Characterisation of TSC1 promoter deletions in tuberous sclerosis complex patients

Tuberous sclerosis complex (TSC), an autosomal dominant disorder, is a multisystem disease with manifestations in the central nervous system, kidneys, skin and/or heart. Most TSC patients carry a pathogenic mutation in either TSC1 or TSC2. All types of mutations, including large rearrangements, nonsense, missense and frameshift mutations, have been identified in both genes, although large rearrangements in TSC1 are scarce. In this study, we describe the identification and characterisation of eight large rearrangements in TSC1 using multiplex ligation-dependent probe amplification (MLPA) in a cohort of 327 patients, in whom no pathogenic mutation was identified after sequence analysis of both TSC1 and TSC2 and MLPA analysis of TSC2. In four families, deletions only affecting the non-coding exon 1 were identified. In one case, loss of TSC1 mRNA expression from the affected allele indicated that exon 1 deletions are inactivating mutations. Although the number of TSC patients with large rearrangements of TSC1 is small, these patients tend to have a somewhat milder phenotype compared with the group of patients with small TSC1 mutations.     

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.     

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

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

Mutational analysis of TSC1 and TSC2 genes in Japanese patients with tuberous sclerosis complex

We have surveyed the mutations of TSC1 and TSC2 from 38 (25 sporadic, 11 familial, and 2 unknown) Japanese patients with tuberous sclerosis complex. In 23 of 38 subjects, we detected 18 new mutations in addition to 4 mutations that had been previously reported. We also found 3 new polymorphisms. The mutations were not clustered on a particular exon in either of the genes. Seven TSC1 mutations found in 3 familial and 4 sporadic cases were on the exons (3 missense, 2 nonsense point mutations, a 1-base insertion, and a 2-bp deletion). Fifteen TSC2 mutations were found in 5 familial cases, 10 sporadic cases, and 1 unknown case. The 12 mutations were on the exons (8 missense, 1 nonsense point mutations, a 1-bp insertion, a 5-bp deletion, and a 4-bp replacement) and 3 point mutations were on the exon–intron junctions. Although the patients with TSC2 mutations tend to exhibit relatively severe mental retardation in comparison to those with TSC1 mutations, a genotype–phenotype correlation could not yet be established. The widespread distribution of TSC1/TSC2 mutations hinders the development of a simple diagnostic test, and the identification of individual mutations does not provide the prediction of prognosis. Received: April 5, 1999 / Accepted: June 12, 1999     

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

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 x 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.      

Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance

We performed a comprehensive analysis for mutations in the TSC1 gene using Southern blot analysis, and SSCP and heteroduplex analysis of amplified exons in 13 families with genetic linkage to the TSC1 region, 22 small families without linkage information, and 126 sporadic patients. 17 unique mutations were identified in 21 patients. Mutations were found in 7/13 (54%) TSC1-linked families, 1/22 (5%) small families without linkage, and 13 of 126 (10%) sporadic cases. The mutations were all chain-terminating, with 14 small deletions, 1 small insertion, and 6 nonsense mutations. In families with mutations, all individuals carrying a mutation met formal diagnostic criteria for TSC, apart from a 3-year-old girl who had inherited a deletion mutation, and who had no seizures, normal intelligence, normal abdominal ultrasound, and hypomelanotic macules only on physical exam. We assessed the incidence and severity of mental retardation in the 13 sporadic patients with TSC1 mutations versus the entire sporadic cohort, and found no significant difference. The observations indicate that TSC1 mutations are all inactivating, suggest that TSC1 disease occurs in only 15–20% of the sporadic TSC population, and demonstrate that presymptomatic TSC does occur.     

Comprehensive mutation analysis of TSC1 using two-dimensional DNA electrophoresis with DGGE

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of benign tumors in multiple organs often causing serious neurologic impairment. To develop a reliable genetic test for TSC, two-dimensional electrophoresis with denaturing gradient gel electrophoresis (2D DGGE) has been developed to detect mutations in TSC1 . The 23 exons of TSC1 were amplified using two rounds of PCR. In the first round, all coding regions of TSC1 were amplified in four fragments ranging in size from 7.4 kb to 9.9 kb. In the second round, 32 fragments representing 23 exons were amplified using primers designed to avoid overlapping fragments and with a GC clamp on one end to optimise melting characteristics. These exon fragments were then separated by size in the first dimension using a polyacrylamide gel, and by melting characteristics in the second dimension using a urea/formamide gradient to yield 32 distinct bands. If a mutation is present, four bands instead of one, are typically observed. During the development of this assay, we analysed 63 patient samples with known TSC1 mutations from prior studies. These 63 patients had 68 known mutations or polymorphisms. With DGGE, all 68 of these were identified (45 point mutations, 3 small insertions, 20 small deletions) and an additional 27 single base variants were discovered. To evaluate the assay, we analysed 19 of these samples in a blinded study. In the blinded analysis, 19/20 (95%) known mutations or polymorphisms were detected. The single missed mutation in the blinded analysis could be identified in retrospect and the assay was modified accordingly. During this study, we identified 2 new mutations (exon 8 and exon 15), a new polymorphism (intron 4), and the first variant identified in a non-coding exon (exon 2).     

Determining the pathogenicity of patient-derived TSC2 mutations by functional characterization and clinical evidence

Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.     

Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype--phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple organs and tissues. TSC is caused by mutations in either the TSC1 or TSC2 gene. We searched for mutations in both genes in a cohort of 490 patients diagnosed with or suspected of having TSC using a combination of denaturing gradient gel electrophoresis, single-strand conformational polymorphism, direct sequencing, fluorescent in situ hybridisation and Southern blotting. We identified pathogenic mutations in 362 patients, a mutation detection rate of 74%. Of these 362 patients, 276 had a definite clinical diagnosis of TSC and in these patients 235 mutations were identified, a mutation detection rate of 85%. The ratio of TSC2:TSC1 mutations was 3.4:1. In our cohort, both TSC1 mutations and mutations in familial TSC2 cases were associated with phenotypes less severe than de novo TSC2 mutations. Interestingly, consistent with other studies, the phenotypes of the patients in which no mutation was identified were, overall, less severe than those of patients with either a known TSC1 or TSC2 mutation.     

Functional assessment of TSC1 missense variants identified in individuals with tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Previously, we demonstrated that pathogenic amino acid substitutions in the N-terminal domain of TSC1 (amino acids 50-224) are destabilizing. Here we investigate an additional 21 unclassified TSC1 variants. Our functional assessment identified four substitutions (p.L61R, p.G132D, p.F158S, and p.R204P) between amino acids 50 and 224 that reduced TSC1 stability and prevented the TSC1-TSC2-dependent inhibition of TORC1. In four cases (20%), our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our new data confirm our previous finding that the N-terminal region of TSC1 is essential for TSC1 function.     

Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation

Tuberous sclerosis complex is an inherited tumour suppressor syndrome, caused by a mutation in either the TSC1 or TSC2 gene. The disease is characterised by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction, and dermatological abnormalities. The TSC1 gene was recently identified and has 23 exons, spanning 45 kb of genomic DNA, and encoding an 8.6 kb mRNA. After screening all 21 coding exons in our collection of 225 unrelated patients, only 29 small mutations were detected, suggesting that TSC1 mutations are under-represented among TSC patients. Almost all TSC1 mutations were small changes leading to a truncated protein, except for a splice site mutation and two in frame deletions in exon 7 and exon 15. No clear difference was observed in the clinical phenotype of patients with an in frame deletion or a frameshift or nonsense mutation. We found the disease causing mutation in 13% of our unrelated set of TSC patients, with more than half of the mutations clustered in exons 15 and 17, and no obvious under-representation of mutations among sporadic cases. In conclusion, we find no support for a genotype-phenotype correlation for the group of TSC1 patients compared to the overall population of TSC patients.     

Missense mutations to the TSC1 gene cause tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Here we investigate the effects of putative TSC1 missense mutations identified in individuals with signs and/or symptoms of TSC on TSC1-TSC2 complex formation and mTOR signalling. We show that specific amino-acid substitutions close to the N-terminal of TSC1 reduce steady-state levels of TSC1, resulting in the activation of mTOR signalling and leading to the symptoms of TSC.     

Analysis of both TSC1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of multiple hamartomas involving many organs. About two-thirds of the cases are sporadic and appear to represent new mutations. With the cloning of two causative genes, TSC1 and TSC2 it is now possible to analyze both genes in TSC patients and identify germline mutations. Here we report the mutational analysis of the entire coding region of both TSC1 and TSC2 genes in 126 unrelated TSC patients, including 40 familial and 86 sporadic cases, by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Mutations were identified in a total of 74 (59%) cases, including 16 TSC1 mutations (5 sporadic and 11 familial cases) and 58 TSC2 mutations (42 sporadic and 16 familial cases). Overall, significantly more TSC2 mutations were found in our population, with a relatively equal distribution of mutations between TSC1 and TSC2 among the familial cases, but a marked underrepresentation of TSC1 mutations among the sporadic cases (P = 0.0035, Fisher's exact test). All TSC1 mutations were predicted to be protein truncating. However, in TSC2 13 missense mutations were found, five clustering in the GAP-related domain and three others occurring in exon 16. Upon comparison of clinical manifestations, including the incidence of intellectual disability, we could not find any observable differences between TSC1 and TSC2 patients. Our data help define the distribution and spectrum of mutations associated with the TSC loci and will be useful for both understanding the function of these genes as well as genetic counseling in patients with the disease.     

Three independent mutations in the TSC2 gene in a family with tuberous sclerosis

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by hamartomas and hamartias in multiple organs. TSC is caused by a wide spectrum of mutations within the TSC1 and TSC2 genes. Here, we report a unique family with three independent pathological mutations in TSC2. A c.1322G>A mutation in exon 12 created a stop codon, whereas a second mutation in exon 23 (c.2713C>T) was a missense change. The third mutation was a 4 base pair deletion in intron 20 of TSC2. We showed that this mutation was responsible for abnormal splicing. The three mutations were most likely de novo, as parents of affected patients did not present any features of TSC. In addition, we showed gonadal mosaicism in a branch of the family. To our knowledge, several independent mutations in TSC2 have never been observed in a single family. The probability of finding a family with three different pathological TSC2 mutations is extremely low. We discuss two main hypotheses that may be raised to explain this recurrence: (i) the TSC2 mutation rate is underestimated. In such a case, the likelihood of finding a family with three independent mutations in TSC2 may not be dramatically low; (ii) a heritable defect in a DNA repair gene (eg, mismatch repair gene) segregating in the family that is unlinked to the TSC2 gene might predispose to the occurrence of multiple TSC2 gene mutations, used as a specific target during embryogenesis.     

Pathogenesis of multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis and association with tuberous sclerosis genes TSC1 and TSC2

Tuberous sclerosis (TSC) is a rare, genetically determined disorder / familial tumor syndrome, currently diagnosed using specific clinical criteria proposed by Gomez, including the presence of multiorgan hamartomas. Pulmonary involvement in TSC is well known as pulmonary lymphangioleiomyomatosis (LAM), which has an incidence of 1-2.3% in TSC patients. LAM has immunohistochemical expression of both smooth-muscle actin and a monoclonal antibody specific for human melanoma, HMB-45. It has recently been reported that multifocal micronodular pneumocyte hyperplasia (MMPH) associated with TSC should be considered as a distinct type of lung lesion, whether it occurs with or without LAM. Two predisposing genes have been found in families affected by TSC; approximately half of the families show linkage to TSC1 at 9q34.3, and the other half show linkage to TSC2 at 16p13.3. TSC genes are considered to be tumor suppressor genes, and mutations in them may lead to abnormal differentiation and proliferation of cells. Tuberin, the TSC2 gene product, has recently been found to be expressed in LAM and MMPH. In this article we discuss the histogenesis and genetic abnormalities of neoplastic lesions associated with TSC, and we review the current understanding of the pathogenesis of pulmonary hamartomatous lesions such as LAM and MMPH in TSC.     

TSC1 involvement in bladder cancer: diverse effects and therapeutic implications

TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small‐molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon‐based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT‐4, HCV29, 97–1. Interestingly, only RT‐4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97–1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP‐AUY922, and we then found synergistic inhibitory effects of NVP‐AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97–1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1‐mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

结节性硬化症家系的基因诊断及产前诊断

目的 对结节性硬化症(tuberous sclerosis complex,TSC)患者进行基因突变检测,并在基因诊断结果明确的基础上应用于产前诊断.方法 应用聚合酶链反应-变性高效液相色谱(polymerase chain reaction-denaturing high-performance liquid chromatography,PCR-DHPLC)、DNA测序技术,对19个家系的21例TSC患者进行TSC1和TSC2基因的突变检测.结果 在19个家系21例患者中发现17种不同的基因突变,其中13种突变未见报道,包括TSCj基因的c.2672delA、c.2672insA和TSC2基因的c.4918insCGCC、c.1143delG、Intron27+1 G＞A、c.1957-1958delAG、Intron5+1 G＞A、c.910insCT、c.2753C＞G、c.4078dupAGCAAGTCCAGCTCCTC、Intron 11-1 G＞A、Intron 14+1 G＞A、c.684 C＞A.对7个家系进行了产前诊断,其中6个家系的胎儿均未发现其家系先证者所具有的突变,胎儿出生后电话随访至1～4岁无TSC的症状出现.而另一家系的胎儿携带有和母亲一样的突变,经遗传咨询后,家属选择了引产.结论 本研究证实的TSC基因突变中,有76.5%(13/17)的突变均未在其他研究中被发现,说明中国人群TSC基因的突变谱可能与其他人群具有较明显的差异;本研究中TSC基因诊断率为89.5%(17/19),提示TSC的发生可能还有其它未知的遗传病因;在有家族史的病例中,TSC1与TSC2有相似的突变比例,而在散发病例中,TSC2的突变更加常见;13种新突变患者的父母均无类似突变,说明TSC致病基因具有较高的自发突变率.     

Germ line MLH1 and MSH2 mutations in Taiwanese Lynch syndrome families: characterization of a founder genomic mutation in the MLH1 gene

This multicenter study evaluated the mutation spectrum and frequencies of the MLH1 and MSH2 genes and determined the occurrence of large genomic deletions in 93 unrelated Taiwanese families that fulfilled the Amsterdam criteria II by denaturing high-performance liquid chromatography analysis, DNA sequencing for aberrant chromatograms, and multiplex ligation-dependent probe amplification analysis. In total, 38 pathogenic mutations (10 large deletions and 28 point mutations or small deletion/insertions) in the MSH2 or MLH1 gene were identified in 61 of the 93 families (66%). Three of the 10 large deletions and 14 of the 28 point mutations or small insertions/deletions have not been reported elsewhere. Three mutations in the MLH1 gene, the MLH1 c.1846_1848delAAG (5 families), deletion exons 11–15 (4 unrelated families), and MLH1 c.793C>T (13 unrelated families), accounted for 35% of all cases with pathogenic mutations. Haplotype analysis indicated that mutant c.793C>T alleles were derived from two distinct common founders that might be inherited from a single ancestor of presumably Chinese origin. As a mutation detection strategy for Taiwanese Lynch syndrome patients, we recommend that diagnosis starts with screening for large genomic deletions and continues by screening for common mutations in exons 10 and 16 of the MLH1 gene prior to searching for small mutations in the remaining exons.     

Exon scanning of the entire TSC2 gene for germline mutations in 40 unrelated patients with tuberous sclerosis

Tuberous sclerosis complex (TSC) is a dominantly inherited multisystem disorder resulting in the development of hamartomatous growths in many organs. Genetic heterogeneity has been demonstrated linking the familial cases to either TSC1 at 9q34.3, or TSC2 at 16p13.3. About two-thirds of the TSC cases are sporadic and appear to represent new mutations. While both genes are thought to account for all familial cases, with each representing approximately 50% of the mutations, the proportion of sporadic cases with mutations in TSC1 and TSC2 is yet to be determined. We have examined the entire coding sequence of the TSC2 gene in 20 familial and 20 sporadic cases and identified a total of twenty-one mutations representing 50% and 55% of familial and sporadic cases respectively. Our rate of mutation detection is significantly higher than other published reports. Twenty out of 21 mutations are novel and include 6 missense, 6 nonsense, 5 frameshifts, 2 splice alterations, a 34 bp deletion resulting in abnormal splicing, and an 18 bp deletion which maintains the reading frame. The mutations are distributed throughout the coding sequence with no specific hot spots. There is no apparent correlation between mutation type and clinical severity of the disease. Our results document that at least 50% of sporadic cases arise from mutations in the TSC2 gene. The location of the mutations described here, particularly the missense events, should be valuable for further functional analysis of this tumor suppressor protein. Hum Mutat 12:408–416, 1998. © 1998 Wiley-Liss, Inc.