Novel mutations detected in the TSC2 gene from both sporadic and familial TSC patients
Novel mutations detected in the TSC2 gene from both sporadic and familial TSC patientsPeter J. Wilson, Vijaya Ramesh, Arthur Kristiansen, Catherine Bove, Serguisy Jozwiak1, David J. Kwiatkowski2, M. Priscilla Short3 and Jonathan L. Haines*
Molecular Neurogenetics Unit, MGH East, Building 149, 13th Street, Charlestown, MA 02129, USA, 1Division of Child Neurology, Children's Health Center, Warsaw, Poland, 2Division of Experimental Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA and 3Department of Pediatric Neurology, 5841 South Maryland Ave, Chicago, IL 60637, USA
Received September 27, 1995;Revised and Accepted November 17, 1995
Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by hamartomas in one or more organs, including the brain, skin, heart and kidneys. Linkage studies have shown locus heterogeneity with one TSC gene mapped to chromosome 9q34 and a second to 16p13.3. The gene on 16p13.3, TSC2, has been cloned and shown to encode a 5.5 kb transcript that is widely expressed. To facilitate the search for mutations in the TSC2 gene product, tuberin, we have designed an RT-PCR-based assay system to scan the expressed coding region of the TSC2 gene in lymphoblasts. Using 34 overlapping PCR assays we performed single-strand conformation polymorphism analysis of DNA from 26 apparently sporadic TSC cases, two TSC families non-informative for linkage analysis and two confirmed chromosome 16-linked TSC families. Of the 60 chromosomes scanned, 14 showed abnormal SSCP mobility shifts. Using direct PCR sequencing we have identified five missense mutations, one 3 bp in-frame deletion and one 2 bp frameshift deletion, one nonsense mutation, one 29 bp tandem duplication and five silent nucleotide changes that are likely to be polymorphisms. There is no apparent clustering of mutations within TSC2. The diversity of mutation types argues that TSC2 may not act in a classic tumor suppressor fashion. In addition, we saw no specific correlation between the different mutations and clinical severity or expression. These data confirm that TSC2 is indeed the relevant gene, and that a substantial number of sporadic cases arise from mutations in the TSC2 gene.
Tuberous sclerosis complex (TSC) is a multisystem disorder characterized by the widespread development of tumor-like growths known as hamartomas usually occurring in the brain, eyes, skin, kidneys, lungs, heart and skeleton (1 ,2 ). It is the unpredictable distribution of these lesions that is thought to result in the broad range of clinical phenotypes seen in this disease even within the same family (3 ). Seizures are the most common neurologic symptom of TSC occurring in 80-90% of all cases (3 ). In addition to the lesions and seizures, delayed psychomotor development and behavioral problems, especially of an autistic type, have been reported (4 ).
TSC is inherited as an autosomal dominant disease with an incidence of approximately 1 in 10 000 in the population (5 ); however, between one-half to three-quarters of patients appear as sporadic cases (6 ,7 ). Linkage studies have shown locus heterogeneity for TSC, with disease determining genes on chromosomes 9 (TSC1) and 16 (TSC2) (8 -15 ). Frequent loss of heterozygosity for alleles in 16p13.3 and rare loss in 9q34 has been observed in hamartomas from TSC patients indicating that a second somatic mutation may be required to produce the TSC phenotype at the cellular level (16 -18 ). This observation is consistent with the TSC1 and TSC2 genes acting as growth suppressors in a manner similar to other tumor suppressor genes (19 -21 ).
The TSC2 gene in chromosome 16p13.3 has been isolated by positional cloning, and large intragenic deletion mutations within this gene have been demonstrated in several TSC cases (22 ). The 5.5 kb TSC2 transcript encodes a 1784 amino acid protein (tuberin) with little similarity to other known genes. To optimize the search for mutations in the TSC2 gene, we have designed a set of 34 overlapping PCR assays that encompass the entire expressed TSC2 gene in lymphoblasts (exons 25 and 31A are alternatively spliced in lymphoblasts and so were not specifically examined in this study) (23 ,24 ). Using this system we have scanned the TSC2 gene in 26 apparently sporadic TSC cases, two TSC families of unknown linkage and patients from two chromosome 16-linked TSC families and have identified multiple independent mutations that appear to be distributed randomly across the TSC2 gene.
Using cDNA derived from lymphoblasts of both patients and controls three primary overlapping PCR products were generated to encompass the expressed TSC2 gene, consisting of a 2074, 2388 and 1533 bp PCR product. Utilizing the primary products as templates, secondary PCR was performed with 34 overlapping primer sets (Table 1 ) designed to yield product sizes optimal for single-strand conformation polymorphism (SSCP) analysis. All assays were run under a standardized set of amplification conditions, varying only the annealing temperature and the addition of 10% DMSO to optimize results.
The appropriate PCR products from the observed SSCP shifts were PCR amplified and prepared for direct DNA sequencing. Five of the shifts, including two chromosome 16-linked familial patients (TSC-001 and TSC-028) and one of the unknown linkage TSC families (TSC-382), revealed missense mutations (Table 2 ). These changes were all unique and resulted in non-conservative amino acid substitutions. To determine if these changes were related to the disease we carried out SSCP on 50 normal controls using the relevant exons and observed no abnormal mobility shifts when compared with the mutants (primers were designed from intronic sequence communicated by J. Sampson). The mutations that were observed in chromosome 16-linked families were further confirmed by observing that these changes segregated with other affected family members (Fig. 4 ). Two of these changes occurred in the same exon, exon 37 (Table 2 ).
TSC2 mutations in both sporadic and familial patients
Patient
Exon
DNA sequence
Codon
Consequence
alteration
change
TSC-001
E29
3616 C-T
Arg1199Trp
Missense
TSC-028
E12
1365 G-C
Met449Iso
Missense
TSC-115
E37
5084 C-A
Ala1689Glu
Missense
TSC-117
E37
5075 C-T
Pro1186Leu
Missense
TSC-382
E16
1849 C-T
Arg611Trp
Missense
TSC-037
E14
1531 C-T
Arg505X
Nonsense
TSC-086
E10
1112 to 1113
Iso365fs >
Frameshift
del 2 bp (TC)
385X
TSC-077
E33
4474 to 4476
Phe1486
In frame deletion
del 3 bp (TTC)
TSC-422
E34
4519-4547
Leu1510fs>
Frameshift
29 bp tandem duplication
1541X
Numbering of bases showing alteration is given relative to the cDNA sequence with the initiator ATG beginning at base 19. All coding sequence bases are given in upper case. For deletions, the span of deleted bases (numbered as above) is given, followed by the deletion size (`del'). For deletions of less than 5 bp, the deleted bases are also named.Original amino acid and position of the residues in the protein (numbered from the initiator Met as 1) is followed by new amino acid for missense mutation, X for nonsense mutation, or fs for frameshift, followed by the position of the next in frame stop codon.
Table 4 presents a summary of the clinical findings in each of the patients with identified mutations. The only consistent finding is the occurrence of seizures in every patient. It should be noted that the clinical findings even within the two chromosome 16-linked TSC families and unknown linkage TSC families vary substantially.
Both sporadic and familial patients were identified through neurology clinics, referrals from the National Tuberous Sclerosis Association (NTSA) and private physicians. All individuals were clinically examined by a physician knowledgeable about the clinical criteria for TSC, and medical records were obtained and reviewed by one of us (MPS). Additional tests including renal ultrasound, head and abdominal CT scan, retinal examination, and/or brain MRI were obtained when possible. Only individuals meeting standard diagnostic criteria (3 ) for TSC were considered as affected in our analyses.
Blood samples were collected and transformed into lymphoblastoid cell lines using Epstein-Barr virus as previously described (44 ). The ethnic origin of both the TSC patients and controls used in this study was Caucasian. Genomic DNA and cytoplasmic RNA were prepared from the cell lines using standard methods.
Approximately 40 µg total RNA was used in the cDNA synthesis reaction which was as follows, initially 500 ng of random hexamers was added to the total RNA and this was incubated at 70oC for 10 min and placed directly on ice. To this reaction was then added 20 µl 5*first-strand synthesis buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 3 µl 10 mM dNTPs, 1 µl BSA (1mg/µl), 6 µl 0.1 M DTT, 2 µl RNasin (Promega) and 2 µl Superscript II Rnase H-reverse transcriptase (200 units/µl, Gibco BRL) made up to a final volume of 100 µl. The reaction was mixed and incubated at room temperature for 10 min, it was then placed at 42oC for 50 min and then heat inactivated at 70oC for 10 min. Finally, the reaction was incubated with 2 µl RNase H (2.5 units/µl; Gibco BRL) for 20 min at 37oC. The cDNA reaction was then extracted once with phenol/chloroform and then once with chloroform/isoamyl alcohol. The supernatant was then ethanol precipitated with an equal vol. 4 M NH4 acetate and 2 vols absolute ethanol. The cDNA pellet was then resuspended in 20 µl TE buffer.
Three primary overlapping PCR products were generated to encompass the entire coding region of the TSC2 gene (Table 1 , primers in bold italics). PCRs were done as follows; 10 µl 10*PCR buffer (100 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 500 mM KCl, 0.1% gelatin), 2 µl 2 mM dNTPs, 10 pmol of each primer, 2 U Taq polymerase (Hoffman LaRoche), 10% DMSO and 1 µl cDNA (see previous section), reactions were made up to 100 µl with water, cycles were as follows; the first two overlapping products (2074 and 2388 bp) conditions for 30 cycles were: 92oC for 1 min, 67oC for 2 min and 70oC for 3 min, after an initial denaturation step of 92oC for 2 min. The remaining primary PCR (1533 bp product) was done under the same conditions, but with an annealing temperature of 65oC.
SSCP analysis was performed according to the procedure of Orita et al. (45 ) with minor modifications. One µl of the primary PCR product (in some cases a 1 in 10 dilution was made) was used for all the nested sets of primers (Table 1 ). Each 25 µl reaction contained 70 µM of dATP, dCTP, dGTP, dTTP, 2.5 pmol of each primer, 0.2 U Taq polymerase (Hoffman La Roche), 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% gelatin and 0.2 µl [alpha]-33P-dATP [Amersham; 10 µCi/ml]. Amplification was carried out for 30 cycles as follows; 92oC for 1 min, 60-70oC for 1 min and 70oC for 1 min, after an initial denaturation step at 92oC for 2 min. (NB: where the annealing temperature was 70oC, the annealing/extension step was combined for 1.5 min.) One µl of the labelled amplified DNA was diluted into 9 µl 0.1% SDS and 10 mM EDTA, and an equal vol. of loading dye (95% formamide, 0.5 M EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) was added. The samples were denatured for 10 min at 95oC and placed immediately on ice prior to loading, the samples were separated on a 0.5*MDE gel according to the manufacturer's specifications (AT Biochem.), typically this was at 6-8 W for 14 h at room temperature. The gels were dried and then exposed to Kodak X-AR film overnight at room temperature.
For DNA sequencing PCR amplifications were performed in 100 µl volumes as described for SSCP analysis except for the addition of 200 µM dNTPs, 10 pmol of each primer and the omission of a radioactive nucleotide. The PCR product was directly sequenced without purification. Essentially, 2-5 µl (of the 100 µl reaction) was incubated with 1 unit exonuclease I (USB) and 2 units of shrimp alkaline phosphatase (USB) in a 9 µl reaction vol. at 37oC for 15 min and then 80oC for 15 min. The appropriate primer was added and annealed at 95oC for 3 min and the reaction was placed immediately on ice. The sequencing reactions were performed by the dideoxy chain termination method using Sequenase (T7 DNA polymerase, Amersham USB) under the conditions recommended by the supplier. Both strands of PCR amplified products with SSCP mobility shifts were analyzed in all cases. When it became necessary to separate the alleles, the PCR products were cloned into pCR script (Stratagene) according to the manufacturer's specifications. If the mutation occurred in a defined exon (both boundaries characterized) then the exon was amplified from the patients genomic DNA as an additional confirmation of the mutation. The intronic primer sequences used in this study are available upon request.
We would like to thank Karen Pulaski and Chris Sterner for technical help and discussions. This work was supported by NIH grants RO1-NS24279, NS31535 and a grant from the NTSA. PJW is the Manuel R. Gomez research fellow of the NTSA. We thank Arun Kumar for sharing his results prior to publication. We would also like to thank the families and patients for giving of both time and themselves to make this work possible.
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