The routine diagnosis of heritable skin and connective tissue disorders is complicated by the fact that in this group of disorders, clinical manifestations may result from genetic or phenotypic heterogeneity and the existence of new genes and/or novel disease subtypes. Autozygosity mapping (AM) has been proven to be a useful adjunct in the molecular diagnosis of homozygous autosomal recessive (AR) diseases. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using genomewide single nucleotide polymorphism (SNP) array-based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases (84.7%) were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analyzed with an EB-targeted next-generation sequencing (NGS) panel. This approach identified mutations in five additional cases, initially undiagnosed due to presence of compound heterozygosity, deep intronic mutations, or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 out of 46 cases (95.7%) diagnosed genetically. The yield of 84.7% using AM-guided sequencing was remarkably similar to that of the independent use of our previously reported NGS targeted panel, in which potential causative variants were identified in 76 of 91 (83.5%) families with EB. Thus, AM is an expedient and cost efficient approach to identify mutated genes consanguineous families.

656 skin biopsies with positive direct immunofluorescence from the UK and overseas were studied over a 2-year period. The length of time biopsies had remained in Michel’s medium at pH 7.0 in various diseases (pemphigoid, pemphigus, linear IgA disease, epidermolysis bullosa acquisita, lupus erythematosus, vasculitis, amyloid, lichen planus and dermatitis herpetiformis) was analysed. We concluded that direct immunofluorescence remained positive at 6 months and that Michel’s medium is a reliable long-term maintenance medium for skin biopsies.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.

Conflicto de intereses: Los autores declaran no tener conflictos de intereses Imágenes: Los autores declaran haber obtenido las imágenes con el permiso de los pacientes Política de derechos y autoarchivo: se permite el autoarchivo de la versión post-print (SHERPA/RoMEO) Licencia CC BY-NC-ND. Licencia Creative Commons Atribución-NoComercial-SinDerivar 4.0 Internacional Universidad de Salamanca. Su comercialización está sujeta al permiso del editor MANIFESTACIONES CLÍNICAS DEL SÍNDROME DE RAMSAY-HUNT EN UNA SERIE DE 20 CASOS RAMÍREZ-SALAS JE ET AL.

Conflicto de intereses: Los autores declaran no tener conflictos de intereses Imágenes: Los autores declaran haber obtenido las imágenes con el permiso de los pacientes Política de derechos y autoarchivo: se permite el autoarchivo de la versión post-print (SHERPA/RoMEO) Licencia CC BY-NC-ND. Licencia Creative Commons Atribución-NoComercial-SinDerivar 4.0 Internacional Universidad de Salamanca. Su comercialización está sujeta al permiso del editor PALABRAS CLAVE: vértigo visual; rehabilitación vestibular; habituación; equilibrio; dependencia visual; estabilidad postural.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin disease caused by loss of function mutations in the gene encoding type VII collagen (COL7A1). RDEB is characterized by trauma induced wounds and excessive scarring, and one of the most frequent complications in RDEB is the development of fatal squamous cell carcinoma (SCC). Our published and unpublished data show that TGF-beta signaling is increased in RDEB, and others have shown that TGF-beta is a major disease modifier. We previously demonstrated that the matricellular protein thrombospondin-1 (TSP1) is increased in RDEB fibroblasts and that COL7A1 expression modulates TSP1. Here we investigated the role of TSP1 in TGF-beta activation in RDEB primary fibroblasts. Knock-down of TSP1 reduced phosphorylation of SMAD3 (a downstream target of TGF-beta signaling) in RDEB patient fibroblasts, but not in non-RDEB normal breast fibroblasts. Over-expression of COL7A1 in RDEB fibroblasts reduced intracellular TSP1 and phosphorylated SMAD3. Furthermore, peptide inhibition of TSP1 binding to the TGF-beta e latency associated protein complex decreased nuclear localization of phosphorylated SMAD3 in tissue engineered extracellular matrix (ECM) from RDEB fibroblasts compared with controls and to a similar extent as observed after SB-431542 inhibition of the TGF-beta type I receptor. Decreased nuclear phosphorylated SMAD3 correlated with a decrease in collagen fiber formation in tissue engineered ECM from RDEB fibroblasts treated with the peptide TSP1 inhibitor, as evaluated by picrosirius red staining followed by analyses of birefringent collagen fibrillar deposits using a polarizing microscope. These data suggest TSP1 is a major activator of TGF-beta signaling in RDEB and identify TSP1 as a potent therapeutic target.

Mutations in the gene encoding collagen VII (C7) cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). This disease is characterized by severe skin fragility and non-healing wounds aggravated by scarring and fibrosis. The altered skin architecture in RDEB also allows for development of fatal squamous cell carcinoma. We previously demonstrated that the matricellular protein thrombospondin-1 (TSP1) is increased in RDEB fibroblasts and that C7 expression modulates TSP1. Because TGF-beta is increased in RDEB and correlates with disease severity, and TSP1 is shown to activate TGF-beta, we investigated the role of TSP1 in TGF-beta activation in RDEB patient cells. Knock-down of TSP1 reduced phosphorylation of SMAD3 (a downstream target of TGF-beta signaling) in RDEB patient fibroblasts. In contrast, no reduced SMAD3 phosphorylation was observed in control cells. Over-expression of C7 in RDEB fibroblasts reduced TSP1 and phosphorylation of SMAD3. Furthermore, inhibition of TSP1 binding to the latency-associated protein (LAP)/ TGF-beta complex decreased fibrosis in engineered extracellular matrix derived from RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. Using immuno-precipitation, proximity ligation assay and real-time, labelfree analysis of binding interactions, we demonstrate that C7 binds TSP1, thereby likely preventing TSP1-LAP association and subsequent TGF-beta activation. Our study suggests a novel mechanism for increased TGF-beta signaling in the absence of C7 in RDEB patient skin. Moreover, our data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment in RDEB patients.

Introduction Breast cancer (BC) is the most frequent neoplasia among women and one of the leading causes of cancer-related death worldwide. It is a clinically heterogeneous disease, with the triple negative (TN) as the most aggressive subtype. Epigenetic alterations are common molecular abnormalities in cancer, including changes in DNA methylation, microRNA profiling and post-translational modifications of histones. Aberrant DNA methylation has been recognised as one of the most common epigenetic modifications in BC. Methylation of certain genes is associated with pathological features, differential response to therapy or metastases during follow-up, being also a predictor of survival in BC; but little is known about its involvement in TN BC. Our objective was to identify the methylome of TN BC compared to that of non-neoplastic mammary tissue. Material and methods A 450k-methylation array was performed to identify differentially methylated genes in 8 TN BC samples (T) and 5 non-neoplastic (N) tissues from reduction mammoplasties. For that, DNA was extracted, bisulphite-converted, whole-genome amplified and hybridised to Infinium HumanMethylation450 BeadChip array. The level of DNA methylation at each CpG locus (b-value) was calculated as M/(M+U), ranging from 0 (100% unmethylated, U) to 1 (100% methylated, M). Differentially expressed probes were obtained by limma t-test (False Discovery Rate<0.05). Those probes with delta b-value (Db) >0.2 and located in CpG islands in the 5´UTR, 1500-200 bp upstream the transcription start site or in the first exon, were considered. ADAM12, VWCE and TSPAN9 genes were selected for further validation by pyrosequencing in a series of 30 T and 20 n, using a PyroMark Q24 after PCR amplification.Protein expression was explored by immunohistochemistry. Results and discussions A total of 700 hypermethylated and 349 hypomethylated probes in T compared to N, corresponding to 15 and 7 hyper-and hypomethylated genes, respectively, were found to fulfil our criteria. ADAM12, VWCE and TSPAN9 genes showed lower methylation levels in T than in N (p<0.05). The increased protein expression in T compared to N was confirmed, highlighting the causal role of the hypomethylation in the regulation of the expression of these genes. No association was found between these alterations and clinicopathological variables. Conclusion This is the first description of ADAM12, VWCE and TSPAN9 hypomethylation and overexpression in TN BC compared to non-neoplastic mammary tissues.