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John Wood, Glyn Chidlow, Robert Casson, Shiwani Sharma, Kathryn Burdon, Jamie Craig; HUMAN OCULAR DISTRIBUTION OF PRODUCTS OF THE POAG-ASSOCIATED CDKN2B-AS1 GENE CLUSTER REGION. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1606.
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A recent study identified that there were highly significant associations of common single nucleotide polymorphisms near the CDKN2B-AS1 gene region at the 9p21 locus with POAG (Burdon, KP et al, Nature Genetics 2011, 43:574-578). This region houses the CDKN2B/p15INK4B-CDKN2A/p16INK4A-p14ARF tumour suppressor gene cluster and is adjacent to the S-methyl-5'-thioadenosine phosphorylase (MTAP) gene. In an attempt to understand the ocular function of these genes and, therefore, how they may be involved in the pathogenesis of POAG, we studied the distribution patterns of their gene products within human ocular tissues.
We utilized immunohistochemistry to identify localization patterns for p15INK4B, p16INK4A, p14ARF and MTAP proteins within the human eye. We employed several antibodies and used appropriate positive control tissues for determination of optimal labeling and therefore the correct ocular distribution pattern for each protein. Where appropriate, we tested specificity of antibodies using Western blot analyses of recombinant proteins.
MTAP was detected in control malignant pleural mesothelioma sections and in western blot studies using positive recombinant protein. The best antibody (Proteintech Group) yielded positive labeling in corneal epithelium, trabecular meshwork and retinal astrocytes and Muller cells. P16INK4A was appropriately identified in positive control cervical adenocarcinoma sections by three antibodies tested, each of which also recognized the recombinant protein. No labeling was detected in ocular tissues by these antibodies. Positive P14ARF labeling was also identified by two antibodies in cellular nucleoli in cervical adenocarcinoma sections; however, no specific labeling could be detected in human eyes. A single antibody (Cell Signaling Technology) was able to specifically detect recombinant p15INK4B by Western blot and in positive skin squamous cells. This antibody labeled cell nuclei in the retinal inner nuclear and ganglion cell layers and glial columns in the optic nerve.
Analysis of the distribution of gene products from or near the 9p21 gene region in the human eye indicated that resting levels of P16INK4A/P14ARF were too low to detect but that both MTAP and p15INK4B were present in different cells. This work provides a framework for future studies that will seek to explore how these gene products may influence the pathogenesis of POAG.
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