in patients with TSC-LAM appears to be almost identical to that of several studies on mutation analysis in patients with TSC (Au et al 1998;Jones et al 1997;Kwiatkowska et al 1998;Van Bakel et al 1997;Wilson et al 1996;Yamashita et al 2000) but lower than that of other studies (Cheadle et al 2000;Dabora et al 2001;Jones et al 1999;Niida et al 1999;Strizheva et al 2001). The detection rate varies widely from 37% to 83%, depending on the screening method used.…”

DNA Copy Number Losses in Human Neoplasms

“…146 Mutations including large deletions in TSC2 (tuberous sclerosis 2), located at 16p13.3, are found in patients with tuberous sclerosis, indicating its role to act as a tumor suppressor. 147 E-cadherin, the CDH1 gene (16q22.1, cadherin 1) has been suggested to act as a tumor/invasion suppressor for sporadic infiltrative lobular breast carcinomas. 148 H-cadherin, the CDH13 gene (16q24.2-q23, cadherin 13), has been reported to be inactivated due to deletions and hypermethylations in lung cancer.…”

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

“…Previously established methods to identify small mutations in the TSC genes include single-stranded conformation gel analysis (SSCP) [Au et al 1998;Jones et al, 1999], heteroduplex analysis (HD) [Jones et al, 1999], two-dimensional electrophoresis with denaturing gradient gel electrophoresis (DGGE; for TSC1 only) [Dabora et al, 1998], protein truncation test (PTT) [van Bakel et al, 1997;Benit et al, 1999;Mayer et al, 1999], and the recently developed denaturing high-performance liquid chromatography (DHPLC) [Choy et al, 1999;Dabora et al, 2001]. For large deletions and rearrangements, pulsed-field gel electrophoresis, Southern blot analysis [European Tuberous Sclerosis Consortium, 1993;Longa et al, 2001], and long-range PCR (for TSC2 only) [Dabora et al, 2000] have been described.…”