Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila

Abstract

Individuals carrying the same pathogenic mutation can present with a broad range of disease outcomes. While some of this variation arises from environmental factors, it is increasingly recognized that the background genetic variation of each individual can have a profound effect on the expressivity of a pathogenic mutation. In order to understand this background effect on disease-causing mutations, studies need to be performed across a wide range of backgrounds. Recent advancements in model organism biology allow us to test mutations across genetically diverse backgrounds and identify the genes that influence the expressivity of a mutation. In this study, we used the Drosophila Genetic Reference Panel, a collection of ∼200 wild-derived strains, to test the variability of the retinal phenotype of the Rh1(G69D) Drosophila model of retinitis pigmentosa (RP). We found that the Rh1(G69D) retinal phenotype is quite a variable quantitative phenotype. To identify the genes driving this extensive phenotypic variation, we performed a genome-wide association study. We identified 106 candidate genes, including 14 high-priority candidates. Functional testing by RNAi indicates that 10/13 top candidates tested influence the expressivity of Rh1(G69D). The human orthologs of the candidate genes have not previously been implicated as RP modifiers and their functions are diverse, including roles in endoplasmic reticulum stress, apoptosis and retinal degeneration and development. This study demonstrates the utility of studying a pathogenic mutation across a wide range of genetic backgrounds. These candidate modifiers provide new avenues of inquiry that may reveal new RP disease mechanisms and therapies.

Figure 1.

Severity of the Rh1^G69D retinal phenotype depends on genetic background. (A) The eye sizes for 173 DGRP/Donor F1 crosses. There is a very strong strain effect on the severity of the retinal degeneration as measured by eye size (P < 2.2 × 10⁻¹⁶). Median ± SD. (B) Examples of the qualitative differences in eye phenotype across the size spectrum shown in (A). Small = three strains with the smallest eyes. Medium = strains with eye size from the middle of the distribution. Large = three strains with the largest eyes. RAL# = the strain number of the DGRP strain.

Figure 2

Functional analysis of candidate genes. (A) The eye sizes for RNAi KD of candidate genes on the Rh1^G69D background. White = AttP control; red = significantly different in size from AttP control (P); blue = not significantly different in size but shows clear qualitative change in eye phenotype (see B); gray = not significantly different from AttP control. In the box plots, the boxes represent the interquartile range, the whiskers represent 1.5 × interquartile range and open circles are outliers. See Supplementary Material, Table S7 for values and significance. (B) Representative examples of eyes from functional tests. All eyes are from flies carrying Rh1^G69D. Control is on the left. RNAi KD eyes are shown in the order that they appear in (A). Note that hppy and Cdk5 KD eyes appear qualitatively different from the control, but do not show a difference in measured eye size. The control strain shown in (A and B) is the 60 100 AttP strain. This strain has the same genetic background for all the RNAi strains except CG2004, and thus serves as their control. CG2004 is on a different genetic background; its appropriate control strain is 60 000 AttP. Since we found that the two AttP control strains are indistinguishable in eye phenotype in the Rh1^G69D background (Supplementary Material, Fig. S9), for simplicity, only the results for 60 100 AttP are shown here.