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Background\ud Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease affecting multiple body systems with wide variability in presentation. In 2013, Pediatric Neurology published articles outlining updated diagnostic criteria and recommendations for surveillance and management of disease manifestations. Advances in knowledge and approvals of new therapies necessitated a revision of those criteria and recommendations.\ud Methods\ud Chairs and working group cochairs from the 2012 International TSC Consensus Group were invited to meet face-to-face over two days at the 2018 World TSC Conference on July 25 and 26 in Dallas, TX, USA. Before the meeting, working group cochairs worked with group members via e-mail and telephone to (1) review TSC literature since the 2013 publication, (2) confirm or amend prior recommendations, and (3) provide new recommendations as required.\ud Results\ud Only two changes were made to clinical diagnostic criteria reported in 2013: “multiple cortical tubers and/or radial migration lines” replaced the more general term “cortical dysplasias,” and sclerotic bone lesions were reinstated as a minor criterion. Genetic diagnostic criteria were reaffirmed, including highlighting recent findings that some individuals with TSC are genetically mosaic for variants in TSC1 or TSC2. Changes to surveillance and management criteria largely reflected increased emphasis on early screening for electroencephalographic abnormalities, enhanced surveillance and management of TSC-associated neuropsychiatric disorders, and new medication approvals.\ud Conclusions\ud Updated TSC diagnostic criteria and surveillance and management recommendations presented here should provide an improved framework for optimal care of those living with TSC and their families.

Summary Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling., Graphical Abstract, Highlights • G3BPs act non-redundantly in the TSC-mTORC1 signaling axis • G3BPs reside at the lysosomal surface and inhibit mTORC1 • The TSC complex requires G3BPs as its lysosomal tether • G3BP1 deficiency phenocopies TSC complex loss in cancer cells and neurons, Distinct from their contributions to stress granules, G3BPs regulate mTORC1 activity through spatial control of the TSC complex.

Background\ud The severity of Tuberous Sclerosis Complex (TSC) can vary among affected individuals. Complications of TSC can be life threatening, with significant impact on patients’ quality of life. Management may vary dependent on treating physician, local and national policies, and funding. There are no current UK guidelines. We conducted a Delphi consensus process to reach agreed guidance for the management of patients with TSC in the UK.\ud \ud Methods\ud We performed a literature search and reviewed the 2012/13 international guideline for TSC management. Based on these, a Delphi questionnaire was formed. We invited 86 clinicians and medical researchers to complete an online survey in two rounds. All the people surveyed were based in the UK. Clinicians were identified through the regional TSC clinics, and researchers were identified through publications. In round one, 55 questions were asked. In round two, 18 questions were asked in order to obtain consensus on the outstanding points that had been contentious in round one. The data was analysed by a core committee and subcommittees, which consisted of UK experts in different aspects of TSC. The Tuberous Sclerosis Association was consulted.\ud \ud Results\ud 51 TSC experts took part in this survey. Two rounds were required to achieve consensus. The responders were neurologists, nephrologists, psychiatrist, psychologists, oncologists, general paediatricians, dermatologist, urologists, radiologists, clinical geneticists, neurosurgeons, respiratory and neurodisability clinicians.\ud \ud Conclusions\ud These new UK guidelines for the management and surveillance of TSC patients provide consensus guidance for delivery of best clinical care to individuals with TSC in the UK.

In tuberous sclerosis (TSC)–associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2+/− mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2+/− mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.

Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem genetic disorder characterized by benign tumors in multiple organs, including the skin, brain, kidneys, and lungs and occasional malignant tumors. Hamartomas in the brain, retina, and sometimes other organs also occur (1–3). The estimated prevalence is 1:600–1:10,000 live births in the general population (4–6). Patients present at different ages with different manifestations, and varying degrees of organ involvement (Figure 1). CNS manifestations of TSC mainly present in childhood, affect around 85% of patients (8), frequently resulting in epilepsy refractory to treatment, intellectual impairment, autistic spectrum disorder, attention deficit hyperactivity disorder, and behavioral problems (1–3). Renal angiomyolipomas (AMLs) occur in ~80% of patients (9); kidney disease is the leading cause of death in adults with TSC (10). TSC is complex and highly varied (Figure 1) necessitating careful coordination of care, which is lacking for most patients in the UK. Some TSC manifestations are rarer; e.g., subependymal giant cell astrocytoma (SEGA) occurs in around 20–24% of patients (11, 12) (Figure 2). The major unsolved problem in TSC is refractory epilepsy and TSC-associated neuropsychiatric disorders (TAND); of which preliminary evidence suggests refractory epilepsy is a major cause (13–15). TSC, like other complex rare diseases, is a major burden to patients, families and healthcare systems. Optimizing care will alleviate some of this while waiting for medical research to deliver a cure. Classically, a clinical diagnosis of TSC is made by identifying major and minor features (Table 1) (1, 16). With wider availability of genetic testing, identification of pathogenic mutations in TSC1 or TSC2 is now sufficient to establish a diagnosis, regardless of the presence of clinical features (1, 16), and is particularly useful in confirming a suspected diagnosis, as many clinical TSC manifestations are infrequent in young patients (1, 16). The approval of the mTORC1 inhibitor—everolimus—for the treatment of AMLs, SEGA, and refractory epilepsy represents a significant advance in the potential management of the disease (17–19). Whilst not licensed in Europe, the Federal Drugs Agency (FDA) have also approved sirolimus for use in pulmonary lymphangioleiomyomatosis (LAM) (18). Refractory seizures adversely affect early development (20). Furthermore, appropriate early treatment of infantile spasms with vigabatrin has been shown to reduce the long-term impact of the neurological and neuropsychiatric aspects of TSC on patients (13, 14). A retrospective UK cohort study linking Clinical Practice Research Datalink (CPRD) to Hospital Episode Statistics (HES) data identified 334 patients with TSC revealed a much lower frequency of complications than would be expected from previous research; the disparity possibly reflecting under-recognition, and hence suggestive of inadequate medical care (7). It is clear from these findings, and the observation that many new patients referred to TSC clinics have never had holistic systematic monitoring, that many patients receive inadequate care. In the UK, about 1000 TSC families are known to the UK Tuberous Sclerosis Association, known as the TSA (Patient organization), and a similar number (usually the same families) attend UK specialist TSC clinics. Therefore, in most other cases, the quality of care delivered is unknown. Given the range of organ systems affected by TSC, its treatment requires coordination across a number of medical specialties over a patient's lifetime (Table 2). Currently in the UK, 16 centers host specialist TSC clinics—but most UK TSC patients are not currently managed within them. These specialist clinics have often been founded by enthusiastic clinicians but are frequently inadequately funded. The transition from pediatric to adult services can be particularly challenging in the absence of a systematic service. In Wales, a specialist TSC clinic that has been established through a partnership, between a pharmaceutical company and the NHS, awaits the development of a fully sustainable commissioning model. In Northern Ireland, a TSC clinic has been running since 1995, and directly reviews the majority of TSC patients in the region. In the UK, specialized service specifications are in place for adults and children with genetic disorders such as cystic fibrosis and inherited metabolic disorders. These are funded by NHS England, the Department of Health, Social Services and Public Safety in Northern Ireland, and Welsh Health Specialized Services Committee in Wales. However, no similar service or service specification is yet available for TSC patients. We propose a comprehensive, holistic model of care—to manage patients that present with a range of manifestations, requiring specialist management from a wide range of specialties (Figures 1, 2).

Understanding the development and function of the nervous system is one of the foremost aims of current biomedical research. The nervous system is generated during a relatively short period of intense neurogenesis that is orchestrated by a number of key molecular signalling pathways. Even subtle defects in the activity of these molecules can have serious repercussions resulting in neurological, neurodevelopmental and neurocognitive problems including epilepsy, intellectual disability and autism. Tuberous sclerosis complex (TSC) is a monogenic disease characterised by these problems and by the formation of benign tumours in multiple organs, including the brain. TSC is caused by mutations in the TSC1 or TSC2 gene leading to activation of the mechanistic target of rapamycin (mTOR) signalling pathway. A desire to understand the neurological manifestations of TSC has stimulated research into the role of the mTOR pathway in neurogenesis. In this review we describe TSC neurobiology and how the use of animal model systems has provided insights into the roles of mTOR signalling in neuronal differentiation and migration. Recent progress in this field has identified novel mTOR pathway components regulating neuronal differentiation. The roles of mTOR signalling and aberrant neurogenesis in epilepsy are also discussed. Continuing efforts to understand mTOR neurobiology will help to identify new therapeutic targets for TSC and other neurological diseases.

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a multi-system genetic disorder characterised by the development of benign growths and diverse clinical manifestations, varying in severity, age at onset and with high clinical burden.AIMS: This longitudinal study aims to describe the broad spectrum of clinical manifestation profiles in a large, representative cohort of TSC patients in the UK in order to better understand disease complexity.METHODS: TSC patients in the Clinical Practice Research Datalink (CPRD) and linked Hospital Episodes Statistics (CPRD-HES) were retrospectively identified between 1987 and 2013. Available history was extracted for each patient and clinical diagnosis, procedure and medication records reviewed. A random selection of patients from the CPRD-HES was used as a Comparator cohort.RESULTS: Three hundred and thirty-four TSC patients with a mean (SD) age of 30.3 (18.6) years were identified (53% female). TSC was diagnosed at mean age 3.2 (4.2) years. Epilepsy and psychiatric manifestations were reported frequently in paediatric (77% and 55%, respectively) and adult patients (66% and 68%, respectively). The prevalence of manifestations in the TSC cohort was markedly higher versus the Comparator cohort. The majority of paediatric (46%) and adult TSC patients (62%) developed clinical manifestations affecting at least three organ systems and forty-nine distinctive organ system manifestation profiles were identified.CONCLUSIONS: TSC patients present with multiple and complex clinical manifestations and profiles that necessitate the co-ordinated action of a multidisciplinary team in order to improve the quality and efficiency of care.

Epilepsy affects 75% to 90% of people with tuberous sclerosis, a multisystem genetic disorder. Although seizures can occur for the first time at any age, onset in infancy or childhood is usual. Around 30% of patients present with infantile spasms that often respond well to treatment with vigabatrin. Later seizures may occur as specific patterns, such as in Lennox-Gastaut syndrome, or with combinations of seizures including focal and multifocal seizures, and drop attacks. Most patients have two or more seizure types. Seizure control using current antiepileptic drugs is often unsatisfactory, leading to frequent polypharmacy. Epilepsy surgery has a place in the management of some patients. Mutations in the TSC1 and TSC2 genes that cause tuberous sclerosis lead to hyperactivation of signaling via the mammalian target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1 have recently been shown to be effective treatments for some manifestations of tuberous sclerosis; they are now being assessed as potential novel antiepileptic drugs in tuberous sclerosis and related disorders.

Tuberous sclerosis (TSC) is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR. Tumour responses in TSC patients to rapamycin, an allosteric inhibitor of mTOR, or its analogs are partial and reversible probably due to feedback activation of Akt. In this study, we examined the efficacy of GSK2126458, an ATP-competitive dual inhibitor of PI3K/mTOR, in comparison to rapamycin for treatment of renal tumours in genetically engineered Tsc2+/- mice. We found that both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 also significantly reduced the number and size of all lesions (cystic, papillary and solid) although to a lesser extent compared to rapamycin. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Furthermore, GSK2126458 and rapamycin suppressed proliferation of tumour cells. Importantly, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are therefore needed to test whether rapamycin in combination with GSK2126458 could promote apoptosis and thus improve therapy of TSC-associated renal tumours.

Nearly all patients with tuberous sclerosis complex (TSC) develop renal angiomyolipomas, although the tumor cell of origin is unknown. We observed decreased renal angiomyolipoma development in patients with TSC2- polycystic kidney disease 1 deletion syndrome and hypertension that were treated from an early age with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers compared with patients who did not receive this therapy. TSC-associated renal angiomyolipomas expressed ANG II type 1 receptors, platelet-derived growth factor receptor-β, desmin, α-smooth muscle actin, and VEGF receptor 2 but did not express the adipocyte marker S100 or the endothelial marker CD31. Sera of TSC patients exhibited increased vascular mural cell-secreted peptides, such as VEGF-A, VEGF-D, soluble VEGF receptor 2, and collagen type IV. These findings suggest that angiomyolipomas may arise from renal pericytes. ANG II treatment of angiomyolipoma cells in vitro resulted in an exaggerated intracellular Ca2+response and increased proliferation, which were blocked by the ANG II type 2 receptor antagonist valsartan. Blockade of ANG II signaling may have preventative therapeutic potential for angiomyolipomas.

Metformin, a substrate of several poly-specific organic cation transporters, is a widely used biguanide for the treatment of type II diabetes. Recent studies suggest that metformin attenuates mTORC1 signalling by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) in the presence or absence of a functional hamartin/tuberin (TSC1/TSC2) complex. Metformin has also been reported to inhibit mTORC1 independent of AMPK through p53-dependent regulated in development and DNA damage responses 1 (REDD1) or by inhibiting Rag GTPases. These observations suggest that metformin could have therapeutic potential for tuberous sclerosis, an inherited disorder characterised by the aberrant activation of mTORC1 and the development of tumours in many organs, including the kidneys. In this study, we investigated the effect of metformin on renal lesions in a Tsc1(+/-) mouse model of tuberous sclerosis. Continuous treatment of metformin for 9 months at doses of up to 600 mg/kg/day had no significant effect on renal lesions in nine treated mice compared to 10 controls. Metformin treatment appeared to attenuate mTORC1 signalling in Tsc1(+/-) kidney tissues but not in renal tumours. Surprisingly, the expression of the organic cation transporters Slc22a1, Slc22a2 and Slc22a3 essential for the cellular uptake of metformin was highly suppressed in renal tumours. Treatment of cultured cells derived from a Tsc1-associated renal tumour with 5-aza-2-deoxycytidine or trichostatin A greatly increased the expression of these genes. These data suggest that the epigenetic suppression of the organic cation transporters in Tsc-associated mouse renal tumours may contribute to the lack of response to metformin treatment.

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder characterized by the development of diverse clinical manifestations. The complexity of this disease is likely to result in substantial challenges and costs in disease management throughout the patient's lifetime. This retrospective database study aims to quantify healthcare resources utilized by TSC patients.TSC patients in the Clinical Practice Research Datalink linked to the Hospital Episodes Statistics database were identified between January 1987 and June 2013. Analyses were conducted over the most recent 3-year period of data and stratified by pediatric (18) and adult patients. Prescriptions, procedures, diagnostic tests, and healthcare encounters were reported in comparison with a matched comparator cohort. Costs and key economic drivers by primary organ system manifestations were also examined.A total of 286 patients with TSC were identified and consistently reported 2-fold greater resource use than the matched presumably healthy controls. Despite this comparatively greater resource use, half of TSC patients did not record any procedures, and 20% of patients did not record any diagnostic tests; however, inpatient hospitalizations were greater for the TSC cohort (3.1 vs 1.3), but length of stay was comparable. TSC patients had costs totaling £12,681 per patient over the 3-year period, a figure 2.7-fold greater than the total costs in the comparator cohort (£4,777). Costs for patients with specific primary manifestations were even greater, with brain manifestations incurring £22,139 per affected patient. Kidney and nervous system manifestations were the main cost drivers.The economic burden of TSC and its impact on NHS healthcare resources is mostly attributable to the broad spectrum of manifestations that develop within multiple organ systems. TSC patients may benefit from co-ordinated care based on their requirement for high numbers of healthcare visits across specialties.

Renal-related disease is the most common cause of tuberous sclerosis complex (TSC)-related death in adults, and renal angiomyolipomas can lead to complications that include chronic kidney disease (CKD) and hemorrhage. International TSC guidelines recommend mammalian target of rapamycin (mTOR) inhibitors as first-line therapy for management of asymptomatic, growing angiomyolipomas >3 cm in diameter. This review discusses data regarding patient outcomes that were used to develop current guidelines for embolization of renal angiomyolipomas and presents recent data on 2 available mTOR inhibitors - sirolimus and everolimus - in the treatment of angiomyolipoma. TSC-associated renal angiomyolipomas can recur after embolization. Both sirolimus and everolimus have shown effectiveness in reduction of angiomyolipoma volume, with an acceptable safety profile that includes preservation of renal function with long-term therapy. The authors propose a hypothesis for mTORC1 haploinsufficiency as an additional mechanism for CKD and propose that preventive therapy with mTOR inhibitors might have a role in reducing the number of angiomyolipoma-related deaths. Because mTOR inhibitors target the underlying pathophysiology of TSC, patients might benefit from treatment of multiple manifestations with one systemic therapy. Based on recent evidence, new guidelines should be considered that support the earlier initiation of mTOR inhibitor therapy for the management of renal angiomyolipomas to prevent future serious complications, rather than try to rescue patients after the complications have occurred.

Background Tuberous sclerosis complex (TSC) is a genetic disorder affecting about 1 in 6000 people and is characterised by the development of tumours in many organs, including the skin and kidneys, and by a range of neurological and neuropsychiatric manifestations. TSC-associated neuropsychiatric disorders (TAND) occur in the majority of those with TSC, and they have a significant impact on patients and their families, given the everyday impact of TAND on education, employment, family and social life. The potential benefits of better treatment for TAND therefore include reduction in health care demands and wider benefits for patients and their carers. Methods/design We have planned a single-centre, two-arm, individually randomised, phase II, double-blind, placebo-controlled trial of everolimus versus placebo in the treatment of neurocognitive problems in patients with tuberous sclerosis. Everolimus is a licensed medicine in this patient group, but for a different target of effect. The present trial is a proof-of-principle study developed to provide effect size estimates which may be used to inform the design of subsequent trials. Forty-eight patients aged 16–60 years with tuberous sclerosis who have an IQ >60 and a significant deficit (at least −2 SD) in one or more primary outcome measures will be randomly allocated in a ratio of 2:1 to receive everolimus or placebo, respectively. Participants will be assessed for eligibility and then be started on study medication 4 weeks later. They will then be randomised and receive placebo or everolimus for 24 weeks. Neurocognitive and safety assessments will be carried out at baseline and weeks 4, 12, 24 and 36. Discussion This study is designed to determine the effect sizes of treatment with everolimus or placebo for 6 months on specific neurocognitive functions—recall memory (verbal and non-verbal) and executive function—in people affected by TSC who have significant deficits in these functions. These data will provide new evidence to determine whether larger-scale trials are indicated and to explore suitable outcome measures and analytical methods for neurocognitive trial design. Trial registration ISRCTN09739757. Registered on 28 Dec 2011. Electronic supplementary material The online version of this article (doi:10.1186/s13063-016-1446-6) contains supplementary material, which is available to authorized users.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that affects multiple organ systems and is caused by loss-of-function mutations in one of two genes: TSC1 or TSC2. The disorder can affect both adults and children. First described in depth by Bourneville in 1880, it is now estimated that nearly 2 million people are affected by the disease worldwide. The clinical features of TSC are distinctive and can vary widely between individuals, even within one family. Major features of the disease include tumours of the brain, skin, heart, lungs and kidneys, seizures and TSC-associated neuropsychiatric disorders, which can include autism spectrum disorder and cognitive disability. TSC1 (also known as hamartin) and TSC2 (also known as tuberin) form the TSC protein complex that acts as an inhibitor of the mechanistic target of rapamycin (mTOR) signalling pathway, which in turn plays a pivotal part in regulating cell growth, proliferation, autophagy and protein and lipid synthesis. Remarkable progress in basic and translational research, in addition to several randomized controlled trials worldwide, has led to regulatory approval of the use of mTOR inhibitors for the treatment of renal angiomyolipomas, brain subependymal giant cell astrocytomas and pulmonary lymphangioleiomyomatosis, but further research is needed to establish full indications of therapeutic treatment. In this Primer, we review the state-of-the-art knowledge in the TSC field, including the molecular and cellular basis of the disease, medical management, major knowledge gaps and ongoing research towards a cure.

OBJECTIVE: Transgenic mouse models of tuberous sclerosis (TSC) develop renal cysts, cystadenomas, solid adenomas and carcinomas. Identification and characterisation of these lesions in vivo may help in TSC pre-clinical trials. This study was to evaluate T2 weighted MRI for assessment of renal lesions in two Tsc mouse models. MATERIALS AND METHODS: Tsc1(+/-), Tsc2(+/-) and wild type mice were subjected to a first MRI scan at 12 months of age and a second scan 2 months later. One Tsc2(+/-) mouse was treated with rapamycin for two months after the initial scan. Immediately following the second scan, mice were sacrificed and MRI images were compared to renal histological findings. RESULTS: MRI identified all types of Tsc-associated renal lesions in both Tsc1(+/-) and Tsc2(+/-) mice. The smallest detectable lesions were

Aims The Tuberous Sclerosis 2000 Study is the first comprehensive longitudinal study of tuberous sclerosis (TS) and aims to identify factors that determine prognosis. Mode of presentation and findings at initial assessments are reported here. Methods Children aged 0–16 years newly diagnosed with TS in the UK were evaluated. Results 125 children with TS were studied. 114 (91%) met clinical criteria for a definite diagnosis and the remaining 11 (9%) had pathogenic TSC1 or TSC2 mutations. In families with a definite clinical diagnosis, the detection rate for pathogenic mutations was 89%. 21 cases (17%) were identified prenatally, usually with abnormalities found at routine antenatal ultrasound examination. 30 cases (24%) presented before developing seizures and in 10 of these without a definite diagnosis at onset of seizures, genetic testing could have confirmed TS. 77 cases (62%) presented with seizures. Median age at recruitment assessment was 2.7 years (range: 4 weeks–18 years). Dermatological features of TS were present in 81%. The detection rate of TS abnormalities was 20/107 (19%) for renal ultrasound including three cases with polycystic kidney disease, 51/88 (58%) for echocardiography, 29/35 (83%) for cranial CT and 95/104 (91%) for cranial MRI. 91% of cases had epilepsy and 65% had intellectual disability (IQ

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.

Purpose: Renal angiomyolipomas are a frequent manifestation of tuberous sclerosis and sporadic lymphangioleiomyomatosis (LAM). These disorders are associated with mutations of TSC1 or TSC2 that lead to overactivation of mTOR complex 1 (mTORC1), suggesting an opportunity for targeted therapy by using mTORC1 inhibitors. This study investigated the efficacy and safety of the mTORC1 inhibitor sirolimus for treatment of renal angiomyolipomas in patients with these disorders. Experimental Design: In this multicenter phase 2 nonrandomized open label trial, 16 patients with tuberous sclerosis or sporadic LAM and renal angiomyolipoma(s) were treated with oral sirolimus for up to 2 years. Steady-state blood levels were 3 to 10 ng/mL. The primary outcome was change in size of renal angiomyolipomas measured by MRI and assessed by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Secondary outcomes included safety, neurocognitive function, and pulmonary function. Results: The response rate, by RECIST criteria, was 50%. Summated angiomyolipoma diameters were reduced in all 16 patients and by 30% or more in eight (all from the per protocol group of 10). Forty-one of 48 angiomyolipomas were smaller at the last measurement than at baseline. Most shrinkage occurred during the first year of treatment. There was little change in pulmonary function. Recall memory improved in seven of eight patients with tuberous sclerosis. Adverse events were consistent with the known toxicities of sirolimus. Conclusions: This study showed sustained regression of renal angiomyolipomas in patients with tuberous sclerosis or sporadic LAM receiving 2 years of sirolimus treatment. Possible effects on pulmonary function and neurocognition require further investigation. Clin Cancer Res; 17(12); 4071–81. ©2011 AACR.

To the Editor: Tuberous sclerosis is an autosomal dominant disorder characterized by hamartomatous growths in many organs and caused by inherited mutations of the TSC1 or TSC2 gene. Acquired (somat...

Background Tuberous sclerosis complex is a multisystem genetic disease, caused by mutation in the TSC1 or TSC2 genes, associated with many features, including intellectual disability (ID). We examined psychometric profiles of patients with TSC1 or TSC2 mutations and tested whether different mutation types were associated with different degrees of intellectual ability. Methods One hundred subjects with known TSC1 / TSC2 mutations were assessed using a range of IQ or developmental quotient (DQ) measures. Effects of mutations on TSC1/TSC2 proteins were inferred from sequence data and published biochemical studies. Results Most individuals with TSC1 mutations fell on a normal distribution identical to the general population, with ∼10% showing profound ID. Of individuals with TSC2 mutations, 34% showed profound ID, and the remainder a pattern of IQ/DQ more variable and shifted to the left than in TSC1 or the general population. Truncating TSC1 mutations were all predicted to be subject to nonsense-mediated mRNA decay. Mutations predicted to result in unstable protein were associated with less severe effects on IQ/DQ. There was a statistically significant negative correlation between length of predicted aberrant C-terminal tails arising from frameshift mutations in TSC1 and IQ/DQ; for TSC2 a positive but not statistically significant correlation was observed. Conclusion We propose a model where (i) IQ/DQ correlates inversely with predicted levels and/or deleterious biochemical effects of mutant TSC1 or TSC2 protein, and (ii) longer aberrant C-terminal tails arising from frameshift mutations are more detrimental for TSC1 and less for TSC2. Predictions of the model require replication and biochemical testing.

Purpose The tuberous sclerosis (TSC) genes TSC1 and TSC2 encode the protein products hamartin and tuberin, respectively, and are putative tumour suppressor genes. Germ-line mutation of either TSC gene leads to the development of the heritable disorder TSC. This disorder is characterized by the development of hamartomas in many organs and is associated with the proliferative lung disease, lymphangioleiomyomatosis, the brain tumour giant cell astrocytoma and occasionally with renal cell carcinoma. However, the TSC genes have not been studied in breast cancer. The current study investigated the expression of the TSC gene products and the potential mechanisms of their aberrancy in human breast cancer cells and tissues. Experimental design and results Using immunohistochemical analysis, both hamartin and tuberin were found to be strongly stained in normal mammary epithelial cells and weakly in stromal cells. In invasive tumour tissues, however, the staining of both proteins were to be markedly reduced (P < 0.01). At message level, although normal and tumour tissues expressed both TSC products, the transcript levels of tuberin was significantly lower in tumour tissues compared with normal tissues (P < 0.05). There was no statistical difference between node negative and node positive tumours with both hamartin and tuberin. Tumours from patients who developed recurrence and died from breast cancer had significantly low levels of tuberin compared with those who remained disease free (P = 0.03 and 0.05, respectively). Likewise, hamartin levels were significantly lower in patients with metastasis, recurrence and mortality, when compared with those remained disease free (P = 0.001, 0.041 and 0.003, respectively). Using methylation specific PCR, the TSC1 promoter was found to be heavily methylated in ZR751, MDA MB 435, and BT549, but not in MCF-7 which expressed highly level of hamartin. TSC1 promoter methylation was also seen in most breast tumours, but only in a limited number of normal tissues. The methylation of TSC2 promoter appears to be less frequent. MDA MB 468, MDA MB 483, MDA MB 435S and weakly MDA MB 435 were found to have methylated TSC2 promoter. In breast tissues, however, a very small number of samples were found to have methylation of the TSC2 promoter. Conclusion TSC1 genes are aberrantly expressed in human breast cancer cell lines and breast tumour tissues and their promoters are seen to be methylated in breast tumour tissues. The expression of TSC1 is associated with an unfavourable clinical outcome in patients with breast cancer.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or the TSC2 genes and characterized by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsc1 in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1-) has a recessive embryonic lethal phenotype. We found that a significant number (approximately 27%) of heterozygous (Tsc1+/-) mice on the C57BL/6 background died before weaning (P = 0.014) and show that these mice die in the post-natal period (P = 0.033), normally at 1-2 days, from unknown causes. Forty-four percent (7/16) of Tsc1+/- mice on a C3H background developed macroscopically visible renal lesions as early as 3-6 months, increasing to 95% (37/39) by 15-18 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent (16/20) of Tsc1+/- mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs wereor = 5 mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasized to the lungs. We detected loss of the wild-type Tsc1 allele and elevated levels of p-mTOR and p-S6 in lesions from Tsc1+/- mice. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models.

Chordomas are rare sacrococcygeal/sacral, sphenooccipital/clivus, and spinal tumors whose molecular etiology remains relatively understudied. As several anecdotal reports had described chordomas in individuals with tuberous sclerosis complex (TSC), a multisystem hamartoma syndrome, we hypothesized that the genes that cause TSC may have an etiological role in chordomas. In two cases of sacrococcygeal chordomas in individuals with TSC, one with a germ-line TSC2 mutation and the other with a germ-line TSC1 mutation, we confirmed somatic inactivation of the corresponding wild-type allele by loss of heterozygosity analysis and immunohistochemistry. These data provide the first evidence of a pathogenic role by TSC genes in sacrococcygeal chordomas.

The tuberous sclerosis complex genes TSC1 and TSC2 were first identified by positional cloning strategies in the heritable human disorder tuberous sclerosis. They encode previously unknown proteins, termed hamartin and tuberin respectively, that form a functional complex. The phenotypic manifestations of tuberous sclerosis are extremely diverse and suggest normal roles for TSC1 and TSC2 in regulating the growth, proliferation, migration and differentiation of many cell types. Investigations of TSC1 and TSC2 in a number of model organisms and cell-culture systems have provided new insights into the mechanisms through which these roles are effected. Most promisingly, the hamartin–tuberin complex has been shown to function as a negtive regulator of the insulin receptor/phosphoinositide 3-kinase/S6 kinase pathway. Drugs that act to inhibit this pathway may have therapeutic potential for tuberous sclerosis and the related disorder lymphangioleiomyomatosis.

Somatic mosaicism is a frequent phenomenon in mendelian disorders that exhibit a high proportion of new mutations; however, mutant alleles present at low frequency are difficult to detect and characterize. We have previously shown that denaturing high-performance liquid chromatography (DHPLC) can detect TSC1 and TSC2 mutations in tuberous sclerosis patients with low-level somatic mosaicism, even when direct sequencing cannot identify the causative lesion. Characterization of these mutations traditionally involves extensive sequencing of cloned products. To overcome this limitation, we have utilized DHPLC with an in-line fraction collector to isolate low-level heteroduplex peaks that can be directly sequenced to reveal the mutation. We have successfully applied this technique to resolve the mutations 2724-1G>C in TSC1and 1462-28del42bp, 1774del4bp, and N1643K (4947C>G) in TSC2, which were present in only 6.5–17% of the patients' alleles. We have also applied this technique to successfully resolve seven somatic APC mutations in colorectal tumor samples that were previously undetectable by direct PCR product sequencing. This method may simplify many of the currently challenging goals in mutation detection. Hum Mutat 21:112–115, 2003. © 2003 Wiley-Liss, Inc.

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Tuberous sclerosis (TSC) is an inherited disorder best known for its association with severe learning difficulties, epilepsy, behavioural problems, skin and renal pathology. Lymphangioleiomyomatosis (LAM), characterized by alveolar smooth muscle proliferation and cystic destruction of parenchyma, occurs as an infrequent symptomatic pulmonary complication in TSC and as a very rare sporadic disease in those without signs of TSC. Considered a generalized and progressive cystic lung disease that is difficult to treat with a poor prognosis, it has been reported almost exclusively in women, most commonly presenting with dyspnoea and pneumothorax in those of childbearing age. We investigated the clinical features and prognosis of LAM in patients with TSC including the effects of treatment, stratified by the method of diagnosis of LAM (i.e. histological or radiological). We found histological proof of diagnosis in 10 of 21 patients with TSC and symptomatic lung disease, onset in childhood in four, three males with LAM, individuals with apparently focal disease, great variation in clinical course and no clear treatment benefit. In those with TSC, symptomatic LAM is infrequent but causes a significant morbidity and mortality. It was not possible to detect predisposing factors, other than being female. Males with apparent LAM should be rigorously investigated.

Tuberous sclerosis complex is an autosomal dominant disorder that occurs owing to inactivating mutations in either TSC1 or TSC2. Tuberous sclerosis complex-related tumors in the brain, such as subependymal giant cell astrocytoma, and in the kidney, such as angiomyolipoma, can cause significant morbidity and mortality. Recently, randomized clinical trials (EXIST-1 and EXIST-2) of everolimus for each of these tuberous sclerosis complex-associated tumors demonstrated the benefit of this drug, which blocks activated mammalian target of rapamycin complex 1. Here we report on the spectrum of mutations seen in patients treated during these trials and the association between mutation and response. TSC2 mutations were predominant among patients in both trials and were present in nearly all subjects with angiomyolipoma in whom a mutation was identified (97%), whereas TSC1 mutations were rare in those subjects (3%). The spectrum of mutations seen in each gene was similar to those previously reported. In both trials, there was no apparent association between mutation type or location within each gene and response to everolimus. Everolimus responses were also seen at a similar frequency for the 16–18% of patients in each trial in whom no mutation in either gene was identified. These observations confirm the strong association between TSC2 mutation and angiomyolipoma burden seen in previous studies, and they indicate that everolimus response occurs regardless of mutation type or location or when no mutation in TSC1 or TSC2 has been identified.

Objectives—To assess the process involved in obtaining ethical approval for a single-centre study involving geographically dispersed subjects with an uncommon genetic disorder. Design—Observational data of the application process to 53 local research ethics committees (LRECs) throughout Wales, England and Scotland. The Multicentre Research Ethics Committee (MREC) for Wales had already granted approval. Results—Application to the 53 LRECs required 24,552 sheets of paper and took two months of the researcher's time. The median time taken for approval was 39 days with only seven (13%) of committees responding within the recommended 21 days. In at least nineteen cases (36%) a subcommittee considered the application. Thirty-three committees (62%) accepted the proposal without amendments but, of the remainder, four (8%) requested changes outside of the remit of LRECs. Discussion—Difficulties still exist with the system for obtaining ethical approval for studies involving a single centre but with patients at multiple sites, as is often required for genetic observational research. As such studies differ from true multicentre studies, it may be advantageous to develop a separate and specific process of application to ensure that resources are not unnecessarily expended in the quest for ethical approval. Key Words: Research ethics • MREC • LREC

The genetic events involved in the aetiology of non-clear-cell renal cell carcinoma (RCC) and a proportion of clear cell RCC remain to be defined. Germline mutations of the TSC1and TSC2genes cause tuberous sclerosis (TSC), a multi-system hamartoma syndrome that is also associated with RCC. We assessed 17 sporadic clear cell RCCs with a previously identified VHLmutation, 15 clear-cell RCCs without an identified VHLmutation and 15 non-clear-cell RCCs for loss of heterozygosity (LOH) at chromosomes 9q34 and 16p13.3, the chromosomal locations of TSC1and TSC2. LOH was detected in 4/9, 1/11 and 3/13 cases informative at both loci. SSCP analysis of the whole coding region of the retained allele did not reveal any cases with a detectable intragenic second somatic mutation. Furthermore, RT-PCR analysis of TSC1and TSC2on total RNA from 8 clear-cell RCC cell lines confirmed expression of both TSC genes. These data indicate that biallelic inactivation of TSC1or TSC2is not frequent in sporadic RCC and suggests that the molecular mechanisms of renal carcinogenesis in TSC are likely to be distinct. © 2001 Cancer Research Campaignhttp://www.bjcancer.com http://www.bjcancer.com

Over the past decade, there has been considerable progress in understanding the molecular genetics of tuberous sclerosis, a disorder characterised by hamartomatous growths in numerous organs. We review this progress, from cloning and characterising TSC1 and TSC2, the genes responsible for the disorder, through to gaining insights into the functions of their protein products hamartin and tuberin, and the identification and engineering of animal models. We also present the first comprehensive compilation and analysis of all reported TSC1 and TSC2 mutations, consider their diagnostic implications and review genotype/phenotype relationships.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple tissues and organs. TSC exhibits locus heterogeneity with genes at 9q34 (TSC1) and 16p13.3 (TSC2) that have 21 and 41 coding exons, respectively. The mutational spectrum at both loci is wide and previous studies have shown that 60%-70% of cases are sporadic and represent new mutations. We have formatted denaturing high performance liquid chromatography (DHPLC) for rapid screening of all coding exons of TSC1 and TSC2. DHPLC analysis detected likely disease-causing mutations in 103 of 150 unrelated cases (68%), compared with 92/150 (61%) and 87/150 (58%) for single-strand conformation polymorphism analysis (SSCP) and conventional heteroduplex analysis (HA), respectively. Capital, consumable and labour costs were determined for each exon screening procedure. Estimated costs per patient sample depended on throughput, particularly for DHPLC, where a high proportion of costs are fixed, and were pounds sterling 257, pound sterling 216 and pound sterling 242 for DHPLC, SSCP and HA, respectively, assuming a throughput of 252 samples per year, or pound sterling 354, pound sterling 233 and pound sterling 259, assuming a throughput of 126 samples per year. DHPLC had the advantages of increased sensitivity and reduced labour costs when compared with more traditional approaches to exon screening but, unless expensive DHPLC equipment is being efficiently utilised for a very high proportion of the time available, overall costs are slightly higher.

Thirty patients with tuberous sclerosis (from 29 different families) were examined for evidence of macroscopically visible pitted enamel hypoplasia. Of 23 patients with permanent teeth, 11 (48%) showed multiple enamel pits (mean 4.6 pits, range 3-9), but none were seen in six patients with deciduous teeth. Five of 563 controls (0.88%) had similar pitted enamel hypoplasia. Simple dental examination may be a useful adjunct in the assessment of patients with permanent teeth when a diagnosis of tuberous sclerosis is being considered, but is less likely to be helpful in the pre-school child.

Hereditary renal carcinoma in the Eker rat is an excellent example of predisposition to a specific cancer being transmitted as a dominant trait. Recently, we identified a germline mutation of the tuberous sclerosis 2 (Tsc2) gene in the Eker rat. In the present study, we analyzed the upstream region of the Tsc2 gene. A novel leader exon (exon 1a) in a CpG island was found, and core promoter activity was identified in a 242-bp region of this island. Exon 1a and the promoter region were conserved in the human TSC2 gene. In addition, a rat homolog of a gene found upstream of TSC2 in human has been identified, indicating that the genomic organization around Tsc2/TSC2 is conserved between the two species. Characterization of the 5' region of Tsc2 and TSC2 will facilitate studies of the regulation of the gene and its disregulation in tumorigenesis.

The tuberous sclerosis 2 (TSC2) gene is thought to function as a growth suppressor in sporadic and TSC-associated hamartomas and tumors. Clusters of dysplastic glial cells are a common feature of cortical tubers and subependymal nodules in tuberous sclerosis patients. In an effort to identify TSC2 gene alterations in sporadic gliomas, we detected a novel polymorphism adjacent to the 3′splice site of intron 4. We evaluated the distribution of this variant allele in a series of 244 patients with glial tumors, including 55 gangliogliomas, 31 pilocytic astrocytomas (WHO grade I), 50 astrocytomas (WHO grades II and III), and 108 glioblastomas (WHO grade IV). The allelic distribution in the general population was estimated by examining 381 healthy blood donors. This rare allele appeared in the control population and in the patients with astrocytic gliomas with a virtually identical frequency (8.14%, and 8.20%, respectively). The frequency of the rare allele in gangliogliomas, however, was significantly higher (15.5%; p = 0.024). The fact that both gangliogliomas and cortical tubers in tuberous sclerosis contain neuronal and astrocytic elements and may resemble each other histologically suggests that the TSC2 gene may be involved in the development of these tumors. The rare allele of the TSC2 gene emerges as a candidate for a predisposing factor for the formation of sporadic gangliogliomas.

Germ-line mutations of the TSC2 tumour suppressor gene have been identified in humans with tuberous sclerosis and in the Eker rat. Tuberin, the human TSC2 gene product, has a small region of homology with rap1GAP and stimulates rap1 GTPase activity in vitro, suggesting that one of its cellular roles is to function as a GTPase activating protein (GAP). We have undertaken a comparative analysis of the TSC2 gene in human and the pufferfish, Fugu rubripes. In addition to the GAP domain, three other regions of the proteins are highly conserved (peptide sequence similarity >80%). These regions are likely to represent further functional domains. To facilitate analysis of mutations within these domains we have determined the genomic structure of the human TSC2 gene. It comprises 41 exons, including exon 31 which was absent from the originally described spliceoform of the human TSC2 transcript and was identified following exon prediction from Fugu genomic sequence. These findings support the proposal of the Fugu genome as a tool for human gene analysis.

Renal involvement is common in tuberous sclerosis. Angiomyolipomas and cysts are found in approximately 50 and 30% of patients respectively, and often occur together. Tuberous sclerosis also appears to be associated with a small but increased risk of renal cell carcinoma. Recent studies have begun to elucidate the molecular genetic mechanisms underlying the renal manifestations of this systemic autosomal dominant disorder.

Tuberous sclerosis is an autosomal-dominant disorder characterised by seizures, intellectual disability, autistic spectrum disorder and the growth of benign tumours in multiple organs including the brain, skin, heart and kidneys. It is caused by mutations in either the TSC1 gene on 9q34 or the TSC2 gene on 16p13.3. The gene products, TSC1 and TSC2, interact and as a heterodimer act to integrate multiple of stimuli such as mitogenic stimulation, oxygen and energy levels into the control of diverse cell processes. One of the best characterised downstream targets of TSC1/TSC2 is mammalian target of rapamycin complex 1 (mTORC1), which regulates proteins synthesis, metabolism, autophagy and cell division amongst other cellular processes. Clinical trials in patients with tuberous sclerosis using mTOR inhibitors have shown promising results. Key Concepts: Tuberous sclerosis is an autosomal-dominant multisystem disorder. Tuberous sclerosis is caused by mutations in either of two genes TSC1 or TSC2. One of the key functions of the TSC1/TSC2 protein complex is to integrate signals from multiple stimuli to regulate cellular processes such as growth and division via the mammalian target of rapamycin (mTOR) pathway. Knowledge of the molecular pathology of tuberous sclerosis has been translated into clinical trials using mTOR inhibitors to treat manifestations of the condition. Keywords: tuberous sclerosis; TSC1; TSC2; mTOR; sirolimus

Tuberous sclerosis (TSC) is a heterogeneous trait. Since 1990, linkage studies have yielded putative TSC loci on chromosomes 9, 11, 12 and 16. Our current analysis, performed on 14 Dutch and British families, reveals only evidence for loci on chromosome 9q34 (TSC1) and chromosome 16p13 (TSC2). We have found no indication for a third locus for TSC, linked or unlinked to either of these chromosomal regions. The majority of our families shows linkage to chromosome 9. We have refined the candidate region for TSC1 to a region of approximately 5 cM between ABL and ABO.

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. Hum Mutat 32: 424-435, 2011. (C) 2011 Wiley-Liss, Inc.

Tuberous sclerosis (TSC) is an autosomal dominant multisystem disorder with loci assigned to chromosomes 9 and 16. Using pulsed-field gel electrophoresis (PFGE), we identified five TSC-associated deletions at 16p13.3. These were mapped to a 120 kb region that was cloned in cosmids and from which four genes were isolated. One gene, designated TSC2, was interrupted by all five PFGE deletions, and closer examination revealed several intragenic mutations, including one de novo deletion. In this case, Northern blot analysis identified a shortened transcript, while reduced expression was observed in another TSC family, confirming TSC2 as the chromosome 16 TSC gene. The 5.5 kb TSC2 transcript is widely expressed, and its protein product, tuberin, has a region of homology to the GTPase-activating protein GAP3.

Previous linkage studies in tuberous sclerosis have implicated three disease determining loci at 9q, 11q, and 12q. We have collated phenotypic and genotypic data on 1622 members of 128 families with tuberous sclerosis in order to evaluate simultaneously the evidence for these putative loci. Affection status in the family members has been reassessed using uniform diagnostic criteria and genotypic data extensively checked before analysis under alternative models of locus heterogeneity. One tuberous sclerosis determining locus, accounting for approximately 50% of the families studied, has been found to map in the region of D9S10 on 9q34 but no evidence has been found to support the existence of major loci on 11q or 12q. A locus, or loci, elsewhere in the genome is likely to account for tuberous sclerosis in most non-chromosome 9 linked families.

Failure in the regulation of mTOR (mammalian target of rapamycin) appears to be critical to the pathogenesis of the inherited disorder tuberous sclerosis and the related lung disease LAM (lymphangioleiomyomatosis). Both diseases are caused by mutations of TSC1 or TSC2 (TSC is tuberous sclerosis complex) that impair GAP (GTPase-activating protein) activity of the TSC1–TSC2 complex for Rheb, leading to inappropriate activity of signalling downstream of mTORC1 (mTOR complex 1). mTOR inhibitors are already used in a variety of clinical settings including as immunosuppressants, anticancer agents and antiproliferative agents in drug-eluting coronary artery stents. They also represent candidate therapies directed to the underlying molecular pathology in tuberous sclerosis and LAM. Phase I/II clinical trials of the mTORC1 inhibitor rapamycin have demonstrated reduction in size of tuberous-sclerosis- and LAM-associated renal tumours (angiomyolipomas) and some evidence for reversible improvement in lung function in patients with LAM. A case series of tuberous-sclerosis-associated brain tumours were also reported to shrink during rapamycin therapy. An important, although variable, feature of the tuberous sclerosis phenotype is learning difficulty. Recent studies in mouse models carrying heterozygous Tsc2 mutations demonstrated improvement in memory and learning deficits following treatment with rapamycin. These promising pre-clinical and early human trials are being followed by larger-scale randomized control trials of mTOR inhibitors for treatment of renal, lung and brain manifestations of TSC1- and TSC2-associated disease.

Tuberous sclerosis (TSC) is a multisystem autosomal dominant hamartosis whose genetics is complicated by reduced penetrance and widely varying clinical expression. Results of linkage analyses have variously suggested two different locations for a TSC gene. A collaborative dataset has been assembled to clarify the issue of genetic heterogeneity. We have now analyzed the data from a combined sample of 111 families. Using Ott's HOMOG programs, we completed three tests of homogeneity: (1) for chromosome 9q, (2) for chromosome 11q, and (3) for the combined 9q and 11q data. For test 1 the chi-square (1 df) was 21.54 (p less than 0.001), for test 2 the chi-square (1 df) was 0.13 (p greater than 0.35), and for test 3 the chi-square (2 df) was 37.61 (p less than 0.0001). Additionally, we examined the combined data for evidence that a third, as yet unlinked locus exists. Results of this last test were suggestive but not significant. Clearly loci for TSC are present on both chromosomes 9q and 11q. The maximum likelihood estimate of the proportion of chromosome 9q-linked families is 0.38, for chromosome 11q-linked families is 0.47, and for the unlinked type 0.15. Alternative explanations for these latter families include chance sampling of recombinants, nongenetic phenocopies, or misclassification.

After our initial reports 3-5 supporting a locus for tuberous sclerosis (TSC) on 9q, linkage analysis was undertaken in eight large multigeneration TSC families using nine polymorphic markers. Six of the markers were from the distal long arm of chromosome 9 and three from the long arm of chromosome 11. The data as a whole supported a TSC locus on distal 9q, the peak lod score on multipoint analysis being 3.77 6 cM proximal to the Abelson oncogene locus (ABL). However, analysis of 2-point lod scores using the HOMOG programs revealed significant evidence for genetic heterogeneity (p = 0.01), tight linkage to ABL being highly unlikely in one family. After exclusion of the unlinked family multipoint, analysis gave a peak lod score of 6.1 in the vicinity of ABL. The family unlinked to ABL showed no recombinants with two chromosome 11 probes, but it was too small to provide significant evidence for linkage. Genetic heterogeneity in TSC as demonstrated by this study will complicate efforts to clone the genes by reverse genetics and will severely hamper the use of linked probes for carrier detection and prenatal diagnosis.

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Author(s): Povey, S; Attwood, J; Janssen, LA; Burley, M; Smith, M; Flodman, P; Morton, NE; Edwards, JH; Sampson, JR; Yates, JR

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by widespread hamartosis. Preliminary evidence of linkage between the TSC locus and markers on chromosome 9q34 was established, but subsequently disputed. More recently, a putative TSC locus on chromosome 11 has been suggested and genetic heterogeneity seems likely. Here we describe an approach combining multipoint linkage analysis and heterogeneity tests that has enabled us to obtain significant evidence for locus heterogeneity after studying a relatively small number of families. Our results support a model with two different loci independently causing the disease. One locus (TSC1) maps in the vicinity of the Abelson oncogene at 9q34 and a second locus (TSC2) maps in the region of the anonymous DNA marker Lam L7 and the dopamine D2 receptor gene at 11q23.