Abstract
Purpose:
To identify microRNAs (miRNAs) involved in primary open-angle glaucoma (POAG), using genetic data. MiRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression. Genetic variants in miRNAs or miRNA-binding sites within gene 3′-untranslated regions (3′UTRs) are expected to affect miRNA function and contribute to disease risk.
Methods:
Data from the recent genome-wide association studies on intraocular pressure, vertical cup-to-disc ratio (VCDR), cupa area and disc area were used to investigate the association of miRNAs with POAG endophenotypes. Putative targets of the associated miRNAs were studied according to their association with POAG and tested in cell line by transfection experiments for regulation by the miRNAs.
Results:
Of 411 miRNA variants, rs12803915:A/G in the terminal loop of pre–miR-612 and rs2273626:A/C in the seed sequence of miR-4707 were significantly associated with VCDR and cup area (P values < 1.2 × 10−4). The first variant is demonstrated to increase the miR-612 expression. We showed that the second variant does not affect the miR-4707 biogenesis, but reduces the binding of miR-4707-3p to CARD10, a gene known to be involved in glaucoma. Moreover, of 72,052 miRNA-binding-site variants, 47 were significantly associated with four POAG endophenotypes (P value < 6.9 × 10−6). Of these, we highlighted 10 variants that are more likely to affect miRNA-mediated gene regulation in POAG. These include rs3217992 and rs1063192, which have been shown experimentally to affect miR-138-3p– and miR-323b-5p–mediated regulation of CDKN2B.
Conclusions:
We identified a number of miRNAs that are associated with POAG endophenotypes. The identified miRNAs and their target genes are candidates for future studies on miRNA-related therapies for POAG.
Primary open-angle glaucoma (POAG), the most common optic neuropathy, is the leading cause of irreversible blindness, affecting approximately 60 million individuals worldwide.
1,2 The disease is characterized by progressive loss of retinal ganglion cells and optic nerve degeneration that can be secondary to elevated intraocular pressure (IOP).
3 The optic nerve damage is characterized by an increase in cup size, which is the central area of the optic disc. Cup enlargement can be measured by the vertical cup-to-disc ratio (VCDR), comparing the vertical diameter of the cup with vertical diameter of the total optic disc.
4 The VCDR ranges from 0 to 1; a ratio above 0.7 or an asymmetry between eyes above 0.2 is considered as suspect for glaucoma in the clinical setting.
5 POAG is presumed to be a complex progressive neurodegenerative disorder caused by multiple genetic as well as environmental factors.
2 Previous genome-wide association studies (GWASs) have revealed a number of susceptibility loci for POAG by studying the disease directly or its endophenotypes including IOP and optic disc parameters (VCDR, cup area, and disc area).
5,6 Most of the associated variants identified by GWASs are located in noncoding regions of the genome and their mechanistic contributions to POAG and its endophenotypes remain poorly understood.
5,6
MicroRNAs (miRNAs) are small noncoding RNAs, consisting of 19 to 22 nucleotides, that posttranscriptionally regulate gene expression.
7 There are strong indications that miRNAs play important roles in the pathogenesis of POAG.
8–11 For example, miR-29b and miR-24 are involved in gene regulation in trabecular meshwork cells.
12 Moreover, the miRNA expression levels have been linked to maintaining the balance of the aqueous humor, the change in the trabecular meshwork, and the apoptosis of the retinal ganglion cells.
13–15 A number of miRNAs (e.g., miR-200c, miR-204, miR-183, and miR-182) are also reported as potential diagnostic biomarkers or therapeutic targets for glaucoma.
9,16 The biogenesis of miRNAs is a multistep coordinated process.
17,18 In the nucleus, miRNA genes are initially transcribed as long primary transcripts. Further processing and cleavage by the RNase
Drosha and
Dicer enzymes generate mature miRNAs.
17,18 The mature miRNAs are subsequently incorporated into the RNA-induced silencing complex (RISC) to interact with the 3′-untranslated region (3′UTR) of target mRNAs, resulting in mRNA degradation or translational repression.
7,18 Genetic variants in miRNA-encoding sequences can have profound effects on miRNA biogenesis and function.
19,20 In addition, polymorphisms located in miRNA-binding sites within the 3′UTR of target genes are expected to affect miRNA-mediated gene regulation.
20,21 Previous studies
19,21–23 have shown that such miRNA-related variants contribute to complex disease risk; a candidate variant association study
24 has also recently reported the association between genetic variation in miR-182 and POAG. In this study, we applied an in silico study on the existing GWAS of IOP and optic disc parameters
5 and performed in vitro experiments to identify miRNAs and target genes that may play a role in POAG.
Interaction Analysis Between an miRNA and Its Target Genes Using the Rotterdam Study Data
Functional Annotation of miRNA-Binding-Site Variants Associated With Glaucoma Endophenotypes
The authors thank the Rotterdam Study participants, the staff involved with the Rotterdam Study, and the participating general practitioners and pharmacists. They also thank the IGGC consortium for making the GWAS summary statistics data publicly available.
Supported by Glaucoomfonds, Oogfonds, Landelijke Stichting voor Blinden en Slechtzienden and Novartis Foundation (Uitzicht grant 2015-37). A.I. Iglesias was supported by a grant from National Institutes of Health (NIH), National Eye Institute (NEI), (1 R01 EY024233-03). The Rotterdam Study is supported by Erasmus MC (Erasmus Medical Center Rotterdam), the Erasmus University Rotterdam, the Netherlands Organization for Scientific Research (NWO), the Netherlands Organization for Health Research and Development (ZonMW), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, and the Ministry of Health, Welfare and Sports.
Disclosure: M. Ghanbari, None; A.I. Iglesias, None; H. Springelkamp, None; C.M. van Duijn, None; M.A. Ikram, None; A. Dehghan, None; S.J. Erkeland, None; C.C.W. Klaver, None; M.A. Meester-Smoor, None
Tin Aung,
1–3 Kathryn P. Burdon,
4 Ching-Yu Cheng,
1–3 Jessica N. Cooke Bailey,
5 Jamie E. Craig,
6 Angela J. Cree,
7 Paul J. Foster,
8 Christopher J. Hammond,
9 Alex W. Hewitt,
10,11 René Höhn,
12,13 Pirro G. Hysi,
9 Jost Jonas,
14 Anthony P. Khawaja,
8,15 Andrew J. Lotery,
7 Stuart MacGregor,
16 David A. Mackey,
17 Paul Mitchell,
18 Louis R. Pasquale,
19,20 Francesca Pasutto,
21 Norbert Pfeiffer,
22 Ananth C. Viswanathanm,
8 Veronique Vitart,
23 Eranga N. Vithana,
1 Jie Jin Wang,
18 Janey L. Wiggs,
19 Robert Wojciechowski,
24–26 Tien Yin Wong,
1–3 and Terri L. Young
27
1Singapore Eye Research Institute, Singapore National Eye Centre, Singapore.
2Ophthalmology & Visual Sciences Academic Clinical Program (Eye ACP), Duke-NUS Medical School, Singapore.
3Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
4Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.
5Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio, United States.
6Department of Ophthalmology, Flinders University, Adelaide, Australia.
7Clinical & Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.
8NIHR Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom.
9Department of Twin Research and Genetic Epidemiology, King's College London, United Kingdom.
10Centre for Eye Research Australia, University of Melbourne, Department of Ophthalmology, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
11School of Medicine, Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.
12Department of Ophthalmology, University Medical Center Mainz, Mainz, Germany.
13Department of Ophthalmology, Inselspital, University Hospital Bern, University of Bern, Switzerland.
14Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University of Heidelberg, Mannheim, Germany.
15Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom.
16Statistical Genetics, QIMR Berghofer Medical Research Institute, Brisbane, Australia.
17Lions Eye Institute, Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia, Australia.
18Centre for Vision Research, Department of Ophthalmology and Westmead Institute for Medical Research, University of Sydney, Sydney, New South Wales, Australia.
19Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
20Channing Division of Network Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States.
21Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
22Department of Ophthalmology, University Medical Center Mainz, Mainz, Germany.
23Institute of Genetics and Molecular Medicine, Medical Research Council Human Genetics Unit, University of Edinburgh, Edinburgh, United Kingdom.
24Computational and Statistical Genomics Branch, National Human Genome Research Institute (NIH), Baltimore, Maryland, United States.
25Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States.
26Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, Maryland, United States.
27Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States.