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Benjamin Bakall, Heather T. Daggett, Rebecca M. Johnston, Peter M. Brzeskiewicz, William Kimberling, Edwin M. Stone; Mutation Analysis In A Large Cohort Of Individuals With Usher Syndrome. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4544. doi: https://doi.org/.
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Usher syndrome is the most common cause of combined deaf-blindness. Mutations in Usher syndrome genes are also responsible for a significant fraction of nonsyndromic deafness and nonsyndromic retinitis pigmentosa. Identification of disease-causing genetic variants in patients with Usher syndrome is important to strengthen the clinical diagnosis, improve the accuracy of genetic counseling and facilitate selection of individuals for future clinical trials. There are currently nine genes and two additional genetic loci associated with Usher syndrome, and strategies for effectively and cost-efficiently screening for causative variants in these genes are still in evolution.
One or more of three different genotyping methods were used to screen the genomic DNA of 1269 patients with the clinical diagnosis of Usher syndrome. Specifically, 454 were screened using the SNPlex platform, 773 with TaqMan OpenArray and 765 with Fluidigm. The mutations included in each of the screening panels were selected on the basis of their previously observed frequencies, their molecular compatibility with each of the screening methods, and their absence from a cohort of 100 ethnically matched normal individuals. Forty-seven different variants were included in the SNPlex assay, 30 in the TaqMan OpenArray assay and 45 in the Fluidigm assay. These variants were distributed among six genes: MYO7A, CDH23, PCDH15, USH1C, USH2A, and USH3A. All variants identified by the screening panels were confirmed with Sanger sequencing.
The SNPlex assay correctly identified one or more mutations in 33% of the patients evaluated using that platform, OpenArray identified variants in 30%, and Fluidigm identified variants in 32%. There was a much higher rate of false positive results for SNPlex (21.2%) compared to OpenArray (12.8%) and Fluidigm (11.6%). For all methods combined, one or more disease-causing variants were identified in 423 of the 1269 patients (33.3%).
All three screening methods performed well for detecting known disease-causing variants although the SNPlex platform had a higher false positive rate than the other two. For a genetically heterogeneous disease like Usher syndrome, high-throughput screening of common disease-causing variants is faster and more cost-effective than Sanger sequencing for the initial phase of a comprehensive mutation screening strategy. Although Sanger sequencing is still required for identification of rarer alleles, it can be limited to a single gene (a nine-fold reduction of work) when a patient’s first allele is identified using an allele-specific method.<!--Copyright (c) 2006 Microsoft Corporation. All rights reserved.-->
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