Portal de Periódicos da CAPES

Insights from a Desmoplakin Mutation Identified in Lethal Acantholytic Epidermolysis Bullosa

2010; Elsevier BV; Volume: 130; Issue: 11 Linguagem: Inglês

10.1038/jid.2010.189

1523-1747

Ryan P. Hobbs, Sandra Y. Han, Paul A. van der Zwaag, Maria C. Bolling, Jan D.H. Jongbloed, Marcel F. Jonkman, Spiro Getsios, Amy S. Paller, Kathleen J. Green,

Autoimmune Bullous Skin Diseases

desmoplakin intermediate filament lethal acantholytic epidermolysis bullosa By linking tension-bearing intermediate filaments (IFs) to sites of robust cell–cell adhesion at desmosomes, desmoplakin (DP) has an essential role in maintaining integrity of tissues that experience mechanical stress such as skin and heart (Green and Simpson, 2007Green K.J. Simpson C.L. Desmosomes: new perspectives on a classic.J Invest Dermatol. 2007; 127: 2499-2515Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Two DP isoforms are present in epithelia, resulting from alternative splicing of one gene. The C-terminal IF-binding domain in each isoform is identical, containing three plakin-repeat domains (A, B, and C), separated by flexible linker regions, and followed by a 68-residue tail involved in regulating DP-IF specificity. These motifs cooperate to anchor IFs to desmosomes (Stappenbeck et al., 1993Stappenbeck T.S. Bornslaeger E.A. Corcoran C.M. et al.Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.J Cell Biol. 1993; 123: 691-705Crossref PubMed Scopus (149) Google Scholar; Meng et al., 1997Meng J.J. Bornslaeger E.A. Green K.J. et al.Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments.J Biol Chem. 1997; 272: 21495-21503Crossref PubMed Scopus (135) Google Scholar; Choi et al., 2002Choi H.J. Park-Snyder S. Pascoe L.T. et al.Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure.Nat Struct Biol. 2002; 9: 612-620PubMed Google Scholar; Fontao et al., 2003Fontao L. Favre B. Riou S. et al.Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus.Mol Biol Cell. 2003; 14: 1978-1992Crossref PubMed Scopus (85) Google Scholar). Over 50 mutations have been identified throughout the desmoplakin gene (DSP) associated with autosomal-dominant or -recessive disorders affecting the skin, heart, hair, and nails (http://www.arvcdatabase.info; Bolling and Jonkman, 2009Bolling M.C. Jonkman M.F. Skin and heart: une liaison dangereuse.Exp Dermatol. 2009; 18: 658-668Crossref PubMed Scopus (45) Google Scholar). Lethal acantholytic epidermolysis bullosa (LAEB) is an autosomal-recessive disease of severe skin and mucosal fragility caused by premature DSP termination. In the first reported LAEB case, the resultant truncated protein lacked the IF-binding domain and consequently lost keratin anchorage at the desmosomal plaque (Jonkman et al., 2005Jonkman M.F. Pasmooij A.M. Pasmans S.G. et al.Loss of desmoplakin tail causes lethal acantholytic epidermolysis bullosa.Am J Hum Genet. 2005; 77: 653-660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The second LAEB case reported a 5-base pair deletion causing frameshift and premature termination in exon 20. No tissue was available for immunofluorescence or biochemical analysis so the effect on protein function is unknown (Bolling et al., 2010Bolling M.C. Veenstra M.J. Jonkman M.F. et al.Lethal acantholytic epidermolysis bullosa due to a novel homozygous deletion in DSP: expanding the phenotype and implications for desmoplakin function in skin and heart.Br J Dermatol. 2010; 162: 1388-1394Crossref PubMed Scopus (35) Google Scholar). Here, we describe a previously unreported DSP mutation resulting in LAEB without apparent cardiac involvement. Biochemical analysis of patient keratinocytes (KCs) sheds light on possible compensatory mechanisms that may allow embryonic survival. A full-term girl, born to consanguineous parents, showed virtually total denudement of skin (Figure 1a) and mucosae, absent fingernails and toenails (Figure 1b), and total alopecia (Figure 1c). Denuded sites re-epithelialized within days without residual scarring, but blistering recurred rapidly. Cardiac echocardiograms and chest X-rays were normal, but the airway filled with extensive sloughed mucosa. Histological sections revealed suprabasal acantholysis or cell clusters in a single acantholytic basal cell layer (arrow, Figure 1d). Immunofluorescence microscopy revealed staining for all basement membrane zone markers, with plectin and cytokeratin staining a discontinuous single row of basal KCs adherent to the basement membrane. DP staining was absent (data not shown). Based on the poor prognosis, the parents withdrew support and the baby expired on day 26 from airway obstruction. Autopsy was refused. After parental institutional review board-approved informed consent in adherence to the Declaration of Helsinki Principles, mutation screening was performed on genomic DNA isolated from peripheral blood. The complete genomic DNA coding sequence of DSP was amplified and directly sequenced as described (Whittock et al., 1999Whittock N.V. Ashton G.H. Dopping-Hepenstal P.J. et al.Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency.J Invest Dermatol. 1999; 113: 940-946Crossref PubMed Scopus (117) Google Scholar). The proband showed a homozygous single nucleotide deletion (DSP:c.7248delT) in exon 24, which was heterozygous in parental genomic DNA (data not shown). Consistent with a C-terminal mutation, an anti-DP C-terminal antibody (NW6), failed to reveal the punctate pattern characteristic of desmosome staining at cell–cell interfaces in cultured patient KCs (Figure 2a). Surprisingly, an N-terminal antibody (NW161) only rarely detected faint DP punctae between cells (Figure 2a). While the desmosomal cadherin, desmoglein-2, was absent from intercellular borders (data not shown), E-cadherin, β-catenin, and plakoglobin staining were better preserved, consistent with a less-pronounced effect on adherens junctions (Figure 2b). DP was undetectable in patient KC lysates, regardless of antibody domain specificity, without reduction of other desmosomal components (Figure 2c). The identified point mutation predicts truncation (p.Phe2416LeufsX14) within plakin-repeat domain B (Figure 1e). Although quantitative real-time RT-PCR analysis indicated reduced DSP transcript levels in patient KCs compared to normal human epidermal KCs, the difference was not statistically significant (Figure 2e). These data support the idea that reduced DP expression in patient KCs is controlled either translationally or through degradation pathways, the latter that was proposed for a recent case of cutaneous disease without evidence of heart defects resulting from virtually complete loss of plakoglobin in the skin (Cabral et al., 2010Cabral R.M. Liu L. Hogan C. et al.Homozygous mutations in the 5′ region of the JUP gene result in cutaneous disease but normal heart development in children.J Invest Dermatol. 2010; 130: 1543-1550Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Interestingly, LAEB KCs exhibit reduced E-cadherin and elevated N-cadherin protein levels compared to multiple normal human epidermal KCs isolates of the same passage number (Figure 2d and Supplementary Figure S1 online). In addition, the extracellular matrix glycoprotein fibronectin was elevated in LAEB lysates (Figure 2d). Furthermore, plakoglobin and, to a lesser extent, β-catenin were observed in the nucleus of LAEB KCs (Figure 2b), where they may initiate transcription events upon the loss of DP expression (Garcia-Gras et al., 2006Garcia-Gras E. Lombardi R. Giocondo M.J. et al.Suppression of canonical Wnt/beta-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy.J Clin Invest. 2006; 116: 2012-2021Crossref PubMed Scopus (459) Google Scholar). We propose that elevated N-cadherin and fibronectin may facilitate re-epithelialization in the patient's skin (Clark, 1990Clark R.A. Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin.J Invest Dermatol. 1990; 94: 128S-134SAbstract Full Text PDF PubMed Google Scholar; Peinado et al., 2004Peinado H. Portillo F. Cano A. Transcriptional regulation of cadherins during development and carcinogenesis.Int J Dev Biol. 2004; 48: 365-375Crossref PubMed Scopus (483) Google Scholar), and further speculate that loss of DP might trigger a "cadherin switch," a process that occurs during epithelial–mesenchymal transition (Nishimura and Takeichi, 2009Nishimura T. Takeichi M. Remodeling of the adherens junctions during morphogenesis.Curr Top Dev Biol. 2009; 89: 33-54Crossref PubMed Scopus (122) Google Scholar), but has not previously been linked with desmosome molecules. Although it was not possible to assess whether similar elevation of N-cadherin occurred in cardiac tissue, compensation by this intercalated disc protein might help counter any potential loss of DP in the heart. Download .pdf (.1 MB) Help with pdf files Supplementary Information A consistent genotype–phenotype correlation linking DSP mutations to disease has not yet emerged. DP truncations and/or point mutations may variably cause cardiac and/or skin syndromes (Supplementary Table S1 online). In the cases of LAEB, severe skin fragility is observed when mutations cause complete loss of DP C-terminal expression. However, less severe cases of skin fragility/woolly hair and palmoplantar keratoderma, with or without cardiomyopathy, have been reported in patients when (1) at least one copy of full-length DP I is expressed, but with a point mutation (Whittock et al., 2002Whittock N.V. Wan H. Morley S.M. et al.Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome.J Invest Dermatol. 2002; 118: 232-238Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar; Alcalai et al., 2003Alcalai R. Metzger S. Rosenheck S. et al.A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair.J Am Coll Cardiol. 2003; 42: 319-327Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar; Mahoney et al., 2010Mahoney M.Y. Sadowski S. Brennan D. et al.Compound heterozygous desmoplakin mutations result in a phenotype with a combination of myocardial, skin, hair, and enamel abnormalities.J Invest Dermatol. 2010; 130: 968-978Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar); (2) DP I expression is lost, but at least one copy of full-length DP II is expressed (Uzumcu et al., 2006Uzumcu A. Norgett E.E. Dindar A. et al.Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome.J Med Genet. 2006; 43: e5Crossref PubMed Google Scholar; Asimaki et al., 2009Asimaki A. Syrris P. Ward D. et al.Unique epidermolytic bullous dermatosis with associated lethal cardiomyopathy related to novel desmoplakin mutations.J Cutan Pathol. 2009; 36: 553-559Crossref PubMed Scopus (21) Google Scholar); or (3) homozygous DP C-terminus truncation occurs downstream of plakin-repeat domain B (Norgett et al., 2000Norgett E.E. Hatsell S.J. Carvajal-Huerta L. et al.Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.Hum Mol Genet. 2000; 9: 2761-2766Crossref PubMed Scopus (608) Google Scholar). Thus, it seems likely that presence of at least one full-length copy of a DP isoform, whether DP I or DP II, may keep patients from developing early lethality as seen in LAEB. In contrast, near-complete loss of DP (described herein) would be predicted to result in severe impairment of both epidermal and cardiac integrity (Gallicano et al., 2001Gallicano G.I. Bauer C. Fuchs E. Rescuing desmoplakin function in extra-embryonic ectoderm reveals the importance of this protein in embryonic heart, neuroepithelium, skin and vasculature.Development. 2001; 128: 929-941PubMed Google Scholar). In utero survival and the lack of observed cardiac involvement in the patient of this study could reflect compensatory mechanisms that stabilized adhesion, re-epithelialization, and cardiac integrity. We thank the patient and her family for participation in this study and greatly appreciate the technical assistance provided by David Quach at Northwestern University. This work was supported by National Institutes of Health grants R01 AR43380 and R01 AR41836 (KJG), the Northwestern Skin Disease Research Center (P30AR057216), and the Vlinderkind Foundation (MJ). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid