Molecular genetic investigation of the variability of the GTPase activating protein- (GAP-) related domain of the tuberous sclerosis-2 (TSC2) gene in TSC patients and healthy subjects [Elektronische Ressource] / vorgelegt von Karin Louise Zügge geb. Gierke

Karin Gierke

Sujets

• Gesundheit

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Zentrum für Kinderheilkunde und Jugendmedizin Albert-Ludwigs-Universität Freiburg, Germany          MOLECULAR GENETIC INVESTIGATION OF THE VARIABILITY OF THE GTPase ACTIVATING PROTEIN- (GAP-) RELATED DOMAIN OF THE TUBEROUS SCLEROSIS - 2 (TSC2) GENE IN TSC PATIENTS AND HEALTHY SUBJECTS     Inaugural - Dissertation  zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br.         Vorgelegt 2002 von Karin Louise Zügge geb. Gierke geboren in Madison, Wisconsin/U.S.A.

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Dekan: Prof. Dr. med. J. Zentner

1. Gutachter: Prof. Dr. med. L.B. Zimmerhackl

2. Gutachter: Prof. Dr. med. E. Schulz

Jahr der Promotion: 2004

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Table of Contents   1 Introduction 1-12 1.1 History 1 1.2 Clinical characteristics 3 1.3 Pathogenesis 5 1.4 Genetics 6 1.5 Protein products 7 1.6 The clinical variability predicament 10 1.7 The Freiburg-Heidelberg project 11 1.8 Objectives and research questions 12 2 Materials and Methods 13-22  2.1 Materials 13-15 2.1.1 Apparatus and equipment 13 2.1.2 Buffers and solutions 13 2.1.3 Chemicals, enzymes, kits and size markers 14 2.1.4 Databases and software 14 2.1.5 Media and cell cultures 14 2.1.6 Oligonucleotides and DNA-sequencing 15 2.2 Methods 15-22 2.2.1 Probands 15 2.2.1.1 TSC patients and families 15 2.2.1.2 Healthy population 15 2.2.2 Cell culture and transformation from peripheral blood samples 16 2.2.3 Freezing of cells 16 2.2.4 DNA extraction 17  

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2.2.5 PCR 2.2.6 PCR product analysis 2.2.7 SSCP 2.2.8 Purification of the PCR product 2.2.9 Sequencing 2.2.10 Sequence analysis

18 19 20 21 21 22 3 Results 23-31 3.1 Patient group 23 3.1.1 Amplification of exons 23 3.1.2 Mutational screening 23 3.1.3 Sequencing results 24 3.1.3.1 Mutations 24 3.1.3.2 Polymorphisms 26 3.1.4 Exon 40 and quality control 26 3.2 Control group of healthy probands 28 3.2.1 Exon amplification 28 3.2.2 Mutational screening 28 3.2.3 Sequencing results and numerical comparison to patient group 29 3.3 The Freiburg-Heidelberg project 30 4 Discussion 32-43 4.1 Patient group 32 4.1.1 PCR products 32 4.1.2 SSCP 32 4.1.3 Sequencing 33 4.1.3.1 Mutations 33 4.1.3.2 Polymorphisms 35   Table of Contents

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iii 4.1.3.3 Quality control and exon 40 36 4.2 Control group of healthy probands 36 4.2.1 Exon amplification and mutational screening 37 4.2.2 Sequencing results and numerical comparison to patient group 37 4.3 Screening the whole gene 38 4.3.1 Number and type of mutations found 38 4.3.2 Genotype-phenotype correlation 39 4.3.3 Clinical variability 40 4.3.4 TSC1 and TSC2 frequency ratios 41 4.4 Conclusions 42 4.5 Future 43

5 Summary  6 Zusammenfassung  7 Appendix  8 Abbreviations and symbols  9 Literature  10 Acknowledgments  11 Curriculum vitae  12 Publications

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1 Introduction   Tuberous sclerosis complex (TSC) is not a disease that summons a definite picture to mind for most of us. It is, however, one of the most common genetic illnesses with a frequency of approximately 1:6000 (Kwiatkowska et al., 1999; Gomez et al, 1999). 1  defect in one of two genes, TSC1 or TSC2, causes the disorder. A A wide variety of clinical manifestations and several different names make TSC such a commonly unfamiliar disease. TSC is a complex hereditary syndrome with a broad array of clinical characteristics and thus various faces. The one unifying characteristic of TSC is the development of benign localized tumors (hamartomas) in various tissues. The basis of the nametuberous sclerosis is given by the cortical tubers found in the brain. Further typical manifestations include renal angiomyolipomas and cysts, cardiac rhabdomyomas, facial angiofibromas and pigment disorders in the skin. The severity of manifestation is as variable as the tissues affected by the disease. The scale goes from a few irregularities in the skin to severe multi-organ TSC with epilepsy, behavior disorders and mental retardation, multiple kidney lesions and renal failure. (Roach et al., 1998)  1.1 History   Not only do the varying manifestations of TSC make this a hard disease to characterize, but also the various names given TSC over the centuries illustrate the complexity of the disorder. The earliest documented description of a likely TSC case is accredited to von Recklinghausen in 1862. He performed an autopsy on a newborn child, who died shortly after birth, in which he described multiple heart tumors and hardened areas in the brain. Bourneville described TSC as a form of epilepsy in Paris in 1880, and thus TSC is also known as Maladie de Bourneville in France. Pringle described the skin manifestation of Adenoma sebaceum 1885, making TSC in synonymous with Pringles Disease in England. (Gomez et al., 1999) These early scholars did not correlate their findings to other manifestations or make the connection that these might all belong to the same disorder. It was Vogt

 1  Some examples of well known inherited disorders include: galactosemia (1:50000), adrenogenital syndrome (1:10000), phenylketonuria (1:7000), hypothyroidism (1:4000), neurofibromatosis (von Recklinghausen, NF1 = 1:4000) and cystic fibrosis (1:2000). (von Harnack, 1994) 

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who first described a trias of facial angiofibromas, epileptic seizures and intellectual handicap that could also include heart and kidney tumors in 1908. The establishment of autosomal dominant inheritance was based on the report of a family with affected members in three generations by H. Berg in 1913. The term phakomatoses (neurocutaneous dysplasias) for diseases such as TSC and neurofibromatosis was proposed in 1921 by van der Hoeve. Von Hippel-Lindau and Sturge-Weber syndromes were later included in the list of phakomatoses. Accurate nomenclature and the term tuberous sclerosis were proposed to replace the poorly chosenepiloia  by Critchley and Earl in 1932. (Gomez et al., 1999) Over the course of the twentieth century, modern medical technology has brought great progress in diagnostics. The introduction of computer tomography of the head in 1973 and the use of ultraviolet light to detect skin lesions has increased the diagnosis of both patients and asymptomatic family members. Magnetic resonance imaging is proving to be an even better diagnostic tool for identifying cortical tubers before they calcify. These methods along with careful clinical examination allow for a certain diagnosis of TSC -- a decisive factor in the increasing awareness that this is a relatively common disorder. (Gomez et al., 1999) Recent events and progress in genetic research promise new possibilities for molecular diagnostics and hope for future treatment advances. Research impulses were given and a consensus in diagnostic criteria was established at the 1991 New York Academy of Science TSC meeting (Roach et al., 1998). The identification of TSC2 on chromosome 16 in 1993 (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and of TSC1 on chromosome 9 in 1997 (van Slegtenhorst et al., 1997) have provided for new possibilities in molecular screening. Advances in molecular diagnostic methods are currently being explored to simplify the as yet very lengthy process of screening all exons of both large genes. Molecular genetic identification of the disease causing mutation is becoming increasingly significant for patients and their families, because this information is essential for adequate genetic counseling. Despite the many current and the hope for future advances, the clinical diagnosis of TSC remains of central importance for the individual patient and treatment of TSC continues to emphasize symptomatic measures.   

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 Introduction

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1.2 Clinical characteristics  It is difficult to diagnose TSC unless clinicians follow strict diagnostic criteria and thoroughly examine their patients. Table 1 shows the revised diagnostic criteria and also demonstrates that almost any organ or system can be affected. A definite TSC diagnosis requires that the patient exhibit either two major features or one major and two minor features. (Roach et al., 1998) 

Major FeaturesMinor Features Facial angiofibromas or forehead plaque Multiple dental enamel pits Nontraumatic ungual or periungual Hamartomatous renal polyps fibroma Hypomelanotic macules (3 or more) Bone cysts Shagreen patch (connective tissue Cerebral white matter radial migration naevus) lines Multiple retinal nodular hamartomas Gingival fibromas Cortical tuber Nonrenal hamartoma Subependymal nodule Retinal achromatic patch Subependymal giant cell astrocytoma Confetti skin lesions Cardiac rhabdomyoma (single or Multiple renal cysts multiple) Lymphangiomyomatosis Renal angiomyolipoma  Table 1: and minor features for diagnosis of TSC. Almost every organ or Major tissue type can be affected by TSC. At least two major features or one major and two minor features are required for definite diagnosis of TSC. (Adapted from Roach et al., 1998)  Yet another varying factor in the complex of tuberous sclerosis is the time at which the lesions emerge. Typically, cardiac rhabdomyomas arise in the perinatal period and decline later in life. Cortical tubers also occur in early childhood, which correlates to the severity of central nervous involvement, epilepsy and intellectual disability. Various lesions in the skin can occur at very different ages. Hypomelanotic macules are present at birth or appear in the first months of life and the first angiofibromas can be seen around three years of age. In contrast, subungual fibromas can appear much later, between 15 and 60 years of age. Figure 1 demonstrates the time course of tuberous sclerosis lesions. (Kwiatkowski and Short, 1994)   

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Cortical Tubers

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Subependymal Nodules

Facial Lesions

Renal Cysts, Angiomyolipoma

approx. 50 years Age in years

Subungual Fibromas

     Cardiac  Rhabdo-  myomas     Relative   level of  expression        birth    Figure 1: Time course of TSC manifestations. Relative levels of expression versus age at manifestation are plotted on an arbitrary scale. Some lesions arise early (e.g. cardiac rhabdomyomas and cortical tubers) while others appear later in life (e.g. subungual fibromas). (Kwiatkowski and Short, 1994)  The course and prognosis of TSC is very dependent on the organs affected. Involvement of the central nervous system (CNS) in particular is a sign of a more serious condition. Although it must be emphasized that not all TSC patients are mentally retarded, over 50 % of children with TSC are autistic and 50 % of children with infantile seizures have TSC (Kwiatkowski and Short, 1994). It is clear that these symptoms in early childhood are cause for great concern and lead to a determined quest for a diagnosis in these cases (Hunt and Dennis, 1987). This may lead to a possible overinclusion of individuals with severe and profound intellectual handicap (Baker et al., 1998). In contrast, renal involvement may not be noticed until an early death occurs and angiomyolipomas are discovered upon autopsy -- they are found in about two thirds of all TSC kidneys (Zimmerhackl et al., 1994). Adding to the complexity of tuberous sclerosis are the asymptomatic and mild cases. Clinically obvious cases are easily detected and reported, but many mild cases are not. Particularly normally intelligent persons with TSC may be missed by epidemiological studies (Gillberg et al., 1994). For this reason, researchers assume TSC to be more common than initially expected, and its current predicted prevalence is 1:6000 (Kwiatkowska et al., 1999). The asymptomatic cases also explain the   Introduction

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difficulty in genetic counseling. Parents with a child, who seems to be a sporadic TSC case, are told that there is a 2 % recurrence risk in subsequent children (Osborne et al., 1991, Rose et al., 1999). It is hoped that greater awareness of the clinical aspects of TSC and the intense efforts to fully resolve its genetic mechanisms will help clinicians more fully advise and treat their patients.  1.3 Pathogenesis   Though TSC is characterized by high variability, the typical manifestation is the development of hamartomas -- lesions that display abnormal tissue differentiation. These tumors are rarely malignant, meaning they do not grow invasively and do not metastasize. Lesions are as a norm localized and the surrounding tissue usually remains healthy. Rare malignant forms of hamartomas are known as hamartoblastomas. Localized lesions are responsible for TSC symptoms in the organs in which they occur, or they may occasionally be asymptomatic. (summarized in Gomez et al., 1999) Histological examination of hamartomas shows tissue reminiscent of neuronal cells. It is assumed that TSC is based upon a disturbance in neuronal cell migration and differentiation, in particular neural cells derived from the neural crest. For example, the major feature of hypomelanotic macules in the skin represent a local pigment disorder in melanocytes, which are derived from the neural crest cells. (summarized in Rott et al., 1999) The migration of neural crest cells in various tissues is one possible explanation for the ability of TSC to occur in almost any organ. TSC lesions have been often described in the CNS, kidney, skin, heart, lung, retina, bone, and teeth. Skeletal muscle, peripheral nervous system and thymus involvement have as yet not been described. Despite these tendencies, there is as yet no common trend in the emergence of hamartomas that can predict the course of the disease. (summarized in Rott et al., 1999) An argument for the theory that TSC lesions arise from a single cell is demonstrated in the example of angiomyolipomas. A progenitor cell in which one of the TSC genes is defective can give rise to the three different cell types (blood vessel, smooth muscle and fat) found in angiomyolipomas (Young and Povey, 1998). The unicellular origin is confirmed by the finding of non-random X-inactivation in tumors from female TSC patients (Green et al, 1996).

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Hamartomas also demonstrate loss of heterozygosity, which is evidence for the tumor suppressor function of the two TSC genes (Carbonara et al., 1994 and Henske et al., 1996). The loss of both alleles of a gene coding for a tumor suppressor is required for loss of growth control (Lewin 1998). This loss of the healthy allele means that TSC behaves recessively at the cellular level. Patients have been studied, who were heterozygous for TSC1 or TSC2 markers, but whose angiomyolipoma tissue were homozygous for these markers (Young and Povey, 1998). Depending on when and where the loss of the healthy allele occurs, the clinical outcome of the disease will therefore vary.  1.4 Genetics   TSC is autosomal dominantly inherited, but more than 50 % of patients with TSC have sporadic mutations (Verhoef et al., 1999). TSC is genetically heterogeneous, meaning that a defect in one of two genes (TSC1 and TSC2) and their products (hamartin and tuberin, respectively) cause the disorder. In 1993 the TSC2 gene was identified on chromosome 16p13.3 (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and TSC1 followed in 1997 on chromosome 9q34.3 (van Slegtenhorst et al., 1997). TSC1 spans 45 kb of genomic DNA and contains 23 exons. The TSC2 genomic DNA is 43 kb long and consists of 41 exons. The location of TSC2 is particularly interesting. It is located directly next to the gene affected in polycystic kidney disease, PKD-1, and kidney cysts are also one of the possible clinical features of TSC (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). PKD-1 lies directly centromeric to TSC2 and the genes are separated by only 60 base pairs. Another neighbor of TSC2 on chromosome 16 is CYLD-1 at 16q12-13, which, when mutated, results in autosomal dominant cylindromatosis (Verhoef et al., 1998). CYLD causes skin lesions clinically similar to the typical TSC facial angiofibromas. These diseases, in some cases clinically very similar to TSC, further complicate the diagnosis of this complex. Recent initial evidence for the location of genes that might be involved in autism also revealed linkage to chromosome 16p near TSC2 (International Molecular Genetic Study of Autism Consortium, 1998). Autism and other psychiatric and behavioral disorders including attention deficit hyperactivity disorder, aggression and anxiety are often comorbid with TSC (Smalley, 1998). The chromosomal locations of TSC1 and TSC2 can be seen in figure 2.

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 Introduction