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Collagen XVII Causes a Phenotype with Predominant Features of Epidermolysis Bullosa Simplex
Deletion of the Cytoplasmatic Domain of BP180/Collagen XVII Causes a Phenotype with Predominant Features of Epidermolysis Bullosa Simplex Marcel Huber, Michaela Floeth,* Luca Borradori,² Heike SchaÈcke,* Elizabeth L. Rugg,³ E. Birgitte Lane,³ Edgar Frenk, Daniel Hohl, and Leena Bruckner-Tuderman*
Department of Dermatology, CHUV-DHURDV, Lausanne, Switzerland; *Department of Dermatology, University Hospital MuÈnster, MuÈnster, Germany; ²Department of Dermatology, CMU-DHURDV, Geneva, Switzerland; ³CRC Cell Structure Research Group, Cancer Research Campaign Laboratories, Department of Anatomy and Physiology, University of Dundee, Dundee, U.K.
BP180/collagen XVII cDNA revealed a 1172 bp deletion corresponding to an in-frame deletion from Ile18 to Asn-407 from the intracellular domain of the polypeptide. Mutation analysis of the COL17A1 gene disclosed a paternal nonsense mutation, R1226X, and a large maternal genomic deletion extending from intron 2 to intron 15, but no mutations in basal keratin genes. These ®ndings underline the functional importance of the intracellular BP180/collagen XVII domain for the interaction of hemidesmosomes with keratin intermediate ®laments and for the spatial stability of basal keratinocytes, and provide a functional explanation for the epidermolysis-bullosasimplex-like phenotype. Further, the data demonstrate that defects in a given gene can cause unexpected phenotypes of epidermolysis bullosa categories, depending on the function of the affected protein domain. Key words: bullous pemphigoid/genodermatoses/mutation. J Invest Dermatol 118:185±192, 2002
BP180/collagen XVII is a hemidesmosomal transmembrane molecule serving as cell-surface receptor. Mutations in its gene cause junctional epidermolysis bullosa. Here, we report a patient with mutations in the gene for BP180/collagen XVII, COL17A1, but predominant phenotypic features of epidermolysis bullosa simplex. At birth, the proband presented with bullous lesions on the trunk, face, and hands. Ultrastructurally, hemidesmosomes were fairly normal, but the attachment of intermediate ®laments with the hemidesmosomal plaques appeared to be impaired. Blister formation demonstrated both intraepidermal and junctional cleavage. Immuno¯uorescence staining with antibodies to keratins, several hemidesmosomal proteins, and the extracellular domain of BP180/collagen XVII showed normal staining patterns, whereas an antibody against the intracellular domain of BP180/collagen XVII yielded a negative immuno¯uorescence signal. Analysis of
T
Hintner and Wolff, 1982; Fine et al, 2000). At the ultrastructural level, cleavage occurs in the lamina lucida or at the lamina lucida/ basal cell interface. Hemidesmosomes and the associated anchoring ®laments are rudimentary or absent. Immuno¯uorescence staining of EB skin with antibodies to the structural components of the basement membrane zone is the key diagnostic tool used to determine the EB category (Fine et al, 2000). In JEB, the staining shows hemidesmosome components in the blister roof and lamina densa components in the blister base. In addition, immuno¯uorescence studies reveal abnormal expression of BP180/collagen XVII (Jonkman et al, 1995; McGrath et al, 1995; Pohla-Gubo et al, 1995) or of laminin 5 (Jonkman et al, 1995) in JEB skin. BP180/collagen XVII, initially identi®ed as a major target of IgG autoantibodies in bullous pemphigoid patients, is a transmembrane glycoprotein with a type II orientation, i.e., the amino-terminal domain localizes to the cytoplasm and the extracellular carboxyterminal end spans the lamina lucida of the basal membrane zone (Li et al, 1993; Masunaga et al, 1997). It is a structural component of hemidesmosomes in strati®ed and other complex epithelia, such as skin, mucous membranes, and the eyes. Recent cell transfection and yeast-two hybrid experiments have demonstrated that the intracellular domain of BP180/collagen XVII interacts with b4 integrin and BP230, and the juxtamembranous extracellular
he name epidermolysis bullosa (EB) refers to the mechanically induced detachment of the epidermis from the dermis, into the blister roof. Based on ultrastructural criteria, EB is divided into three main categories according to the precise level of blister formation: simplex, junctional, and dystrophic subtypes (Fine et al, 2000). In EB simplex (EBS), a disease caused by keratin or plectin mutations (Corden and McLean, 1996; Uitto et al, 1996), the tissue separation takes place within the basal keratinocytes, whereas in junctional EB (JEB) the cleavage occurs along the lamina lucida of the basement membrane zone. JEB is a heterogeneous group of autosomal recessive bullous disorders (Darling et al, 1997; Pulkkinen and Uitto, 1998). The patients are clinically characterized by life-long blistering of skin and mucous membranes, diffuse alopecia, partial absence of eyelashes, eyebrows, pubic and axillary hair, nail dystrophy, and dental anomalies (Hashimoto et al, 1976;
Manuscript received February 20, 2001; revised July 19, 2001; accepted for publication September 3, 2001. Reprint requests to: Dr. Daniel Hohl, CHUV-Service de dermatologie, HoÃpital de Beaumont, 1011 Lausanne, Switzerland. Email: [email protected] Abbreviations: BP180, bullous pemphigoid 180; EB, epidermolysis bullosa. 0022-202X/01/$15.00
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 185
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domain with a6 integrin. These interactions are important in the formation and stabilization of the hemidesmosomes (Hopkinson et al, 1995; Borradori et al, 1997; Hopkinson and Jones, 2000). Recently, a number of mutations in the COL17A1 gene, encoding BP180/collagen XVII, have been described (Pulkkinen and Uitto, 1998; 1999). The majority of them are premature termination codons resulting in accelerated mRNA decay, but in some cases missense mutations have been found (McGrath et al, 1996; Schumann et al, 1997; Floeth et al, 1998). A small number of patients with a nonlethal JEB phenotype show mutations in the genes coding for laminin 5 (Jonkman et al, 1996; Pulkkinen et al, 1998) with normal BP180/collagen XVII expression. Finally, a family with bigenic inheritance, with mutations in both the COL17A1 and LAMB3 genes, was recently identi®ed (Floeth and Bruckner-Tuderman, 1999). Here we report a striking case presenting with characteristic phenotypic features of mainly EBS. At the molecular level, the phenotype was caused by compound heterozygous mutations in the COL17A1 gene. The paternal mutation caused a premature stop codon at position 1226, and the maternal mutation was a large genomic deletion extending from intron 2 through intron 15. This leads to in-frame skipping of exons 3±15, a gene segment that encodes for a large portion of the cytoplasmatic domain of BP180/ collagen XVII. MATERIALS AND METHODS Clinical case The male proband of this study was born at the thirtyninth week of gestation after an uneventful pregnancy. He was the offspring of noncosanguinous healthy parents and had two older maternal half-brothers who were clinically unaffected. At birth, he was noted to have disseminated isolated and grouped blisters on the scalp, face, hands, feet, buttocks and scrotum, nail beds and buccal mucosa (Fig 1A). No other abnormalities or developmental defects were noted. With advancing age, blisters tended to occur on physically stressed skin of the face, hands, and feet, and healed initially without scarring (Fig 1B). At the age of 5 mo milia were noted on the left heel. Currently, this 3-yold patient shows disseminated blistering and mild atrophic scarring on knees and elbows, as well as bilateral thickening of big and second toe nails. Otherwise, the patient shows normal skin, adnexal, and mucosal structures, i.e., no signs of alopecia, loss of eyelashes, enamel defects, or caries. Informed consent was obtained from the parents prior to further investigations. Human in vivo studies were approved by the local research ethics review committee. Electron microscopy Electron microscopic examination of a skin biopsy from clinically unaffected skin was performed using standard methods (Frenk et al, 1996). In short, 1 mm3 nonlesional skin samples were ®xed in 2.5% glutaraldehyde, post®xed in osmium tetraoxide, and embedded in Epon. Ultrathin sections were stained in uranyl acetate and lead citrate and observed using a Phillips CM10 electron microscope. Cell culture Punch biopsies obtained from the patient and normal volunteers were used to establish primary keratinocytes on lethally irradiated murine 3T3 ®broblasts in keratinocyte complete medium (Dulbecco's modi®ed Eagle's medium/Ham's F12 3:1, 10% fetal bovine serum, hydrocortisone 0.4 mg per ml, choleratoxin 0.1 nM, insulin 5 mg per ml, adenine 20 mg per ml, apotransferrin 5 mg per ml, triiodothyronine 2 nM, epidermal growth factor 10 ng per ml, penicillin 100 U per ml, and streptomycin 0.1 mg per ml) (Rheinwald and Green, 1975; 1977; Green et al, 1979). Keratinocytes at passages 2±6 were used for the experiments. Cell extracts used for immunoblot analysis were isolated from keratinocytes cultured in serum-free keratinocyte growth medium (Sigma, Buchs, Switzerland) containing 50 mg of ascorbic acid per ml for 48 h before harvest. Isolation of RNA and northern blot analysis Total RNA was isolated using the guanidine-thiocyanate method (Chomczynski and Sacchi, 1987). Denaturing of RNA gels using 13 mg of total RNA per lane and transfer to Zetaprobe membrane (Bio-Rad, Hercules, CA) were carried out as described earlier (Huber et al, 1997). Membranes were hybridized with 32P-labeled 5¢ (residues 97±636) and 3¢ (residues 3665± 4402) probes of the BP180/collagen XVII cDNA (GenBank M91669). Final washes were performed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate, pH 7, at 65°C for 30 min.
Figure 1. Clinical features of the patient. (A) Presence of grouped blisters on the buttock at birth. (B) Patient at the age of 7 mo showing blisters at different stages after trauma, which were healing without scarring.
Southern blot analysis Genomic DNA was digested with XhoI (Roche Biochemicals, Rotkreuz, Switzerland) according to the recommendations of the supplier. The digested DNA was electrophoresed and transfered to Zetaprobe membrane (Bio-Rad, Switzerland). Membranes were probed with a 32P-labeled fragment of exon 2 of the COL17A gene (nucleotides 103955±104205; GenBank AL138761). After overnight incubation at 65°C, the membrane was washed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate, pH 7, at 65°C for 30 min and then exposed for 7 d to XAR-5 autoradiographic ®lm. Cloning of cDNA by reverse transcription polymerase chain reaction (RT-PCR) To analyze cDNA fragments from the patient and unaffected individuals, total RNA from cultured keratinocytes was used for RT-PCR using the Advantage cDNA PCR kit (Clontech, Palo Alto, CA) with the primers BP7 (5¢-ATTCCTTCAAAACCTCAAACC-3¢; nucleotides 72±92 of BP180/collagen XVII cDNA M91669) and BP8 (5¢-GCTCCTCCTGGCTGTCACTGT-3¢; nucleotides 1116± 1095 of BP180/collagen XVII cDNA M91669). Reaction conditions were as follows: 94°C for 2 min, and then 35 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 3 min. Ampli®ed fragments were cloned into the pGEM-Teasy vector (Promega, Madison, WI) and subjected to sequence analysis. Protein concentrations Protein content was determined with the Bradford assay (Bio-Rad) using bovine serum albumin as standard (Bradford, 1976). Mutation detection Genomic DNA was isolated from peripheral blood using the QIAamp blood kit (Qiagen, Basel, Switzerland). PCR ampli®cation of all COL17A1 (GenBank U76564±U76604) exons was carried out as described previously (Gatalica et al, 1997). Ampli®cation of COL17A1 exon 51 was performed using the following primers: sense
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Figure 2. Immuno¯uorescence staining for the intracellular domain of BP180/collagen XVII is absent in the patient's skin. Frozen sections from control and patient skin were incubated with antibodies against the intracellular and extracellular BP180/collagen XVII domains. (A) Staining of normal control skin with antibodies to the extracellular NC16a domain of BP180/collagen XVII. (B) Strongly reduced staining of the patient's skin with the NC16a domain antibodies in one skin biopsy. The faint staining is localized in the roof of a small blister (the star denotes the blister cavity), indicating junctional blister formation. (C) Positive staining of the patient's skin with the NC16a antibodies in a second skin biopsy. In this specimen, the signal is observed on the ¯oor of the blister, indicating intraepidermal splitting. (D) Staining of control skin with antibody 1A8c against the intracellular domain of BP180/collagen XVII. (E) Negative staining of patient's skin with antibody 1A8c. (F) Positive staining of patient's skin with antibody 1D1 against the extracellular domain of BP180/ collagen XVII.
primer 5¢-TTTCTCTCCTCCCATCACCC-3¢ and antisense primer 5¢TGTCCCTTTAAGTGCCTCCC-3¢. The size of the PCR product was 374 bp. Heteroduplex analysis was performed according to Ganguly et al (1993), and heteroduplex-forming PCR products were sequenced with an automated sequencer (Genome Express, Grenoble, France). The deletion was de®ned using PCR ampli®cation of COL17A1 exons 2±16 directly from genomic DNA. The primers used were sense primer 5¢-TTATCAGCTTCAACAGTGGG-3¢ and antisense primer 5¢CATCATCTGGTCCATTAGCC-3¢. The PCR ampli®cation of normal control DNA showed a number of large products, but the ampli®cation of the patient's DNA generated a distinct band of about 1500 bp, which was further characterized by southern blotting. The hybridization was performed with a BP180/collagen XVII cDNA fragment spanning exons 14±17, which was labeled with digoxigenin (Digoxigenin DNA Labeling Kit, Roche, Mannheim, Germany). The detection was performed with alkaline-phosphatase-labeled digoxigenin antibodies and CDP-Star substrate. The dideoxynucleotide sequencing of the 1500 bp fragment was performed with nested PCR and automated sequencing in both directions. For detection of the entire sequence, additional intronic primers were used for ampli®cation: sense primer 5¢-TTATGTGTCAAAGAATCCCAAAGGTC-3¢ (designated TH2up) and antisense primer 5¢TTGTTCCTGAGTCCGTGATGC-3¢ (TH16do). The sequence was veri®ed by digesting the fragment with EcoRI/PvuII and cloning into the EcoRI/EcoRV sites of the pBIISK+ vector (Stratagene, La Jolla, CA), followed by automated sequencing. For sequencing of intron 2 in normal controls, the sense primer was TH2up and the antisense primer was 5¢-GGAAGGGTGCTCCTGGTAAGTC-3¢ (NK3do). For sequencing of intron 15 in normal controls, the sense primer was 5¢AACCACATTTCTTGACGATGCC-3¢ (NK15up) and the antisense primer was TH16do. PCR ampli®cation and sequence analysis of keratin genes K5 and K14 were carried out as described previously (Shemanko et al, 2000). Antibodies and immunodetection of proteins Antigen mapping (Hintner et al, 1981) was performed using the following antibodies for immuno¯uorescence staining of 5 mm cryosections of the patient's and control skin: cytokeratins (Dako, Hamburg, Germany), BP230 (a high titer autoantiserum), a BP180/collagen XVII antibody raised against the recombinant NC16a domain (Schumann et al, 2000), BP180/collagen XVII antibodies 1A8c and 1D1 (kind gift of Dr. K. Owaribe, Nagoya University, Nagoya), a6 and b4 integrins (Gibco Life Technologies, Eggenstein, Germany), collagen IV (Dako), laminin 5 (kind gift from Dr. R. Burgeson, CBRC, Harvard Medical School, Cambridge, MA), and collagen VII (Bruckner-Tuderman et al, 1995). For immunoblotting, keratinocytes were extracted with a buffer containing 0.1 M NaCl, 20 mM Tris±HCl, pH 7.4, 1% Nonidet P-40, and proteinase inhibitors (Schumann et al, 1997; SchaÈcke et al, 1998), and the proteins were separated on a 4.5±15% sodium dodecyl sulfate polyacrylamide gel. Proteins in the culture medium were precipitated with chloroform/methanol and subjected to immunoblotting in a similar
manner. As antibodies, polyclonal antibodies against a recombinant fragment (ecto-1) spanning about 200 of the most carboxy-terminal amino acids of BP180/collagen XVII, the NC16a antibody against the recombinant NC16a domain, and the antibody Ab-Col15-2 against the recombinant Col15 domain (SchaÈcke et al, 1998; Schumann et al, 2000; Tasanen et al, 2000) were used.
RESULTS Immuno¯uorescence analysis reveals absence of BP180/ collagen XVII intracellular domain The patient's clinical symptoms at birth were compatible with EB. This was re¯ected by histology showing that during blister formation epidermal±dermal tissue separation could occur either at the level of the lamina lucida of the basement membrane or within the basal keratinocytes (Fig 2). Further investigation with antigen mapping of the patient's skin demonstrated that staining patterns for keratins K5 and K14, a6b4 integrin, BP230, collagen IV, plectin, and laminin 5 were normal. In contrast, the signals with antibodies against BP180/ collagen XVII were unanticipated. Monoclonal antibody 1A8c against the intracellular domain exhibited no staining at all, but antibodies against the extracellular domain showed normal signals in most regions of the epidermal basement membrane zone (Fig 2). These ®ndings were suggestive of aberrant expression of BP180/ collagen XVII in the patient's skin. As the EBS-like features were prominent, cultured keratinocytes derived from the patient's skin were stained with antibodies against keratins K5 and K14. No balllike aggregates typical for cultured EBS cells were found in the proband's keratinocytes, however, suggesting that keratin intermediate ®laments were formed normally (data not shown). Keratin intermediate ®laments are abnormally attached to hemidesmosomes Electron microscopic examination of the proband's dermo-epidermal junction revealed hemidesmosomes of varying width and structure. Within the basal keratinocytes, the keratin intermediate ®lament bundles did not appear to properly attach to the hemidesmosomal cytoplasmic inner plaque. The other morphologic structures of the basement membrane zone, i.e., the lamina lucida, lamina densa, and the anchoring ®brils, appeared normal (Fig 3). No full-length BP180/collagen XVII mRNA or protein is found in cultured keratinocytes from the patient Northern blot analysis of total RNA obtained from cultured normal keratinocytes demonstrated a 6 kb mRNA band with BP180/ collagen XVII cDNA probes from the 5¢ and 3¢ end (Fig 4). In contrast, when total RNA from the patient's keratinocytes was
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Figure 3. Electron microscopy reveals abnormalities in hemidesmosome and keratin intermediate ®lament structures. Heterogeneous hemidesmosomes of varying size and structure were found in the proband's skin. Moreover, the keratin intermediate ®lament bundles do not extend into the hemidesmosomes. The other ultrastructural units of the basement membrane zone, lamina lucida, lamina densa, and the anchoring ®brils appeared normal. Bar: 0.25 mm.
hybridized with a 5¢ end probe that spans the intracellular domain of the protein, no signal was found. As a 3¢ probe revealed a diminished size of BP180/collagen XVII mRNA, the ®ndings were suggestive for an internal deletion within the 5¢ segment (Fig 4). Immunoblot analysis of keratinocyte extracts and media was performed to visualize a putative truncated polypeptide corresponding to the short mRNA observed with northern blotting. Antibodies recognizing the extracellular domain BP180/collagen XVII revealed two bands in extracts of normal keratinocytes, with molecular weights of 180 and 120 kDa (Fig 5). The 180 kDa band corresponds to the full-length transmembrane molecule, and the 120 kDa band to the soluble, shed ectodomain (SchaÈcke et al, 1998). In culture medium, only the 120 kDa ectodomain was present. Analysis of the patient's keratinocytes showed the presence of the 120 kDa band in the medium and cell extracts, whereas the 180 kDa transmembrane form was absent. Instead, the cell extract contained a polypeptide of approximately 130 kDa (Fig 5). This truncated polypeptide was recognized by antibodies to the NC16a domain and to the distal C-terminus of the molecule, indicating that the entire ectodomain was present. In contrast, antibodies to the intracellular domain did not recognize it. Thus, the protein results were in agreement with the mRNA analysis and implicated a mutation that produced an in-frame deletion in the 5¢ end of the mRNA and yielded a BP180/collagen XVII polypeptide chain that was shortened by about 50 kDa in its intracellular domain. Nevertheless, the truncated molecule was anchored to the cell plasma membrane and shed in an apparently normal manner. Mutational analysis demonstrates a large genomic deletion from intron 3 to intron 15 due to a 42 bp sequence homology The initial search for mutations in the keratin genes K5 and K14 did not reveal any sequence abnormalities. RT-PCR analysis of the patient's keratinocyte RNA using the BP180/ collagen XVII primers BP7 and BP8 (see Materials and Methods) revealed a 500 bp band, compared with a 1600 bp band obtained with control RNA (data not shown). Cloning and sequencing of the 500 bp band revealed a large deletion in the cDNA extending from nucleotide position 158 to 1329, which corresponded
Figure 4. Northern blot analysis shows a large deletion in the 5¢ part of the BP180/collagen XVII mRNA. Total RNA was isolated from keratinocytes cultured in high calcium medium established from the patient (lanes 1, 4) and from normal control individuals (lanes 2, 3, 5, 6). The northern blot was hybridized with a BP180/collagen XVII 5¢ probe (nucleotides 97±636, GenBank M91669) in lanes 1±3, or with a BP180/collagen XVII 3¢ probe (nucleotides 3555±4402, GenBank M91669) in lanes 4±6. As loading control glyceraldehyde-3-phosphatedehydrogenase was used to ensure equal amounts of RNA.
precisely to the omission of exons 3 through 15. This translates to an in-frame deletion in the polypeptide from Ile-18 to Asn-407, thus encompassing a major part of the intracellular domain of the BP180/collagen XVII polypeptide. Heteroduplex analysis of genomic DNA of the patient provided evidence for two different genetic aberrations in the COL17A1 gene. Dideoxynucleotide sequencing revealed a heterozygous Cto-T transition at position 3781 leading to the premature termination codon R1226X (Fig 6B). The mutation eliminated the consensus sequence for TaqI restriction enzyme, and veri®cation by TaqI digestion showed that it was paternally inherited (Fig 6A, B). PCR analysis with primers placed in exons 2 and 16 generated a 1500 bp amplimer with the patient's and his mother's DNA, but not with DNA from the father or from normal controls. This
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Figure 5. Immunoblot analysis reveals that patient keratinocytes produce only BP180/collagen XVII of 130 kDa molecular weight. Proteins extracted from cultured cells and culture medium were immunoblotted with the NC16a antibody. Lane 1: Both full-length BP180/collagen XVII (upper arrowhead) and the shed ectodomain (lower arrowhead) were present in cell extracts from normal keratinocytes (Co). Lane 2: The shed ectodomain was precipitated from the medium of normal keratinocytes. Lane 3: The cell extracts of the patient's keratinocytes (EB) contained mutant BP180/collagen XVII polypeptide of approximately 130 kDa (arrow) and the shed ectodomain (lower arrowhead). Lane 4: The soluble ectodomain was present in the medium of the patient's keratinocytes (lower arrowhead). Identical results were obtained when using antibodies against the carboxy-terminal region or against the Col15 domain (see Materials and Methods). On the right, the migration positions of molecular weight standards, from top to bottom 200 kDa, 111 kDa, 80 kDa, and 46 kDa, are shown.
suggested a maternally inherited deletion that reached from intron 2 to intron 15. Subsequent sequence analysis of normal controls disclosed an identical 42 nucleotide stretch in both introns 2 and 15. In the proband's DNA, only one 42 bp stretch was present and it was ¯anked at the 5¢ end by a normal intron 2 sequence and at the 3¢ end by a normal intron 15 sequence (Fig 6C). Searching the COL17A sequence (GenBank AL138761) with the 42 bp sequence shows indeed that introns 2 and 15 contain this sequence (nucleotides 103161±103203 and 82167±82209); in both cases this sequence is part of the highly conserved AluSg repeats. Southern blot analysis using XhoI-digested DNA and a BP180/ collagen XVII exon 2 probe revealed, as predicted by the COL17A sequence (GenBank AL138761), a 27 kbp fragment in normal DNA and two bands at 27 and 6 kbp in patient DNA (Fig 7). This argues that an Alu-mediated mutational event had occurred that removed the 21 kbp stretch of DNA between the two 42 bp repeats. The deletion was not found in either 150 normal chromosomes or 28 chromosomes of other unrelated EB patients, indicating that it does not represent a neutral polymorphism. Taken together, the mutation analysis, combined with RNA and protein assays, showed that the patient was hemizygous for the deletion. Like many mutations leading to premature termination codons, R1226X led to nonsense-mediated mRNA degradation, as both northern blots and immunoblots failed to demonstrate the presence of full-length BP180/collagen XVII mRNA or polypeptide chains. DISCUSSION BP180/collagen XVII, a type II transmembrane protein, presumably functions as a cell-adhesion receptor providing a connection between extracellular matrix ligands and the intracellular keratinocyte intermediate ®lament network. Support for this role emerged from its abnormal expression caused by mutations in the COL17A1 gene in JEB patients (Pulkkinen and Uitto, 1999). Absence of BP180/collagen XVII as a consequence of null mutations leads to severe epidermal dysadhesion, whereas missense mutations causing structural abnormalities in the extracellular domain lead to
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unfolding of the triple helix, susceptibility to proteolytic degradation, and functional insuf®ciency (Tasanen et al, 2000). In this study, we investigated an EB patient with phenotypic features of mainly EBS. This analysis provides genetic evidence for the function of BP180/collagen XVII as linker molecule between the keratin intermediate ®lament network and the extracellular matrix proteins at the dermal±epidermal junction. Whereas the expression of several hemidesmosomal proteins and their ligands, e.g., a6b4 integrin, BP230, collagen IV, plectin, laminin 5, and keratins K5 and K14, was normal, the expression of BP180/ collagen XVII was aberrant. The absence of the intracellular domain of BP180/collagen XVII was evident by immuno¯uorescence, immunoblot, and northern blot analysis, whereas the extracellular segment of the polypeptide was present normally in the patient's skin. Genetic analysis revealed compound heterozygosity of the proband for two different aberrations in the COL17A1 gene. The paternal defect R1226X is a recurrent mutation that has been described in both homozygous and heterozygous forms (Jonkman et al, 1997; Schumann et al, 1997; Floeth et al, 1998; Floeth and Bruckner-Tuderman, 1999). The premature termination codon presumably leads to nonsensemediated mRNA decay (Schumann et al, 1997; Floeth et al, 1998; Floeth and Bruckner-Tuderman, 1999) as northern blots showed no evidence for the expression of normal size mRNA. The proband's cells contained a 130 kDa truncated polypeptide; however, it was not clear whether the shortened length resulted from a deletion or, potentially, from a C-terminal truncation caused by the premature termination codon. Immunoblotting with a panel of domain-speci®c BP180/collagen XVII antibodies identi®ed only one kind of truncated chain, which spanned the entire ectodomain but lacked a segment from the intracellular domain, thus excluding the possibility of a C-terminally truncated BP180/collagen XVII polypeptide. Thus, the proband was functionally homozygous (hemizygous) for the maternal mutation. This resulted in skipping of exons 3±15 and elimination of amino acids 18±407 from the BP180/collagen XVII polypeptide, generating an intracellular domain that consisted of only 76 amino acids instead of the normal 466 residues. This ®nding was consistent with the negative immuno¯uorescence and immunoblot ®ndings obtained with monoclonal antibody 1A8c, the epitope of which has been mapped to the region between the amino acids 113±201 of BP180/collagen XVII (Borradori et al, 1997). The truncated molecule is stable, and both mRNA and protein expression levels seemed to be equivalent to those in normal individuals. The 21 kbp genomic deletion extends from intron 2 to intron 15. Both introns contain the same perfectly matched 42 nucleotide long region (5¢-aactcctgacctcgtgatctgcccgcctcggcctcccaaagt-3¢), which is part of a larger (approximately 300 bp) repeat homologous to the Sg member of the Alu repeat family (GenBank AL138761). Sequences similar to these two Alu repeats are not found elsewhere in the COL17A1 gene. Both Alu repeats have the same orientation and display a sequence similarity of 84%, which is even higher around the 42 bp region of homology. Introns 2 and 15 contain in addition to the Alu repeats other interspersed repetitive elements such as (GT)n (TGTA)n, MER41B, and MER5A repeats. Therefore, it seems likely that the genomic deletion is due to a recombination event that occurred between the two AluSg repeats using the 42 bp stretch as crossover point. Alu-mediated genomic rearrangements have been shown to be involved in other inherited diseases such as Fanconi anemia (Morgan et al, 1999), familial hypercholesterolemia (Lehrman et al, 1987), hereditary angiedema (Stoppa-Lyonnet et al, 1990), and pseudoxanthoma elasticum (Ringpfeil et al, 2001), as well as in genetic predisposition for colon and breast cancer (NystroÈm-Lahti et al, 1995; Puget et al, 1999). Moreover, tandem duplication mediated by Alu-dependent recombination in somatic tissue has been reported in cases of acute myeloid leukemia (Strout et al, 1998). It is likely that secondary structures formed by the regions of homology and components of
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Figure 6. Pedigree and mutation analysis in the COL17A1 gene. (A) The pedigree of the family demonstrates that the mutation R1226X (black areas) and the genomic deletion (dotted areas) of the proband (1) occurred in a compound heterozygous manner, because the father (3) is the carrier of R1226X and the mother (2) of the deletion. (B) Sequence analysis of the patient's PCR product containing exon 51 showed a C-toT transition at nucleotide position 3781, designated R1226X. A restriction endonuclease site for Taq1 is eliminated by the mutation. Accordingly, the 342 bp PCR product (upper band) of a normal control (C1) and the mother (2) were cleaved into a 185 bp (lower band) and a 157 bp fragment, whereas the proband (1), the father (3), and a previously characterized unrelated patient with the same mutation (C2) showed an additional uncleaved band of 342 bp. (C) The maternal mutation was a large genomic deletion, extending from intron 2 to intron 15. In normal controls, an identical 42 bp sequence was found within both introns. In the proband's DNA, this 42 bp sequence is present once and is ¯anked at the 5¢ end by the sequence corresponding to normal intron 2 (underlined) and at the 3¢ end by the sequence corresponding to normal intron 15 (doubly underlined).
the DNA replication and recombination machinery may play a role in the generation and subsequent deletion of loop structures. Recent cell biologic studies provide explanations for the biologic and clinical phenotype of the present proband. They have shown that the intracellular domain of BP180/collagen XVII contains sequences important for the localization of the protein into hemidesmosomes and its interaction with other hemidesmosomal components, such as the intracellular domain of the b4 integrin subunit and the N-terminal region of BP230 (Hopkinson et al, 1995; Borradori et al, 1997; Aho and Uitto, 1998; Schaapveld et al, 1998; Hopkinson and Jones, 2000). There is strong evidence that an N-terminal 401 amino acid stretch of BP180/collagen XVII contains binding sites to the N-terminal region of BP230 and the C-terminal half of the cytoplasmic domain of b4 integrin (Geerts et al, 1999; Koster et al, 1999; Hopkinson and Jones, 2000). In addition, the cytoplasmic domain of BP180/collagen XVII may also associate with plectin (Aho and Uitto, 1997). Therefore, the deletion of the region Ile18±Asn407 is expected to impair the association of BP180/collagen XVII not only with b4 integrin and BP230 but also with plectin. Both plectin and BP230 are cytoplasmic components of hemidesmosomes that are believed to attach the keratin cyto-
skeleton to the hemidesmosomal plaque and, by means of interactions with the cytoplasmic domains of BP180/collagen XVII and b4 integrin, to the plasma membrane (Guo et al, 1995; Uitto et al, 1996; Andra et al, 1997; Borradori and Sonnenberg, 1999). Therefore, it is likely that the poor linkage of intermediate ®laments to hemidesmosomes observed by electron microscopy in the proband's skin results from an impaired incorporation and/or stabilization of BP230 and plectin into hemidesmosomes as a result of perturbed interactions with the cytoplasmic domain of BP180/ collagen XVII. Moreover, the reduced attachment of keratin ®laments to the basal plasma membrane might account for the increased mechanical fragility of keratinocytes, thus explaining the blistering arising in the basal layer and the clinical EBS-like phenotype. The extracellular domain of BP180/collagen XVII appeared to be expressed normally as shown by immunoblot analysis. In concert, antibodies to the extracellular domain of BP180/collagen XVII showed linear immuno¯uorescence staining along the basement membrane zone of the patient's skin, a staining pattern indistinguishable from normal controls. Nevertheless, as also junctional cleavage was seen in the proband's skin, dermoepidermal cohesion must be impaired as well. It is likely that the
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Figure 7. Southern blot analysis of the maternal deletion. Genomic DNA from the patient (lane 1) and a normal individual (lane 2) was digested with XhoI and hybridized with a BP180/collagen XVII exon 2 probe. The arrowhead marks the start of the electrophoresis. Arrows mark the position of the molecular weight markers of 21 kbp, 5.2 kbp, 4.3 kbp, 3.5 kbp, and 2 kbp (top to bottom).
large deletion of the cytoplasmic domain BP180 not only compromises molecular associations important for the stabilization, and thus function, of hemidesmosomes, but also has an impact on the interaction of the extracellular domain of BP180/collagen XVII with extracellular matrix protein(s). The genetic, biochemical, and clinical data obtained here extend the spectrum of the phenotypic heterogeneity of EB. They demonstrate for the ®rst time that defects in a given gene can give rise to clinical and biologic phenotypes characteristic of different EB categories, depending on the function of the protein domain affected by the mutation. This work also underlines the importance of using intracellular-domain-speci®c BP180/collagen XVII antibodies for assessment by antigen mapping of EB patients to avoid faulty diagnosis. The authors thank S. Aebischer, M. Schubert, and A. Wissel for excellent technical assistance. This work was supported by grant 31-45943.95 (Swiss National Science Foundation) to D.H., by grant 31-56727.99 (Swiss National Science Foundation) and by the Fondation TeÂleÂthon Action Suisse (Aubonne, Switzerland) to L.B., by grants Br 1475/1±2 and SFB 293-B3 from the Deutsche Forschungsgemeinschaft, and by EU contract BMH-4-97-2062 to L.B-T.
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J Biol Chem 272:21018±21026, 1997 Jonkman MF, de Jong MC, Heeres K, et al: 180-kD bullous pemphigoid antigen (BP180) is de®cient in generalized atrophic benign epidermolysis bullosa. J Clin Invest 95:1345±1352, 1995 Jonkman MF, de Jong MC, Heeres K, et al: Generalized atrophic benign epidermolysis bullosa. Either 180-kd bullous pemphigoid antigen or laminin5 de®ciency. Arch Dermatol 132:145±150, 1996 Jonkman MF, Scheffer H, Stulp R, et al: Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 88:543±551, 1997 Koster J, Favre B, Geerts D, Sonnenberg A, Borradori L: Characterization of domains within bullous pemphigoid antigen 230 important for the assembly of hemidesmosomes. Arch Dermatol Res 91:120, 1999 Lehrman MA, Goldstein JL, Russell DW, Brown MS: Duplication of seven exons in LDL receptor gene caused by Alu±Alu recombination in a subject with familial hypercholesterolemia. Cell 48:827±835, 1987 Li K, Tamai K, Tan EM, Uitto J: Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5¢-end of the gene and the 3¢-untranslated region of the mRNA. J Biol Chem 268:8825±8834, 1993 Masunaga T, Shimizu H, Yee C, Borradori L, Lazarova Z, Nishikawa T, Yancey KB: The extracellular domain of BPAG2 localizes to anchoring ®laments and its carboxyl terminus extends to the lamina densa of normal human epidermal basement membrane. J Invest Dermatol 109:200±206, 1997 McGrath JA, Gatalica B, Christiano AM, et al: Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa. 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Schaapveld RQ, Borradori L, Geerts D, et al: Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/ plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180. J Cell Biol 142:271±284, 1998 SchaÈcke H, Schumann H, Hammami-Hauasli N, Raghunath M, BrucknerTuderman L: Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain. J Biol Chem 273:25937± 25943, 1998 Schumann H, Hammami-Hauasli N, Pulkkinen L, et al: Three novel homozygous point mutations and a new polymorphism in the COL17A1 gene: relation to biological and clinical phenotypes of junctional epidermolysis bullosa. Am J Hum Genet 60:1344±1353, 1997 Schumann H, Baetge J, Tasanen K, Wojnarowska F, Schacke H, Zillikens D, Bruckner-Tuderman L: The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am J Pathol 156:685±695, 2000 Shemanko CS, Horn HM, Keohane SG, et al: Laryngeal involvement in the Dowling±Meara variant of epidermolysis bullosa simplex with keratin mutations of severely disruptive potential. Br J Dermatol 142:315±320, 2000 Stoppa-Lyonnet D, Carter PE, Meo T, Tosi M: Clusters of intragenic Alu repeats predispose the human C1 inhibitor locus to deleterious rearrangements. Proc Natl Acad Sci USA 87:1551±1555, 1990 Strout M, Marcucci G, Bloom®eld C, Caligiuri M: The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc Natl Acad Sci USA 95:2390± 2395, 1998 Tasanen K, Eble JA, Aumailley M, et al: Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain col15. J Biol Chem 275:3093± 3099, 2000 Uitto J, Pulkkinen L, Smith FJ, McLean WH: Plectin and human genetic disorders of the skin and muscle. The paradigm of epidermolysis bullosa with muscular dystrophy. Exp Dermatol 5:237±246, 1996
Department of Dermatology, CHUV-DHURDV, Lausanne, Switzerland; *Department of Dermatology, University Hospital MuÈnster, MuÈnster, Germany; ²Department of Dermatology, CMU-DHURDV, Geneva, Switzerland; ³CRC Cell Structure Research Group, Cancer Research Campaign Laboratories, Department of Anatomy and Physiology, University of Dundee, Dundee, U.K.
BP180/collagen XVII cDNA revealed a 1172 bp deletion corresponding to an in-frame deletion from Ile18 to Asn-407 from the intracellular domain of the polypeptide. Mutation analysis of the COL17A1 gene disclosed a paternal nonsense mutation, R1226X, and a large maternal genomic deletion extending from intron 2 to intron 15, but no mutations in basal keratin genes. These ®ndings underline the functional importance of the intracellular BP180/collagen XVII domain for the interaction of hemidesmosomes with keratin intermediate ®laments and for the spatial stability of basal keratinocytes, and provide a functional explanation for the epidermolysis-bullosasimplex-like phenotype. Further, the data demonstrate that defects in a given gene can cause unexpected phenotypes of epidermolysis bullosa categories, depending on the function of the affected protein domain. Key words: bullous pemphigoid/genodermatoses/mutation. J Invest Dermatol 118:185±192, 2002
BP180/collagen XVII is a hemidesmosomal transmembrane molecule serving as cell-surface receptor. Mutations in its gene cause junctional epidermolysis bullosa. Here, we report a patient with mutations in the gene for BP180/collagen XVII, COL17A1, but predominant phenotypic features of epidermolysis bullosa simplex. At birth, the proband presented with bullous lesions on the trunk, face, and hands. Ultrastructurally, hemidesmosomes were fairly normal, but the attachment of intermediate ®laments with the hemidesmosomal plaques appeared to be impaired. Blister formation demonstrated both intraepidermal and junctional cleavage. Immuno¯uorescence staining with antibodies to keratins, several hemidesmosomal proteins, and the extracellular domain of BP180/collagen XVII showed normal staining patterns, whereas an antibody against the intracellular domain of BP180/collagen XVII yielded a negative immuno¯uorescence signal. Analysis of
T
Hintner and Wolff, 1982; Fine et al, 2000). At the ultrastructural level, cleavage occurs in the lamina lucida or at the lamina lucida/ basal cell interface. Hemidesmosomes and the associated anchoring ®laments are rudimentary or absent. Immuno¯uorescence staining of EB skin with antibodies to the structural components of the basement membrane zone is the key diagnostic tool used to determine the EB category (Fine et al, 2000). In JEB, the staining shows hemidesmosome components in the blister roof and lamina densa components in the blister base. In addition, immuno¯uorescence studies reveal abnormal expression of BP180/collagen XVII (Jonkman et al, 1995; McGrath et al, 1995; Pohla-Gubo et al, 1995) or of laminin 5 (Jonkman et al, 1995) in JEB skin. BP180/collagen XVII, initially identi®ed as a major target of IgG autoantibodies in bullous pemphigoid patients, is a transmembrane glycoprotein with a type II orientation, i.e., the amino-terminal domain localizes to the cytoplasm and the extracellular carboxyterminal end spans the lamina lucida of the basal membrane zone (Li et al, 1993; Masunaga et al, 1997). It is a structural component of hemidesmosomes in strati®ed and other complex epithelia, such as skin, mucous membranes, and the eyes. Recent cell transfection and yeast-two hybrid experiments have demonstrated that the intracellular domain of BP180/collagen XVII interacts with b4 integrin and BP230, and the juxtamembranous extracellular
he name epidermolysis bullosa (EB) refers to the mechanically induced detachment of the epidermis from the dermis, into the blister roof. Based on ultrastructural criteria, EB is divided into three main categories according to the precise level of blister formation: simplex, junctional, and dystrophic subtypes (Fine et al, 2000). In EB simplex (EBS), a disease caused by keratin or plectin mutations (Corden and McLean, 1996; Uitto et al, 1996), the tissue separation takes place within the basal keratinocytes, whereas in junctional EB (JEB) the cleavage occurs along the lamina lucida of the basement membrane zone. JEB is a heterogeneous group of autosomal recessive bullous disorders (Darling et al, 1997; Pulkkinen and Uitto, 1998). The patients are clinically characterized by life-long blistering of skin and mucous membranes, diffuse alopecia, partial absence of eyelashes, eyebrows, pubic and axillary hair, nail dystrophy, and dental anomalies (Hashimoto et al, 1976;
Manuscript received February 20, 2001; revised July 19, 2001; accepted for publication September 3, 2001. Reprint requests to: Dr. Daniel Hohl, CHUV-Service de dermatologie, HoÃpital de Beaumont, 1011 Lausanne, Switzerland. Email: [email protected] Abbreviations: BP180, bullous pemphigoid 180; EB, epidermolysis bullosa. 0022-202X/01/$15.00
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 185
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domain with a6 integrin. These interactions are important in the formation and stabilization of the hemidesmosomes (Hopkinson et al, 1995; Borradori et al, 1997; Hopkinson and Jones, 2000). Recently, a number of mutations in the COL17A1 gene, encoding BP180/collagen XVII, have been described (Pulkkinen and Uitto, 1998; 1999). The majority of them are premature termination codons resulting in accelerated mRNA decay, but in some cases missense mutations have been found (McGrath et al, 1996; Schumann et al, 1997; Floeth et al, 1998). A small number of patients with a nonlethal JEB phenotype show mutations in the genes coding for laminin 5 (Jonkman et al, 1996; Pulkkinen et al, 1998) with normal BP180/collagen XVII expression. Finally, a family with bigenic inheritance, with mutations in both the COL17A1 and LAMB3 genes, was recently identi®ed (Floeth and Bruckner-Tuderman, 1999). Here we report a striking case presenting with characteristic phenotypic features of mainly EBS. At the molecular level, the phenotype was caused by compound heterozygous mutations in the COL17A1 gene. The paternal mutation caused a premature stop codon at position 1226, and the maternal mutation was a large genomic deletion extending from intron 2 through intron 15. This leads to in-frame skipping of exons 3±15, a gene segment that encodes for a large portion of the cytoplasmatic domain of BP180/ collagen XVII. MATERIALS AND METHODS Clinical case The male proband of this study was born at the thirtyninth week of gestation after an uneventful pregnancy. He was the offspring of noncosanguinous healthy parents and had two older maternal half-brothers who were clinically unaffected. At birth, he was noted to have disseminated isolated and grouped blisters on the scalp, face, hands, feet, buttocks and scrotum, nail beds and buccal mucosa (Fig 1A). No other abnormalities or developmental defects were noted. With advancing age, blisters tended to occur on physically stressed skin of the face, hands, and feet, and healed initially without scarring (Fig 1B). At the age of 5 mo milia were noted on the left heel. Currently, this 3-yold patient shows disseminated blistering and mild atrophic scarring on knees and elbows, as well as bilateral thickening of big and second toe nails. Otherwise, the patient shows normal skin, adnexal, and mucosal structures, i.e., no signs of alopecia, loss of eyelashes, enamel defects, or caries. Informed consent was obtained from the parents prior to further investigations. Human in vivo studies were approved by the local research ethics review committee. Electron microscopy Electron microscopic examination of a skin biopsy from clinically unaffected skin was performed using standard methods (Frenk et al, 1996). In short, 1 mm3 nonlesional skin samples were ®xed in 2.5% glutaraldehyde, post®xed in osmium tetraoxide, and embedded in Epon. Ultrathin sections were stained in uranyl acetate and lead citrate and observed using a Phillips CM10 electron microscope. Cell culture Punch biopsies obtained from the patient and normal volunteers were used to establish primary keratinocytes on lethally irradiated murine 3T3 ®broblasts in keratinocyte complete medium (Dulbecco's modi®ed Eagle's medium/Ham's F12 3:1, 10% fetal bovine serum, hydrocortisone 0.4 mg per ml, choleratoxin 0.1 nM, insulin 5 mg per ml, adenine 20 mg per ml, apotransferrin 5 mg per ml, triiodothyronine 2 nM, epidermal growth factor 10 ng per ml, penicillin 100 U per ml, and streptomycin 0.1 mg per ml) (Rheinwald and Green, 1975; 1977; Green et al, 1979). Keratinocytes at passages 2±6 were used for the experiments. Cell extracts used for immunoblot analysis were isolated from keratinocytes cultured in serum-free keratinocyte growth medium (Sigma, Buchs, Switzerland) containing 50 mg of ascorbic acid per ml for 48 h before harvest. Isolation of RNA and northern blot analysis Total RNA was isolated using the guanidine-thiocyanate method (Chomczynski and Sacchi, 1987). Denaturing of RNA gels using 13 mg of total RNA per lane and transfer to Zetaprobe membrane (Bio-Rad, Hercules, CA) were carried out as described earlier (Huber et al, 1997). Membranes were hybridized with 32P-labeled 5¢ (residues 97±636) and 3¢ (residues 3665± 4402) probes of the BP180/collagen XVII cDNA (GenBank M91669). Final washes were performed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate, pH 7, at 65°C for 30 min.
Figure 1. Clinical features of the patient. (A) Presence of grouped blisters on the buttock at birth. (B) Patient at the age of 7 mo showing blisters at different stages after trauma, which were healing without scarring.
Southern blot analysis Genomic DNA was digested with XhoI (Roche Biochemicals, Rotkreuz, Switzerland) according to the recommendations of the supplier. The digested DNA was electrophoresed and transfered to Zetaprobe membrane (Bio-Rad, Switzerland). Membranes were probed with a 32P-labeled fragment of exon 2 of the COL17A gene (nucleotides 103955±104205; GenBank AL138761). After overnight incubation at 65°C, the membrane was washed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate, pH 7, at 65°C for 30 min and then exposed for 7 d to XAR-5 autoradiographic ®lm. Cloning of cDNA by reverse transcription polymerase chain reaction (RT-PCR) To analyze cDNA fragments from the patient and unaffected individuals, total RNA from cultured keratinocytes was used for RT-PCR using the Advantage cDNA PCR kit (Clontech, Palo Alto, CA) with the primers BP7 (5¢-ATTCCTTCAAAACCTCAAACC-3¢; nucleotides 72±92 of BP180/collagen XVII cDNA M91669) and BP8 (5¢-GCTCCTCCTGGCTGTCACTGT-3¢; nucleotides 1116± 1095 of BP180/collagen XVII cDNA M91669). Reaction conditions were as follows: 94°C for 2 min, and then 35 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 3 min. Ampli®ed fragments were cloned into the pGEM-Teasy vector (Promega, Madison, WI) and subjected to sequence analysis. Protein concentrations Protein content was determined with the Bradford assay (Bio-Rad) using bovine serum albumin as standard (Bradford, 1976). Mutation detection Genomic DNA was isolated from peripheral blood using the QIAamp blood kit (Qiagen, Basel, Switzerland). PCR ampli®cation of all COL17A1 (GenBank U76564±U76604) exons was carried out as described previously (Gatalica et al, 1997). Ampli®cation of COL17A1 exon 51 was performed using the following primers: sense
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Figure 2. Immuno¯uorescence staining for the intracellular domain of BP180/collagen XVII is absent in the patient's skin. Frozen sections from control and patient skin were incubated with antibodies against the intracellular and extracellular BP180/collagen XVII domains. (A) Staining of normal control skin with antibodies to the extracellular NC16a domain of BP180/collagen XVII. (B) Strongly reduced staining of the patient's skin with the NC16a domain antibodies in one skin biopsy. The faint staining is localized in the roof of a small blister (the star denotes the blister cavity), indicating junctional blister formation. (C) Positive staining of the patient's skin with the NC16a antibodies in a second skin biopsy. In this specimen, the signal is observed on the ¯oor of the blister, indicating intraepidermal splitting. (D) Staining of control skin with antibody 1A8c against the intracellular domain of BP180/collagen XVII. (E) Negative staining of patient's skin with antibody 1A8c. (F) Positive staining of patient's skin with antibody 1D1 against the extracellular domain of BP180/ collagen XVII.
primer 5¢-TTTCTCTCCTCCCATCACCC-3¢ and antisense primer 5¢TGTCCCTTTAAGTGCCTCCC-3¢. The size of the PCR product was 374 bp. Heteroduplex analysis was performed according to Ganguly et al (1993), and heteroduplex-forming PCR products were sequenced with an automated sequencer (Genome Express, Grenoble, France). The deletion was de®ned using PCR ampli®cation of COL17A1 exons 2±16 directly from genomic DNA. The primers used were sense primer 5¢-TTATCAGCTTCAACAGTGGG-3¢ and antisense primer 5¢CATCATCTGGTCCATTAGCC-3¢. The PCR ampli®cation of normal control DNA showed a number of large products, but the ampli®cation of the patient's DNA generated a distinct band of about 1500 bp, which was further characterized by southern blotting. The hybridization was performed with a BP180/collagen XVII cDNA fragment spanning exons 14±17, which was labeled with digoxigenin (Digoxigenin DNA Labeling Kit, Roche, Mannheim, Germany). The detection was performed with alkaline-phosphatase-labeled digoxigenin antibodies and CDP-Star substrate. The dideoxynucleotide sequencing of the 1500 bp fragment was performed with nested PCR and automated sequencing in both directions. For detection of the entire sequence, additional intronic primers were used for ampli®cation: sense primer 5¢-TTATGTGTCAAAGAATCCCAAAGGTC-3¢ (designated TH2up) and antisense primer 5¢TTGTTCCTGAGTCCGTGATGC-3¢ (TH16do). The sequence was veri®ed by digesting the fragment with EcoRI/PvuII and cloning into the EcoRI/EcoRV sites of the pBIISK+ vector (Stratagene, La Jolla, CA), followed by automated sequencing. For sequencing of intron 2 in normal controls, the sense primer was TH2up and the antisense primer was 5¢-GGAAGGGTGCTCCTGGTAAGTC-3¢ (NK3do). For sequencing of intron 15 in normal controls, the sense primer was 5¢AACCACATTTCTTGACGATGCC-3¢ (NK15up) and the antisense primer was TH16do. PCR ampli®cation and sequence analysis of keratin genes K5 and K14 were carried out as described previously (Shemanko et al, 2000). Antibodies and immunodetection of proteins Antigen mapping (Hintner et al, 1981) was performed using the following antibodies for immuno¯uorescence staining of 5 mm cryosections of the patient's and control skin: cytokeratins (Dako, Hamburg, Germany), BP230 (a high titer autoantiserum), a BP180/collagen XVII antibody raised against the recombinant NC16a domain (Schumann et al, 2000), BP180/collagen XVII antibodies 1A8c and 1D1 (kind gift of Dr. K. Owaribe, Nagoya University, Nagoya), a6 and b4 integrins (Gibco Life Technologies, Eggenstein, Germany), collagen IV (Dako), laminin 5 (kind gift from Dr. R. Burgeson, CBRC, Harvard Medical School, Cambridge, MA), and collagen VII (Bruckner-Tuderman et al, 1995). For immunoblotting, keratinocytes were extracted with a buffer containing 0.1 M NaCl, 20 mM Tris±HCl, pH 7.4, 1% Nonidet P-40, and proteinase inhibitors (Schumann et al, 1997; SchaÈcke et al, 1998), and the proteins were separated on a 4.5±15% sodium dodecyl sulfate polyacrylamide gel. Proteins in the culture medium were precipitated with chloroform/methanol and subjected to immunoblotting in a similar
manner. As antibodies, polyclonal antibodies against a recombinant fragment (ecto-1) spanning about 200 of the most carboxy-terminal amino acids of BP180/collagen XVII, the NC16a antibody against the recombinant NC16a domain, and the antibody Ab-Col15-2 against the recombinant Col15 domain (SchaÈcke et al, 1998; Schumann et al, 2000; Tasanen et al, 2000) were used.
RESULTS Immuno¯uorescence analysis reveals absence of BP180/ collagen XVII intracellular domain The patient's clinical symptoms at birth were compatible with EB. This was re¯ected by histology showing that during blister formation epidermal±dermal tissue separation could occur either at the level of the lamina lucida of the basement membrane or within the basal keratinocytes (Fig 2). Further investigation with antigen mapping of the patient's skin demonstrated that staining patterns for keratins K5 and K14, a6b4 integrin, BP230, collagen IV, plectin, and laminin 5 were normal. In contrast, the signals with antibodies against BP180/ collagen XVII were unanticipated. Monoclonal antibody 1A8c against the intracellular domain exhibited no staining at all, but antibodies against the extracellular domain showed normal signals in most regions of the epidermal basement membrane zone (Fig 2). These ®ndings were suggestive of aberrant expression of BP180/ collagen XVII in the patient's skin. As the EBS-like features were prominent, cultured keratinocytes derived from the patient's skin were stained with antibodies against keratins K5 and K14. No balllike aggregates typical for cultured EBS cells were found in the proband's keratinocytes, however, suggesting that keratin intermediate ®laments were formed normally (data not shown). Keratin intermediate ®laments are abnormally attached to hemidesmosomes Electron microscopic examination of the proband's dermo-epidermal junction revealed hemidesmosomes of varying width and structure. Within the basal keratinocytes, the keratin intermediate ®lament bundles did not appear to properly attach to the hemidesmosomal cytoplasmic inner plaque. The other morphologic structures of the basement membrane zone, i.e., the lamina lucida, lamina densa, and the anchoring ®brils, appeared normal (Fig 3). No full-length BP180/collagen XVII mRNA or protein is found in cultured keratinocytes from the patient Northern blot analysis of total RNA obtained from cultured normal keratinocytes demonstrated a 6 kb mRNA band with BP180/ collagen XVII cDNA probes from the 5¢ and 3¢ end (Fig 4). In contrast, when total RNA from the patient's keratinocytes was
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Figure 3. Electron microscopy reveals abnormalities in hemidesmosome and keratin intermediate ®lament structures. Heterogeneous hemidesmosomes of varying size and structure were found in the proband's skin. Moreover, the keratin intermediate ®lament bundles do not extend into the hemidesmosomes. The other ultrastructural units of the basement membrane zone, lamina lucida, lamina densa, and the anchoring ®brils appeared normal. Bar: 0.25 mm.
hybridized with a 5¢ end probe that spans the intracellular domain of the protein, no signal was found. As a 3¢ probe revealed a diminished size of BP180/collagen XVII mRNA, the ®ndings were suggestive for an internal deletion within the 5¢ segment (Fig 4). Immunoblot analysis of keratinocyte extracts and media was performed to visualize a putative truncated polypeptide corresponding to the short mRNA observed with northern blotting. Antibodies recognizing the extracellular domain BP180/collagen XVII revealed two bands in extracts of normal keratinocytes, with molecular weights of 180 and 120 kDa (Fig 5). The 180 kDa band corresponds to the full-length transmembrane molecule, and the 120 kDa band to the soluble, shed ectodomain (SchaÈcke et al, 1998). In culture medium, only the 120 kDa ectodomain was present. Analysis of the patient's keratinocytes showed the presence of the 120 kDa band in the medium and cell extracts, whereas the 180 kDa transmembrane form was absent. Instead, the cell extract contained a polypeptide of approximately 130 kDa (Fig 5). This truncated polypeptide was recognized by antibodies to the NC16a domain and to the distal C-terminus of the molecule, indicating that the entire ectodomain was present. In contrast, antibodies to the intracellular domain did not recognize it. Thus, the protein results were in agreement with the mRNA analysis and implicated a mutation that produced an in-frame deletion in the 5¢ end of the mRNA and yielded a BP180/collagen XVII polypeptide chain that was shortened by about 50 kDa in its intracellular domain. Nevertheless, the truncated molecule was anchored to the cell plasma membrane and shed in an apparently normal manner. Mutational analysis demonstrates a large genomic deletion from intron 3 to intron 15 due to a 42 bp sequence homology The initial search for mutations in the keratin genes K5 and K14 did not reveal any sequence abnormalities. RT-PCR analysis of the patient's keratinocyte RNA using the BP180/ collagen XVII primers BP7 and BP8 (see Materials and Methods) revealed a 500 bp band, compared with a 1600 bp band obtained with control RNA (data not shown). Cloning and sequencing of the 500 bp band revealed a large deletion in the cDNA extending from nucleotide position 158 to 1329, which corresponded
Figure 4. Northern blot analysis shows a large deletion in the 5¢ part of the BP180/collagen XVII mRNA. Total RNA was isolated from keratinocytes cultured in high calcium medium established from the patient (lanes 1, 4) and from normal control individuals (lanes 2, 3, 5, 6). The northern blot was hybridized with a BP180/collagen XVII 5¢ probe (nucleotides 97±636, GenBank M91669) in lanes 1±3, or with a BP180/collagen XVII 3¢ probe (nucleotides 3555±4402, GenBank M91669) in lanes 4±6. As loading control glyceraldehyde-3-phosphatedehydrogenase was used to ensure equal amounts of RNA.
precisely to the omission of exons 3 through 15. This translates to an in-frame deletion in the polypeptide from Ile-18 to Asn-407, thus encompassing a major part of the intracellular domain of the BP180/collagen XVII polypeptide. Heteroduplex analysis of genomic DNA of the patient provided evidence for two different genetic aberrations in the COL17A1 gene. Dideoxynucleotide sequencing revealed a heterozygous Cto-T transition at position 3781 leading to the premature termination codon R1226X (Fig 6B). The mutation eliminated the consensus sequence for TaqI restriction enzyme, and veri®cation by TaqI digestion showed that it was paternally inherited (Fig 6A, B). PCR analysis with primers placed in exons 2 and 16 generated a 1500 bp amplimer with the patient's and his mother's DNA, but not with DNA from the father or from normal controls. This
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Figure 5. Immunoblot analysis reveals that patient keratinocytes produce only BP180/collagen XVII of 130 kDa molecular weight. Proteins extracted from cultured cells and culture medium were immunoblotted with the NC16a antibody. Lane 1: Both full-length BP180/collagen XVII (upper arrowhead) and the shed ectodomain (lower arrowhead) were present in cell extracts from normal keratinocytes (Co). Lane 2: The shed ectodomain was precipitated from the medium of normal keratinocytes. Lane 3: The cell extracts of the patient's keratinocytes (EB) contained mutant BP180/collagen XVII polypeptide of approximately 130 kDa (arrow) and the shed ectodomain (lower arrowhead). Lane 4: The soluble ectodomain was present in the medium of the patient's keratinocytes (lower arrowhead). Identical results were obtained when using antibodies against the carboxy-terminal region or against the Col15 domain (see Materials and Methods). On the right, the migration positions of molecular weight standards, from top to bottom 200 kDa, 111 kDa, 80 kDa, and 46 kDa, are shown.
suggested a maternally inherited deletion that reached from intron 2 to intron 15. Subsequent sequence analysis of normal controls disclosed an identical 42 nucleotide stretch in both introns 2 and 15. In the proband's DNA, only one 42 bp stretch was present and it was ¯anked at the 5¢ end by a normal intron 2 sequence and at the 3¢ end by a normal intron 15 sequence (Fig 6C). Searching the COL17A sequence (GenBank AL138761) with the 42 bp sequence shows indeed that introns 2 and 15 contain this sequence (nucleotides 103161±103203 and 82167±82209); in both cases this sequence is part of the highly conserved AluSg repeats. Southern blot analysis using XhoI-digested DNA and a BP180/ collagen XVII exon 2 probe revealed, as predicted by the COL17A sequence (GenBank AL138761), a 27 kbp fragment in normal DNA and two bands at 27 and 6 kbp in patient DNA (Fig 7). This argues that an Alu-mediated mutational event had occurred that removed the 21 kbp stretch of DNA between the two 42 bp repeats. The deletion was not found in either 150 normal chromosomes or 28 chromosomes of other unrelated EB patients, indicating that it does not represent a neutral polymorphism. Taken together, the mutation analysis, combined with RNA and protein assays, showed that the patient was hemizygous for the deletion. Like many mutations leading to premature termination codons, R1226X led to nonsense-mediated mRNA degradation, as both northern blots and immunoblots failed to demonstrate the presence of full-length BP180/collagen XVII mRNA or polypeptide chains. DISCUSSION BP180/collagen XVII, a type II transmembrane protein, presumably functions as a cell-adhesion receptor providing a connection between extracellular matrix ligands and the intracellular keratinocyte intermediate ®lament network. Support for this role emerged from its abnormal expression caused by mutations in the COL17A1 gene in JEB patients (Pulkkinen and Uitto, 1999). Absence of BP180/collagen XVII as a consequence of null mutations leads to severe epidermal dysadhesion, whereas missense mutations causing structural abnormalities in the extracellular domain lead to
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unfolding of the triple helix, susceptibility to proteolytic degradation, and functional insuf®ciency (Tasanen et al, 2000). In this study, we investigated an EB patient with phenotypic features of mainly EBS. This analysis provides genetic evidence for the function of BP180/collagen XVII as linker molecule between the keratin intermediate ®lament network and the extracellular matrix proteins at the dermal±epidermal junction. Whereas the expression of several hemidesmosomal proteins and their ligands, e.g., a6b4 integrin, BP230, collagen IV, plectin, laminin 5, and keratins K5 and K14, was normal, the expression of BP180/ collagen XVII was aberrant. The absence of the intracellular domain of BP180/collagen XVII was evident by immuno¯uorescence, immunoblot, and northern blot analysis, whereas the extracellular segment of the polypeptide was present normally in the patient's skin. Genetic analysis revealed compound heterozygosity of the proband for two different aberrations in the COL17A1 gene. The paternal defect R1226X is a recurrent mutation that has been described in both homozygous and heterozygous forms (Jonkman et al, 1997; Schumann et al, 1997; Floeth et al, 1998; Floeth and Bruckner-Tuderman, 1999). The premature termination codon presumably leads to nonsensemediated mRNA decay (Schumann et al, 1997; Floeth et al, 1998; Floeth and Bruckner-Tuderman, 1999) as northern blots showed no evidence for the expression of normal size mRNA. The proband's cells contained a 130 kDa truncated polypeptide; however, it was not clear whether the shortened length resulted from a deletion or, potentially, from a C-terminal truncation caused by the premature termination codon. Immunoblotting with a panel of domain-speci®c BP180/collagen XVII antibodies identi®ed only one kind of truncated chain, which spanned the entire ectodomain but lacked a segment from the intracellular domain, thus excluding the possibility of a C-terminally truncated BP180/collagen XVII polypeptide. Thus, the proband was functionally homozygous (hemizygous) for the maternal mutation. This resulted in skipping of exons 3±15 and elimination of amino acids 18±407 from the BP180/collagen XVII polypeptide, generating an intracellular domain that consisted of only 76 amino acids instead of the normal 466 residues. This ®nding was consistent with the negative immuno¯uorescence and immunoblot ®ndings obtained with monoclonal antibody 1A8c, the epitope of which has been mapped to the region between the amino acids 113±201 of BP180/collagen XVII (Borradori et al, 1997). The truncated molecule is stable, and both mRNA and protein expression levels seemed to be equivalent to those in normal individuals. The 21 kbp genomic deletion extends from intron 2 to intron 15. Both introns contain the same perfectly matched 42 nucleotide long region (5¢-aactcctgacctcgtgatctgcccgcctcggcctcccaaagt-3¢), which is part of a larger (approximately 300 bp) repeat homologous to the Sg member of the Alu repeat family (GenBank AL138761). Sequences similar to these two Alu repeats are not found elsewhere in the COL17A1 gene. Both Alu repeats have the same orientation and display a sequence similarity of 84%, which is even higher around the 42 bp region of homology. Introns 2 and 15 contain in addition to the Alu repeats other interspersed repetitive elements such as (GT)n (TGTA)n, MER41B, and MER5A repeats. Therefore, it seems likely that the genomic deletion is due to a recombination event that occurred between the two AluSg repeats using the 42 bp stretch as crossover point. Alu-mediated genomic rearrangements have been shown to be involved in other inherited diseases such as Fanconi anemia (Morgan et al, 1999), familial hypercholesterolemia (Lehrman et al, 1987), hereditary angiedema (Stoppa-Lyonnet et al, 1990), and pseudoxanthoma elasticum (Ringpfeil et al, 2001), as well as in genetic predisposition for colon and breast cancer (NystroÈm-Lahti et al, 1995; Puget et al, 1999). Moreover, tandem duplication mediated by Alu-dependent recombination in somatic tissue has been reported in cases of acute myeloid leukemia (Strout et al, 1998). It is likely that secondary structures formed by the regions of homology and components of
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Figure 6. Pedigree and mutation analysis in the COL17A1 gene. (A) The pedigree of the family demonstrates that the mutation R1226X (black areas) and the genomic deletion (dotted areas) of the proband (1) occurred in a compound heterozygous manner, because the father (3) is the carrier of R1226X and the mother (2) of the deletion. (B) Sequence analysis of the patient's PCR product containing exon 51 showed a C-toT transition at nucleotide position 3781, designated R1226X. A restriction endonuclease site for Taq1 is eliminated by the mutation. Accordingly, the 342 bp PCR product (upper band) of a normal control (C1) and the mother (2) were cleaved into a 185 bp (lower band) and a 157 bp fragment, whereas the proband (1), the father (3), and a previously characterized unrelated patient with the same mutation (C2) showed an additional uncleaved band of 342 bp. (C) The maternal mutation was a large genomic deletion, extending from intron 2 to intron 15. In normal controls, an identical 42 bp sequence was found within both introns. In the proband's DNA, this 42 bp sequence is present once and is ¯anked at the 5¢ end by the sequence corresponding to normal intron 2 (underlined) and at the 3¢ end by the sequence corresponding to normal intron 15 (doubly underlined).
the DNA replication and recombination machinery may play a role in the generation and subsequent deletion of loop structures. Recent cell biologic studies provide explanations for the biologic and clinical phenotype of the present proband. They have shown that the intracellular domain of BP180/collagen XVII contains sequences important for the localization of the protein into hemidesmosomes and its interaction with other hemidesmosomal components, such as the intracellular domain of the b4 integrin subunit and the N-terminal region of BP230 (Hopkinson et al, 1995; Borradori et al, 1997; Aho and Uitto, 1998; Schaapveld et al, 1998; Hopkinson and Jones, 2000). There is strong evidence that an N-terminal 401 amino acid stretch of BP180/collagen XVII contains binding sites to the N-terminal region of BP230 and the C-terminal half of the cytoplasmic domain of b4 integrin (Geerts et al, 1999; Koster et al, 1999; Hopkinson and Jones, 2000). In addition, the cytoplasmic domain of BP180/collagen XVII may also associate with plectin (Aho and Uitto, 1997). Therefore, the deletion of the region Ile18±Asn407 is expected to impair the association of BP180/collagen XVII not only with b4 integrin and BP230 but also with plectin. Both plectin and BP230 are cytoplasmic components of hemidesmosomes that are believed to attach the keratin cyto-
skeleton to the hemidesmosomal plaque and, by means of interactions with the cytoplasmic domains of BP180/collagen XVII and b4 integrin, to the plasma membrane (Guo et al, 1995; Uitto et al, 1996; Andra et al, 1997; Borradori and Sonnenberg, 1999). Therefore, it is likely that the poor linkage of intermediate ®laments to hemidesmosomes observed by electron microscopy in the proband's skin results from an impaired incorporation and/or stabilization of BP230 and plectin into hemidesmosomes as a result of perturbed interactions with the cytoplasmic domain of BP180/ collagen XVII. Moreover, the reduced attachment of keratin ®laments to the basal plasma membrane might account for the increased mechanical fragility of keratinocytes, thus explaining the blistering arising in the basal layer and the clinical EBS-like phenotype. The extracellular domain of BP180/collagen XVII appeared to be expressed normally as shown by immunoblot analysis. In concert, antibodies to the extracellular domain of BP180/collagen XVII showed linear immuno¯uorescence staining along the basement membrane zone of the patient's skin, a staining pattern indistinguishable from normal controls. Nevertheless, as also junctional cleavage was seen in the proband's skin, dermoepidermal cohesion must be impaired as well. It is likely that the
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Figure 7. Southern blot analysis of the maternal deletion. Genomic DNA from the patient (lane 1) and a normal individual (lane 2) was digested with XhoI and hybridized with a BP180/collagen XVII exon 2 probe. The arrowhead marks the start of the electrophoresis. Arrows mark the position of the molecular weight markers of 21 kbp, 5.2 kbp, 4.3 kbp, 3.5 kbp, and 2 kbp (top to bottom).
large deletion of the cytoplasmic domain BP180 not only compromises molecular associations important for the stabilization, and thus function, of hemidesmosomes, but also has an impact on the interaction of the extracellular domain of BP180/collagen XVII with extracellular matrix protein(s). The genetic, biochemical, and clinical data obtained here extend the spectrum of the phenotypic heterogeneity of EB. They demonstrate for the ®rst time that defects in a given gene can give rise to clinical and biologic phenotypes characteristic of different EB categories, depending on the function of the protein domain affected by the mutation. This work also underlines the importance of using intracellular-domain-speci®c BP180/collagen XVII antibodies for assessment by antigen mapping of EB patients to avoid faulty diagnosis. The authors thank S. Aebischer, M. Schubert, and A. Wissel for excellent technical assistance. This work was supported by grant 31-45943.95 (Swiss National Science Foundation) to D.H., by grant 31-56727.99 (Swiss National Science Foundation) and by the Fondation TeÂleÂthon Action Suisse (Aubonne, Switzerland) to L.B., by grants Br 1475/1±2 and SFB 293-B3 from the Deutsche Forschungsgemeinschaft, and by EU contract BMH-4-97-2062 to L.B-T.
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