Insights from a Desmoplakin Mutation Identified in 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, 2007). 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., 1993; Meng et al., 1997; Choi et al., 2002; Fontao et al., 2003).

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, 2009). 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., 2005). 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., 2010). 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.

Figure 1. Clinical features of lethal acantholytic epidermolysis bullosa (LAEB) and desmoplakin (DP) schematic highlighting sites of mutations.

At birth, patient had (a) virtually total skin denudement with underlying erythema, (b) absent toenails, and (c) total alopecia. (d) Hematoxylin and eosin (H&E) sections of a skin biopsy show only a single, discontinuous row of basal keratinocytes (KCs; arrows). (e) Desmoplakin schematic showing gene exons, protein domains, and protein sites of homozygous or compound heterozygous mutations identified from patients with either LAEB (p.I958LfsX5 (Bolling et al., 2010); p.R1934X/p.L203GfsX28 (Jonkman et al., 2005)), cardiocutaneous disorders similar to Carvajal/Naxos syndrome (p.K2542SfsX18 (Norgett et al., 2000); p.R1267X (Uzumcu et al., 2006); p.Q673X/Q1446X (Asimaki et al., 2009); p.G2375R (Alcalai et al., 2003); p.T2104QfsX11/p.A2655D (Mahoney et al., 2010)), or skin fragility/wooly hair syndrome (p.C809X/N287K, p.Q664X/R2366C (Whittock et al., 2002)). Bar = 20 μm.

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., 1999). 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, b-catenin, and plakoglobin staining were better preserved, consistent with a less-pronounced effect on adherens junctions (Figure 2b).

Figure 2. Immunostaining and biochemical analysis of lethal acantholytic epidermolysis bullosa (LAEB) keratinocytes (KCs).

KCs immunostained with antibodies targeting (a) desmoplakin (DP) N terminus (NW161) or C terminus (NW6), (b) E-cadherin (HECD-1), b-catenin (C2206), or plakoglobin (1407) (bar = 30 μm). Lysates were separated on 7.5% SDS-PAGE gel, transferred to nitrocellulose, and probed using antibodies targeting (c) DP N terminus, DP C terminus, DP I-specific (DP2.17), plakoglobin, or desmoglein-2 (4B2), (d) and E-cadherin, N-cadherin (Invitrogen, Carlsbad, CA; cat. no. 33-3900), or fibronectin (Sigma, St Louis, MO; cat. no. F3648). Tubulin (12G10) used as loading control. Primary antibodies listed in parenthesis. (e) Quantitation of DP mRNA levels based on quantitative real-time RT-PCR analysis from three independent experiments in triplicate. DP primers used were the following: forward 5′-ACCAGAACCAGAACACCATC-3′ and reverse 5′-GGGCAAAACACTCATCCAATTC-3′. Error bars derived from standard deviation of C_t values. P-value determined by unpaired Student's t-test.

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 RTPCR 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., 2010).

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., 2006). We propose that elevated N-cadherin and fibronectin may facilitate re-epithelialization in the patient's skin (Clark, 1990; Peinado et al., 2004), and further speculate that loss of DP might trigger a “cadherin switch,” a process that occurs during epithelial–mesenchymal transition (Nishimura and Takeichi, 2009), 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.

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., 2002; Alcalai et al., 2003; Mahoney et al., 2010); (2) DP I expression is lost, but at least one copy of full-length DP II is expressed (Uzumcu et al., 2006; Asimaki et al., 2009); or (3) homozygous DP C-terminus truncation occurs downstream of plakin-repeat domain B (Norgett et al., 2000). 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., 2001). 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.

Abbreviations

DP

desmoplakin

IF

intermediate filament

LAEB

lethal acantholytic epidermolysis bullosa