2 Tuberous Sclerosis Complex

TSC (OMIM#191100) is an autosomal dominant disorder characterized by a broad phenotypic spectrum including epilepsy, mental retardation, skin lesions and tumors in various organs. The broad phenotypic spectrum reflected the development of hamartomas in multiple organs throughout the body (Schwartz et al., 2008). The incidence of TSC has been reported to be 1 in approximately 6000 (Osborne et al., 1991). However, its true incidence is not known because of a number of undiagnosed cases consisting mostly of mildly affected or asymptomatic individuals (Osborne et al., 1991). Two-thirds of TSC patients have sporadic mutations and only about one-thirds is familial. The genes in which abnormalities are found are called TSC1 and TSC2. Both genes have been studied by multigenerational linkage analysis (Fryer et al., 1987 and Kandt et al., 1992). TSC1 is located at position 9q34, and encodes a protein called hamartin, with an mRNA transcript of 8.6 kb containing 23 exons and encompassing 55 kb of DNA (van Slegtenhorst et al., 1997). TSC2 is located at position 16p13.3, and encodes a protein called tuberin, with an mRNA transcript of 5.5 kb, containing 41 exons and encompassing 40 kb of DNA (Eur TS Consortium, 1993).


Introduction
TSC (OMIM#191100) is an autosomal dominant disorder characterized by a broad phenotypic spectrum including epilepsy, mental retardation, skin lesions and tumors in various organs. The broad phenotypic spectrum reflected the development of hamartomas in multiple organs throughout the body (Schwartz et al., 2008). The incidence of TSC has been reported to be 1 in approximately 6000 (Osborne et al., 1991). However, its true incidence is not known because of a number of undiagnosed cases consisting mostly of mildly affected or asymptomatic individuals (Osborne et al., 1991). Two-thirds of TSC patients have sporadic mutations and only about one-thirds is familial. The genes in which abnormalities are found are called TSC1 and TSC2. Both genes have been studied by multigenerational linkage analysis (Fryer et al., 1987 andKandt et al., 1992). TSC1 is located at position 9q34, and encodes a protein called hamartin, with an mRNA transcript of 8.6 kb containing 23 exons and encompassing 55 kb of DNA . TSC2 is located at position 16p13.3, and encodes a protein called tuberin, with an mRNA transcript of 5.5 kb, containing 41 exons and encompassing 40 kb of DNA (Eur TS Consortium, 1993).

Clinical diagnosis of tuberous sclerosis complex
There are major and minor clinical features ( Table 1) that enable clinicians to clinically diagnose TSC. The clinical diagnosis is made when two major features, or one major and two minor ones, can be shown (Table 2). Sometimes, an antenatal diagnosis can be made based on fetal ultrasound and MRI, which show cardiac and brain lesions (Roach et al., 1998). Most patients are diagnosed in infancy or early childhood. A definitive diagnosis of TSC can be made when two major features or one major feature plus two minor features are demonstrated (Hyman, 2000 andRoach, 1998). Additional diagnostic categories include probable TSC when one major feature and one minor feature are present, and possible TSC when either one major feature or two or more minor features are present (Hyman, 2000 andRoach, 1998). However, as hamartomas are individually rare in the population without TSC, the presence of hamartomas in two different organ systems is considered by some clinicians to be sufficient for the diagnosis (O'Callaghan, 2000). Multiple, randomly distributed pits in dental enamel (Mennel et al., 2007) Dental pits are pits in the enamel of the permanent teeth. They occur more frequently in individuals with TSC than in people who do not have TSC.  (Xu et al., 1996) Most lesions are best seen on proton densityweighted images as bright spots.

MRI (brain) 15%
Shagreen patch (connective tissue nevus) (Schwartz et al., 2007) Slightly  (Mennel et al., 2007) These small fibrous nodules of the oral cavity are most commonly evident on the gingiva, especially in the anterior segment of the upper jaw but also on the buccal mucosa and dorsal surface of the tongue.

Oral (gums) 32%
Multiple retinal nodular hamartomas  Retinal hamartoma is a common finding in tuberous sclerosis, but the symptomatic changes of this lesion have rarely been described.

Epilepsy in tuberous sclerosis complex
Epilepsy was once included in tuberous sclerosis triad along with mental retardation and adenoma sebaceum (Provenzale, 1991). Although removed from the diagnostic criteria, epilepsy remains a dominant feature in tuberous sclerosis, covering up to 60% -90% of TSC cases. Genetic factors play important contributions in the manifestation of epilepsy in TSC. I t h a s r e c e n t l y b e e n d e s c r i b e d t h a t i n a c t i v a t i o n o f TSC2 causes more severe epilepsy phenotype than inactivation of TSC1 in a mouse model of tuberous sclerosis and suggested that the difference in phenotype may be related to the degree to which TSC1 and TSC2 inactivation causes abnormal mTOR activation (Zheng, 2010). Based on clinical features alone, TSC patients may experience a wide variety of ictal symptoms. Two syndromes are usually noted: infantile spasms (Christophea et al., 2000), which then evolve into partial or mixed epilepsies (Dabora et al., 2001) and partial seizures, typically starting later in childhood. The high incidence of infantile spasms in TSC has long been emphasized. Infantile spasms have been reported to be the presenting symptoms in up to 69% of patients with TSC. Infantile spasms in TSC usually have their onset between 4 to 6 months of age Fukushima et al 2001). TSC has been found in 7%-25% of infants with symptomatic West syndrome. West syndrome classically consists of the clinical-electroencephalographic triad of spasms (the seizure type), hypsarrhythmia and mental deficiencies. The main types of spasms (flexor, extensor, mixed) may occur in infant with TSC, but focal features such as head turning, nystagmus, tonic eye deviation or unilateral limb movement differentiate them from classical infantile spasms. Partial seizures predominate with the increasing age Jambeque et al., 1991). Location of tubers on MRI often correlates with EEG discharges. Tubers consist of dysplastic neurons and glial cells that distort the normal cortical architecture, causing them to be highly epileptogenic (Christopher et al., 2000). Jambaque and collegues found that an initial presentation with infantile spasms, refractory seizures and mental retardation or behaviour disorder were all more likely in children with greater numbers of cortical tubers. The classic interictal EEG pattern of patients with epileptic spasm is hypsarrhythmia. Hypsarrhythmia was originally defined by Gibbs and Gibbs in 1950 as completely chaotic and disorganized background pattern consisting of high amplitude slow waves and spikes that are asynchronous, non-rhythmic and variable in duration and topography. The spikes usually alternate randomly between focal, multifocal and generalized discharges at different moments within a brief record. It is most pronounced in slow-wave sleep. Ictal recordings of spasms can demonstrate a focal increase in spikes and polyspikes at the onset, with an abrupt generalized slow followed by electro decrement or generalized lower amplitude fast activity coincident with the spasm itself. Curatolo has theorized the following sequence of events. Electrographic onset of spasms is more common from the posterior temporal and occipital regions than from other locations. Subsequent partial seizures tend to arise from the frontal or anterior temporal regions. Drug selection should be tailored to both clinical and EEG attributes observed. Antiepileptic drug monotherapy is used whenever possible. Vigabatrin may be especially effective for infantile spasms in patients with TSC, with some series reporting complete control occurring in about 95% of patients (Aicardi, 1996 andHancock, 1999). Vigabatrin produces its antiepileptic effect by irreversibly inhibiting the enzyme GABA-aminotransferase. This results in increased brain and spinal fluid concentration of the inhibitory neurotransmitter GABA. Unfortunately, recent reports of visual-field constriction associated with vigabatrin therapy may limit its use and may prevent from becoming an approved treatment in United States and other countries (Krauss et al., 1998). Other evidence from randomized controlled studies includes using ACTH and corticosteroids for infantile spasms. Chronic use of benzodiazepines and barbiturates should be avoided if possible owing to their cognitive and behavioural adverse effects. Other medications useful to treat seizures in TSC include lamotrigine, felbamate, topiramate, carbamazepine and levetiracetam. When anticonvulsant options have been exhausted, alternative treatment such as ketogenic diet may be tried. If severely disabling seizures are present with consistent electroclinical and imaging data suggesting a confined area of seizure onset, surgical treatment should be considered. The most common surgical procedures offered to TSC patient include topectomies, gyrectomies or wider lobar resection as well as multiple subpial transection. Vagal nerve stimulation is a surgical option restricted to TSC patients with intractable epilepsy who fail to meet the criteria for resective surgery.

Molecular diagnosis of tuberous sclerosis complex
Despite the comprehensive criteria for clinical diagnosis of TSC, molecular analyses of both causing genes remain of importance. Mutation analysis in TSC patients is useful 1) to confirm a clinical diagnosis of TSC, especially in young patients in whom many clinical features have yet to develop, 2) in families with sporadic cases of TSC, mutation analysis may provide reassurance that the rest of the family members do not carry the mutation. However, such testing does not provide complete reassurance in regard to the possibility of having the second child with TSC, even when the parents do not appear to carry the mutation, and 3) to perform prenatal diagnosis, in families with either a child or a parent with a known mutation.

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Novel Aspects on Epilepsy 16 5. TSC1 and TSC2 genes variation spectrum *R/C = Reported/Concluded. "Reported Pathogenicity" refers to the pathogenicity as published by the authors in the original paper or as submitted to the LOVD-TSC database if the data has not been published. "Concluded Pathogenicity" refers to the pathogenicity that the curators of the database have assigned to the variant. (Personal Communication with the LOVD-TSC Database Curator; Dr. Rosemary Ekong). "-" = no known pathogenicity; "-?" = probably no pathogenicity; "+" = pathogenic; "+?" = probably pathogenic; "?" = unknown pathogenicity. *R/C = Reported/Concluded. "Reported Pathogenicity" refers to the pathogenicity as published by the authors in the original paper or as submitted to the LOVD-TSC database if the data has not been published. "Concluded Pathogenicity" refers to the pathogenicity that the curators of the database have assigned to the variant. (Personal Communication with the LOVD-TSC Database Curator; Dr. Rosemary Ekong). "-" = no known pathogenicity; "-?" = probably no pathogenicity; "+" = pathogenic; "+?" = probably pathogenic; "?" = unknown pathogenicity.  , 2000). This may reflect the low frequency of TSC1 diseases. b. Several large studies reported that TSC1 mutations are presented with less severe phenotype than TSC2 mutations. In 1991, Osborne and colleagues outlined that although TSC incidence is reported as 1 in approximately 6000, the true incidence of TSC is not known because of a number of undiagnosed cases consisting mostly of mildly affected or asymptomatic individuals. It is probable that this portion of patients harbored TSC1 mutations, accounting for the small number of TSC1 mutations found until today. c. TSC2 coding region is about 1.5 times longer than TSC1 and has approximately twice the number of splice sites, affording a proportionally increased opportunity for all manner of small mutations.

Molecular pathogenesis of TSC
Up to 85% of TSC cases are due to mutations in either TSC1 or TSC2 genes which lead to a truncated protein with a loss of function mechanism. Investigation of somatic mutations in a variety of TSC hamartomas supports classification of the TSC1 and TSC2 as tumor suppressor genes (Cheadle et al., 2000). Mutations in TSC1 and TSC2 affect neuronal proliferation, differentiation, and migration (Crino et al., 1999). The identification of the TSC1 and TSC2 genes and their encoded proteins, hamartin and tuberin respectively, has aided in understanding the molecular pathogenesis of TSC where hamartomatous formation is the outcome. Both hamartin and tuberin are widely expressed in normal tissues including brain, liver, and kidney. Hamartin is highly expressed in G0-arrested cells and throughout the ongoing cell cycle (Crino, 2004). Alterations in tuberin expression have been reported in patients with TSC. Immunoreactivity of tuberin is reduced in the brain with TSC. Loss of hamartin and tuberin formation due to TSC1 and TSC2 mutations can enhance proliferation of neural and astrocytic precursor cells and increased in cell size characteristic of dysplastic neurons and giant cells. When either of the TSC1 or TSC2 genes is inactivated, G1 is shortened and tissues become hypertropic (Potter et al., 2001). Over-expression of either hamartin or tuberin can lengthen G1 and inhibits cell proliferation (Tapon et al., 2001). Many studies indicated that hamartin and tuberin, encoded by TSC1 and TSC2 genes respectively, function as a complex. The complex has a stable interaction with stoichiometry of 1:1. The tight binding interaction between the two proteins formed a tumour suppressor heterodimer (Kwiatkowaki, 2008). Hamartin and tuberin have been found to physically associate with one another in vivo. Disruption in either one of the two genes may result in a truncated protein with the loss in controlling the cell growth and proliferation. The 130 kDa hamartin contains a putative transmembrane domain. Hamartin's membrane bound protein and two coiled-coil domains are necessary for its association with tuberin van Slegtenhorst et al., 1998).
Although TSC1-or TSC2-specific functions are possible, it seems that the predominant biochemical activity of these proteins is exerted by an equimolar complex, which regulates the state of GTP-loading of the rheb GTPase, and thereby regulates mTOR activation in the cell. As most hamartomas in TSC develop through a two-hit inactivation mechanisms (Knudson's hypothesis for tumor suppressor genes, including TSC1 and TSC2), it appears likely that somatic mutations in TSC1 are less common than those in TSC2, just as the rate of germline mutation in TSC1 is much lower than that in TSC2. Thus, fewer and/or less severe clinical manifestations would be seen in TSC1 patients. LOH is very common within TSC hamartomas, except for cardiac or brain. In both organs, study says that wildtype hamartin and tuberin are present. LOH is an event by which within the affected cells, the genomic DNA loss its heterozygosity, becoming homozygous for the mutation. In other cells (unaffected cells) the genomic DNA shows heterozygous for the mutation. To analyze the occurrence of LOH it is necessary to perform mutation analysis on the genomic DNA extracted from the affected cells as well as from the unaffected cells.
After the discovery of TSC1 and TSC2 and their encoded proteins, several downstream protein cascades that might be affected by the pathogenesis of the disease, such as the pathway of mTOR (mammalian target of rapamycin), were identified.

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Novel Aspects on Epilepsy 20 Figure 1 illustrated the roles of hamartin and tuberin in cell metabolism describing that disruption in either of both genes may result in loss of the control of cell growth and proliferation. Hamartin-tuberin complex inhibits the mTOR which is a key regulator in the signalling pathway of cell proliferation and organ size (Kwiatkowski, 2008). It has been reported that the complex regulates mTOR via hydrolysis of Rheb-GTP into its inactive GDP bound state, Rheb-GDP (Rosner et al., 2004 and. Tuberin and hamartin form an intracellular complex which activates GTPase, reducing stimulation of mTOR. mTOR detects signals of nutrient availability, hypoxia, or growth factor stimulation, and is part of many cell processes, such as cell-cycle progression, transcription and translation control, and nutrient uptake. It phosphorylates, among other proteins, S6K1 and eukaryotic translation initiation factor 4E-BP1. S6K1 is a kinase that activates ribosomal subunit protein S6, leading to ribosome recruitment and protein translation. 4E-BP1 inhibits activity of eukaryotic translation initiation factor 4E (eIF4E) and, when phosphorylated by mTOR, releases eIF4E from its control. The complex inhibits mTOR by acting as a GAP toward Rheb, which promotes hydrolysis of Rheb-GTP, converting it to an inactive GDP bound state. Without its active GTP bound state, Rheb cannot stimulate mTOR-mediated signalling to downstream components S6K1 and 4E-BP1. The mechanism is reversed with the presence of amino acids which activates Rheb-GEF. RhebGEF converts Rheb-GDP to its active Rheb-GTP and promotes mTOR signalling. Akt inactivates TSC tumor suppressor complex by phosphorylation of TSC2 . Common TSC2 mutations result in the loss of the GAP domain of tuberin through Cterminal truncations, whereas some point mutations are clustered within the GAP domain. It is also reported that, an intact GAP domain of tuberin is crucial for association with hamartin in the formation of tuberin-hamartin heterodimers. The heterodimers will inhibit Rheb-induced mTOR signalling and can also function as a GAP toward Rheb. Higher proportion of the active GTP bound form of Rheb can likely be found within TSC patients. It is the result of non-functional tuberin-hamartin heterodimers where the genes failed to encodes for a functional protein (van Slegtenhorst et al., 1998). The 200kDa (1806 amino acids) tuberin is homologous to the GTPase activating proteins (GAP) rap1GAP and mSpa1 where it contains relatively hydrophobic N-terminal domain and conserved 163 amino acids region close to the C-terminus. Rap1GAP is the member of Ras-related protein and functions in regulation of DNA synthesis and cell-cycle transition. The GAP activity of functional tuberin can regulate the effects of Rap1 on G to S phase transition during cell division. Thus, it implies that TSC2 mutations may result in constitutive activation of Rap1 (Wienecke et al., 1996 andWienecke et al., 1997). Tuberin also has been demonstrated to interact with rab5. Rab5 is a cytosolic protein, is an effector for the endosomal small GTPase and therefore involved in endocytic fusion events (Stenmark et all., 1995). Consistently with the finding, tuberin has also been shown to act as a GTPase activating protein for rab5 and reduce the fluid-phase endocytosis (Xiao et al., 1997). As for 130 kDa (1164 amino acids) hamartin, it has hydrophilic protein with no significant homology to tuberin or other known vertebrate protein. Van Slegtenhorst and colleagues have investigated the association between endogenous hamartin and tuberin and they found out that both proteins play a closely related role (van Slegtenhorst et al., 1998). The methods used in the study suggest that inactivation of hamartin and tuberin may prevent the formation of a functional protein complex.
Hamartin was recently identified as an interactor with the cytoskeletal proteins, ERM family (Lamb et al., 2000). The function loss can alternatively compromise neural migration via interaction with ERM or actin binding proteins. It was also shown to activate the GTPase Rho and regulates focal adhesion and stress fiber formation. Hamartin activates the GTPase Rho via the overlapping region of Rhos's amino acid and hamartin's tuberin-interaction domain (Lamb et al., 2000). The dysregulation of signalling by the Rho family of GTPase is said to have a critical role in cancer cell migration, invasion, and metastasis (Clark et al., 2000;Evers et al., 2000;Royal et al., 2000 andSchmitz et al., 2000). It has also been recently shown that the functional complex interact with G2/M cyclindependant kinase 1 and its regulatory cyclins. Thus, mutation in either both genes may alter the kinetics of cell divisions (Catania et al., 2001). The functional heteromeric complex of hamartin and tuberin also plays important role in modulating the pathways of insulin receptor-or insulin-like growth factor-mediated signalling. The pathway functions downstream of the cell signalling molecule Akt, also play roles in regulating cell growth and potentially cell size.

Studies on therapeutics options for TSC
At present, the management of TSC is symptomatic. Some of TSC manifestations have been subjected to drug therapies but they are still in the developmental stage (Yates et al., 2006). Table 5 summarized several drugs under investigation for their efficacy towards Tuberous Sclerosis Complex. The discovery of mTOR (mammalian target of rapamycin) pathway upregulation in tuberous-sclerosis-associated tumours presents new possibilities for treatment strategies. A TSC mouse treated with rapamycin, also known as sirolimus, was found to have its learning and memory deficits improved (Ehninger et al., 2008). Sirolimus is a macrolide antibiotic that acts as an mTOR kinase inhibitor. It is isolated from Streptomyces hygroscopicus. Sirolimus and its analogs have been shown to make the dysregulated mTOR pathway return to normal in cells that lack TSC1 or TSC2. Several results from in-vitro or in-vivo animal studies suggest that sirolimus or its analogues might be effective in the treatment of various manifestations of tuberous sclerosis such as skin lesions (Rauktys et al., 2008), lymphangioleiomyomatosis (Goncharova et al., 2006 andBissler et al., 2008), renal angiomyolipomas (Lee et al., 2006;Herry et al., 2007 andWienecke et al., 2006), renal-cell carcinoma (Robb et al., 2007), subependymal giant-cell astrocytomas (Franz et al., 2006) or even polycystic kidney disease (Weimbs et al., 2006). However, angiomyolipomas increased in volume after the therapy was discontinued, and some patients taking sirolimus experienced serious adverse events (Bissler et al., 2008;Herry et al., 2007 andWienecke et al., 2006). Recently, other classes of drugs have also been found to be possible therapeutic options for TSC. Interferon gamma and interferon alpha interact with mTOR, leading to deactivation of the translational repressor 4E-BP1, which could be beneficial for the treatment of tuberous sclerosis (Kaur et al., 2007). Other classes of drugs ranging from those which can alter amino acids metabolism, inhibit VEGF signalling and inhibit microtubules were also studied. Presence or absence of amino acids is an important regulator of mTOR pathway signalling (Avruch et al., 2006). For example, L-asparaginase, a hydrolase enzyme and one of the most important agents used in multidrug chemotherapy for the treatment of cancer. It is mainly used to treat human leukemic cells in acute lymphoblastic leukemic. L-asparaginase has been found to www.intechopen.com Novel Aspects on Epilepsy 22 reduce the levels of mTOR pathway's target p70 S6 kinase and 4EBP-1 (Iiboshi et al., 1999). The reduction indicates that L-Asparaginase can be a possible therapeutic option for TSC. VEGF is thought to play important role in the pathogenesis of TSC since tumors associated with TSC are vascular. TSC2 has also been found to have association with increased levels of VEGF in cultured cells (Brogarolas et al., 2002). Since VEGF signalling is important in the TSC pathogenensis, combination of VEGF inhibitors with mTOR inhibitor analogs may provide a promising treatment. Sorafenib is one of VEGF inhibitors. It is an oral targeted kinase inhibitor that blocks VEGF-R. In TSC tumor preclinical study by Lee and colleagues, combination of Sorafenib and Sirolimus was found more effective than single agent (Lee et al., 2009). Other VEGF inhibitor is Sunitinib which also inhibit platelet derived VEGF-R. It is a receptor tyrosine kinase inhibitor. Bevacizumab, a recombinant humanised monoclonal antibody, is also a VEGF-R inhibitor. Both Sunitinib and Bevacizumab produce inhibitory effects to VEGF-R signalling pathway and may be useful for TSC treatment.