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D. A. Simpson, P. Crowe, G. J. McKay, J. O'Neil, G. R. Clark, G. Silvestri; Development of a Retinitis Pigmentosa (RP) Genotyping Microarray and Detection of Known and Novel Mutations in a Cohort of Northern Irish RP Patients. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1691. doi: https://doi.org/.
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Retinitis Pigmentosa (RP) is extremely genetically heterogeneous. Although 20 genes have been implicated in non-syndromic recessive RP to date, it is very difficult to identify the causative mutation when a new patient presents. Although screens for all known mutations have recently become available, they cannot detect novel changes. Therefore, in order to maximise the ability to detect RP mutations, we have developed a microarray capable of resequencing reported mutations and the exons within which they are found.
A database of all reported mutations which cause recessive or X-linked RP was compiled and an Affymetrix customseq resequencing array designed to screen these and adjacent sequences. Several genes implicated in Leber Congenital Amaurosis were also included. In total 30 kbp from 22 genes were tiled on the array. Approximately 100 amplicons spanning the regions of intest were amplified from each patient DNA sample. These were pooled, fragmented, labelled and hybridised to the genechip according to the manufacturer’s protocol. Results were analysed using the GeneChip DNA Analysis Software (GDAS, Affymetrix).
An average call rate of 91% was achieved for all the sequences analysed. A total of nine known mutations in RGR, USH2A and CRB1 were confirmedin 35 recessive or sporadic RP patients. A number of novel sequence variants were also detected.
This microarray platform provides a rapid and effective screen for RP mutations and provides a new tool to include in screening strategies. Technological improvements will facilitate fuller coverage of recently discovered genes in subsequent genechip designs, although PCR amplification remains a limiting factor. It is anticipated that the improved detection of RP mutations will facilitate genotype:phenotype correlations, better prognosis and application of therapeutic interventions such as gene therapy.
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