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• W2085192679 abstract "Integrin α6β4 is a hemidesmosomal transmembrane molecule involved in maintaining basal cell-matrix adhesion through interaction of the large intracytoplasmic tail of the β4 subunit with the keratin intermediate filament network, at least in part through its binding with plectin and BP180/type XVII collagen. Here we report a patient with predominant features of epidermolysis bullosa simplex due to a mutation in the integrin β4 gene. The patient, a 49-y-old female, had mild blistering of hands and feet from birth on, dystrophy of the nails with onychogryposis, and enamel hypoplasia. She had no alopecia and no history of pyloric atresia. Electron microscopy and antigen mapping of a skin blister revealed that the level of separation was intraepidermal, low in the basal keratinocytes through the attachment plaque of the hemidesmosome. Immuno-fluorescence microscopy revealed absent binding of monoclonal antibody 450–11 A against the third fibronectin III repeat on the intracellular domain of integrin β4, whereas binding was reduced with monoclonal antibodies recognizing epitopes on amino-terminal and carboxy-terminal ends of the polypeptide. At the molecular level the phenotype was caused by a novel 2 bp deletion 4733delCT in ITGB4, resulting in in-frame skipping of exon 36 and a deduced 50 amino acid deletion (1450–1499) within the third fibronectin type III repeat in the cytoplasmic domain of the integrin β4 polypeptide. Immunoblot analysis demonstrated a 5 kDa shorter β4 polypeptide. The 4733delCT mutation was heterozygously present in the DNA. The patient is also expected to be heterozygous for a null allele, as no full-size protein was detected in vitro and the epitope 450–11 A was absent in vivo. These data show that deletion of the third fibronectin type III repeat in the cytoplasmic domain of integrin β4, which is thought to interact with BP180/type XVII collagen, is clinically pathogenic and results in a mild phenotype with predominant features of epidermolysis bullosa simplex. Integrin α6β4 is a hemidesmosomal transmembrane molecule involved in maintaining basal cell-matrix adhesion through interaction of the large intracytoplasmic tail of the β4 subunit with the keratin intermediate filament network, at least in part through its binding with plectin and BP180/type XVII collagen. Here we report a patient with predominant features of epidermolysis bullosa simplex due to a mutation in the integrin β4 gene. The patient, a 49-y-old female, had mild blistering of hands and feet from birth on, dystrophy of the nails with onychogryposis, and enamel hypoplasia. She had no alopecia and no history of pyloric atresia. Electron microscopy and antigen mapping of a skin blister revealed that the level of separation was intraepidermal, low in the basal keratinocytes through the attachment plaque of the hemidesmosome. Immuno-fluorescence microscopy revealed absent binding of monoclonal antibody 450–11 A against the third fibronectin III repeat on the intracellular domain of integrin β4, whereas binding was reduced with monoclonal antibodies recognizing epitopes on amino-terminal and carboxy-terminal ends of the polypeptide. At the molecular level the phenotype was caused by a novel 2 bp deletion 4733delCT in ITGB4, resulting in in-frame skipping of exon 36 and a deduced 50 amino acid deletion (1450–1499) within the third fibronectin type III repeat in the cytoplasmic domain of the integrin β4 polypeptide. Immunoblot analysis demonstrated a 5 kDa shorter β4 polypeptide. The 4733delCT mutation was heterozygously present in the DNA. The patient is also expected to be heterozygous for a null allele, as no full-size protein was detected in vitro and the epitope 450–11 A was absent in vivo. These data show that deletion of the third fibronectin type III repeat in the cytoplasmic domain of integrin β4, which is thought to interact with BP180/type XVII collagen, is clinically pathogenic and results in a mild phenotype with predominant features of epidermolysis bullosa simplex. junctional epidermolysis bullosa with pyloric atresia Epidermolysis bullosa (EB) is a heterogeneous group of bullous disorders of the skin and mucous membranes characterized by detachment of epithelium after minor friction. EB is classified into three main categories according to the level of blister formation: simplex, junctional, and dystrophic (Fine et al., 2000Fine J.D. Eady R.A. Bauer E.A. et al.Revised classification system for inherited epidermolysis bullosa. Report of the Second International Consensus Meeting on Diagnosis and Classification of Epidermolysis Bullosa.J Am Acad Dermatol. 2000; 42: 1051-1066Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In EB simplex (EBS) the skin separates above the plasma membrane within the basal cells, whereas in junctional EB (JEB) the cleavage occurs below the plasma membrane within the lamina lucida of the epidermal basement membrane zone (BMZ). EBS is caused by mutations in the KRT5 and KRT14 genes coding for the basal cell keratins 5 and 14 (Corden and McLean, 1996Corden L.D. McLean W.H. Human keratin diseases: hereditary fragility of specific epithelial tissues.Exp Dermatol. 1996; 5: 297-307Crossref PubMed Scopus (186) Google Scholar) and in the PLEC1 gene coding for the hemidesmosomal plaque protein plectin (Uitto et al., 1996Uitto J. Pulkkinen L. Smith F.J. McLean W.H. 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 (89) Google Scholar). JEB is a heterogeneous group of autosomal recessive blistering disorders caused by mutations in genes encoding structural components of the hemidesmosome, lamina lucida, and lamina densa. Mutations underlying JEB have been identified in six different genes: the three genes that encode the α3, β3, and γ2 polypeptides of the laminin 5 complex (LAMA3, LAMB3, and LAMC2) (Nakano et al., 2002Nakano A. Chao S.C. Pulkkinen L. Murrell D. Bruckner-Tuderman L. Pfendner E. Uitto J. Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs non-Herlitz phenotypes.Hum Genet. 2002; 110: 41-51Crossref PubMed Scopus (114) Google Scholar), the COL17A1 gene, coding for type XVII collagen, also known as the 180 kDa bullous pemphigoid antigen (BP180) (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 Dermatol. 1998; 7: 46-64Crossref PubMed Scopus (89) Google Scholar), and the genes encoding integrin α6β4 (ITGA6 and ITGB4) (Nakano et al., 2001Nakano A. Pulkkinen L. Murrell D. et al.Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta4 integrin gene (ITGB4) and genotype/phenotype correlations.Pediatr Res. 2001; 49: 618-626Crossref PubMed Scopus (82) Google Scholar). The last two components, type XVII collagen and integrin α6β4, are transmembrane adhesion molecules of the hemidesmosome, which bridge the intracellular hemidesmosomal plaque with the extracellular basement membrane matrix. Although complete ablation of either transmembrane protein results in junctional cleavage below the plasma membrane, it is conceivable that abnormalities of the cytoplasmic domain may result in intracellular separation consistent with a simplex type of split level, and indeed, for type XVII collagen this was recently reported in a patient with a genomic deletion in COL17A1 corresponding to an in-frame deletion from the intracellular domain of the polypeptide (Huber et al., 2002Huber M. Floeth M. Borradori L. et al.Deletion of the cytoplasmatic domain of BP180/collagen XVII causes a phenotype with predominant features of epidermolysis bullosa simplex.J Invest Dermatol. 2002; 118: 185-192Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This patient not only had a predominant simplex type of cleavage but also a milder clinical phenotype with features of EBS that lacked the abnormalities more typical for JEB, such as alopecia, loss of eyelashes, and enamel defects. In this study we report a patient in whom deletion of a cytoplasmic domain of integrin β4 also causes EBS. Integrin β4 is predominantly expressed in stratified squamous and transitional epithelia, and within the cutaneous BMZ is localized to hemidesmosomes, which rely on the presence of this integrin for their formation. The α6β4 heterodimer is involved in maintaining basal cell–matrix adhesion through interaction of the large intracytoplasmic tail of the β4 subunit with the keratin intermediate filament network, at least in part through its binding with plectin and BP180/type XVII collagen. Also, the extracellular domain of β4 integrin serves as a receptor for laminin 5 and interacts with the BP180, both of them being components of the lamina lucida. For a review see Borradori and Sonnenberg, 1999Borradori L. Sonnenberg A. Structure and function of hemidesmosomes: more than simplex adhesian complexes.Invest Dermatol. 1999; 112: 411-418Crossref PubMed Scopus (451) Google Scholar. Complete ablation of integrin β4 due to homozygous ITGB4 null alleles results in the lethal form of JEB associated with pyloric atresia (JEB-PA, MIM 226730). Although there are nonlethal forms of JEB due to ITGB4 missense mutations, almost all are associated with pyloric atresia (Nakano et al., 2001Nakano A. Pulkkinen L. Murrell D. et al.Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta4 integrin gene (ITGB4) and genotype/phenotype correlations.Pediatr Res. 2001; 49: 618-626Crossref PubMed Scopus (82) Google Scholar). There is only one report of a case with JEB due to ITGB4 mutations that was not associated with pyloric atresia (Inoue et al., 2000Inoue M. Tamai K. Shimizu H. Owaribe K. Nakama T. Hashimoto T. McGrath J.A. A homozygous missense mutation in the cytoplasmic tail of β4 integrin, G931D, that disrupts hemidesmosome assembly and underlies non-Herlitz junctional epidermolysis bullosa without pyloric atresia? [letter].J Invest Dermatol. 2000; 114: 1061-1064Crossref PubMed Scopus (36) Google Scholar). The patient was homozygous for the G931D missense mutation in the cytoplasmic tail of integrin β4. This patient had the typical clinical features of generalized atrophic benign JEB with progressive alopecia, tooth defects, and nail dystrophy. The level of separation in JEB-PA due to integrin β4 mutations is mostly within the lamina lucida, but often epithelial remnants are present on the blister floor that are thought to be “torn-off” from the basal keratinocytes (McMillan et al., 1998McMillan J.R. McGrath J.A. Tidman M. Eady R.A.J. Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa.J Invest Dermatol. 1998; 110: 132-137Crossref PubMed Scopus (47) Google Scholar). Actually, “pseudojunctional” splits very low in the basal cells have been reported in JEB-PA patients with mutations in the β4 gene (Mellerio et al., 1998Mellerio J.E. Pulkkinen L. McMillan J.R. et al.Pyloric atresia–junctional epidermolysis bullosa syndrome: mutations in the integrin beta4 gene (ITGB4) in two unrelated patients with mild disease.Br J Dermatol. 1998; 139: 862-871Crossref PubMed Scopus (44) Google Scholar;Pulkkinen et al., 1998aPulkkinen L. Kim D.-U. Uitto J. Epidermolysis bullosa with pyloric atresia: novel mutations in the β4 integrin gene (ITGB4).Am J Pathol. 1998; 152: 157-166PubMed Google Scholar), as well as in knock-out mice with ablation of the ITGA6 or ITGB4 genes (Georges-Labouesse et al., 1996Georges-Labouesse E. Messaddeq N. Yehia G. Cadalbert L. Dierich A. Le Meur M. Absence of integrin α6 leads to epidermolysis bullosa and neonatal death in mice.Nat Genet. 1996; 13: 370-373Crossref PubMed Scopus (448) Google Scholar;Van der Neut et al., 1996Van der Neut R. Krimpenfort P. Calafat J. Niessen C.M. Sonnenberg A. Epithelial detachment due to absence of hemidesmosomes in integrin β4 null mice.Nat Genet. 1996; 13: 366-369Crossref PubMed Scopus (344) Google Scholar). Here we report a remarkable EB case presenting with localized blistering of the hands and feet without pyloric atresia due to a mutation in the integrin β4 gene. The phenotype had predominantly features of EBS with intraepidermal blister formation. At the molecular level the phenotype was caused by a novel 2 bp deletion 4733delCT in ITGB4, resulting in in-frame skipping of exon 36 and deletion of a cytoplasmic domain of the integrin β4 polypeptide. The patient, a 49-y-old female, had from birth on blister formation of the hands and feet after minor trauma. She is the only child of a nonconsanguineous marriage and there is no family history of EB. She did not conceive. Although the whole skin was fragile, such as after the use of adhesive plaster, blistering was mainly restricted to the hands (Figure 1a) and feet (Figure 1b). Blistering frequency increased during the summer. The skin of the body was intact and normally pigmented. Subungual blister formation from early infancy on had led to thickening of nail plates (pachyonychia), distal onycholysis, and excessive curvature in longitudinal and transverse directions (onygogryphosis) (Figure 1a). The toenails were dystrophic (Figure 1b). The soles showed hyperkeratosis with circumscript callous formation. Her teeth were characterized by the absence of enamel since appearance and were all restored with crowns by the age of 15. She had normal scalp hair, eye lashes, and eye brows, and secondary hair was present. The right conjunctiva showed increased vascular injection. There was no history of pyloric atresia or other gastrointestinal or urinary problems related to EB. Skin biopsies were taken from healthy looking skin, from lesional skin (left little finger), and from a freshly rubbed blister. Electron microscopy was performed as previously described (Jonkman et al., 1992Jonkman M.F. De Jong M.C.J.M. Heeres K. Sonnenberg A. Expression of integrin α6β4 in junctional epidermolysis bullosa.J Invest Dermatol. 1992; 99: 489-496Abstract Full Text PDF PubMed Google Scholar). For immuno-fluorescence microscopy on tissue samples, unfixed cryostat sections of snap-frozen skin biopsies were processed as previously described (Jonkman et al., 1992Jonkman M.F. De Jong M.C.J.M. Heeres K. Sonnenberg A. Expression of integrin α6β4 in junctional epidermolysis bullosa.J Invest Dermatol. 1992; 99: 489-496Abstract Full Text PDF PubMed Google Scholar). The following monoclonal antibodies were used: pan-keratin with CK1 (Dako), laminin 5 with GB3, uncein with 19-DEJ-1, type VII collagen with LH7:2, BP180/type collagen XVII endodomain with 1A8c, BP180/type collagen XVII ectodomain with 1D1 and NCC-Lu-226 (Yamada et al., 1996Yamada T. Endo R. Tsukagoshi K. et al.Aberrant expression of a hemidesmosomal protein, bullous pemphigoid antigen 2, in human squamous cell carcinoma.Lab Invest. 1996; 75: 589-600PubMed Google Scholar), plectin/HD1 with HD121, BP230 with 815, integrin α3 with J143, integrin β1 with TS2/16, integrin α6 with GoH3, integrin β4 ectodomain with 58XB4 and 439–9B, and the integrin β4 endodomain with 450–11 A (Kennel et al., 1991Kennel S.J. Falcioni R. Wesley J.W. Microdistribution of specific rat monoclonal antibodies to mouse tissues and human tumor xenografts.Cancer Res. 1991; 51: 1529-1536PubMed Google Scholar) and clone 7 (BD Biosciences). More precisely, the epitope of the 450–11A monoclonal antibody is located within the segment of the third fibronectin III (FNIII) repeat (codons 1458–1541) on the β4 endodomain (personal communication, Dr. Sonnenberg), whereas clone 7 recognizes an epitope at the carboxy-terminal end. In combination with primary mouse monoclonal antibodies we used Alexa 488-conjugated goat antihuman IgG (Molecular Probes Europe, Leiden, The Netherlands) as secondary step. Preparation of cell extracts and immunoblotting was as described before (Van Leusden et al., 2001Van Leusden M.R. Pas H.H. Gedde-Dahl Jr, T. Sonnenberg A. Jonkman M.F. Truncated type XVII collagen expression in a patient with non-Herlitz junctional epidermolysis bullosa as a consequence of a homozygous splice site mutation.Lab Invest. 2001; 81: 887-894Crossref PubMed Scopus (13) Google Scholar). For detection of integrin β4 we used goat polyclonal antibody N-20 against the extracellular amino-terminus (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal antibody 450–11A against the intracellular domain. Bound goat antibody was detected by subsequent incubation with alkaline-phosphatase-conjugated rabbit antigoat IgG and alkaline-phosphatase-conjugated goat antirabbit IgG, and bound monoclonal mouse antibody by goat antimouse IgG followed by alkaline-phosphatase-conjugated rabbit antigoat IgG. Keratinocytes were isolated from skin biopsies and cultured in serum-free medium (BioWhittaker, Verviers, Belgium) as described previously (Jonkman et al., 1997Jonkman M.F. Scheffer H. Stulp R.P. et al.Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion.Cell. 1997; 88: 543-551Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Total RNA was extracted from cells cultured for three passages using Trizol (Gibco, Glasgow, U.K.), according to the manufacturer's protocol. DNA was extracted from cultured keratinocytes briefly as follows: cells were rinsed twice with phosphate-buffered saline and lyzed in the culture flask with 150 mm NaCl, 10 mm ethylenediamine tetraacetic acid (EDTA) pH 8.0, 1% sodium dodecyl sulfate, with gentle shaking for a few minutes. The viscous supernatant was transferred to a clean tube, proteinase K was added to 100 μg per ml, and the lysate was incubated at 60°C for 16 h. After extraction with phenol/chloroform high molecular weight DNA was precipitated with ethanol or 2-propanol, washed with 70% ethanol, and dissolved overnight in 10 mM Tris–HCl, 0.1 mM EDTA pH 8.0, under continuous shaking. Nucleotide numbering, used throughout this study for primer position and the site of mutation, is based on the 41 exons containing mRNA (including exons 33 and 35) and is relative to the start site of translation (accession number NM_000213). Amino acid numbering is based on the integrin β4A splice variant expressed in keratinocytes that lacks translation of exons 33 and 35 (accession number NP_000204). Aliquots of total RNA from patient and control cultured keratinocytes were reverse transcribed with M-MLV Reverse Transcriptase (GibcoBRL) and oligo(dT) priming. Subsequently, ITGB4 cDNA was amplified with overlapping primer sets (Table I) and analyzed on agarose gel. The mRNA sequence from the public domain, used for primer development and nucleotide numbering, was basically GenBank accession no. X53587, with the 159 nucleotide exon 35 sequence from GenBank X52186 (nucleotides 4358–4515) inserted at position 4744–4745 of X53587. The multiple bands in the patient's cDNA due to aberrant splicing were isolated from gel and cloned in vector pCRII (TOPOTM TA-cloning Kit, Invitrogen). Different clones were isolated, analyzed by restriction digestion and agarose gel electrophoresis, and subjected to automated sequencing. In addition, genomic DNA was PCR amplified and sequenced directly.Table IITGB4 PCR primer setsaPCR primer sets used for amplification of cDNA and genomic DNA. Primer names indicate the relative cDNA sequence position, with the start site of translation chosen as position 1 (cDNA nucleotide numbering is with exons 33 and 35 inclusive).SetSense primerExonAntisense primerExonSize (bp)bSizes in bp: sets 1–9 for cDNA amplification; set i35 for PCR on genomic DNA.1m155F: 5′-tgcagccccatctcctag10817R: 5′-catcagcctcatagtggaag897320671F: 5′-ggatctcaggcaacctggat71616R: 5′-tcatactcgcagaactgacc1394531485F: 5′-ctctctgagtgacattcagc132212R: 5′-aggcacagtacttccagcat1872742091F: 5′-tgtcctggtgcacaagaaga172667R: 5′-catcagcactgtgtccacaa2457652488F: 5′-gagaacctgctgaagcctga212880R: 5′-cagctgcttctcgtcgtcat2539262701F: 5′-cttacagagaagcaggtgga243694R: 5′-agacgacattgaaggccaga3099373569F: 5′-gcgactatgagatgaaggtg294395R: 5′-actggtcgtggtcatcctgt3461684053F: 5′-tgacgttctacgctctccat324954R: 5′-ggaacacgtaggagtggttg3753294837F: 5′-tacagtgtggagtaccagct365711R: 5′-aaggatggagtagctgagga41874i354666F: 5′-cgagactctataatcctggc354802R: 5′-cagctcactctgagagatgt36622a PCR primer sets used for amplification of cDNA and genomic DNA. Primer names indicate the relative cDNA sequence position, with the start site of translation chosen as position 1 (cDNA nucleotide numbering is with exons 33 and 35 inclusive).b Sizes in bp: sets 1–9 for cDNA amplification; set i35 for PCR on genomic DNA. Open table in a new tab Examination of skin specimens by electron microscopy showed a normal number (81 per 40 μm BMZ, normal 71) of hypoplastic hemidesmosomes (average diameter 117 nm, normal ±200 nm) (Figure 2a) in uninvolved skin. The inner cytoplasmic plaque was absent in 30% (normal ±10%) of the hemidesmosomes, coinciding with poor attachment of intermediate tonofilaments. Biopsies of affected skin and of a blister induced by rubbing showed a plane of separation above the plasma membrane through the hemidesmosomal plaque. Blistering started with vacuolization in the cytoplasm of the basal cells (Figure 2b). Type VII collagen, laminin 5, the ectodomains and endodomains of BP180/type XVII collagen, the ectodomains of integrin α6, and the ectodomains and endodomains of the β4 subunit lined the blister floor (data not shown). BP230 (Figure 3a) was predominantly present in the blister floor, whereas keratin (Figure 3b) and plectin/HD1 (Figure 3c) were mainly found in the roof of the blister. The cleavage level was thus extremely low in the basal cell separating plectin that is localized on the attachment plaque furthest inwards from the plasma membrane (Shimizu et al., 1999Shimizu H. Masunaga T. Kurihara Y. et al.Expression of plectin and HD1 epitopes in patients with epidermolysis bullosa simplex associated with muscular dystrophy.Arch Dermatol Res. 1999; 291: 531-537Crossref PubMed Scopus (10) Google Scholar) from those localized closer to the plasma membrane (BP230, integrin α6β4, and BP180/type XVII collagen) (Ishiko et al., 1993Ishiko A. Shimizu H. Kikuchi A. Ebihara T. Hashimoto T. Nishikawa T. Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.J Clin Invest. 1993; 91: 1608-1615Crossref PubMed Scopus (158) Google Scholar;Sato et al., 1998Sato M. Shimizu H. Ishiko A. et al.Precise ultrastructural localization of in vivo deposited IgG antibodies in fresh perilesional skin of patients with bullous pemphigoid.Br J Dermatol. 1998; 138: 965-971Crossref PubMed Scopus (9) Google Scholar). α6β4 An antibody directed against the third FNIII repeat on the intracellular domain of integrin β4 failed to detect any epitope, even in nonlesional skin (Figure 4a), strongly indicating ITGB4 as the site of mutation. Staining for the extracellular part of integrin β4 with monoclonal antibodies 58XB4 (Figure 4b) and 439–9B was moderately reduced along the epidermal BMZ, whereas staining for the intracellular tail with monoclonal antibody clone 7 was in a normal linear pattern severely reduced. Expression of integrin α6 was moderately reduced and confined to the epidermal BMZ (Figure 4c). The expression of uncein, the monoclonal marker for JEB, was severely reduced along the epidermal BMZ (not shown). Plectin, BP230, integrin α3β1, and BP180/type XVII collagen (Figure 4d) were normally distributed along the epidermal BMZ. Immunoblots from proteins extracted from cultured keratinocytes showed that patient cells synthesize integrin β4 of reduced size as shown by polyclonal antibody N-20 directed against the extracellular domain (Figure 5). As expected, antibody 450–11 A, having its epitope within the third FNIII repeat in the cytoplasmic domain, failed to detect any protein on immunoblot. RNA was extracted from keratinocytes derived from patient skin biopsies and cultured for a few passages, and the ITGB4 mRNA was analyzed by RT-PCR with nine overlapping primer sets. Table I summarizes the primer sequences and positions. Comparison with control keratinocyte RNA showed no abnormal transcripts except for the amplicon encompassing exon 36: three bands were visible on agarose gel, one with apparently normal length, and two shorter PCR products compared to the control amplification. The shortest band was the most predominant one (Figure 6). PCR fragments were isolated from gel, subcloned into plasmids, and sequenced. The shortest fragment appeared to be generated by skipping of exon 36, as the sequence was reading from exon 34 directly into exon 37 (ITGB4 exon 35 normally is absent in keratinocytes, as is exon 33). The in-frame skipping of the 150 bp of exon 36 results in deletion of aa-1450 to aa-1499 corresponding to the beginning of the third fibronectin repeat (1458–1541) in the intracellular domain of integrin β4 (Figure 7). The sequence of a clone containing an insert of apparently normal length, from the upper band (Figure 6), revealed a 2 bp (CT) deletion in the sequence of exon 36 at position 4733 (CTGACTG(ct)ΔGGTGTG). PCR products from the band with intermediate size were unclonable; these fragments most probably were heteroduplex PCR products, as an intermediate band with similar size could be generated when a mixture of two clones, one with upper band insert and one with lower band insert, was subjected to PCR (results not shown). Sequence analysis of PCR amplified genomic DNA confirmed the 2 bp deletion (ITGB4: 4733delCT) and also showed that the patient was heterozygous for this mutation.Figure 7Molecular defect associated with the mutation 4733delTC. Schematic presentation of the integrin β4A chain in keratinocytes. The 4733delTC (italic) is shown in exon 36, and the 50 residues deletion eliminating part of the third FNIII-like repeat. sp, signal peptide; tm, transmembrane domain; CS, connecting segment; 1, 2, 3, and 4, FNIII-like repeats.View Large Image Figure ViewerDownload (PPT) The heterozygous presence of the 2 bp deletion in exon 36 of ITGB4 in the patient was most probably accompanied by a null allele. RT-PCR and subsequent sequence analysis revealed aberrant splicing of ITGB4 exon 36. Sequencing of a clone not deleted for exon 36 identified a 2 bp deletion at position 4733 in exon 36. Sequence analysis of genomic DNA showed that this mutation was heterozygously present in the patient. The mutation leads to a frame shift and a premature termination codon (PTC) at the second position of exon 37. The majority of RT-PCR product resulted from mRNA with skipped exon 36. Exon 36 is 150 bp in size and deletion does not destroy the open reading frame but leads to a 50 amino acid deletion in the cytoplasmic domain, involving the third FNIII repeat (Figure 7). These findings fit with the absent binding of monoclonal antibody 450–11A, which recognizes an epitope within the third FNIII repeat of β4 to patient's epidermis and keratinocyte extract. In our RT-PCR analysis of the patient's mRNA the band with skipped exon 36 was the major product. It is likely that the 2 bp deletion, leading to a PTC, is rescued by alternative splicing of exon 36. The presence of the carboxy-terminal end of the protein in the skin of the patient is in line with an in-frame skip. Immunoblot analysis indeed showed that the patient's cells produced the β4 protein of reduced size. No full-size protein was detected on immunoblots and, given the low intensity of the upper RT-PCR band, consisting partially if not mostly of 2 bp deleted sequences, the patient is also expected to be heterozygous for a null allele. Attempts to specifically amplify cDNA from this allele by using nondeletion-site primers failed. The search for the second mutation failed in that the analysis of genomic DNA was unable to disclose any pathogenic sequence variants other than the heterozygous 2 bp deletion (Professor J. McGrath, personal communication). Nonetheless, the contribution of the other allele to protein production may be considered as very low. Moderation of phenotypic severity by skipping of exons containing nonsense mutations has been observed before (Morisaki et al., 1993Morisaki H. Morisaki T. Newby L.K. Holmes E.W. Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect.J Clin Invest. 1993; 91: 2275-2280Crossref PubMed Scopus (60) Google Scholar). Maintenance of the open reading frame by ribosome-mediate scanning of pre-mRNA transcripts for PTCs serves as an additional level of scrutiny during splice site selection (Dietz and Kendzior, 1994Dietz H.C. Kendzior Jr, R.J. Maintenance of an open reading frame as an additional level of scrutiny during splice site selection.Nat Genet. 1994; 8: 183-188Crossref PubMed Scopus (129) Google Scholar), and indeed, in-frame skipping of exons containing PTCs has been reported in the COL7A1 and LAMB3 genes in dystrophic and junctional forms of epidermolysis bullosa (McGrath et al., 1999McGrath J.A. Ashton G.H. Mellerio J.E. Salas-Alanis J.C. Swensson O. McMillan J.R. Eady R.A. Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations.J Invest Dermatol. 1999; 113: 314-321Crossref PubMed Scopus (62) Google Scholar). mRNA rescue may also depend on activation of the illegitimate splicing event during development, explaining the amelioration with aging of the clinical phenotyp" @default.
• W2085192679 created "2016-06-24" @default.
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• W2085192679 date "2002-12-01" @default.
• W2085192679 modified "2023-10-14" @default.
• W2085192679 title "Deletion of a Cytoplasmic Domain of Integrin β4 Causes Epidermolysis Bullosa Simplex1" @default.
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