Abstract
Exfoliation syndrome (XFS) is the most common recognizable cause of open-angle glaucoma worldwide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) of 1,484 cases and 1,188 controls from Japan and followed up the most significant findings in a further 6,901 cases and 20,727 controls from 17 countries across 6 continents. We discovered a genome-wide significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (odds ratio (OR) = 1.16, P = 3.36 × 10−11). Although we also confirmed overwhelming association at the LOXL1 locus, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on the ancestry group (Japanese: ORA allele = 9.87, P = 2.13 × 10−217; non-Japanese: ORA allele = 0.49, P = 2.35 × 10−31). Our findings represent the first genetic locus outside of LOXL1 surpassing genome-wide significance for XFS and provide insight into the biology and pathogenesis of the disease.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
Change history
09 March 2015
In the version of this article initially published online, the affiliation of author Nicole Weisschuh was incorrect. Her correct affiliation is the Institute for Ophthalmic Research, Department of Ophthalmology, Tübingen, Germany. The error has been corrected for the print, PDF and HTML versions of this article.
27 April 2015
In the version of this article initially published, the name of author Afsaneh Naderi Beni was misspelled. The error has been corrected in the HTML and PDF versions of the article.
References
Schlötzer-Schrehardt, U. & Naumann, G.O. Ocular and systemic pseudoexfoliation syndrome. Am. J. Ophthalmol. 141, 921–937 (2006).
Ritch, R. & Schlotzer-Schrehardt, U. Exfoliation syndrome. Surv. Ophthalmol. 45, 265–315 (2001).
Thorleifsson, G. et al. Common sequence variants in the LOXL1 gene confer susceptibility to exfoliation glaucoma. Science 317, 1397–1400 (2007).
Chen, H. et al. Ethnicity-based subgroup meta-analysis of the association of LOXL1 polymorphisms with glaucoma. Mol. Vis. 16, 167–177 (2010).
Fingert, J.H. et al. LOXL1 mutations are associated with exfoliation syndrome in patients from the midwestern United States. Am. J. Ophthalmol. 144, 974–975 (2007).
Hayashi, H., Gotoh, N., Ueda, Y., Nakanishi, H. & Yoshimura, N. Lysyl oxidase–like 1 polymorphisms and exfoliation syndrome in the Japanese population. Am. J. Ophthalmol. 145, 582–585 (2008).
Fan, B.J. et al. DNA sequence variants in the LOXL1 gene are associated with pseudoexfoliation glaucoma in a U.S. clinic-based population with broad ethnic diversity. BMC Med. Genet. 9, 5 (2008).
Yang, X. et al. Genetic association of LOXL1 gene variants and exfoliation glaucoma in a Utah cohort. Cell Cycle 7, 521–524 (2008).
Hewitt, A.W. et al. Ancestral LOXL1 variants are associated with pseudoexfoliation in Caucasian Australians but with markedly lower penetrance than in Nordic people. Hum. Mol. Genet. 17, 710–716 (2008).
Pasutto, F. et al. Association of LOXL1 common sequence variants in German and Italian patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma. Invest. Ophthalmol. Vis. Sci. 49, 1459–1463 (2008).
Ozaki, M. et al. Association of LOXL1 gene polymorphisms with pseudoexfoliation in the Japanese. Invest. Ophthalmol. Vis. Sci. 49, 3976–3980 (2008).
Fan, B.J. et al. LOXL1 promoter haplotypes are associated with exfoliation syndrome in a U.S. Caucasian population. Invest. Ophthalmol. Vis. Sci. 52, 2372–2378 (2011).
Wolf, C. et al. Lysyl oxidase–like 1 gene polymorphisms in German patients with normal tension glaucoma, pigmentary glaucoma and exfoliation glaucoma. J. Glaucoma 19, 136–141 (2010).
Lemmelä, S. et al. Association of LOXL1 gene with Finnish exfoliation syndrome patients. J. Hum. Genet. 54, 289–297 (2009).
Aragon-Martin, J.A. et al. Evaluation of LOXL1 gene polymorphisms in exfoliation syndrome and exfoliation glaucoma. Mol. Vis. 14, 533–541 (2008).
Chen, L. et al. Evaluation of LOXL1 polymorphisms in exfoliation syndrome in a Chinese population. Mol. Vis. 15, 2349–2357 (2009).
Mossböck, G. et al. Lysyl oxidase–like protein 1 (LOXL1) gene polymorphisms and exfoliation glaucoma in a Central European population. Mol. Vis. 14, 857–861 (2008).
Challa, P. et al. Analysis of LOXL1 polymorphisms in a United States population with pseudoexfoliation glaucoma. Mol. Vis. 14, 146–149 (2008).
Ramprasad, V.L. et al. Association of non-synonymous single nucleotide polymorphisms in the LOXL1 gene with pseudoexfoliation syndrome in India. Mol. Vis. 14, 318–322 (2008).
Nakano, M. et al. Novel common variants and susceptible haplotype for exfoliation glaucoma specific to Asian population. Sci. Rep. 4, 5340 (2014).
Mori, K. et al. LOXL1 genetic polymorphisms are associated with exfoliation glaucoma in the Japanese population. Mol. Vis. 14, 1037–1040 (2008).
Williams, S.E. et al. Major LOXL1 risk allele is reversed in exfoliation glaucoma in a black South African population. Mol. Vis. 16, 705–712 (2010).
Krumbiegel, M. et al. Genome-wide association study with DNA pooling identifies variants at CNTNAP2 associated with pseudoexfoliation syndrome. Eur. J. Hum. Genet. 19, 186–193 (2011).
Rioux, J.D. et al. Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease. Nat. Genet. 29, 223–228 (2001).
Westra, H.J. et al. Systematic identification of trans eQTLs as putative drivers of known disease associations. Nat. Genet. 45, 1238–1243 (2013).
Boyle, A.P. et al. Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 22, 1790–1797 (2012).
Ward, L.D. & Kellis, M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 40, D930–D934 (2012).
Schlötzer-Schrehardt, U., Kortje, K.H. & Erb, C. Energy-filtering transmission electron microscopy (EFTEM) in the elemental analysis of pseudoexfoliative material. Curr. Eye Res. 22, 154–162 (2001).
Reinhardt, D.P., Ono, R.N. & Sakai, L.Y. Calcium stabilizes fibrillin-1 against proteolytic degradation. J. Biol. Chem. 272, 1231–1236 (1997).
Willer, C.J., Li, Y. & Abecasis, G.R. METAL: fast and efficient meta-analysis of genomewide association scans. Bioinformatics 26, 2190–2191 (2010).
Purcell, S., Cherny, S.S. & Sham, P.C. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 19, 149–150 (2003).
Tsou, W.L., Soong, B.W., Paulson, H.L. & Rodriguez-Lebron, E. Splice isoform–specific suppression of the CaV2.1 variant underlying spinocerebellar ataxia type 6. Neurobiol. Dis. 43, 533–542 (2011).
Lee, M.C. et al. Expression of the primary angle closure glaucoma (PACG) susceptibility gene PLEKHA7 in endothelial and epithelial cell junctions in the eye. Invest. Ophthalmol. Vis. Sci. 55, 3833–3841 (2014).
Smith, R.S. et al. Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum. Mol. Genet. 9, 1021–1032 (2000).
Acknowledgements
The authors thank the staff and participants of all studies for their important contributions. We thank K.-K. Heng, X.-Y. Chen, H.-M. Soo, S.-Q. Mok, A. Jamuth, N. Foxworth and M. Elbl for technical assistance. This research was funded by the Biomedical Research Council, Agency for Science, Technology and Research, Singapore. J.L.W. acknowledges support from US National Institutes of Health/National Eye Institute grants (NIH/NEI R01 EY020928 and NIH/NEI P30 EY014104). S.W.M.J. acknowledges support from grant EY11721 from the US National Institutes of Health/National Eye Institute and is an investigator of the Howard Hughes Medical Institute. L.R.P. acknowledges support from a Harvard Medical School Distinguished Ophthalmology Scholar Award and the Harvard Glaucoma Center of Excellence. J.H.F. acknowledges support from US National Institutes of Health/National Eye Institute grants (EY023512 and EY018825). Z.Y. acknowledges support from the National Natural Science Foundation of China (81025006 and 81170883), as well as from the Department of Science and Technology of Sichuan Province, China (2012SZ0219 and 2011jtd0020). M.S. acknowledges support from Robert Bosch Stiftung (Stuttgart, Germany) and the German Cancer Consortium (DKTK), Germany. The Australian case cohort was funded by grants from the Ophthalmic Research Institute of Australia and National Health and Medical Research Council (NHMRC) project 535044. The Thessaloniki Eye Study was cofunded by the European Union (European Social Fund) and Greek national funds under act ‘Aristia’ of the operational program ‘Education and Lifelong Learning’ (Supplementary Note). Blue Mountains Eye Study (BMES) GWAS and genotyping costs were supported by the Australian NHMRC (Canberra, Australia; NHMRC project grants 512423, 475604 and 529912) and the Wellcome Trust, UK, as part of the Wellcome Trust Case Control Consortium 2 (A. Viswanathan, P. McGuffin, P. Mitchell, F. Topouzis, P. Foster; grants 085475/B/08/Z and 085475/08/Z). K.P.B. is an NHMRC Senior Research Fellow, and J.E.C. is an NHMRC Practitioner Fellow. M.A.B. is an NHMRC Principal Research Fellow. A.W.H. is an NHMRC Peter Doherty Fellow.
Author information
Authors and Affiliations
Consortia
Contributions
T.A., M.O. and C.-C.K. conceived the project. M.O., T.M., R.R.A., A.H., S.N., J.E.C., A.W.H., D.A.M., P.M., J.J.W., Y.S.A., J.C.Z., Y.N., T.Z., M.P., L.J., Y.X.W., S.W., D.P., P.G.S., Y.I., R.S.K., M.U., S. Manabe, K.H., S. Kazama, R.I., Y.M., K. Miyata, K.S., T.H., E.C., K.I., S.I., A.Y., M.Y., Y.K., M.A., T.O., T. Sakurai, T. Sugimoto, H.C., K.Y., S.Y.A., E.A.O., S.A.A.-O., O.O., L.A.-J., S.A.S., Y.Y., Ç.O., M.R.K., A.N.B., S.Y., E.L.A., E.K.-J., U.L., P.C., R.M.R., A.Z., T.C., R. Ramakrishnan, K.N., R.V., P.Z., X.C., D.G.-V., S.A.P., R.H., S.-L.H., U.-C.W.-L., C.M., U.S.-S., S. Moebus, N. Weisschuh, R.S., A.G., I.L., J.G.C., M.C., Q.Y., V.V., P. Founti, A.C., A.L., E.A., A.L.C., M.R.W., D.J.R., I.M.-B., K. Mori, S. Heegaard, W.L.M.A., J.B.J., L.X., J.M.L., F.L., N. Wang, P. Frezzotti, S. Kinoshita, J.H.F., M.I., D.P.E., L.R.P., T.K., J.L.W., F.T., N.Y. and R. Ritch conducted patient recruitment and phenotyping. Z.L., S.U., M.K., K.P.B., M.A.B., J.J.W., Y.G., K.-Y.T., L.H., P.S., W.Y.M., S.Q.P., B.Z., J.S., N.Z., Z.Y. and S.V. performed genotyping experiments. J.M.H., A.S.Y.C., M.C.L., E.N.V., G.R.H. and S.W.M.J. led and performed immunohistochemistry and immunofluorescence experiments. Z.L., K.P.B., R.A.F., P.L., K.K.A.-A., L.A.S., L.H., K.S.S., J.N.F., M.N., F.M., N.G., M.M., S.U., M.K., Y.Y.T., J.H.K., A.E.A.K., S. Herms, Y.L., K.T., B.Z., J.S., N.Z., S.V., Z.Y., G.R.H., P.S., A.C.O., F.P. and A.G. performed analysis. E.N.V., T.Y.W., C.Y.C., P.S., A.M.H., M.M.N., B.C., E.S., M.S. and A.R. contributed genetic and genotyping data from control populations. The manuscript was drafted by C.-C.K., with critical input from T.A., R.R.A., L.R.P., J.L.W., F.P., F.T., M.D., S.W.M.J., R. Ritch and M.A.H. The manuscript was approved by all authors. C.-C.K. was responsible for obtaining financial support for this study.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
A full list of members and affiliations appears in the Supplementary Note.
A full list of members and affiliations appears in the Supplementary Note.
Integrated supplementary information
Supplementary Figure 2 Manhattan plots for the genome-wide association study of exfoliation syndrome.
(a) Discovery-phase (1,484 cases and 1,188 controls) data only. (b) Meta-analysis of the discovery and validation (stage 1) phases comprising up to 4,112 cases and 10,135 controls. CACNA1A rs4926244 was brought forward for replication (stage 2).
Supplementary Figure 3 Regional association analysis of the CACNA1A locus.
The genomic locations of the genes flanking CACNA1A are also shown. The locations for rs4926244, rs121908212 (T666M) and rs121908217 (R583Q) are shown in the context of CACNA1A. P values on the y axis are truncated at P < 1 × 10–10.
Supplementary Figure 4 Regional association plot of the CACNA1A locus for SNP markers imputed using cosmopolitan population haplotypes based on data from 2,535 individuals from 26 distinct populations around the world obtained from the 1000 Genomes Project Phase 3 release (June 2014) for reference panel construction.
No imputed genetic marker showed evidence of association surpassing the directly genotyped rs4926244. A total of 17 imputed SNPs at the CACNA1A locus show P < 0.001 for association with exfoliation syndrome. The individual SNP IDs and their pairwise correlation with rs4926244 are shown in Supplementary Table 5. The dashed vertical lines mark regions of recombination flanking rs4926244 and the 17 SNPs correlated with it.
Supplementary Figure 5 Forest plot for the association between LOXL1 rs4886776 and exfoliation syndrome in the GWAS discovery and stage 1 validation sample collections.
The black lines denote the 95% confidence intervals of the odds ratios for each collection. The blue diamonds denote the 95% confidence intervals for summary observations for Japanese samples, non-Japanese samples and the overall meta-analysis.
Supplementary Figure 6 Analysis of CACNA1A expression in human ocular tissues.
(a) The CACNA1A-specific 250-bp RT-PCR product was seen in anterior sclera (AS), cornea (C), lens capsule (LC), iris (I), trabecular meshwork (TM), retina and retinal pigment epithelium (R), choroid (CH) and the optic nerve (ON). An RT-PCR product was not observed for the optic nerve head (ONH). The amplification product shown was from PCR that used CACNA1A primers F4 (5′-CAGAGCAAGGCCAAGAAGC-3′) and R4 (5′-CTTGTTCCGGACTCCATGTG-3′). The ubiquitously expressed gene ACTB was used as the normalizing control. A no-template sample acted as the negative control (NC) to ensure non-contamination of the RT-PCR reaction mix. M denotes the molecular-weight marker. (b) Whole-cell lysates from ARPE19, NPCE, HTM, HelaS, MCF7 and HEK293 cells were analyzed for CACNA1A expression. Two CACNA1A protein bands, at ~275 kDa and ~250 kDa, were observed (arrows), which likely correspond to the 2,506- and 2,261-residue CaV2.1_V2 and CaV2.1_V1 isoforms, respectively (NCBI reference sequences NP_001120694.1 and NP_001120693.1). Human ocular tissue–derived cell lines (ARPE19, NPCE and HTM) displayed higher CACNA1A expression levels than non-ocular tissue–derived cell lines (HelaS, MCF7 and HEK293). Among the ocular cell lines, ARPE19 and HTM expressed higher levels of the larger 275-kDa protein, whereas the NPCE cell line expressed higher levels of the smaller 250-kDa protein.
Supplementary Figure 7 Immunofluorescence analysis of CACNA1A distribution within ocular tissues.
(a) CACNA1A staining was detected in the corneal epithelium (Epi), cornea endothelium (Endo) and corneal stroma. (b) Ocular CACNA1A protein was found to be differentially expressed in tissues involved in the aqueous humor outflow pathways, with the highest level of immunoreactivity in the ciliary muscle (CM), followed by the iris dilator muscle (IDM), ciliary processes (CP), trabecular meshwork (TM) and Schlemm's canal (SC). No CACNA1A was detected in the sclera. (c) CACNA1A was expressed most abundantly in the iris sphincter muscle (ISM), followed by iris pigmented epithelium (IPE) and iris stroma. (d) CACNA1A expression was also observed in the lens epithelium (Lens Epi) but not in the lens fibers. (e) Strong immunofluorescence labeling of CACNA1A in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) was observed. Ubiquitous labeling of other retinal layers such as the nerve fibers layer (NFL), rods and cones layer (RCL) and retinal pigment epithelium (RPE) was also detected.
Supplementary Figure 8 Immunohistochemistry analysis of CACNA1A in human ocular tissues.
(a–c) XFS human eyes. CACNA1A immunohistochemistry staining (1:100k; Abcam; red chromophobe) of three XFS globes. The ciliary body (a,b) and ciliary processes (a–c) show positive staining at low magnification (20×). Asterisks highlight the non-staining exfoliative material along the zonules and ciliary processes. Zonules show focal staining for CACNA1A (arrow). (d,e) XFS human eyes. CACNA1A immunohistochemistry showing positive staining of the non-pigmented ciliary epithelium (NPCE) and pigmented ciliary epithelium (PCE) and ciliary body smooth muscles (40×). Asterisks highlight the non-staining exfoliative material. (f,g) Normal human eyes. CACNA1A immunohistochemistry showing positive staining of the non-pigmented ciliary epithelium (NPCE) and pigmented ciliary epithelium (PCE) and ciliary body smooth muscles.
Supplementary Figure 9 Immunolocalization of CACNA1A in the lens, cornea, retina and optic nerve in both non-XFS and XFS eyes.
(a,b) Microscopy observations (CACNA1A IHC panel, 40×) in both non-XFS and XFS eyes show positive immunoreactivity in the anterior lens epithelium, corneal epithelial cells, stromal keratocytes and corneal endothelial cells. The optic nerve glia and vascular endothelial cells also show positive CACNA1A staining in both non-XFS and XFS eyes. In non-XFS retina, strong diffuse immunoreactivity is seen in the photoreceptor inner segments (IS), inner nuclear layer (INL) and outer nuclear layer (ONL) and in the nerve fiber layer (NFL) of non-XFS globes in comparison to XFS globes, where focal and patchy immunostaining of the IS, ONL, INL and NFL is observed. (c) CACNA1A-positive immunoreactivity is seen in the anterior iris border, iris stromal cells, the iris dilator and sphincter pupillae as well as the pigmented iris epithelium in both XFS and non-XFS irides (Iris panel, CACNA1A IHC panels). Exfoliated material does not show positive staining with CACNA1A (asterisk, CACNA1A IHC XFS2 panel) in contrast to its LOXL1-positive immunoreactivity (asterisk, LOXL1 IHC XFS1 panel).
Supplementary Figure 10 CACNA1A expression in adult mouse anterior ocular tissues.
Cells in the mouse lens, iris, ciliary body and cornea express CACNA1A as detected by immunofluorescent labeling. (a) High magnification of the lens showed epithelial cells (LE) and cortical fiber cells (CFC) that expressed CACNA1A (red). As judged by the absence of CACNA1A immunoreactivity, other areas of the lens did not exhibit CACNA1A expression (data not shown). (b) Expression of CACNA1A (red) is observed in distinct regions of the iris. B6(Cg)-Tyrc-2J/J mice were used to allow assessment in the absence of pigmentation. Labeling by CACNA1A and smooth muscle actin (ACTA2; green) show strong overlap (yellow) in the dilator muscle (arrowhead), which separates the anterior iris stroma (S) and the iris pigmented epithelium (PE). Low CACNA1A levels were detected in the sphincter muscle (asterisk). Subsets of cells in the S and PE also express CACNA1A (arrows). (c) The mouse iris is continuous with the ciliary body (arrowhead). The vascular stroma of the ciliary body (CB) is labeled by endomucin (EMCN; green) and does not co-label with CACNA1A (red). A subset of cells in the ciliary epithelium also expresses CACNA1A (arrow). The iridocorneal angle in this image is compressed owing to sectioning artifact (asterisk). CACNA1A expression was considered weak or absent in the trabecular meshwork. (d) CACNA1A (red) is expressed in the corneal epithelium (EP) but not in the corneal endothelium (EN). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.
Supplementary Figure 11 Ancestry analysis of the GWAS discovery data set using principal components.
(a) Plot showing the ancestry of the XFS cases and controls from Japan relative to the international HapMap panels. (b) Plot showing direct genetic ancestral matching between the Japanese XFS cases and controls.
Supplementary Figure 12 Quantile-quantile plot of the GWAS discovery stage P values.
No genomic inflation was observed.
Supplementary Figure 13 Quantile-quantile plot of the discovery-stage P values contrasting patients with exfoliation syndrome without glaucoma (n = 788) against patients presenting with exfoliation syndrome accompanied by glaucoma (n = 594).
A total of 102 exfoliation syndrome cases did not have the glaucoma subdiagnosis.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–13, Supplementary Tables 1–10 and Supplementary Note. (PDF 2493 kb)
Rights and permissions
About this article
Cite this article
Aung, T., Ozaki, M., Mizoguchi, T. et al. A common variant mapping to CACNA1A is associated with susceptibility to exfoliation syndrome. Nat Genet 47, 387–392 (2015). https://doi.org/10.1038/ng.3226
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng.3226
This article is cited by
-
Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome
BMC Genomics (2023)
-
A Comparison of Genomic Advances in Exfoliation Syndrome and Primary Open-Angle Glaucoma
Current Ophthalmology Reports (2021)
-
Is pseudoexfoliation glaucoma a neurodegenerative disorder?
Journal of Biosciences (2021)
-
Association of IL-10 gene promoter polymorphisms with susceptibility to pseudoexfoliation syndrome, pseudoexfoliative and primary open-angle glaucoma
BMC Medical Genetics (2020)
-
De novo variants in an extracellular matrix protein coding gene, fibulin-5 (FBLN5) are associated with pseudoexfoliation
European Journal of Human Genetics (2019)
