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

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 (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P \, < \, 1.19\times {10}^{-8}$$\end{document}P<1.19×10−8). Substantial genetic sharing between PCV and typical nAMD is noted (rg = 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; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P=1.05\times {10}^{-6}$$\end{document}P=1.05×10−6) 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.

For all study subjects, venous blood was collected to extract DNA.For GWAS, DNA samples were genotyped using Illumina OmniExpress-12 v1 for 520 cases on 714,255 variants and Human610-Quad BeadChip for 1,044 controls on 573,871 variants.We acknowledge the funding from the Direct Grants of the Chinese University of Hong Kong (4054119, CPP; and 2015.1.045,LJC; Hong Kong).

Japan
Cases: All AMD cases were of Japanese descent recruited from the Department of Ophthalmology at Kyoto University Hospital, Fukushima Medical University Hospital, and the Kobe City Medical Center General Hospital.All participants underwent a comprehensive ophthalmic examination, including determination of bestcorrected visual acuity, intraocular pressure measurement, indirect ophthalmoscopy, slit-lamp biomicroscopy with a contact lens, and OCT examination (Spectralis, Heidelberg Engineering, Heidelberg, Germany; and/or Cirrus, Carl Zeiss Meditec, Dublin, California, USA).Subsequently, fluorescein angiography and ICG angiography were performed on each patient on fundus photographs, using a confocal laser scanning system (Heidelberg Retina Angiography II, Heidelberg Engineering, Heidelberg, Germany).Venous blood was collected to extract DNA for genotyping.Controls: The control subjects were cataract patients without signs of AMD in both eyes and were recruited from the Department of Ophthalmology at Kyoto University Hospital, the Ozaki Eye Hospital, Mizoguchi Eye Clinic, the Japanese Red Cross Otsu Hospital, and Nagahama City Hospital.
DNA samples of 997 cases and 1,174 controls were genotyped using Illumina OmniExpress-12 v1 on 714,255 variants.We acknowledge grants-in-aid for scientific research (No. 24249082) from the Japan Society for the Promotion of Science, Tokyo, Japan.

Korea
Cases: Unrelated subjects of native Korean descent, aged 50 years or older, were recruited from 6 University Hospital-based tertiary retinal care centres, including the Seoul National University Bundang Hospital in Seongnam, Seoul National University Hospital in Seoul, Kyungpook National University Hospital, and Yeungnam University Hospital in Daegu, as well as Kosin University Hospital and Busan Paik Hospital in Busan, Korea.Each AMD patient was examined based on a standardized protocol to capture clinical, imaging and functional data.Based on clinical and ocular examination results, patients were categorized according to the AREDS classification system 5 .Late AMD was diagnosed and evaluated with fundus photographs, fluorescein and ICG angiography, and was categorized into typical neovascular AMD, polypoidal choroidal vasculopathy and geographic atrophy according to examination findings.Subjects with geographic atrophy were excluded from the current study.A 10 − 20  sample of venous blood was collected to extract DNA for genotyping.The standard ocular examinations included dilated fundus examination, fundus photography, OCT (Spectralis, Heidelberg Engineering, Heidelberg, Germany; and/or Cirrus, Carl Zeiss Meditec, Dublin, California, USA) fundus fluorescein angiography and ICG angiography (Heidelberg Retina Angiography, Heidelberg Engineering, Heidelberg, Germany).Controls: The hospital-based controls were from Seoul National University Bundang Hospital and other institutions in Korea.The average age was around 70 years (SD = 9).Subjects without any sign of AMD were enrolled as controls.They had no drusen and pigment abnormalities in the fundus photograph and/or optical coherence tomography.DNA samples of 529 cases and 940 controls were genotyped using Illumina OmniExpress-12 v1 on 714,255 variants.We acknowledge the Seoul National University Bundang Hospital Research Grant Fund (grant no.03-2009-008), and National Research Foundation of Korea (NRF-2009-0072603, NRF-2012R1A1A2008943, NRF-2014R1A2A1A09005824) grants funded by the Ministry of Education, Science and Technology, Korea.Supplementary Figure 3: Quantile-quantile (QQ) plots for Asian nAMD GWAS and subtypes.
GWAS discovery phase for nAMD contains 5,124 subjects.Meta-analyses of 4 cohorts for nAMD and two subtypes (PCV and typical nAMD) included a total of 8,621, 7,048, and 7,066 subjects, separately.We excluded variants with imputation quality indicator  2 < 0.5 and minor allele frequency < 0.1% and kept only variants that exist in at least 3 out of 4 studies.Genomic inflation factor  for the discovery phase and meta-analysis for nAMD, PCV, and typical nAMD GWAS are 0.99, 1.04, 1.06 and 1.02, separately.We determined  by taking the median of  2 statistics for all variants and dividing it by the median of simulated  2 statistics at one degreeof-freedom.

GWAS discovery phase for nAMD
Meta-analysis for nAMD