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Meta-Analysis
. 2017 Jul;49(7):993-1004.
doi: 10.1038/ng.3875. Epub 2017 May 29.

Genetic Association Study of Exfoliation Syndrome Identifies a Protective Rare Variant at LOXL1 and Five New Susceptibility Loci

Tin Aung  1   2   3 Mineo Ozaki  4   5 Mei Chin Lee  1   6 Ursula Schlötzer-Schrehardt  7 Gudmar Thorleifsson  8 Takanori Mizoguchi  9 Robert P Igo Jr  10 Aravind Haripriya  11 Susan E Williams  12 Yury S Astakhov  13 Andrew C Orr  14   15 Kathryn P Burdon  16   17 Satoko Nakano  18 Kazuhiko Mori  19 Khaled Abu-Amero  20   21 Michael Hauser  1   22   23 Zheng Li  24 Gopalakrishnan Prakadeeswari  25 Jessica N Cooke Bailey  10 Alina Popa Cherecheanu  26   27 Jae H Kang  28 Sarah Nelson  29 Ken Hayashi  30 Shin-Ichi Manabe  30 Shigeyasu Kazama  31 Tomasz Zarnowski  32 Kenji Inoue  33 Murat Irkec  34 Miguel Coca-Prados  35   36   37 Kazuhisa Sugiyama  38 Irma Järvelä  39 Patricio Schlottmann  40 S Fabian Lerner  41 Hasnaa Lamari  42 Yildirim Nilgün  43 Mukharram Bikbov  44 Ki Ho Park  45 Soon Cheol Cha  46 Kenji Yamashiro  47   48 Juan C Zenteno  49   50 Jost B Jonas  51   52 Rajesh S Kumar  53 Shamira A Perera  1   2 Anita S Y Chan  1   2   6 Nino Kobakhidze  54 Ronnie George  55 Lingam Vijaya  55 Tan Do  56 Deepak P Edward  57   58 Lourdes de Juan Marcos  59   60 Mohammad Pakravan  61 Sasan Moghimi  62 Ryuichi Ideta  63 Daniella Bach-Holm  64 Per Kappelgaard  64 Barbara Wirostko  65 Samuel Thomas  65 Daniel Gaston  15 Karen Bedard  15 Wenda L Greer  15 Zhenglin Yang  66   67 Xueyi Chen  68 Lulin Huang  69   70 Jinghong Sang  52 Hongyan Jia  52 Liyun Jia  52   71 Chunyan Qiao  52 Hui Zhang  52 Xuyang Liu  72 Bowen Zhao  52   71 Ya-Xing Wang  71 Liang Xu  52 Stéphanie Leruez  73 Pascal Reynier  74 George Chichua  54 Sergo Tabagari  75 Steffen Uebe  76 Matthias Zenkel  7 Daniel Berner  7 Georg Mossböck  77 Nicole Weisschuh  78 Ursula Hoja  7 Ulrich-Christoph Welge-Luessen  7 Christian Mardin  7 Panayiota Founti  79 Anthi Chatzikyriakidou  80 Theofanis Pappas  79 Eleftherios Anastasopoulos  79 Alexandros Lambropoulos  80 Arkasubhra Ghosh  81 Rohit Shetty  53 Natalia Porporato  82 Vijayan Saravanan  25 Rengaraj Venkatesh  83 Chandrashekaran Shivkumar  84 Narendran Kalpana  85 Sripriya Sarangapani  86 Mozhgan R Kanavi  87 Afsaneh Naderi Beni  61 Shahin Yazdani  61 Alireza Lashay  62 Homa Naderifar  62 Nassim Khatibi  62 Antonio Fea  88 Carlo Lavia  88 Laura Dallorto  88 Teresa Rolle  88 Paolo Frezzotti  89 Daniela Paoli  90 Erika Salvi  91 Paolo Manunta  92 Yosai Mori  93 Kazunori Miyata  93 Tomomi Higashide  38 Etsuo Chihara  94 Satoshi Ishiko  95 Akitoshi Yoshida  96 Masahide Yanagi  97 Yoshiaki Kiuchi  97 Tsutomu Ohashi  98 Toshiya Sakurai  99 Takako Sugimoto  5 Hideki Chuman  5 Makoto Aihara  100 Masaru Inatani  101 Masahiro Miyake  47 Norimoto Gotoh  102 Fumihiko Matsuda  102 Nagahisa Yoshimura  47   103 Yoko Ikeda  19 Morio Ueno  19 Chie Sotozono  19 Jin Wook Jeoung  45 Min Sagong  46 Kyu Hyung Park  104 Jeeyun Ahn  105 Marisa Cruz-Aguilar  49 Sidi M Ezzouhairi  42 Abderrahman Rafei  106 Yaan Fun Chong  1 Xiao Yu Ng  1 Shuang Ru Goh  1 Yueming Chen  1 Victor H K Yong  1 Muhammad Imran Khan  107 Olusola O Olawoye  108   109 Adeyinka O Ashaye  108   109 Idakwo Ugbede  110 Adeola Onakoya  111   112 Nkiru Kizor-Akaraiwe  113   114 Chaiwat Teekhasaenee  115 Yanin Suwan  115 Wasu Supakontanasan  115 Suhanya Okeke  113   114 Nkechi J Uche  114   116   117 Ifeoma Asimadu  113 Humaira Ayub  118 Farah Akhtar  119 Ewa Kosior-Jarecka  32 Urszula Lukasik  32 Ignacio Lischinsky  120 Vania Castro  121 Rodolfo Perez Grossmann  122 Gordana Sunaric Megevand  123 Sylvain Roy  123 Edward Dervan  124 Eoin Silke  124 Aparna Rao  125 Priti Sahay  125 Pablo Fornero  126 Osvaldo Cuello  126 Delia Sivori  41 Tamara Zompa  127 Richard A Mills  16 Emmanuelle Souzeau  16 Paul Mitchell  128 Jie Jin Wang  128 Alex W Hewitt  17   129 Michael Coote  129 Jonathan G Crowston  129 Sergei Y Astakhov  13 Eugeny L Akopov  13 Anton Emelyanov  13   130 Vera Vysochinskaya  130 Gyulli Kazakbaeva  44 Rinat Fayzrakhmanov  44 Saleh A Al-Obeidan  20 Ohoud Owaidhah  57 Leyla Ali Aljasim  57 Balram Chowbay  131   132   133 Jia Nee Foo  24   134 Raphael Q Soh  24 Kar Seng Sim  24 Zhicheng Xie  24 Augustine W O Cheong  24 Shi Qi Mok  24 Hui Meng Soo  24 Xiao Yin Chen  24 Su Qin Peh  24 Khai Koon Heng  24 Rahat Husain  2 Su-Ling Ho  135 Axel M Hillmer  24 Ching-Yu Cheng  1   2   3   6 Francisco A Escudero-Domínguez  59 Rogelio González-Sarmiento  60   136 Frederico Martinon-Torres  137   138 Antonio Salas  139   140 Kessara Pathanapitoon  141 Linda Hansapinyo  141 Boonsong Wanichwecharugruang  142 Naris Kitnarong  143 Anavaj Sakuntabhai  144   145 Hip X Nguyn  56 Giang T T Nguyn  56 Trình V Nguyn  56 Werner Zenz  146 Alexander Binder  146 Daniela S Klobassa  146 Martin L Hibberd  24   147 Sonia Davila  24 Stefan Herms  148   149   150 Markus M Nöthen  148   151 Susanne Moebus  152 Robyn M Rautenbach  153 Ari Ziskind  153 Trevor R Carmichael  12 Michele Ramsay  154 Lydia Álvarez  35   36 Montserrat García  35   36 Héctor González-Iglesias  35   36 Pedro P Rodríguez-Calvo  35   36 Luis Fernández-Vega Cueto #  35   36 Çilingir Oguz  155 Nevbahar Tamcelik  156 Eray Atalay  1   156 Bilge Batu  156 Dilek Aktas  157 Burcu Kasım  34 M Roy Wilson  158 Anne L Coleman  159 Yutao Liu  160 Pratap Challa  22 Leon Herndon  22 Rachel W Kuchtey  161 John Kuchtey  161 Karen Curtin  65 Craig J Chaya  65 Alan Crandall  65 Linda M Zangwill  162 Tien Yin Wong  1   2   3 Masakazu Nakano  163 Shigeru Kinoshita  19   164 Anneke I den Hollander  107   165 Eija Vesti  166 John H Fingert  167   168 Richard K Lee  169 Arthur J Sit  170 Bradford J Shingleton  171 Ningli Wang  52   71 Daniele Cusi  172 Raheel Qamar  173   174 Peter Kraft  175 Margaret A Pericak-Vance  176 Soumya Raychaudhuri  177   178   179   180   181 Steffen Heegaard  182   183 Tero Kivelä  184 André Reis  75 Friedrich E Kruse  7 Robert N Weinreb  162 Louis R Pasquale  28   185 Jonathan L Haines  10   186 Unnur Thorsteinsdottir  8   187 Fridbert Jonasson  187   188 R Rand Allingham  1   22 Dan Milea  1   2   6 Robert Ritch  189 Toshiaki Kubota  18 Kei Tashiro  163 Eranga N Vithana  1   3 Shazia Micheal  165 Fotis Topouzis  79 Jamie E Craig  16 Michael Dubina  13   130 Periasamy Sundaresan  190 Kari Stefansson  8   187 Janey L Wiggs  185 Francesca Pasutto  76 Chiea Chuen Khor  1   24   191
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Free PMC article
Meta-Analysis

Genetic Association Study of Exfoliation Syndrome Identifies a Protective Rare Variant at LOXL1 and Five New Susceptibility Loci

Tin Aung et al. Nat Genet. .
Free PMC article

Abstract

Exfoliation syndrome (XFS) is the most common known risk factor for secondary glaucoma and a major cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A, have previously been associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results across populations, and to identify new variants associated with XFS. We identified a rare protective allele at LOXL1 (p.Phe407, odds ratio (OR) = 25, P = 2.9 × 10-14) through deep resequencing of XFS cases and controls from nine countries. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10-8). We identified association signals at 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

Figures

Figure 1
Figure 1
LOXL1 p.Y407F regulates ECM synthesis and improves cellular adhesion. (a) Schematic diagram of LOXL1 indicating the protein domain positions for the variants evaluated in this study. b) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and elastin (green). Cell nuclei are stained in blue. The heat map for elastin indicates the intensity of elastin staining from red (increased expression) to purple (decreased expression). c) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and collagen IV (green). Cell nuclei are stained in blue.. The heat map for collagen IV indicates the intensity of collagen IV staining from red (increased expression) to purple (decreased expression). d) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and fibrillin 1 (green). Cell nuclei are stained in blue. The heat map for fibrillin 1 indicates the intensity of fibrillin 1 staining from red (increased expression) to purple (decreased expression). e) Cumulative average of impedance values (as a surrogate for cellular adhesion strength) measured over 35h post nucleofection of HLECs overexpressing the four tested LOXL1 haplotypes. Data represent mean ± s.e.m. of four independent experiments. ** represents P<0.01 when compared against the rare, protective LOXL1 p.407F-carrying G-A-T haplotype. The four haplotypes tested were LOXL1-(G-A-T), - (G-A-A), -(T-G-A) and -(G-G-A). This experiment was further validated in Supplementary Figure 7.
Figure 2
Figure 2
Manhattan plot of the results from the GWAS discovery plus replication meta-analysis comprising 13,620 XFS cases and 109,837 controls. Genetic markers are plotted according to chromosomal location on the horizontal axis and statistical significance on the vertical axis. SNP markers at seven independent loci surpass genome-wide significance (defined as P<5×10−8). They are LOXL1, CACNA1A, POMP, TMEM136, AGPAT1, SEMA6A and RBMS3.
Figure 3
Figure 3
Expression of POMP protein in ocular tissues of normal human donor eyes and donor eyes with XFS, as determined by Western blotting and immunohistochemistry. Immunofluorescence labelling of normal eye tissues shows punctate POMP immune-positivity (green fluorescence) in the cytoplasm of the corneal epithelium (A), the corneal endothelium (B), limbal epithelium and stromal cells (C), trabecular meshwork endothelium (D), ciliary epithelium (E), and retinal cell layers (F). Reduced POMP protein expression levels in iris and ciliary body tissues of XFS eyes compared to age matched controls are shown by Western blot analysis (G), and by immunofluorescence labelling of iridal (H,L) and ciliary epithelia (J,M) as well as vascular endothelia in the iris (K,N). Reduced staining intensity in XFS tissues is associated with LOXLI-positive exfoliation material accumulations (red immunofluorescence) on the surface of the iris pigment epithelium (L), ciliary epithelium (M) and iris blood vessel walls (N). Western blot (cropped images) and densitometry analysis shows reduced POMP protein expression in iris and ciliary body tissue lysates of XFS eyes compared to control eyes (G). Data are shown as the POMP/B-actin ratio (n=6 for each group; mean ± standard deviation; *P<0.01; **P<0.005); uncropped versions of all Western blots are shown in Supplementary Figure 16. (BV blood vessel, CE ciliary epithelium, CoE corneal epithelium, DM Descemet membrane, GCL retinal ganglion cell layer, INL inner nuclear layer, IPE iris pigment epithelium, LE limbal epithelium, ONL outer nuclear layer, SC Schlemm’s canal, ST stroma, TM trabecular meshwork; DAPI nuclear counterstain in blue; scale bars = 100 μm in C,D,F and 20 μm in A,B,E,H-N).
Figure 4
Figure 4
Expression of TMEM136 protein in ocular tissues of normal human donor eyes and donor eyes with XFS, as determined by Western blotting and immunohistochemistry. Immunofluorescence labelling of normal eye tissues shows cytoplasmic TMEM136 immunopositivity (green fluorescence) in limbal blood vessels (A), trabecular meshwork and Schlemm’s canal endothelium (B), walls of aqueous veins (arrows) (C), blood vessels of the iris (arrows) (D), blood vessels and epithelia of the ciliary body (E), and retinal blood vessels and cell layers (F). Reduced TMEM136 protein expression levels in iris and ciliary body tissues of XFS eyes compared to age matched controls are shown by Western blot analysis (G), and by immunofluorescence labelling of iridal (H,L) and ciliary epithelia (J,M) as well as vascular endothelia in the iris (K,N). Reduced staining intensity in XFS tissues is associated with LOXL1- positive exfoliation material accumulations (red immunofluorescence) on the surface of the iris pigment epithelium (L), ciliary epithelium (M) and iris blood vessel walls (N). Western blot (cropped images) and densitometry analysis shows reduced TMEM136 protein (isoform 1 at 28 KD and isoform 3 at 31 KD) expression in iris and ciliary body tissue lysates of XFS eyes compared to control eyes (G). Data are shown as the TMEM136/B-actin ratio (mean ± standard deviation; n=6 for each group; *P<0.01; **P<0.005); uncropped versions of all Western blots are shown in Supplementary Figure 17. (AV aqueous vein, BV blood vessel, CE ciliary epithelium, DIL dilator muscle, GCL retinal ganglion cell layer, INL inner nuclear layer, IPE iris pigment epithelium, LE limbal epithelium, ONL outer nuclear layer, SC Schlemm’s canal, ST stroma, TM trabecular meshwork; DAPI nuclear counterstain in blue; scale bars = 200 μm in A, 100 μm in B-F and 20 μm in H-N).

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