Genotype/Phenotype Correlations in Tuberous Sclerosis Complex

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Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of widespread hamartomatous lesions in various organs, including brain, skin, kidneys, heart, and eyes. Central nervous system is almost invariably involved, with up to 85% of patients presenting with epilepsy, and at least half of patients having intellectual disability or other neuropsychiatric disorders including autism spectrum disorder. TSC is caused by the mutation in one of the 2 genes TSC1, at 9q34, and TSC2, at 16p13.3. They respectively encode for hamartin and tuberin, which form an intracellular complex inhibiting the mammalian target of rapamycin. Mammalian target of rapamycin overactivation following the genetic defect determines the cell growth and proliferation responsible for TSC-related lesions, as well as the alterations in neuronal excitability and synaptogenesis leading to epilepsy and neuropsychiatric disorders. A causative mutation for the disorder is identified in about 85% of patients with a clinical diagnosis of TSC. Mosaicism and technology limits likely explain most of the no mutation identified cases. This review confirms that patients with TSC2 mutations considered as a group usually present a more severe phenotype, characterized by higher number of tubers, earlier age at seizure onset and higher prevalence of intellectual disability. However, the clinical phenotype of the disease presents a high variability, thus making the prediction of the phenotype on an individual basis still challenging. The increasing application of new molecular techniques to subjects with TSC has the potential to significantly reduce the rate of patients with no mutation demonstrated and to identify an increasing higher number of mutations. This would hopefully allow a better characterization of higher risk mutations, which might help clinicians to plan individualized surveillance plans. Furthermore, the increasing availability of disease registries to collect clinical and genetics data of patients help to define more valid and clinically oriented genotype or phenotype correlations.

Introduction

Tuberous Sclerosis Complex (TSC, MIM#191100) is an autosomal dominant disorder characterized by the development of widespread hamartomatous lesions in various organs, including brain, skin, kidneys, heart, and eyes.1 The incidence is of 1 in 5800 to 1 in 10000 newborns.2, 3, 4 Clinical manifestations in TSC present a significant age-dependency, with some signs and symptoms being present above all during infancy, and others appearing later during childhood or even adulthood.1

Central nervous system (CNS) involvement is almost invariably present, and it is the cause of the major burden for patients and their families.5 From a neuropathological point of view, the hallmark of the disease is represented by the so-called cortical tubers, subependymal nodules (SEN) and subependymal giant cell astrocytomas (SEGA). Their location and tendency to grow can determine the obstruction of cerebrospinal fluid flow thus leading to obstructive hydrocephalus, which still represents a major cause of morbidity and mortality in patients affected by TSC.6 White matter is also consistently involved in TSC, both in the form of radial migration lines, as well as with the presence of diffuse abnormalities revealed by the application of diffusion techniques during brain imaging.7, 8 The structural lesions of CNS are associated with neurological signs and symptoms, such as epilepsy, and neuropsychiatric disorders.5 Epilepsy affects up to 85% of patients with TSC, with 80% of them presenting seizures in the first 3 years of life and nearly 67% in the first 12 months.9 Early onset epilepsy is associated with a higher risk of refractory seizures and neurocognitive sequelae.5 Patients with TSC are also at a higher risk for different cognitive and behavioral disturbances. In particular, intellectual disability (ID) is present in about half of patients, ranging from severe to mild impairment.5 Cognitive impairment in TSC is a multifactorial condition, but early age at seizure onset and mutation on TSC2 gene appear to be among the most important risk factors.10 Neuropsychiatric disorders, such as autism and attention deficit hyperactivity disorder (ADHD), are highly represented in TSC patients, at a higher rate than in the general population. All these disorders are now grouped under the acronym TAND, meaning tuberous sclerosis-associated neuropsychiatric disorders, and although significant advances have been made in the knowledge of TSC in the last decades, they still appear to be under-recognized and sometimes under-treated.11

The most common nonneurological characteristics of tuberous sclerosis are dermatological, renal, pulmonary, cardiac, and ophthalmological manifestations.5 Renal manifestations are the second most significant cause of morbidity and mortality in TSC following CNS involvement.1 Patients with TSC may present with renal angiomyolipomas (AMLs) and renal cysts, up to a polycystic kidney disease. Cardiac involvement usually occurs very early in the course of the disease, with cardiac rhabdomyomas being evident already in the second trimester of pregnancy and sometimes leading to a prenatal diagnosis of TSC.5 Pulmonary lymphangioleiomyomatosis (LAM) represents another significant source of TSC-related morbidity, and it almost exclusively affects women from the second or third decade of life.12

TSC is caused by mutations in either 1 of the 2 tumor suppressor genes, TSC1 at 9q34 coding for hamartin/TSC1 (130 kDa) and TSC2 at 16p13.3 coding for tuberin/TSC2 (200 kDa).13, 14 During the last 20 years, a long road has been made toward a better characterization of the disease.TSC1 and TSC2 form an intracellular GTPase activating protein (GAP), normally inhibiting the mammalian target of rapamycin (mTOR) signaling pathway.15 mTOR controls multiple downstream effectors, which regulate protein synthesis and other processes related to cell growth, proliferation, metabolism, and cell survival and its overactivation following TSC1/TSC2 mutations determines an increase in cell growth and proliferation, thus representing the molecular basis for all TSC-related lesions.15 Furthermore, preclinical and clinical evidence now supports a key role for mTOR pathway in altering neuronal excitability and synaptogenesis, thus leading to an increased susceptibility to seizures, autism spectrum disorder (ASD) and ID.16 Clarifying the role of the 2 genes in mTOR pathway has provided new understandings into basic cell biology leading to the development of promising new therapies based on the use of specific mTOR inhibitors.15 The diagnosis of TSC has always been considered as a clinical one, with the presence of major and minor criteria to help clinicians in defining the presence of a definite, possible, or probable diagnosis.17 However, the significant progresses made in the recent years on the genetics and physiopathology of the disease led to the need of a modification of the diagnostic algorithm, expressed by the new diagnostic criteria, published after an International Consensus Conference held in Washington in 2012.18 The 2 main modifications compared to previous criteria have been the elimination of the “probable” diagnosis of TSC, and the introduction of a genetic diagnostic criterion. Indeed, although clinical features continue to be the principal diagnostic tool, the presence of a recognized pathogenic mutation in 1 of the 2 genes TSC1 or TSC2 is now sufficient for a definite diagnosis.18 A mutation is considered pathogenic when it clearly determines a loss of function of the TSC1 or TSC2 proteins, or prevents protein synthesis.18 Also missense mutations can be pathogenic, but their role on protein function should be established by functional assessment (www.lovd.nl/TSC1, www.lovd/TSC2).18, 19, 20 By contrast, different variants of TSC1 or TSC2 genes with uncertain significance cannot be considered as pathogenic, and are therefore not sufficient to achieve a definite diagnosis of TSC.18 It is important to underline that 10%-25% of patients have no mutation identified (NMI) by conventional genetic testing, but the lack of identification of a causative genetic mutation does not exclude the diagnosis.18

From a clinical point of view, the disease is characterized by a wide phenotypic variability, with some patients presenting very few signs and symptoms, and others suffering from very severe manifestations. This is also evident in the same family and even in monozygotic twins, with the same mutation leading to very different clinical expression.21, 22 During the last years, many new mutations have been described.

The aim of this article is to provide a clinically oriented and up-to-date review of the current knowledge on the genotype-phenotype correlations in TSC.

Section snippets

Search Strategy and Selection Criteria

Information in this paper is mainly based on peer-reviewed medical publications from 2000-2015 (PubMed). Selection criteria are the novelty and importance of studies, and their relevance to child neurologists. Search terms included “tuberous sclerosis,” “genetics,” “genotype,” and “phenotype.” Only articles published in English were reviewed. All articles were read by the authors, and references were reviewed to identify any additional relevant studies. Significant historical references have

Mutations of TSC1 and TSC2 Genes

A free, open international database is available online, listing all the TSC1 and TSC2 mutations reported by geneticists and clinicians, sometimes associated with a phenotype description, thus allowing a partial genotype/phenotype correlation. Up to now, a total of 705 unique DNA variants have been reported for TSC1 (http://chromium.lovd.nl/LOVD2/TSC/ home.php?select_db=TSC1) and 1976 for TSC2 (http://chromium.lovd.nl/LOVD2/TSC/ home.php?select_db=TSC2). There are no mutation hotspots on the 2

Familial Cases

One-third of TSC cases are familial with two-thirds being sporadic.24 Different studies suggested a higher proportion of TSC1 mutations in familial cases.25, 26, 27, 28 However, subsequent observations pointed out that the estimated frequency of TSC1 vs TSC2 mutations in familial cases may have been biased because of the small numbers of families studied and suggested that TSC2 mutations are more frequent in both familial and sporadic case.29

Mutational Spectrum of TSC1

The TSC1 gene is located on 9q34, consists of 53284 nucleotides (nt) from nt position 134756557-134809841 and is composed of 23 exons. TSC1 encodes the gene protein hamartin of 130 kDa (March 2006, Human Genome Assembly, genome.ucsc.edu). The coding sequence counts for 21 exons, specifically from exon 3, in which there is the initial codon (ATG), to exon 23. Exon 2 is alternatively spliced but it has no effect on the encoded protein.14 Exon 15, the largest exon of TSC1, and exon 16 are the sites

Mutational Spectrum of TSC2

The TSC2 gene is located on 16p13, consists of 40723 nt from nt position 2037991-2078714 and is composed of 42 exons. TSC2 encodes for tuberin, a guanosine triphosphatase–activating protein of 200 kDa.13 For TSC2 there is alternative splicing of moderate complexity of exon 25, 3 bp of exon 26 and exon 31.32 TSC2 contains the GAP domain from exons 34-38. The GAP domain has been shown to stimulate the GTPase activity of GPAse Rhe, the activator of mTORCH. In TSC2, all kinds of mutations have been

No Mutation Identified

Different hypothesis have been raised to explain the existence of subjects with TSC and NMI. First of all, the existence of a third gene has been hypothesized long ago,33 although this hypothesis has never been confirmed nor definitely excluded. Furthermore, constitutional epigenetic modifications leading to transcriptional silencing may occur.33, 34 Finally, it should be considered that conventional genetic testing cannot be able to detect mutations occurring in intronic and regulatory

Mosaicism in TSC

Mosaicism derives by a first TSC1 or TSC2 mutation occurring during embryogenesis, and it can be somatic (generalized) or germline (confined gonadal).32 Mosaicism prevalence in TSC is difficult to ascertain since we might not be able to detect it all the times it occurs; however, 2 large series reported a prevalence of 28% and 15%, mostly in patients with large genomic deletions on TSC2,31, 32, 35 whereas in subjects with small mutations, it has been reported at lower frequencies (10%).36 The

Genetic Analysis and Detection Rate

In the last years, genetic diagnosis of TSC could benefit from the introduction, even in the clinical setting, of a new technique called next generation sequencing (NGS), a massively parallel sequencing method with the potential of increasing the detection rates of TSC1/2 mutation in subjects with TSC. A first application of this technique in 38 individuals with TSC and NMI, led to the identification of heterozygous mutations missed by previous analysis in 13% of cases and mosaicism in 6%.37

Correlations Between Genotype and TSC-Associated Brain Pathology

Cortical tubers have a very high prevalence in patients with TSC, being present in about 90% of patients.18 Although in many studies there is no statistically significant difference of the prevalence of tubers in different mutation groups, a trend toward a major representation in patients with TSC2 mutation is identifiable, with a mean of 91% of TSC2 patients presenting this kind of cortical dysplasia, compared with 76% of TSC1 and 73% of NMI.23, 29, 42, 47, 52, 54 Even if a clear relationship

Neurological Manifestations

Table 2 summarizes the main studies reporting the prevalence of the different neuropsychiatric manifestations and neuropathologic lesions according to the mutational status of the patients.

Clinical Characteristics

ID is a frequent feature in TSC with a reported prevalence between 40% and 70%; it is estimated that about half of patients with TSC present a normal intellectual level, with the remainder suffering from various degrees of impairment.5 A limit of most studies assessing the prevalence of ID in TSC population is that cognitive ability was often estimated clinically, or through indirect methods, such as level of schooling, without the administration of standardized assessments. A bimodal

Neuropsychiatric Disorders Tuberous Sclerosis-Associated Neuropsychiatric Disorders (TAND)

Although the presence of neuropsychiatric symptoms in TSC was already highlighted in the first description of this disease made by Bourneville,67 tuberous sclerosis-associated neuropsychiatric disorders manifestations continue to be under-recognized by clinicians dealing with patients with TSC.11 It is still difficult to ascertain the exact prevalence of the different neuropsychiatric disorders in TSC, since most of the studies use different and often not standardized diagnostic methods.68 This

Nonneurological Manifestations

Table 3 reports the different rates of systemic manifestations of TSC according to the mutational status of the patients.

Clinical Characteristics

Pulmonary LAM is a progressive lung disease characterized by cystic destruction of pulmonary parenchyma and almost exclusively affects women.12 It is a significant source of morbidity and mortality in patients with TSC, and it affects about 47% of women with TSC.5

Although it may be asymptomatic, it can cause dyspnea, spontaneous pneumothorax, chylous effusion, and haemoptysis, and has the potential to lead to respiratory insufficiency requiring lung transplantation.12, 76

Genotype/Phenotype Correlations

There is a strong

Current Perspectives and Future Directions

After the discovery of the putative genes of TSC, great expectations from the scientific community were that a deeper knowledge of the genetic basis of the disease would lead to a better characterization of the clinical aspects of the disease. Unfortunately, despite the continuous advances in molecular testing techniques, the great variability of the clinical manifestations makes the prediction of the phenotype on an individual basis still challenging.29, 42 Therefore, although genetic

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    Paolo Curatolo and Romina Moavero received funding from the European Community’s Seventh Framework Programme, (FP7/2007-2013) under Grant Agreement no. 602391 (www.epistop.eu).

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