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Connexin channels in congenital skin disorders.

ABSTRACT: Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of how connexin functions are affected. Increasing evidence suggests that the skin diseases produced by connexin mutations result from dominant gains of function. In palmoplantar keratoderma with deafness, the connexin 26 mutations transdominantly alter the function of wild-type connexin 43 and create leaky heteromeric hemichannels. In keratitis-ichthyosis-deafness syndrome, different connexin 26 mutations can either form dominant hemichannels with altered calcium regulation or increased calcium permeability, leading to clinical subtypes of this syndrome. It is only with detailed understanding of these subtle functional differences that we can hope to create successful pathophysiology driven therapies for the connexin skin disorders.

SUBMITTER: Lilly E

PROVIDER: S-EPMC4779425 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Connexin channels in congenital skin disorders.

Lilly Evelyn E Sellitto Caterina C Milstone Leonard M LM White Thomas W TW

Seminars in cell & developmental biology 20160113

Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of h ...[more]

PMID: 26775130

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Project description:Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.

Project description:The application of genome sequencing in patients with intellectual disability and congenital anomalies (ID/CA) typically identifies causative mutations only in a minority of cases. We hypothesized that some cases of ID/CA are caused by epigenetic aberrations that dysregulate normal genome function, and that these would be missed by conventional sequencing approaches. We performed Illumina 450k DNA methylation profiling in ~500 individuals with ID/CA, who were negative for causative mutations by microarray, exome- and/or whole genome sequencing. We screened patient methylation profiles for epimutations absent in ~1,500 controls, and validated these by bisulfite sequencing, revealing that epimutations represent large methylation changes specifically on one allele. Parental studies show that approximately half represent de novo events, a rate significantly higher than that seen in controls. We identified seven recurrent events, two of which (FMR1, MEG3) have known disease associations, validating our method for detecting pathogenic epimutations. From large-scale sequencing, population and expression studies we conclude that epimutations are: (i) Frequently associated with extreme outlier and mono-allelic gene expression, with an impact comparable to loss-of-function mutations; (ii) Generally conserved across multiple tissues within an individual, validating the use of blood DNA to study ID/CA; (iii) Can occur secondary to cis-linked regulatory mutations, providing a rationale for interpreting non-coding genetic variants; (iv) Occur sporadically with a remarkably high de novo rate, and (v) Exhibit non-Mendelian inheritance, likely being reset between generations by epigenetic reprogramming during embryogenesis. We conclude that epimutations underlie 5-10% of patients with ID/CA who are refractory to conventional mutation screening, and propose that epigenome profiling represents a promising method for the study of human disease that complements sequence-based approaches.

Dataset Information

Connexin channels in congenital skin disorders.

Publications

Connexin channels in congenital skin disorders.

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