Evolution of genome diagnostics in epidermolysis bullosa: Unveiling the power of next-generation sequencing.

BACKGROUND: Genome diagnostics is considered gold standard diagnostics for epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous group of rare disorders characterized by blistering and wounding of mucocutaneous tissues. EB is caused by pathogenic variants in genes encoding proteins of the dermo-epidermal junction. Accurate genetic diagnosis of EB is crucial for prognostication, counselling and precision-medicine. Genome diagnostics for EB started in 1991 with the introduction of Sanger sequencing (SS), analysing one gene at a time. In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel. Several studies have shown a beneficial role for NGS in EB diagnostics, but its true benefit has not been quantified. OBJECTIVES: To determine the benefit of NGS in EB by systematically evaluating the performance of different genome diagnostics used over time based on robust data from the Dutch EB Registry. METHODS: The diagnostic performances of SS and NGS were systematically evaluated in a retrospective observational study including all index cases with a clinical diagnosis of EB in whom genome diagnostics was performed between 01 January 1994 and 01 January 2022 (n = 308), registered at the Dutch EB Expertise Centre. RESULTS: Over time, a genetic diagnosis was made in 289/308 (94%) EB cases. The diagnostic yield increased from 89% (SS) to 95% (NGS). Most importantly, NGS significantly reduced diagnostic turnaround time (39 days vs. 211 days, p < 0.001). The likelihood of detecting variants of uncertain significance and additional findings increased from 5% and 1% (SS) to 22% and 13% (NGS) respectively. CONCLUSIONS: Our study quantifies the benefit of NGS-based methods and demonstrate they have had a major impact on EB diagnostics through an increased diagnostic yield and a dramatically decreased turnaround time (39 days). Although our diagnostic yield is high (95%), further improvement of genome diagnostics is urgently needed to provide a genetic diagnosis in all EB patients.

Patients suffering from dystrophic epidermolysis bullosa are prone to developing autoantibodies against skin proteins: A longitudinal confirmational study.

Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.

[Eosinophilic dermatoses]. / Eosinophile Dermatosen.

Eosinophilic dermatoses are a heterogeneous group of rare diseases that histopathologically display the defining pattern of an eosinophil-rich dermal infiltrate. In these eosinophilic dermatoses, a histopathologic pattern called flame figures, which result from degranulation of eosinophils in the tissue, can be observed. Although eosinophil granulocytes can also be detected in other dermatoses such as atopic dermatitis, urticaria, prurigo and bullous pemphigoid, the eosinophil-rich infiltrate is decisive for classic eosinophilic dermatoses. Accordingly, eosinophilic dermatoses include hypereosinophilic syndrome, eosinophilic fasciitis, granuloma faciale, pustular sterile eosinophilia, and angiolymphoid hyperplasia with eosinophilia. These eosinophilic dermatoses display clinical different patterns and are discussed in this article, as well as the interesting eosinophils and novel therapeutic options.

European guidelines (S3) on diagnosis and management of mucous membrane pemphigoid, initiated by the European Academy of Dermatology and Venereology - Part I.

This guideline on mucous membrane pemphigoid (MMP) has been elaborated by the Task Force for Autoimmune Blistering Diseases of the European Academy of Dermatology and Venereology (EADV) with a contribution of physicians from all relevant disciplines and patient organizations. It is a S3 consensus-based guideline encompassing a systematic review of the literature until June 2019 in the MEDLINE and EMBASE databases. This first part covers methodology, the clinical definition of MMP, epidemiology, MMP subtypes, immunopathological characteristics, disease assessment and outcome scores. MMP describes a group of autoimmune skin and mucous membrane blistering diseases, characterized by a chronic course and by predominant involvement of the mucous membranes, such as the oral, ocular, nasal, nasopharyngeal, anogenital, laryngeal and oesophageal mucosa. MMP patients may present with mono- or multisite involvement. Patients' autoantibodies have been shown to be predominantly directed against BP180 (also called BPAG2, type XVII collagen), BP230, laminin 332 and type VII collagen, components of junctional adhesion complexes promoting epithelial stromal attachment in stratified epithelia. Various disease assessment scores are available, including the Mucous Membrane Pemphigoid Disease Area Index (MMPDAI), the Autoimmune Bullous Skin disorder Intensity Score (ABSIS), the 'Cicatrising Conjunctivitis Assessment Tool' and the Oral Disease Severity Score (ODSS). Patient-reported outcome measurements (PROMs), including DLQI, ABQOL and TABQOL, can be used for assessment of quality of life to evaluate the effectiveness of therapeutic interventions and monitor disease course.

European Guidelines (S3) on diagnosis and management of mucous membrane pemphigoid, initiated by the European Academy of Dermatology and Venereology - Part II.

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.

IgE autoantibodies in serum and skin of non-bullous and bullous pemphigoid patients.

BACKGROUND: Non-bullous pemphigoid (NBP) is a pemphigoid variant which frequently resembles other pruritic skin diseases. In contrast with bullous pemphigoid (BP), blisters are absent. In BP, previous studies showed that IgE autoantibodies may be involved in its pathogenesis. IgE-activated mast cells, basophils and eosinophils may participate in BP by inducing pruritus and possibly blister formation, although the differential role of IgE in NBP compared with BP has not yet been described. OBJECTIVE: To assess IgE in serum and skin of NBP and BP patients. METHODS: We examined total IgE and pemphigoid-specific IgE in the serum of 68 NBP and 50 BP patients by enzyme-linked immunosorbent assay (ELISA). Sera of 25 pemphigus patients and 25 elderly patients with pruritus were included as controls. Skin biopsies of 14 NBP and 14 BP patients with the highest IgE titres to NC16A were stained for IgE by immunofluorescence techniques. RESULTS: Total IgE was elevated in 63% of NBP and 60% of BP patients, and in 20% of pemphigus controls, as well as 60% of elderly controls. IgE ELISAs were more frequently positive in BP than in NBP (NC16A 18% vs. 9%, P = 0.139; BP230 34% vs. 22%, P = 0.149). IgE ELISAs for NC16A and BP230 were positive in 8% and 20% of elderly controls, respectively, while all pemphigus controls were negative. Two of 28 biopsies (7%; one NBP, one BP) showed linear IgE along the basement membrane zone, while in most biopsies (71% NBP; 86% BP) IgE was bound to dermal cells. CONCLUSION: Since IgE was present in the serum and skin of both NBP and BP patients, this supports IgE-dependent mechanisms common to both diseases, such as pruritus. However, it remains to be elucidated whether IgE contributes to blister formation in BP.

[Skin-coloured vulvar papules]. / Huidkleurige vulvaire papels.

We report a 43-year-old woman with asymptomatic vulvar papules. Histopathology showed ducts with luminal differentiation and eosinophil debris with a tadpole shape. Based on these findings, the diagnosis vulvar syringoma was made. Syringoma are benign eccrine sweat gland tumour mostly located on lower eyelids and cheeks. Vulvar syringoma are rare.

Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases.

BACKGROUND: Antilaminin-332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin-332. At present no commercial assay is available to demonstrate antilaminin-332 antibodies, and diagnosis relies on in-house techniques with limited sensitivities. OBJECTIVES: In order to move, keratinocytes cultured in vitro secrete laminin-332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin-332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin-332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints. METHODS: Normal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin-332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin-332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera. RESULTS: IgG of all antilaminin-332 pemphigoid sera (n = 16) bound to laminin-332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt-split skin analysis, demonstrating that it is a very sensitive technique. CONCLUSIONS: The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies. What's already known about this topic? Antilaminin-332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies. The diagnosis of antilaminin-332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin-332 antibodies and can be confirmed only in specialized laboratories. Keratinocytes in culture need laminin-332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin-332 autoantibodies in patient serum using the native laminin-332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies.

Pemphigoid variants affecting the skin.

Pemphigoid diseases are autoimmune subepidermal blistering diseases affecting the skin and mucous membranes, which are caused by autoantibodies targeting structural hemidesmosomal proteins or hemidesmosome-associated proteins. Variants of pemphigoid can be differentiated based on targeted antigens and clinical aspects. In this review, we will discuss pemphigoid variants that predominantly affect the skin, and provide clinicians with clues to diagnosis.

Foreign body reaction triggered by cytotoxic T lymphocyte-associated protein 4 blockade 25 years after dermal filler injection.

Foreign body reactions are regularly seen as a late complication of cosmetic treatment with synthetic dermal fillers. Often this foreign body reaction is triggered by a systemic infection, but other systemic triggers are also reported. In this case report, we present a woman in her 60s who was treated with ipilimumab for metastatic melanoma. After two courses of treatment she developed painless facial nodules. A foreign body reaction to dermal fillers was suspected because the patient had received cosmetic treatment with dermal fillers 25 years previously. This reaction was confirmed by excision and histological examination. In the absence of other known triggers, this case revealed immunotherapy (ipilimumab) and subsequent activation of the adaptive immune system as potential triggers of foreign body reactions to dermal fillers. Immunotherapy is increasingly used as anticancer treatment for an increasing number of tumour types. Furthermore, synthetic dermal fillers have frequently been used in the past. Therefore, physicians should be aware of this late-occurring complication of synthetic filler treatment in patients who develop skin lesions during immunotherapy.