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 In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel.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.

[18F]Sodium Fluoride PET has the potential to identify active formation of calcinosis cutis in limited cutaneous systemic sclerosis.

OBJECTIVES: Calcinosis cutis affects 20-40% of patients with systemic sclerosis (SSc). When calcinosis cutis becomes clinically apparent, it is irreversible in most cases. Detection of active calcification formation might allow early disease-modifying interventions. We assessed the feasibility of visualizing active calcifications using [18F]Sodium Fluoride ([18F]NaF) PET/low-dose CT (LDCT) in SSc patients with calcinosis cutis. METHODS: In this cross-sectional, observational pilot study patients underwent a whole body [18F]NaF PET/LDCT. All patients met the 2013 ACR/EULAR SSc criteria and had clinically detectable calcinosis cutis. (Sub)cutaneous calcifications were described by three investigators. RESULTS: Nine female patients were included (median age 59.0 years [IQR 51.5-70.5]). [18F]NaF uptake was mostly visible in the fingers (n=7) and knees (n=5). [18F]NaF PET showed calcifications in the fingers of 3 patients where calcifications were undetected on LDCT and in the clinic. Ninety-seven percent of [18F]NaF positive lesions was visible on LDCT. Of all lesions visible on LDCT, 70% was also visible on [18F]NaF PET. CONCLUSION: Imaging of active calcifications in SSc is feasible using [18F]NaF PET/LDCT. Seventy percent of calcifications on LDCT were [18F]NaF PET positive. Although these findings require replication, [18F]NaF PET/LDCT may detect active calcification formation, being potentially suitable for early disease-modifying interventions.

Proposal for a 6-step approach for differential diagnosis of neonatal erythroderma.

The broad differential diagnosis of neonatal erythroderma often poses a diagnostic challenge. Mortality of neonatal erythroderma is high due to complications of the erythroderma itself and the occasionally severe and life-threatening underlying disease. Early correct recognition of the underlying cause leads to better treatment and prognosis. Currently, neonatal erythroderma is approached on a case-by-case basis. The purpose of this scoping review was to develop a diagnostic approach in neonatal erythroderma. After a systematic literature search in Embase (January 1990 - May 2020, 74 cases of neonatal erythroderma were identified, and 50+ diagnoses could be extracted. Main causes were the ichthyoses (40%) and primary immunodeficiencies (35%). Congenital erythroderma was present in 64% (47/74) of the cases, predominantly with congenital ichthyosis (11/11; 100%), Netherton syndrome (12/14, 86%) and Omenn syndrome (11/23, 48%). Time until diagnosis ranged from 102 days to 116 days for cases of non-congenital erythroderma and congenital erythroderma respectively. Among the 74 identified cases a total of 17 patients (23%) died within a mean of 158 days and were related to Omenn syndrome (35%), graft-versus-host disease (67%) and Netherton syndrome (18%). Disease history and physical examination are summarized in this paper. Age of onset and a collodion membrane can help to narrow the differential diagnoses. Investigations of blood, histology, hair analysis, genetic analysis and clinical imaging are summarized and discussed. A standard blood investigation is proposed, and the need for skin biopsies with lympho-epithelial Kazal-type related Inhibitor staining is highlighted. Overall, this review shows that diagnostic procedures narrow the differential diagnosis in neonatal erythroderma. A 6-step flowchart for the diagnostic approach for neonatal erythroderma during the first month of life is proposed. The approach was made with the support of expert leaders from international multidisciplinary collaborations in the European Reference Network Skin-subthematic group Ichthyosis.

[Medicaments and oral healthcare. Medicaments potentially inducing angioedema and/or urticaria]. / Serie: Medicamenten en mondzorg. Medicamenten die angio-oedeem en/of urticaria kunnen induceren.

Angioedema stems from increased vasodilation and vascular permeability, resulting in extravasation of fluid. Hereditary and acquired types of angioedema can be distinguished, with 3 and 4 subtypes, respectively. Groups of medicaments potentially inducing angioedema are, among others: ACE inhibitors, angiotensin II receptor blockers, dipeptidyl peptidase-4 inhibitors, thrombocyte aggregation inhibitors and immunosuppressive agents. Urticaria is characterised by red, slightly raised swellings, usually associated with a strong itching sensation and can be subdivided in an acute and a chronic type. Mast cells in the uppermost layer of the skin or the mucous membranes release a lot of histamine, increasing the dilation and permeability of blood capillaries, resulting in extravasation of fluid. Medicaments potentially inducing urticaria are, among others, the following groups: analgesics, anaesthetics, antibiotics, antidepressants, antihistamines, antihypertensives, antifungals, corticosteroids, H2 blockers, cancer medicaments, muscle relaxants, thrombocyte aggregation inhibitors and vaccines. Medical history and being alert when administering and prescribing anaesthetics, analgesics and antibiotics are very important in the prevention or treatment of angioedema and/or urticaria.

Novel insights into the epidemiology of epidermolysis bullosa (EB) from the Dutch EB Registry: EB more common than previously assumed?

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of rare and incurable genetic disorders characterized by fragility of the skin and mucosae, resulting in blisters and erosions. Several epidemiological studies in other populations have been carried out, reporting varying and sometimes inconclusive figures, highlighting the need for standardized epidemiological analyses in well-characterized cohorts. OBJECTIVES: To evaluate the epidemiological data on EB in the Netherlands, extracted from the molecularly well-characterized cohort in the Dutch EB Registry. METHODS: In this observational study all EB-patients that were based in the Netherlands and captured in the Dutch EB Registry between 1988 and 2018 were included. The epidemiological outcomes were based on complete diagnostic data (clinical features, immunofluorescence, electron microscopy and mutation analysis), with longitudinal follow-up. RESULTS: A total of 464 EB-patients (287 families) were included. The incidence and point-prevalence of EB in the Netherlands were 41.3 per million live births and 22.4 per million population, respectively. EB Simplex (EBS), Junctional EB (JEB), Dystrophic EB (DEB) and Kindler EB were diagnosed in 45.7%, 18.8%, 34.7% and 0.9% of the EB-patients, respectively, with an incidence and point-prevalence of 17.5 and 11.9 (EBS), 9.3 and 2.1 (JEB), 14.1 and 8.3 (DEB), 0.5 and 0.2 (Kindler EB). In 90.5% of the EB-patients the diagnosis was genetically confirmed. During the investigated time period 73 EB-patients died, 72.6% of whom as a direct consequence of their EB. CONCLUSION: The epidemiological outcomes of EB in the Netherlands are high, attributed to a high detection rate in a well-organized set-up, indicating that EB might be more common than previously assumed. These epidemiological data help to understand the extensive need for (specialized) medical care of EB-patients and is invaluable for the design and execution of therapeutic trials. This study emphasizes the importance of thorough reporting systems and registries worldwide.

[Medicaments and oral healthcare. Hyperpigmentation of oral soft tissues due to afamelanotide]. / Serie: Medicamenten en mondzorg. Hyperpigmentatie van de orale slijmvliezen door afamelanotide.

The medicament afamelanotide is an analogue of endogenous ?-melanocyte-stimulating hormone. It promotes cutaneous pigmentation, providing protection from sunlight. In dermatology, afamelanotide seems to establish therapeutic results for polymorphic light eruption, solar urticaria, erythropoietic protoporphyria, Hailey-Hailey disease, vitiligo and acne vulgaris. Afamelanotide is available for non-medical use to realise quick and easy skin tanning. Adverse effects of afamelanotide mentioned in the scientific literature are development and aggravation of melanocytic naevi, degeneration of melanocytic naevi to melanomas, melanonychia, systemic toxicity, rhabdomyolysis, posterior reversible encephalopathy syndrome, priapism and hyperpigmentation of oral soft tissues. Furthermore, numerous adverse effects of afamelanotide have been reported to the Netherlands pharmacovigilance centre LAREB as well as numerous adverse effects due to overdosage of afamelanotide to the National Poisons Information Centre. Dentists should be alert to hyperpigmentation of oral soft tissues due to afamelanotide.

Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.

BACKGROUND: Several new genes and clinical subtypes have been identified since the publication in 2014 of the report of the last International Consensus Meeting on Epidermolysis Bullosa (EB). OBJECTIVES: We sought to reclassify disorders with skin fragility, with a focus on EB, based on new clinical and molecular data. METHODS: This was a consensus expert review. RESULTS: In this latest consensus report, we introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Other disorders with skin fragility, where blisters are a minor part of the clinical picture or are not seen because skin cleavage is very superficial, are classified as separate categories. These include peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility. Because of the common manifestation of skin fragility, these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. CONCLUSIONS: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and genetic features of EB. What is already known about this topic? Epidermolysis bullosa (EB) is a group of genetic disorders with skin blistering. The last updated recommendations on diagnosis and classification were published in 2014. What does this study add? We introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Clinical and genetic aspects, genotype-phenotype correlations, disease-modifying factors and natural history of EB are reviewed. Other disorders with skin fragility, e.g. peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility are classified as separate categories; these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Linked Comment: Pope. Br J Dermatol 2020; 183:603.

Mutations in KRT5 and KRT14 cause epidermolysis bullosa simplex in 75% of the patients.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis that may be caused by mutations in the genes KRT5 and KRT14 encoding the basal epidermal keratins 5 (K5) and 14 (K14). Three main clinical subtypes of EBS exist, differing in onset, distribution and severity of skin blistering. Previous reports of KRT5 and KRT14 mutations suggest a correlation between the location of the mutation and the severity of the associated EBS phenotype. OBJECTIVES: The prevalence of KRT5/KRT14 mutations and the genotype-phenotype correlation in the largest tissue-confirmed EBS population is investigated. METHODS: KRT5 and KRT14 genomic DNA and cDNA sequences of 76 clinically well-defined unrelated EBS probands were amplified and then subjected to direct sequencing and product length analysis. Immunofluorescence microscopy on patients' skin biopsies with antibodies against K5 and K14 was performed to study protein expression. RESULTS: In 57 of 76 (75%) probands 41 different KRT5 and KRT14 mutations were identified, of which 12 were novel. Mutations affecting the highly conserved helix boundary motifs of the rod domains of K5 and K14, and the K14 helix initiation motif in particular, were associated with the severest, EBS Dowling-Meara, phenotype. In 21 EBS probands (37%) the mutation was de novo. In 19 probands (25%) KRT5 or KRT14 mutations were excluded. CONCLUSIONS: The phenotype-genotype correlation observed in this large EBS population underscores the importance of helix boundary motifs for keratin assembly. Only three-quarters of biopsy-confirmed EBS probands have KRT5 or KRT14 mutations, indicating genetic heterogeneity in EBS. Alternative gene candidates are discussed.

A novel mutation in the L12 domain of keratin 1 is associated with mild epidermolytic ichthyosis.

BACKGROUND: Epidermolytic ichthyosis (EI), previously termed bullous congenital ichthyosiform erythroderma or epidermolytic hyperkeratosis, is a clinically heterogeneous genodermatosis caused by mutations in the genes encoding the suprabasal keratins 1 and 10. Classical EI is clinically characterized by severe neonatal erythroderma, blistering and fragile skin in infancy, quickly subsiding with subsequent development of generalized scaling hyperkeratosis. We report three Dutch families with palmoplantar keratoderma and mild blistering, but without neonatal erythroderma and generalized scaling. A novel heterozygous missense mutation in the linker L12 domain of KRT1:c.1019A>G, p.Asp340Gly was found associated with this phenotype in these families. OBJECTIVES: To investigate the effects of the novel KRT1:p.Asp340Gly and the one other previously reported KRT1:p.Asp340Val mutations on keratinocyte cytoskeleton formation and stress resistance. METHODS: Wild-type and mutant pEGFP-KRT1 fusion constructs were transfected into HaCaT cells and exposed to hypo-osmotic shock. Haplotyping and genealogical studies were performed to investigate the possibility of a common founder for p.Asp340Gly. RESULTS: Cells transfected with either one of the keratin 1 L12 domain mutations showed significantly increased tonofilament aggregation. The haplotype around the KRT1 gene was shared in all affected family members of two families and a common founder was traced. CONCLUSIONS: Our study supports the pathogenicity of the keratin 1 L12 domain mutations in vitro. These mutations are associated with a milder EI phenotype with pronounced palmoplantar keratoderma, and without neonatal erythroderma and scaling. The KRT1:p.Asp340Gly mutation in the Dutch families is likely to have arisen from a common founder.

Lethal acantholytic epidermolysis bullosa due to a novel homozygous deletion in DSP: expanding the phenotype and implications for desmoplakin function in skin and heart.

Desmoplakin is the major linker in desmosomes in epithelia and myocardium, anchoring intermediate filaments by the C-terminus to plakoglobin and plakophilin in the desmosomal plaque. Mutations in the gene DSP encoding desmoplakin have been associated with various phenotypes affecting skin and/or heart. One of these phenotypes, lethal acantholytic epidermolysis bullosa (LAEB), is characterized by extensive postnatal shedding of epidermis leading to early demise and is caused by recessive mutations in the gene DSP resulting in truncation of the desmoplakin C-terminus. Here we describe two infants born to the same consanguinous parents who suffered extensive epidermal dislodgment and died shortly after birth. In addition, universal alopecia, anonychia, malformed ears and cardiomyopathy were observed. As the clinical diagnosis was LAEB, DSP mutation analysis was performed. A homozygous deletion (c.2874del5) abrogating the donor splice site of exon 20 was found. The deletion is predicted to cause read-through in intron 20 with subsequent recognition of a premature termination codon, resulting in desmoplakin lacking its rod domain and C-terminus (p.Lys959MetfsX5). Electron microscopic analysis of skin biopsies showed absence of the desmosomal inner dense plaque and lack of tonofilament insertion. This is the second report of LAEB. These findings suggest DSP mutations as the aetiology of LAEB and cardiomyopathy as part of the phenotype. Furthermore, they indicate that in addition to the desmoplakin C-terminus, the rod domain is dispensable for intrauterine development but is essential for the inner dense plaque of desmosomes.

Acquired palmoplantar keratoderma and immunobullous disease associated with antibodies to desmocollin 3.

We present a case of immunobullous disease with an impressive acquired palmoplantar keratoderma (PPK) and unique antigenicity. The palms of the patient showed hyperkeratotic ridges with a tripe pattern that decreased with the amelioration of the immunobullous condition. The histopathology of perilesional skin (blister) demonstrated eosinophilic spongiosis and suprabasal blistering as in pemphigus vulgaris. In palmar skin, acantholysis, intraepidermal pustules, papillomatosis and marked hyperkeratosis were observed. Direct and indirect immunofluorescence displayed intraepidermal intercellular IgG staining as well as linear IgG staining along the epidermal basement membrane zone. Immunochemical assays revealed IgG antibodies to the desmosomal protein desmocollin 3 and to the hemidesmosomal proteins BP230 and LAD-1. Affinity-purified antidesmocollin 3 serum IgG bound to monkey oesophagus in the typical pemphigus pattern. Desmocollins are transmembrane proteins of the desmosome. Desmosome diseases may cause hereditary PPK. In our patient with acquired PPK, we hypothesize that the antibodies to desmocollin 3 were, apart from their role in eliciting the pemphigus-like blistering disease, also implicated in the pathogenesis of the PPK.