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Four Novel Plectin Gene Mutations in Japanese Patients with Epidermolysis Bullosa with Muscular Dystrophy Disclosed by Heteroduplex Scanning and Protein Truncation Tests

1999; Elsevier BV; Volume: 112; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.1999.00461.x

1523-1747

Yasuko Takizawa, Hiroshi Shimizu, Takeji Nishikawa, Fatima Rouan, Mitsuru Kawai, Masako Udono, Leena Pulkkinen, Jouni Uitto,

Polysaccharides and Plant Cell Walls

Epidermolysis bullosa with muscular dystrophy (EB-MD) is a distinct variant of EB caused by mutations in the plectin gene (PLEC1). In this study, we have examined two Japanese patients with EB-MD using heteroduplex scanning or a protein truncation test for mutation detection analysis. The results revealed that both patients were compound heterozygotes for novel PLEC1 mutations (Q1936X/Q1053X and R2421X/12633ins4), which all caused premature termination of translation of the corresponding polypeptides. These cases, which demonstrate the utility of two complementary mutation detection strategies, add to the repertoire of plectin mutations in EB-MD. Epidermolysis bullosa with muscular dystrophy (EB-MD) is a distinct variant of EB caused by mutations in the plectin gene (PLEC1). In this study, we have examined two Japanese patients with EB-MD using heteroduplex scanning or a protein truncation test for mutation detection analysis. The results revealed that both patients were compound heterozygotes for novel PLEC1 mutations (Q1936X/Q1053X and R2421X/12633ins4), which all caused premature termination of translation of the corresponding polypeptides. These cases, which demonstrate the utility of two complementary mutation detection strategies, add to the repertoire of plectin mutations in EB-MD. epidermolysis bullosa with muscular dystrophy protein truncation test Epidermolysis bullosa with muscular dystrophy (EB-MD) is a distinct variant of EB, inherited in an autosomal recessive pattern (for a recent review, seeUitto et al., 1996Uitto J. Pulkkinen L. Smith F.J.D. McLean W.H.I. Plectin and human genetic disorders of the skin and muscle. The paradigm of epidermolysis bullosa with muscular dystrophy.Exp Dermatol. 1996; 5: 237-246Crossref PubMed Scopus (91) Google Scholar). Clinically, the affected individuals demonstrate blistering of the skin and mucous membranes, usually noted at birth or shortly thereafter. These cutaneous changes are associated with nail dystrophy and enamel hypoplasia. Characteristically, later in life a progressive muscle weakness ensues, but the time of onset of the muscle involvement is highly variable. In some cases, the muscle weakness has been noted during the early infancy, whereas in others it is not recognized until the second or third decade of life. The reasons for the variability of onset of muscular dystrophy are unclear, but in most cases the muscle involvement results in profound weakness, and the patients ultimately become wheelchair bound. Electron microscopy of the skin in patients with EB-MD has demonstrated tissue separation in the lower portion of the basal keratinocytes, just above the attachment plaques of the hemidesmosomes (McLean et al., 1996McLean W.H.I. Pulkkinen L. Smith F.J.D. et al.Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.Genes Dev. 1996; 10: 1724-1735Crossref PubMed Scopus (284) Google Scholar;Pulkkinen et al., 1996Pulkkinen L. Smith F.J.D. Shimizu H. et al.Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy.Hum Mol Genet. 1996; 5: 1539-1546Crossref PubMed Scopus (139) Google Scholar;Smith et al., 1996Smith F.J.D. Eady R.A.J. Leigh I.M. et al.Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.Nat Genet. 1996; 13: 450-457Crossref PubMed Scopus (333) Google Scholar). Consequently, EB-MD was initially classified as a simplex variant of EB, but more recent molecularly based classification has designated it as a hemidesmosomal variant (Pulkkinen and Uitto, 1998Pulkkinen L. Uitto J. Hemidesmosomal variants of epidermolysis bullosa. Mutations in the α6β4 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes.Exp Derm. 1998; 7: 46-64Crossref PubMed Scopus (96) Google Scholar). Initial immunofluorescence revealed absent or moderately attenuated staining with antibodies recognizing plectin or its variant HD1, a multifunctional attachment protein present in the dermal–epidermal junction (Gache et al., 1996Gache Y. Chavanas S. Lacour J.P. Wiche G. Owaribe K. Meneguzzi G. Ortonne J.-P. Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.J Clin Invest. 1996; 97: 2289-2298Crossref PubMed Scopus (211) Google Scholar). This protein is also expressed in the sarcolemma and the Z-lines of the muscle, and the expression in patients with EB-MD in the muscle was also shown to be negative (Chavanas et al., 1996Chavanas S. Pulkkinen L. Gache Y. et al.A homozygous nonsense mutation in the PLEC1 gene in patients with epidermolysis bullosa simplex with muscular dystrophy.J Clin Invest. 1996; 90: 2196-2200Crossref Scopus (91) Google Scholar;Smith et al., 1996Smith F.J.D. Eady R.A.J. Leigh I.M. et al.Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.Nat Genet. 1996; 13: 450-457Crossref PubMed Scopus (333) Google Scholar). These observations suggested that plectin is a potential candidate gene/protein system at fault in EB-MD. Subsequent cloning of the human plectin gene (PLEC1) allowed development of mutation detection strategies that were applied to EB-MD (Liu et al., 1996Liu C.-G. Maercker C. Castanon M.J. Hauptmann R. Wiche G. Human plectin. Organization of the gene, sequence analysis, and chromosomal location (8q24).Proc Natl Acad Sci USA. 1996; 93: 4278-4283Crossref PubMed Scopus (117) Google Scholar;McLean et al., 1996McLean W.H.I. Pulkkinen L. Smith F.J.D. et al.Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.Genes Dev. 1996; 10: 1724-1735Crossref PubMed Scopus (284) Google Scholar). These initial strategies consisted of polymerase chain reaction (PCR) amplification of genomic sequences, followed by heteroduplex scanning and direct automated nucleotide sequencing (McLean et al., 1996McLean W.H.I. Pulkkinen L. Smith F.J.D. et al.Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.Genes Dev. 1996; 10: 1724-1735Crossref PubMed Scopus (284) Google Scholar;Pulkkinen et al., 1996Pulkkinen L. Smith F.J.D. Shimizu H. et al.Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy.Hum Mol Genet. 1996; 5: 1539-1546Crossref PubMed Scopus (139) Google Scholar;Smith et al., 1996Smith F.J.D. Eady R.A.J. Leigh I.M. et al.Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.Nat Genet. 1996; 13: 450-457Crossref PubMed Scopus (333) Google Scholar). Subsequently, a protein truncation test (PTT) for the two large 3′ exons (32 and 33) of the plectin gene was developed (Dang et al., 1998Dang M. Pulkkinen L. Smith F.J.D. McLean W.H.I. Uitto J. Novel compound heterozygous mutations in the plectin gene in epidermolysis bullosa with muscular dystrophy and the use of protein truncation test for detection of premature termination codon mutations.Lab Invest. 1998; 78: 195-204PubMed Google Scholar). Utilization of these strategies enabled specific mutations in the plectin gene to be disclosed, and the majority of the mutations were stop codon or premature termination codon-causing mutations within exon 32 (for a summary, seePulkkinen and Uitto, 1998Pulkkinen L. Uitto J. Hemidesmosomal variants of epidermolysis bullosa. Mutations in the α6β4 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes.Exp Derm. 1998; 7: 46-64Crossref PubMed Scopus (96) Google Scholar). In this study, we have examined two Japanese patients with EB-MD, and we were able to identify novel plectin mutations in each of the four alleles. Patient 1The proband is a 9 y old Japanese male, the third child of clinically normal parents, with two older brothers who were clinically normal The proband's father and the maternal grandfather were first cousins (Figure 1a) Blistering and erosions of the skin were noted at birth, together with nail dystrophy General muscle weakness was noted during infancy, and the muscle weakness progressed so that at 9 y of age he had major difficulties in walking He also has tooth decay and nail dystrophy, as well as urethral strictures that require frequent hospitalizationsDiagnostic transmission electron microscopy of the patient's skin revealed blistering at the level of the lower portion of the basal keratinocytes Immunofluorescence staining of the skin with antibodies for plectin (5B3 and 10F6; kindly provided by Dr Gerhard Wiche, University of Vienna, BioCenter, Vienna) or HD1 (121, E2, K15, and 156; kindly provided by Dr Katsushi Owaribe, Nagoya University, Nagoya, Japan) was entirely negative Staining of the proband's skin with a battery of other antibodies, including those recognizing laminin 5, type VII collagen, α6β4 integrin, BPAG1, BPAG2, and uncein epitopes, were positive (data not shown)Patient 2The proband is a 33 y old Japanese male, the second child of unrelated, clinically normal, parents The older brother was also clinically unaffected Blistering of the skin was first noted within a few days of birth, and the patient also exhibited nail dystrophy and partial scarring alopecia First signs of muscle weakness in the proximal extremities were noted at the age of 5 y The muscle weakness became pronounced at the age of 17, and the patient was unable to walk at the age of 22 Immunofluoresence staining of the proband's skin with the same antibodies for plectin and HD1, as were used in case 1, was completely negative, whereas a variety of other basement membrane zone antibodies resulted in positive staining (data not shown) DNA was isolated from peripheral blood from the two probands and their immediate family members. DNA from unrelated healthy individuals served as control. In case 1, the mutation detection strategy consisted of PCR amplification of exonic sequences, and the PCR products were subjected to heteroduplex analysis by conformation-sensitive gel electrophoresis (Ganguly et al., 1993Ganguly A. Rock M.J. Prockop D.J. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (619) Google Scholar). PCR amplification of exons 25 and 32, which were shown to contain the mutations in this case (see Results), was performed with the following primer pairs. Exon 25: sense, 5′-AGGACCGGCTGATGGCTGAG-3′; anti-sense, 5′-AAGACAGGGAGTGGGAAGC-3′. Exon 32 (nucleotides 5786–6191): sense, 5′-CGCAACACAAGGCTGACATC-3′; anti-sense, 5′-TCCTCCACGTTGGCTTTCAG-3′. The PCR products were subjected to direct automated nucleotide sequencing (PE Applied Biosystems, Foster City, CA). In case 2, PTT was applied to cover two large 3′ exons, numbers 32 and 33, by PCR amplification in two or three overlapping fragments, respectively. The forward primers (see below) were created to include the 5′ sequence of the corresponding fragment preceded by a T7 promoter sequence (underlined) and a eukaryotic translation initiation codon (bolded) (Hogervorst et al., 1995Hogervorst F.B. Cornelis R.S. Bout M. et al.Rapid detection of BRCA1 mutations by the protein truncation test.Nat Genet. 1995; 10: 208-212Crossref PubMed Scopus (280) Google Scholar;Dang et al., 1998Dang M. Pulkkinen L. Smith F.J.D. McLean W.H.I. Uitto J. Novel compound heterozygous mutations in the plectin gene in epidermolysis bullosa with muscular dystrophy and the use of protein truncation test for detection of premature termination codon mutations.Lab Invest. 1998; 78: 195-204PubMed Google Scholar). The reverse primers 32 were created to correspond to the 3′ intronic sequences with the addition of a downstream stop codon or they contained the natural stop codon (double underlined). The reverse primer for exon 33 was designed to correspond to the intronic sequences 3′ and to include a naturally occuring stop codon at the end of exon 33. Specifically, the primers were: (L; exon 32) 5′-GCTAATACGACTCACTATAGGAACAGACCACCATGTTCCGCGAGCTGGCCGAGG-3′; (R; exon 32) 5′-TCAACCCACCAAAGCAGATCC-3′; (L; exon 33) 5′-GCTAATACGACTCACTATAGGAACAGACCACCATGCTGGGCTTCCACCTTCCC-3′; (R; exon 33) 5′-GTGTGAGTGGCAGGTAGAAGG-3′. The PCR conditions were: 94°C, 5 min; followed by 94°C, 45 s; 60°C, 30 s; 68°C, 2.5 min (38 cycles). Four hundred nanograms of genomic DNA and 20 pmol of each primer were used in a 50 μl PCR reaction mixture containing 10 mM dNTP, 1.75 mM MgCl2, and 3.5 U of Expand Long Template PCR System polymerase enzyme (Boehringer, Indianapolis, IN). Genomic segments corresponding to these exons were PCR amplified, and the PCR products were used directly as templates in a coupled transcription/translation reticulocyte lysate system reaction with S35-labeled methionine, according to the supplier's protocol (TnT/T7; Promega, Madison, WI). The radiolabeled translation products were examined on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were fixed with 40% methanol/10% acetic acid, dried under vacuum, and exposed on X-OMAT AR film (Eastman-Kodak, Rochester, NY) for 14–16 h at room temperature. When evidence for a truncated polypeptide was observed, the corresponding PCR products were subjected to direct nucleotide sequencing. Two Japanese patients with EB-MD were subjected to mutation detection analysis using strategies described in Materials and Methods. In case 1, scanning of plectin genomic sequences by conformation-sensitive gel electrophoresis (CSGE) revealed heteroduplex bands in the case of exons 25 and 32 (Figure 1b). Specifically, CSGE of exon 32 revealed heteroduplex bands in the case of the proband (III-3), his oldest brother (III-1), and the father (II-1), whereas the mother (II-2) and the middle brother (III-2) showed homoduplex band only, similar to that noted in an unrelated healthy control (C). Direct nucleotide sequencing of the PCR products indicated that the individuals demonstrating the heteroduplex band were heterozygous for a C-to-T transition in nucleotide position 5806 (Figure 1c). This nucleotide substitution results in change of a codon for glutamine (CAG) to a stop codon (TAG), and this mutation was designated as Q1936X. This mutation was predicted to truncate the protein polypeptides so that the molecule lacks the carboxy-terminal end of the rod domain and the entire carboxy-terminal intermediate filament-binding domain. The rod domain of plectin may be important in plectin dimerization, as antibodies directed against epitopes within this domain have been found to disrupt self-association of plectin (Foisner et al., 1991Foisner R. Feldman B. Sander I. Wiche G. Monoclonal antibody mapping of structural and functional plectin epitopes.J Cell Biol. 1991; 112: 397-405Crossref PubMed Scopus (49) Google Scholar). Scanning of exon 25 by CSGE revealed heteroduplex bands in the case of the proband (III-3), as well as the middle brother (III-2) and the proband's mother (II-2) (Figure 1b). Sequencing of the corresponding PCR products revealed a C-to-T transition at position 3157 (Figure 1d). This nucleotide substitution also results in a change of a codon for glutamine (CAG) to a stop codon (TAG), and this mutation was designated as Q1053X. This mutation eliminates most of the functional domains of the plectin polypeptide, including coiled-coil rod domain and carboxy-terminal intermediate filament-binding domain. Thus, the Proband 1 is a compound heterozygote for two stop codon mutations, Q1936X/Q1053X. In case 2, an initial search for mutations by CSGE was unyielding, and subsequently, PTT was used as an alternate strategy to examine exons 32 and 33. Protein products obtained from coupled transcription/translation of PCR fragment spanning the 3′ side of exon 32 revealed evidence for synthesis of a shortened polypeptide. Specifically, the full-length protein product was expected to be ≈78 kDa, whereas in the case of the proband, a shortened polypeptide of ≈66 kDa was noted (Figure 2b, lanes 1 and 2, respectively). Sequencing of the mutated region of the PCR product corresponding to the truncated polypeptide revealed a C-to-T transition at nucleotide position 7261, which resulted in substitution of a codon for arginine (CGA) by a stop codon (TGA) (Figure 1c) within the carboxy-terminal end of the rod domain. Direct sequencing of the corresponding segment in other family members revealed that the father (I-1) and the older brother (II-1) were heterozygous carriers of this mutation. Examination of exon 33 sequences by PTT revealed evidence for a shortened polypeptide. PCR amplification spanning the 3′ side of exon 33 showed single bands of 2457 bp for the proband and the control on a 0.8% agarose gel (data not shown). Protein products of the proband's DNA revealed a shortened polypeptide of ≈44 kDa, the full-length protein product being estimated to be ≈81 kDa (Figure 2b, lanes 3 and 4). Direct nucleotide sequencing of the corresponding PCR product revealed a 4 bp insertion (AGAC) at nucleotide position 12633 (Figure 2c). This insertion resulted in a frameshift that caused a 39-amino acid missense sequence, followed by a premature termination codon (TGA). Direct nucleotide sequencing of the corresponding segment in members of the nuclear family revealed that the mother was a heterozygous carrier of this mutation 12633ins4, whereas the father and the older brother had corresponding wild-type allele only. The 12633ins4 is the first mutation found in exon 33 of the plectin gene. This mutation results in truncation of the end of the carboxy-terminal globular domain, which plays an important role in association with intermediate filaments (Wiche et al., 1993Wiche G. Gromov D. Donovan A. Castanon M.J. Fuchs E. Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association.J Cell Biol. 1993; 121: 607-619Crossref PubMed Scopus (114) Google Scholar). Thus, the proband in family 2 was a compound heterozygote for nonsense and premature termination codon-causing mutations, R2421X/12633ins4, in the plectin gene. Previously nine distinct mutations have been disclosed in the plectin gene in patients with EB-MD (Pulkkinen and Uitto, 1998Pulkkinen L. Uitto J. Hemidesmosomal variants of epidermolysis bullosa. Mutations in the α6β4 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes.Exp Derm. 1998; 7: 46-64Crossref PubMed Scopus (96) Google Scholar). The addition of the four novel PLEC1 mutations detected in this study significantly contributes to the repertoire of the EB-MD database. Among the eight previous cases, only one, reported byDang et al., 1998Dang M. Pulkkinen L. Smith F.J.D. McLean W.H.I. Uitto J. Novel compound heterozygous mutations in the plectin gene in epidermolysis bullosa with muscular dystrophy and the use of protein truncation test for detection of premature termination codon mutations.Lab Invest. 1998; 78: 195-204PubMed Google Scholar, was a compound heterozygote, whereas the remaining cases were homozygous for the mutations. Interestingly, in case 1 of our study, the father and the maternal grandfather were first cousins, yet the maternal and paternal mutations of the proband were different. Two independent, complementary mutation detection strategies were employed in this study. In the first case, a strategy based on PCR amplification of PLEC1 genomic sequences, followed by heteroduplex scanning and automated nucleotide sequencing, was utilized. This strategy allows expedient scanning of exonic sequences for heterozygous sequence variants (Ganguly et al., 1993Ganguly A. Rock M.J. Prockop D.J. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (619) Google Scholar), but PCR amplification of segments of large exons, such as exons 32 and 33 in PLEC1, is difficult due to the highly repetitive nature of the plectin gene that is particularly GC rich (McLean et al., 1996McLean W.H.I. Pulkkinen L. Smith F.J.D. et al.Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.Genes Dev. 1996; 10: 1724-1735Crossref PubMed Scopus (284) Google Scholar). Furthermore, all sequence variants do not result in the formation of detectable heteroduplexes under the CSGE conditions utilized, as illustrated by case 2 in this study. Secondly, PTT was applied to examine exons 32 and 33 for premature termination codon-causing mutations in case 2. This technique utilized PCR fragments of up to 1.5 kb, thus requiring fewer PCR reactions. A general weakness of PTT is that it will not detect missense mutations or common polymorphisms, and it may not detect small in-frame insertions or deletions, such as a previously described homozygous 9 bp deletion in a Japanese patient with EB-MD (Pulkkinen et al., 1996Pulkkinen L. Smith F.J.D. Shimizu H. et al.Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy.Hum Mol Genet. 1996; 5: 1539-1546Crossref PubMed Scopus (139) Google Scholar). In such cases, heteroduplex scanning is more likely to find the mutations. Nevertheless, use of these complementary technologies was clearly successful in disclosing mutations in the plectin gene and can be applied to further cases. Identification of additional mutations in PLEC1 in patients with EB-MD will be helpful in further delineation of genotype/phenotype correlations. This aspect is particularly important in view of the fact that all EB-MD patients evaluated thus far have cutaneous blistering noted at birth or shortly thereafter, whereas the onset of muscle involvement is highly variable, ranging from early infancy to the third decade of life (Uitto et al., 1996Uitto J. Pulkkinen L. Smith F.J.D. McLean W.H.I. Plectin and human genetic disorders of the skin and muscle. The paradigm of epidermolysis bullosa with muscular dystrophy.Exp Dermatol. 1996; 5: 237-246Crossref PubMed Scopus (91) Google Scholar). Thus expansion of the mutation database is critical for prognostication of the phenotypic progression of this devastating genetic disease involving the skin and the muscle. We thank Drs. Gerhard Wiche and Katsushi Owaribe for providing antibodies. Carol Kelly provided excellent assistance. This study was supported by Grants-in-Aid for Scientific Research (Nos. 05404036 and 07457191) from the Ministry of Education, Science, and Culture of Japan, and by Keio Gijuku Academic Development Funds (HS), as well as by the United States Public Health Service, National Institutes of Health grant PO1-AR38923 (JU).