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Genetic and Biological Characterization of Keratin 5 and 14 Mutations in EBS

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Genetic and Biological Characterization of Keratin 5 and 14 Mutations in EBS Aus der Universitäts-Hautklinik der Albert-Ludwigs-Universität Freiburg i.Br. Genetic and biological characterization of keratin 5 and 14 mutations in EBS INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert- Ludwigs-Universität Freiburg i.Br. Vorgelegt 2007 von Yow-Ren Chang geboren in Chang-Hua, Taiwan 2 Dekan Prof. Dr. med. Christoph Peters 1.Gutachter Prof. Dr. med. Leena Bruckner-Tuderman 2.Gutachter PD Dr. Deborah Morris-Rosendahl Jahr der Promotion 2007 3 Part of this work has been published in: Has C, Chang YR , Volz A, Hoeping D, Kohlhase J, Bruckner-Tuderman L. Novel keratin 14 mutations in patients with severe recessive epidermolysis bullosa simplex. J Invest Dermatol. 2006;126(8):1912-4. 4 Contents Abbreviations......................................................................................................................... 6 1. Abstract – Zusammenfassung............................................................................................ 7 2. Introduction........................................................................................................................ 9 2.1. Keratins and keratin filaments .................................................................................... 9 2.2. Keratin mutations and epidermolysis bullosa simplex ............................................. 13 Aims of the study............................................................................................................. 20 3. Materials and methods..................................................................................................... 21 3.1. Materials ................................................................................................................... 21 Technical equipment.................................................................................................... 21 Reagents....................................................................................................................... 21 Buffers and solutions ................................................................................................... 22 Enzymes....................................................................................................................... 22 Primary antibodies ....................................................................................................... 22 Secondary antibodies ................................................................................................... 22 Ready to use kits.......................................................................................................... 22 3.2 Patients....................................................................................................................... 24 3.3 Mutation detection ..................................................................................................... 24 Isolation of gDNA ....................................................................................................... 24 Amplification of gDNA fragments by PCR ................................................................ 24 Agarose gel electrophoresis.........................................................................................26 DNA sequencing.......................................................................................................... 26 Restriction enzyme digestion.......................................................................................27 3.4 Indirect immunofluorescence of skin cryosections ................................................... 28 3.5 Bioinformatics ........................................................................................................... 29 4. Results.............................................................................................................................. 30 4.1. Keratin mutations and their consequences................................................................ 30 Clinical features ........................................................................................................... 30 Indirect immunofluorescence ......................................................................................30 Mutation survey ........................................................................................................... 30 Novel and recurrent KRT5 and KRT14 mutations .......................................................33 Polymorphisms ............................................................................................................ 34 4.2. Genotype – phenotype correlations ..........................................................................36 Recessive EBS ............................................................................................................. 36 Novel deletion / insertion mutations in EBS ............................................................... 38 Novel missense mutations ...........................................................................................41 EBS with mottled pigmentation and EBS with circinate erythema............................. 44 5 5. Discussion........................................................................................................................ 48 Recessive epidermolysis bullosa simplex.................................................................... 49 Novel deletion / insertion mutations in EBS ............................................................... 51 Novel missense mutations in EBS...............................................................................52 Recurrent mutations in EBS ........................................................................................54 Outlook ........................................................................................................................ 55 6. Reference List.................................................................................................................. 56 Curriculum Vitae ................................................................................................................. 62 Acknowledgments ............................................................................................................... 63 6 Abbreviations BMZ basement membrane zone DEBS dominant epidermolysis bullosa simplex EBS epidermolysis bullosa simplex EBS-DM epidermolysis bullosa simplex Dowling-Meara EBS-K epidermolysis bullosa simplex Koebner EBS-MP epidermolysis bullosa simplex with mottled pigmentation EBS-WC epidermolysis bullosa simplex Weber-Cockayne HIP helix initiation peptide HTP helix termination peptide IIF indirect immunofluorescence IFs intermediate filaments K5 keratin 5 protein K14 keratin 14 protein KIF keratin intermediate filament KRT Keratin gene KRT5 and KRT14 genes encoding for K5 and K14, respectively PCR polymerase chain reaction PTC premature termination codon REBS recessive epidermolysis bullosa simplex 7 1. Abstract – Zusammenfassung Abstract Epidermolysis bullosa simplex (EBS) represents a heterogeneous group of inherited blistering diseases with epidermal cytolysis due to fragility of the basal layer of epidermal keratinocytes. EBS has a predominantly autosomal dominant mode of inheritance. The clinical spectrum of EBS ranges from relatively mild blistering of the hands and feet, to generalized blistering, which can be fatal. Keratins are obligate heterodimer proteins that form the intermediate filament cytoskeleton of all epithelial cells. Genetic defects in the epidermal expression of keratins are known to form the basis of EBS. It results from mutations in either the keratin 5 (KRT5 ) or keratin 14 ( KRT14 ) gene, which consist of 9 and 8 exons, respectively. We performed mutation analysis and indirect immunofluorescence (IIF) staining of the skin in 20 patients with the clinical diagnosis of EBS to determine the molecular basis of the disease. Genomic DNA was extracted from peripheral blood leukocytes, then amplified by PCR followed by restriction enzyme analysis, direct automated DNA sequencing and analysis with sequence analysis software. IIF of skin cryosections was performed with antibodies to keratin 5 and keratin 14. In this study 18 different mutations were identified, 10 of them novel. We found two novel homozygous KRT14 nonsense mutations in 2 patients with recessive EBS. We observed certain correlations between the genotype and the phenotype in our patients, but the results also show that it is not possible to predict the EBS phenotype only based on the location of the mutation. Finally, we discuss possible implications of the novel mutations on protein structure and summarize the spectrum of mutations reported so far in EBS. These findings should be helpful for genetic counselling and may contribute to the spectrum of the EBS mutation database and pave the way for gene therapy. 8 Zusammenfassung Epidermolysis bullosa simplex (EBS) ist eine heterogene Gruppe vererbter, blasenbildender Erkrankungen, mit epidermaler Zytolyse aufgrund von Fragilität der basalen Schicht der Keratinozyten. EBS wird vorwiegend autosomal dominant vererbt. Das klinische Spektrum der EBS reicht von relativ milden Blasen an Händen und Füßen zu generalisierter Blasenbildung. Keratine sind obligate heterodimere Proteine, die das intermediäre Filament-Zytoskelet der epithelialen Zellen bilden. Genetische Defekte der epidermalen Expression von Keratinen sind die Ursache von EBS. Sie resultiert aus Mutationen im Keratin 5 ( KRT5 ) oder Keratin
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