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Experimental dermatology • Review article Type XVII collagen gene mutations in junctional epidermolysis bullosa and prospects for gene therapy J. W. Bauer and C. Lanschuetzer Department of Dermatology, General Hospital Salzburg, Austria Summary Non-Herlitz junctional epidermolysis bullosa (nH-JEB) is caused predominantly by mutations leading to premature stop codons on both alleles of the type XVII collagen gene (COL17A1). The analysis of mutations in this gene has provided a means of correlating genotype with phenotype of nH-JEB patients. The phenotype of nH-JEB is characterized by generalized blistering of skin and mucous membranes with atrophic scarring and nail dystrophy. Atrophic alopecia is a distinct feature of nH-JEB patients, but one that is not associated with the severity of the disease at other sites. Enamel hypoplasia and pitting of the teeth are also characteristic for nH-JEB and can be used to facilitate the correct diagnosis in children with a blistering skin disease. Analysis of the biological consequences of mutations in the COL17A1 gene has shown that most patients lack type XVII collagen mRNA due to nonsense-mediated mRNA decay. Patients with these mutations can therefore be a target for corrective gene therapy using vectors coding for full-length type XVII collagen. Proof of principle for this approach has recently been demonstrated. The analysis of naturally occurring phenomena of gene correction in the COL17A1 gene provides evidence for other mechanisms of gene correction in genetic diseases. For example, exclusion of an exon carrying a mutation can lead to a milder phenotype of nH-JEB than predicted by the original mutation. In addition, we have gained data suggesting that COL17A1 exons harbouring pathogenic mutations can also be repaired by trans-splicing, i.e. aligning corrected RNA sequences to introns in the vicinity of faulty exons in the COL17A1 premtRNA. Introduction Epidermolysis bullosa (EB) is the term applied to a heterogeneous group of inherited skin disorders in which minor trauma leads to blistering of the skin and mucous membranes.1 Depending on the level of tissue cleavage EB has been divided into three main groups: EB simplex with blister formation occurring by disruption of the basal keratinocytes, junctional EB (JEB) with cleavage in the lamina lucida and dystrophic EB with cleavage below the lamina densa of the dermo-epidermal basement Correspondence: Johann Bauer, Department of Dermatology, General Hospital Salzburg, Muellner Hauptstr. 48, A-5020 Salzburg, Austria. Tel.: +43 662 4482 3042. Fax: +43 662 4482 3044. E-mail: [email protected] Accepted for publication 4 September 2002 membrane zone. Most JEB patients belong to two main groups: Herlitz JEB and generalized atrophic benign EB (GABEB). Patients diagnosed with the former disease usually die within the first 2 years of life, whereas the latter diagnosis is associated with a better prognosis and a tendency of further improvement during life. Initial observations describing reduced expression of bullous pemphigoid antigen 2 (also termed type XVII collagen) in patients suffering from GABEB2,3 were followed by the identification of mutations in the gene COL17A1 coding for type XVII collagen in GABEB patients.4 Since then a large number of mutations in the COL17A1 gene have been published (Table 1). In addition, it has been shown that the phenotype of GABEB can also result from mutations in the genes coding for the polypeptide chains of laminin 5.5 Recently, the term GABEB has been merged into the group of non-Herlitz JEB (nH-JEB).6 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60 53 Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer Affected exons ⁄ introns Reference Mutations leading to stop codons R1226X ⁄ 4150insG R1226X ⁄ 1706delA 4003delTC ⁄ 4003delTC 3514ins25 4003delTC ⁄ Q1403X 4003delTC ⁄ G803X 2944del5 ⁄ 2944del5 2944del5 ⁄ Q1023X 1706delA ⁄ R1226X 2342delG ⁄ 2342delG Q1016X ⁄ Q1016X R1226X ⁄ R1226X R145X 520delAG ⁄ 520delAG 2965delG ⁄ 2965delG 2666delTT G258X ⁄ G258X 3674insT ⁄ 4426insC 2881delA ⁄ 2881delA 4003delTC ⁄ 4003delTC partially 4080insGG R795X ⁄ R795X exon 51 ⁄ exon 52 exon 51 ⁄ exon 18 exon 52 ⁄ exon 52 exon 48 exon 52 ⁄ exon 53 exon 52 ⁄ exon 34 exon 43 ⁄ exon 43 exon 43 ⁄ exon 45 exon 18 ⁄ exon 51 exon 30 ⁄ exon 30 exon 45 ⁄ exon 45 exon 51 ⁄ exon 51 exon 8 exon 7 ⁄ exon 7 exon 43 ⁄ exon 43 exon 37 exon11 ⁄ exon11 exon 54 ⁄ exon 50 exon 41 ⁄ exon 41 exon 52 ⁄ exon 52 ⁄ exon 52 exon 33 ⁄ exon 33 4 35 36 17 37 37 7 7 23 38 13 13 13 39 39 39 40 41 42 24 16 Acceptor splice site mutations 2441–2 A fi G ⁄ 2441–2 A fi G 2441–1 G fi T 3053–1 G fi C intron 31 ⁄ intron 31 intron 31 intron 44 43 44 27 Donor splice site mutations 3871+1 G fi C 4261+1 G fi C ⁄ 4261+1 G fi C intron 51 intron 52 ⁄ intron 52 27 28 Missense mutations G627V R1303Q ⁄ R1303Q V1026M G539E G633D S265C ⁄ S265C exon exon exon exon exon exon 16 13 41 37 14 45 In-frame skipping mutation Ile18del389 (158del1172) exon 2–exon 15 11 Digenic mutation L855X ⁄ R1226X plus R635X. (LAMB3 gene) exon 37 ⁄ exon 51 46 23 52 ⁄ exon 52 46 18 23 11 ⁄ exon 11 Table 1 Published mutations in the COL17A1 gene. Each line documents the first report of a distinct mutation in COL17A1. According to the convention, mutations are reported as either the change in amino acid (R1226X, for example, indicates that the codon for arginine at number 1226 is replaced by a nonsense codon) or a change in nucleotide sequence (4150insG, for example, indicates an insertion of a guanine at position 4150 in the complementary DNA). In one family, mutations in the LAMB3 and the COL17A1 gene have been found (digenic mutation). The mutations have been sorted by the year of appearance in the literature. Analysis of genotype)phenotype correlation The COL17A1 gene comprises 56 distinct exons, which span approximately 52 kb of the genome on the long arm of chromosome 10. It encodes a polypeptide, the a1(XVII) chain, consisting of an intracellular globular domain, a transmembranous segment, and an extracel- 54 lular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids.7 The mutations reported in the COL17A1 gene have allowed the establishment of a mutation database, which facilitates the analysis of the effects of specific mutations on the clinical presentation of nH-JEB (Fig. 1). Stop codon mutations or mutations leading to 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60 Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer Figure 1 Schematic representation of the type XVII collagen polypeptide, and positions of the mutations disclosed in the corresponding gene, COL17A1, in families with nH-JEB. The type XVII collagen polypeptide, as deduced from the corresponding cDNA sequence, consists of intracellular, transmembranous and extracellular domains. The amino-terminus is localized in the keratinocyte, the extracellular domain traverses the lamina lucida of the cutaneous basement membrane zone and the carboxy-terminus is situated in the lamina densa of the cutaneous basement membrane zone. The arrows indicate the approximate position of mutations along the type XVII collagen polypeptide chain. Mutations leading to stop codons are depicted above the polypeptide; all other mutations are shown below. The figure is not drawn to scale. downstream stop codons on both alleles are associated with the original ‘GABEB’ phenotype.8 This phenotype is characterized by blisters occurring since birth, which heal with atrophic hyperpigmented or hypopigmented scars. There is no severe scarring or exuberant granulation tissue. Nail dystrophy is seen in most of the patients to a varying degree and some patients also present with EB naevi.9 Mucosal involvement is variable but less severe than in dystrophic EB (for a comprehensive review of ‘GABEB’ see reference 10). In one family, a phenotype comparable to EB simplex has been described. The underlying mutation in that case deleted a major part of the intracellular domain of type XVII collagen.11 A distinct clinical sign of nH-JEB is atrophic, incomplete universal alopecia. However, one patient has been described who has the characteristic clinical, ultrastructural and biochemical features of nH-JEB without alopecia.12 nH-JEB in another patient with normal hair and a mild phenotype with acral blistering has been associated with the homozygous missense mutation R1303Q.13 Patients with compound heterozygosity for the stop codon mutation R145X and the missense mutation G633D have been shown to present as a mild form of nH-JEB including normal scalp hair, but missing axillary and pubic hair.14 A remarkable variety in the expression of alopecia has been noted in the reports of Mazzanti et al.15 and Ruzzi et al.16 describing Italian families supported by appropriate clinical, ultrastructural and molecular data. In these families homozygosity for the nonsense mutation R795X leads to a mild phenotype of nH-JEB by exclusion of the faulty exon 33. There is a spectrum of severity of the alopecia ranging from almost complete universal alopecia to just absent pubic and sparse axillary hair, suggesting that additional factors influence hair loss in these families. Therefore, in general, alopecia cannot be used as a marker of disease severity in nH-JEB. Another feature, which is unique to nH-JEB, is enamel hypoplasia and pitting of the teeth associated with severe caries. These dental abnormalities are 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60 55 Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer present in patients with stop codon mutations or mutations leading to downstream stop codons on both alleles of COL17A1 or LAMB3 (coding for the b chain of laminin 5) and can also be caused by hemizygosity for the dominant negative missense mutation G627V.17 The teeth phenotype can be used as a guide to the correct diagnosis in children with a blistering skin disease.18 Gene therapy Gene therapy describes the possibility of correcting the consequences of a genetic defect on the molecular level. Different methods are currently being tested for application in EB gene therapy (Table 2). Most commonly, the correct expression of a defective gene in the skin has been achieved by using retroviral delivery systems. These retroviruses infect keratinocytes and stably integrate DNA into the host genome. For the COL17A1 gene, phenotypic reversion of the ultrastructural characteristics of nH-JEB keratinocytes has been demonstrated using this method.19 Nevertheless, in the field of gene therapy there remain multiple issues that have not yet been convincingly resolved and prevent immediate translation to patients. First, there is need to target keratinocyte stem cells to achieve sustained expression of the transgene. This issue is complicated by difficulties in identifying keratinocyte stem cells by distinctive markers, although recently better methods have been elaborated that identify stemcell-like fractions in mouse keratinocyte populations.20 Table 2 Current approaches in EB gene therapy. Method of transduction Method of correction Retrovirus cDNA** PAC*** Clon Transient Transient Transposase Integrase Gene Trans-splicing Ribozyme cDNA cDNA Gene* Reference COL17A1 LAMB3 ITGB4 COL7A1 COL17A1 KRT14 LAMB3 COL7A1 19 47, 48 49 50 34 A B C *Genes: COL17A1, coding for type XVII collagen; LAMB3, coding for the b-chain of laminin 5; ITGB4, coding for b4 integrin; COL7A1, coding for type VII collagen; KRT14, coding for keratin 14. **cDNA, complementary DNA. ***PAC, P1-based artificial chromosome. A McLean I and Terron A. J Invest Dermatol 2002; 119: 268. B Ortiz S et al. J Invest Dermatol 2002;119: 230. C Ortiz S et al. J Invest Dermatol 2002; 119: 343. 56 In addition, it has been shown that through ex vivo selection using clonal analysis transduced keratinocytes can be obtained that have the capacity to replicate for more than 30 generations and are able to reconstitute a fully differentiated epidermis on nude mice for more than 40 weeks.21 Second, the ex vivo approaches to gene therapy necessitate the transfer of transduced epidermal sheets back to the patient’s skin using surgical procedures with perhaps the potential for scarring, infection or delayed wound healing. Possibly these hurdles can be overcome through technical advances such as programmed diathermosurgery (Timedsurgery)22 or laser surgery. Finally, transduction of keratinocytes with vectors containing normal fulllength cDNA copies of a defective gene is not indicated in autosomal dominant forms of EB. In these forms of EB, technologies such as ribozyme therapy or antisense oligonucleotides are more relevant and might be used to reduce the amounts of defective protein. Analysis of naturally occurring phenomena of gene correction in the COL17A1 gene pave the way for alternative approaches to medical gene correction. In 1997 Jonkman et al.23 reported a patient with patches of normal appearing skin in a symmetrical leaf-like pattern on the upper extremities. The underlying mutations in the COL17A1 gene were identified as R1226X paternally and 1706delA maternally. In the clinically unaffected areas of skin, about 50% of the basal cells were expressing type XVII collagen protein, albeit at a reduced level. The reason for the re-expression of type XVII collagen was a mitotic gene conversion surrounding the maternal mutation, thus overriding the heterozygous mutation. These observations suggest that less than 50% of full-length type XVII collagen is necessary to correct the phenotypic expression of nH-JEB. However, in a second nH-JEB patient, in whom the homozygous mutation 4003delTC was partially corrected by the somatic mosaic back mutation 4080insGG, no phenotypic correction, i.e. no clinically normal skin, could be observed. It is likely that the 25 incorrect amino acids between the deletion and insertion mutations prevented functional correction in this patient.24 In a third example, a partly successful naturally occurring attempt at gene correction by modulation of splicing reactions has been described in patients with the homozygous mutation R785X.16 In these patients, exclusion of exon 33 harbouring this mutation leads to an unusual mild phenotype, although there is only a 5% level of detectable type XVII collagen protein. Similar in-frame skipping of exons has also been reported for patients with mutations in the COL7A1 and LAMB3 genes.25 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60 Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer Future perspectives In recent years data have accumulated that indicate that the spliceosome can be a target for attempting gene correction. The spliceosome of a cell is the site where small-nuclear ribonucleoproteins facilitate removal of introns from premtRNA (the so-called splicing reaction) and therefore control the correct assembly of exons in mature RNA. If mutations disrupt the normal sequence of a splice site, the spliceosome may use other intronic or exonic splice sites (so-called cryptic splice sites) usually leading to out-of-frame transcripts, and rarely to in-frame transcripts. In this case, antisense oligonucleotide technology can be used to suppress the use of cryptic splice sites. This approach has already been tested successfully for the suppression of aberrant splicing in patients with thalassaemia.26 This technology could be applied in type XVII collagen-deficient EB patients, who have a splice site mutation producing at least one alternative in-frame transcript.27,28 In this case suppression of splice sites leading to out-of-frame transcripts could redirect the splicing machinery to the in-frame splice reactions, therefore producing polypeptides that have only small changes in protein composition. Another mechanism of ‘corrective’ splicing is the activation of naturally occurring splice variants to exclude faulty exons (see above) or to include missing exons. This approach has been described for inclusion of exon 7 in the survival motor neurone 2 gene in vitro. By this mechanism spinal muscular atrophy patients could be protected from the effects of loss of function in the survival motor neurone 1 gene.29 Figure 2 SMaRT can reprogramme exons at the 3¢, 5¢ or within the gene. The COL17A1 premtRNA (target) is shown by a rod. The premtRNA is the intermediate product of the RNA polymerases that transcribe the DNA of the gene into premtRNA in the nucleus. Shortly after transcription the premtRNA still contains the intronic sequences, which are cleaved by a process called splicing, i.e. before the mature RNA enters the cytoplasm for translation. For clarity, the introns surrounding the targeted exon, exon 52, are depicted separately. The position of a common Austrian mutation 4003delTC in exon 52 is indicated. The Pre-Therapeutic-Molecule (PTM) is introduced into the keratinocyte harbouring the mutation in the COL17A1 gene through a given transduction process. This RNA usually consists of a binding region complementary to an intronic sequence and the exon(s) to be corrected. In the upper panel the binding region is complementary to intronic sequences in intron 51, preceding exon 52. The remaining part of the PTM in the upper panel consists of RNA sequences containing exons 52–56. PTMs successfully binding to intron 51 in COL17A1 premtRNA are replacing exon 52 harbouring the 4003delTC mutation and the remaining C-terminus of COL17A1 RNA. This reaction is called 3¢ trans-splicing. In the middle panel the PTM replaces exon 52 to the N-terminal end (5¢ trans-splicing). Also exon 52 alone can be replaced by trans-splicing (lower panel). 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60 57 Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer Modulation of splicing activities can also be attempted by trans-splicing. Trans-splicing refers to a process whereby an intron of one premtRNA interacts with an intron of a second premtRNA, enhancing the recombination of splice sites between these two premtRNAs. A practical application of targeted trans-splicing to modify specific target RNAs has been used in group I ribozyme-based mechanisms. For example, using the Tetrahymena group I ribozyme, targeted trans-splicing has been achieved in human erythrocyte precursors to alter mutant b-globin transcripts from individuals with sickle cell disease.30 A related approach is spliceosome-mediated RNA trans-splicing (SMaRT). SMaRT uses the introns preceding or following an exon harbouring a mutation as a hinge to introduce a corrected species of RNA into a given premtRNA (see Fig. 2). Therefore, this technique reduces the size of a corrective insert into a viral vector depending on the position of the mutation. This fact is of particular interest in genes coding for large proteins involved in EB, i.e. type VII collagen (9.2 kB mRNA) and plectin (14.2 kB mRNA). Proof of principle for SMaRT has first been demonstrated in the b-human chorionic gonadotrophin gene 6.31 Its applicability in the correction of genetic diseases has been expanded by work showing correction of the predominant cystic fibrosis transmembrane conductance regulator (CFTR) mutation deltaF508 in a CFTR-minigene construct in vitro32 and in a CFTR mouse model in vivo.33 Recently, it has been demonstrated that also keratinocytes allow for trans-splicing reactions in the COL17A1 gene34 and are therefore a potential therapeutic target for SMaRT. Conclusion Cloning of the COL17A1 gene and detection of mutations in this gene have facilitated the establishment of the correct diagnosis and prognostic factors for nH-JEB patients. Identification of the mutations in these patients is the basis for correcting the genetic defects in the COL17A1 gene. Analysis of naturally occurring phenomena of gene correction in the COL17A1 gene has suggested that gene therapy is feasible to correct the effects of such mutations. It has already been shown that the reversal of phenotypic features of type XVII collagen-deficient nH-JEB keratinocytes is achievable by retroviral transduction. Besides introducing full-length COL17A1 cDNA into keratinocytes, modulation of splicing reactions in keratinocytes might also play a role in gene therapy for inherited dermatological diseases. 58 References 1 Fine J, Bauer E, McGuire J, Moshell A, eds. Epidermolysis Bullosa. Baltimore and London: Johns Hopkins University Press, 1999. 2 Pohla-Gubo G, Lazarova Z, Giudice GJ et al. 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Key points • Most mutations in the COL17A1 gene lead to premature stop codons associated with nonsense-mediated mRNA decay. • Missense mutations in the COL17A1 gene are associated with a milder phenotype. • Incomplete alopecia universalis is a distinct feature for nH-JEB, but is not a marker of overall severity. • Enamel hypoplasia and pitting of the teeth is a distinct clinical feature of nH-JEB that can be used to distinguish nH-JEB from other EB subtypes. 60 • Phenotypic reversion of the ultrastructural features of nH-JEB has been reported by retroviral expression of type XVII collagen. • Modulation of RNA splicing represents a new option for gene correction in keratinocytes. 2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60