Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (102)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jones, A. C.
Right arrow Articles by Cheadle, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, A. C.
Right arrow Articles by Cheadle, J. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 2155-2161  

Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis
Introduction
Results
   Mutations at the TSC1 locus
   Mutations at the TSC2 locus
   TSC1 and TSC2 mutations in familial and sporadic cases
   Mental handicap in TSC1 and TSC2 cases
   Mutation type and mental handicap
Discussion
MATERIALS AND Methods
   Patients
   Pulsed field and Southern analysis
   PCR
   SSCP and heteroduplex analysis
Acknowledgements
References

Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis

Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis

Alistair C. Jones, Claire E. Daniells, Russell G. Snell, Maria Tachataki, Shelley A. Idziaszczyk, Michael Krawczak, Julian R. Sampson*, Jeremy P. Cheadle

Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, UK

Received August 7, 1997; Revised and Accepted September 3, 1997

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomatous growths in many organs. Sixty to seventy percent of cases are sporadic and appear to represent new mutations. TSC exhibits locus heterogeneity: the TSC2 gene is located at 16p13.3 whilst the TSC1 gene, predicted to encode a novel protein termed hamartin, has recently been cloned from 9q34. With the exception of a contiguous gene deletion syndrome involving TSC2 and PKD1, TSC1 and TSC2 phenotypes have been considered identical. We have now comprehensively defined the TSC1 mutational spectrum in 171 sequentially ascertained, unrelated TSC patients by single strand conformation polymorphism and heteroduplex analysis of all 21 coding exons, and by assaying a restriction fragment spanning the whole locus. Mutations were identified in 9/24 familial cases, but in only 13/147 sporadic cases. In contrast, a limited screen revealed TSC2 mutations in two of the familial cases and in 45 of the sporadic cases. Thus TSC1 mutations were significantly under-represented among sporadic cases (Fisher's exact p-value = 3.12 × 10-4). Both large deletions and missense mutations were common at the TSC2 locus whereas most TSC1 mutations were small truncating lesions. Mental retardation was significantly less frequent among carriers of TSC1 than TSC2 mutations (odds ratio 5.54 for sporadic cases only, 6.78 ± 1.54 when a single randomly selected patient per multigeneration family was also included). No correlation between mental retardation and the type of mutation was found. We conclude that there is a reduced risk of mental retardation in TSC1 as opposed to TSC2 disease and that consequent ascertainment bias, at least in part, explains the relative paucity of TSC1 mutations in sporadic TSC.

INTRODUCTION

Tuberous sclerosis (TSC) is a multisystem disorder characterised by the widespread development of hamartomatous growths in many tissues and organs. The brain, eyes, kidneys, heart and skin are all frequently involved and the lungs, skeleton and endocrine glands are occasionally affected (1). Lesions in the brain are associated with the most frequent severe manifestations which include seizures, mental handicap and a variety of behavioural disorders (1,2). TSC is transmitted as an autosomal dominant trait but [sim]60-70% of cases are sporadic, apparently representing new mutations (3,4).

The pathogenesis of TSC has been poorly understood and recent efforts to establish the primary underlying defect have focused on positional cloning of the causative genes. Genetic linkage studies have demonstrated loci at 9q34 (TSC1) (5) and 16p13.3 (TSC2) (6) and [sim]50% of multigeneration families show segregation of the trait with either locus (7). Positional cloning of the TSC2 gene in 1993 was substantially facilitated by the characterisation of large deletions at the TSC2 locus (8). Similar rearrangements have not been reported at the TSC1 locus, and the TSC1 gene was only identified after extensive sequence analysis of its candidate region (9).

The TSC2 gene encodes a 200 kDa protein, tuberin, which contains a GTPase activating protein (GAP) domain. Hamartin, the 130 kDa predicted product of the TSC1 gene, is a novel protein and shows no homology to tuberin or other known vertebrate proteins. Loss of heterozygosity across the TSC1 and TSC2 chromosomal regions and intragenic somatic mutations affecting wild-type TSC1 or TSC2 alleles in TSC associated hamartomas indicate that the genes act as tumour suppressors (10-12). More recently, the tumour suppressor properties of TSC2 have been formally demonstrated by transgene expression in the Eker rat, a naturally occurring animal model which carries a germline TSC2 mutation and develops renal and other tumours (13).

Table 1 Mutations identified in TSC1
Patient
no.		 Location

 Mutation

 Nucleotide
alteration		 Type of
mutation		 Sporadic (S)/
familial
CL3 Intron 3 328-2 A[rarr]G A[rarr]G at 328-2 splicing F
327 Exon 5 S91X C[rarr]A at 493 nonsense F
280 Exon 5 W103X G[rarr]A at 530 nonsense S
215 Exon 7 747delTA 2 bp deletion frameshift F
256 Exon 10 S334X C[rarr]A at 1222 nonsense S
CL1 Intron 10 1250+1 G[rarr]A G[rarr]A at 1250+1 splicing F
322 Exon 11 1331insA 1 bp insertion frameshift S
312 Exon 15 R509X C[rarr]T at 1746 nonsense S
141 Exon 15 1801delAG 2 bp deletion frameshift F
176 Exon 15 2105delAAAG 4 bp deletion frameshift F
250 Exon 15 2122delAC 2 bp deletion frameshift S
224 Exon 17 2323delAGTT 4 bp deletion frameshift S
CL5 Exon 17 2324dupGTTACTC 7 bp duplication frameshift F
319 Exon 17 2365delG 1 bp deletion frameshift S
365 Exon 17 A726E C[rarr]A at 2398 missense S
198 Exon 18 R786X C[rarr]T at 2577 nonsense S
314 Exon 18 R786X C[rarr]T at 2577 nonsense S
207 Exon 19 2632del14bp 14 bp deletion frameshift S
74 Exon 20 S836X C[rarr]G at 2728 nonsense F
CL2 Exon 21 2871insT 1 bp insertion frameshift F
108 Exon 21 2887insA 1 bp insertion frameshift S
161 Exon 21 2895delAG 2 bp deletion frameshift S
The mutations in patients 312, 141,176, 250 and CL5 have been reported previously (9).

With the exception of a contiguous gene deletion syndrome involving TSC2 and PKD1 at 16p13.3 (14,15), significant differences between TSC1 and TSC2 phenotypes have not been demonstrated (7). The identification of the TSC1 gene has now made it possible to more critically evaluate the TSC1 and TSC2 phenotypes in sporadic as well as familial cases.

To provide insight into the distribution and spectrum of mutations at the recently identified TSC1 locus, we investigated 171 sequentially ascertained and unrelated patients with TSC, 147 sporadic cases and 24 cases with an affected parent ± other affected relatives. We assayed the TSC1 locus for large rearrangements using pulsed field gel electrophoresis and screened all 21 coding exons using both SSCP and heteroduplex analysis. We noted a significant under-representation of TSC1 mutations among sporadic, but not familial, cases of TSC. Phenotypic analysis showed that mental handicap was significantly less common among patients with TSC1 mutations, providing a likely explanation for their relative paucity among sporadic cases.

RESULTS

Mutations at the TSC1 locus

No abnormalities at the TSC1 locus were detected using pulsed field gel electrophoresis. In 22 cases likely disease-causing mutations were identified by SSCP (18 cases) and/or heteroduplex analysis (20 cases) (Fig. 1). In 19/22 cases the mutations were predicted to be truncating (seven with nonsense substitutions and 12 with small rearrangements altering the reading frame). Two further mutations involved invariant nucleotides at splice sites and both of these segregated with TSC in TSC1-linked families and one missense mutation (A726E) was observed (Table 1 and Fig. 2). This was considered likely to be pathogenic since it occurred in a sporadic case and neither unaffected parent carried the change and analysis with five polymorphic microsatellite markers was consistent with biological parenthood. Eleven novel polymorphisms were observed in affected individuals and normal controls, including three missense changes within the coding region, M322T (1186 T[rarr]C) with a rare allele frequency of 15% and the unique variants H732Y (2415 C[rarr]T) and G1035S (3324 G[rarr]A) and eight other variants, 958+3 A[rarr]G (unique), 1251-9 A[rarr]G (unique), 1554+13 C[rarr]T (unique), 1555-55 C[rarr]G (unique), E445 (1556 A[rarr]G) (rare allele frequency 14%), 2847-4 (T)17-21 (five alleles of 17-21 repeats with allele frequencies of 1.6-57%), A943 (3050 C[rarr]T) (rare allele frequency 10%) and E1094 (3503 G[rarr]A) (unique).

Mutations at the TSC2 locus

Nineteen large rearrangements were identified at the TSC2 locus by pulsed field gel electrophoresis (15 cases) and Southern analysis (four cases). Eighteen were substantial deletions removing all or part of the gene and one case had an [sim]600 kb inversion which disrupted the gene. Fifteen of these mutations have been previously reported (8,14,15). The unreported mutations were three intragenic deletions (two of [sim]5 kb and [sim]1 kb occurring de novo in sporadic cases and an [sim]10 kb deletion in a familial case) and a de novo deletion of >30 kb which involved the 5[prime] end of the TSC2 gene plus the adjacent OCTS3 gene in a further sporadic case. SSCP and heteroduplex analysis were completed for 11 of the 41 exons of the TSC2 gene (exons 1, 16, 25, 31, 34-39 and 41 which together comprise [sim]25% of the coding region). Likely disease-causing mutations were identified in 28 cases, 23 of which are reported elsewhere (16; Jones et al. manuscript in preparation). Three of the unreported mutations were small frameshift changes in exon 41, 5425 insT, 5358 del32bp and 5406 insC. Each of these mutations was predicted to extend the open reading frame into the 3[prime]UTR of the adjacent PKD1 gene. The other novel mutations were two splice site mutations flanking exon 39, 5087-2 A[rarr]G and 5178 delTG, both of which were shown to represent de novo mutations in sporadic cases.

TSC1 and TSC2 mutations in familial and sporadic cases

Exons 1 and 2 are non-coding leader exons and were not analysed.TSC1 and TSC2 mutations were not equally distributed between sporadic and familial cases. Of the 47 cases with a TSC2 mutation, 45 were sporadic (i.e. no clinically affected parent or sibling) and two were familial. In contrast, 13 of the 22 patients with TSC1 mutations were sporadic cases and nine were familial cases. Thus, TSC1 mutations were significantly under-represented among sporadic cases (Fisher's exact p-value = 3.12 × 10-4).


Figure 1 Detection and characterisation of TSC1 mutations and polymorphisms. (A) Three alterations in exon 17 revealed on a triple-loaded SSCP gel. The associated mutations are: lane 3b, H732Y; lane 6c, 2365 delG; lane 11b, 2323delAGTT. (B) Three mutations in exon 21 revealed on a quadruple-loaded heteroduplex gel. The associated mutations are: lane2b, 2895 delAG; lane 8d, 2871 insT; lane 9a, 2887 insA. (C) Sequencing of the missense mutation A726E (C[rarr]A at 2398). The mutation was shown to be a de novo change in the sporadic case.


Figure 2 Distribution of mutations at the TSC1 locus.

Mental handicap in TSC1 and TSC2 cases

To determine whether major differences between TSC1 and TSC2 associated phenotypes might be responsible for the difference in frequency of TSC1 and TSC2 mutations in sporadic and familial cases, we assessed the prevalence of mental handicap in these groups (Table 2). Differences were expressed as the odds ratio m2(1 - m1)/m1(1 - m2) where m1 and m2 denote the prevalence of mental handicap among carriers of TSC1 or TSC2 mutations, respectively. Mental handicap was significantly more common in sporadic cases with TSC2 mutations (33/45) as opposed to TSC1 mutations (4/13; odds ratio 5.54, Fisher's exact p-value = 0.011). However, ascertainment of sporadic cases may be biased towards a more severe phenotype than in familial cases, leading to underestimation of the true magnitude of the phenotypic difference between TSC1 and TSC2 cases as a whole. Therefore, the mean odds ratio (± s.d.) was determined as expected when one affected individual from each multiplex family was randomly added to the patient sample. When probands were included (which might again bias both samples towards more severe phenotypes), the mean odds ratio was 6.78 ± 1.54, increasing to 8.79 ± 0.82 when probands were excluded. In both cases, the odds ratio (i.e. the excess of mental handicap in cases with TSC2 mutations) would have been found significantly larger than unity (Fisher's exact p-value <0.05) in any resampling round. No significant difference was noted in the frequency of mental handicap in male (19/30) and female (16/28) sporadic cases or male (5/27) and female (5/31) familial cases.

Mutation type and mental handicap

We looked for any association between mental handicap and mutation type at the TSC2 locus. However, no significant difference in the frequency of mental handicap was found between cases with large rearrangements detected by pulsed field analysis (13/19) and small mutations detected by SSCP or heteroduplex analysis (19/28), or between cases with truncating or deletion mutations (21/30) and missense mutations (9/15). At the TSC1 locus, we noted that mental handicap was seen in patients with truncating mutations towards the 3[prime] end of the gene (6/14 cases with mutations in exons 15-23), but not in patients with truncating mutations towards the 5[prime] end of the gene (0/7 cases with mutations in exons 3-14). However, this trend did not reach statistical significance.

DISCUSSION

Previous linkage studies have suggested that the TSC phenotype cosegregates with TSC1 and TSC2 in similar proportions of affected multiplex families (7). However, TSC1 mutations were found in only a small proportion of sporadic cases during a search which finally confirmed the identity of TSC1 (9). We have now undertaken an extensive assessment of TSC1 mutations and have confirmed their relative paucity among sporadic compared with familial cases. Given the different mutational spectra we observed at the TSC1 and TSC2 loci, differences in locus specific mutation rates might contribute to the under representation of TSC1 mutations among sporadic cases. Differences in severity of the TSC1 and TSC2 phenotypes could also be a contributing factor.

We evaluated the frequency of mental handicap among patients with TSC1 and TSC2 mutations because this is a major and easily documented aspect of phenotypic severity in TSC and is usually apparent from an early age (1). Although mental handicap occurred in both TSC1 and TSC2 associated disease, it was significantly less common among patients with TSC1 mutations. This was true whether sporadic cases were considered alone or together with familial cases. The number of mentally handicapped familial cases was too small for independent statistical analysis. One previous study (7) has addressed the question of phenotype severity in TSC1 and TSC2 disease and included an assessment of mental handicap. No significant difference in the frequency of mental handicap was found between TSC1 and TSC2 families. However, only families suitable for linkage analysis were assessed and these were likely to be biased toward mild phenotypes since severely affected individuals rarely reproduce. Further studies are now warranted to evaluate mental handicap in a larger group of multiplex families and to assess other aspects of the TSC phenotype in TSC1 and TSC2 cases. The trend we noted for 3[prime], but not 5[prime], truncating mutations of TSC1 to be associated with mental handicap also requires further investigation. Mutations which leave the 5[prime] end of the gene intact might create mutant products capable of exerting a dominant-negative effect, as has been suggested for other tumour suppressors such as APC (17,18).

Because of the different types of mutation identified at the TSC1 and TSC2 loci, we considered the possibility that the nature of the mutation might have a significant bearing on the TSC phenotype. However, we found no association between mutation type and mental handicap. In the present study we did not comprehensively screen the TSC1 locus by Southern analysis in all patients (60/171 were tested and were normal), but we and other members of the TSC1 Consortium screened 250 unrelated patients by Southern analysis and found no rearrangements (9). It is therefore unlikely that the under-representation of TSC1 mutations in sporadic cases is the result of undetected deletions at the TSC1 locus.

Table 2 TSC1 and TSC2 mutations in familial and sporadic TSC
  Cases studied TSC1 mutations TSC2 mutations
Familial  24  9  2
Sporadic
(mentally handicapped)		 147

 13 (4)

 47 (32)

Now that both TSC1 and TSC2 have been identified, comprehensive molecular genetic diagnostics for TSC can be developed. The differences we have documented in the nature of TSC1 and TSC2 mutations and in their distribution among sporadic and familial cases will be helpful in devising efficient strategies for this. The preponderance of small truncating mutations at the TSC1 locus suggests that the in vitro detection of truncated protein products may represent an appropriate method for detecting TSC1 mutations. It appears that only a small proportion of these are recurrent. We detected the same nonsense mutation (R786X) in two unrelated sporadic cases and this mutation has also been detected previously in one additional sporadic and one familial case (9). Specific assays for this mutation, and for R509X and 2105delAAAG, which were detected in the current and the previous series (9), might be incorporated into a diagnostic protocol. In contrast to the TSC1 mutational spectrum, large deletions and missense mutations are frequent at the TSC2 locus. Detection of a large proportion of TSC2 mutations will continue to require a combination of complimentary approaches. Family history appears to be a major factor determining the probability of TSC1 and TSC2 mutations and will also be helpful when deciding which locus to investigate first in a given family.

The functional relationships between the TSC1 and TSC2 gene products are not yet known. The TSC2 gene contains a region of homology with the GTPase activating protein rap1GAP (9,16) and the possibility of a signalling role mediated by Rap1 (19) or involvement in control of endocytosis mediated via Rab5 (20) have been suggested. In contrast, the TSC1 product, hamartin, only shows homology with a putative yeast protein of unknown function and not with any known vertebrate proteins (9). Hamartin does contain a predicted coiled-coil domain and a search for interactions with tuberin or other members of the pathways already implicated as involving tuberin may prove fruitful. A fuller understanding of the implied roles of hamartin and tuberin in neuronal migration and differentiation may eventually provide insights into the differing risks of mental retardation that we have observed in patients with TSC1 and TSC2 mutations.

MATERIALS AND Methods

Patients

One hundred and seventy one unrelated patients with TSC were sequentially ascertained at the Institute of Medical Genetics, Cardiff, as part of ongoing studies into clinical and genetic aspects of the condition. One hundred and forty seven were sporadic cases and 24 were familial cases with an affected parent ± other affected relatives. Three of the familial cases were from clearly TSC1 linked families and two were from TSC2 linked families. In the remainder linkage status was uncertain or not investigated. We have also specifically ascertained samples from patients with tuberous sclerosis and renal cystic disease for an independent study (15). Patients who were ascertained for that study (because of their specific phenotype) were not included in the present series. All patients fulfilled the diagnostic criteria recommended by the Diagnostic Criteria Committee of the National Tuberous Sclerosis Association (21). Mental handicap was considered to be present when formal developmental assessment had revealed an overall developmental quotient under 70, when unassisted mainstream schooling was impossible because of learning difficulties (not behavioural problems) or where an adult was institutionalised or required supervision in the community.

Pulsed field and Southern analysis

High molecular weight DNA for pulsed field gel electrophoresis was prepared from peripheral blood leukocytes in agarose plugs as described (8). We assayed a 420 kb MluI fragment spanning the TSC1 locus (9) and 350 kb ClaI and 130 kb MluI fragments spanning the TSC2 locus (14,15). Plugs were digested with the appropriate enzymes according to the manufacturer's recommendations and restriction fragments were resolved using a BioRad CHEF DRII apparatus and 1% agarose gels. Blotting and hybridisation were performed using standard methods (22). Probes used to screen for rearrangements have been described previously and were 90F5, 151G9 and 148E8 flanking the TSC1 locus (9) and H2, CW21, BFS2 and SM6 flanking the TSC2 locus (14). Mutations were characterised by hybridisation with further probes as described (8,14,15). DNA for Southern analysis was prepared by standard methods (22), digested with EcoRI (both loci), TaqI and PstI (TSC2 locus only) and probed with TSC1 and TSC2 cDNA clones. Samples yielding variant restriction patterns were subject to further restriction analysis to characterise the mutations involved as has been described (8,15).

PCR

DNA was extracted from peripheral blood or from lymphoblastoid cell lines by standard methods. PCR primers were designed with the aid of the `Oligo Analysis' software package. PCR was carried out in 50 [mu]l reaction volumes containing 100 ng genomic DNA, 25 pmole primers, 0.2 mM dNTP, 5 [mu]l reaction buffer (100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), and 1 U AmpliTaq Gold Polymerase (Cetus). Cycling parameters were 94°C 10 min, followed by 32-33 cycles of 54-58°C 1 min, 72°C 1 min, 94°C 30 s, and a final step of 72°C 10 min. The complete coding sequence (exons 3-23) and intron/exon boundaries of the TSC1 gene were amplified as 26 fragments (Table 3). Conditions for the amplification of exons 16 and 34-38 of TSC2 have been described (16; Jones et al., manuscript in preparation). Exons 1, 25, 31, 39 and 41 of TSC2 were amplified using the primers 1F 5[prime]-CAGAGGTGTTGCTCAGATGTCCC-3[prime], 1R 5[prime]-ATTTCCCTCTAGCCTAGCAAAGA-3[prime] (253 bp), 25F 5[prime]-CCCTCC- ACTGGCTTGTTCTCC-3[prime], 25R 5[prime]-CGGGCAAGACGATGAG- GTCAT-3[prime] (305 bp), 31F 5[prime]-CACGGGGCCTGTGCTCTCTG-3[prime], 31R 5[prime]-CAATGGAGGCAGACGGACCAT-3[prime] (259 bp), 39F 5[prime]-CCCTGGGCCTGGCGTGACC-3[prime], 39R 5[prime]-GCAGGGGTGAGCTCACTAC-3[prime] (200 bp), 41F 5[prime]-GCCAGCCTCCCAGACTTACTG-3[prime], 41R 5[prime]-GACTGCAATCTGTGCCTC- TATGT-3[prime] (313 bp). The 2847-4 (T)17-21 repeat was typed by end-labelling AJ40 with [[gamma]-33P]dATP and sizing of the PCR products by comparison with an M13 sequencing ladder run on a 6% polyacrylamide gel. Evidence for biological paternity and maternity was assessed using highly polymorphic microsatellite repeats on chromosomes 4 (D4S43), 6 (D6S250), 7 (D7S636), 15 (LS6-1, GABRB3, IR4-3R) and 16 (D16S665). The 5358 del32bp mutation was characterised by cloning the PCR product into pGEM-T (Promega), followed by transformation into JM109, and sequencing by standard double stranded methods.

Table 3 Primers used for the amplification of the TSC1 coding region (exons 3-23)
Exon

 Primer

 Sequence

 Product
size
3 AJ63 5[prime]-GGGGCCATTTAGTGACTGT-3[prime] 299 bp
  AJ62 5[prime]-GCAGGATTCTAGTGGCTCTAA-3[prime]  
4 AJ61 5[prime]-TAGGATGTTAAGGGGAATAAGT-3[prime] 241 bp
  AJ60 5[prime]-CTCAGGACAAGTTGCACAGT-3[prime]  
5 AJ59 5[prime]-CTTCATACATTCATGTGAGGACT-3[prime] 277 bp
  AJ58 5[prime]-CCTTGCTTTAAGTTGCCTAAA-3[prime]  
6 AJ57 5[prime]-CAGTGTTTAGAGCCTCTTCAT-3[prime] 283 bp
  AJ56 5[prime]-AAAGCATTCACCTCACAGG-3[prime]  
7 AJ55 5[prime]-GCTGTTTTGCACTCCTCAAT-3[prime] 307 bp
  AJ54 5[prime]-CCCTGTCTGCCGTTAAATAC-3[prime]  
8 AJ53 5[prime]-CACAAACATTCAGCCCTTTAT-3[prime] 197 bp
  AJ52 5[prime]-CTCAACAGGGATTACCTCCTA-3[prime]  
9 AJ32 5[prime]-TGGCACTGAGTTGACACTCT-3[prime] 315 bp
  AJ31 5[prime]-CAAATAATGTTTTCCAGAGACA-3[prime]  
10 AJ30 5[prime]-CACACTAACCCCCTGTGTTC-3[prime] 238 bp
  AJ29 5[prime]-TTCCCAACCACATACTAAATCT-3[prime]  
11 AJ28 5[prime]-AACCTCGTGGATGACTTAGC-3[prime] 268 bp
  AJ27 5[prime]-AACAGCAAGTGGTCCCTTAG-3[prime]  
12 AJ26 5[prime]-AATAGTTGGGCTCAGTGTTCAT-3[prime] 281 bp
  AJ25 5[prime]-CCCATTGCATTTTAGGTCAG-3[prime]  
13 AJ24 5[prime]-CAACATTTTTCGTCTTGTGA-3[prime] 170 bp
  AJ23 5[prime]-ACATATAACCCAATTAGAAGAGG-3[prime]  
14 AJ22 5[prime]-TGTCCAGCCTTCTCTGTTCA-3[prime] 278 bp
  AJ21 5[prime]-GAGCGAGGGTCAGGTTTTAT-3[prime]  
15 AJ6 5[prime]-GAATACCGACTGCCATTTCT-3[prime] 303 bp
  AJ5 5[prime]-AGGGCTTTCATCAGCACTG-3[prime]  
  AJ4 5[prime]-GCAAGCCTTTACTCCCATAG-3[prime] 276 bp
  AJ3 5[prime]-GGCACACCATCTTCCTCTG-3[prime]  
  AJ2 5[prime]-CAGCCCATCATTTTGTCATC-3[prime] 256 bp
  AJ1 5[prime]-AGGTGGGAGTGTGAAGAATG-3[prime]  
16 AJ20 5[prime]-TTTTGACCACAAGGAAGTGAT-3[prime] 217 bp
  AJ19 5[prime]-GGACAGAAAGGGCAACAAG-3[prime]  
17 AJ18 5[prime]-GGCTTGATTGAACCATCTGTA-3[prime] 312 bp
  AJ17 5[prime]-CTCGGCTGCTGTGCTTTAT-3[prime]  
18 AJ16 5[prime]-CCTGTGTTGGAAGACAGCTAA-3[prime] 283 bp
  AJ15 5[prime]-ACTGCTCTCCGGCATTCTC-3[prime]  
19 AJ14 5[prime]-GCCTGTTGGTGTTCCTCAAA-3[prime] 210 bp
  AJ13 5[prime]-AATGTTAGCAAATGGTGTTTCA-3[prime]  
20 AJ12 5[prime]-GCTGATTCCCTGTTTAATGAC-3[prime] 276 bp
  AJ11 5[prime]-GCCATGTGGGAGACATACTG-3[prime]  
21 AJ39 5[prime]-TTCAGGAAGTAGAAATGATGA-3[prime] 265 bp
  AJ9 5[prime]-AGATACAGACCAGCCAGAAT-3[prime]  
  AJ10 5[prime]-AAAATGGATACAGCATGTTTA-3[prime] 111 bp
  AJ40 5[prime]-TTCTAGCTCTTTCCGATAGG-3[prime]  
22 AJ8 5[prime]-TCAAACTCCAGGCAAGGTAA-3[prime] 296 bp
  AJ7 5[prime]-CAGCTTAGTCCCAAGGTCAT-3[prime]  
23 AJ38 5[prime]-CCTCCGAATGTGGACAGTC-3[prime] 298 bp
  AJ37 5[prime]-CAGACGCTTCTCCCATAGTC-3[prime]  
  AJ36 5[prime]-GGCAGTAGTGGAAGCAGAGG-3[prime] 295 bp
  AJ35 5[prime]-CCAAGTCTTTGCCCAGTTCT-3[prime]  
  AJ34 5[prime]-CATGACCAGTAGCCTTTCTGA-3[prime] 234 bp
  AJ33 5[prime]-GCATTCACACCTCCTGTTCT-3[prime]  

SSCP and heteroduplex analysis

SSCP was performed on 3 [mu]l PCR product diluted 1:10 with formaldehyde containing 0.0125% bromophenol blue and 0.75% ficoll 400. Samples were denatured at 94°C 5 min, snap cooled on ice and triple loaded (2 h intervals) on a MDE gel (Flowgen). Electrophoresis was performed in 0.6× TBE at 6 W for 18 h at room temperature. Gels were blotted onto Hybond N (Amersham) and hybridised with 32P labelled PCR product, washed and exposed to film. Heteroduplex analysis was performed by mixing 5 [mu]l aliquots of two PCR products with 0.6 [mu]l 0.1 M EDTA, denaturing at 94°C 5 min, then slowly cooling to 37°C. One microlitre of electrophoresis dye (Hoefer) was added to each of the samples which were then quadruple loaded (1 h intervals) on an MDE gel and run at 4.5 W for 15 h at room temperature. Products were visualised by standard silver staining (23). PCR products of samples displaying variant banding patterns were sequenced using either the Sequenase PCR Product Sequencing kit or the ThermoSequenase cycle sequencing kit (Amersham).

ACKNOWLEDGEMENTS

J. Maynard and A. John provided protocols on multi-sample heteroduplex analysis, N. Williams provided advice on microtitre PCR, and S. Tomkins and clinical colleagues helped with provision of samples. This work was supported by grants from the Tuberous Sclerosis Association (GB), the National Tuberous Sclerosis Association (USA), the Welsh Scheme for the Development of Health and Social Research, the Deutsche Forschungsgemeinschaft and the Medical Research Council.

REFERENCES

1. Gomez, M.R. (1988) Tuberous Sclerosis, 2nd edition. Raven Press, New York.

2. Smalley, S., Tanguay, P., Smith, M. and Gutierrez, G. (1992) Autism and tuberous sclerosis. J. Autism Dev. Disord., 22, 339-355. MEDLINE Abstract

3. Sampson, J.R., Scahill, S.J., Stephenson, J.B.P., Mann, L. and Connor, J.M. (1989) Genetic aspects of tuberous sclerosis in the west of Scotland. J. Med. Genet., 26, 28-31. MEDLINE Abstract

4. Osborne, J.P., Fryer, A. and Webb, D. (1991) Epidemiology of tuberous sclerosis. Ann. N.Y. Acad. Sci. 615, 125-127. MEDLINE Abstract

5. Fryer, A.E., Chalmers, A., Connor, J.M., Fraser, I., Povey, S., Yates, A.D., Yates, J.R.W. and Osborne, J.P. (1987) Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet i, 659-661.

6. Kandt, R.S., Haines, J.L., Smith, M., Northrup, H., Gardner, R.J.M., Short, M.P., Dumars, K., Roach, E.S., Steingold, S., Wall, S., Blanton, S.H., Flodman, P., Kwiatkowski, D.J., Jewell, A., Weber, J.L., Roses, A.D. and Pericak-Vance, M.A. (1992) Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. Nature Genet., 2, 37-41. MEDLINE Abstract

7. Povey, S., Burley, M.W., Attwood, J., Benham, F., Hunt, D., Jeremiah, S.J., Franklin, D., Gillet, G., Malas, S., Robson, E.B., Tippett, P., Edwards, J.H., Kwiatkowski, D.J., Super, M., Mueller. R., Fryer A., Clarke, A., Webb D. and Osborne J. (1994) Two loci for tuberous sclerosis: one on 9q34 and one on 16p13. Ann. Hum. Genet., 58,107-127. MEDLINE Abstract

8. The European Chromosome 16 Tuberous Sclerosis Consortium (1993) Identification and characterisation of the tuberous sclerosis gene on chromosome 16. Cell, 75,1305-1315.

9. The TSC1 Consortium. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277, 805-808.

10. Green, A.J., Smith, M. and Yates, J.R.W. (1993) Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patients. Nature Genet., 6,193-196.

11. Carbonara, C., Longa, L., Grosso, E., Borrone, C., Garre, M.G., Brisigotti, M., Migone, M. (1994) 9q34 loss of heterozygosity in a tuberous sclerosis astrocytoma suggests a growth suppressor-like activity also for the TSC1 gene. Hum. Mol. Genet., 3, 829-1832.

12. Henske, L.P., Scheithauer, B.W., Short, M.P., Wollman, R., Nahmias, J., Hornigold, N., van Slegtenhorst, M., Welsh, C.T. and Kwiatkowski D.J. (1996) Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am. J. Hum. Genet., 59,400-496. MEDLINE Abstract

13. Kobayashi, T., Mitani, H., Takahashi, R.I., Hirabayashi, M., Ueda, M., Tamura, H. and Hino, O. (1997) Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild type TSC2 transgene. Proc. Natl. Acad. Sci. USA, 94, 3990-3993. MEDLINE Abstract

14. Brook-Carter, P.T., Peral, B., Ward, C.J., Thompson, P., Hughes, J., Maheshwar, M., Nellist, M., Gamble, V., Harris, P.C. and Sampson, J.R. (1994) Deletion of the TSC2 and PKD1 genes associated with severe infantile polycystic kidney disease-a contiguous gene syndrome. Nature Genet., 8, 328-332. MEDLINE Abstract

15. Sampson, J.R., Maheshwar, M.M., Aspinwall, R. Thompson, P., Cheadle, J.P., Ravine, D., Roy, S., Haan, E., Bernstein, J. and Harris, P.C. (1997) Renal cystic disease in tuberous sclerosis: role of the polycystic kidney disease 1 gene. Am. J. Hum. Genet. in press.

16. Maheshwar, M.M., Cheadle, J.P., Jones A.C., Myring, J., Fryer A.E., Harris P.C. and Sampson, J.R. (1997) The GAP related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum. Mol. Genet. 6, 1991-1996.

17. Spiro, L., Olschwang, S,. Groden, J., Robertson, M., Samowitz, W., Joslyn, G., Gelbert, L., Thliveris, A., Carlson, M., Otterud, B., Lynch, H., Watson, P., Lynch, P., Laurentpuig, P., Burt, R., Highes, J.P., Thomas, G., Leppert, M. and White, R. (1993) Alleles of the APC gene: an attenuated form of familial polyposis. Cell, 75, 951-957.

18. Su, L., Johnson, K.A., Smith, K.J., Hill, D.E., Vogelstein, B. and Kinzler, K.W. (1993) Association between wild-type and mutant APC gene products. Cancer Res., 53, 2728-2731. MEDLINE Abstract

19. Wienecke, R., Konig, A. and DeClue, J.E. (1995) Identification of tuberin, the tuberous sclerosis 2 product - tuberin possesses specific rap1GAP activity. J. Biol. Chem., 270, 16409-16414. MEDLINE Abstract

20. Xiao, G.-H., Shoarinejad, F., Jin, F., Golemis, E.A. and Yeung, R.S. (1997) The tuberous sclerosis 2 gene product functions as a rab5a GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem., 272, 6097-6100.

21. Roach, E.S., Smith, M., Huttenlocher, P., Bhat, M., Alcorn, D. and Hawley, L. (1992) Report of the Diagnostic Criteria Committee of the National Tuberous Sclerosis Association. J. Child Neurol., 7,221-224. MEDLINE Abstract

22. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbour Laboratory Press.

23. Abernathy, C.R., Rasmussen, S.A., Stalker, H.J., Zori, R., Driscoll, D.J., Williams, C.A., Kousseff, B.G. and Wallace, M.R. (1997) NF1 mutation analysis using a combined heteroduplex/SSCP approach. Hum. Mutat. 9, 548-554. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +44 1222 743922; Fax:+44 1222 747603; Email: wmgjrs@cardiff.ac.uk

This page is maintained by OUP admin. Last updated Sat Oct 18 13:41:19 BST 1997. Part of the OUP Journals World Wide Web service.
Copyright Oxford University Press, 1997


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
D A Muzykewicz, A Sharma, V Muse, A L Numis, J Rajagopal, and E A Thiele
TSC1 and TSC2 mutations in patients with lymphangioleiomyomatosis and tuberous sclerosis complex
J. Med. Genet., July 1, 2009; 46(7): 465 - 468.
[Abstract] [Full Text] [PDF]

Home page
 J Child NeurolHome page
L.D. Hamiwka and E.C. Wirrell
Comorbidities in Pediatric Epilepsy: Beyond ``Just'' Treating the Seizures
J Child Neurol, June 1, 2009; 24(6): 734 - 742.
[Abstract] [PDF]

Home page
 J Child NeurolHome page
G. Ramantani, P. Niggemann, G. Hahn, A. Nake, R. Fahsold, and M. A. Lee-Kirsch
Unusual Radiological Presentation of Tuberous Sclerosis Complex With Leptomeningeal Angiomatosis Associated With a Hypomorphic Mutation in the TSC2 Gene
J Child Neurol, March 1, 2009; 24(3): 333 - 337.
[Abstract] [PDF]

Home page
 INT J SURG PATHOLHome page
M. Bisceglia, C. Galliani, I. Carosi, A. Simeone, and D. Ben-Dor
Tuberous Sclerosis Complex With Polycystic Kidney Disease of the Adult Type: the TSC2/ADPKD1 Contiguous Gene Syndrome
International Journal of Surgical Pathology, October 1, 2008; 16(4): 375 - 385.
[Abstract] [PDF]

Home page
 Hum Mol GenetHome page
L. S. Pymar, F. M. Platt, J. M. Askham, E. E. Morrison, and M. A. Knowles
Bladder tumour-derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms
Hum. Mol. Genet., July 1, 2008; 17(13): 2006 - 2017.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
F. E. Jansen, O. Braams, K. L. Vincken, A. Algra, P. Anbeek, A. Jennekens-Schinkel, D. Halley, B. A. Zonnenberg, A. van den Ouweland, A. C. van Huffelen, et al.
Overlapping neurologic and cognitive phenotypes in patients with TSC1 or TSC2 mutations
Neurology, March 18, 2008; 70(12): 908 - 915.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
P. B. Crino, K. L. Nathanson, and E. P. Henske
The Tuberous Sclerosis Complex.
N. Engl. J. Med., September 28, 2006; 355(13): 1345 - 1356.
[Full Text] [PDF]

Home page
 J Child NeurolHome page
V. Wong
Study of the Relationship Between Tuberous Sclerosis Complex and Autistic Disorder
J Child Neurol, March 1, 2006; 21(3): 199 - 204.
[Abstract] [PDF]

Home page
 J Child NeurolHome page
P. Prather and P. J. de Vries
Behavioral and Cognitive Aspects of Tuberous Sclerosis Complex
J Child Neurol, September 1, 2004; 19(9): 666 - 674.
[Abstract] [PDF]

Home page
 J Child NeurolHome page
K.-S. Au, A. T. Williams, M. J. Gambello, and H. Northrup
Molecular Genetic Basis of Tuberous Sclerosis Complex: From Bench to Bedside
J Child Neurol, September 1, 2004; 19(9): 699 - 709.
[Abstract] [PDF]

Home page
 J. Med. Genet.Home page
K Mayer, M Goedbloed, K van Zijl, M Nellist, and H-D Rott
Characterisation of a novel TSC2 missense mutation in the GAP related domain associated with minimal clinical manifestations of tuberous sclerosis
J. Med. Genet., May 1, 2004; 41(5): e64 - e64.
[Full Text] [PDF]

Home page
 J. Med. Genet.Home page
J C Lewis, H V Thomas, K C Murphy, and J R Sampson
Genotype and psychological phenotype in tuberous sclerosis
J. Med. Genet., March 1, 2004; 41(3): 203 - 207.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
M. A. Knowles, T. Habuchi, W. Kennedy, and D. Cuthbert-Heavens
Mutation Spectrum of the 9q34 Tuberous Sclerosis Gene TSC1 in Transitional Cell Carcinoma of the Bladder
Cancer Res., November 15, 2003; 63(22): 7652 - 7656.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Neuroradiol.Home page
W. Mukonoweshuro, I. D. Wilkinson, and P. D. Griffiths
Proton MR Spectroscopy of Cortical Tubers in Adults with Tuberous Sclerosis Complex
AJNR Am. J. Neuroradiol., November 1, 2001; 22(10): 1920 - 1925.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
S L DABORA, A A NIETO, D FRANZ, S JOZWIAK, A VAN DEN OUWELAND, and D J KWIATKOWSKI
Characterisation of six large deletions in TSC2 identified using long range PCR suggests diverse mechanisms including Alu mediated recombination
J. Med. Genet., November 1, 2000; 37(11): 877 - 883.
[Full Text]

Home page
 Arch. Dis. Child.Home page
F. J O'Callaghan and J. P Osborne
Recent advances: Advances in the understanding of tuberous sclerosis
Arch. Dis. Child., August 1, 2000; 83(2): 140 - 142.
[Full Text] [PDF]

Home page
 Arch NeurolHome page
M. H. Hyman and V. H. Whittemore
National Institutes of Health Consensus Conference: Tuberous Sclerosis Complex
Arch Neurol, May 1, 2000; 57(5): 662 - 665.
[Full Text] [PDF]

Home page
 J. Med. Genet.Home page
P. J DE VRIES and P. F BOLTON
Genotype-phenotype correlations in tuberous sclerosis
J. Med. Genet., May 1, 2000; 37(5): 3e - 3.
[Full Text]

Home page
 J. Med. Genet.Home page
F. J K O'CALLAGHAN, M. NOAKES, and J. P OSBORNE
Renal angiomyolipomata and learning difficulty in tuberous sclerosis complex
J. Med. Genet., February 1, 2000; 37(2): 156 - 157.
[Full Text]

Home page
 J. Biol. Chem.Home page
M. Nellist, M. A. van Slegtenhorst, M. Goedbloed, A. M. W. van den Ouweland, D. J. J. Halley, and P. van der Sluijs
Characterization of the Cytosolic Tuberin-Hamartin Complex. TUBERIN IS A CYTOSOLIC CHAPERONE FOR HAMARTIN
J. Biol. Chem., December 10, 1999; 274(50): 35647 - 35652.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
P. B. Crino and E. P. Henske
New developments in the neurobiology of the tuberous sclerosis complex
Neurology, October 22, 1999; 53(7): 1384 - 1384.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
A. C. Jones, J. Austin, N. Hansen, B. Hoogendoorn, P. J. Oefner, J. P. Cheadle, and M. C. O'Donovan
Optimal Temperature Selection for Mutation Detection by Denaturing HPLC and Comparison to Single-Stranded Conformation Polymorphism and Heteroduplex Analysis
Clin. Chem., August 1, 1999; 45(8): 1133 - 1140.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Neuroradiol.Home page
Baron and A. Barkovich
MR Imaging of Tuberous Sclerosis in Neonates and Young
AJNR Am. J. Neuroradiol., May 1, 1999; 20(5): 907 - 916.
[Abstract] [Full Text]

Home page
 BMJHome page
F. J K O'Callaghan
Tuberous sclerosis
BMJ, April 17, 1999; 318(7190): 1019 - 1020.
[Full Text]

Home page
 J. Neurol. Neurosurg. PsychiatryHome page
O. Hurko and T. T Provost
Neurology and the skin
J. Neurol. Neurosurg. Psychiatry, April 1, 1999; 66(4): 417 - 430.
[Full Text]

Home page
 J. Med. Genet.Home page
M. van Slegtenhorst, S. Verhoef, A. Tempelaars, L. Bakker, Q. Wang, M. Wessels, R. Bakker, M. Nellist, D. Lindhout, D. Halley, et al.
Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation
J. Med. Genet., April 1, 1999; 36(4): 285 - 289.
[Abstract] [Full Text]

Home page
 NEJMHome page
J. Kwiatkowska, J. Wigowska-Sowinska, D. Napierala, R. Slomski, and D. J. Kwiatkowski
Mosaicism in Tuberous Sclerosis as a Potential Cause of the Failure of Molecular Diagnosis
N. Engl. J. Med., March 4, 1999; 340(9): 703 - 707.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
N. Satake, T. Kobayashi, E. Kobayashi, K. Izumi, and O. Hino
Isolation and Characterization of a Rat Homologue of the Human Tuberous Sclerosis 1 Gene (Tsc1) and Analysis of Its Mutations in RatRenal Carcinomas
Cancer Res., February 1, 1999; 59(4): 849 - 855.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. E. Clurman and P. Porter
New insights into the tumor suppression function of P27Kip1
PNAS, December 22, 1998; 95(26): 15158 - 15160.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (102)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jones, A. C.
Right arrow Articles by Cheadle, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, A. C.
Right arrow Articles by Cheadle, J. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?