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D.T. Hartong, T.L. McGee, N. Gorji, E.L. Berson, T.P. Dryja; Search for Recessive Retinitis Pigmentosa Genes Using Microarray Analysis of RNA Expression Levels in Lymphoblasts . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5521.
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© ARVO (1962-2015); The Authors (2016-present)
Recessive retinitis pigmentosa (RP) is often caused by nonsense mutations that lead to low mRNA levels as a result of nonsense–mediated decay. Some RP genes are expressed at detectable levels in leukocytes as well as the retina. We designed a microarray–based method to find recessive RP genes based on low lymphoblast mRNA levels.
We established lymphoblast cell lines from 18 unrelated index patients with recessive RP as well all of their affected siblings (1 sibship with 4 affected members, 1 sibship with 3 affected members, 8 with 2 affected members, and 8 isolates) and 4 controls. The patients had no mutations in the following previously identified recessive RP genes: USH2A, TULP1, RPE65, PDE6A, PDE6B and CNGA1 (at least 4 of these 5 genes were screened in each index patient). To date, RNA was isolated from cell lines of 21 patients (five sibpairs, one 4–sibship family and 7 isolates) and 4 controls, and hybridized on Affymetrix genechip Human Genome U133Plus2.0 containing probes from all known genes and EST transcripts. After normalization, expression levels of the 6 individual sibships were compared to the other samples; significance was tested using the Student t–test.
Approximately 37% of the probes were expressed at levels above noise; all others were excluded from further analysis. For each sibship, we found 1–17 candidate genes with the following properties: 1) average mRNA expression in the members of a sibship was < 50% the average expression across all other samples; and 2) for every probe associated with that gene, the difference in expression between the affected sibs and all other samples was statistically significant at p–value < 0.05 after a Bonferroni correction for multiple analyses. These genes will be evaluated further with segregation analysis and direct sequencing.
Microarray analyses of RNA expression in lymphoblasts reveal a small set of genes in each recessive RP sibship that are expressed at levels significantly lower than normal. We are currently searching for pathogenic mutations in these candidate genes.
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