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H. Khanna, A. I. den Hollander, C. Murga-Zamalloa, I. Lopez, F. P. M. Cremers, R. K. Koenekoop, A. Swaroop; Truncated CEP290/NPHP6 Protein Variants in Lymphocytes of Leber Congenital Amaurosis Patients With Mutations in CEP290: A Potential Diagnostic Test and Implications Into the Pathogenesis of Associated LCA. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1652.
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LCA is a genetically heterogeneous disorder with at least eight causative genes. Mutations in CEP290 have been shown to be a frequent cause of LCA. As CEP290 is widely expressed, the purpose of this study was to develop a screening strategy to predict potential CEP290 mutations in LCA and design a model system to examine the pathophysiology of CEP290-mediated retinopathy in patients.
Lymphocytes were collected from LCA patients that are homozygous or heterozygous for the most commonly observed CEP290 mutation, c.2991+1655A>G. Proteins extracted from the lymphocytes and Epstein -Barr Virus transformed lymphoblastoid cell lines of normal controls and patients were subjected to SDS-PAGE, followed by immunoblotting using anti-CEP290 antibody.
While a single immunoreactive bd of the expected size (~290 kDa) was consistently detected in lymphocytes of normal individuals, we observed multiple immunoreactive bands in patient samples. These protein bands likely reflect truncated (p.Cys998X) CEP290 protein and/or its degradation products. We also detect a full-length CEP290 band of 290 kDa in all patients, consistent with the RT-PCR studies.
Our studies demonstrate that the involvement of CEP290 as a causative gene in LCA patients can be detected by immunoblot analysis of lymphocyte protein extracts. The expression of full-length CEP290 in LCA patients, in addition to the truncated protein variants, supports the hypothesis that retinal degeneration in the rd16 mouse and LCA patients is caused by haplo-insufficiency, whereas complete loss of CEP290 function is associated with more severe Joubert Syndrome involving kidney and cerebellar disease as well. Additional studies are needed to ascertain whether immunoblotting can be used as an efficient diagnostic method to identify LCA patients with CEP290 mutations.
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