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BIBTEX RIS

Natural Occurrence of Autoantibodies against Basement Membrane Proteins in Epidermolysis Bullosa

2021; Elsevier BV; Volume: 142; Issue: 7 Linguagem: Inglês

10.1016/j.jid.2021.10.030

ISSN

1523-1747

Autores

Antoni Gostyński, Gilles F.H. Diercks, M.J. Escámez, Nisha Suyien Chandran, R. de Lucas, Adela García‐Martín, Marcela Del Río, Jeroen Bremer, Maria C. Bolling, Álvaro Meana, Sara Llames, Enno Schmidt, Ralf J. Ludwig, Marcel F. Jonkman, Hendri H. Pas, Anna M.G. Pasmooij,

Tópico(s)

Nail Diseases and Treatments

Resumo

Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by lifelong trauma-induced blistering of the skin and mucosa and extracutaneous manifestations. Autoantibodies to a structural protein of the epidermal basement membrane zone (BMZ) such as dystonin (BP230), plectin, type XVII collagen (COL17/BP180), laminin-332, or type VII collagen (COL7) result in the same level of blister formation as in EB subtypes caused by mutations in their coding gene, such as in EB simplex (DST and PLEC), junctional EB (JEB) (COL17A1, LAMA3, LAMB3, or LAMC2), and dystrophic EB (DEB) (COL7A1) (Goletz et al., 2017Goletz S. Zillikens D. Schmidt E. Structural proteins of the dermal-epidermal junction targeted by autoantibodies in pemphigoid diseases.Exp Dermatol. 2017; 26: 1154-1162Crossref PubMed Scopus (52) Google Scholar; Has et al., 2020Has C. Bauer J.W. Bodemer C. Bolling M.C. Bruckner-Tuderman L. Diem A. et al.Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.Br J Dermatol. 2020; 183: 614-627Crossref PubMed Scopus (208) Google Scholar). The innate and adaptive immune systems are designed not to recognize the host's own cells and proteins owing to natural immunological tolerance and negative selection of host-specific T lymphocytes in the central lymphatic organs. However, the lack of one of the proteins due to inherited mutations can interfere with this process. When the missing protein is introduced later in life, it can be recognized as dangerous, and an immune response can occur (Alberts et al., 2002Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular biology of the cell.4th ed. Garland Publishing Science, New York City, NY2002Google Scholar; Siprashvili et al., 2016Siprashvili Z. Nguyen N.T. Gorell E.S. Loutit K. Khuu P. Furukawa L.K. et al.Safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa.JAMA. 2016; 316: 1808-1817Crossref PubMed Scopus (118) Google Scholar). Four previous publications presented results of serological tests, ELISA, and indirect immunofluorescence (IIF) on monkey esophagus in patients with EB (DEB and EB simplex) (Annicchiarico et al., 2015Annicchiarico G. Morgese M.G. Esposito S. Lopalco G. Lattarulo M. Tampoia M. et al.Proinflammatory cytokines and antiskin autoantibodies in patients with inherited epidermolysis bullosa.Medicine (Baltimore). 2015; 94: e1528Crossref PubMed Scopus (39) Google Scholar; Esposito et al., 2016Esposito S. Guez S. Orenti A. Tadini G. Scuvera G. Corti L. et al.Autoimmunity and cytokine imbalance in inherited epidermolysis bullosa.Int J Mol Sci. 2016; 17: 1625Crossref Scopus (25) Google Scholar; Tampoia et al., 2013Tampoia M. Bonamonte D. Filoni A. Garofalo L. Morgese M.G. Brunetti L. 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 (29) Google Scholar; Woodley et al., 2014Woodley D.T. Cogan J. Wang X. Hou Y. Haghighian C. Kudo G. et al.De novo anti-type VII collagen antibodies in patients with recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2014; 134: 1138-1140Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Circulating antibodies against BMZ proteins were present in the serum, and the authors suggested the need for further ex vivo experiments to assess their pathogenicity. Although three publications lacked direct immunofluorescence (DIF) on a skin biopsy specimen and IIF on salt split skin (SSS) for detection of tissue-bound autoantibodies, Woodley et al., 2014Woodley D.T. Cogan J. Wang X. Hou Y. Haghighian C. Kudo G. et al.De novo anti-type VII collagen antibodies in patients with recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2014; 134: 1138-1140Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar additionally performed DIF and IIF on SSS in patients with DEB with a positive ELISA. Treatment approaches for EB are being investigated, and progress has been recently made; however, they can be threatened by pre-existent circulating antibodies (Eichstadt et al., 2019Eichstadt S. Barriga M. Ponakala A. Teng C. Nguyen N.T. Siprashvili Z. et al.Phase 1/2a clinical trial of gene-corrected autologous cell therapy for recessive dystrophic epidermolysis bullosa.JCI Insight. 2019; 4e130554Crossref PubMed Scopus (47) Google Scholar; Gaucher et al., 2020Gaucher S. Lwin S.M. Titeux M. Abdul-Wahab A. Pironon N. Izmiryan A. et al.EBGene trial: patient preselection outcomes for the European GENEGRAFT ex vivo phase I/II gene therapy trial for recessive dystrophic epidermolysis bullosa.Br J Dermatol. 2020; 182: 794-797Crossref PubMed Scopus (16) Google Scholar; Hirsch et al., 2017Hirsch T. Rothoeft T. Teig N. Bauer J.W. Pellegrini G. De Rosa L. et al.Regeneration of the entire human epidermis using transgenic stem cells.Nature. 2017; 551: 327-332Crossref PubMed Scopus (381) Google Scholar). In these studies, their presence was assessed before transplantation only by ELISA, IIF, and western blot. In the study of Eichstadt et al., 2019Eichstadt S. Barriga M. Ponakala A. Teng C. Nguyen N.T. Siprashvili Z. et al.Phase 1/2a clinical trial of gene-corrected autologous cell therapy for recessive dystrophic epidermolysis bullosa.JCI Insight. 2019; 4e130554Crossref PubMed Scopus (47) Google Scholar, DIF was performed but only on the transplanted sites after the transplantation and not before. IgG deposition was found in one of the transplanted sites in one of the treated subjects at 3 months and 2 years after transplantation; however, circulating antibodies were only detectable at months 1 and 3 and until month 6 after transplantation. Therefore, they suggested that the humoral immune response was provoked by the transplantation site rather than that the circulating antibodies were pre-existing. For the diagnosis of pemphigoid diseases, Meijer et al., 2019Meijer J.M. Diercks G.F.H. de Lang E.W.G. Pas H.H. Jonkman M.F. Assessment of diagnostic strategy for early recognition of bullous and nonbullous variants of pemphigoid.JAMA Dermatol. 2019; 155: 158-165Crossref PubMed Scopus (38) Google Scholar recently proposed that DIF and IIF on SSS and not ELISA or blot are essential, and therefore, these techniques should be used to illustrate whether pre-existing antibodies can bind to the skin (Schmidt and Zillikens, 2009Schmidt E. Zillikens D. Diagnosis and clinical severity markers of bullous pemphigoid.F1000 Med Rep. 2009; 1: 15Crossref PubMed Google Scholar). Because these data are missing in the literature, we have investigated skin biopsies and serum of 37 patients with EB with a wide variety of techniques, including DIF and IIF on SSS to assess the presence of circulating antibodies. Of the 37 patients, 12 were affected with JEB due to mutations in LAMB3 and COL17A1, and 25 were affected with DEB due to mutations in COL7A1 (Table 1). A total of 10 of the 37 included patients had revertant mosaicism (6 with JEB and 4 with recessive DEB [RDEB]) (Supplementary Table S1), that is, healthy, natural skin patches due to correcting somatic mutations that occurred during embryo development or later in life (Pasmooij et al., 2012Pasmooij A.M. Jonkman M.F. Uitto J. Revertant mosaicism in heritable skin diseases: mechanisms of natural gene therapy.Discov Med. 2012; 14: 167-179PubMed Google Scholar). We analyzed the already stored punch biopsies from 35 of the 37 patients. Serum samples from all the patients were obtained with permission from medical ethical committees in the Netherlands (University Medical Center Groningen 2013/317) and Spain (Code Hospital Universitario La Paz: PI1359 and PI1595). All patients or their parents provided written informed consent. Furthermore, 14 sera from 13 patients with severe burn wounds were used as the control for ELISA, blotting, and IIF. For a detailed methods description, see previous publications (Groth et al., 2011Groth S. Recke A. Vafia K. Ludwig R.J. Hashimoto T. Zillikens D. et al.Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid.Br J Dermatol. 2011; 164: 76-82Crossref PubMed Scopus (63) Google Scholar; Vodegel et al., 2004Vodegel R.M. de Jong M.C. Meijer H.J. Weytingh M.B. Pas H.H. Jonkman M.F. Enhanced diagnostic immunofluorescence using biopsies transported in saline.BMC Dermatol. 2004; 4: 10Crossref PubMed Scopus (65) Google Scholar). The age of the patients at the time of biopsy and serum sampling varied from 0 to 61 years (Supplementary Table S1) for the patients with EB and from 6 to 86 years for the burn wound patients (Supplementary Table S2). DIF was performed on all available skin specimens (1‒3 biopsies per patient) to detect human IgG and IgA. Furthermore, we performed IIF on two substrates, monkey esophagus, and SSS; keratinocyte footprint assay for laminin-332 (Giurdanella et al., 2020Giurdanella F. Nijenhuis A.M. Diercks G.F.H. Jonkman M.F. Pas H.H. Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases.Br J Dermatol. 2020; 182: 373-381Crossref PubMed Scopus (10) Google Scholar); and ELISA for COL17 (NC16A), BP230, and COL7. In addition, immunoblot was performed on keratinocyte cell extract to detect antibodies against BP230, COL17, and LAD-1 (Groningen, The Netherlands) (Pas, 2001Pas H.H. Immunoblot assay in differential diagnosis of autoimmune blistering skin diseases.Clin Dermatol. 2001; 19: 622-630Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), on dermal extract to detect antibodies against COL7, and on the recombinant C-terminus of laminin γ1 (Lübeck, Germany) to detect antibodies against the p200 protein. ELISA for the NC16A domain of COL17, BP230, and COL7 were performed in two different laboratories in Groningen (The Netherlands) and in Lübeck (Germany) using MBL and Euroimmun kits, respectively.Table 1Detailed Data Per Patient of all Tests Conducted for AutoantibodiesPatientDatabase NRMutation (DNA)Mutation (Protein)Protein Expression in Nonblistered SkinBiopsyDirect IFIIFKFALam332ELISA MBP GroningenELISA Euroimmune LübeckImmunoblot GroningenImmunoblot LübeckNREB TypeMOSSSBP180BP230COL7BP180BP230COL7BP180BP230LADCOL7DermExtrLam- γ1COL17A1 Patients1JEB-intermedEB02501COL17A1: c.2237delG / c.2237delGp.Gly746Alafs∗53 / p.Gly746Alafs∗53Negative (COL17)––––18179–––––––––2JEB-intermedEB02601COL17A1: c.3676C>T / c.1601delAp.Arg1226∗ / p.Asp534Alafs∗19Negative (COL17)–BMZ––––––––––––––3JEB-intermedEB03502COL17A1: c.2237delG / c.3676C>Tp.Gly746Alafs∗53 / p.Arg1226∗Negative (COL17)––––––––––––––––4JEB-intermedEB13401COL17A1: c.1365delC / c.3600_3601delCTCOL17A1: c.1260delC / c.3495_c.3496delCTp.Thr421Leufs∗72 / p.Ser1166Leufs∗6Negative (COL17)––––––––––––––––5JEB-intermedEB20801COL17A1: c.2237delG / c.2237delGp.Gly746Alafs∗53 / p.Gly746Alafs∗53Negative (COL17)–BMZ––––––––––––––6JEB-intermedEB08601COL17A1: c.1772-2 A>C / c.3327delTin-frame exon skipping / p.Pro1110Argfs∗21Negative (COL17)––IgA roof––––––––Weakly positive––––7JEB-locEB16801COL17A1: c.4320delT / c.4320delTp.Gln1442Lysfs∗70 / p.Gln1442Lysfs∗70Strongly reduced (COL17)––––––––––––––––8JEB-locEB09801COL17A1: c.2251C>T / c.3327delTp.Gln751∗ / p.Pro1110Argfs∗21Strongly reduced (COL17)––––––––––––––––LAMB3 Patients9JEB-intermedEB02901LAMB3: c.628G>A / c.628G>Ap.Glu210Lys / p.Glu210LysStrongly reduced (lam332)–BMZ––––––––––––––10JEB-intermedEB29901LAMB3: c.1106dup / c.1106dupp.Ala370Serfs∗13 / p.Ala370Serfs∗13Normal (lam332)–––Positive––––––––––––11JEB- intermedEB13201LAMB3: c.1063T>C / c.1903C>Tp.Cys355Arg / p.Arg635∗Strongly reduced (lam332)––––––––––––––––12JEB-locEB25401LAMB3: c.628G>A / c.1903C>Tp.Glu210Lys / p.Arg635∗Strongly reduced (lam332)––––––––––––––––COL7A1 patients13RDEB-sevEB26501 (P116)COL7A1: c.887delG / c.6527dupp.Gly296Valfs∗5 / p.Gly2177Trpfs∗113Strongly reduced (type XII collagen)–ECS––––––2442––––––14RDEB-sevEB29701 (P108)COL7A1: c.2142A>G / c.6527dupAltered splicing resulting in out-of-frame transcript p.Gly2177Trpfs∗113Negative (COL7)–––––––––––––PositiveEBA/p200Positive15RDEB-sevEB02201COL7A1: c.3G>T / c.4997dupStart-lost / p.Pro1668fAlafs∗4Negative (COL7)––––29196–––––––––16RDEB-sevEB07701COL7A1: c.925_944dup / c.1264dupp.Ile315Metfs∗12 / p.Arg422Profs∗19Negative (COL7)––––6037132340–––––––17RDEB-sevEB09001COL7A1: c.4767delA / c.4767delAp.Asp1590Thrfs∗120 / p.Asp1590Thrfs∗120Negative (COL7)––––––––––––––––18RDEB-sevP10COL7A1: c.6527dup / c.6527dupp.Gly2177Trpfs∗113 / p.Gly2177Trpfs∗113Strongly reduced (COL7)Not tested–––1012–1643030–Dubious––––19RDEB-sevP31COL7A1: c.6527dup / c.6527dupp.Gly2177Trpfs∗113 / p.Gly2177Trpfs∗113Strongly reduced (COL7)––––16––––––Dubious––––20RDEB-sevP46COL7A1: c.325_326insCG / c.3277-1G>Cp.Glu109Alafs∗39 / Altered splicing resulting in out-of-frame transcriptsStrongly reduced (COL7)–BMZ––483514––––Positive––––21RDEB-sevP52COL7A1: c.7756dup / c.7930-1G>Cp.Gln2586Profs∗12 / In-frame exon skippingNegative (COL7)––––––––––––––EBA/p200–22RDEB-sevP104COL7A1: c.6618+1G>A / c.6618+1G>Ap.K2206_G2207insMSL_E220Gfs∗86/ p.K2206_G2207insMSL_E220Gfs∗86Altered splicing resulting in out-of-frame transcripts / Altered splicing resulting in out-of-frame transcriptsStrongly reduced (COL7)––––39––30––––––EBA/p200–23RDEB-sevP14COL7A1: c.6527dup / c.336C>Gp.Gly2177Trpfs∗113 / p.Tyr112∗Negative (COL7)IgG––––––––25––––––24RDEB-sevP18COL7A1: c.6527dup / c.2984dupp.Gly2177Trpfs∗113 / p.Gly996Trpfs∗44Negative (COL7)Not tested–––––––2573––––––25RDEB-sevP61COL7A1: c.6527dup / c.6527dupp.Gly2177Trpfs∗113 / p.Gly2177Trpfs∗113Strongly reduced (COL7)––––9640–608364––––––26RDEB-sevP104.ICOL7A1: c.6618+1G>A / c.6618+1G>Ap.K2206_G2207insMSL_E220Gfs∗86/ p.K2206_G2207insMSL_E220Gfs∗86Altered splicing resulting in out-of-frame transcripts / Altered splicing resulting in out-of-frame transcripts?Strongly reduced (COL7)––––––––––––––––27RDEB-sevP78COL7A1: c.6527dup / c.5130_5131insCTCACp.Gly2177Trpfs∗113 / p.Thr1711Leufs∗132Negative (COL7)––IgAFloor–24208–––––––––28RDEB-sevEB26001 (P17)COL7A1: c.6527dup / c.6527dupp.Gly2177Trpfs∗113 / p.Gly2177Trpfs∗113Strongly reduced (COL7)––––––––––––––––29RDEB-sevEB02401COL7A1: c.6508C>T / c.6508C>Tp.Gln2170∗ / p.Gln2170∗Negative (COL7)IgG/IgA/C3c to revertant skinBMZIgGfloor–9–10––27––––––30RDEB-sevEB06401COL7A1: c.1573C>T / c.6508C>Tp.Arg525∗ / p.Gln2170∗Negative (COL7)––––911––––––––––31RDEB-intermedEB00901COL7A1: c.2699G>A / c.7237G>Ap.Trp900∗ / p.Gly2413ArgMinimally reduced (COL7)––––2212––––––––––32RDEB-intermedEB34701COL7A1: c.5272G>A / (unknown)p.Gly1758Arg / unknownMinimally reduced (COL7)––––––––––––––––33RDEB-intermedP36COL7A1: c.6527dup / c.7300G>Ap.Gly2177Trpfs∗113 / p.Gly2434ArgReduced (COL7)––––––––––––––––34RDEB-intermedP204COL7A1: c.2722_2723delCA / c.5188 C>Tp.Gln908Valfs∗45 / p.Arg1730∗Reduced (COL7)––––10––––43––––––35RDEB- intermedP120COL7A1: c.5576_5577delAA / c.4012G>Ap.Lys1858Argfs∗12 / p.Gly1338ArgStrongly reduced (COL7)––––––––––––––––36RDEB- inversaEB04701COL7A1: c.8083G>A / c.8083G>Ap.Gly2695Ser / p.Gly2695SerMinimally reduced (COL7)––––16––154––––––––37DDEBP50COL7A1: c.6182G>T / c.=p.Gly2061Val / p.=Reduced (COL7)–––––––––––Weakly positive––––Abbreviations: BMZ, basement membrane zone; COL, collagen; DDEB, dominant dystrophic epidermolysis bullosa; DermExtr, dermal extract; EB, epidermolysis bullosa; ECS, extracellular space; IF, immunofluorescence; IIF, indirect immunofluorescence; JEB-intermed, junctional epidermolysis bullosa intermediate; JEB-loc, junctional epidermolysis bullosa localized; KFA, keratinocyte footprint assay; Lam, laminin; MO, monkey esophagus; NR, number; RDEB-intermed, recessive dystrophic epidermolysis bullosa intermediate; RDEB-sev, recessive dystrophic epidermolysis bullosa severe; SSS, salt split skin.Only positive results above the cut-off value are given. Cut-off values for ELISA as used and evaluated by our diagnostic laboratories: For MBP, COL17 NC16A: >9 positive, = 9 doubtful, < 9 negative; BP230: > 20 positive, 9‒20 doubtful, < 9 negative; COL7: > 6 positive, = 6 doubtful, < 6 negative; For Euroimmun, COL17 NC16A: > 20 positive , BP230: > 20 positive, COL7: > 20 positive. Positive results are indicated in bold, and doubtful results are indicated in italic. Open table in a new tab Abbreviations: BMZ, basement membrane zone; COL, collagen; DDEB, dominant dystrophic epidermolysis bullosa; DermExtr, dermal extract; EB, epidermolysis bullosa; ECS, extracellular space; IF, immunofluorescence; IIF, indirect immunofluorescence; JEB-intermed, junctional epidermolysis bullosa intermediate; JEB-loc, junctional epidermolysis bullosa localized; KFA, keratinocyte footprint assay; Lam, laminin; MO, monkey esophagus; NR, number; RDEB-intermed, recessive dystrophic epidermolysis bullosa intermediate; RDEB-sev, recessive dystrophic epidermolysis bullosa severe; SSS, salt split skin. Only positive results above the cut-off value are given. Cut-off values for ELISA as used and evaluated by our diagnostic laboratories: For MBP, COL17 NC16A: >9 positive, = 9 doubtful, < 9 negative; BP230: > 20 positive, 9‒20 doubtful, < 9 negative; COL7: > 6 positive, = 6 doubtful, < 6 negative; For Euroimmun, COL17 NC16A: > 20 positive , BP230: > 20 positive, COL7: > 20 positive. Positive results are indicated in bold, and doubtful results are indicated in italic. The most important finding of our study is that only two patients (2 of 35, 5.7%) showed linear binding of IgG along the BMZ in DIF. Both patients, #23 and #29, have severe RDEB and were negative for COL7 staining, although patient #29 also had a proven revertant patch. Patient #23 was negative for all serological tests except for one of the ELISA's for COL7 (Table 1). Because patient #23 was negative for the COL7 protein with LH7.2 in the skin, the IgG in the DIF was either not directed to COL7 or it is possible that the patient expresses small amounts of truncated COL7 protein to which the IgG is directed. Patient #29 DIF showed 3+ IgG staining to the BMZ in the revertant skin and negative in the mutant skin. Serology revealed dermal binding of IgG in SSS and positive anti-COL7 autoantibodies in both ELISAs, consistent with a diagnosis of EB acquisita (Figure 1 and Table 1). Both patients did not report any noticeable change of skin phenotype that would indicate the manifestation of EB acquisita, and in the case of patient #29, his revertant skin patch did not blister, even after inducing mechanical trauma (minimal skin rub test) (Figure 1b). This suggests that his general blistering was caused by RDEB and not by circulating autoantibodies as in a case published by Guerra et al., 2018Guerra L. Condorelli A.G. Fortugno P. Calabresi V. Pedicelli C. Di Zenzo G. et al.Epidermolysis bullosa (EB) acquisita in an adult patient with previously unrecognized mild dystrophic EB and biallelic COL7A1 mutations.Acta Derm Venereol. 2018; 98: 411-415Crossref PubMed Scopus (7) Google Scholar, where EB acquisita occurred in a patient with a mild DEB phenotype. In 22 of the 37 patients (59.5%), we found at least one positive serological test (Table 1), and in three other patients, we found at least one serological test that was doubtful, meaning that 67.5% (25/37) of our cohort had circulating antibodies against BMZ proteins. Interestingly, the proportion of patients with at least one positive or doubtful serological test was highest in the severe RDEB subgroup (83%, 14/18 positive and 1/18 doubtful) than in patients with JEB (50%, 4/12 positive and 2/12 doubtful) and in patients with other types of DEB (57%, 4/7 positive). However, the number of patients with JEB and other types of DEB was limited. Furthermore, besides patient #29, none of the other patients showed binding of IgG to the BMZ in SSS, whereas only two patients showed IgA binding (patient #6, positive and patient #27, doubtful), thereby resulting in 3 of 37 (8.1%) with positive or doubtful IIF on SSS. These positive findings on DIF and/or SSS, although only in three patients, are in contrast with the findings of Woodley et al., 2014Woodley D.T. Cogan J. Wang X. Hou Y. Haghighian C. Kudo G. et al.De novo anti-type VII collagen antibodies in patients with recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2014; 134: 1138-1140Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar. In their RDEB cohort, 11 of 22 patients had a positive ELISA. However, none of these 11 patients had a positive DIF or SSS. Finally, it is remarkable that several of the patients have circulating autoantibodies against the protein that they are thought to be lacking owing to their mutations. This might indicate that patients are never truly null and that these patients still express a very small amount of the deficient protein, albeit in a truncated or altered form. An alternative explanation could be that these patients have revertant areas, which have not yet been identified. In our cohort, we found positive Euroimmun ELISA for NC16A in 5 of 37 patients (13.5%) and positive MBP ELISA for NC16A in 12 of 37 patients (32.4%). van Beek et al., 2014van Beek N. Dohse A. Riechert F. Krull V. Recke A. Zillikens D. et al.Serum autoantibodies against the dermal-epidermal junction in patients with chronic pruritic disorders, elderly individuals and blood donors prospectively recruited.Br J Dermatol. 2014; 170: 943-947Crossref PubMed Scopus (15) Google Scholar described that ELISA for NC16A in the elderly (aged >70 years) was positive in about 6.5% and 3.5% for Euroimmun and MBP ELISAs, respectively. This suggests more positive reactions in the patients with EB than in the published elderly group. An important question is why circulating autoantibodies are found so frequently in patients with EB and especially in patients with severe RDEB, which do not seem to be clinically relevant. Esposito et al., 2016Esposito S. Guez S. Orenti A. Tadini G. Scuvera G. Corti L. et al.Autoimmunity and cytokine imbalance in inherited epidermolysis bullosa.Int J Mol Sci. 2016; 17: 1625Crossref Scopus (25) Google Scholar showed that patients with EB and especially those with RDEB have higher levels of proinflammatory cytokines than the levels in the control population. In addition, a recent review by Huitema et al., 2021Huitema L. Phillips T. Alexeev V. Igoucheva O. Immunological mechanisms underlying progression of chronic wounds in recessive dystrophic epidermolysis bullosa.Exp Dermatol. 2021; 30: 1724-1733Crossref PubMed Scopus (8) Google Scholar states that there is evidence that patients with RDEB have an underlying immunity defect. The high number of positive ELISAs in patients with EB may thus be caused by exposure to self-antigens due to repeated skin damage combined with a chronic immunological response or underlying immunity defect because all serological tests were negative in 13 patients with severe acute burn used as controls (data not shown). However, the exact reason is still unknown and warrants further investigation. To summarize, the clinical relevance of autoantibodies in EB is disputable, especially those detected by ELISA or immunoblot because in the majority of patients, no in vivo binding of antibodies could be shown. Furthermore, because more than half of our cohort had a positive serological test without apparent clinical meaning, an exclusion for the therapy trials for EB based on ELISA causes a risk of omitting possible candidates. We suggest DIF combined with IIF on SSS because these methods have a better diagnostic and prognostic value. However, in EB, the clinical significance of reactivity even in DIF and/or IIF on SSS remains uncertain. No datasets were generated or analyzed during this study. Antoni Gostyński: http://orcid.org/0000-0002-1091-2914 Gilles F.H. Diercks: http://orcid.org/0000-0001-8053-216X Maria-José Escamez: http://orcid.org/0000-0002-5434-1885 Nisha Suyien Chandran: http://orcid.org/0000-0001-8225-0035 Raúl de Lucas: http://orcid.org/0000-0001-7587-267X Adela Garcia-Martin: http://orcid.org/0000-0001-7054-7907 Marcela Del Rio: http://orcid.org/0000-0003-2910-7189 Jeroen Bremer: http://orcid.org/0000-0002-7550-6386 Maria C. Bolling: http://orcid.org/0000-0003-2086-9363 Alvaro Meana: http://orcid.org/0000-0001-9434-6042 Sara G. Llames: http://orcid.org/0000-0001-5456-1389 Enno Schmidt: http://orcid.org/0000-0002-1206-8913 Ralf Ludwig: http://orcid.org/0000-0002-1394-1737 Marcel F. Jonkman: http://orcid.org/0000-0002-8471-9340 Hendri H. Pas: http://orcid.org/0000-0001-8823-2591 Anna M.G. Pasmooij: http://orcid.org/0000-0003-0641-3829 The authors state no conflict of interest. This study was funded by the Dutch Butterfly Child Foundation (Stichting Vlinderkind). We gratefully acknowledge all the patients and families for their participation in this study. In addition, we are thankful for the great assistance provided by the research technicians from the University Medical Center Groningen (The Netherlands): Miranda Nijenhuis, Duco Kramer, Gonnie Meijer, and Janny Zuiderveen. MFJ, founder of the Center of Blistering Diseases in Groningen, is deceased (14 January 2019). Conceptualization: AG, AMGP, GFHD, HHP, MFJ, NSC; Data Curation: AMGP, JB, MCB; Formal Analysis: AG, GFHD, NSC; Investigation: AG, AGM, AM, AMGP, GFHD, ES, MDR, MJE, NSC, RDL, RL, SGL; Methodology: AG, AMGP, HHP, MJE, NSC; Supervision: AMGP, ES, HHP, RL; Validation: AMGP, GFHD; Writing - Original Draft Preparation: AG, AMGP; Writing - Review and Editing: AG, AM, AMGP, GFHD, HHP, JB, MCB, MFJ, MJE, NSC, SGL Supplementary Table S1Included patients with age at the time of serum and biopsy sampling, and identification of revertant mosaicismPatientDatabase NRMutation (DNA)Mutation (Protein)Protein Expression in Nonblistered SkinAgeRevertant Mosaicism IdentifiedNREB typeSerum SamplingBiopsy SamplingCOL17A1 Patients1JEB-intermedEB02501COL17A1: c.2237delG / c.2237delGp.Gly746Alafs∗53/p.Gly746Alafs∗53Negative (type XVII collagen)6159Yes2JEB-intermedEB02601COL17A1: c.3676C>T/c.1601delAp.Arg1226∗/p.Asp534Alafs∗19Negative (type XVII collagen)4644Yes3JEB-intermedEB03502COL17A1: c.2237delG/c.3676C>Tp.Gly746Alafs∗53/p.Arg1226∗Negative (type XVII collagen)5548Yes4JEB-intermedEB13401COL17A1: c.1365delC/c.3600_3601delCTCOL17A1: c.1260delC/c.3495_c.3496delCTp.Thr421Leufs∗72/p.Ser1166Leufs∗6Negative (type XVII collagen)118Yes5JEB-intermedEB20801COL17A1: c.2237delG/c.2237delGp.Gly746Alafs∗53/p.Gly746Alafs∗53Negative (type XVII collagen)5247Yes6JEB-intermedEB08601COL17A1: c.1772-2 A>C/c.3327delTin-frame exon skipping/p.Pro1110Argfs∗21Negative (type XVII collagen)140No7JEB-locEB16801COL17A1: c.4320delT/c.4320delTp.Gln1442Lysfs∗70/p.Gln1442Lysfs∗70Strongly reduced (type XVII collagen)4637No8JEB-locEB09801COL17A1: c.2251C>T/c.3327delTp.Gln751∗/p.Pro1110Argfs∗21Strongly reduced (type XVII collagen)4734NoLAMB3 patients9JEB-intermedEB02901LAMB3: c.628G>A/c.628G>Ap.Glu210Lys/p.Glu210LysStrongly reduced (lam332)7171Yes10JEB-intermedEB29901LAMB3: c.1106dup/c.1106dupp.Ala370Serfs∗13/p.Ala370Serfs∗13Normal (lam332)6157No11JEB- intermedEB13201LAMB3: c.1063T>C/c.1903C>Tp.Cys355Arg/p.Arg635∗Strongly reduced (lam332)3839No12JEB-locEB25401LAMB3: c.628G>A/c.1903C>Tp.Glu210Lys/p.Arg635∗Strongly reduced (lam332)00NoCOL7A1 Patients13RDEB-sevEB26501 (P116)COL7A1: c.887delG/c.6527dupp.Gly296Valfs∗5/p.Gly2177Trpfs∗113Strongly reduced (type XII collagen)2625Yes14RDEB-sevEB29701 (P108)COL7A1: c.2142A>G/c.6527dupAltered splicing resulting in out-of-frame transcript p.Gly2177Trpfs∗113Negative (type VII collagen)2525Yes15RDEB-sevEB02201COL7A1: c.3G>T/c.4997dupStart-lost/p.Pro1668fAlafs∗4Negative (type VII collagen)2117No16RDEB-sevEB07701COL7A1: c.925_944dup/c.1264dupp.Ile315Metfs∗12/p.Arg422Profs∗19Negative (type VII collagen)3520No17RDEB-sevEB09001COL7A1: c.4767delA/c.4767delAp.Asp1590Thrfs∗120/p.Asp1590Thrfs∗120Negative (type VII collagen)162No18RDEB-sevP10COL7A1: c.6527dup/c.6527dupp.Gly2177Trpfs∗113/p.Gly2177Trpfs∗113Strongly reduced (type VII collagen)9Not testedNo19RDEB-sevP31COL7A1: c.6527dup/c.6527dupp.Gly2177Trpfs∗113/p.Gly2177Trpfs∗113Strongly reduced (type VII collagen)2120No20RDEB-sevP46COL7A1: c.325_326insCG/c.3277-1G>Cp.Glu109Alafs∗39/Altered splicing resulting in out-of-frame transcriptsStrongly reduced (type VII collagen)4848No21RDEB-sevP52COL7A1: c.7756dup/c.7930-1G>Cp.Gln2586Profs∗12/In-frame exon skippingNegative (type VII collagen)5045No22RDEB-sevP104COL7A1: c.6618+1G>A/c.6618+1G>Ap.K2206_G2207insMSL_E220Gfs∗86/ p.K2206_G2207insMSL_E220Gfs∗86Altered splicing resulting in out-of-frame transcripts/Altered splicing resulting in out-of-frame transcriptsStrongly reduced (type VII collagen)2828No23RDEB-sevP14COL7A1: c.6527dup/c.336C>Gp.Gly2177Trpfs∗113/p.Tyr112∗Negative (type VII collagen)77No24RDEB-sevP18COL7A1: c.6527dup/c.2984dupp.Gly2177Trpfs∗113/p.Gly996Trpfs∗44Negative (type VII collagen)13Not testedNo25RDEB-sevP61COL7A1: c.6527dup/c.6527dupp.Gly2177Trpfs∗113/p.Gly2177Trpfs∗113Strongly reduced (type VII collagen)60No26RDEB-sevP104.ICOL7A1: c.6618+1G>A/c.6618+1G>Ap.K2206_G2207insMSL_E220Gfs∗86/ p.K2206_G2207insMSL_E220Gfs∗86Altered splicing resulting in out-of-frame transcripts/Altered splicing resulting in out-of-frame transcripts?Strongly reduced (type VII collagen)3532No27RDEB-sevP78COL7A1: c.6527dup/c.5130_5131insCTCACp.Gly2177Trpfs∗113/p.Thr1711Leufs∗132Negative (type VII collagen)50No28RDEB-sevEB26001 (P17)COL7A1: c.6527dup/c.6527dupp.Gly2177Trpfs∗113/p.Gly2177Trpfs∗113Strongly reduced (type VII collagen)4443Yes29RDEB-sevEB02401COL7A1: c.6508C>T/c.6508C>Tp.Gln2170∗/p.Gln2170∗Negative (type VII collagen)2527Yes30RDEB-sevEB06401COL7A1: c.1573C>T/c.6508C>Tp.Arg525∗/p.Gln2170∗Negative (type VII collagen)2927No31RDEB-intermedEB09001COL7A1: c.2699G>A/c.7237G>Ap.Trp900∗/p.Gly2413ArgMinimally reduced (type VII collagen)3026No32RDEB-intermedEB34701COL7A1: c.5272G>A/(unknown)p.Gly1758Arg/unknownMinimally reduced (type VII collagen)4343No33RDEB-intermedP36COL7A1: c.6527dup/c.7300G>Ap.Gly2177Trpfs∗113/p.Gly2434ArgReduced (type VII collagen)148No34RDEB-intermedP204COL7A1: c.2722_2723delCA/c.5188 C>Tp.Gln908Valfs∗45/p.Arg1730∗Reduced (type VII collagen)2221No35RDEB-intermedP120COL7A1: c.5576_5577delAA/c.4012G>Ap.Lys1858Argfs∗12/p.Gly1338ArgStrongly reduced (type VII collagen)40No36RDEB-inversaEB04701COL7A1: c.8083G>A/c.8083G>Ap.Gly2695Ser/p.Gly2695SerMinimally reduced (type VII collagen)279No37DDEBP50COL7A1: c.6182G>T/c.=p.Gly2061Val/p.=Reduced (type VII collagen)1717NoAbbreviations: DDEB, dominant dystrophic epidermolysis bullosa; DEB, dystrophic epidermolysis bullosa; EB, epidermolysis bullosa; JEB, junctional epidermolysis bullosa; JEB-intermed, junctional epidermolysis bullosa intermediate; JEB-loc, junctional epidermolysis bullosa localized; NR, number; RDEB-intermed, recessive dystrophic epidermolysis bullosa intermediate; RDEB-inversa, recessive dystrophic epidermolysis bullosa inversa; RDEB-sev, recessive dystrophic epidermolysis bullosa severe.The age at serum and biopsy sampling is indicated per patient with EB. In 10 patients, revertant mosaicism was identified (6 JEB and 4 DEB). In patients in which revertant mosaicism was not identified, it cannot be completely excluded that these patients may have a revertant area that has remained unnoticed until now. Open table in a new tab Supplementary Table 2The Age at Serum Sampling Per Patient with Burn WoundsBurn Wound PatientNRPercentage of Body Surface AffectedAgeSerum Sampling3865703980unknown40246741Unknown2042807643Unknownunknown44503845626467041479045489053499086509546Abbreviation: NR, number.In addition, the percentage of affected skin area is indicated. Open table in a new tab Abbreviations: DDEB, dominant dystrophic epidermolysis bullosa; DEB, dystrophic epidermolysis bullosa; EB, epidermolysis bullosa; JEB, junctional epidermolysis bullosa; JEB-intermed, junctional epidermolysis bullosa intermediate; JEB-loc, junctional epidermolysis bullosa localized; NR, number; RDEB-intermed, recessive dystrophic epidermolysis bullosa intermediate; RDEB-inversa, recessive dystrophic epidermolysis bullosa inversa; RDEB-sev, recessive dystrophic epidermolysis bullosa severe. The age at serum and biopsy sampling is indicated per patient with EB. In 10 patients, revertant mosaicism was identified (6 JEB and 4 DEB). In patients in which revertant mosaicism was not identified, it cannot be completely excluded that these patients may have a revertant area that has remained unnoticed until now. Abbreviation: NR, number. In addition, the percentage of affected skin area is indicated.

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