Identification and Computational Analysis of Novel Pathogenic Variants in Pakistani Families with Diverse Epidermolysis Bullosa Phenotypes.

Epidermolysis bullosa (EB) includes a group of rare gesnodermatoses that result in blistering and erosions of the skin and mucous membranes. Genetically, pathogenic variants in around 20 genes are known to alter the structural and functional integrity of intraepidermal adhesion and dermo-epidermal anchorage, leading to four different types of EB. Here we report the underlying genetic causes of EB phenotypes segregating in seven large consanguineous families, recruited from different regions of Pakistan. Whole exome sequencing, followed by segregation analysis of candidate variants through Sanger sequencing, identified eight pathogenic variants, including three novel (ITGB4: c.1285G>T, and c.3373G>A; PLEC: c.1828A>G) and five previously reported variants (COL7A1: c.6209G>A, and c.1573C>T; FERMT1: c.676insC; LAMA3: c.151insG; LAMB3: c.1705C>T). All identified variants were either absent or had very low frequencies in the control databases. Our in-silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Intriguingly, we report the first case of a recessively inherited form of rare EBS-Ogna associated with a homozygous variant in the PLEC gene. Our study highlights the clinical and genetic diversity of EB in the Pakistani population and expands the mutation spectrum of EB; it could also be useful for prenatal diagnosis and genetic counseling of the affected families.

WebPalestra: epidermólise bolhosa

Conceito Epidermólise Bolhosa (EB). Definindo os 4 tipos e cuidados iniciais com o recém nascido. Orientações básicas quanto aos curativos adequados e cadastro Debra Brais.

WebPalestra: epidermólise bolhosa: o que há de novo, atualizações e pesquisas

Pesquisas em desenvolvimento e mais atuais sobre Epidermolise Bolhosa, com destaque para o transplante de medula óssea, diagnóstico genético e terapias gênicas.

Next-generation sequencing through multigene panel testing for the diagnosis of hereditary epidermolysis bullosa in Chinese population.

Epidermolysis bullosa (EB) is a heritable blistering disorder. We performed a next-generation sequencing-based multigene panel test and successfully predicted 100% of the EB types, including, 36 EB simplex (EBS), 13 junctional EB (JEB), 86 dystrophic EB (DEB), and 3 Kindler EB. Chinese JEB and recessive DEB (RDEB) patients have relatively mild phenotypes; for severe type separately accounts for 45.5% and 23.8%, respectively. We identified 96 novel and 49 recurrent pathogenic variants in 11 genes, although we failed to detect the second mutation in one JEB and five RDEB patients. We identified one novel p.E475K mosaic mutation in the clinically normal mother of one out of 13 EBS patients with KRT5 mutations, one recurrent p.G2034R mosaic mutation, and one novel p.G2043R mosaic mutation in the clinically normal relatives of two out of 19 dominant DEB patients. This study shows that next-generation technology could be an effective tool in diagnosing EB.

Inherited epidermolysis bullosa: New diagnostics and new clinical phenotypes.

Inherited epidermolysis bullosa (EB) is a group of heterogeneous genetic disorders characterized by skin fragility. EB comprises a large spectrum of phenotypes, ranging from severe cutaneous and extracutaneous involvement caused by lack of key adhesion proteins, to mild cutaneous fragility caused by subtle molecular defects. Disease-causing variants in 20 different genes account for the genetic and allelic heterogeneity of EB. Here, we discuss the development of laboratory methods that enabled these discoveries and the clinical and molecular features of some new EB entities elucidated during the past 5-6 years.

Mutations in PLOD3, encoding lysyl hydroxylase 3, cause a complex connective tissue disorder including recessive dystrophic epidermolysis bullosa-like blistering phenotype with abnormal anchoring fibrils and type VII collagen deficiency.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

The Position of Targeted Next-generation Sequencing in Epidermolysis Bullosa Diagnosis.

The precise classification of epidermolysis bullosa (EB) into 4 main types and more than 30 subtypes is based on the level of skin cleavage, as well as clinical and molecular features, and is crucial for early prognostication, case management, genetic counselling and prenatal or pre-implantation diagnosis. We report here the molecular pathology of 40 consecutive cases of suspected EB, which were investigated by immunofluorescence mapping (IFM) and/or by a targeted next-generation sequencing (NGS) multi-gene panel. IFM correctly established the EB subtype in 76% of cases, while the molecular pathology was completely elucidated in 90% of cases by the targeted NGS multi-gene panel. Thirteen previously unreported mutations in EB genes were identified. In cases with unclear clinical and IFM findings, mutations were found by NGS in previously unexpected genes. IFM was useful in delivering fast results in newborns, and in indicating the consequences of the variants of uncertain significance on protein level. This study underscores the efficacy of the strategy of combining targeted NGS with IFM in resolving unusual EB phenotypes. It also suggests that, despite technological advances, careful clinical evaluation and deep phenotyping remains a crucial factor that dictates successful diagnosis of EB.

[Epidermolysis bullosa].

Epidermolysis bullosa (EB) is a rare genodermatosis. A new classification system is presented, distinguishing the subtypes of EB, and this system is based on the phenotype, mode of inheritance, ultrastructure, immunofluorescence findings, and specific mutation(s) present. EB is inherited in an autosomal dominant or -recessive fashion. Clinical manifestations vary in severity and character according to subtype. The severity ranges from mild localized to life-threatening. Available treatment is mainly symptomatic with therapeutic treatment in an experimental stage.

Cardiomyopathy in Patients With Hereditary Bullous Epidermolysis. / Miocardiopatía en pacientes con epidermólisis ampollosa hereditaria.

INTRODUCTION AND OBJECTIVE: In recent decades, an association has been reported between epidermolysis bullosa (EB) and dilated cardiomyopathy (DC). DC is typically in an advanced phase when detected, leading to a poorer prognosis. Our objective was to determine the prevalence of DC in patients with EB seen in Hospital San Joan de Déu in Barcelona, Spain, between May 1986 and April 2015. METHODS: This was a descriptive, cross-sectional chart-review study in which we recorded the type and main subtypes of EB and the presence or absence of DC. RESULTS: Fifty-seven patients with EB were found, 19 with EB simplex, 10 with junctional EB, 27 with dystrophic EB (14 dominant dystrophic and 13 recessive dystrophic), and just 1 with Kindler syndrome. DC was detected in only 2 patients with recessive dystrophic EB. Twenty-three patients had presented factors that could have had a causal relationship with the potential onset of DC. CONCLUSION: DC is a possible complication of EB, particularly in recessive dystrophic EB. Periodic follow-up should be performed to make an early diagnosis and start treatment.

Molecular pathology of the basement membrane zone in heritable blistering diseases:: The paradigm of epidermolysis bullosa.

Epidermolysis bullosa (EB), a phenotypically heterogeneous group of skin fragility disorders, is characterized by blistering and erosions with considerable morbidity and mortality. Mutations in as many as 18 distinct genes expressed at the cutaneous basement membrane zone have been shown to be associated with the blistering phenotype, attesting to the role of the corresponding proteins in providing stable association of the epidermis to the dermis through adhesion at the dermo-epidermal basement membrane zone. Thus, different forms of EB have been highly instructive in providing information on the physiological functions of these proteins as integral components of the supramolecular adhesion complexes. In addition, precise information of the underlying genes and distinct mutations in families with EB has been helpful in subclassification of the disease with prognostic implications, as well as for prenatal testing and preimplantation genetic diagnosis. Furthermore, knowledge of the types of mutations is a prerequisite for application of allele-specific treatment approaches that have been recently developed, including read-through of premature termination codon mutations and chaperone-facilitated intracellular transport of conformationally altered proteins to proper physiologic subcellular location. Collectively, EB serves as a paradigm of heritable skin diseases in which significant progress has been made in identifying the underlying genetic bases and associated aberrant pathways leading from mutations to the phenotype, thus allowing application of precision medicine for this, currently intractable group of diseases.

The Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI): grading disease severity and assessing responsiveness to clinical change in epidermolysis bullosa.

BACKGROUND: The lack of validated outcome measures for epidermolysis bullosa (EB) presents major barriers to evaluating disease severity and comparing the efficacy of therapies. The Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) was recently introduced as a valid and reliable instrument for EB; however, its interpretation for use in clinical practice and clinical trials is yet to be defined. OBJECTIVE: To assess the interpretability of the EBDASI in classifying patients according to disease severity and clinical response. METHODS: A total of 53 outpatients with EB at two interstate institutions were prospectively evaluated. At each visit, the principal dermatologist completed the EBDASI and global assessments of disease severity and change. Classifications for mild, moderate and severe disease using the EBDASI were determined using receiver operating characteristic curves. Minimal clinically important differences for the EBDASI activity subscale were calculated and compared with the standard error of measurement. RESULTS: Total EBDASI score ranges of 0-42, 43-106 and 107-506 corresponded to mild, moderate and severe disease respectively. Reduction in EBDASI activity scores of greater than 9 indicated clinically significant improvement. An increase of 3 in the activity score indicated deterioration. CONCLUSION: The EBDASI is a responsive tool and may be useful in characterizing disease severity and response. The cut-offs proposed in this study provide the first practical guide for interpreting the EBDASI, further supporting its use for longitudinal patient assessment and in clinical trials.

[Hereditary epidermolysis bullosa: French national guidelines (PNDS) for diagnosis and treatment]. / Épidermolyses bulleuses héréditaires : protocole national de diagnostic et de soins (PNDS).

Hereditary epidermolysis bullosa (EB) is a heterogeneous group of rare genetic diseases characterized by fragile skin and/or mucous membrane, and it may be either local or generalized. It is caused by mutations in genes encoding different proteins involved mainly in the structure and function of the dermal-epidermal junction. Nineteen genes have so far been identified. They are classified by level of skin cleavage (from top to bottom) into four groups: EB simplex, junctional EB, dystrophic EB and Kindler syndrome. Clinically suspected diagnosis is confirmed by immunohistochemical examination of a skin biopsy at specialized centres in order to determine the level of cleavage and the deficient protein. This first step may be followed by genetic analysis. The severity of the disease is highly variable, ranging from localized forms with little effect on quality of life to rapidly lethal forms. In generalized severe forms, the extent and chronicity of lesions, as well as mucosal involvement, can lead to systemic complications: malnutrition, pain, joint contractures, chronic inflammation, amyloidosis, cutaneous squamous cell carcinoma. Some specific forms are associated with other cutaneous signs (nail involvement, alopecia, hyperpigmentation, palmoplantar keratoderma) or extracutaneous involvement (muscular dystrophy or pyloric atresia). No curative treatment of EB is available today. EB requires multidisciplinary medical care, nursing, psychological and social management. This is best provided by a specialized network, involving reference centres, centres of expertise and daily caregivers. The goal of treatment is the prevention and treatment of lesions with specific non-adherent dressings and the prevention, detection and treatment of complications. It is essential not to traumatize the skin (bandaging, friction, etc.). Protein, gene or cell replacement therapy, and allogeneic bone marrow, cord blood or pluripotent stem-cell transplantation are currently being assessed. The aim of these French recommendations (national diagnostic and treatment protocol [PNDS]) is to provide healthcare professionals with guidance on the course of EB and on optimal patient management.

Epidemiology of Inherited Epidermolysis Bullosa Based on Incidence and Prevalence Estimates From the National Epidermolysis Bullosa Registry.

Importance: Accurate estimation of the incidence and prevalence of each subtype of epidermolysis bullosa (EB) is essential before clinical trials can be designed and sufficient funding allocated by government agencies and third-party insurers for the care of these individuals. Objective: To determine the incidence and prevalence of inherited EB stratified by subtype in the United States during a 16-year period. Design, Setting, and Participants: Prospective cross-sectional and longitudinal study. Data were obtained from 3271 patients consecutively enrolled in the National Epidermolysis Bullosa Registry from January 1, 1986, through December 31, 2002, using a detailed instrument created with the assistance of the National Institutes of Health. Analyses were performed in January 1999 and April 2015. Participants were patients of all ages with EB. Main Outcomes and Measures: Extensive clinical and laboratory data were collected on patients who were subclassified and serially revalidated based on published diagnostic recommendations by an international panel of experts. Pertinent to this report, estimates were made of the incidence and prevalence during 2 time frames. Results: During the first 5 years of funding of the registry, the overall incidence and prevalence of inherited EB were 19.60 and 8.22 per 1 million live births, respectively. When reassessed over the entire 16 years of the study, the prevalence rose to 11.07, whereas the overall incidence remained unchanged at 19.57 cases. Changes were also observed within some disease subsets as increased numbers of patients were identified, recruited, followed up longitudinally, and resubclassified as needed over time. For example, in 2002, the prevalence of EBS overall and localized EBS had increased considerably by 30.4% and 25.5%, respectively, whereas the prevalence of generalized intermediate EBS declined by 76.7% as a result of later subclassification of some of those patients into other subtypes. In contrast, no significant change was noted in the overall prevalence of JEB or generalized severe JEB, although there was a 73.0% decline in the prevalence of generalized intermediate JEB. Conclusions and Relevance: Precise estimates of the incidence and prevalence of each major subtype of inherited EB in the United States are now available that should assist investigators in choosing which subtypes are amenable to properly designed, large-scale, clinical trials.

Newborn with severe epidermolysis bullosa: to treat or not to treat?

Epidermolysis bullosa (EB) is an inherited skin disease with four main subtypes that cannot be distinguished clinically at birth. All subtypes may present with widespread life-threatening blisters and fragile skin, making treatment and handling of the newborn with EB challenging. The prognosis of EB depends on the subtype, and therefore maximum treatment is necessary until the final diagnosis is known. In this case, it took 2 weeks before a final diagnosis was reached. In the meantime, we had several ethical discussions on the treatment level. The most important issues were management of pain and nutrition. For immediate pain relief, intranasal fentanyl worked best and gabapentin was successfully used for chronic pain. The feeding difficulties were handled first by a nasogastric feeding tube. Later a normal feeding bottle proved to be adequate.

The Frequency of Signs of Meibomian Gland Dysfunction in Children with Epidermolysis Bullosa.

PURPOSE: To determine the frequency of meibomian gland dysfunction (MGD) in children with epidermolysis bullosa (EB). DESIGN: Hospital-based cross-sectional study. PARTICIPANTS: One hundred five children with different forms of EB. METHODS: Prospective ophthalmic examination of children with EB presenting over seventeen months including meibomian gland assessment using a recognized classification. MAIN OUTCOME MEASURES: Frequency of MGD. RESULTS: One hundred five children were recruited, 8.6% with junctional EB, 34.3% with simplex EB, 34.3% with autosomal recessive dystrophic EB, and 22.9% autosomal dominant dystrophic EB. Mean age was 7.42 years (range, 0.08-17.75 years). Ninety-two children (87.62%) demonstrated 1 or more features of MGD. CONCLUSIONS: Most children with EB exhibit signs of MGD. To the best of our knowledge, this is the first prospective ocular surface evaluation in children with EB to include lid margin evaluation using a recognized classification system. Our findings help explain some of the ocular surface anomalies seen in children with EB.

[Current approaches to the morphologic diagnosis of different types of congenital epidermolysis bullosa]. / Sovremennye podkhody k morfologicheskoi diagnostike razlichnykh tipov vrozhdennogo bulleznogo epidermoliza.

Congenital epidermolysis bullosa (CEB) is an extensive group of hereditary skin diseases, the differential diagnosis of which is a challenge due to the rarity of this pathology and the diversity of its clinical manifestations. The determination of the type of CEB makes it possible to estimate its prognosis and to facilitate a prenatal diagnosis. AIM: to optimize the morphological diagnosis of different types of CEB. MATERIAL AND METHODS: 28 skin biopsies from 14 patients with different types of CEB were investigated. The investigators performed routine histological examination of skin fragments taken from a bullous area and immunofluorescence antigen mapping using the indirect immunofluorescence test (IIFT) with antibodies against structural proteins of the dermal-epidermal junction (laminin α3, ß3, and γ2 chains, keratins 5 and 14, types VII and XVII collagen, α6 and ß4 integrin subunits, desmoplakin, plectin, kindlin-1, and plakophillin) of the apparently unaffected skin. The intact skin of healthy individuals, which had been obtained during cosmetic operations, was used as controls in IIFT. RESULTS: Immunofluorescence antigen mapping could determine the type of CEB in all cases and in 86% of cases identify the protein, the impaired production of which was responsible for the development of the disease. CONCLUSION: Immunofluorescence antigen mapping is an integral part of the comprehensive morphological diagnosis of CEB, acting as an intermediate between the morphological verification of CEB diagnosis and the targeted search for mutations by a molecular genetic method.

Using immunofluorescence (antigen) mapping in the diagnosis and classification of epidermolysis bullosa: a first report from Iran.

BACKGROUND: Immunofluorescence antigen mapping (IFM), is a newly introduced technique for diagnosis and classification of epidermolysis bullosa (EB) disease. The precise level of skin cleavage can be determined using monoclonal antibodies to EB-specific basement membrane zone protein. OBJECTIVE: To apply IFM technique in diagnosis and classification of EB and to identify utility and limitation of this method in our clinical setting. METHODS: IFM was done according to a described protocol by Pohla-Gubo et al. Monoclonal antibodies used for antigen mapping were against cytokeratin 5, cytokeratin 14, α6 integrin, ß4 integrin, laminin 332, Collagen IV, and Collagen VII. RESULTS: IFM was done for 95 referred patients, compromising 49 females and 46 males, aged 5 days to 45 years (mean = 9.5 years). Ninety cases were diagnosed with EB and classified as follows: EB simplex: (n = 13), junctional EB (n = 14), dystrophic EB (n = 62), and Kindler syndrome (n = 1). Diagnosis was not made in five cases as their specimens contained no blister. Confirmatory genetic analysis was done for five junctional cases from two families with clinical features of laryngo-onycho-cutaneous syndrome. Genetic molecular studies showed nonsense mutations in the last codon of exon 39 of the laminin α3a (LAMA3) gene (p.Gln57X) and a donor splice site mutation in LAMA3 (IVS57+5G>A) in the first and second family, respectively. CONCLUSION: IFM technique is relatively simple to perform, and interpretation of the results is not sophisticated. The proportion of inconclusive results will be decreased if the specimens contain freshly induced blister.

Evaluation of internal consistency of the epidermolysis bullosa oropharyngeal severity score (EBOS).

OBJECTIVE: To evaluate the internal consistency of the epidermolysis bullosa oropharyngeal severity score (EBOS). MATERIALS AND METHODS: Data from 92 patients of varying EB types/sub-types already described in a previous multi-center study were re-analyzed via the coefficient Cronbach's α (CR-α). Additionally, the corrected item total correlation between each item and the items' overall score with Pearson's product-moment correlation (ρ) was calculated. RESULTS: The alpha coefficient for the mean total score of 17 items is 0.941. The inter-observer reliability for disease severity score was excellent for oral medicine specialist (α = 0.924) and dermatologist (α = 0.916) and the intra-observer reliability was good at Time 1 (α = 0.895) and Time 2 (α = 0.897). The analysis of CR-α per single item revealed that alpha was greater than 0.904 for disease activity and 0.743 for structural damage, after the elimination of four items for oral medicine specialist and greater than 0.898 for disease activity and 0.769 for structural damage after the elimination of five items for dermatologist. Similarly the analysis of the corrected items-EBOS correlation showed that the same items do not correlate very well (ρ < 0.4) with the overall EBOS. CONCLUSIONS: The EBOS turned out to have a strong and reliable internal consistency, as the majority of the EBOS' items were consistent with each other.

Correlation between nutritional, hematological and infectious characteristics and classification of the type of epidermolysis bullosa of patients assisted at the Dermatology Clinic of the Hospital Universitário de Brasília.

Epidermolysis bullosa comprises a group of phenotypically different genodermatosis, hereditary or acquired, characterized by skin fragility and subsequent formation of blisters in response to mechanical trauma, and which may also affect mucous membranes. This study aimed to analyze the relation between the nutritional, hematologic, infectious characteristics and the type of epidermolysis bullosa, through a descriptive case study based on data from medical records of 10 patients with epidermolysis bullosa assisted regularly at the Dermatology Clinic of the Hospital Universitário de Brasília. The old classification of the type of epidermolysis bullosa, weight and height, blood count, white blood cell count, platelet count and description of the type and frequency of secondary infections during the service were considered. We verified a predominance of iron deficiency anemia, chronic leukocytosis, thrombocytosis, chronic malnutrition, low height for age and thinness, and people with epidermolysis bullosa simplex exhibited appropriate relation between height/age and BMI/age. The non-specific skin infection was the most prevalent in both sexes. The severity of the type of epidermolysis bullosa and frequency of secondary infections did not form a directly proportional relation. The absence of direct proportion in all cases between the type of epidermolysis bullosa and the analysis parameters suggest a possible significant interference from other aspects such as the extent of the affected skin area, extracutaneous type of engagement and specific genetic mutation. The inclusion of these factors in the new classification proposed by Fine et al can contribute significantly to a better correlation of clinical parameters and appropriate preventive and therapeutic approaches.