Genomic Convergence to Prioritize Candidate Genes for Primary Open–Angle Glaucoma: Analysis of Ciliary Body and Retina Transcriptomes

Abstract

Purpose: : Eleven GLC1 loci have been mapped for primary open–angle glaucoma (POAG), but only 3 genes have been characterized: TIGR/myocilin, OPTN and WDR36. We have previously performed linkage analysis on 24 French–Canadian POAG families. Five of them displayed haplotypes compatible with segregation of a disease–causing gene at either GLC1B, GLC1C or GLC1D. The relatively small sizes of these families hampered confinement of the disease loci. We exploited genomic convergence, a multistep approach that combines gene expression with genomic linkage, to identify and prioritize candidate genes for POAG within these loci.

Methods: : Serial analysis of gene expression (SAGE) was performed on 1 retina of an asymptomatic 76 year old male. Affymetrix microarrays (HG U133 plus 2.0) done in triplicate were used to probe 47 400 transcripts in retina and ciliary body. Tissues were obtained from 1 asymptomatic 67 year old male, 1 asymptomatic 75 year old female and 1 76 year old POAG female.

Results: : 21 025 different species of SAGE tags were detected in a total of 46 096 sequenced tags. Unspecific tags matching to 2 or more transcripts and tags detected 4 times or less, were not considered for further analysis. The remaining tags corresponded to 784 transcripts of which 9 mapped within 1 of the 8 GLC1 loci encoding a POAG gene not yet characterized. These 9 transcripts were encoded by 7 genes of which 2 mapped at GLC1B. In our microarrays experiment, 684 and 421 transcripts were differentially expressed, respectively, in the retina and ciliary body of our patient as compared to controls. The transcripts that mapped to 1 of the 8 GLC1 loci, for which a POAG gene remain to be identified, corresponded to 30 genes. Five of these genes localized within GLC1B, 3 within GLC1C and 2 within GLC1D. None of the 7 genes identified by SAGE corresponded to the 30 characterized by microarrays. The most interesting genes were REEP1α (GLC1B), TFDP2α, ZBTB38α (GLC1C) and ENY2α (GLC1D).

Conclusions: : Two complementary procedures were exploited to prioritize candidate genes. SAGE was used to identify enriched genes whereas microarrays were exploited to assess differentially expressed genes in ocular tissues. These transcriptomic analyses of the retina and ciliary body revealed 37 genes that mapped within GLC1 loci encoding unknown glaucoma genes. Twelve of these genes mapped at GLC1B, 1C or 1D and therefore represent primary candidate genes in our families.

Keywords: candidate gene analysis • genetics • retina