Plectin Mutations Underlie Epidermolysis Bullosa Simplex in 8% of Patients

MC Bolling et al. Plectin Mutations in Basal EBS

loss of IL36RN activity is not associated with a significant increase of PV risk. As our study was focused on IL36RN, we cannot exclude the possibility that mutations in functionally related genes (e.g., IL36A, IL36B, and IL36G) may be associated with the disease. Thus, our findings warrant further genetic investigations into the role of IL-36 cytokines in human PV.

1

Division of Genetics and Molecular Medicine, King’s College London, London, UK; 2 Department of Dermatology, University of Glasgow, Glasgow, UK and 3Queen Mary, University of London, Barts and The London School of Medicine and Dentistry, London, UK 4 These authors contributed equally to this work E-mail: [email protected] or [email protected]

SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

CONFLICT OF INTEREST The authors state no conflict of interest.

REFERENCES

ACKNOWLEDGMENTS We acknowledge support from the Department of Health via the NIHR comprehensive Biomedical Research Centre award to GSTT NHS Foundation Trust in partnership with King’s College London and KCH NHS Foundation Trust. This work was supported by the National Psoriasis Foundation, USA (Discovery Grant to FC) and the Medical Research Council (grant G0601387 to RCT and JNB). DB’s PhD studentship is funded by the Psoriasis Association. SKM is supported by the NIHR through the Academic Clinical Fellowship scheme. 1

1

Dorottya M. Berki , Satveer K. Mahil , A. David Burden2, Richard C. Trembath1,3, Catherine H. Smith1, Francesca Capon1,4 and Jonathan N. Barker1,4

Adzhubei IA, Schmidt S, Peshkin L et al. (2010) A method and server for predicting damaging missense mutations. Nat Methods 7:248–9 Blumberg H, Dinh H, Dean C Jr et al. (2010) IL-1RL2 and its ligands contribute to the cytokine network in psoriasis. J Immunol 185:4354–62 Blumberg H, Dinh H, Trueblood ES et al. (2007) Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation. J Exp Med 204:2603–14 Carrier Y, Ma HL, Ramon HE et al. (2011) Interregulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis. J Invest Dermatol 131: 2428–37 Johnston A, Xing X, Guzman AM et al. (2011) IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in

psoriasis and promotes keratinocyte antimicrobial peptide expression. J Immunol 186:2613–22 Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protocols 4:1073–81 Marrakchi S, Guigue P, Renshaw BR et al. (2011) Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis. N Engl J Med 365:620–8 Navarini AA, Valeyrie-Allanore L, Setta-Kaffetzi N et al. (2013) Rare variations in IL36RN in severe adverse drug reactions manifesting as acute generalized exanthematous pustulosis. J Invest Dermatol 133:1904–7 Onoufriadis A, Simpson MA, Pink AE et al. (2011) Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. Am J Hum Genet 89:432–7 Setta-Kaffetzi N, Navarini AA, Patel VM et al. (2013) Rare pathogenic variants in IL36RN underlie a spectrum of psoriasis-associated pustular phenotypes. J Invest Dermatol 133: 1366–9 Strange A, Capon F, Spencer CC et al. (2010) A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 42:985–90 Tortola L, Rosenwald E, Abel B et al. (2012) Psoriasiform dermatitis is driven by IL-36mediated DC-keratinocyte crosstalk. J Clin Invest 122:3965–76

Plectin Mutations Underlie Epidermolysis Bullosa Simplex in 8% of Patients Journal of Investigative Dermatology (2014) 134, 273–276; doi:10.1038/jid.2013.277; published online 18 July 2013

TO THE EDITOR Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis characterized by an intraepidermal split through the cytoplasm of basal keratinocytes, which is mainly caused by dominant-negative mutations in the genes encoding keratins 5 and 14 (Coulombe et al., 1991; Lane et al., 1992). Mutation analysis of KRT5 and KRT14 in a large biopsy-confirmed EBS population in the Netherlands, however, revealed that in 25% of unrelated cases no

mutations could be identified in these genes (Bolling et al., 2011). A similar percentage of EBS cases with wild-type KRT5 and KRT14 genes was reported for the EBS population in the United Kingdom (Rugg et al., 2007). A missense mutation, Arg2000Trp, in PLEC, encoding the hemidesmosomal protein plectin, which connects the basal keratins to the hemidesmosomal plaque, was associated in cases with dominant EBS of hands and feet (EBS-Ogna) (KossHarnes et al., 2002; Kiritsi et al., 2013).

Abbreviation: EBS, epidermolysis bullosa simplex Accepted article preview online 17 June 2013; published online 18 July 2013

In this study, we investigated the frequency of PLEC mutations in biopsyproven EBS probands that lacked mutations in KRT5 and KRT14. The study was performed according to the Declaration of Helsinki principles, and informed consent was obtained from the patients. PLEC mutation analysis was performed in 16 Dutch probands with a biopsyproven EBS in which mutations in KRT5 and KRT14 had been excluded. PCR amplification of all exons and adjacent intronic sequences of PLEC (GenBank NM_000445) was performed using primers located in the flanking introns. Primers resulting in overlapping PCR products were used for the large exons www.jidonline.org

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MC Bolling et al. Plectin Mutations in Basal EBS

Table 1. Characteristics of the PLEC mutations found in this study and associated immunofluorescence microscopy findings in patient skin biopsies Proband

Mutation c.1

Mutation p.2

Inheritance

Domain

Conservation

1 (EB184)

5998C4T (CGG4TGG)

Arg2000Trp

AD

Rod

M, d, zf, fr

HD121 k, 10F6 absent in basal cell layer, and reduced throughout epidermis

2 (EB149)

5998C4T (CGG4TGG)

Arg2000Trp

Sp

Rod

M, d, zf, fr

HD121 k, 10F6 absent in basal cell layer, reduced throughout epidermis

3 (EB229)

5998C4T (CGG4TGG)

Arg2000Trp

AD

Rod

M, d, zf, fr

HD121 k, 10F6 absent in basal cell layer, and reduced throughout epidermis

4 (EB241)

5998C4T (CGG4TGG)

Arg2000Trp

AD

Rod

M, d, zf, fr

HD121 k, 10F6 absent in basal cell layer, reduced throughout epidermis

5 (EB146)

8668A4T (ACGoTCG)

Thr2890Ser

AD

Plakin-repeat domain 1

M, d, zf

HD121Bnormal, 10F6 slightlykpanepidermal

6 (EB203)

10579C4T (CGC4TGC)

Arg3527Cys

AD

Plakin-repeat domain 3

M, d

HD121Bnormal, 10F6 slightlykpanepidermal

Immunofluorescence

Abbreviations: AD, autosomal dominant; d, dog; fr, frog; M, mouse; Sp, sporadic; zf, zebrafish. Numbering according to GenBank NM_000445.3 with one being the adenine from the ATG start codon. 2 GenBank NP_000436.2 (isoform 1c). 1

31 and 32 (information about primers and amplification conditions is available on request). Mutations found were excluded in at least 150 matched healthy control DNA samples. In 6 of the 16 probands (38%), we found three autosomal-dominant PLEC missense mutations (Table 1, Supplementary Files S1 and S2 online). The mutations segregated with the phenotype were not known as single-nucleotide polymorphisms and were absent in control DNA samples, thus strongly indicating pathogenicity. The missense mutations affected wellconserved residues (Supplementary File S2 online). Four unrelated probands (EB184, proband 1; EB149, proband 2; EB229, proband 3; and EB241, proband 4) carried the original Ogna mutation PLEC:c.5998C4T, p.Arg2000Trp. In EB149, the mutation was de novo. In probands 1, 3 and 4, the mutation segregated with the phenotype in an autosomal-dominant fashion in the family. In proband 5, a refugee from Iran, a new heterogygous missense mutation c.8668A4T, p.Thr2890Ser was detected. An affected brother carried the mutation, whereas an unaffected son did not carry the mutation. In proband 6, another new heterozygous missense mutation was detected: c.10597C4T, p.Arg3527Cys. This mutation was found in his affected mother as well. The unaffected siblings and father did not carry the mutation. Both new PLEC 274

mutations are therefore dominant. The clinical features (Figure 1a, Supplementary File S1 online) of seasonal blisters of hands, feet, and lower legs are consistent with previous reports on EBSOgna (Gedde-Dahl, 1971; Kiritsi et al., 2013). Cardiological and neurological examination excluded cardiomyopathy and muscular dystrophy in all probands. Electron microscopic analysis revealed abnormal hemidesmosomes and an intraepidermal split just above the hemidesmosomal plaque (Supplementary File S3b online). Immunofluorescence plectin staining with antibody HD121 in skin samples of probands 1–4 (p.Arg2000Trp) was reduced along the epidermal basement membrane zone, and the faint pericellular epidermal staining was absent (Supplementary File S3 online). In contrast, skin of probands 5 (p.Thr2890Ser) and 6 (p.Arg3527Cys) showed normal HD121 staining. Staining of plectin with 10F6 was absent in the basel cell layer in probands 1–4 or reduced in probands 5 and 6 (Table 1, Figure 1b). Expression of hemidesmosomal components type XVII collagen and integrin b4 was normal in all probands (data not shown). In hemidesmosomes, plectin is an important anchoring protein for basal keratins to integrin b4 (for review see Sonnenberg and Liem (2007)). Plectin dimerizes with its central a-helical

Journal of Investigative Dermatology (2014), Volume 134

coiled-coil rod domain that stabilizes hemidesmosomes (Koster et al., 2004). Disruptive amino acid substitutions in the rod domain, like the Arg4Trp substitution at position 2,000 (large, basic, and positively charged arginine to hydrophobic and neutral tryptophan), interfere with dimer formation and make it more vulnerable to proteolysis (Walko et al., 2011; Winter and Wiche, 2012). Mutations p.Thr2890Ser and p.Arg 3527Cys are located in the plakin-repeat domains PRD1 and PRD3, respectively (Figure 1c). These substitutions predict considerable alterations to protein folding. Both mutations do not directly affect the known intermediate filamentbinding site that resides in PRD5-PRD6 (Steinbock et al., 2000). However, all PRDs are believed to be tightly packed to provide structural rigidity (Janda et al., 2001). In addition, a phosphorylation site and binding sites for several cell signaling proteins have been identified in the plectin C-terminus (Osmanagic-Myers and Wiche, 2004). Threonine residues are often involved in phosphorylation. Alignment of the six PRDs revealed that in some PRDs the residue corresponding to Thr2890 within the 38-amino acid sequence is sometimes a serine (Choi et al., 2002). However, the full segregation of the phenotype with the mutation within the family and the exclusion of the mutation in over 300 healthy control alleles strongly suggest pathogenicity.

MC Bolling et al. Plectin Mutations in Basal EBS

* 10F6 control

10F6 proband 1

10F6 proband 5

Exon nr 2–8

9–30

31

32 CH1-CH2

Actin-binding domain consisting of two calponin homology (CH) domains Globular plakin domain with spectrin repeats

NH2

COOH

CH1-CH2

Central coiled-coil rod domain R2000W

T2890S

R3527C C-terminal plakin repeats with intermediate filament-binding motif

Figure 1. Epidermolysis bullosa simplex due to dominant PLEC mutations. (a) Patients 5 (left) and 6 (right) had hemorrhagic and serous blisters (arrows) on the foot and thickening discolored toenails. (b) Plectin staining with antibody 10F6 of control (left), proband 1 with Ogna mutation (middle), and proband 5 with C-terminal mutation (right) reveals that the Ogna mutation reduces staining, especially in the basal cell layer (arrows), more than the C-terminal mutation does. Bar ¼ 50 mm. (c) Schematic representation of the plectin protein with the mutations depicted below the protein. GenBank accession number NM_000436.3.

Immunofluorescence staining with anti-plectin antibody 10F6 in skin with the Ogna mutation Arg2000Trp shows severe reduction in the basal cell layer staining (Koss-Harnes et al., 1997). The Ogna mutation renders plectin’s 190nm-long coiled-coil rod domain more vulnerable to cleavage by calpains (Walko et al., 2011). In agreement, the new C-terminal mutations in probands 5 and 6 apparently do not affect plectin staining as much as the Ogna mutation. Therefore, normal HD121 antigen mapping does not exclude EBS-plectin. In conclusion, we showed that in six of the 16 (38%) EBS probands with wildtype KRT5 and KRT14 genes (8% of a total EBS population of 78 probands), dominant PLEC missense mutations underlie non-syndromic EBS, making

PLEC the third most candidate gene to screen for mutations in EBS patients. Furthermore, we add two mutations to the known PLEC:p.Arg2000Trp mutation causing EBS.

Medicine, Colleges of Life Sciences and Medicine, Dentistry and Nursing, University of Dundee, Dundee, UK E-mail: [email protected]

SUPPLEMENTARY MATERIAL

CONFLICT OF INTEREST The authors state no conflict of interest.

Marieke C. Bolling1, Jan D.H. Jongbloed2, Ludolf G. Boven2, Gilles F.H. Diercks1, Frances J.D. Smith3, W.H. Irwin McLean3 and Marcel F. Jonkman1 1

Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2 Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands and 3Epithelial Genetics Group, Division of Molecular

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES Bolling MC, Lemming HH, Jansen GH et al. (2011) Mutations in KRT5 and KRT14 cause epidermolysis bullosa simplex in 75% of the patients. Br J Dermatol 164:637–44 Choi HJ, Park-Snyder S, Pascoe LT et al. (2002) Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure. Nat Struc Biol 9:612–20 Coulombe PA, Hutton ME, Letai A et al. (1991) Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66:1301–11

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Y Sawada et al. Decreased Expression of AchE in CUHA

Gedde-Dahl T Jr (1971) Epidermolysis Bullosa: A Clinical, Genetic and Epidemiological Study. The Johns Hopkins University Press: Baltimore, MD Janda L, Damborsky J, Rezniczek GA et al. (2001) Plectin repeats and modules: strategic cysteines and their presumed impact on cytolinker functions. Bioessays 23:1064–9 Kiritsi D, Pigors M, Tantcheva-Poor I et al. (2013) Epidermolysis bullosa simplex Ogna revisited. J Invest Dermatol 133:270–3 Koss-Harnes D, Hoyheim B, Anton-Lamprecht I et al. (2002) A site-specific plectin mutation causes dominant epidermolysis bullosa simplex Ogna: two identical de novo mutations. J Invest Dermatol 118:87–93 Koss-Harnes D, Jahnsen FL, Wiche G et al. (1997) Plectin abnormality in epidermolysis bullosa simplex Ogna: non-responsiveness of basal

keratinocytes to some anti-rat plectin antibodies. Exp Dermatol 6:41–8 Koster J, van Wilpe S, Kuikman I et al. (2004) Role of binding of plectin to the integrin beta4 subunit in the assembly of hemidesmosomes. Mol Biol Cell 15:1211–23 Lane EB, Rugg EL, Navsaria H et al. (1992) A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356:244–6

Sonnenberg A, Liem RK (2007) Plakins in development and disease. Exp Cell Res 313: 2189–203 Steinbock FA, Nikolic B, Coulombe PA et al. (2000) Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin’s intermediate filament-binding domain. J Cell Sci 113:483–91

Osmanagic-Myers S, Wiche G (2004) PlectinRACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity. J Biol Chem 279:18701–10

Walko G, Vukasinovic N, Gross K et al. (2011) Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna. PLoS Genet 7:e1002396

Rugg EL, Horn HM, Smith FJ et al. (2007) Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations. J Invest Dermatol 127:574–80

Winter L, Wiche G (2012) The many faces of plectin and plectinopathies: pathology and mechanisms. Acta Neuropathol 125: 77–93

Decreased Expression of Acetylcholine Esterase in Cholinergic Urticaria with Hypohidrosis or Anhidrosis Journal of Investigative Dermatology (2014) 134, 276–279; doi:10.1038/jid.2013.244; published online 11 July 2013

TO THE EDITOR Cholinergic urticaria (CholU) is a rare condition that is clinically characterized by pinpoint-sized, highly pruritic wheals. CholU is occasionally associated with depressed sweating, as reported under the name of hypohidrosis (incomplete lack of sweating) or anhidrosis (complete lack of sweating) (CholU with hypohidrosis or anhidrosis (CUHA) (Bito et al., 2012)). We have recently shown that CUHA patients develop wheals exclusively on the hypohidrotic area where the expression of cholinergic receptor M3 (CHRM3) is incompletely decreased in sweat gland epithelial cells and mast cells, whereas the patients did not exhibit wheals in the anhidrotic area where CHRM3 expression is completely absent (Sawada et al., 2010). Acetylcholine esterase (AchE) is a processing enzyme of acetylcholine, and disordered AchE might contribute to the pathogenesis of CholU (Magnus and Thompson, 1956). However, the mechanism underlying the reduction of AchE release in CholU remains

unelucidated. Moreover, there have been a considerable number of reports demonstrating that T lymphocytes infiltrate around eccrine glands in CholU patients and that the systemic corticosteroid therapy improves sweating, as well as urticaria (Nakazato et al., 2004). We therefore investigated the eccrine gland expression of AchE and CHRM3 and characterized skininfiltrating lymphocytes/mast cells and attracting chemokines. Enrolled in this study were nine CUHA patients, three CholU patients, and five healthy controls (Table 1). CUHA and CholU were diagnosed on the basis of typical episodes of small pinpoint wheals following exercise and sweating (Black et al., 1996), and were confirmed by our provocation test showing the development of numerous small wheals after exercise. Sweating was evaluated with starch-iodine test. The study design was approved by the review board of University of Occupational and Environmental Health and was conducted according to the

Abbreviations: AchE, acetylcholine esterase; AD, atopic dermatitis; CHRM3, cholinergic receptor M3; CholU, cholinergic urticaria; CUHA, cholinergic urticaria with hypohidrosis or anhidrosis; RD, red density Accepted article preview online 7 June 2013; published online 11 July 2013

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Declaration of Helsinki guidelines. Measurements in this study were performed after written informed consent had been obtained from these patients. Skin biopsy specimens were taken from the patients and healthy control subjects. Significantly higher numbers of lymphocytes (Figure 1a) and mast cells (Figure 1b) infiltrated around eccrine glands in CUHA patients than in controls and CholU patients. The lymphocytes consisted of a mixture of CD4 þ and CD8 þ T cells and a mixture of CXCR3 þ (mostly Th1 cells) and CCR4 þ T cells (mostly Th2 cells) in all cases. The deparaffinized sec tions were immunohistochemically stained with anti-AchE, anti-CHRM3, anti-CXCL9/MIG, anti-CXCR10/IP-10, antiCXCL11/I-TAC, anti-CCL2/MCP-1, antiCCL3/MIP-1a, anti-CCL5/RANTES, anti-CCL17/TARC, and anti-CCL22/ MDC antibodies. Digitalized specimens were exported to JPG files by the NDP view software (Hamamatsu Photonics, Hamamatsu, Japan), and the staining intensity was expressed as ‘‘red density’’ (RD) (Hino et al., 2010; Sawada et al., 2010). The expression levels of CCL2/ MCP-1, CCL5/RANTES, and CCL17/ TARC were highest in CUHA and