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Arch Dermatol Res (2000) 292 : 159–163

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O R I G I N A L PA P E R

J. Yu-Yun Lee · Ching Li · Sheau-Chiou Chao · Leena Pulkkinen · Jouni Uitto

A de novo glycine substitution mutation in the collagenous domain of COL7A1 in dominant dystrophic epidermolysis bullosa

Received: 1 September 1998 / Revised: 23 November 1999 / Accepted: 26 November 1999

Abstract Dystrophic epidermolysis bullosa (DEB) is a hereditary mechanobullous disorder characterized by fragility of the skin and mucous membrane due to abnormalities of anchoring fibrils. Both dominant and recessive DEB have been shown to be caused by mutations in COL7A1, the gene encoding type VII collagen which is the major component of anchoring fibrils. De novo mutation in dominant DEB is rare. In this study, we report a novel de novo glycine substitution mutation in COL7A1 in a Chinese female patient presenting with mild DEB. In search of the mutation, we scanned the entire COL7A1 using polymerase chain reaction (PCR) amplification of all exons of COL7A1, followed by heteroduplex analysis and direct sequencing of the PCR products that exhibited heteroduplex pattern. A G-to-A transition at nucleotide position 6082 within exon 73 of COL7A1was detected. The mutation converted a glycine to an arginine (G2028R) within the triple-helical domain of type VII collagen. It was confirmed that the mutation was present only in the proband. Haplotype analyses suggested that the case arose as a de novo occurrence of autosomal dominant DEB.

J. Y.-Y. Lee (쾷) · S.-C. Chao Department of Dermatology, National Cheng-Kung University Hospital, 138 Sheng-Li Rd., Tainan, Taiwan, R.O.C. e-mail: [email protected], Tel.: +886-6-2766180, Fax: +886-6-2004326 C. Li Department of Pathology, National Cheng-Kung University Hospital, Tainan, Taiwan, R.O.C. L. Pulkkinen · J. Uitto Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, USA L. Pulkkinen · J. Uitto Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, USA

Key words COL7A1 · De novo mutation · Dystrophic epidermolysis bullosa

Introduction Dystrophic epidermolysis bullosa (DEB) is a group of hereditary mechanobullous disorders characterized by blistering and scarring of the skin and mucosae induced by minor trauma, and associated nail dystrophy [1, 2]. The fragility of the skin or mucous membranes is attributed to morphologic alteration, scarcity, or even complete absence of the anchoring fibrils, the attachment structures extending from the basement membrane zone to the underlying dermis [3]. Blisters occur below the basement membrane zone at the level of the anchoring fibrils. Anchoring fibrils are composed predominantly of type VII collagen which is a homotrimer consisting of three identical proα1 (VII) chains [4]. Both dominant and recessive DEB have been linked to COL7A1 and, to date, over 100 pathogenetic mutations have been detected within COL7A1 in different variants of DEB [5–13]. For the great majority of DEB families, the COL7A1 mutations have been specific to the individual families and only a few recurrent mutations have been reported [13, 14]. In DEB, there is considerable phenotypic as well as genetic variability. After examining the COL7A1 mutations in DEB, certain correlations between genotypes and phenotypes have been suggested [15]. In the majority of the cases with the severe, Hallopeau-Siemens type of recessive DEB (HS-RDEB) characterized by mutilating scarring of the skin and complete absence of the anchoring fibrils, the characteristic genetic defects consist of premature termination codon mutations (PTC) in both COL7A1 alleles, resulting in truncated type VII collagen polypeptides which are unable to assemble into functional anchoring fibrils [5]. The milder, mitis form of RDEB is frequently caused by a missense mutation or an in-frame deletion in one or both alleles, mutations which are less disruptive to the protein [6, 16, 17]. In dominantly inherited forms of DEB, the majority of the reported mutations

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have involved glycine substitutions in the triple-helical domain of COL7A1 [18–22], although a limited number of cases have displayed splice-site mutations [23, 24]. The glycine substitution apparently destabilizes the triple helix of the collagen. Because the type VII collagen is a homotrimer, only one-eighth of the collagen molecule will consist of three normal proα1 (VII) polypeptides in a patient with dominant DEB. Thus the mutated polypeptide will result in a dominantly inherited, relatively mild disease through dominant negative interference in the formation of type VII collagen. De novo mutation involving glycine substitution in DEB is a rare event. The first such mutation was reported by Dunnill et al. in recessive DEB [7]. Kon et al. reported the first de novo glycine substitution mutation in dominant DEB in a Japanese patient with mild disease [22]. The patient was shown to have a heterozygous G2079E mutation in exon 75. In this report, we describe another de novo glycine substitution mutation involving exon 73 of COL7A1 in a Chinese girl presenting with a mild form of DEB.

Fig. 2 Family pedigree of the patient

Materials and methods The patient was a 17-month-old Chinese female patient who presented with recurrent vesicles on the knees, shins, toes and fingers which she had had since birth. The blisters healed with scarring and milia (Fig. 1). In addition, nail atrophy was noted in the big toes and a ring finger. There was no mucosal involvement. She was the only one affected in the family (Fig. 2). The parents were not related. Histopathology of a skin lesion revealed a reepithelialized subepidermal blister. Electron microscopy showed poorly formed anchoring fibrils beneath the lamina densa (Fig. 3). Based on these findings, a diagnosis of DEB was made. Because no other family member was affected, a mitis form of RDEB and dominant DEB due to a de novo mutation were considered. PCR amplification and heteroduplex analyses Genomic DNA was extracted from peripheral blood from the proband and other members of the family. DNA samples were then subjected to mutation screening by amplification of segments of COL7A1 spanning all 118 exons of the gene using primers syn-

Fig. 3 Electron microscopy reveals absence or poorly formed anchoring fibrils (arrows) underneath the lamina densa (× 19,950)

thesized on the basis of intronic sequences (GenBank L23982) [25, 26]. Specifically, the primers used to amplify the 286-bp product containing exon 73 were: upstream primer, 5′-GGGTGTAGCTGTACAGCCAC-3′ and downstream primer, 5′-CCCTCTTCCCTCACTCTCCT-3′. For PCR amplification, approximately 250 ng of genomic DNA, 20 pmol of each primer, 0.5 mM MgCl2, 20 µmol of each dNTP and 1.25 U of Taq polymerase (GIBCO BRL, Life Technologies, Gaithersburg, Md.) were used in a total volume of 50 µl. The amplification conditions were 94 °C for 5 min, followed by 40 cycles of 94 °C for 45 s, 59 °C for 45 s and 70 °C for 60 s, and extension at 72 °C for 10 min in an OmniGene thermal cycler (Marsh Scientific, Rochester, N.Y.). The PCR products were examined on 2% agarose gel and 3–10 µl of the samples were prepared for heteroduplex analysis using conformation-sensitive gel electrophoresis (CSGE) as described by Ganguly et al. [27]. The PCR products demonstrating shifted bands were subjected to direct automated sequencing (ABI Advanced Biotechnologies, Columbia, Md.). Verification of the mutations

Fig. 1 The feet, especially the toes, show blisters, scarring and milia. Note that the big toenails are atrophic

Since the G-to-A transition at nucleotide 6082 within exon 73 abolishes an enzyme restriction site for SmaI, the PCR products generated above were subjected to SmaI digestion according to the

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A

C

B

Fig. 4 A–C Identification and verification of the mutation in DEB. A Heteroduplex analysis by conformation-sensitive gel electrophoresis of PCR amplicon of exon 73 of COL7A1 in individuals identified on the top of each lane corresponding to Fig. 2. Note that in the affected individual, a heteroduplex band (→), in addition to the homoduplex band, is detected. B Direct sequencing of the PCR product from the proband (left) and the mother (right) demonstrates a G-to-A transition at position 6126 in the proband. As a result, the codon for glycine (GGG) is changed to a codon for arginine (AGG). C Digestion of the PCR products of exon73 with SmaI reveals that the unaffected individuals are homozygous for 148-bp, 93-bp and 45-bp fragments. The affected individual bearing the mutation shows an additional band with the predicted size of 193 bp, indicating heterozygosity for the G-to-A nucleotide substitution (NC normal control, M marker)

manufacturer’s recommendation (Boehringer-Mannheim, Germany). The digestion products were analyzed on 2% agarose gels. Paternity testing Analysis of microsatellites (D1S213, D1S498, D1S206, D1S218, D1S207, D1S199,D4S405, D5S436and D6S441) was performed by using fluorescence-labeled primers according to the manufacturer’s recommendation (PE Applied Biosystems, Foster City, Calif.). PCR products were analyzed on ABO377 sequencer together with internal standards using Genescan software. The size of the microsatellite alleles were determined by Genotyper soft-

ware. The likelihood of paternity was calculated based on allelic frequencies in the general population assuming a prior probability of 50% [28].

Results CSGE of the PCR products of exon 73 revealed a distinct heteroduplex pattern with the proband’s DNA, while the DNA samples of the parents and the three siblings showed a homoduplex band only (Fig. 4 A). Direct DNA sequencing of the products from the mother and the proband showed a heterozygous G-to-A transition at nucleotide 6082 within the exon 73 of the proband (Fig. 4 B). The substitution converted a glycine (GGG) to arginine (AGG). This mutation (G2028R) was confirmed by the loss of a SmaI restriction site. Digestion of the 286-bp PCR products with SmaI revealed three bands (148 bp, 93 bp and 45 bp) in the unaffected members and the normal control (Fig. 4 C). The proband’s DNA showed an additional band of 193 bp, indicating heterozygosity for this mutation. The loss of this SmaI restriction site was not found in 12 other unrelated Taiwanese DEB families or in 66 alleles from 33 healthy, unrelated Taiwanese controls. These findings suggest that G2028R is not a common polymorphism and is a pathogenic mutation specific for this fam-

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The findings indicate that the patient was heterozygous for a de novo mutation resulting in a glycine substitution, presumably causing dominant negative interference and the resultant clinical phenotype of a mild DEB

Discussion

Fig. 5 Exclusion of nonpaternity. A battery of microsatellite markers with known allelic frequencies in the general population were used to genotype the proband and her parents, as shown. The results confirmed paternity with 99.999946% certainty

ily. Scanning of the entire COL7A1 revealed another heteroduplex pattern occurring in the PCR product spanning exons 103-104 with the patient’s and her father’s DNA. Direct sequencing of the exon 103-104 PCR products demonstrated a G-to-A transition at nucleotide 30303 located in the middle of the intron between the two exons. This mutation was most likely a polymorphism. Nonpaternity was excluded by the use of a panel of microsatellite markers (Fig. 5). Specifically, microsatellite markers corresponding to chromosomes 1, 4, 5 and 6 were used to analyze the DNA from the proband and her parents. Based on allelic frequencies of the markers in the general population, the probability of paternity was calculated to be 99.999946%, thus excluding nonpaternity in this family. Haplotype analysis of the region spanning COL7A1 locus further revealed that the proband had inherited the same paternal allele as the two other unaffected siblings but had inherited a maternal allele that was different from those two siblings (not shown). Both direct sequencing of the maternal DNA and restriction enzyme digestion with SmaI showed that the mother did not carry the same G-to-A transition at nucleotide 6082 (Fig. 4 B).

We have described the clinicopathological findings in a mild case of DEB and detected a de novo glycine substitution mutation in the collagenous domain of COL7A1. The mutation, G2028R, replaces a glycine residue in the collagenous subdomain consisting of 71 uninterrupted GlyX-Y repeats, and could destabilize the critical triple helical conformation of the collagenous domain. This mutation is located 50 amino acids downstream from the major 39 amino acid noncollagenous “hinge” region in the collagenous domain. Based on the observation that the positions of the imperfections and interruptions in the type VII collagen are well conserved through evolution, Christiano et al. [18] have speculated that the stability of the triple helical segment adjacent to the unstable nonhelical interruption is critical for the function of type VII collagen. As more and more COL7A1 mutations have been delineated, it appears that this hypothesis is correct. In the most recent report [15] the distribution of 97 COL7A1 mutations along the type VII collagen polypeptide is depicted. The mutations in dominant DEB have involved glycine substitution mutations in the triple helical domains. Interestingly, among the 26 such mutations, half of them spanning from G1982 W to G2079 E are noted to cluster immediately downstream from the noncollagenous “hinge” region. The mutation in our case is also located in this region. De novo mutations in dominant DEB appear to be rare. The first case was reported by Kon et al. in 1997 [22]. We now report another case of de novo mutation in dominant DEB. Both cases show similar features, including mild clinical manifestations and glycine substitution mutations located close to the 39 amino acid noncollagenous interruption. Whether this particular region is a “hot zone” for such de novo mutation remains to be seen. The genetic confirmation of a de novo mutation in DEB has an impact in genetic counseling for patients with relatively mild DEB when the patient appears to be the only person affected in the family. The question is whether the patient has mitis recessive DEB or dominant DEB. In recessive DEB, the risk of clinically unaffected parents producing an affected child in the subsequent pregnancy is 25% and the patient having affected offspring is about as low as in the general population. While the risk is practically zero and 50%, respectively, in the case of de novo dominant DEB. However, if the mutation is germline in one of the parents, the risk of having an affected child will depend on the percentage of mutated cells in the germline. Since recessive DEB and dominant DEB cannot be distinguished on the basis of clinical and ultrastructural findings, in clinical practice dominant DEB derived from de novo mutation or parental germline mosaicism is often considered if there is no history of consanguineous mar-

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riage. Mutation analyses have demonstrated that most patients with mild DEB are compound heterozygotes or have homozygous missense mutations inherited in a recessive manner [15]. Since de novo mutation in dominant DEB is rare, it appears appropriate to consider each “new” case as RDEB at genetic counseling, unless proven to be a dominant mutation by molecular analysis. Acknowlegements This study was supported by Chinese National Science Grant 86-2314B-006, and by the United States Public Health Service, National Institutes of Health grant P01-AR38923.

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