Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

Using this pattern recognition by direct immunofluorescence microscopy we discovered several cases of EBA which would otherwise have been erroneously diagnosed as a form of pemphigoid or linear IgA disease.

SummaryBackground Paraneoplastic pemphigus (PNP) is a multiorgan disease characterized by antibodies against plakins, desmogleins and the a2-macroglobulin-like-1 (A2ML1) protein, in association with an underlying neoplasm. Accurate diagnosis relies on the demonstration of these autoantibodies in serum. Objectives To evaluate the value of different laboratory techniques in the serological diagnosis of PNP. Methods We performed immunoblotting, envoplakin (EP) enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IIF) on rat bladder, radioactive immunoprecipitation and a nonradioactive combined immunoprecipitation-immunoblot assay. Additional assays included BP180 ELISA and BP230 ELISA. We included the sera of 19 patients with PNP and 40 control subjects. Results The sensitivities were 63% for anti-EP ELISA, 74% for rat bladder IIF, 89% for immunoblotting, 95% for radioactive immunoprecipitation and 100% for nonradioactive immunoprecipitation. Specificities ranged from 86% to 100%. The BP180 and BP230 ELISAs had low sensitivity and specificity for PNP. The combination of rat bladder IIF and immunoblot showed 100% sensitivity and specificity. The analysis of sequential PNP sera showed that antibody titres may decrease over time, possibly resulting in negative outcomes for EP ELISA and rat bladder IIF studies. Conclusions The detection of autoantibodies against EP and periplakin, or A2ML1 by immunoprecipitation is most sensitive for PNP. The combination of rat bladder IIF and immunoblotting is equally sensitive and highly specific, and represents an alternative valuable and relatively easy approach for the serological diagnosis of PNP.

This guideline has been initiated by the task force Autoimmune Blistering Diseases of the European Academy of Dermatology and Venereology, including physicians from all relevant disciplines and patient organizations. It is a S3 consensus‐based guideline that systematically reviewed the literature on mucous membrane pemphigoid (MMP) in the MEDLINE and EMBASE databases until June 2019, with no limitations on language. While the first part of this guideline addressed methodology, as well as epidemiology, terminology, aetiology, clinical presentation and outcome measures in MMP, the second part presents the diagnostics and management of MMP. MMP should be suspected in cases with predominant mucosal lesions. Direct immunofluorescence microscopy to detect tissue‐bound IgG, IgA and/or complement C3, combined with serological testing for circulating autoantibodies are recommended. In most patients, serum autoantibodies are present only in low levels and in variable proportions, depending on the clinical sites involved. Circulating autoantibodies are determined by indirect IF assays using tissue substrates, or ELISA using different recombinant forms of the target antigens or immunoblotting using different substrates. The major target antigen in MMP is type XVII collagen (BP180), although in 10–25% of patients laminin 332 is recognized. In 25–30% of MMP patients with anti‐laminin 332 reactivity, malignancies have been associated. As first‐line treatment of mild/moderate MMP, dapsone, methotrexate or tetracyclines and/or topical corticosteroids are recommended. For severe MMP, dapsone and oral or intravenous cyclophosphamide and/or oral corticosteroids are recommended as first‐line regimens. Additional recommendations are given, tailored to treatment of single‐site MMP such as oral, ocular, laryngeal, oesophageal and genital MMP, as well as the diagnosis of ocular MMP. Treatment recommendations are limited by the complete lack of high‐quality randomized controlled trials.

Large-scale electron microscopy ("nanotomy") allows straight forward ultrastructural examination of tissue, cells, organelles, and macromolecules in a single data set. Such data set equals thousands of conventional electron microscopy images and is freely accessible (www.nanotomy.org). The software allows zooming in and out of the image from total overview to nanometer scale resolution in a 'Google Earth' approach. We studied the life-threatening human autoimmune blistering disease pemphigus, using nanotomy. The pathomechanism of cell-cell separation (acantholysis) that underlies the blistering is poorly understood. Ultrastructural examination of pemphigus tissue revealed previously unreported findings: (i) the presence of double-membrane structures between cells in all pemphigus types; (ii) the absence of desmosomes around spontaneous blisters in pemphigus foliaceus (PF); (iii) lower level blistering in PF when force induced; and (iv) intercellular widening at non-acantholytic cell layers. Thus, nanotomy delivers open-source electron microscopic maps of patient tissue, which can be analyzed for additional anomalies from any computer by experts from different fields.

The frequency of EBA is about one in 18 among patients with sAIBD. The clinical phenotype in two of three cases is inflammatory, thus mimicking other sAIBDs, e.g. bullous pemphigoid, mucous membrane pemphigoid, or linear IgA disease. The yield of diagnosed EBA cases almost triples when serration pattern analysis is used by direct immunofluorescence microscopy on skin biopsy.

A low dose of rituximab is an effective and safe treatment for pemphigus. Relapses may occur, mostly at the end of the second year. Cost-effectiveness studies with a long follow-up are required to determine the proper dosage of this expensive drug in pemphigus.

IMPORTANCE A substantial number of patients with bullous pemphigoid do not develop skin blisters and may not have received the correct diagnosis. Diagnostic criteria and an optimal diagnostic strategy are needed for early recognition and trials.OBJECTIVES To assess the minimal requirements for diagnosis of bullous and nonbullous forms of pemphigoid and to evaluate the optimal diagnostic strategy. DESIGN, SETTING, AND PARTICIPANTSThis paired, multivariable, diagnostic accuracy study analyzed data from 1125 consecutive patients with suspected pemphigoid who were referred to the Groningen Center for Blistering Diseases from secondary and tertiary care hospitals throughout the Netherlands. Eligible participants were patients with paired data on at least (1) a skin biopsy specimen for the direct immunofluorescence (DIF) microscopy test;(2) indirect immunofluorescence on a human salt-split skin substrate (IIF SSS) test; and(3) 1 or more routine immunoserologic tests administered between January 1, 2002, and May 1, 2015. Samples were taken from patients at the time of first diagnosis, before introduction of immunosuppressive therapy, and within an inclusion window of a maximum of 4 weeks. Data analysis was conducted from October 1, 2015, to December 1, 2017. MAIN OUTCOMES AND MEASURESPairwise DIF, IIF SSS, IIF on monkey esophagus, BP180 and BP230 enzyme-linked immunosorbent assays, and immunoblot for BP180 and BP230 tests were performed. The results were reported in accordance with 2015 version of the Standards for Reporting Diagnostic Accuracy. RESULTSOf the 1125 patients analyzed, 653 (58.0%) were women and 472 (42.0%) were men, with a mean (SD) age of 63.2 (19.9) years. In total, 343 participants received a pemphigoid diagnosis, with 782 controls. Of the 343 patients, 74 (21.6%, or 1 in 5) presented with nonbullous pemphigoid. The DIF microscopy was the most sensitive diagnostic test (88.3% [n = 303]; 95% CI, 84.5%-91.3%), whereas IIF SSS was less sensitive (77.0% [n = 263]; 95% CI, 72.2%-81.1%) but was highly specific (99.9%; 95% CI, 99.3%-100%) and complemented most cases with negative DIF findings. Results of the BP180 NC16A enzyme-linked immunosorbent assay did not add diagnostic value for initial diagnosis in multivariable logistic regression analysis of combined tests. These findings lead to the proposed minimal criteria for diagnosing pemphigoid: (1) pruritus and/or predominant cutaneous blisters, (2) linear IgG and/or C3c deposits (in an n-serrated pattern) by DIF on a skin biopsy specimen, and (3) positive epidermal side staining of IgG by IIF SSS on a serum sample; this proposal extends bullous pemphigoid with the unrecognized nonbullous form.CONCLUSIONS AND RELEVANCE Both DIF and IIF SSS tests should be performed for diagnosis of the bullous and nonbullous variants of pemphigoid, and the BP180 NC16A enzyme-linked immunosorbent assay is recommended as an add-on test for disease activity monitoring.