APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans

Abstract

Myopia is the most common vision disorder and the leading cause of visual impairment worldwide. However, gene variants identified to date explain less than 10% of the variance in refractive error, leaving the majority of heritability unexplained ("missing heritability"). Previously, we reported that expression of APLP2 was strongly associated with myopia in a primate model. Here, we found that low-frequency variants near the 5'-end of APLP2 were associated with refractive error in a prospective UK birth cohort (n = 3,819 children; top SNP rs188663068, p = 5.0 × 10-4) and a CREAM consortium panel (n = 45,756 adults; top SNP rs7127037, p = 6.6 × 10-3). These variants showed evidence of differential effect on childhood longitudinal refractive error trajectories depending on time spent reading (gene x time spent reading x age interaction, p = 4.0 × 10-3). Furthermore, Aplp2 knockout mice developed high degrees of hyperopia (+11.5 ± 2.2 D, p < 1.0 × 10-4) compared to both heterozygous (-0.8 ± 2.0 D, p < 1.0 × 10-4) and wild-type (+0.3 ± 2.2 D, p < 1.0 × 10-4) littermates and exhibited a dose-dependent reduction in susceptibility to environmentally induced myopia (F(2, 33) = 191.0, p < 1.0 × 10-4). This phenotype was associated with reduced contrast sensitivity (F(12, 120) = 3.6, p = 1.5 × 10-4) and changes in the electrophysiological properties of retinal amacrine cells, which expressed Aplp2. This work identifies APLP2 as one of the "missing" myopia genes, demonstrating the importance of a low-frequency gene variant in the development of human myopia. It also demonstrates an important role for APLP2 in refractive development in mice and humans, suggesting a high level of evolutionary conservation of the signaling pathways underlying refractive eye development.

Fig 1. APLP2 expression is associated with myopic phenotype in the monkey model of myopia.

(A) Gene set enrichment analysis (GSEA) identified genes differentially expressed in the retina of monkeys with refractive errors induced by form-deprivation. Expression patterns of these genes exhibited statistically significant associations with phenotype “myopia” versus “hyperopia”. The heat map shows genes with the highest positive correlation with either the myopic or hyperopic phenotype. The expression level for each gene was normalized across the samples such that the mean was 0 and the standard deviation (SD) was 3. Expression levels greater than the mean are shaded in red, and those bellow the mean are shaded in blue. The scale (left) indicates SDs above or below the mean. (B) Graph showing the distribution of the GSEA correlation (ranking metric) scores for the 119 differentially expressed genes. Ranking metric score reflects the strength of correlation between a gene’s expression pattern and either the myopic or hyperopic phenotype. Positive values indicate a positive correlation with hyperopia and a negative correlation with myopia (i.e., downregulation in myopia and overexpression in hyperopia). Negative values indicate a positive correlation with myopia and a negative correlation with hyperopia (i.e., overexpression in myopia and downregulation in hyperopia). Arrows identify APLP2, which was found to be overexpressed in myopia, suppressed in hyperopia, had strong positive association with myopic phenotype and was negatively correlated with hyperopia (ranking metric score -0.63). These analyses were carried out using gene expression data previously reported by Tkatchenko et al. [53].

Fig 2. Association between genetic variants at the APLP2 locus and refractive error in children and adults.

The y-axis of all graphs indicates the observed log₁₀ (P-values) for single-marker association tests from GWAS for refractive error, for SNPs within 100 kb of the APLP2 gene in children (n = 3,819) participating in the ALSPAC study (A, B) and adults (n = 45,756) participating in the CREAM consortium sample (C, D). Region plots for all SNPs examined (A, C) show genomic position on the x-axis (build hg19 coordinates) while the colour coding indicates LD (r²) with the lead SNP estimated from CEU individuals in HapMap Phase 2, and the right-hand y-axis indicates the recombination rate. Quantile-quantile plots (B, D) display expected log₁₀ (P-values) on the x-axis.

Fig 3. APLP2 genotype and reading behaviour interact to influence refractive eye development in children.

Refractive development in ALSPAC participants (n = 5,200) was modelled over the 8–15 year age range. Models included as a predictor variable either rs188663068 genotype (A, C, D) or a binary term categorizing children as spending a “high” or “low” amount of time reading at age 8½ years (B). Analyses used the full sample (A), those with information available on time spent reading (B), or a stratified sample consisting of the low (C) or high (D) readers.

Fig 4. Aplp2 regulates refractive eye development in the mouse.

(A) Effect of targeted deletion of Aplp2 on refractive eye development in the mouse. Aplp2 knockout mice (generated on C57BL/6J background) develop high degrees of hyperopia (+11.5 ± 2.2 D, p < 1.0 × 10⁻⁴) compared to both heterozygous (-0.8 ± 2.0 D, p < 1.0 × 10⁻⁴) and wild-type (+0.3 ± 2.2 D, p < 1.0 × 10⁻⁴) littermates. Refractive errors were measured at P35 (age when refractive errors stabilize in mice) using automated infrared photorefractor. Red horizontal bars, mean. (B) Effect of targeted deletion of Aplp2 on susceptibility to experimental myopia in mice. Lack of Aplp2 expression had a negative dose-dependent effect on susceptibility to myopia in mice. Visual form deprivation (VFD) induced -1.2 ± 0.6 D of myopia (p = 3.0 × 10⁻²) in the Aplp2 knockouts compared to -5.7 ± 1.1 D (p < 1.0 × 10⁻⁴) in heterozygous and -11.0 ± 1.7 D (p < 1.0 × 10⁻⁴) in wild-type littermates. VFD was carried out for 21 days from P24 through P45 and refractive status of the deprived eyes versus control eyes was measured using an automated infrared photorefractor (Methods). Red horizontal bars, mean. (C) Effect of targeted deletion of Aplp2 on visual acuity in mice. Visual acuity in Aplp2 knockouts was not significantly different from that in the heterozygous and wild-type littermates (F(2, 20) = 0.6, p = 0.58). Error bars, s.d.; n = 13. (D) Effect of targeted deletion of Aplp2 on contrast sensitivity in mice. Lack of Aplp2 resulted in a dose-dependent reduction in contrast sensitivity (F(12, 120) = 3.6, p = 1.5 × 10⁻⁴). Error bars, s.d.; n = 13. Both visual acuity and contrast sensitivity were measured at P80 using a mouse virtual optomotor system.

Fig 5. Analysis of scotopic electroretinograms in the Aplp2 knockout mice.

(A-E) Effect of targeted deletion of Aplp2 on the a-wave and b-wave. Lack of Aplp2 causes a dose-dependent decrease in the b-wave amplitude (F(2, 18) = 6.9, p = 6.0 × 10⁻³). The b-wave implicit time was increased in the Aplp2 knockouts compared to both heterozygous and wild-type littermates (F(2, 18) = 6.1, p = 9.6 × 10⁻³). Lack of Aplp2 did not have significant impact on either a-wave amplitude or a-wave implicit time (F(2, 18) = 0.8, p = 0.47, amplitude; F(2, 18) = 2.6, p = 0.1, implicit time). (F-H) Effect of targeted deletion of Aplp2 on oscillatory potentials. The amplitude of the oscillatory potentials (OP) exhibited a dose-dependent decrease in the Aplp2 knockout mice, while the OP implicit time was increased in both heterozygous and knockout animals compared to the wild-type littermates. OP1 amplitude: F(2, 18) = 3.6, p = 5.0 × 10⁻²; OP2 amplitude: F(2, 18) = 15.6, p = 1.0 × 10⁻⁴; OP3 amplitude: F(2, 18) = 20.5, p < 1.0 × 10⁻⁴; OP4 amplitude: F(2, 18) = 9.7, p = 1.0 × 10⁻³; OP5 amplitude: F(2, 18) = 1.9, p = 0.2; OP1 implicit time: F(2, 18) = 7.2, p = 5.0 × 10⁻³; OP2 implicit time: F(2, 18) = 10.9, p = 8.0 × 10⁻⁴; OP3 implicit time: F(2, 18) = 20.9, p < 1.0 × 10⁻⁴; OP4 implicit time: F(2, 18) = 17.7, p < 1.0 × 10⁻⁴; OP5 implicit time: F(2, 18) = 4.5, p = 3.0 × 10⁻². Error bars, s.d.; n = 7.

Fig 6. Analysis of Aplp2 expression in the retina.

(A) Double staining with antibodies to Chx10 (red), which label bipolar cells, and Aplp2 (green) demonstrate that Aplp2 is expressed in the bipolar cells of the retina. (B) Double staining with antibodies to Pax6 (red), which label amacrine cells, and Aplp2 (green) demonstrate that Aplp2 is expressed in the amacrine cells of the retina. Expression of Aplp2 is also observed in the ganglion cell layer. (C) Analysis of Aplp2 expression in the retina at the mRNA level using in situ hybridization. In situ revealed that Aplp2 is expressed in the inner nuclear and ganglion cell layers of the retina. (D) Double staining with antibodies to glycine (red) and Aplp2 (green) revealed that Aplp2 is strongly expressed in the glycinergic amacrines. Arrows show two glycinergic amacrines with high levels of Aplp2 expression. (E) Double staining with antibodies to GABA (red) and Aplp2 (green) demonstrated that Aplp2 is not expressed in the GABAergic amacrines. Arrows show a glycinergic amacrine with strong expression of Aplp2 and an Aplp2-negative GABAergic amacrine. Blue, cell nuclei counterstained with DAPI. GABA, gamma-Aminobutyric acid; GABAA, GABAergic amacrine; GCL, ganglion cell layer; Gly, glycine; GlyA, glycinergic amacrine; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.