Abstract
Purpose:
To investigate the contribution of extracellular HSPA5 (aka GRP78) in human atrophic age-related macular degeneration using novel systems genetic methods, mathematical modeling and immunostaining techniques.
Methods:
To determine the cellular localization and contribution of extracellular HSPA5, single and double immunostaining in human normal and AMD retinas were performed using well-characterized antibodies. Quantitative trait locus (QTL) mapping was used to map the genomic region(s) that regulate Hspa5 in a BXD genetic reference panel. Multiple partial correlation and heatmap analyses were performed to define the relationship between the expression of Hspa5 and all genes within the mouse genome. The top candidate genes were identified and further evaluated using SNP screening.
Results:
In non-AMD retinas, extracellular HSPA5 was localized intensely around cones within the macular area; however it was scattered in the peripheral retina. In contrast, in AMD retinas, there were very few HSPA5 immunopositive cones, especially in the macula. In addition, the immunolabeling profiles were aberrant. Our mapping studies in BXD mice indicate a single highly significant trans-eQTL with an LRS of 18.4 on Chr2:169-175Mb and a suggestive trans-eQTL on Chr15:75-100Mb, both of which suggest that other genes control Hspa5 expression either directly or indirectly. Partial correlation analyses and heatmaps confirmed the QTLs on Chrs 2 and 15. The best candidate extracellular genes in those intervals were B4galt5, Dpm1, Sulf2, Fbln1, Glt8d3 and Pmm1. Direct correlation analyses showed that expression of Sulf2, Pmm1 and Fbln1 were significantly correlated with Hspa5. Only Sulf2 had a non-synonymous SNP, which makes it our top candidate for controlling Hspa5 expression.
Conclusions:
The presence of HSPA5 in the interphotoreceptor matrix around cones in healthy human retinas and its absence in atrophic AMD retinas supports our assertion that HSPA5 may play a role in cone structural integrity in the macula. Integrated approaches using mouse and human analyses suggest that HSPA5 may be a locus for AMD progression and/or susceptibility. Our studies further suggest that Sulf2 is the best candidate gene for controlling Hspa5 expression in the retina.
Keywords: 585 macula/fovea •
536 gene modifiers •
554 immunohistochemistry