Identification of candidate genes for human retinal degeneration loci using differentially expressed genes from mouse photoreceptor dystrophy models

Abstract

Purpose: Retinal degeneration (RD) is a complex mechanism that appears to involve many biologic processes including oxidative stress, apoptosis, and cellular remodeling. Currently there are 51 mapped, but not identified, RD human disease loci.

Methods: To assign possible disease genes to RD loci, we have used a comparative genomics procedure that incorporates microarray gene expression data of three independent mouse models for photoreceptor dystrophy (rd1, rd2, and constant light-damage in BALB/c mice), human ortholog data, and databases of known chromosomal locations involved in human RD. Immunohistochemistry and enzyme activity assays were used to further characterize a candidate gene product.

Results: Our analysis yielded candidate genes for four mapped, but unsolved, human chromosomal locations and confirmed two previously identified monogenic disease loci for human RD, thus validating our approach. PLA2G7 (phospholipase A2, group VII; PAF-AH, Lp-PLA2), a candidate for a dominant form macular dystrophy (Benign Concentric Annular Macular Dystrophy [BCMAD]), was selected for further study. The PLA2G7 enzyme is known to mediate breakdown of oxidatively damaged phospholipids, a contributor to oxidative stress in the retina. PLA2G7 protein was enriched in mouse photoreceptor inner and outer segments. In the rd1, rd2, and BALB/c mice exposed to constant light, retinal tissue activity levels, but not plasma levels, were significantly reduced at the onset of photoreceptor cell death.

Conclusions: We have shown that this comparative genomics approach verified existing RD genes as well as identified novel RD candidate genes. The results on the characterization of the PLA2G7 protein, one of the novel RD genes, suggests that retinal tissue PLA2G7 levels may constitute an important risk factor for BCMAD. In summary, this reverse mapping approach, using accepted mouse models of human disease and known human RD loci, may prove useful in identifying possible novel disease candidates for RD and may be applicable to other human diseases.

Figure 1

Pla2g7 mRNA and activity levels, analyzing levels from P10 rd1, P21 rd2, and 48 h light-exposed BALB/c animals and their respective age-matched controls. A: Pla2g7 mRNA levels were plotted from Appendix 1. Retina Pla2g7 mRNA levels are significantly reduced in all three genotypes when compared to controls. Data are expressed as mean±SD of the two arrays analyzed per genotype. B: Tissue retina Pla2g7 levels as measured in a calorimetric assay using 2-thio platelet activating factor (PAF) as substrate, revealed that activity levels in retinas from the three genotypes correlated well with the respective reduced amount of mRNA found in the tissue. Data are expressed as mean±SEM of at least three, independent samples in unit of activity (μmol/min/mg of protein). C: Plasma Pla2g7 levels measured in mandibular blood samples revealed that the two genetic mutations (rd1 and rd2) or the environmental stress (constant light) did not influence systemic, plasma-derived Pla2g7 activity. Data are expressed as mean±SEM of at least three independent samples in unit of activity (μmol/min/mL of plasma). In the graph, red indicates control and blue indicates experimental. The following abbreviations were used: light-damage (LD) and not significant (n.s.)

Figure 2

Pla2g7 localization. Immunohistochemistry was performed in juvenile C57BL/6 (P17) frozen sections (A), using no primary antibody conditions as the negative control (B). Pla2g7 was found to be localized throughout the retina. Relatively elevated levels were found in the photoreceptor inner and outer segments, whereas moderate staining was found in the two plexiform layers, as well as the inner nuclear layer (INL) and the retinal ganglion cell (RGC) layer. For each image, the corresponding DIC image is provided. The following abbreviations were used: retinal pigment epithelium (RPE), outer segments (OS), inner segments (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and RGC: retinal ganglion cells (RGC). Scale bar in (A) represents 20 μm.