Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase.

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca(2+) ATPase) was adsorbed on a solid supported membrane and then activated with Ca(2+) and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca(2+) jumps in the absence of ATP and following ATP jumps in the presence of Ca(2+). The related charge movements are attributed to Ca(2+) reaching its binding sites in the ground state of the enzyme (E(1)) and to its vectorial release from the enzyme phosphorylated by ATP (E(2)P). The Ca(2+) concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H(+) in exchange for Ca(2+) at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca(2+) and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca(2+)/H(+) exchange is lower in the E(1) than in the E(2)P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca(2+) affinity produced by ATP utilization and results in the countertransport of two Ca(2+) and two H(+) within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca(2+) binding domain, are involved in the observed Ca(2+)/H(+) exchange

Chapter Patient-generated evidence in Epidermolysis Bullosa (EB): Development of a questionnaire to assess the Quality of Life

Epidermolysis Bullosa (EB) is a group of genetic conditions that cause fragile and blistering skin. Although there are different types of EB, which differ in severity, their signs and symptoms overlap. As a result of this disorder, patients face an unbearable burden in their lives, and their Quality of Life (QoL) is negatively affected at every life cycle stage. Nevertheless, the assessment of the quality of life of these patients is scanty. This project aims to develop a patient-centered questionnaire to assess the QoL of EB patients. This tool will be a valid aid for clinicians to understand patients better and identify the areas that need more attention; moreover, it will allow them to follow the patients over time and evaluate the impact of any treatments. The methodological process to develop the questionnaire consisted of two phases: firstly, a critical review of scientific literature was performed; secondly, a pseudo-Delphi study was carried out. A multidisciplinary panel (including patients, caregivers, and clinicians) actively participated in round tables to discuss the main areas of interest. Starting from this initial set of areas and through the repetition of Delphi (up to three rounds), a gradual refinement of the statements was carried out to define a list of items to be included in an easy-to-use but meaningful questionnaire. The final patient-centered questionnaire is thus able to measure the QoL beyond the physical symptoms and the clinical evolution of the disease, encompassing functional autonomy, psycho-emotional state, social relations and the working field

Clotrimazole inhibits the Ca2+-ATPase (SERCA) by interfering with Ca2+ binding and favoring the E2 conformation.

Clotrimazole (CLT) is an antimycotic imidazole derivative that is known to inhibit cytochrome P-450, ergosterol biosynthesis and proliferation of cells in culture, and to interfere with cellular Ca(2+) homeostasis. We found that CLT inhibits the Ca(2+)-ATPase of rabbit fast-twitch skeletal muscle (SERCA1), and we characterized in detail the effect of CLT on this calcium transport ATPase. We used biochemical methods for characterization of the ATPase and its partial reactions, and we also performed measurements of charge movements following adsorption of sarcoplasmic reticulum vesicles containing the ATPase onto a gold-supported biomimetic membrane. CLT inhibits Ca(2+)-ATPase and Ca(2+) transport with a K(I) of 35 mum. Ca(2+) binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca(2+) are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of P(i) in the absence of Ca(2+) is only minimally inhibited. It is concluded that CLT inhibits primarily Ca(2+) binding and, consequently, the Ca(2+)-dependent reactions of the SERCA cycle. It is suggested that CLT resides within the membrane-bound region of the transport ATPase, thereby interfering with binding and the conformational effects of the activating cation

The phenotypic and genotypic spectra of ichthyosis with confetti plus novel genetic variation in the 3' end of KRT10: from disease to a syndrome

Ichthyosis with confetti (IWC) is a genodermatosis caused by dominant negative mutations in the gene encoding keratin 10 (KRT10). We investigated clinical and genetic details of a substantial number of patients with IWC in order to define major and minor criteria for diagnosis of this rare disorder.; Parallel clinical investigation of 6 patients with IWC revealed a novel spectrum of phenotypes. We found several features that qualify as major criteria for diagnosis, which are clearly and consistently associated with the condition. These included malformation of ears, hypoplasia of mammillae, and dorsal acral hypertrichosis. Genetic analysis of patients revealed several different frameshift mutations in intron 6 or exon 7 of KRT10. Analysis of this locus in 17 unrelated control individuals revealed 2 novel polymorphisms of KRT10.; We present for the first time to our knowledge the spectrum of clinical variability of IWC in 6 patients with confirmed mutations in KRT10. From this, we have extracted major and minor criteria to aid early and correct clinical diagnosis. Ectodermal malformations, present in all patients, suggest a novel classification of IWC as a syndrome. There is remarkable genetic variation at the IWC disease locus within control individuals from the general population

Assessment of the risk and characterization of non-melanoma skin cancer in Kindler syndrome: study of a series of 91 patients.

BACKGROUND: Kindler Syndrome (KS) is a rare genodermatosis characterized by skin fragility, skin atrophy, premature aging and poikiloderma. It is caused by mutations in the FERMT1 gene, which encodes kindlin-1, a protein involved in integrin signalling and the formation of focal adhesions. Several reports have shown the presence of non-melanoma skin cancers in KS patients but a systematic study evaluating the risk of these tumors at different ages and their potential outcome has not yet been published. We have here addressed this condition in a retrospective study of 91 adult KS patients, characterizing frequency, metastatic potential and body distribution of squamous cell carcinoma (SCC) in these patients. SCC developed in 13 of the 91 patients. RESULTS: The youngest case arose in a 29-year-old patient; however, the cumulative risk of SCC increased to 66.7% in patients over 60 years of age. The highly aggressive nature of SCCs in KS was confirmed showing that 53.8% of the patients bearing SCCs develop metastatic disease. Our data also showed there are no specific mutations that correlate directly with the development of SCC; however, the mutational distribution along the gene appears to be different in patients bearing SCC from SCC-free patients. The body distribution of the tumor appearance was also unique and different from other bullous diseases, being concentrated in the hands and around the oral cavity, which are areas of high inflammation in this disease. CONCLUSIONS: This study characterizes SCCs in the largest series of KS patients reported so far, showing the high frequency and aggressiveness of these tumors. It also describes their particular body distribution and their relationship with mutations in the FERMT-1 gene. These data reinforce the need for close monitoring of premalignant or malignant lesions in KS patients

Neurofibromin Deficiency and Extracellular Matrix Cooperate to Increase Transforming Potential through FAK-Dependent Signaling

Simple SummaryNeurofibromatosis type 1 is a genetic disease that predisposes to tumors of the nervous system, primarily the neurofibroma. Plexiform neurofibromas (Pnfs) are of the greatest concern because of location, size, and frequent progression to malignancy. Although research is making great progress, the lack of in-depth understanding of the molecular mechanisms driving neoplastic progression results in the absence of prognostic indicators and therapeutic targets. We document that cell-cell cooperativity and the dynamics of the extracellular matrix play important roles in the growth and transformation of Pnf cells, directly through the cooperation of RAS and focal adhesion kinase (FAK) signaling. In turn, we found that treatment of Pnf cells with both MEK and FAK inhibitors is effective in abolishing the transforming ability of these cells.Plexiform neurofibromas (Pnfs) are benign peripheral nerve sheath tumors that are major features of the human genetic syndrome, neurofibromatosis type 1 (NF1). Pnfs are derived from Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1(+/-) milieu and thus are variably lacking in the key Ras-controlling protein, neurofibromin (Nfn). As these SCs are embedded in a dense desmoplastic milieu of stromal cells and abnormal extracellular matrix (ECM), cell-cell cooperativity (CCC) and the molecular microenvironment play essential roles in Pnf progression towards a malignant peripheral nerve sheath tumor (MPNST). The complexity of Pnf biology makes treatment challenging. The only approved drug, the MEK inhibitor Selumetinib, displays a variable and partial therapeutic response. Here, we explored ECM contributions to the growth of cells lacking Nfn. In a 3D in vitro culture, NF1 loss sensitizes cells to signals from a Pnf-mimicking ECM through focal adhesion kinase (FAK) hyperactivation. This hyperactivation correlated with phosphorylation of the downstream effectors, Src, ERK, and AKT, and with colony formation. Expression of the GAP-related domain of Nfn only partially decreased activation of this signaling pathway and only slowed down 3D colony growth of cells lacking Nfn. However, combinatorial treatment with both the FAK inhibitor Defactinib (VS-6063) and Selumetinib (AZD6244) fully suppressed colony growth. These observations pave the way for a new combined therapeutic strategy simultaneously interfering with both intracellular signals and the interplay between the various tumor cells and the ECM

Protocol for the phase 2 EDELIFE trial investigating the efficacy and safety of intra-amniotic ER004 administration to male subjects with X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a rare genetic disorder characte-rised by abnormal development of the skin and its appendages, such as hair and sweat glands, the teeth, and mucous glands of the airways, resulting in serious, sometimes life-threatening complications like hyperthermia or recurrent respiratory infections. It is caused by pathogenic variants of the ectodysplasin A gene

Ectodermal dysplasias: Classification and organization by phenotype, genotype and molecular pathway

An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non‐syndromic traits of the causative gene (e.g., non‐syndromic hypodontia or missing teeth associated with pathogenic variants of EDA “ectodysplasin”). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g., EDA, WNT “wingless‐type,” TP63 “tumor protein p63”) or the components of complex molecular structures (e.g., connexins, keratins, cadherins)

Mutational Spectrum of the ABCA12 Gene and Genotype-Phenotype Correlation in a Cohort of 64 Patients with Autosomal Recessive Congenital Ichthyosis

Autosomal recessive congenital ichthyosis (ARCI) is a non-syndromic congenital disorder of cornification characterized by abnormal scaling of the skin. The three major phenotypes are lamellar ichthyosis, congenital ichthyosiform erythroderma, and harlequin ichthyosis. ARCI is caused by biallelic mutations in ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, NIPAL4, PNPLA1, SDR9C7, SULT2B1, and TGM1. The most severe form of ARCI, harlequin ichthyosis, is caused by mutations in ABCA12. Mutations in this gene can also lead to congenital ichthyosiform erythroderma or lamellar ichthyosis. We present a large cohort of 64 patients affected with ARCI carrying biallelic mutations in ABCA12. Our study comprises 34 novel mutations in ABCA12, expanding the mutational spectrum of ABCA12-associated ARCI up to 217 mutations. Within these we found the possible mutational hotspots c.4541G>A, p.(Arg1514His) and c.4139A>G, p.(Asn1380Ser). A correlation of the phenotype with the effect of the genetic mutation on protein function is demonstrated. Loss-of-function mutations on both alleles generally result in harlequin ichthyosis, whereas biallelic missense mutations mainly lead to CIE or LI

Estudio clínico y molecular en una familia con displasia ectodérmica hipohidrótica autosómica dominante

Hypohidrotic ectodermal dysplasia (HED) is a rare disease characterized by deficiency in development of structure derived from the ectoderm and is caused by mutations in the genes EDA, EDAR, or EDARADD. Phenotypes caused by mutations in these three may exhibit similar clinical features, explained by a common signaling pathway. Mutations in EDA gene cause X linked HED, which is the most common form. Mutations in EDAR and EDARADD genes cause autosomal dominant and recessive form of HED. The most striking clinical findings in HED are hypodontia, hypotrichosis and hypohidrosis that can lead to episodes of hyperthermia. We report on clinical findings in a child with HED with autosomal dominant inheritance pattern with a heterozygous mutation c.1072C>T (p.Arg358X) in the EDAR gene. A review of the literature with regard to other cases presenting the same mutation has been carried out and discussed