T-cell activation and bacterial infection in skin wounds of recessive dystrophic epidermolysis bullosa patients.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4high population into CD4high CD25+ FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/Treg-mediated immuno-suppression in RDEB.

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Whole-Transcriptome Analysis by RNA Sequencing for Genetic Diagnosis of Mendelian Skin Disorders in the Context of Consanguinity.

BACKGROUND: Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences. METHODS: We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis. RESULTS: We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel. CONCLUSIONS: Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.

Ancestral patterns of recessive dystrophic epidermolysis bullosa mutations in Hispanic populations suggest sephardic ancestry.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genodermatosis caused by mutations in the gene coding for type VII collagen (COL7A1). More than 800 different pathogenic mutations in COL7A1 have been described to date; however, the ancestral origins of many of these mutations have not been precisely identified. In this study, 32 RDEB patient samples from the Southwestern United States, Mexico, Chile, and Colombia carrying common mutations in the COL7A1 gene were investigated to determine the origins of these mutations and the extent to which shared ancestry contributes to disease prevalence. The results demonstrate both shared European and American origins of RDEB mutations in distinct populations in the Americas and suggest the influence of Sephardic ancestry in at least some RDEB mutations of European origins. Knowledge of ancestry and relatedness among RDEB patient populations will be crucial for the development of future clinical trials and the advancement of novel therapeutics.

Birth cohort-specific trends of sun-related behaviors among individuals from an international consortium of melanoma-prone families.

BACKGROUND: Individuals from melanoma-prone families have similar or reduced sun-protective behaviors compared to the general population. Studies on trends in sun-related behaviors have been temporally and geographically limited. METHODS: Individuals from an international consortium of melanoma-prone families (GenoMEL) were retrospectively asked about sunscreen use, sun exposure (time spent outside), sunburns, and sunbed use at several timepoints over their lifetime. Generalized linear mixed models were used to examine the association between these outcomes and birth cohort defined by decade spans, after adjusting for covariates. RESULTS: A total of 2407 participants from 547 families across 17 centers were analyzed. Sunscreen use increased across subsequent birth cohorts, and although the likelihood of sunburns increased until the 1950s birth cohort, it decreased thereafter. Average sun exposure did not change across the birth cohorts, and the likelihood of sunbed use increased in more recent birth cohorts. We generally did not find any differences in sun-related behavior when comparing melanoma cases to non-cases. Melanoma cases had increased sunscreen use, decreased sun exposure, and decreased odds of sunburn and sunbed use after melanoma diagnosis compared to before diagnosis. CONCLUSIONS: Although sunscreen use has increased and the likelihood of sunburns has decreased in more recent birth cohorts, individuals in melanoma-prone families have not reduced their overall sun exposure and had an increased likelihood of sunbed use in more recent birth cohorts. These observations demonstrate partial improvements in melanoma prevention and suggest that additional intervention strategies may be needed to achieve optimal sun-protective behavior in melanoma-prone families.

Bullosis Diabeticorum: A Neglected Bullous Dermatosis.

A 75-year-old African-American man presented with a 3-year history of painless, fluid-filled blisters, for which his primary care physician had treated him with doxycycline, cephalexin, and topical corticosteroids, with no significant improvement. The blisters had ruptured spontaneously and healed with scarring. He denied antecedent trauma. His medical history was remarkable for insulin-dependent type 2 diabetes mellitus, hypertension, hypercholesterolemia, primary cutaneous melanoma status-post excision, and breast cancer status-post mastectomy and chemotherapy. Physical examination revealed nontender bullae, measuring up to 4 cm × 3 cm and containing serous fluid, on the anterior portion of both tibias (Figure 1). The Nikolsky sign was negative. There was no evidence of surrounding inflammation. A biopsy revealed subepidermal bullae formation with sparse inflammatory infiltrate (Figure 2). Direct and indirect immunofluorescence studies were negative for immunoglobulin (Ig) G, IgA, IgM, complement C3, C5b-9, and fibrinogen deposition. Culture of the bullous fluid was negative.

Mutations in the cholesterol transporter gene ABCA5 are associated with excessive hair overgrowth.

Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5' donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth.

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.

Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.

Characterization of individuals at high risk of developing melanoma in Latin America: bases for genetic counseling in melanoma.

PURPOSE: CDKN2A is the main high-risk melanoma-susceptibility gene, but it has been poorly assessed in Latin America. We sought to analyze CDKN2A and MC1R in patients from Latin America with familial and sporadic multiple primary melanoma (SMP) and compare the data with those for patients from Spain to establish bases for melanoma genetic counseling in Latin America. METHODS: CDKN2A and MC1R were sequenced in 186 Latin American patients from Argentina, Brazil, Chile, Mexico, and Uruguay, and in 904 Spanish patients. Clinical and phenotypic data were obtained. RESULTS: Overall, 24 and 14% of melanoma-prone families in Latin America and Spain, respectively, had mutations in CDKN2A. Latin American families had CDKN2A mutations more frequently (P = 0.014) than Spanish ones. Of patients with SMP, 10% of those from Latin America and 8.5% of those from Spain had mutations in CDKN2A (P = 0.623). The most recurrent CDKN2A mutations were c.-34G>T and p.G101W. Latin American patients had fairer hair (P = 0.016) and skin (P < 0.001) and a higher prevalence of MC1R variants (P = 0.003) compared with Spanish patients. CONCLUSION: The inclusion criteria for genetic counseling of melanoma in Latin America may be the same criteria used in Spain, as suggested in areas with low to medium incidence, SMP with at least two melanomas, or families with at least two cases among first- or second-degree relatives.Genet Med 18 7, 727-736.

The Importance of Esophagography in Patients With Recessive Dystrophic Epidermolysis Bullosa.

OBJECTIVE: The purpose of this study was to assess esophageal damage in patients with recessive dystrophic epidermolysis bullosa (RDEB) with or without dysphagia. SUBJECTS AND METHODS: Fourteen patients with either severe generalized or another generalized form of RDEB recruited through a research and support foundation were evaluated for obstructive esophageal lesions by means of barium esophagography. RESULTS: All patients, even those without dysphagia, had at least one stenosis; five patients had two stenoses. Stenotic lesions occurred most often (74%) in the upper third of the esophagus. CONCLUSION: Esophageal stenosis is a common complication in patients with RDEB, even when they do not have dysphagia. We recommend regular esophagographic examinations of all patients with RDEB.

Fatal Outcome from Brain Vascular Lesions in a Neonate with Blue Rubber Bleb Nevus Syndrome.

The most common cause of death in blue rubber bleb nevus syndrome is gastrointestinal bleeding. Here we present a case of central nervous system bleeding that resulted in death.

Position effect on FGF13 associated with X-linked congenital generalized hypertrichosis.

X-linked congenital generalized hypertrichosis (Online Mendelian Inheritance in Man 307150) is an extremely rare condition of hair overgrowth on different body sites. We previously reported linkage in a large Mexican family with X-linked congenital generalized hypertrichosis cosegregating with deafness and with dental and palate anomalies to Xq24-27. Using SNP oligonucleotide microarray analysis and whole-genome sequencing, we identified a 389-kb interchromosomal insertion at an extragenic palindrome site at Xq27.1 that completely cosegregates with the disease. Among the genes surrounding the insertion, we found that Fibroblast Growth Factor 13 (FGF13) mRNA levels were significantly reduced in affected individuals, and immunofluorescence staining revealed a striking decrease in FGF13 localization throughout the outer root sheath of affected hair follicles. Taken together, our findings suggest a role for FGF13 in hair follicle growth and in the hair cycle.

Lethal Keratitis, Ichthyosis, and Deafness Syndrome Due to the A88V Connexin 26 Mutation.

Keratitis-ichthyosis-deafness syndrome is a well-characterized disease that has been related to mutations in the GJB6 gene. Clinical features such as erythrokeratoderma, palmoplantar keratoderma, alopecia, and progressive vascularizing keratitis, among others, are well known in this entity. In this report we describe a newborn female patient diagnosed with keratitis-ichthyosis-deafness syndrome with a lethal outcome due to sepsis. The patient harbored the mutation A88V that has been previously reported in lethal cases.

[Sjögren's syndrome (SS), a review of the subject and saliva as a diagnostic method]. / Síndrome de Sjögren (SS), revisión del tema y saliva como método diagnóstico.

Sjögren's syndrome is a chronic autoimmune disease whose main clinical manifestation is oral dryness (xerostomia) and ocular dryness (xerophthalmia). It is characterized by progressive mononuclear infiltration of the exocrine glands and can affect a variety of organ systems. The prevalence of primary Sjögren's syndrome varies from 0.01 up to 4.8%; this variability reflects differences in definition, application of diagnostic criteria, and geographic differences in age groups. The etiology of primary Sjögren's syndrome is unknown, but the interaction between genetic and environmental factors (viruses, hormones, vitamins, stress) is important. There are few reported cases of concordance in monozygotic twins, and it is common for patients with primary Sjögren's syndrome to have relatives with other autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, thyroid disease, psoriasis, and multiple sclerosis. Among the most common findings is hypergammaglobulinemia. Elevated levels of γ-globulins contain autoantibodies directed against nonspecific antigens such as rheumatoid factor, antinuclear antibodies, and cellular antigens SS-A/Ro and SS-B/La. Regarding diagnosis, there have been 11 different published criteria for Sjögren's syndrome since 1965; none have been approved by the American College of Rheumatology or the European League Against Rheumatism. The current criteria were published in 2012 jointly with the progressive advance in the knowledge of the human salivary proteome that has gained wide acceptance in Sjögren's syndrome, with the possibility of using saliva as a useful tool in both diagnosis and prognosis in this field because the analysis of salivary proteins may reflect the state of locally underlying disease of the salivary glands, which are the target organs in this disease.

Chromoblastomycosis by Cladophialophora carrionii associated with squamous cell carcinoma and review of published reports.

Chromoblastomycosis is a subcutaneous, chronic, granulomatous mycosis that occurs more frequently in tropical and subtropical countries. We describe a case of a 63-year-old male patient with diagnosis of chromoblastomycosis by Cladophialophora carrionii with an 18-year evolution who developed a lethal squamous cell carcinoma.

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.

Hypertrichosis lanuginosa congenita treated with diode laser epilation during infancy.

We report the case of a girl with hypertrichosis lanuginosa congenita treated with diode laser depilation since the age of 9 months. The treatment was well tolerated, and neither general nor local anesthesia was needed. A reduction of approximately 80% of facial and body hair was noted, which improved her condition significantly.

[Primary cutaneous coccidioidomycosis in an infant]. / Coccidioidomicosis cutánea primaria en infante.

Coccidioidomycosis is a systemic granulomatosis caused by dimorphic fungi Coccidioides immitis, which are endemic of the San Joaquin Valley in California, USA, and C. posadasii found in the southwestern desert of the USA, Mexico, and South America. The primary cutaneous form is extremely infrequent. There have been 25 reported cases in literature, all of them in adults. This is the first case in an infant.