Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE

Abstract

Purpose: To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE).

Methods: Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR.

Results: Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified.

Conclusions: The EyeSAGE database, combining three different gene-profiling platforms including the authors' multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.

Figure 1

Posterior eyecup of a human donor eye. Right eye obtained from a 64-year-old white male donor within 4 hours of death and stored in RNA preservative. The donor had no ocular history of disease. Macular (M) and midperipheral (P) regions, from which 4-mm diameter punches were taken.

Figure 2

Real-time qRT-PCR summaries of known rod-specific (PDE6A), cone-specific (PDE6C and GNAT2), ganglion cell-associated (THY-1), and candidate cone photoreceptor–associated genes (SH3BGRL2, CPLX4, DIRAS2, HR, KIAA1345, ZNF595OS, and UCHL1) in the macula relative to expression in the peripheral retina.

Figure 3

Real-time qRT-PCR summaries of cone-specific (PDE6C and GNAT2), candidate cone photoreceptor (HR, CPLX4, and KIAA1345) and ganglion cell-associated (THY-1) gene expression in regions of the human retina relative to expression in peripheral retina (calculated using the comparative C_T method). PR, photoreceptor layer; Peri, peripheral.

Figure 4

Single human photoreceptor (A) isolation and (B) RT-PCR. (A) Single photoreceptor cells were aspirated into micropipette. (B) Ethidium bromide–stained agarose gel of PCR products amplified in nested RT-PCR reactions of single photoreceptor cDNAs. Rod, rodspecific PDE6A gene primers (124-bp product) plus UCHL1 primers (153-bp product) or plus SH3BGRL2 primers (100-bp product); “cone” cone-specific PDE6C gene primers (195-bp product) plus UCHL1 primers or SH3BGRL2 primers. UCHL1 is expressed in the inner retina and is therefore not detected in either photoreceptor derived cDNA pool. Lanes 1, 2: UCHL1 reactions; lane 3: 100-bp ladder; lanes 4, 5: SH3BGRL2 reactions.

Figure 5

Real-time qRT-PCR summaries of rod-specific (PDE6A), RPE-specific (RDH5), and candidate RPE (EMP3 and MMP25) gene expression in the peripheral retina, RPE/choroid, and RPE cells (RPE-enriched) relative to expression in the peripheral retina.