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provided by Elsevier - Publisher Connector Homozygous Splice Site Mutations in PKP1 Result in Loss of Epidermal 1 Expression and Underlie / in Two Consanguineous Families

Eli Sprecher,à Vered Molho-Pessach,w Arieh Ingber,w Efraim Sagi,w Margarita Indelman,à and Reuven Bergmanà ÃDepartment of and Laboratory of Molecular Dermatology, Rambam Medical Center, Haifa, Israel; wDepartment of Dermatology, Hadassah University Hospital and Faculty of Medicine, Hebrew University, Jerusalem, Israel

During the last years, a growing number of inherited skin disorders have been recognized to be caused by abnormal function of desmosomal proteins. In the present study, we describe the first female individuals affected with the ectodermal dysplasia/skin fragility syndrome (MIM604536), a rare autosomal recessive disease due to mutations in the PKP1 gene encoding plakophilin 1, a critical component of desmosomal plaque. One patient was shown to carry a homozygous splice site mutation in intron 4. The second patient displayed a homozygous recurrent mutation affecting the acceptor splice site of intron 1. Both mutations were associated with intraepidermal separation, widening of intercellular spaces, and abnormal ultrastructure, and were found to result in the absence of immunoreactive plakophilin 1 in the epidermis of the affected individuals. These two cases emphasize the role of molecular genetics in the assessment of congenital blistering in newborns and illustrate the importance of proper desmosomal activity for normal epidermis development and function. Key words: alopecia/blistering///plakophilin J Invest Dermatol 122:647 –651, 2004

Congenital blistering can pose a serious diagnostic chal- skin syndrome and epidermolysis bullosa, and who were lenge to the pediatric dermatologist (Schachner and Press, eventually shown to carry homozygous mutations in PKP1 1983). Among blistering genodermatoses, epidermolysis resulting in lack of plakophilin 1 expression and underlying bullosa a heterogeneous group of inherited skin diseases EDSF. These cases emphasize the complexity of the ranks first in terms of prevalence (Fine et al, 1999). A differential diagnosis of congenital bullous eruptions in number of additional inherited vesiculobullous disorders, newborns and illustrate the power of molecular genetic however, have been recognized to be part of this expanding analysis in the assessment of this group of disorders. spectrum of diseases, including epidermolytic hyperkera- tosis (MIM113800),1 (MIM173650; Kind- ler, 1954), and ectodermal dysplasia/skin fragility (EDSF) Results syndrome (MIM604536; McGrath et al, 1997). EDSF, an autosomal recessive disorder, manifests with Clinical description Family 1 comprised a 6-mo-old skin fragility, , abnormal hair affected female child who was the offspring of healthy first growth, and nail dystrophy and, in some but not all cases, cousins of Arab Moslem origin. She had a healthy brother. with defective sweating (Whittock et al, 2000). Since its She was delivered vaginally after 37 wk of an uneventful initial description in 1997, the syndrome has been shown in pregnancy. A few hours after birth, superficial blisters were four male individuals to result from mutations in the PKP1 noticed on several parts of her body, which suggested the gene encoding plakophilin 1 (PKP1), a critical component diagnosis of staphylococcal scalded skin syndrome. The of the desmosomal plaque (McGrath et al, 1997, 1999; child’s general behavior was normal. She had no fever. She Whittock et al, 2000; Hamada et al, 2002). received parenteral ampicillin and gentamicin without In the present report, we describe two children initially improvement for 4 d. Cultures obtained from several sites suspected of being affected with staphylococcal scalded remained sterile. The child was referred to our outpatient clinics with a clinical diagnosis of epidermolysis bullosa at 10 d of age. On physical examination, numerous erosions Abbreviation: EDSF, Ectodermal dysplasia/skin fragility were evident on the occipital aspect of the scalp (Fig 1a), on 1Klein I, Bergman R, Indelman M, Sprecher E: A newborn the face in a perioral distribution (Fig 1b), and on the presenting with congenital blistering. Int J Dermatol, in press. extensor parts of the limbs. No mucosal lesions or

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 647 648 SPRECHER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 1 Clinical features of ESDF. (a) Sparse, short, and coarse hair on the scalp with numerous skin superficial erosions (family 1 patient). (b) Perioral superficial erosions (family 1 patient). (c) Toenail plate thickening and yellowish discoloration as well as superficial erosions over foot dorsum (family 2 patient). (d) Suprabasal and intraepidermal clefts with a few acantholytic keratinocytes in a skin biopsy obtained from the family 1 patient. The adjacent spinous zone demonstrates widened intercellular spaces (hematoxylin–eosin, original scale bar ¼ 100 mm). (e) Electron micrograph of a skin biopsy obtained from the family 1 patient showing widened intercellular spaces between keratinocytes. Keratinocytes show perinuclear retraction of tonofilaments (scale bar ¼ 2 mm). Few rudimentary without attached tonofilaments are seen between keratinocytes (insert, scale bar ¼ 0.3 mm). palmoplantar involvement were observed and there was normal. Chest computed tomography disclosed a right no evidence of abnormal sweating. The child, however, aortic arch. Examination of a skin biopsy revealed supra- displayed prominent nail dystrophy and severe hypotricho- basal intraepidermal separation with many epidermal cells sis (Fig 1a). The remaining hairs were short, coarse, and curly. with increased eosinophilic cytoplasm as in dyskeratotic Eyebrows and eyelashes were present. Severe pruritus was keratinocytes. Electron microscopic examination of a skin reported by the parents and evident during physical biopsy revealed intraepidermal skin separation and acan- examination. Routine laboratory tests were normal. Echo- tholysis (not shown). cardiography revealed patent foramen ovale with mild left to right shunt. Histopathological examination of a skin biopsy Mutational analysis Since many of the clinical findings stained with hematoxylin–eosin revealed widened intercel- displayed by both patients had previously been reported in lular spaces and suprabasal intraepidermal clefts with association with EDSF (McGrath et al, 1997), we sequenced acantholytic cells (Fig 1d). Electron microscopic examina- the entire coding region of the PKP1 gene including intron– tion of a lesional skin biopsy disclosed, mainly in the exon boundaries. The affected child of family 1 was shown spinous layers, a widening of intercellular spaces with a to carry a homozygous A ! G transition in intron 4 at reduced number of small and often distorted desmosomes, nucleotide position 847-2 (accession number NM_000299) not attached to tonofilaments. Normal-appearing desmo- (Fig 2a). The mutation, termed 847-2A4G, affects the somes were usually not observed. (Fig 1e). Light micro- consensus acceptor splice site of intron 4 and is expected scopic examination of plucked hair was unremarkable. to lead to aberrant splicing. The mutation was shown to be The second patient was a 3-y-old female child belonging carried in a heterozygous state by both parents and to be to a consanguineous family of Arab Moslem origin. Her absent in the patient’s unaffected brother. The family 2 parents were unaware of any familial relationship to family 1. patient displayed a homozygous G ! A transition at nucle- The child presented clinical features strikingly similar to otide position 203-1 (Fig 2a). The 203-1G4A mutation those displayed by the first patient. Two brothers with modifies the consensus acceptor splice site of intron 1 and congenital skin generalized erythema and blistering died at is predicted to result in abnormal splicing too. The mutation the age of 2 wk of sepsis. She was born after an uneventful was shown to be carried in a heterozygous state by both pregnancy. Soon after birth, general erythroderma and skin parents. This mutation has previously been described in an blisters were observed. Nail dystrophy was noted (Fig 1c). EDSF patient of Anglo-Saxon origin (McGrath et al, 1999) She was discharged with a diagnosis of epidermolysis and represents the first recurrent mutation in PKP1 bullosa. Her disease course was initially complicated by described to date. recurrent episodes of pneumonia and sepsis as well as 847-2A4G generates a novel restriction site for FauI marked failure to thrive. During the first year of life, the while 203-1G4A generates a novel recognition site for erythroderma receded and skin blisters occurred less DdeI. Segregation of the mutation in both families was frequently. Itching became prominent and palmoplantar confirmed by PCR-RFLP (Fig 2b, c). The 847-2A4G keratoderma progressively developed. On physical exam- mutation was absent from a panel of 100 control chromo- ination, she displayed skin blisters and erosions over the somes. feet (Fig 1c), the limbs, and around the mouth; sparse, coarse scalp hair; palmoplantar ; and yellow- Consequences of splice site mutations in PKP1 In silico ish and thickened nails (Fig 1c). Routine blood tests were simulation using the Splice Site Prediction by Neural 122 : 3 MARCH 2004 PKP1 NOVEL MUTATIONS 649

Figure 2 Molecular analysis. (a) Mutation identification. Sequence analysis reveals a homozygous splice site mutation (847-2A4G) in intron 4 in the affected individual of family 1, and a homozygous splice site mutation (203-1G4A) in intron 1 in the affected individual of family 2 (arrow, upper panels). The wild-type sequences are given for comparison (lower panels). (b) Verification of 847-2A4G. 847-2A4G creates a novel recognition site for FauI endonuclease. A 527 bp fragment encompassing exon 5 was amplified as previously described (Whittock et al, 2000) and digested with FauI. The affected patient displays a 367 bp fragment that is not shown by her unaffected brother, but is carried in a heterozygous state by both her parents. (c) Verification of 203-1G4A. 203-1G4A creates a novel recognition site for DdeI endonuclease. A 374 bp fragment encompassing exon 2 was amplified as previously described (Whittock et al, 2000) and digested with DdeI. The affected patient displays a novel 237 bp fragment while her heterozygous parents carried both the 237 bp fragment and a wild-type 298 bp fragment. Additional fragments of length 22 bp, 54 bp, and 61 bp are not visible. (d) Effect of 847-2A4GonPKP1 RNA levels. Total RNA was extracted from skin biopsies obtained from family 1 patient (P) or from an unrelated control individual (C) and amplified using primer pairs specific for PKP1 or b-. Patient-derived PKP1 cDNA was less abundant than control-derived PKP1 cDNA. (e) Immunofluorescence study. Staining of skin biopsies demonstrates the absence of immunoreactive PKP1 protein in the skin of the family 1 patient (/) as opposed to normal membranal staining in the epidermis of a healthy unrelated control individual ( þ / þ ). (f) Spectrum of mutations in PKP1. Mutations are indicated along a schematic representation of the plakophilin 1 molecule. Armadillo repeats are represented in orange, and the head domain is depicted in blue.

Network software (http://www.fruitfly.org/seq_tools/splice. PCR product revealed the presence of an additional 26 bp html) revealed a number of possible consequences of the within the mutant cDNA due to the activation of a cryptic 847-2A4G mutation, including exon skipping, integration acceptor splice site located at nucleotide position 847-28 of intron 4 within the mature mRNA, and activation of a within intron 4. As a consequence, a premature termination number of potential cryptic splicing sites. In each case, the codon is introduced 169 bases downstream to the normal resulting mRNA is predicted to carry a premature termina- intron 4 acceptor splice site. RNA was not available from the tion codon, which may either lead to the synthesis of a family 2 patient. A previous study, however, showed that truncated protein or to nonsense-mediated mRNA decay this mutation is likely to result in premature termination of (Frischmeyer and Dietz, 1999). In order to explore these protein translation too (McGrath et al, 1999). different possibilities, we extracted RNA from a skin biopsy To correlate these results at the protein level, we stained obtained from non-lesional skin of the family 1 patient and frozen biopsy sections of both patients with a monoclonal amplified a fragment of the cDNA encompassing exon 5 as antibody directed against PKP1. No immunoreactive PKP1 described in Materials and Methods using primers specific protein could be detected by immunofluorescence in the for PKP1 and b-actin. Patient PKP1 cDNA was less skin of the two EDSF patients as compared with normal skin abundant and of a slightly higher size as compared with (Fig 2e). Thus, the 847-2A4G and 203-1G4A mutations control PKP1 cDNA (Fig 2d). Sequencing of the patient RT- result in plakophilin 1 deficiency. 650 SPRECHER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Discussion thickening associated with significant loss of barrier activity and chronic dermatitis (Chidgey et al, 2001). Thus, the effect A total of four sporadic cases of EDSF have been reported of PKP1 deficiency on epidermal barrier function deserves to date (McGrath et al, 1997, 1999; Whittock et al, 2000; further scrutiny. Hamada et al, 2002). All patients demonstrated skin fragility, EDSF is one of a number of desmosomal genoderma- nail dystrophy, palmoplantar keratoderma, and alopecia, toses described during recent years and which include and all were shown to carry mutations predicted to result in dominantly (e.g., striate palmoplantar keratoderma) as well premature termination codons (Fig 2f). The present cases, as recessively (e.g., Naxos disease) inherited disorders the first female patients with EDSF ever described, perfectly (reviewed in McMillan and Shimizu, 2001). Although fit the general paradigm of EDSF except for the absence of diseases caused by recessive mutations in desmosomal palmoplantar keratoderma and skin thickening in the family proteins share numerous characteristics such as kerato- 1 patient. Since this child is an 8-mo-old only, we cannot derma and alopecia (McGrath et al, 1997; McKoy et al, exclude the possibility that keratoderma will develop in the 2000; Norgett et al, 2000), EDSF is the only one including future, especially once the child becomes more active. skin blistering/fragility as a prominent feature. In contrast, Thus, given the clinical similarities between the present cardiac manifestations are a hallmark of recessive muta- cases and the previously described Japanese and Eur- tions in genes coding for at least two other desmosomal opean patients, it seems that neither sex nor ethnic origin proteins, and (McKoy et al, 2000; significantly influence the phenotypic expression of muta- Norgett et al, 2000; Whittock et al, 2002). Although a patent tions in the PKP1 gene. 203-1G4A is the first reported foramen ovale and a right aortic arch were noticed in our recurrent mutation in PKP1. The 203-1 nucleotide position is patients, no major cardiovascular abnormality was identi- likely to represent a mutational hot spot, since 203-1G4A fied, probably reflecting the tissue-specific pattern of was initially described in a compound heterozygous state in expression of PKP1 (Moll et al, 1997). a child of Anglo-Saxon descent whereas the present case is In summary, we have identified a novel and a recurrent of Arab Moslem origin. mutation in PKP1 underlying EDSF in two female patients. The PKP1 gene codes for two alternatively spliced PKP1 These two cases illustrate the etiological diversity asso- variants: the short variant, PKP1a, is a major component of ciated with congenital skin blistering, emphasize the role the desmosomal plaque in all stratified epithelia, while the of molecular genetics in the assessment of skin fragility long variant, PKP1b, is exclusively found in the nucleus syndromes in children, and demonstrate once again the where it may play a role in signal transduction (Schmidt importance of proper desmosomal activity for epidermis et al, 1997). Keratin intermediate filaments binding to desmo- normal development and function. somal plaque, at least in lower suprabasal epidermal cells, is critically dependent on normal PKP1 function (McGrath et al, 1997). Recent data suggest that the N-terminal Materials and Methods domain of PKP1 stabilizes lateral interactions between Patients and biological materials All participants or their legal desmoplakin molecules, which in turn bind to keratin guardian provided written and informed consent according to a molecules (Kowalczyk et al, 1999). This may account for protocol previously approved by the local Helsinki Committee and recent results demonstrating that PKP1 stabilizes a number by the Committee for Genetic Studies of the Israeli Ministry of of critical components of desmosomal plaque, plays a role Health. Blood samples were drawn from all family members and in the transition of desmosomes from a calcium-dependent DNA was extracted according to standard procedures. Skin biopsies were obtained from the patients and processed for to a calcium-independent state, and regulates keratinocyte histopathology and electron microscopy as previously described motility (South et al, 2003). The absence of PKP1 expres- (Bergman et al, 1997). sion in EDSF, as shown in our two patients, may thus explain abnormal desmosome assembly and aberrant cell– Mutation analysis Genomic DNA of affected individuals was cell adhesion within the epidermis leading to epidermal PCR-amplified using PKP1 specific primer pairs (Whittock et al, acantholysis (Fig 1e). Normal desmosomal function also 2000) with Taq polymerase and Q solution (Qiagen, Valencia, California). Cycling conditions were 951C 5 min followed by 35 seems to be required during hair morphogenesis since cycles at 951C30s,571C–601C45s,721C 90 s, and a final mutations in two desmosomal proteins, desmoglein 4 (Kljuic extension step at 721C for 7 min. Resulting amplicons were gel- et al, 2003) and corneodesmosin (Levy-Nissenbaum et al, purified (QIAquick gel extraction kit, Qiagen) and subjected to 2003), underlie recessive and dominant hypotrichosis bidirectional DNA sequencing using the BigDye terminator system simplex. Thus, alopecia in EDSF may result from impaired on an ABI Prism 3100 sequencer (PE Applied Biosystems, Foster desmosomal function as well. City, California). Severe itching was noticed in our patients and pruritus Reverse-transcription polymerase chain reaction Total RNA was also reported in a previous report of an EDSF patient was extracted from a snap-frozen skin biopsy obtained from non- (Whitock et al, 2000). Pruritus is strongly associated with lesional skin using the RNeasy extraction kit (Qiagen) and amplified disruption of epidermal barrier integrity. Although no clear using the TITAN One Tube RT-PCR kit (Roche Molecular pathomechanistic relationship has been established be- Biochemicals, Mannheim, Germany) and intron-crossing primer tween desmosome dysfunction and pruritus, it is of note pairs specific for PKP1 located in exons 3 and 7, respectively (forward 50-GCGCTTCAGCTCCTACAGCC-30 and reverse 50- that engineered mice, lacking desmocollin 1, a critical com- CGCTACACAGTTCTGGACATAG-30). PCR conditions were: 30 ponent of desmosomal plaque, display a phenotype with min at 501C; 941C for 2 min; 941C for 30 s, 601C for 30 s and striking similarities to EDSF, including skin fragility with 681C for 90 s, for a total of 10 cycles; 941C for 30 s, 601C for 30 s evidence for acantholysis, hair abnormalities, and epidermal and 681C for 90 s þ five additional seconds at each cycle, for a 122 : 3 MARCH 2004 PKP1 NOVEL MUTATIONS 651 total of 25 cycles; a final extension step at 681C for 7 min. The Chidgey M, Brakebusch C, Gustafsson E, et al: Mice lacking desmocollin 1 show resulting amplicons were then amplified using Taq polymerase, Q epidermal fragility accompanied by barrier defects and abnormal solution (Qiagen), and nested primers located in exons 3 and 7, differentiation. J Cell Biol 155:821–832, 2001 respectively (forward 50-CGGCGCAGGCAGCGACATCTG-30 and Fine JD, Johnson LB, Suchindran C, Moshell A, Gedde-Dahl T: The epidemiology reverse 50-CATGAGGGAATCAATGAGC-30; expected size 889 bp). of inherited epidermolysis bullosa. In: Fine JD, Bauer E, McGuire J, Cycling conditions were 951C 5 min followed by 35 cycles at 951C Moshell A (eds). Epidermolysis Bullosa. Baltimore, MA: John Hopkins 30 s, 601C45s,721C 90 s, and a final extension step at 721C for University Press, 1999; p 101–113 Frischmeyer PA, Dietz HC: Nonsense-mediated mRNA decay in health and 7 min. The PCR products were gel-purified (QIAquick gel extraction disease. Hum Mol Genet 8:1893–1900, 1999 kit, Qiagen) and subjected to bidirectional DNA sequencing using Hamada T, South AP, Mitsuhashi Y, et al: Genotype–phenotype correlation in skin the BigDye terminator system on an ABI Prism 377 sequencer (PE fragility-ectodermal dysplasia syndrome resulting from mutations in Applied Biosystems). As a control, we amplified as described plakophilin 1. Exp Dermatol 11:107–114, 2002 above total RNA with primers specific for b-actin (forward 50- Kindler T: Congenital with traumatic bulla formation and progressive CCAAGGCCAACCGCGAGAAGATGAC-30 and reverse 50-AGGG- cutaneous atrophy. Br J Dermatol 66:104–111, 1954 TACATGGTGGTGCCGCCAGAC-30; expected size 587 bp). Kljuic A, Bazzi H, Sundberg JP, et al: Desmoglein 4 in hair follicle differentiation and epidermal adhesion: Evidence from inherited hypotrichosis and acquired pemphigus vulgaris. Cell 113:249–260, 2003 Kowalczyk AP, Hatzfeld M, Bornslaeger EA, et al: The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to Immunofluorescence studies Skin biopsies were snap-frozen in desmosomes. Implications for cutaneous disease. J Biol Chem liquid nitrogen, and 6 mm cryostat sections were mounted on 274:18145–18148, 1999 polylysine-coated slides and air-dried. Slides were incubated with Levy-Nissenbaum E, Betz RC, Frydman M, et al: Hypotrichosis simplex of the a primary mouse anti-plakophilin 1 monoclonal antibody (clone scalp is associated with nonsense mutations in CDSN encoding 10B2; Zymed, San Francisco, California) for 1 h at room tempera- corneodesmosin. Nat Genet 34:151–153, 2003 ture and with a secondary FITC-conjugated goat anti-mouse IgG McGrath JA, Hoeger PH, Christiano AM, et al: Skin fragility and hypohidrotic antibody (Zymed) for 30 min. The sections were then examined ectodermal dysplasia resulting from ablation of plakophilin 1. Br J under an Axioscop2 upright microscope (Zeiss, Thornwood, New Dermatol 140:297–307, 1999 McGrath JA, McMillan JR, Shemanko CS, et al: Mutations in the plakophilin 1 York) and images were processed using Image Pro (Media þ gene result in ectodermal dysplasia/skin fragility syndrome. Nat Genet Cybernetics, Silverspring, Maryland). 17:240–244, 1997 McKoy G, Protonotarios N, Crosby A, et al: Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with We are grateful to the family members for their participation in our palmoplantar keratoderma and woolly hair (Naxos disease). Lancet study. We wish to thank Dr Vered Friedman for services in DNA 355:2119–2124, 2000 sequencing and Dr Ofer Shenkar for his help with the immunofluores- McMillan JR, Shimizu H: Desmosomes: Structure and function in normal and cence studies. This study was supported in part by a grant provided by diseased epidermis. J Dermatol 28:291–298, 2001 the Bureau for Economic Growth, Agriculture, and Trade, Office of Moll I, Kurzen H, Langbein L, Franke WW: The distribution of the desmosomal Economic Growth and Agricultural Development, US Agency for protein, plakophilin 1, in human skin and skin tumors. J Invest Dermatol International Development, under the terms of Award No. TA-MOU- 108:139–146, 1997 01-M21-023. Norgett EE, Hatsell SJ, Carvajal-Huerta L, et al: Recessive mutation in desmoplakin disrupts desmoplakin–intermediate filament interactions DOI: 10.1111/j.0022-202X.2004.22335.x and causes , woolly hair and keratoderma. Hum Mol Genet 9:2761–2766, 2000 Manuscript received August 12, 2003; revised September 30, 2003; Schachner L, Press S: Vesicular, bullous and pustular disorders in infancy and accepted for publication October 13, 2003 childhood. Pediatr Clin North Am 30:609–629, 1983 Schmidt A, Langbein L, Rode M, et al: 1a and 1b: Widespread Address correspondence to: Eli Sprecher, MD PhD, Department of nuclear proteins recruited in specific epithelial cells as desmosomal Dermatology and Laboratory of Molecular Dermatology, Rambam plaque components. Cell Tissue Res 290:481–499, 1997 Medical Center, Haifa, Israel. Email: [email protected] South AP, Wan H, Stone MG, et al: Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability. J Cell Sci 116:3303–3314, 2003 Whittock NV, Haftek M, Angoulvant N, et al: Genomic amplification of the human References plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia/skin fragility syndrome. J Invest Dermatol 115:368–374, 2000 Bergman R, David R, Ramon Y, et al: Delayed postburn blisters: An Whittock NV, Wan H, Morley SM, et al: Compound heterozygosity for non-sense immunohistochemical and ultrastructural study. J Cutan Pathol 24: and mis-sense mutations in desmoplakin underlies skin fragility/woolly 429–433, 1997 hair syndrome. J Invest Dermatol 118:232–238, 2002