Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retinal degenerations: hereditary • gene microarray • gene/expression