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Shunbin Xu, Chithra Muraleedharan, Pei Zhuang, William Garcia, Daniel Montenegro; Disease Causing Mutation Screening in miRNA183/96/182 in Patients with Inherited Retinal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6599.
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microRNAs (miRNAs) are small, non-coding RNAs and represent a newly recognized, important mechanism of gene-expression regulation. However, their roles in inherited retinal dystrophy (IRD) are still largely unknown. Previously, we identified a sensory organ-specific miRNA cluster, the miR-183/96/182 cluster (miR-183/96/182), located at Chr6qA3.3 in mouse and Chr7q32.2 in human. Inactivation of miR-183/96/182 in mice resulted in congenital syndromic IRD. We hypothesize that mutations in miR-183/96/182 in human may cause IRD. We will test this hypothesis in this project.
Based on similarity of disease symptoms to phenotypes in the knockout mouse model, genomic DNA samples of patients with various IRD, including Congenital Stationary Night Blindness (CSNB), Usher syndrome, Cone-rod dystrophy, Stargardt disease, Achromatopsia/Blue Cone Monochromacy, Occult Macular Dystrophy, Pattern Dystrophy and Adult Onset Foveomacular Dystrophy, Retinitis Pigmentosa and other retinal dystrophy, and normal subjects are obtained from the National Ophthalmic Genotyping and Phenotyping Network (eyeGENE®), a part of NEI, NIH. miR-183/96/182 is amplified in two amplicons by nested PCR. Amplification products are gel-purified using Qiaquick Gel Extraction kit (Qiagen) and are subjected to Sanger sequencing on both forward and reverse strands at the Applied Genomics Technology Center, WSU.
We have received more than 1100 patient samples. Samples with X-linked IRD or known disease-causing mutations are excluded from the screening. Among 66 patient samples screened by far, three sequence variants are found, two in pre-miR-182 and one in pre-miR-96, in four patient samples. Among these, two sequence variants in pre-miR-182 are known single nucleotide polymorphisms (SNPs). However, the mutation in pre-miR-96 is a novel variant. Whether the new variant affects miRNA biogenesis and contributes to the disease is under investigation.
We established the methodology of robust amplification and sequencing of miR-183/96/182 and efficient identification of sequence variants. Further characterization of the new variants and screening in other patient samples may identify mutations in miR-183/96/182 responsible for IRD. This research will uncover new molecular mechanism of IRD and provide new gene diagnosis and therapeutic target for gene therapy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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