Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing read-through and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1

Designing Genes that Encode Proteins for Biomedical Applications

DNA is a crucial component of all known life. It encodes in genes the structure of the proteins necessary to perform many of the functions in a cell. Proteins are biological polymers consisting of a chain of amino acids. The specific sequence of the amino acids determines the structure and therefore function of the protein. The sequence of the amino acids of a protein is coded in DNA via triplets of the nucleotide bases known as codons, which each can represent only one amino acid. However, an amino acid can be represented by more than one codon, so there are many combinations of DNA that can code for any given protein. The efficiency of expressing a protein from a gene can be affected by the DNA sequence, so to optimize protein production, we want an optimal sequence of DNA. Particularly, we would like to be able to design ab initio, optimized genes that code for protein-based materials for biological applications. We are working to develop a computer program to generate DNA code from an amino acid sequence. The ultimate goal is to optimize the sequence by using codons in an efficient way and removing unwanted patterns in the gene.https://engagedscholarship.csuohio.edu/u_poster_2016/1047/thumbnail.jp

Molecular Mechanisms of Junctional Epidermolysis Bullosa: Col15 Domain Mutations Decrease the Thermal Stability of Collagen XVII

Mutations in the collagen XVII gene, COL17A1, are associated with junctional epidermolysis bullosa. Most COL17A1 mutations lead to a premature termination codon (PTC), whereas only a few mutations result in amino acid substitutions or deletions. We describe here two novel glycine substitutions, G609D and G612R, and a splice site mutation resulting in a deletion of three Gly–X–Y amino acid triplets. In order to investigate the molecular pathomechanisms of non-PTC mutations, G609D and G612R and two previously known substitutions, G627V and G633, and deletion of the amino acids 779–787 were introduced into recombinant collagen XVII. The thermal stability of the mutated collagens was assessed using trypsin digestions at incremental temperatures. All the four glycine substitutions significantly destabilized the ectodomain of collagen XVII, which manifested as 16°C–20°C lower Tm (midpoint of the helix-to-coil transition). These results were supported by secondary structure predictions, which suggested interruptions of the collagenous triple helix within the largest collagenous domain, Col15. In contrast, deletion of the three full Gly–X–Y triplets, amino acids 779–787, had no overall effect on the stability of the ectodomain, as the deletion was in register with the triplet structure and also generated compensatory changes in the NC15 domain

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

Kindler syndrome: extension of FERMT1 mutational spectrum and natural history

Mutations in the FERMT1 gene (also known as KIND1), encoding the focal adhesion protein kindlin-1, underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with an intriguing progressive phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. Herein we review the clinical and genetic data of 62 patients, and delineate the natural history of the disorder, for example, age at onset of symptoms, or risk of malignancy. Although most mutations are predicted to lead to premature termination of translation, and to loss of kindlin-1 function, significant clinical variability is observed among patients. There is an association of FERMT1 missense and in-frame deletion mutations with milder disease phenotypes, and later onset of complications. Nevertheless, the clinical variability is not fully explained by genotype-phenotype correlations. Environmental factors and yet unidentified modifiers may play a role. Better understanding of the molecular pathogenesis of KS should enable the development of prevention strategies for disease complications.Contract grant sponsors: International Kindler Syndrome; The German Federal Ministry for Education and Research; The Excellence Initiative of the German federal and stage government and Freiburg Institute for Advanced Studies, School of Life Sciences (to L.B.T); The Italian Ministry of Heat

Combinatorial omics analysis reveals perturbed lysosomal homeostasis in collagen VII-deficient keratinocytes

The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under- addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro- inflammatory proteins in primary DEB keratinocytes. Increased TGF-β signaling due to loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte- driven, progressive fibrosis

Monoallelic Mutations in the Translation Initiation Codon of KLHL24 Cause Skin Fragility

The genetic basis of epidermolysis bullosa, a group of genetic disorders characterized by the mechanically induced formation of skin blisters, is largely known, but a number of cases still remain genetically unsolved. Here, we used whole-exome and targeted sequencing to identify monoallelic mutations, c.1A>G and c.2T>C, in the translation initiation codon of the gene encoding kelch-like protein 24 (KLHL24) in 14 individuals with a distinct skin-fragility phenotype and skin cleavage within basal keratinocytes. Remarkably, mutation c.1A>G occurred de novo and was recurrent in families originating from different countries. The striking similarities of the clinical features of the affected individuals point to a unique and very specific pathomechanism. We showed that mutations in the translation initiation codon of KLHL24 lead to the usage of a downstreamtranslation initiation site with the same reading frame and formation of a truncated polypeptide. The pathobiology was examined in keratinocytes and fibroblasts of the affected individuals and via expression of mutant KLHL24, and we found mutant KLHL24 to be associated with abnormalities of intermediate filaments in keratinocytes and fibroblasts. In particular, KLHL24 mutations were associated with irregular and fragmented keratin 14. Recombinant overexpression of normal KLHL24 promoted keratin 14 degradation, whereas mutant KLHL24 showed less activity than the normal molecule. These findings identify KLHL24 mutations as a cause of skin fragility and identify a role for KLHL24 in maintaining the balance between intermediate filament stability and degradation required for skin integrity.Peer reviewe