Genotype-Phenotype Correlation in Junctional Epidermolysis Bullosa: Signposts to Severity.

Junctional epidermolysis bullosa (JEB) is a rare autosomal recessive genodermatosis with a broad spectrum of phenotypes. Current genotype-phenotype paradigms are insufficient to accurately predict JEB subtype and characteristics from genotype, particularly for splice site variants, which account for over a fifth of disease-causing variants in JEB. This study evaluated the genetic and clinical findings from a JEB cohort, investigating genotype-phenotype correlations through bioinformatic analyses and comparison with previously reported variants. Eighteen unique variants in LAMB3, LAMA3, LAMC2, or COL17A1 were identified from 17 individuals. Seven had severe JEB, 9 had intermediate JEB, and 1 had laryngo-onycho-cutaneous syndrome. Seven variants were previously unreported. Deep phenotyping was completed for all intermediate JEB cases and demonstrated substantial variation between individuals. Splice site variants underwent analysis with SpliceAI, a state-of-the-art artificial intelligence tool, to predict resultant transcripts. Predicted functional effects included exon skipping and cryptic splice site activation, which provided potential explanations for disease severity and in most cases correlated with laminin-332 immunofluorescence. RT-PCR was performed for 1 case to investigate resultant transcripts produced from the splice site variant. This study expands the JEB genomic and phenotypic landscape. Artificial intelligence tools show potential for predicting the functional effects of splice site variants and may identify candidates for confirmatory laboratory investigation. Investigation of RNA transcripts will help to further elucidate genotype-phenotype correlations for novel variants.

Epidermolysis bullosa.

Epidermolysis bullosa (EB) is an inherited, heterogeneous group of rare genetic dermatoses characterized by mucocutaneous fragility and blister formation, inducible by often minimal trauma. A broad phenotypic spectrum has been described, with potentially severe extracutaneous manifestations, morbidity and mortality. Over 30 subtypes are recognized, grouped into four major categories, based predominantly on the plane of cleavage within the skin and reflecting the underlying molecular abnormality: EB simplex, junctional EB, dystrophic EB and Kindler EB. The study of EB has led to seminal advances in our understanding of cutaneous biology. To date, pathogenetic mutations in 16 distinct genes have been implicated in EB, encoding proteins influencing cellular integrity and adhesion. Precise diagnosis is reliant on correlating clinical, electron microscopic and immunohistological features with mutational analyses. In the absence of curative treatment, multidisciplinary care is targeted towards minimizing the risk of blister formation, wound care, symptom relief and specific complications, the most feared of which - and also the leading cause of mortality - is squamous cell carcinoma. Preclinical advances in cell-based, protein replacement and gene therapies are paving the way for clinical successes with gene correction, raising hopes amongst patients and clinicians worldwide.

Window to the Unknown: Using Storytelling to Identify Learning Needs for the Intrinsic Competencies Within an Online Needs Assessment.

INTRODUCTION: Needs assessments are important for developing online educational resources, but they frequently do not capture learning needs in the intrinsic physician competencies. Storytelling exercises, by promoting critical reflection and emphasizing values and context, may assist curriculum developers in identifying emergent knowledge gaps in these areas that are initially unknown to learners. METHODS: We developed an online curriculum for thrombosis and hemostasis based on an open-access online needs assessment comprised of a topic list, case scenarios, and storytelling exercise. In the storytelling exercise, learners described 1) a difficult clinical case and 2) why that case was difficult. In this qualitative descriptive study, we performed a secondary thematic analysis of this storytelling data, coded for the CanMEDS 2015 intrinsic roles. Two investigators independently coded transcripts with iterative comparison. RESULTS: A total of 143 respondents completed the storytelling exercise. All responses yielded a gap in medical expertise, while 25 (17.5%) described an additional intrinsic role. Learning needs in all six intrinsic roles were identified. The most commonly cited learning needs were in the leader (recognizing how resource allocation impacts health care), communicator (communicating knowledge with patients), and collaborator (unclear communication between providers) roles. These excerpts were notable for how they expressed the complexity and affective components of medicine. CONCLUSIONS: Storytelling exercises can highlight context, attitudes, and relationships that provide depth to needs assessments. These narratives are a novel method of identifying gaps in intrinsic physician competencies that are initially unknown by learners (Johari window). These emergent intrinsic learning needs may be used to enrich learner-centered curricula.

RNA surveillance by uridylation-dependent RNA decay in Schizosaccharomyces pombe.

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3'-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5'-NxAUUAAAA-3' RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.

In utero development of epidermolysis bullosa acquisita.

We report the case of an infant born with perioral vesicles that rapidly spread to involve his mouth and the majority of his body. Histopathology, immunofluorescence, and enzyme-linked immunohistochemistry assays confirmed a diagnosis of epidermolysis bullosa acquisita (EBA). His mother had no history of EBA, and serum indirect immunofluorescence was negative. The patient improved rapidly with local wound care and oral dapsone.

Drug-induced linear IgA bullous dermatosis in a patient with a vancomycin-impregnated cement spacer.

Linear IgA bullous dermatosis (LABD) is an autoimmune blistering rash caused by IgA autoantibodies against the epidermal basement membrane zone. It commonly is drug induced, often in association with systemic vancomycin. We report a case of a previously healthy 77-year-old man who developed a diffuse macular rash and hemorrhagic bullae on the left leg 10 days after placement of a vancomycin-impregnated cement spacer (VICS) during a revision knee arthroplasty and initiation of postoperative treatment with intravenous (IV) vancomycin. The lesions initially were limited to the leg in which the hardware was placed, but the patient later developed painful palmoplantar and oropharyngeal blisters as well as edematous, erythematous plaques on the back and buttocks. A punch biopsy from a lesion on the left thigh revealed neutrophil-rich subepidermal bullae, and immunofluorescence revealed linear IgA and C3 deposition along the dermoepidermal junction, confirming a diagnosis of LABD. This report illustrates the importance of considering antibiotic-impregnated cement spacers, which frequently are used to manage prosthetic joint infections, as potential culprits in patients with cutaneous eruptions.

The Massive Online Needs Assessment (MONA) to inform the development of an emergency haematology educational blog series.

BACKGROUND: Online educational resources are criticized as being teacher-centred, failing to address learner's needs. Needs assessments are an important precursor to inform curriculum development, but these are often overlooked or skipped by developers of online educational resources due to cumbersome measurement tools. Novel methods are required to identify perceived and unperceived learning needs to allow targeted development of learner-centred curricula. OBJECTIVES: To evaluate the feasibility of performing a novel technique dubbed the Massive Online Needs Assessment (MONA) for the purpose of emergency haematology online educational curricular planning, within an online learning community (affiliated with the Free Open Access Medical education movement). METHODS: An online survey was launched on CanadiEM.org using an embedded Google Forms survey. Participants were recruited using the study website and a social media campaign (utilizing Twitter, Facebook, Blogs, and a poster) targeting a specific online community. Web analytics were used to monitor participation rates in addition to survey responses. RESULTS: The survey was open from 20 September to 10 December 2016 and received 198 complete responses representing 6 medical specialties from 21 countries. Most survey respondents identified themselves as staff physicians (n = 109) and medical trainees (n = 75). We identified 17 high-priority perceived needs, 17 prompted needs, and 10 topics with unperceived needs through our MONA process. CONCLUSIONS: A MONA is a feasible, novel method for collecting data on perceived, prompted, and unperceived learning needs to inform an online emergency haematology educational blog. This methodology could be useful to the developers of other online education resources.

Erythematous plaques and papules on a premature infant.

A 2240 gram boy was born at 33.2 weeks gestation with nonblanching, deeply erythematous plaques and papules on the back, flanks, and scalp (Figure 1). His mother was GBS positive and on antibiotic suppression for prior cutaneous MRSA and urinary tract infections. Intrapartum intravenous Penicillin G was administered, and the amniotic sac was artificially ruptured 4 hours prior to delivery to facilitate labor. The delivery was uncomplicated without concern for chorioamnionitis, but the patient initially required CPAP for respiratory distress with 1-minute and 5-minute Apgar scores of 7 and 8, respectively. A skin punch biopsy is shown (Figure 2).

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.

Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2).

The nontemplated addition of single or multiple nucleotides to RNA transcripts is an efficient means to control RNA stability and processing. Cytoplasmic RNA adenylation and the less well-known uridylation are post-transcriptional mechanisms regulating RNA maturation, activity, and degradation. Gld2 is a member of the noncanonical poly(A) polymerases, which include enzymes with varying nucleotide specificity, ranging from strictly ATP to ambiguous to exclusive UTP adding enzymes. Human Gld2 has been associated with transcript stabilizing miRNA monoadenylation and cytoplasmic mRNA polyadenylation. Most recent data have uncovered an unexpected miRNA uridylation activity, which promotes miRNA maturation. These conflicting data raise the question of Gld2 nucleotide specificity. Here, we biochemically characterized human Gld2 and demonstrated that it is a bona fide adenylyltransferase with only weak activity toward other nucleotides. Despite its sequence similarity with uridylyltransferases (TUT4, TUT7), Gld2 displays an 83-fold preference of ATP over UTP. Gld2 is a promiscuous enzyme, with activity toward miRNA, pre-miRNA, and polyadenylated RNA substrates. Apo-Gld2 activity is restricted to adding single nucleotides and processivity likely relies on additional RNA-binding proteins. A phylogeny of the PAP/TUTase superfamily suggests that uridylyltransferases, which are derived from distinct adenylyltransferase ancestors, arose multiple times during evolution via insertion of an active site histidine. A corresponding histidine insertion into the Gld2 active site alters substrate specificity from ATP to UTP.

Autoimmune bullous diseases with skin and eye involvement: Cicatricial pemphigoid, pemphigus vulgaris, and pemphigus paraneoplastica.

Autoimmune blistering diseases are a heterogeneous group of disorders that mostly affect the skin and mucous membranes. Occasionally, other organ systems may be involved, depending on the unique pathophysiology of each disease. Cicatricial pemphigoid, pemphigus vulgaris, and paraneoplastic pemphigus are distinct entities, but all have the potential to have cutaneous and ocular involvement. Awareness and early recognition of ocular involvement in these diseases is important given the increased risk for vision loss and blindness with delay in management. Several skin diseases may be associated with involvement of the external eye. The most common autoimmune diseases are cicatricial pemphigoid, pemphigus vulgaris, and paraneoplastic pemphigus.

Systemic granulocyte colony-stimulating factor (G-CSF) enhances wound healing in dystrophic epidermolysis bullosa (DEB): Results of a pilot trial.

BACKGROUND: Chronic nonhealing wounds are the norm in patients with inherited epidermolysis bullosa (EB), especially those with dystrophic EB (DEB). A possible benefit in wound healing after subcutaneous treatment with granulocyte colony-stimulating factor (G-CSF) was suggested from an anecdotal report of a patient given this during stem cell mobilization before bone-marrow transplantation. OBJECTIVE: We sought to determine whether benefit in wound healing in DEB skin might result after 6 daily doses of G-CSF and to confirm its safety. METHODS: Patients were assessed for changes in total body blister and erosion counts, surface areas of selected wounds, and specific symptomatology after treatment. RESULTS: Seven patients with DEB (recessive, 6; dominant, 1) were treated daily with subcutaneous G-CSF (10 µg/kg/dose) and reevaluated on day 7. For all patients combined, median reductions of 75.5% in lesional size and 36.6% in blister/erosion counts were observed. When only the 6 responders were considered, there were median reductions of 77.4% and 38.8% of each of these measured parameters, respectively. No adverse side effects were noted. LIMITATIONS: Limitations include small patient number, more than 1 DEB subtype included, and lack of untreated age-matched control subjects. CONCLUSIONS: Subcutaneous G-CSF may be beneficial in promoting wound healing in some patients with DEB when conventional therapies fail.

Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.

BACKGROUND: Several new targeted genes and clinical subtypes have been identified since publication in 2008 of the report of the last international consensus meeting on diagnosis and classification of epidermolysis bullosa (EB). As a correlate, new clinical manifestations have been seen in several subtypes previously described. OBJECTIVE: We sought to arrive at an updated consensus on the classification of EB subtypes, based on newer data, both clinical and molecular. RESULTS: In this latest consensus report, we introduce a new approach to classification ("onion skinning") that takes into account sequentially the major EB type present (based on identification of the level of skin cleavage), phenotypic characteristics (distribution and severity of disease activity; specific extracutaneous features; other), mode of inheritance, targeted protein and its relative expression in skin, gene involved and type(s) of mutation present, and--when possible--specific mutation(s) and their location(s). LIMITATIONS: This classification scheme critically takes into account all published data through June 2013. Further modifications are likely in the future, as more is learned about this group of diseases. CONCLUSION: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and molecular features of each EB subtype, and has sufficient flexibility incorporated in its structure to permit further modifications in the future.

Cumulative life course impairment by epidermolysis bullosa.

Inherited epidermolysis bullosa, which encompasses at least 30 distinctive genetic diseases, may be associated with marked functional impairment, the result of the presence of severe cutaneous and extracutaneous manifestations or complications in some of its subtypes.

Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.

Patients with the genetic skin blistering disease recessive dystrophic epidermolysis bullosa (RDEB) develop aggressive cutaneous squamous cell carcinoma (cSCC). Metastasis leading to mortality is greater in RDEB than in other patient groups with cSCC. Here we investigate the dermal component in RDEB using mRNA expression profiling to compare cultured fibroblasts isolated from individuals without cSCC and directly from tumor matrix in RDEB and non-RDEB samples. Although gene expression of RDEB normal skin fibroblasts resembled that of cancer-associated fibroblasts, RDEB cancer-associated fibroblasts exhibited a distinct and divergent gene expression profile, with a large proportion of the differentially expressed genes involved in matrix and cell adhesion. RDEB cancer-associated fibroblasts conferred increased adhesion and invasion to tumor and nontumor keratinocytes. Reduction of COL7A1, the defective gene in RDEB, in normal dermal fibroblasts led to increased type XII collagen, thrombospondin-1, and Wnt-5A, while reexpression of wild type COL7A1 in RDEB fibroblasts decreased type XII collagen, thrombospondin-1, and Wnt-5A expression, reduced tumor cell invasion in organotypic culture, and restricted tumor growth in vivo. Overall, our findings show that matrix composition in patients with RDEB is a permissive environment for tumor development, and type VII collagen directly regulates the composition of matrix proteins secreted by dermal and cancer-associated fibroblasts.

Inherited epidermolysis bullosa: recent basic and clinical advances.

PURPOSE OF REVIEW: This review highlights key findings, both clinical and basic, that have been published in the field of inherited epidermolysis bullosa within the past few years. RECENT FINDINGS: New epidermolysis bullosa phenotypes, genotypes and modes of transmission have been identified, resulting in a revised classification system. Detailed evidence-based data are now available on the risk of extracutaneous complications in each of the major epidermolysis bullosa subtypes. Studies are now underway to try to better explain the biological aggressiveness of squamous cell carcinomas arising in epidermolysis bullosa skin. Cell and animal models have been refined and used to ascertain the feasibility of gene replacement therapy, stem cell transplantation, and treatment with injected allogeneic fibroblasts or recombinant type VII collagen. As a result, clinical trials are now being pursued to test each of these in humans. SUMMARY: Epidermolysis bullosa is caused by mutations in at least 14 genes, leading to a broad spectrum of entities, each of which has its own relative risk for the development of specific extracutaneous complications and/or premature death. Intensive research, both basic and clinical, is bringing us closer to more effective treatments and possibly even a cure.