De Novo Anti-Type VII Collagen Antibodies in Patients with Recessive Dystrophic Epidermolysis Bullosa
2013; Elsevier BV; Volume: 134; Issue: 4 Linguagem: Inglês
10.1038/jid.2013.475
ISSN1523-1747
AutoresDavid T. Woodley, Jon Cogan, Xinyi Wang, Yingping Hou, Cyrus Haghighian, Gail Kudo, Douglas R. Keene, Mei Chen,
Tópico(s)Nail Diseases and Treatments
Resumoanchoring fibril type VII collagen cartilage matrix protein dermal–epidermal junction direct immunofluorescence epidermolysis bullosa acquisita fibronectin type III-like repeat indirect immunofluorescence N-terminal noncollagenous domain of type VII collagen C-terminal noncollagenous domain of type VII collagen premature termination codon recessive dystrophic epidermolysis bullosa RDEB inversa RDEB generalized other RDEB severe, generalized triple helical A domain of von Willebrand factor TO THE EDITOR The two main layers of human skin are held together by structures at the dermal–epidermal junction (DEJ) called anchoring fibrils (AFs). Without properly functioning AFs, the adherence between the epidermis and dermis is compromised. Clinically, this translates into skin fragility and skin bullae. AFs are composed of type VII collagen (C7) that has a central triple helical domain (TH) flanked by a 145-kDa non-collagenous amino-terminal domain (NC1) and a 30-kDa carboxyl-terminal domain (NC2) (Burgeson, 1993Burgeson R.E. Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Scopus (236) Google Scholar). AFs and C7 are perturbed in recessive dystrophic epidermolysis bullosa (RDEB), a disease characterized clinically by skin fragility, skin bullae, scarring, and nail loss (Fine et al., 2008Fine J.D. Eady R.A. Bauer E.A. et al.The Classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2008; 58: 931-950Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar). RDEB is caused by mutations in the COL7A1 gene encoding C7. Over 700 mutations have been identified in DEB patients (Wertheim-Tysarowska et al., 2012Wertheim-Tysarowska K. Sobczyska-Tomaszewska A. Kowalewski C. et al.The COL7A1 mutation database.Hum Mutat. 2012; 33: 327-331Crossref PubMed Scopus (52) Google Scholar). According to a recent consensus report, RDEB is classified as RDEB, severe, generalized (RDEB-sev, gen), RDEB, generalized, other (RDEB-O), and RDEB inversa (RDEB-I) (Fine et al., 2008Fine J.D. Eady R.A. Bauer E.A. et al.The Classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2008; 58: 931-950Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar). There is also an acquired type of EB called epidermolysis bullosa acquisita (EBA). EBA patients are born with normal skin and then during middle age, they inappropriately generate IgG antibodies against their C7 and AFs (Yaoita et al., 1981Yaoita H. Briggaman R.A. Lawley T.J. et al.Epidermolysis bullosa acquisita: ultrastructural and immunological studies.J Invest Dermatol. 1981; 76: 288-292Abstract Full Text PDF PubMed Scopus (221) Google Scholar; Woodley et al., 1984Woodley D.T. Briggaman R.A. O'Keefe E.J. et al.Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita.N Engl J Med. 1984; 310: 1007-1013Crossref PubMed Scopus (435) Google Scholar), leading to skin fragility, trauma-induced blisters, and scarring reminiscent of hereditary RDEB. The conventional wisdom in Dermatology is that patients with genetic RDEB may have a clinical phenotype resembling EBA, but that they have no auto-antibodies against C7. In this study, we identified 22 patients with bona fide RDEB, and characterized their mutations and their disease phenotype clinically, pathologically, ultrastructurally, and immunologically. We sought to determine whether any of these RDEB patients had anti-C7 antibodies in their sera or skin. As summarized in Table 1, 13 of the patients were classified as RDEB-sev, gen (patients 1–13) with COL7A1 mutations that created premature termination codons (PTCs) due to nonsense or splice-site mutations (Spl), small insertions, or deletions. Another nine RDEB patients (patients 14–22) had missense mutations (Mis) in one allele of COL7A1 predicting glycine or arginine substitutions in the TH domain. Six patients (patients 14–19) had mutations associated with RDEB-I. Three patients had RDEB-O (patients 20–22). Of the 22 sequenced RDEB patients, 32 mutant alleles were identified. Nearly one-third (10 of 32) of these mutations have not been previously reported.Table 1Summary of the clinical and mutational analysis of RDEB patientsPatient IDPatient ageAllele 1/allele 2Mutation locationConsequencesClinical diagnosis124G2517KfsX3/G2517KfsX3TH/THPTC/PTCRDEB-sev,gen26c.356_357delCA/c.356_357delCACMP/CMPPTC/PTCRDEB-sev,gen310c.356_357delCA/c.356_357delCACMP/CMPPTC/PTCRDEB-sev,gen427c.4172dupC/c.4182-4188dup7TH/THPTC/PTCRDEB-sev,gen525c.5048-5051dup4/c.6501G-ATH/THPTC/in-frame DelRDEB-sev,gen624c.2993-5_3007dup20/IVS64+4A>GFn3/THPTC/SplRDEB-sev,gen736c.2993-5_3007dup20/IVS64+4A>GFn3/THPTC/SplRDEB-sev,gen811R578X/R578XFn3/Fn3PTC/PTCRDEB-sev,gen95P1523HfsX187/IVS85-1G>TTH/THPTC/SplRDEB-sev,gen103R613X/R1683XFn3/THPTC/PTCRDEB-sev,gen1134c.7787delG/c.7787delGTH/THPTC/PTCRDEB-sev,gen1227IVS17-2delA/R2814XFn3/acidicSpl/PTCRDEB-sev,gen1322R236X/IVS85-1G>AFn3/THPTC/SplRDEB-sev,gen1437R2069C/6501G-ATH/THMis/in-frame DelRDEB-I1523R578X/G1907DFn3/THPTC/MisRDEB-I1628IVS66+1G>C/G2719ATH/THPTC/MisRDEB-I1762R2069C/IVS5+1G>ATH/CMPMis/PTCRDEB-I1811G1907D/c.6311_6312delCTTH/THMis/PTCRDEB-I1938G1907D/R1933XTH/THMis/PTCRDEB-I204c.4919delG/G2366VTH/THPTC/MisRDEB-O2145c.3582-3583delAG/G1782RVWA/THPTC/MisRDEB-O2231G2233S/IVS64-2_-1delAGTH/THMis/SplRDEB-OAbbreviations: CMP, cartilage matrix protein; Del, deletion; Fn3, fibronectin type III-like repeats; Mis, missense; RDEB-sev, gen, RDEB, severe, generalized (formally Hallopeau-Simens RDEB); RDEB-O, RDEB, generalized, other (formerly non-Hallopeau-Simens RDEB); RDEB-I, inversa type of RDEB; PTC, premature termination codon; Spl, splicing; TH, triple helical domain; VWA, A domain of von Willebrand factor (VWF-A).Newly identified mutations are indicated in bold. Open table in a new tab Abbreviations: CMP, cartilage matrix protein; Del, deletion; Fn3, fibronectin type III-like repeats; Mis, missense; RDEB-sev, gen, RDEB, severe, generalized (formally Hallopeau-Simens RDEB); RDEB-O, RDEB, generalized, other (formerly non-Hallopeau-Simens RDEB); RDEB-I, inversa type of RDEB; PTC, premature termination codon; Spl, splicing; TH, triple helical domain; VWA, A domain of von Willebrand factor (VWF-A). Newly identified mutations are indicated in bold. We assessed the level of C7 expression at the DEJ of their skin by immunofluorescence staining of peri-lesional skin with a rabbit-anti-NC1 antibody (Chen et al., 1997Chen M. Petersen M.J. Li H.L. et al.Ultraviolet A irradiation upregulates type VII collagen expression in human dermal fibroblasts.J Invest Dermatol. 1997; 108: 125-128Abstract Full Text PDF PubMed Scopus (34) Google Scholar). As summarized in Table 1 and Supplementary Figure S1 online, nine patients (patients 14–22) expressed C7 at the same level as skin from normal human subjects. The other RDEB patients had reduced (patients 1, 4–7, 9, 10, 12, 13) or no expression of C7 (patients 2, 3, 8, 11). Download .pdf (5.53 MB) Help with pdf files Supplementary Information AFs were evaluated by transmission electron microscopy for density and morphology. As summarized in Table 2 and Supplementary Figure S2 online, RDEB patients had reduced density or complete absence of AFs. When AFs were observed, they appeared attenuated in size or had an abnormal morphology.Table 2Summary of C7 expression and AFs in recessive dystrophic epidermolysis bullosa patients' skin and anti-C7 antibodies in the bloodPatient IDC7 expression at DEJAF by electron microscopyNC1/NC2 ELISAC7 ELISAC7 western blotEpitopeDensityMorphology1Reduced+Very thin and wispy±++NC1/NC22Absent0Absent----3Absent0Absent----4Reduced0Absent----5Reduced++Thin, rarely arching+++NC1/NC26Reduced+++Thin, rarely arching+++NC1/NC27Reduced++Thin, rarely banded, rarely arching----8Absent+Short, rudimentary+++NC1/NC29Reduced++Straight, non-banded+++NC1/NC210Reduced+Thin, mild arching----11Absent+Short, rudimentary----12Reduced+Thin and wispy-++TH13Reduced+Thin and wispy----14Normal++++Few banded, arching, looped----15Normal+++Non-banded, arching----16Normal+++++Banded, arching-++TH17Normal++++Thin, arching, looped----18Normal+++Non-banded, some arching+-+NC1/NC219Normal++++Banded, arching-++TH20Normal+Very thin and straight+++NC1/NC221Normal++++Thin and wispy, occasionally mild arching+++NC1/NC222Normal+++Thin, wispy, occasional arching-++THNHSNormal+++++Thick, banded, arching, looping----EBA---+++NC1/NC2Abbreviations: AF, anchoring fibril; DEJ, dermal–epidermal junction; EBA, epidermolysis bullosa acquisita; NHS, normal human skin; TH, triple helical domain.C7 expression at the DEJ was determined by immunofluorescence staining of cyrosections with an anti-NC1 antibody. AFs were evaluated by transmission electron microscopy, with the density indicated (0 indicates that no AFs were identified; five stars indicate normal density). The morphology of the individual AFs is qualitatively accessed from worst to best: absent, short or rudimentary, thin or wispy, arching, looping, banded, thick. Normal individuals have a 5 plus density with thick, banded, arching, and looping AFs. ELISA was performed with either a commercially available MBL kit that uses a mixture of immobilized NC1 and NC2 domains as the target substrate or our recently developed assay that uses full-length, recombinant human C7 as the target substrate. Immunoblot analysis was performed using purified recombinant C7. Open table in a new tab Abbreviations: AF, anchoring fibril; DEJ, dermal–epidermal junction; EBA, epidermolysis bullosa acquisita; NHS, normal human skin; TH, triple helical domain. C7 expression at the DEJ was determined by immunofluorescence staining of cyrosections with an anti-NC1 antibody. AFs were evaluated by transmission electron microscopy, with the density indicated (0 indicates that no AFs were identified; five stars indicate normal density). The morphology of the individual AFs is qualitatively accessed from worst to best: absent, short or rudimentary, thin or wispy, arching, looping, banded, thick. Normal individuals have a 5 plus density with thick, banded, arching, and looping AFs. ELISA was performed with either a commercially available MBL kit that uses a mixture of immobilized NC1 and NC2 domains as the target substrate or our recently developed assay that uses full-length, recombinant human C7 as the target substrate. Immunoblot analysis was performed using purified recombinant C7. To determine whether RDEB patients have anti-C7 antibodies, we subjected our RDEB patients' sera to two different anti-C7 antibody ELISAs and immunoblot analysis. One commercially available ELISA utilizes NC1 and NC2 domains as the target substrate. The second ELISA is one we developed and employs full-length C7 as the target substrate. We used 13 EBA sera as positive controls and sera from 17 normal subjects as negative controls to establish the assay. The ELISA results are shown in Supplementary Figures S3 and S4 online and summarized in Table 2. With the commercial ELISA, 7 of 22 RDEB patient sera (patients 5, 6, 8, 9, 18, 20, 21) showed reactivity with values above the threshold. Similarly, in the full-length C7 ELISA, 11 of 22 patients exhibited reactivity. Using the full-length C7 ELISA allowed us to identify sera from four RDEB patients (patients 12, 16, 19, 22) that exclusively recognized the TH domain. These sera were further analyzed by immunoblotting against purified C7 (Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar). As summarized in Table 2 and Supplementary Figure S5 online, there is 100% correlation between ELISA and immunoblot results. To determine whether RDEB sera recognize C7 in the skin, we performed indirect immunofluorescence staining using salt-split human skin as substrate (Woodley et al., 1984Woodley D.T. Briggaman R.A. O'Keefe E.J. et al.Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita.N Engl J Med. 1984; 310: 1007-1013Crossref PubMed Scopus (435) Google Scholar). None of the sera from these 11 patients bound to C7 on the dermal side of salt-split skin (data not shown). In addition, direct immunofluorescence of the 11 patients' skin did not detect any anti-C7 antibody deposits (data not shown), suggesting that the anti-C7 antibodies in their sera are likely non-pathogenic. This study provides evidence that 12 of 22 bona fide RDEB patients have low-level circulating anti-C7 autoantibodies that do not bind to the patients' skin. A previous smaller study found that one of seven RDEB patients exhibited anti-C7 antibodies by ELISA (Pendaries et al., 2010Pendaries V. Gasc G. Titeux M. et al.Immune reactivity to type VII collagen: implications for gene therapy of recessive dystrophic epidermolysis bullosa.Gene Therapy. 2010; 17: 930-937Crossref PubMed Scopus (34) Google Scholar). In accordance with our data herein, a recent study of 17 RDEB patients showed that 15 of 17 of the patients exhibited anti-C7 antibodies (Tampoia et al., 2013Tampoia M. Bonamonte D. Filoni A. et al.Prevalence of specific anti-skin autoantibodies in a cohort of patients with inherited epidermolysis bullosa.Orphanet J Rare Dis. 2013; 8: 132Crossref PubMed Scopus (31) Google Scholar). DIF on the RDEB patients, however, was not performed in either of these two studies. Although our RDEB patients had varying types of COL7A1 mutations, the expression of C7 in the DEJ of their skin ranged from none to the same as normal skin. The generation of anti-C7 antibodies in our RDEB cohort did not correlate with the expression of C7 in the patients' skin, the type of COL7A1 mutation, the patients' age, or the classification of RDEB. It is interesting to note that a correlation between anti-C7 antibodies and the Birmingham EB severity score was observed (Tampoia et al., 2013Tampoia M. Bonamonte D. Filoni A. et al.Prevalence of specific anti-skin autoantibodies in a cohort of patients with inherited epidermolysis bullosa.Orphanet J Rare Dis. 2013; 8: 132Crossref PubMed Scopus (31) Google Scholar). All therapies for RDEB including cell therapy, protein therapy, and vector therapy will involve exposure of the patient to new domains of C7 and the potential to generate anti-C7 autoantibodies (Chen et al., 2002Chen M. Kasahara N. Keene D.R. et al.Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.Nat Genet. 2002; 32: 670-675Crossref PubMed Scopus (164) Google Scholar, Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar; Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar, Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Intradermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo.Mol Ther. 2004; 10: 318-326Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar; Wong et al., 2008Wong T. Gammon L. Liu L. et al.Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2008; 128: 2179-2189Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar; Wagner et al., 2010Wagner J.E. Ishida-Yamamoto A. McGrath J.A. et al.Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.N Engl J Med. 2010; 363: 629-639Crossref PubMed Scopus (266) Google Scholar). The presence of anti-C7 antibodies in some RDEB patients prior to treatment should be taken into consideration when selecting and evaluating patients involved in clinical trials. Written informed consent was obtained from the 22 subjects with RDEB. The study was approved by the University of Southern California Institutional Review Boards and was conducted according to the Declaration of Helsinki Principles. This work was supported by NIH grants RO1 AR47981 to MC, and RC4AR060535 and RO1 AR33625 to MC and DTW. We thank Sara Tufa for technical support of transmission electron microscopy. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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