Contribution of common and rare variants to Asian neovascular age-related macular degeneration subtypes

Abstract

Neovascular age-related macular degeneration (nAMD), along with its clinical subtype known as polypoidal choroidal vasculopathy (PCV), are among the leading causes of vision loss in elderly Asians. In a genome-wide association study (GWAS) comprising 3,128 nAMD (1,555 PCV and 1,573 typical nAMD), and 5,493 controls of East Asian ancestry, we identify twelve loci, of which four are novel ([Formula: see text]). Substantial genetic sharing between PCV and typical nAMD is noted (r_g = 0.666), whereas collagen extracellular matrix and fibrosis-related pathways are more pronounced for PCV. Whole-exome sequencing in 259 PCV patients revealed functional rare variants burden in collagen type I alpha 1 chain gene (COL1A1; [Formula: see text]) and potential enrichment of functional rare mutations at AMD-associated loci. At the GATA binding protein 5 (GATA5) locus, the most significant GWAS novel loci, the expressions of genes including laminin subunit alpha 5 (Lama5), mitochondrial ribosome associated GTPase 2 (Mtg2), and collagen type IX alpha 3 chain (Col9A3), are significantly induced during retinal angiogenesis and subretinal fibrosis in murine models. Furthermore, retinoic acid increased the expression of LAMA5 and MTG2 in vitro. Taken together, our data provide insights into the genetic basis of AMD pathogenesis in the Asian population.

Fig. 1. Manhattan plot of Manhattan plot of GWAS for neovascular AMD (nAMD) and QQ plot of whole-exome sequencing gene-level analysis for PCV.

a We conducted genome-wide single-variants association analyses for nAMD in 3128 patients (1555 PCV and 1573 typical nAMD) and 5493 controls. The Manhattan plot exhibits ten logarithms of P values for the association, annotating the nearest genes for each genetic locus that reaches genome-wide significance ($P<5\times {10}^{-8}$). The gene names of novel nAMD loci are highlighted in bold. Two loci (COL4A4/COL4A3 and C6orf223/VEGFA) that showed significant association with PCV in our study are underlined. The red dashed horizontal line represents genome-wide significance ($P<5\times {10}^{-8}$), and the blue line indicates the suggestive significance of $P<1\times {10}^{-5}$. The y-axis breaks at around $P=1\times {10}^{-20}$. b We conducted three gene-based analyses: Burden test, Sequence Kernel Association Test (SKAT), and Combined test of burden test and SKAT (SKAT-O) for functional variants at MAF $<2\%$ in 1019 Chinese subjects (259 PCV and 760 controls). Data points shown with distinct hues and shapes represent genes tested for three different tests. The genomic inflation factor ($\lambda$) of each analysis is shown in the figure.

Fig. 2. Genetic effect size in PCV versus typical nAMD.

For the figure on the left, we grouped variants by minor allele frequency ($1-10\%$, $10-25\%$, or $25-50\%$). Minor allele frequency was computed based on our GWAS data in 8621 subjects of East Asian descent. For the figure on the right, variants were grouped by different association P value thresholds ($P\le 5\times {10}^{-8}$, $5\times {10}^{-8}<P\le 1\times {10}^{-5}$, or $1\times {10}^{-5}<P\le 0.05$) in GWAS for both PCV and typical nAMD. We conducted a single-variant test using the firth bias-corrected likelihood-ratio model, incorporating the top four principal components as covariates. ${\chi }^2$ statistics with one degree-of-freedom were used for the single-variant test. We then performed a meta-analysis under an inverse-weighted fixed-effect model, combining data from all cohorts for each phenotype. Each data point represents a variant out of the total 7,911,145 overlapped variants in PCV and typical nAMD GWAS. The black diagonal dashed line represents the same effect size between PCV and typical nAMD. The gray horizontal and vertical dashed lines at the value of 0 separate the figure into four quadrants. Variants in the first and the third quadrants have a consistent direction of effect between the two AMD subtypes.

Fig. 3. Comparative mRNA expression analysis of selected genes in the retinal tissue of developing mice, RPE cells following the laser-induced CNV in mice, and human retinal pigment epithelial (HRPE) cells upon retinoic acid treatment.

a mRNA expression of COL1A1 and gene candidates at GATA5 locus in the retinal tissue of developing mice. Each group included three mice as test subjects. The expression profiles of selected gene targets from the mouse retina were analyzed on postnatal days P4, P7, P14, P21, and P28, as shown on the y-axis of bar plots ($n=3$ animals). Significant differences in gene expression level were observed between P4 and P14, P21, and P28 for Vegfa (P values: 0.0041, $<0.0001$, and $<0.0001$, respectively). For Col1a1, the P value is $<0.001$ for P4 vs. P21. For Mtg2, significant differences were found for P4 vs. P7, P14, P21, and P28 (P value are $0.0001$, $<0.0001$, $<0.0001$, and $<0.0001$, separately). For Lama5, significant differences were observed for P4 vs. P14 (P value is $0.0215$), P4 vs. P21 (P value is $0.0001$), and P4 vs. P28 (P value $<0.0001$). For Cables2, the P value is $0.0005$ for P4 vs. P14. For Col9a3, significant differences were observed for P4 vs. P14 and P4 vs. P28 with P values $<0.0001$ and $=0.0001$, respectively. b Target genes induced in RPE cells following the laser-induced CNV in mice. qRT-PCR analysis of each gene in RPE cells isolated from mice at day 35 following laser-induced CNV. For Col1a1 and Col9a3, experimental groups had four mice each. Lama5’s control group had six mice, and D35 group had four mice. For Cables2 and Mtg2, the control group had six mice, and the D35 group had five mice. The P values for Mtg2, Lama5, Col9a3, Col1a1, and Cables2 are $0.004$, $0.003$, $0.003$, $0.095$, and $0.203$, respectively. c mRNA expression of gene candidates in human retinal pigment epithelial (HRPE) cells upon retinoic acid treatment, compared to induction using dimethyl sulfoxide (DMSO) solvent in four independent experiments. P values for gene candidates are: MTG2 ($0.016$), LAMA5 ($0.048$), COL9A3 ($0.070$), COL1A1 ($0.854$), and CABLES2 ($0.627$). For all independent experiments, the mRNA expressions of lead gene candidates were measured using qRT-PCR method. Source data are provided as a Source Data file. Error bars were presented as mean $\pm$ S.E.M. Statistical significance was determined by one-way ANOVA or two-tailed, unpaired Student’s t test, $*P<0.05$, $**P<0.01$, $***P<0.001$, and $****P<0.0001$.