University of Groningen. Epidermolysis bullosa simplex Bolling, Maria Caroline

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1 University of Groningen Epidermolysis bullosa simplex Bolling, Maria Caroline IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2010 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Bolling, M. C. (2010). Epidermolysis bullosa simplex: new insights in desmosomal cardiocutaneous syndromes. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

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3 Introduction MC Bolling Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

4 Chapter 1 14

5 Introduction The skin in general The skin, our integumentary system, is the largest organ of the body covering a surface of ~ m 2 with ~ cells per mm 2 in an adult human being. 1, 2 Our skin distinguishes, separates, protects and informs us with regard to our surroundings. The basic function of the skin is the maintenance of an internal environment that is optimized to protect and reproduce DNA. 3 Preservation and reproduction of DNA sustains the species, and facilitates the required adaptation to a continuously changing environment by advantageous changes in DNA, a process called evolution. This necessary genetic adaptation sometimes goes awry, leading to mutations and hereditary disease, the topic of this thesis. To serve the basic functions of protecting and regenerating DNA, one of the main requirements of the skin is that it is capable of maintaining an anatomical barrier that protects the inside from the outside, i.e. pathogens, radiation (such as UV), toxic agents, and mechanical stress. 4-6 Simultaneously, this barrier prevents the loss of heat, fluid and electrolytes from the inside. In addition, the skin acts as a temperature regulator, a sensory system, a sexual medium, excretes wastes, and serves in vitamin D synthesis The advanced anatomical structure of the skin is optimized for the performance of these important functions. In the next paragraphs the structure of the skin will be discussed in detail. Structure of the skin The skin comprises of two main layers: the epidermis and the underlying dermis (figure 1). Below the dermis resides the mesodermally derived hypodermis, or subcutis, mainly consisting of loose connective tissue and lobules of fat. 3 The innermost skin layer, the vascularized dermis, provides nutritional and structural support to the overlying epidermis. The dermis contains vasculature, nerves and cells, such as fibroblasts, macrophages, mast cells and dendritic cells, embedded in an extracellular fibrous matrix consisting of collagens and elastin. 12 The embryonic origin of the dermis varies per body site localization Facing the external environment is the ectodermally derived epidermis with the adnexae extending downwards in the dermis: the hair follicle, the sweat gland, and the sebaceous gland. More than 90% of the epidermal cells are keratinocytes, named after their major component: the keratin intermediate filament (IF) protein. 12 Other cells residing in the epidermis are Langerhans cells, melanocytes and the neuroendocrine Merkel cells. 16 The epidermis can be subdivided in four or five (depending on body site) different layers of differentiation: (from inside to outside) the basal layer with the stem cells, the stratum spinosum, the stratum granulosum, the stratum lucidum (only in palmoplantar skin), and eventually the stratum corneum. The stem cells in the basal layer of the interfollicular epidermis, the hair follicles, the sebaceous glands and the eccrine ducts, provide the continuous renewal of the epidermis by dividing (mitosis) and pushing daughter cells upwards, while keeping their own undifferentiated state. The destination of these daughter cells is to migrate upwards in the epidermis, while undergoing the process 15

6 Chapter 1 of terminal differentiation and withdrawing from the cell cycle after a few rounds of division.17, 18 During this complex process of migration and differentiation, keratinocytes mature until they become anucleated and keratin-packed cells that are eventually sloughed-off ( desquamated ) from the uppermost stratum corneum, a process that takes approximately four weeks.19 The process of differentiation is marked by changes in gene expression and the subsequent changes in protein content of the cells, for example keratins.3, 20, 21 stratum corneum stratum granulosum stratum spinosum stratum basale dermis Figure 1. Light microscopic picture of an H&E staining of normal skin. Throughout the epidermis keratinocytes are held together by a dynamic interaction between different cell-cell junctions: desmosomes, adherens junctions, tight junctions and gap junctions. Desmosomes (macula adherens) anchor the stress-bearing keratin filaments of neighbouring cells (see also paragraph The desmosome), while adherens junctions ( zonula adherens ) and tight junctions ( zonula occludens ) associate with actin microfilaments Gap junctions consist of connexins and allow free exchange of various molecules and ions between cells (for review see 26). The epidermis and dermis are connected by the basement membrane zone (BMZ), also called the dermal-epidermal junction, consisting of specialized aggregations of attachment and signalling molecules.27 The basement membrane zone The epidermis is separated from the dermis by the BMZ (figure 2).27 Here, the keratin IF skeleton of the basal keratinocytes is anchored to the dermal matrix by hemidesmosomes, while the actin and microtubule filament systems are anchored to another cell-matrix junction: the focal adhesion (FA). Hemidesmosomes in the skin are ultrastructurally recognizable as electron-dense structures at the base of the basal keratinocytes (figure 2 and 3).28, 29 On the cytoplasmatic 16

7 Introduction site hemidesmosomes have an inner plaque representing the connection between the basal keratins 5 (K5) and 14 (K14) and the cytoplasmatic domains of the plakin proteins plectin and BP230 (also termed bullous pemphigoid antigen-1: BPAG1), and an outer plaque consisting of the cytoplasmatic domains of integrin α6β4 and type XVII collagen (also called BP180 or bullous pemphigoid antigen-2: BPAG2), and the tetraspanin CD , 31 Outer plaque proteins interact with the anchoring filaments (laminin-332, laminin-311, laminin-511 and the extracellular domain of BP180) which traverse the plasma membrane, the sub-basal dense plate and the lamina lucida, and connect to the anchoring fibrils (type IV and type VII collagen) in the lamina densa and the sublamina densa area. These collagens extend into the dermis from where they loop back into the lamina densa, or insert into anchoring plaques. 32 This complex chain of proteins together is called the hemidesmosome-anchoring filament complex or hemidesmosome-stable adhesion complex (figure 3). 27 Figure 2. Illustration of the skin with on the left side the epidermis with the different layers and the dermis below, and on the right side the hemidesmosome-stable adhesion complex connecting the basal cells to the dermal extracellular matrix. Two types of hemidesmosomes can be distinguished: type II hemidesmosomes are found in simple epithelia, such as the intestine and the uroepithelium, and consist of integrin α6β4 and plectin as basic components, while type I hemidesmosomes are found in stratified epithelia, like the skin, and additionally contain CD151, BP230 and BP , 33 Of note, in the skin both 17

8 Chapter 1 type I and type II hemidesmosomes are observed in the same keratinocyte, probably as a reflection of a dynamic multistep process of assembly/disassembly. 33 Besides their role in stable adhesion, hemidesmosomes need to be highly dynamic and capable of rapid disassembly in case cell detachment from the basement membrane is required, like during cell division and migration (see above). 34, 35 The crucial event in hemidesmosome assembly is thought to be the binding of the actin-binding domain (ABD) of plectin to the first pair of fibronectin type-iii (FNIII) repeats and the N-terminal 35 amino acids of the connecting segment (CS) amid the FNIII repeats of integrin β This connection is strengthened by additional interactions between the N-terminal and C-terminal plakin domains of plectin with the CS and C-terminus of integrin β4. Subsequently, BP180 is binding to the plectin N-terminus and the third FNIII repeat of integrin β4. Lastly, BP230 is recruited into the structure by binding to integrin β4 and BP180. Plectin, BP180, and BP230 anchor the basal epidermal keratins K5 and K14. The importance of the initial plectin-integrin β4 binding for hemidesmosome formation, is indicated in vivo by the hypoplastic appearance of hemidesmosomes in patients with integrin β4 mutations that prevent this association, and the existence of type II hemidesmosomes that apparently do not need BP180 and BP230 for formation. 30, 39 Furthermore, in vitro studies preventing the plectin-integrin Figure 3. Schematic representation of the hemidesmosome-stable adhesion complex (right) and the focal adhesion complex (left). Mutations in the genes coding for the proteins depicted in colour, cause the different forms of EB. Courtesy of M.F. Jonkman. 18

9 Introduction β4 interaction revealed severely impaired hemidesmosome formation. 33 The importance of hemidesmosomes for maintaining epithelial integrity is indicated by hemidesmosomal gene knockout mice showing early neonatal death due to skin defects. In addition, human patients with mutations in hemidesmosomal proteins suffer from skin fragility due to loss off epidermaldermal connection, leading to hereditary blistering diseases collectively called epidermolysis bullosa (EB, see paragraph Epidermolysis bullosa) Another structure connecting basal keratinocytes to the dermal matrix is the FA. FAs are large protein complexes that serve to connect the actin and microtubule cytoskeleton to extracellular matrices, like the dermis. FAs are dynamic structures that also function as signal carriers and, besides cell-matrix adhesion, play an important role in cell migration, proliferation, differentiation and survival. In migrating cells, FAs constantly assemble and disassemble through the continuous association and dissociation of many proteins. Integrins are the essential constituents of FAs. 44, 45 Additional proteins on the cytoplasmatic site can bind these integrins, such as vinculin, talin, α-actinin, filamin, and fermitin family homologue-1 (FFH-1, also known as kindlin-1). Autosomal recessive mutations in the gene encoding FFH-1, FERMT1 (or KIND1) are associated with Kindler syndrome (KS, MIM#173650). KS was the first hereditary FA disorder discovered KS is clinically characterized by poikiloderma and trauma-induced blistering, and is considered as a subtype of EB in the latest EB consensus (see paragraph Epidermolysis bullosa) Many questions concerning KS, the function of FFH-1, and how loss-of-function FERMT1 mutations cause the clinical features of KS are yet unsolved. The oldest patient yet reported with KS is described in chapter 5. The study on the clinical features of this disease in conjunction with the tissue morphology and the molecular aetiology can provide better understanding of the role of FAs in health and disease. The desmosome Desmosomes (composition of the Greek words desmos meaning bond, and soma meaning body) are intercellular structures that link the IF cytoskeletons from neighbouring cells and provide intercellular bonding in many stress-bearing tissues, such as skin and heart. Besides their adhesive function, desmosomes also promote maturation of adherens junctions, influence cytoskeleton dynamics and function in differentiation and tissue morphogenesis (reviewed in 22, 24, 52, 53 ). Desmosomal proteins are derived from three gene families: cadherins, armadillo proteins, and plakins (reviewed in 23, 24, 54 ). Cadherins (desmocollins 1-3 and desmogleins 1-4) form the extracellular connections by homophilic (i.e. binding of desmocollin to desmocollin and desmoglein to desmoglein) and heterophilic (i.e. binding of desmocollin to desmoglein, and vice versa) bonding. On the intracellular site, the cytoplasmic tails of cadherins bind to the armadillo proteins plakoglobin and the plakophilins 1-3. These proteins form the outer dense plaque of the desmosome, visible by electron microscopy (EM) (figure 4 and 5). The plakophilins and plakoglobin, in turn, bind to the N-terminus of desmoplakin. Desmoplakin links the 19

10 Chapter 1 cytoplasmatic IFs to the desmosome by its C-terminus and forms the inner dense plaque (see also chapter 8). Lateral interactions and other proteins, like plectin (see paragraph Plectin), strengthen these connections (figure 4). Desmosome Epidermis IDP ODP PM EC Myocardium ODP IDP EC keratin Dsg1/2/3, Dsc1/2/3 Dsg2, Dsc2 desmin plectin(?) DP PKP1/2/3 PG PKP2 PG DP plectin(?) PM Adherens junction plectin(?) actin vinculin α-actinin talin p120 α-catenin PG/ β-catenin E-cadherin N-cadherin DCCS associated shared by epidermis and myocardium, not associated with DCCS (yet) Figure 4. Schematic representation of the desmosome and the adherens junction. On the left side the proteins expressed in the epidermal desmosomes are depicted, and on the right side the desmosomal proteins of myocardium. DCCS, desmosomal cardiocutaneous syndrome; DP, desmoplakin; EC, extracellular; IDP, inner dense plaque; ODP, outer dense plaque; PG, plakoglobin; PM, plasma membrane; PKP, plakophilin. One of the essential, invariable components of desmosomes in all tissues is desmoplakin, a member of the plakin family of proteins. Plakin proteins are generally involved in tethering major cytoskeletal fibers, including IFs, actin and microtubules, to one another and to plasma membrane associated adhesion complexes (for review see 54 ). Desmoplakin consists of an N-terminal plakin domain, a central coiled-coil rod domain, and a C-terminus consisting of three highly homologous plakin repeat domains (PRDs): A, B, and C. 55, 56 As mentioned above, the N-terminus of desmoplakin binds to the desmosomal outer plaque proteins plakoglobin and the plakophilins. The central rod domain has a high α-helical content, and is thought to be involved in dimer formation. Of note, at least two isoforms exist: desmoplakin-i and desmoplakin-ii, 20

11 Introduction 57, 58 the latter lacking most of the rod domain because of alternative splicing of exon 23. Desmoplakin-I is expressed in both skin and heart (and many other desmosome containing tissues), whereas the shorter desmoplakin-ii is mainly expressed in skin, and only at low levels in certain parts of the heart. 59 The major IF-binding site of desmoplakin has been mapped to the linker sequence between PRDs B and C in the C-terminus, although many other low affinity IF binding sites were identified on the other PRDs as well It is thought that a number of weak but simultaneous interactions are required for IF-binding. Not surprisingly other plakins, like plectin and BPAG1e, also contain multiple PRDs and are believed to form dimers as well, thereby multiplying their IF binding sites. 63 The vital importance of desmoplakin for tissue integrity is exemplified by: 1) mouse knockout studies, and 2) human mutations that target the desmoplakin encoding gene DSP. Knockout of DSP in mice turned out to be lethal in early embryogenesis due to defects in extraembryonic tissues. 64 By rescuing desmoplakin in extra-embryonic tissue in mice, the detrimental effects of desmoplakin-knockout for intrauterine development of heart, neuroepithelium, vasculature and skin became apparent. 65 The desmosomes in desmoplakin-null epidermis appeared normal (or perhaps slightly reduced) in size and number. However, all desmosomes lacked the inner dense plaque and IF insertion, and were torn out of the plasma membrane as a whole. In addition, adherens junctions were markedly affected in number and morphology. Re-expression of the desmoplakin N-terminus was sufficient to restore clustering of other desmosomal proteins at cell-cell borders, while additional re-expression of the C-terminus was necessary to restore desmosome-if interaction. 66 In humans a recessive DSP mutation causing truncation of the desmoplakin C-terminus just proximal to PRD-C resulted in dilated cardiomyopathy (DCM), woolly hair, palmoplantar keratoderma and moderate skin fragility, a syndrome called Carvajal syndrome. 67 A truncation of the complete desmoplakin C-terminus from both alleles (and affecting both desmoplakin isoforms) caused the early lethal phenotype of lethal acantholytic epidermolysis bullosa (LAEB, MIM#609638). In skin of the first LAEB patient reported by Jonkman et al., and the LAEB patients described in chapter 8 of this thesis, keratin IFinsertion to the desmosomal plaque was completely lost, and clinically extensive shedding of the epidermis was present leading to early demise. 68 Additional recessive and dominant mutations in various parts of desmoplakin have been associated with striate palmoplantar keratoderma [MIM#612908], skin fragility-woolly hair syndrome [MIM#607655] and arrhythmogenic right ventricular cardiomyopathy (ARVC) [MIM#607450] In general, however, the genotypephenotype correlations for desmoplakin mutations are only partly understood. This is further discussed in chapter 6. 21

12 Chapter 1 Table 1. Desmosomal proteins associated with human genetic disease. Protein (gene) [MIM*] Expression in skin/heart Dominant [MIM#] Recessive [MIM#] Plakoglobin (JUP) [173325] Desmoplakin (DSP) [125647] Desmocollin-2 (DSC2) [125645] Desmocollin-3 (DSC3) [600271] Desmoglein-1 (DSG1) [125670] Desmoglein-2 (DSG2) [125671] Desmoglein-4 (DSG4) [607892] Plakophilin-1 (PKP1) [601975] Plakophilin-2 (PKP2) [602861] Plectin (PLEC1) [601282] Corneodesmosin (CDSN) [602593] Skin+heart ARVC/D 72 [611528] In-frame insertion Naxos disease (woolly hair, ARVC, PPK) 73 [601214] Deletion>PTC Skin+heart ARVC/D 71 [607450]; SPPK 74 [148700] Missense/nonsense/ deletion/insertion/ splice site Skin+heart ARVC/D 76 [610476] Missense/deletion/ insertion/splice site Carvajal syndrome (woolly hair, PPK, DCM) 67 [605676], Naxos-like syndrome (woolly hair, ARVC, PPK) 75 [605676], LAEB 68,this thesis [609638], SF/WH syndrome 69 [607655] Nonsense, frameshift, deletion Naxos-like disease (woolly hair, ARVC, PPK) 77 Deletion>PTC Skin - Hypotrichosis and recurrent skin vesicles 78 [613102] Nonsense>PTC Skin SPPK 79 [148700] Splice site>in-frame deletion - Skin+heart ARVC/D 80, 81 [610193] Missense/deletion/ insertion/splice site/nonsense Skin - Localized autosomal recessive hypotrichosis 82 [607903] Deletion>PTC Skin - Skin fragility-ectodermal dysplasia syndrome 83 [604536] Skin+heart ARVC/D 84 [609040] Missense/nonsense/ deletion/insertion/ splice site - Skin+heart 85, this thesis EBS-Ogna Missense EBS-MD 86 [226670], EBS-PA 87 [612138], EBS-MD-DCM this thesis Deletion/insertion/ [131950] nonsense Skin - Hypotrichosis simplex 88 [146520] Nonsense ARVC/D, arrhythmogenic right ventricular cardiomyopathy/dystrophy; DCM, dilated cardiomyopathy; EBS, epidermolysis bullosa simplex; LAEB, lethal acantholytic epidermolysis bullosa; MD, muscular dystrophy; PA, pyloric atresia; PTC, premature termination codon; SPPK, striate palmoplantar keratoderma. 22

13 Introduction Skin and heart are tissues that are exposed to high levels of mechanical stress. Macroscopically these tissues appear to have very few in common. However, their intercellular structures that have to meet the high demands, the desmosomes, show remarkable similarities (figure 4 and 5). Likewise, mutations in genes coding for desmosomal proteins that are expressed in both human skin and heart will affect either one of these tissues, or both (table 1). The disorders, in which both skin and heart are affected due to mutations in a desmosomal gene, we have named desmosomal cardiocutaneous syndromes (DCCS) (figure 4). epidermis myocardium DP Figure 5. Immunofluorescence microscopic and ultrastructural views (middle) of the desmosomal structures in the epidermis (left) and the myocardium (right). Most left picture is from Green et al.89, and the second picture from the left is from Franke et al.90 (both with permission). The two right most pictures are own material. Desmoplakin staining with antibody Dp2.17, original magnification 40x. In chapter 6 the features of these syndromes, their molecular background, the lessons that may be learned from them, the remaining unsolved questions and the future perspectives are discussed. A careful study of these syndromes leads to a better understanding of the versatile functions of the cell-cell linking structures and their role beyond adhesion. Insight in the complex cell-signalling pathways in which desmosomal proteins are involved, may provide new entries for therapy. In Chapters 7 and 8 clinical, tissue and molecular studies on patients with clinical features of skin fragility and cardiomyopathy due to mutations in desmosomal proteins are described. The effects of mutations in plectin on human skin, skeletal muscle, and cardiac integrity are further investigated in chapter 7. The study in chapter 8 was aimed at elucidating the underlying molecular defects in novel cases of LAEB, in which an intrauterine enlarged heart was observed. Plectin Plectin (gene: PLEC1) is a versatile cytolinker plakin protein associated with IFs, actin, and microtubules in a wide variety of tissues.54, The gene PLEC1 encodes for multiple different plectin isoforms, generated by alternative splicing of the transcript. The different plectin isoforms link IFs to mitochondria, to the nuclear envelope, and to cell-cell and cell-matrix junctions in 23

14 Chapter 1 multiple tissues. Among these different tissues are skin, skeletal muscle, heart and nerve tissue. 40, 91, 95 In the skin, plectin (isoforms 1, 1a, 1c, and 1f) is present in hemidesmosomes, where it anchors the basal keratin IFs K5 and K14 to integrin β4 and BP180 in the hemidesmosomal plaque. Plectin has also been found in desmosomes, where it colocalized with desmoplakin, and is likely to stabilize the IF binding and/or mediate the interaction between desmoplakin and the actin-system. 96 In addition to linking the IF skeleton to desmosomes and hemidesmosomes, plectin is also able to associate with actin stress fibers by its N-terminus. 36, 97, 98 In skeletal muscle (isoforms 1, 1b, 1d, 1f) plectin is a component of the sarcolemma and the Z-discs (or also called Z-lines). 99 At these sites, plectin is associated with the IF protein desmin and assists in linking adjacent myofibrils to one another, and peripheral myofibrils to costameres at the sarcolemma (= muscle cell membrane) (figure 6) Costameres are sarcolemma-associated structures in muscle tissue with homology to hemidesmosomes in skin. Both hemidesmosomes and costameres anchor IFs to the plasma membrane and extracellular matrix. Findings in patients with mutations in plectin and in vitro studies suggest that the localization of plectin at Z-discs is a necessity for the adequate formation of the muscle desmin cytoskeleton. 104 Figure 6. Illustration of the skeletal myocyte. Different plectin isoforms are expressed, which link the desmin-if fibers to the nucleus, the subsarcolemmal costameres, the mitochondria and the Z-discs. DGC complex, dystrophinassociated glycoprotein complex (link between the cytoskeleton and the extracellular matrix). In addition, plectin is thought to be involved in mitochondrial and nuclear localization by linking these structures to the IF skeleton In cardiac tissue plectin has been localized at desmosomelike structures in the intercalated discs (IDs), and at Z-discs. 40, 91, 109 IDs are intercellular connecting structures that attach the cytoskeletons of adjacent cells and, in addition to desmosome-like structures, are composed of gap junctions and adherens junction. IDs allow direct passage of action potentials from one cardiomyocyte to another. Plectin consists of a central rod domain flanked by large globular domains. The C-terminus consists of six conserved and homologous PRDs involved in IF binding. In its N-terminus, plectin harbours two highly conserved calponin-homology domains forming the actin-binding domain (ABD), followed by nine spectrin-repeats (SRs) (figure 7). 54, 110 The ABD is involved in the mutually exclusive binding to actin, the integrin β4 subunit of the obligate dimer integrin α6β4, and the nuclear envelope protein nesprin-3α. 36, 98, 108, 111 The N-terminus 24

15 Introduction additionally contains binding sites for BP180 in hemidesmosomes and several sarcolemmal proteins in muscle cells. 33, 106, 112 The rod domain of plectin has a highly α-helical structure favouring the coiled-coil formation, which promotes parallel and in-register homodimerization, or heterodimerization with other plectin isoforms. 63, 93, 113 Finally, increasing evidence indicates that, besides its role as a cytolinker, plectin also functions as a scaffold for various cell signalling pathways and interacts with non-receptor tyrosine kinases, mitogen-activated and AMPactivated kinases, and protein kinase A and C By alternative splicing, the PLEC1 gene (chromosome 8q24.13-qter) generates multiple plectin isoforms that differ mainly in their N-terminal region. In addition, a PLEC1 RNA-transcript that lacks the complete rod encoding exon 31 has been described as well (this thesis chapter 9). 118, 119 These different plectin isoforms are expressed in a cell-type and -structure specific 95, 118, way. Plectin-knockout mice died in the first postnatal days displaying extensive skin fragility, and muscular and cardiac pathology. 40 Furthermore, muscle-specific plectin knockout mice developed muscular dystrophy (MD) and exercise-induced cardiomyopathy. 124 Exon nr Q305X,1395G>A 1614del4 2820del21 R1189X Q2466X, Q2545X R3029X NH2 Q305X 2731del14 CH1- CH2 COOH NM_ NP_ plectin 1c 1007ins3 R323Q 1541ins del9 Q1408X 4357ins13 Q1450X 4416delC R2465X T2890S 13524ins16 R2421X R3527C K4460X R2351X 12633ins4 R2319X 2719del9 E1614X E2005X del del19 R2000W Q1053X 5083delG 5905del2 5148del8 5899ins8 Q1713X 5866delC 5309insG Q1936X E1804X Q1910X ABD consisting of two CH1- CH2 EBS-MD calponin homology (CH) domains EBS-PA/lethal globular plakin domain with spectrin repeats EBS-Ogna (dominant) central coiled-coil rod domain EBS without MD, diagnosis before age 5 years c-terminal plakin repeats with IF binding motif This thesis Figure 7. Plectin protein with its different domains (explained in the legend below the protein) and all previously reported PLEC1 mutations in humans with the associated phenotypes. Sequence numbering is according to RefSeqs NM_ (mrna) and NP_ (protein). Eight human plectin isoforms, differing in the N-terminal part preceding the actin-binding domain, are reported in GenBank. Furthermore, evidence has been found for the presence of an additional plectin transcript lacking exon 31 encoding the complete rod domain, the presence of which was already shown in animals. 25

16 Chapter 1 Different mutations in the gene PLEC1 in humans lead to various phenotypes (figure 7). The common clinical feature of all these phenotypes is skin blistering with a plane of cleavage just above the inner plaques of hemidesmosomes and through the cytoplasm of the basal keratinocytes, called epidermolysis bullosa simplex (EBS, see also paragraphs Epidermolysis bullosa and Basal epidermolysis bullosa simplex of this chapter). 51 Homozygous and compound heterozygous nonsense and frameshift mutations located outside exon 31, encoding the entire rod domain, lead to loss-of-function/expression of both the full-length and the rodless plectin isoforms, and give rise to early lethal phenotypes due to severe skin and mucosal fragility and/or pyloric atresia (EBS-PA). 87, 125 Association of the EBS skin phenotype with MD has been reported for autosomal recessive nonsense and frameshift mutations mainly located within exon 31, and in-frame mutations elsewhere in PLEC1 (EBS-MD). 86, 126 The persistent expression of the rodless isoform of plectin in these patients is thought to rescue them from early lethality. 119 No recessive missense mutations have been reported except for the one described in chapter 7 of this thesis. One dominant missense mutation in the plectin rod domain has been described in 2002 in a large Norwegian kindred and in a smaller, unrelated German kindred. Affected persons suffered from basal intraepidermal skin fragility without mucosal, pyloric or muscular involvement (non-syndromic EBS). The phenotype was named EBS-Ogna, after the municipality where the common founder of the Norwegian family originated. 85, 127 This phenotype will now be discussed in further detail as this is interesting in the light of the findings in chapter 2 and 3. The phenotype of affected persons in the Norwegian EBS-Ogna kindred was first described by Gedde-Dahl 128 and consisted of skin fragility of mainly the dorsal aspects of the distal extremities and the shins, small traumatic blood blebs of palms and fingers, and serous blistering of mainly hands and feet upon trivial trauma. These features are very similar to the localized subtype of basal EBS (EBS-loc) due to mutations in K5 and K14 (see also paragraph Basal epidermolysis bullosa simplex). The onset and severity varied between affected persons. An inconsistent feature was onychogryphosis of the big toenails, and rarely other nails, which was present in about half of the affected adult family members. The phenotype was originally separated from the EBS-loc and the generalized subtype of basal EBS (EBS-gen), because of the tendency to form blood blebs and the onychogryphosis. No other organ involvement was noted. In this initial report the blister level observed in microscopy of a H&E staining of skin samples of affected EBS-Ogna family members was intraepidermal, in the suprabasal layers. 128 Later reports of EM analyses of patients skin samples revealed that the blisters originated in the most basal areas of the basal keratinocyte cytoplasm, just above, but not within, the hemidesmosome. 85 Hemidesmosomes had normal structured extracellular parts and showed well developed subbasal dense plates and anchoring filaments, whereas the inner plaques were hypoplastic, thin and fragmented. However, some hemidesmosomes appeared completely normal. In fresh blisters the blister floor was covered by basal cell debris, giving it a basal EBS-blister appearance, whereas in older blisters the debris seemed degraded, rendering the blister a more junctional appearance. For the latter observation the term pseudojunctional 26

17 Introduction was introduced. No keratin aggregations, as seen in the Dowling-Meara subtype of basal EBS (EBS-DM), were observed. Earlier reported immunofluorescence analysis of EBS-Ogna skin had shown reduced basal layer staining with the antibodies 10F6 and 5B3 against the plectin rod domain. 127 This particular finding, in addition to the linkage of the phenotype to chromosome 8q24, provided the clue for PLEC1 as the causative gene for EBS-Ogna. 129 A heterozygous plectin missense mutation, p.arg2000trp, was then found, which fully segregated with the phenotype in the Norwegian and the German kindred. 85 Because all plectin mutations reported until 2002 were associated with MD, muscle biopsies of adult EBS-Ogna patients were investigated, but did not show any abnormalities. 85 As many EBS-Ogna generations had passed without additional organ involvement in any of the affected persons, EBS-Ogna thus comprises another nonsyndromic form of EBS. However, since this single report no other autosomal dominant PLEC1 mutations underlying EBS have been reported. Two reasons can be thought of: 1) there are none, or 2) it was not looked for. We favoured the second reason as it seems very unlikely that only one single aminoacid change in the large α-helical rod domain of plectin could cause such a phenotype. Therefore, PLEC1 seemed a good candidate to screen for mutations in our cohort of EBS patients with wild-type KRT5 and KRT14 genes. The results of this study are presented in chapter 3. The presence of plectin in myocardium at cell-cell and cell-matrix adhesion structures, and the cardiomyopathy observed in plectin-knockout mice, suggest that plectin is important for maintenance of cardiac tissue integrity as well. However, the role of PLEC1 mutations in human cardiac disease had not been investigated yet. We have made a start in chapter 7. The keratin cytoskeleton Keratinocytes are named after the proteins they contain, the keratins. Keratins belong to the IF family of proteins, which share common structural, sequence and functional features. The name intermediate filament is deducted from their diameter which is ~10nm, being intermediate between that of the other cytoskeleton proteins, the microfilaments (actin, ~8 nm) and the microtubules (~25 nm). Most IFs are located in the cytoplasm and provide stability and resilience to the body of the cell. IFs called lamins are present in the nucleus, forming the nucleoskeleton, but these are outside the scope of this thesis. 130, 131 In the skin, keratins provide this structural integrity to keratinocytes. Keratins can be subdivided in type I (acidic) and type II (basic) keratins. The general structure of IFs is a highly conserved three-partite structure characterized by nonα-helical globular N- and C-termini with a central α-helical coiled-coil rod domain. 131, 132 The rod domain consists of four α-helical segments (1A, 1B, 2A, and 2B) interrupted by non-helical linker domains: L1, L12, and L2, respectively, and an additional interruption in segment 2B, the socalled stutter region (figure 8). 27

18 Chapter 1 K5/K14 Figure 8. The molecular organization of K5 and K14. K5 and K14 form obligate parallel, HIM L1 L2 L12 st HTM in-register coiled-coil heterodimers in basal H1 1A 1B 2A 2B H2 head rod tail keratinocytes. Keratins consist of a central α-helical rod domain which is interrupted K5 head (167) K5 tail (115) by three non-helical linker domains (L1, L12, L12 st and L2) giving rise to the four subdomains Coil 1A L1 Coil 1B Coil 2A Coil 2B L2 K14 tail (52) 1A, 1B, 1B and 2B. An additional non-helical K14 head (114) stretch in subdomain 2B, the stutter (st), interrupts the α-helix of 2B. The variable and non-helical head and tail domains are folded backwards supporting the dimer. The figure below is from Hermann et al. 131, with permission. HIM, helix initiation motif; HTM, helix termination motif. The α-helices of the subdomains show a characteristic and highly conserved heptad repeat pattern (abcdefg) n in which position a and d are generally occupied by apolar (hydrophobic) residues forming the helix interface which functions as a seam that drives the coiled-coil heterodimer formation. Additional interactions via the usually hydrophilic and charged residues at positions e and g strengthen the binding. Type I and type II keratins form obligate, parallel and in-register coiled-coil heterodimers by means of interactions via the rod domains of both keratins (reviewed in 131, 133 ). The smallest soluble unit in vivo is a tetramer consisting of two antiparallel heterodimers. To form this tetramer, the first two heptads of the α-helical 1A domain of one dimer connect by a head-to-tail overlap to the 2B end of the next dimer in line (A cn mode). These specific rod endings, important for keratin assembly, are called the helix initiation motif (beginning 1A) and the helix termination motif (end of 2B). The anti-parallel formation of the basic tetramer makes keratin filaments apolar. By lateral association the tetramers form unitlength filaments which then assemble into longer filament structures by head-to-tail linking and compaction to a diameter of ~ 10 nm (figure 9). The length and nature of the head- and tail sequences flanking the rod domain vary considerably between different keratins, implying tissue and differentiation specific and/or dependant functions of these domains. These variable head and tail domains are thought not to be directly involved in electrostatic keratin-keratin interaction. However, in vitro studies have shown that removal of the K5 tail domain does impair filament assembly. 134 It is thought that one of the functions of these domains is providing additional stability to the coiled-coil structure. 135 The phenotypes associated with mutations in these domains point to additional functions beyond keratin assembly and maintenance of cytoskeleton integrity (see also paragraph Basal epidermolysis bullosa simplex). Several keratin genes are expressed in skin. In the basal layer K5 (type II IF) and K14 (type I IF) form the basic dimers. In the hair follicle K15, another type I keratin, is expressed that can form dimers with K The major keratins of the suprabasal layers are K1 and K10. In palmoplantar skin an additional heterodimer partner for K1 is expressed: K9. Other epidermal 28

19 Introduction K5 NH K5-K14 parallel heterodimer K5-K14 anti-parallel tetramer K14 COOH Figure 9. Keratin assembly. K5 (type II IF) and K14 (type I IF) form obligate parallel and in-register heterodimers. Two heterodimers align in an anti-parallel fashion and are thought to comprise the basic building block for further assembly, thus making keratin filaments apolar structures. ULF, unit length filaments. Lateral association of tetramers into ULFs Longitudinal assembly of ULFs into filaments keratins are K2 (upper stratum spinosum), and K6A, K6B, K16 and K17 (palmoplantar skin, oral mucosa and epithelial oesophagus, nail bed and adnexae). Except for K15, mutations in all above mentioned keratins have been associated with genetic disease in humans (table 2) (see the Human Intermediate Filament Database: These mutations are mainly autosomal dominant because mutations in one keratin affect its heterodimer partner in a dominant negative manner. An interesting, disease modulating observation about keratins was the finding of functional redundancy of keratins, i.e. the capability of some keratins to perform similar functions as other keratins and thus compensate for loss of these other keratins (further discussed in chapter 9). Functional redundancy of keratins was indicated by several clinical observations, such as the relative mild phenotype of patients with recessive EBS due to loss of K14 of both alleles. This is probably the result of compensatory presence of K15 in basal keratinocytes, or upregulation of K16. Another example is the presence of palmoplantar keratoderma in patients with mutations in K1, whereas patients with mutations in K10, the heterodimer partner of K1, overall do not display palmoplantar keratoderma. The reason for this is most likely the additional expression of another type I keratin, K9, in palmoplantar skin. 138 On the other hand, patients with K9 mutations have palmoplantar keratoderma as well, so apparently K10 cannot compensate in palmoplantar skin in case of K9 mutations. Loss of keratin expression from one allele was thought not to be associated with a phenotype, as carriers of rare recessive KRT14 and KRT10 null mutations did not show skin abnormalities However, the picture is somewhat more complicated, as heterozygous KRT5 mutations predicted to cause loss of expression have recently been associated with Dowling- 29

20 Chapter 1 Degos Disease, and similar heterozygous mutations in KRT14 haven been associated with Naegeli-Franceschetti-Jadassohn syndrome and Dermatopathia Pigmentosa Reticularis (see also paragraph Basal epidermolysis bullosa simplex and table 5). 142, 143 Dominant mutations in KRT5 and KRT14 cause the bullous genodermatosis EBS, and mutations in KRT1 and KRT10 cause epidermolytic ichthyosis (EI). Since the focus of this thesis is partly on the genodermatoses EBS and EI, these will be discussed in further detail below. Table 2. Keratinopathies: human hereditary diseases associated with mutations in keratins Keratin protein [MIM*] First associated disease [MIM #] Reference (1 st mutation report) K1 [139350], K10 [148080] Epidermolytic Ichthyosis (EI) [113800] K2 [600194] Ichthyosis bullosa of Siemens [146800] 147 K3 [148043], K12 [601687] Meesmann corneal dystrophy [122100] 148 K4 [123940], K13 [148065] White-sponge nevus [193900] 149, 150 K5 [148040], K14 [148066] EBS-loc [131800]; -gen [131900]; -DM [131760] K6A [148041], K6B [148042], K16 [148067], K17 [148069] Pachyonychia congenita (PC1, [167200], PC2 [167210]) 154, 155 K8 [148060], K18 [149070] Liver disease (predisposition) [215600] 156, 157 K9 [607606] Epidermolytic palmoplantar keratoderma [144200] 158 K19 [148020] Primary billiary cirrhosis (predisposition) (-) 159 K85 [602767] Ectodermal dysplasia, pure hair-nail type 160 [602032] K86 [601928] Monilethrix [601928] 161 Epidermolysis bullosa Epidermolysis bullosa (EB) is the name for a group of hereditary diseases that have skin fragility upon mild mechanical trauma as a common feature. The term was first used in 1886 and, as diagnostic and research techniques developed and insight in the molecular background of EB increased, a classification system was developed, which over time has been several times extended and adjusted The latest consensus was recently established (Vienna, 2007) and published (table 3). 51 The four major EB types (EBS, junctional EB (JEB), dystrophic EB (DEB), and KS) are distinguished by the level of blistering in the skin observed with immunofluorescence antigen mapping and/or EM analysis. Because immunofluorescence microscopy is the simplest, fastest, least expensive, and more easily available diagnostic technique, this is regarded as the golden standard for diagnosis and identification of the candidate gene(s). Skin of EBS patients reveals 30

21 Introduction an intraepidermal plane of cleavage, JEB skin an intra-lamina lucida plane of cleavage, and DEB skin a sub-lamina densa plane of cleavage. In skin of patients with KS multiple (mixed-type) planes of cleavage may be seen (figure 10). Table 3. EB classification according to the latest consensus 51 Level of skin blistering Major EB type Major EB subtype Targeted proteins Intraepidermal ( epidermolytic ) Intra - lamina lucida ( lucidolytic ) Sub - lamina densa ( dermolytic ) Mixed EBS [MIM#131800, , and ] JEB [MIM#226650, and ] DEB [MIM#131750, and ] Kindler syndrome (KS) [MIM#173650] Suprabasal EBS Basal EBS JEB-Herlitz JEB, other Dominant DEB Recessive DEB - FFH-1 (Kindlin-1) Plakophilin-1; desmoplakin; others? Keratins 5 and 14 (rare: plectin, integrin α6β4, BP180) Laminin-332 Laminin-332, BP180, integrin α6β4 Type VII collagen Type VII collagen suprabasal EBS basal JEB DEB Kindler syndrome Figure 10. The levels of blister formation observed in skin of patients with one of the major EB types. In EBS the plane of cleavage is intraepidermal (suprabasal or basal). In junctional EB (JEB) the blisters cleave the lamina lucida. In dystrophic EB (DEB) blister formation is beneath the lamina densa (sublamina densa), in the dermis. Skin fragility in Kindler syndrome can give rise to various planes of cleavage. Courtesy of M.F. Jonkman. Subtypes for EBS, JEB, and DEB, have been defined based on the mode of inheritance and/or clinical features (table 3). The general EB prevalence reported in different countries varies from 19 per million to 49 per million In the Netherlands, the prevalence of EB was estimated to be approximately 45 per million, with an incidence of newborns with EB per newborns per year. 170 Mutation analysis can provide the ultimate confirmation of disease and mode of inheritance. Mutations in 10 different proteins encoded by 13 different genes underlie the different (sub-)types of EB (table 3). In the last years, new EB-like disorders have been reported. The 2007 EB-consensus used the following foundation for including new entities as forms of EB: 1) friction-induced blistering is a cardinal clinical feature of the disease and there are other clinical features in 31

22 Chapter 1 common with known EB types, 2) they are hereditary, and 3) from a practical perspective, the patients and their families will potentially benefit from the resources available for EB patients. For example, KS (mutations in FERMT1, see paragraph The basement membrane zone) and LOC syndrome (laryncho-onycho-cutaneous syndrome [MIM#245660], mutations in LAMA3 encoding the laminin-α3 chain of laminin-332) are included in the new consensus, as they share many clinical features with other EB types. 46, 171 In contrast, EI due to mutations in KRT1 and KRT10 (see paragraph Epidermolytic ichthyosis), and pachyonychia congenita-1 en -2 (PC1 and PC2) due to mutations in KRT6A, KRT6B, KRT16, and KRT17, were not included, because blistering was not considered to be a consistent feature in these patients and dermatologists generally consider these disorders as different entities. Another adjustment to the previous classification of is the subdivision of EBS in suprabasal and basal EBS. The former EBS has become basal EBS (see also the following paragraph Basal epidermolysis bullosa simplex). Plakophilin-1 deficiency (also known as ectodermal-dysplasia/skin-fragility syndrome of McGrath) and LAEB 68, 172 due to desmoplakin mutations, were included as suprabasal EBS forms. Basal epidermolysis bullosa simplex EBS is one of the major EB-types and characterized by an intraepidermal level of blister formation (figure 10). As mentioned in the previous paragraph, in the latest EB classification EBS has been subdivided into basal and suprabasal EBS, dependent on the level of blistering within the epidermis. A level of blistering within the basal epidermal keratinocyte layer is defined as basal. A blister level above the basal layer is defined as suprabasal (figure 10). 51 Basal EBS is the most frequently occurring type of EB with approximately one case per live births. 162, Basal EBS is predominantly inherited as an autosomal dominant disorder caused by mutations in the genes KRT5 and KRT14 encoding the basal keratins K5 and K These mutations mostly comprise missense mutations and small in-frame deletions or insertions in the rod domain of K5 and K14 exerting a dominant negative affect on the keratin cytoskeleton of the basal epidermal cells. The phenotype in patients with basal EBS may vary significantly considering onset and severity of blistering. Three major EBS subtypes were defined (figure 11, table 4): 1) EBS-localized (EBS-loc, formerly known as EBS Weber Cockayne) is the mildest form, characterized by mild blistering mainly confined to hands and feet, with onset of blistering at the age of around 1-2 years when the child starts to walk, 2) EBS-generalized non-dowling Meara (EBS-gen) with generalized skin fragility throughout life, usually present at birth or starting within the first few days-weeks of life, and 3) EBS Dowling Meara (EBS-DM), the most severe form, with congenital blistering in a circinate, grouped ( herpetiform ) pattern on erythematous skin, usually involving the oral mucosa and the nail bed with subsequent onycholysis. EM analysis of skin biopsies of EBS-DM patients shows characteristic keratin filament aggregations ( clumping ) in basal keratinocytes (figure 11)

23 Introduction Table 4. EBS classification according to the latest consensus 51 Major EB subtypes EBS subtypes* [MIM#] Targeted proteins Lethal acantholytic EB [609638] Desmoplakin Suprabasal EBS Ectodermal dysplasia-skin fragility syndrome [604536] Plakophilin-1 EBS superficialis [607600]? EBS localized (EBS-loc) [131800] K5, K14 EBS generalized other (EBS-gen/EBS-gen-nonDM) [131900] K5, K14 EBS Dowling Meara (EBS-DM) [131760] K5, K14 EBS mottled pigmentation (EBS-MP) [131960] K5 Basal EBS EBS autosomal recessive (EBS-AR) [601001] K14 EBS migratory circinate (EBS-migr) [609352] K5 EBS Ogna (EBS-Og) [131950] Plectin EBS pyloric atresia (EBS-PA) [612138] Plectin, integrin β4 EBS muscular dystrophy (EBS-MD) [226670] Plectin Pseudojunctional EBS [-] BP180, integrin β4 * Rare phenotypes are depicted in italics loc gen DM MP migr DM Figure 11. Clinical features of the different subtypes of basal EBS. Upper row from left to right: loc, gen, and DM. Lower row: MP, migr, and the electron microscopic observation of keratin clumping in EBS-DM. Additional rare EBS variants have been described, such as EBS with mottled pigmentation (EBS-MP), and EBS migratory circinate (EBS-migr) EBS-migr is clinically characterized by a migrating, belt-like erythema with bullous lesions on the edge of the erythema, and additional pigmentary changes. Few cases of autosomal recessive EBS (EBS-AR) due to recessive KRT14 missense and nonsense/frameshift mutations have been described. 141, For a summary of all phenotypes that have been associated with mutations in K5 and K14, see table 5. The above mentioned basal EBS subtypes are all caused by mutations in the genes KRT5 or KRT14 (tables 4 and 5). In addition, two recessive basal EBS-associated syndromes, EBS-PA and EBS-MD, caused 33