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A missense variant in FGD6 confers increased risk of polypoidal choroidal vasculopathy

Nature Genetics volume 48, pages 640–647 (2016)Cite this article

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Abstract

Polypoidal choroidal vasculopathy (PCV), a subtype of 'wet' age-related macular degeneration (AMD), constitutes up to 55% of cases of wet AMD in Asian patients. In contrast to the choroidal neovascularization (CNV) subtype, the genetic risk factors for PCV are relatively unknown. Exome sequencing analysis of a Han Chinese cohort followed by replication in four independent cohorts identified a rare c.986A>G (p.Lys329Arg) variant in the FGD6 gene as significantly associated with PCV (P = 2.19 × 10−16, odds ratio (OR) = 2.12) but not with CNV (P = 0.26, OR = 1.13). The intracellular localization of FGD6-Arg329 is distinct from that of FGD6-Lys329. In vitro, FGD6 could regulate proangiogenic activity, and oxidized phospholipids increased expression of FGD6. FGD6-Arg329 promoted more abnormal vessel development in the mouse retina than FGD6-Lys329. Collectively, our data suggest that oxidized phospholipids and FGD6-Arg329 might act synergistically to increase susceptibility to PCV.

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Figure 1: A variant allele at rs77466370 (p.Arg329Lys) in the FGD6 gene is associated with PCV.
Figure 2: Expression profile of FGD6 mRNA and subcellular localization of FGD6 protein in HRECs.
Figure 3: Assessment of endothelial cell network formation with HRECs using Matrigel matrix.

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Acknowledgements

We thank all the patients with AMD and their families for participating in this study. This work was carried out on behalf of the Genetics of AMD in Asians (GAMA) Consortium. This research project was supported by the National Natural Science Foundation of China (81170883, 81430008 (Z.Y.), 81200723 and 81300802 (L.H.), 81170882 (Y. Shi), 81371030 (H. Zhang), and 81271005 (Xianjun Zhu)); grants from the Department of Science and Technology of Sichuan Province, China (2014SZ0169, 2015SZ0052 (Z.Y.), 2015SZ0060 (Y.L.), 2016HH00X, 2015JQO057 (L.H.), 2014HH0009 (H. Zhang), and 2014JZ0004 (Y. Shi)); research grants 467708 and 468810 from the General Research Fund, Hong Kong; the National Medical Research Council, Singapore (NMRC/TCR/002-SERI/2008 (R626/47/2008TCR), CSA R613/34/2008, NMRC 0796/2003, and STaR/0003/2008); the National Research Foundation of Singapore, Biomedical Research Council, Singapore (BMRC 09/1/35/ 19/616, 08/1/35/19/550, and 10/1/35/19/675); and the Genome Institute of Singapore (GIS/12-AR2105). This research was also supported by the National Medical Research Council (NMRC grants 0796/2003, IRG07nov013, IRG09nov014, NMRC 1176/2008, NIG/1003/2009, CG/SERI/2010, and CSA/033/2012) and the Biomedical Research Council (BMRC 08/1/35/19/550, 09/1/35/19/616, and 10/1/35/19/671) in Singapore; the Bright Focus Foundation, USA (M2011068); and the National Medical Research Council, Singapore (CSA/033/2012).

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Author notes

  1. Lulin Huang and Houbin Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory for Human Disease Gene Study, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China

    Lulin Huang, Houbin Zhang, Zhengfu Tai, Shaoping Deng, Xianjun Zhu, Li Cai, Fang Lu, Yuanfeng Li, Yi Shi, Yin Lin, Bo Gong, Xiaoqi Liu, Jiyun Yang, Fang Hao, Shi Ma, He Lin, Jing Cheng, Yu Zhou, Lin Zhang, Hong Zheng, Dan Jiang, Xiong Zhu & Zhenglin Yang

  2. Institute of Chengdu Biology, Chinese Academy of Sciences, Chengdu, China

    Lulin Huang, Zhengfu Tai & Zhenglin Yang

  3. Sichuan Translational Medicine Hospital, Chinese Academy of Sciences, Chengdu, China

    Lulin Huang, Zhengfu Tai & Zhenglin Yang

  4. Center of Information in Biomedicine, University of Electronic Science and Technology of China, Chengdu, China

    Lulin Huang, Houbin Zhang, Zhengfu Tai, Shaoping Deng, Xianjun Zhu, Yi Shi, Yin Lin, Lin Zhang & Zhenglin Yang

  5. Singapore Eye Research Institute, Singapore National Eye Centre, Singapore

    Ching-Yu Cheng, Zheng Li, Chui-Ming G Cheung, Kar-Seng Sim, Peter D Cackett, Ian Y Yeo, Tien-Ying Wong & Chiea-Chuen Khor

  6. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Ching-Yu Cheng, Zheng Li, Chui-Ming G Cheung, Tien-Ying Wong & Chiea-Chuen Khor

  7. Duke–National University of Singapore Graduate Medical School, Singapore

    Ching-Yu Cheng, Ian Y Yeo & Tien-Ying Wong

  8. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    Feng Wen, Pancy O S Tam, Kar-Seng Sim, Xiongzhe Zhang & Sibo Tang

  9. Department of Ophthalmology, Xinhua Hospital, Shanghai Jiaotong University, Shanghai, China

    Peiquan Zhao & Ping Fei

  10. Joint Shantou International Eye Center, Shantou University and Chinese University of Hong Kong, Shantou, China

    Haoyu Chen & Weiqi Chen

  11. Department of Human Genetics, Genome Institute of Singapore, Singapore

    Zheng Li, Kar-Seng Sim & Chiea-Chuen Khor

  12. Department of Ophthalmology and Visual Sciences, Chinese University of Hong Kong, Hong Kong, China

    Lijia Chen, Timothy Y Y Lai & Chi-Pui Pang

  13. Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan

    Kenji Yamashiro, Masahiro Miyake & Nagahisa Yoshimura

  14. Department of Ophthalmology, Saitama Medical University, Iruma, Japan

    Keisuke Mori

  15. Princess Alexandra Eye Pavilion, Edinburgh, UK

    Peter D Cackett

  16. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan

    Motokazu Tsujikawa & Kohji Nishida

  17. National Health care Group Eye Institute, Tan Tock Seng Hospital, Singapore

    Augustinus Laude

  18. Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan

    Satoshi Inoue

  19. Department of Surgery, Division of Ophthalmology, Kobe University Graduate School of Medicine, Kobe, Japan

    Yoichi Sakurada & Shigeru Honda

  20. Department of Ophthalmology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan

    Hiroyuki Iijima

  21. Department of Ophthalmology, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, China

    Chuntao Lei

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  1. Lulin Huang
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Contributions

Z.Y. designed the study. L.H., C.-Y.C., F.W., P.O.S.T., P.Z., H.C., Z.L., L. Chen, Z.T., K.Y., S.D., Xianjun Zhu, W.C., L. Cai, F.L., Y. Li, C.-M.G.C., Y. Shi, M.M., Y. Lin, B.G., X.L., K.-S.S., J.Y., K.M., X. Zhang, P.D.C., M.T., K.N., F.H., S.M., H.L., J.C., P.F., T.Y.Y.L., S.T., A.L., S.I., I.Y.Y., Y. Sakurada, Y.Z., H.I., S.H., C.L., H. Zheng, D.J., T.-Y.W., C.-C.K., C.-P.P., N.Y., and Z.Y. recruited the participants. L.H., C.-Y.C., Z.L., J.C., P.F., T.Y.Y.L., S.T., A.L., S.I., I.Y.Y., Y. Shi, Y.Z., H.I., S.H., Xiong Zhu, H. Zhang, D.J., T.-Y.W., C.-C.K., C.-P.P., N.Y., and Z.Y. performed the genotyping. L.H., K.Y., C.-C.K., C.-M.G.C., T.-Y.W., and Z.Y. performed the statistical analysis. H. Zhang, Z.T., B.G., L.Z., and Z.Y. performed the immunohistochemistry and gene expression studies. Z.T., Z.Y., H. Zhang, Y. Shi, and Y. Lin performed the tube formation and wound healing assays. H. Zhang and F.L. performed the mouse analysis. The initial draft of the manuscript was written by L.H., H. Zhang, and Z.Y. and edited by T.-Y.W., C.-Y.C., C.-P.P., and C.-C.K. All authors critically revised and gave final approval to this manuscript.

Corresponding author

Correspondence to Zhenglin Yang.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fundus, angiographic features, and OCT of PCV and CNV.

Shown are the typical clinical features of PCV: orange-redlesions at the posterior pole of the retina in fundus photography and indocyanine green angiography (ICGA), demonstrating PCV polypoid expansion of choroid blood vessels. OCT is abnormal in both PCV and CNV. The patients gave written informed consent for publication of these photographs.

Supplementary Figure 2 Gene identification and functional analysis procedure.

First, we performed exome sequencing for the 194 PCV cases, 155 CNV cases, and 1,253 controls. A total of 118,390 coding SNPs with MAF >0.1% was subjected to single-variant association analysis, and 25 nonsynonymous SNPs showed nominal association (P< 0.005) only in PCVandnot in CNV. FGD6 rs77466370 is the only new locus surpassing genome-wide significance (P=4.24 × 10–7) in this study. We also analyzed these loci in other cohorts and ultimately confirmed the association of PCV and rs77466370. Then, we performed functional studies ofFGD6 in PCV pathology.

Supplementary Figure 3 Ancestry analysis of the whole-exome sequencing datasets using principal components.

PCA for ancestry with 1,602 exome-sequenced samples. The x axis shows eigenvector 1 (first principal component), and the y axis shows eigenvector 2 (second principal component).

Supplementary Figure 4 Quantile–quantile plot of P values for the whole-exome sequencing datasets in the discovery stage.

No genomic inflation was observed.

Supplementary Figure 5 100-kb LD map of the FGD6-Arg329 region for Chinese individuals (CHB+CHS) in 1000 Genome Project Phase 3.

(a) LD map of the region at 95,554,000–95,650,000 on chromosome 12 (hg19) in the vicinity of variant Arg329 (95,604,074). (b) Enlargement of the block corresponding to variant Arg329 in a. The yellow block in a highlights the block forvariant Arg329 in the 100-bp region; the yellow arrow indicates the location of variant Arg329.

Supplementary Figure 6 Comparable expression levels of FGD6-Lys329 and FGD6-Arg329 in HRECs infected by virus.

(a) Immunoblot analysis of human retina vein endothelial cells (HRECs) expressing FGD6-K329 and FGD6-R329 after virus infection. (b) Relative intensities of signal for the recombinant FGD6 proteins in the immunoblot analysis in a. GAPDH was used as a loading control. (c,d) Expression of GFP in HRECs cotransfected to express FGD6-K329 (c) and FGD6-R329 (d).

Supplementary Figure 7 Actin cytoskeleton network in HRECs after FGD6 knockdown with siRNA1 or siRNA 2.

Cells were stained with phalloidin (red) and DAPI (blue). Scale bars, 25 μm.

Supplementary Figure 8 Assessment of endothelial network formation in HRECs expressing FGD6-R257 using Matrigel matrix.

(a,b) Morphology of HRECs transfected with viruses expressing three FGD6proteins (a) andquantitative results for eight independent experiments (data expressed as means ± s.d.) (b). (c,d) Morphology of the endothelial network of HRECs transfected with FGD6 siRNAs followed by rescue with FGD6-R257, with or without VEGF treatment (c), and quantitative results (data expressed as means ± s.d.for the three siRNAs combined) (d). P values were calculated by comparing the rescue ability of FGD6-K329 (Fig. 3e) and FGD6-R257 for the three FGD6 siRNAs. Three independent experiments were performed for each siRNA. VEGF was added at a final concentration of 40 ng/ml. A two-tailed Student’s t test was used to compare the two groups.

Supplementary Figure 9 Wound-healing assays of HRECs.

(a,b) Cell migration assay with inhibition of woundhealing by knockdown of FGD6 using siRNAs. HRECs transfected with control or FGD6 siRNAs were subjected to the wound-healing assay. Representative images (a) and quantitative results (b) are shown. The quantitative results shown are the averages of four independent experiments. A Student’s t test was used to compare the two groups. All values are expressed as means±s.d. (c,d) Wound-healing assays for cells transfected with FGD6 siRNA in the presence of virus expressing FGD6-Lys329, FGD6-Arg329, or GFP without VEGF induction (c) or in the presence of viruses expressing FGD6-K329 or FGD6-R329 after VEGF induction (d). Student’s t test was used to compare the two groups.

Supplementary Figure 10 Endogenous expression of FGD6 in response to OxPAPC in HRECs.

Real-time PCR result for endogenous expression of FGD6 in response to OxPAPC. The results shown are presented as the means ± s.e. of three independent experiments.

Supplementary Figure 11 Mouse vitreous was injected with virus at P12 and dissected 4 weeks later.

Shown are whole-mountretinas stained with antibody to collagen IV to label blood vessels. (a,b) Central (a) and peripheral (b) vessels of the retina after injection with virus encoding FGD6-Lys329, FGD6-Arg329, or GFP. (c) An abnormal vasculogenesis phenotype was observed in the central area of the retina receiving virus encoding FGD6-R329 (pink arrow).

Supplementary Figure 12 Local sequence alignment for FGD6 and CFH and functional prediction for the FGD6 p.Lys329Arg variant and the CFH p.Tyr402His variant.

(a) The HomoloGene cluster of the region encompassing p.Lys329Arg in the FGD6 protein among organisms in the NCBI database. (b) Damage prediction for R329 using PolyPhen-2HumVar suggests that changing this amino acid has a benign effect and that this amino acid falls within the flexible region of the protein. (c) Expasy domain prediction for FGD6. (d) The HomoloGene cluster of the region encompassing p.Tyr402His in the CFH protein among mammalian organisms in the NCBI database. (e) Damage prediction for His402 using PolyPhen-2HumVar also implies that changing this amino acid has a benign effect and that this amino acid falls within the flexible region of the protein. (f) Expasy domain prediction for CFH.

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Supplementary Figures 1–12, Supplementary Tables 1–10 and Supplementary Note. (PDF 2682 kb)

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Huang, L., Zhang, H., Cheng, CY. et al. A missense variant in FGD6 confers increased risk of polypoidal choroidal vasculopathy. Nat Genet 48, 640–647 (2016). https://doi.org/10.1038/ng.3546

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