A homozygous nonsense mutation in the PLEC1 gene in patients with epidermolysis bullosa simplex with muscular dystrophy. S Chavanas, … , J P Ortonne, G Meneguzzi J Clin Invest. 1996;98(10):2196-2200. https://doi.org/10.1172/JCI119028. Research Article Plectin is a widely expressed cytomatrix component involved in the attachment of the cytoskeleton to the plasma membrane. We have recently reported that the skin and muscles of three patients affected by epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), a genetic disorder characterized by skin blistering associated with muscle involvement, are not reactive with antibodies specific to plectin. We demonstrated that in the skin, lack of plectin leads to failure of keratin filaments to connect to the plasma membrane via the hemidesmosomes, whereas in the muscle the deficient expression of the molecule correlates with an aberrant localization of desmin in the muscle fibers. In this study we demonstrate that in a MD-EBS kindred with two affected members, the disease results from a homozygous nonsense mutation in the plectin (PLEC1) gene leading to a premature stop codon (CGA to TGA) and decay of the aberrant plectin messenger RNA. The segregation of the mutated allele implicates the mutation in the pathology of the disorder. These results confirm the critical role of plectin in providing cell resistance to mechanical stresses both in the skin and the muscle. Find the latest version: https://jci.me/119028/pdf Rapid Publication A Homozygous Nonsense Mutation in the PLEC1 Gene in Patients with Epidermolysis Bullosa Simplex with Muscular Dystrophy Stéphane Chavanas,* Leena Pulkkinen,§ Yannick Gache,* Frances J.D. Smith,¶ W.H. Irwin McLean,¶ Jouni Uitto,§ Jean Paul Ortonne,*‡ and Guerrino Meneguzzi* *U385 INSERM, Faculté de Médecine, 06107 Nice Cedex 2; ‡Service de Dermatologie, Hôpital Pasteur, 06002 Nice Cedex 1, France; §Departments of Dermatology, Cutaneous Biology, and Biochemistry and Molecular Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107; and ¶CRC Cell Structure Research group, University of Dundee, Dundee, United Kingdom Abstract blisters of the skin. In the majority of cases of EBS, the skin fragility is caused by mutations in keratins 5 and 14 that com- Plectin is a widely expressed cytomatrix component in- promise the function of the intermediate filament cytoskeleton volved in the attachment of the cytoskeleton to the plasma in resisting shear stresses (1–3). We have recently studied a re- membrane. We have recently reported that the skin and cessively inherited form of epidermolysis bullosa simplex asso- muscles of three patients affected by epidermolysis bullosa ciated with late-onset muscular dystrophy (MD-EBS) (4). This simplex with muscular dystrophy (MD-EBS), a genetic dis- disease is characterized by blistering of the skin as the primary order characterized by skin blistering associated with mus- clinical manifestation at birth. Nail dystrophy and muscle cle involvement, are not reactive with antibodies specific to weakness progressively leading to disabling muscular atrophy plectin. We demonstrated that in the skin, lack of plectin and ptosis are other features observed in MD-EBS (5–7). In leads to failure of keratin filaments to connect to the plasma the patients affected by MD-EBS, skin blisters arise by a low membrane via the hemidesmosomes, whereas in the muscle level severing of the basal keratinocytes. The separation be- the deficient expression of the molecule correlates with an tween dermis and epidermis occurs just above the hemidesmo- aberrant localization of desmin in the muscle fibers. In this somes that are dissociated from the keratin filaments and re- study we demonstrate that in a MD-EBS kindred with two main attached to the basal lamina of the dermal-epidermal affected members, the disease results from a homozygous junction at the floor of the blister. This observation suggests a nonsense mutation in the plectin (PLEC1) gene leading to a defective anchorage of the cytoskeletal structures to the inner premature stop codon (CGA to TGA) and decay of the ab- plaque of the hemidesmosome. In accordance with this possi- errant plectin messenger RNA. The segregation of the mu- bility, immunohistochemical analysis of the skin and muscle of tated allele implicates the mutation in the pathology of the our MD-EBS patients demonstrated negative staining with disorder. These results confirm the critical role of plectin in monoclonal antibodies directed against plectin and the hemi- providing cell resistance to mechanical stresses both in the desmosomal protein HD1, whereas all the other proteins of skin and the muscle. (J. Clin. Invest. 1996. 98:2196–2200.) the dermal-epidermal junction tested were reactive (4). Key words: plectin • cytoskeleton • epidermolysis bullosa • Plectin is a widely expressed cytomatrix component in- muscular dystrophy • hemidesmosome volved in the attachment of the cytoskeleton to the plasma cell membrane (8). This high molecular weight protein interacts Introduction with a variety of intermediate filament proteins, fodrin, ␣-spec- trin, microtubule-associated proteins, and nuclear lamins (9). Epidermolysis bullosa simplex (EBS)1 comprises a group of in- In the skeletal muscle, plectin is found in the sarcolemma and herited disorders of the epidermis characterized by a marked in the Z-line structures, and colocalizes with the intermediate fragility of basal keratinocytes resulting in the development of filament protein desmin (10). In the skin it localizes both in the desmosomes and the hemidesmosomes where it associates with cytoplasmic filaments (10, 11). At the dermal–epidermal Address correspondence to Guerrino Meneguzzi, INSERM U385, junction, plectin, as detected by a hemidesmosome-specific U.F.R de Médecine, Av. de Valombrose, 06107 Nice Cedex 2, France. antibody directed against the protein HD1, has been shown Phone: 33-93-37-77-79; FAX: 33-93-81-14-04; E-mail: meneguzz@ to localize to the periphery of the intracellular portion of the unice.fr hemidesmosomal plaque, close to the region where the 230-kD Received for publication 16 July 1996 and accepted in revised form bullous pemphigoid antigen (BPAG1) has been localized (12), 10 September 1996. but also in other tissues not possessing hemidesmosomes, such as the Schwann cells. Several lines of immunohistological evi- 1. Abbreviations used in this paper: ASO, allele-specific oligomer; CSGE, conformation sensitive gel electrophoresis; EBS, epidermoly- dence suggest that plectin and the hemidesmosomal compo- sis bullosa simplex; MD, muscular dystrophy. nent HD1 may be the same protein. The defective expression of plectin and HD1 we observed in the skin and muscles of our J. Clin. Invest. MD-EBS patients therefore substantiates this hypothesis (4). © The American Society for Clinical Investigation, Inc. The altered expression of this versatile cross-linking ele- 0021-9738/96/11/2196/05 $2.00 ment of cytoskeleton correlated well with the altered labeling Volume 98, Number 10, November 1996, 2196–2200 pattern of keratin intermediate filaments and desmin network 2196 Chavanas et al. noticed in the MD-EBS keratinocytes and muscle fibers, re- apparatus. Two parallel membranes were prehybridized in a solution spectively (4). The gene (PLEC1) encoding plectin was there- of 5ϫ SSPE, 0.5% SDS, 5ϫ Denhardt’s, for 30 min at 37ЊC, and then fore a strong candidate gene for the site of mutations in our hybridized for 1 h at 37ЊC with either the 32P-labeled wild type allele- 6 MD-EBS patients. specific oligomer (ASO) (5 ϫ 10 cpm/ml) with the sequence 5Ј- Ј Ј Since the human plectin cDNA has recently been isolated CTGGAGGAGCAGGCCGCGCAA-3 , and the mutated ASO 5 - CTGGAGGAGTAGGCCGCGCAA-3Ј. Filters were washed in 2ϫ and sequenced, and the organization of the corresponding SSPE, 0.1% SDS for 15 min at room temperature followed by 15 min gene has been defined (13, 14), we investigated one of our at the Tm of the oligonucleotides, and then exposed to x-ray film for MD-EBS families for genetic defects affecting the expression autoradiography. The inheritance of the mutation in the family was of this cytoskeletal protein. also confirmed by comparative sequence analysis of the PCR prod- ucts encompassing the mutation site in the different family members. Methods Results Samples. Skin biopsies for cell culture were obtained from nonblis- tered skin of the MD-EBS patient and from healthy unrelated control Previous results indicated that the clinical phenotype of the subjects. The clinical, ultrastructural, and immunofluorescence find- two MD-EBS members of the family C is associated with an ings in this patient (family C in reference 4) have been described pre- impaired expression of plectin (4). In fact, intact skin and mus- viously (4). Keratinocytes were cultured on feeder layers of irradiated cle samples from the two probands stained negatively with a mouse 3T3 cells (15). DNA was extracted from peripheral blood or from secondary cultures of dermal fibroblasts (16). panel of monoclonal antibodies specific for plectin. Absence of Northern analysis. 20 ␮g of total RNA purified from cultured plectin was confirmed by immunoprecipitation of cell extracts MD-EBS and normal keratinocytes (17), was fractionated by electro- obtained from primary skin keratinocytes (4). To further as- phoresis on a 0.8% agarose gel, blotted to a nylon membrane (Amer- sess the expression of PLEC1 in the MD-EBS keratinocytes, sham International, Little Chalfont, UK) and hybridized with 32P-ran- we determined the steady-state level of the mRNA encoding dom-labeled plectin and GAPDH probes (18). The plectin DNA plectin by Northern analysis of RNA isolated from primary probe (1.3 kb) was obtained by PCR amplification of genomic DNA cultures of epidermal keratinocytes obtained from one of the sequences within exon 33 of the human PLEC1 gene (for the PLEC1 probands. Hybridization with a radioactive cDNA probe for cDNA sequences, see GenBank accession no U53204) using control plectin resulted in a markedly reduced, by around 75%, signal genomic DNA as a template and primers (L) 5Ј-AGGCGGAGC- in the MD-EBS patient as compared to a control, after correc- GGGTGCTTG-3Ј and (R) 5Ј-GCCTTCAGCGCCAGGATCTC-3Ј.