March 2012
Volume 53, Issue 3
Free
Genetics  |   March 2012
A Genetic Epidemiologic Study of Candidate Genes Involved in the Optic Nerve Head Morphology
Author Affiliations & Notes
  • Andrea C. Gasten
    From the Departments of Epidemiology, and
  • Wishal D. Ramdas
    From the Departments of Epidemiology, and
    Ophthalmology,
  • Linda Broer
    From the Departments of Epidemiology, and
  • Leonieke M. E. van Koolwijk
    From the Departments of Epidemiology, and
    Glaucoma Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands;
  • M. Kamran Ikram
    From the Departments of Epidemiology, and
    Neurology,
  • Paulus T. V. M. de Jong
    From the Departments of Epidemiology, and
    Department of Ophthalmogenetics, The Netherlands Institute for Neuroscience, RNAAS, Amsterdam, The Netherlands;
    Department of Ophthalmology, Academic Medical Center, Amsterdam, The Netherlands; and
  • Yurii S. Aulchenko
    From the Departments of Epidemiology, and
  • Roger C. Wolfs
    From the Departments of Epidemiology, and
    Ophthalmology,
  • Albert Hofman
    From the Departments of Epidemiology, and
  • Fernando Rivadeneira
    From the Departments of Epidemiology, and
    Internal Medicine, and
  • Andre G. Uitterlinden
    From the Departments of Epidemiology, and
    Internal Medicine, and
  • Ben A. Oostra
    Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands;
  • Hans G. Lemij
    Glaucoma Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands;
  • Caroline C. W. Klaver
    From the Departments of Epidemiology, and
    Ophthalmology,
  • Nomdo M. Jansonius
    From the Departments of Epidemiology, and
    Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
  • Johannes R. Vingerling
    From the Departments of Epidemiology, and
    Ophthalmology,
  • Cornelia M. van Duijn
    From the Departments of Epidemiology, and
  • Footnotes
    2  Contributed equally to this work and therefore should be considered equivalent authors.
  • Corresponding author: Cornelia M. van Duijn, Department of Epidemiology, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands; c.vanduijn@erasmusmc.nl
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1485-1491. doi:10.1167/iovs.11-7384
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      Andrea C. Gasten, Wishal D. Ramdas, Linda Broer, Leonieke M. E. van Koolwijk, M. Kamran Ikram, Paulus T. V. M. de Jong, Yurii S. Aulchenko, Roger C. Wolfs, Albert Hofman, Fernando Rivadeneira, Andre G. Uitterlinden, Ben A. Oostra, Hans G. Lemij, Caroline C. W. Klaver, Nomdo M. Jansonius, Johannes R. Vingerling, Cornelia M. van Duijn; A Genetic Epidemiologic Study of Candidate Genes Involved in the Optic Nerve Head Morphology. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1485-1491. doi: 10.1167/iovs.11-7384.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: The size of the optic nerve head, referred to as disc area (DA), and the vertical cup-disc ratio (VCDR), are clinically relevant parameters for glaucomatous optic neuropathy. Although these measures have a high heritability, little is known about the underlying genes. Previously, the genes SALL1 and SIX1 were found to be genome-wide significantly associated with DA and VCDR. The purpose of the present study was to investigate whether genes encoding protein known to interact with protein encoded by SALL1 and SIX1 are also associated with either DA or VCDR.

Methods.: A total of 38 candidate genes were chosen covering all known proteins interacting with SALL1 and SIX1. These were initially studied in the Rotterdam Study (RS)-I, including 5312 Caucasian subjects characterized for DA and VCDR. Positive findings were further investigated in two independent cohorts (RS-II and RS-III) and finally replicated in a fourth population (ERF). Bonferroni correction was applied to the meta-analyses.

Results.: Three loci were found to be associated with DA. The only locus significant after correcting for multiple testing is located on chromosome 11p13. Three single nucleotide polymorphisms (SNPs) in ELP4, a gene which neighbors and plays a crucial role in the expression of PAX6, show association in meta-analysis of the four cohorts yielding P values of respectively 4.79 × 10−6, 3.92 × 10−6, and 4.88 × 10−6 which is below the threshold dictated by the most conservative Bonferroni correction (P = 5.2 × 10−6).

Conclusions.: This study suggests that the ELP4-PAX6 region plays a role in the DA. Further research to confirm this finding is needed.

One of the major clinical markers associated with the development of glaucomatous optic neuropathy is the vertical cup-disc ratio (VCDR), which is a quantitative measure for the size of the optic cup relative to the optic disc size (disc area; DA). 1 Enlargement of the optic cup is a diagnostic sign of primary open-angle glaucoma (POAG), a leading cause of visual field loss and blindness worldwide. 2 VCDR has been shown to be the most useful clinical sign in the diagnosis of glaucoma; the cup most commonly enlarges vertically in POAG. 1 While the nature of the relationship of DA to POAG has been a subject of debate, VCDR is associated with DA. In that realm, DA has been studied as an endophenotype for POAG and other disorders. 3 Both DA and VCDR are known to be highly heritable (52%–59%), 4,5 and the genes involved in DA are just beginning to be discovered. Previously, we performed a genome-wide association study and found several genes associated with optic disc parameters, including sal-like 1 (SALL1; chromosome 16q12.1) with DA and sine oculis homeobox homolog 1 (SIX1; chromosome 14q22–23) with VCDR. 3 Recently, the latter gene has also been associated with open-angle glaucoma. 6 SALL1 encodes a zinc finger transcriptional repressor involved in organogenesis and may be linked to POAG through its interaction with SIX1. 7 SIX1 is a transcription factor known to activate SALL1 in kidney development. 7 Transcription from the SALL1 promoter has been shown to be strikingly activated by the SIX1 protein. 7 While the Drosophila Six homolog is known to interact within a network of genes including eyeless, eyes absent, and dachshund to induce compound eye organogenesis, Six1 is not expressed in the developing mouse eye. 8 However, the optix subclass of Six genes, comprising Six3 and Six6, may play a role in the vertebrate eye. 8 SIX1 and SALL1 operate within a regulatory complex. Investigations of genes that operate within this complex may reveal the connections between the protein pathways implicated in DA, VCDR, and glaucomatous pathology. 
In the present study we aimed to investigate whether the genes that encode proteins known to interact with the Sall1 and Six1 proteins are associated with either DA or VCDR to further elucidate the operational pathways. For this purpose we tested 38 candidate genes encoding proteins interacting with the proteins encoded by SIX1 and SALL1 and assessed their association with DA and/or VCDR in the Rotterdam Study-I. We replicated our findings in three independent cohorts. 
Methods
Study Populations
This study was conducted in four cohorts from the Netherlands, the Rotterdam Study (RS)-I, RS-II, RS-III, and the Erasmus Rucphen family study (ERF). RS-I is the discovery cohort and a prospective population-based cohort study of 7983 residents aged 55 years and older living in Ommoord, a district of Rotterdam, the Netherlands. 9 Baseline examination for the ophthalmic part took place between 1991 and 1993; follow-up examinations were performed from 1997 to 1999 and from 2002 to 2006. 
The RS-II prospective population-based cohort study comprised 3011 residents aged 55 years and older from the same district of Rotterdam while similarly the RS-III study included 3932 residents, but aged 45 years and older. The rationale and study designs of these cohorts were similar to that of the RS-I. 9 The baseline examination of RS-II took place between 2000 and 2002; follow-up examination was performed from 2004 to 2005. Baseline examination of RS-III took place between 2006 and 2009. 
In RS-I, of 5974 participants who were genotyped, 5107 had reliable baseline optic disc data and another 205 had reliable follow-up disc data resulting in a total of 5312 included participants. From RS-II a total of 2048 out of 2157 genotyped participants were included in the study, of which 90 were based on follow-up data. From RS-III a total of 1966 genotyped participants were included, resulting in a total of 9326 participants. 
Finally, the fourth cohort was ERF, now consisting of 1919 genotyped participants. This is a family-based study of several thousand individuals from a genetic isolate in the southwest of the Netherlands, founded in the mid-18th century by some 150 individuals. 10 Data collection began in June 2002 and finished in February 2005. 
All measurements in these studies were conducted after the Medical Ethics Committee of the Erasmus University Rotterdam had approved the study protocols and all participants had given their written informed consent in accordance with the Declaration of Helsinki. 
Ophthalmic Examination
In RS-I and RS-II digitized stereoscopic imaging of the optic disc was used for the assessment of the optic nerve head (Topcon ImageNet System; Topcon Corporation, Tokyo, Japan), while RS-III and ERF used confocal scanning laser ophthalmoscopy (Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering, Dossenheim, Germany). 3 It has been shown that both instruments are comparable and that there is high correlation for all stereometric parameters. 11  
To determine DA and VCDR with the stereoscopic imaging system (ImageNet System) used for the RS-I and RS-II populations, two trained technicians marked points on the disc margin and near the retinal blood vessels which were used by the stereoscopic imaging system (ImageNet System) to outline the disc margin. The stereoscopic imaging system (ImageNet System) systematically measures a larger DA than HRT. In brief, the mean difference of agreement between HRT and the stereoscopic imaging system (ImageNet System) is −0.05; 95% confidence interval (CI) of the difference is −0.07 to −0.03; limits of agreements −0.25 to 0.16. When measured with both methods there is a systematic difference in DA. 11  
Genotyping and Imputations
Genomic DNA was extracted from whole blood samples using standard methods. 12 Genome-wide single nucleotide polymorphism (SNP) genotyping was performed using an assay (Infinium II; Illumina, Inc., San Diego, CA) on a microarray (HumanHap 550 Genotyping Bead Chips; Illumina Inc). Approximately 2 million SNPs were imputed using release 22 HapMap CEU population (www.hapmap.org) as reference. The imputations were performed using MACH software (http://www.sph.umich.edu/csg/abecasis/MACH/). The quality of imputations was checked by contracting imputed and actual genotypes at 78,844 SNPs not present on an array (Illumina 550K; Illumina, Inc.) in 437 individuals for whom these SNPs were directly typed using an array set (Affymetrix 500K; SeqWright, Inc., Houston, TX). Using the “best guess” genotype for imputed SNPs the concordance rate was 99% for SNPs with R 2 (ratio of the variance of imputed genotypes to the binomial variance) quality measure greater than 0.9; concordance was still high (94%) when R 2 was between 0.5 and 0.9. For imputed SNPs we chose an R 2 value of 0.85 as the minimum for consideration. 
Choice of Candidate Genes
We constructed a list of interacting proteins for SALL1 and SIX1 using internet databases (GeneCards; www.genecards.org). Because SIX3 and SIX6 are expressed during development of the human eye and SIX4 and SIX5 also play a role in the eye, 8 we decided to include all the SIX genes in our analysis. Table 1 provides an overview of the candidate genes investigated in this study. Of note is the overlap of the regions enframing SIX3 and SIX2 on chromosome 2 and SIX6, SIX1 and SIX4 on chromosome 14. 
Table 1.
 
The 38 Genes/35 ROIs for Association with the Traits DA and VCDR
Table 1.
 
The 38 Genes/35 ROIs for Association with the Traits DA and VCDR
Gene Chr Start Gene* End Gene* Start Region End Region ROI SNPs (n) DA† VCDR†
EYA3 1 28300819 28415131 28200819 28515131 EYA3 150
CTBS 1 85018804 85040163 84918804 85140163 CTBS 273 Y Y
MYOG 1 203052257 203055377 202952257 203155377 MYOG 230 Y
SIX3 2 45169037 45172390 45069037 45272390 SIX32 358 Y
SIX2 2 45232324 45236542 45132324 45336542 SIX32
HOXD13 2 176957532 176960666 176857532 177060666 HOXD13 134
EPHA4 2 222282747 222437010 222182747 222537010 EPHA4 281
PAX3 2 223064607 223163700 222964607 223263700 PAX3 282 Y
EPHA3 3 89156674 89531284 89056674 89631284 EPHA3 457 Y
MDFI 6 41606195 41621982 41506195 41721982 MDFI 150 Y
TAF8 6 42018251 42048644 41918251 42148644 TAF8 138 Y
EYA4 6 133562495 133853258 133462495 133953258 EYA4 586 Y
EZR 6 159186773 159240456 159086773 159340456 EZR 262
HOXA13 7 27236499 27239725 27136499 27339725 HOXA13 326
SHH 7 155595558 155604967 155495558 155704967 SHH 299 Y
MNX1 7 156797547 156803347 156697547 156903347 MNX1 151 Y
EYA1 8 72109668 72274467 72009668 72374467 EYA1 500 Y Y
TERF1 8 73921097 73959987 73821097 74059987 TERF1 382
TG 8 133879205 134147143 133779205 134247143 TG 746 Y Y
PAX2 10 102505468 102589695 102405468 102689695 PAX2 231 Y
LBX1 10 102986733 102988717 102886733 103088717 LBX1 169
FGF8 10 103529887 103535827 103429887 103635827 FGF8 43
MYOD1 11 17741110 17743678 17641110 17843678 MYOD1 174
PAX6 11 31806340 31839509 31706340 31939509 PAX6 151 Y
MYF5 12 81110708 81113447 81010708 81213447 MYF5 135
TBX5 12 114791735 114846247 114691735 114946247 TBX5 281 Y Y
DACH1 13 72012098 72441330 71912098 72541330 DACH1 641 Y Y
SLC22A17 14 23815527 23822080 23715527 23922080 SLC22A17 182 Y Y
COCH 14 31343741 31359822 31243741 31459822 COCH 256 Y Y
OTX2 14 57267425 57277184 57167425 57377184 OTX2 246 Y
SIX6 14 60975938 60978525 60875938 61078525 SIX614 531 Y
SIX1 14 61111417 61116155 61011417 61216155 SIX614
SIX4 14 61176256 61190792 61076256 61290792 SIX614
SALL1 16 51169886 51185183 51069886 51285183 SALL1 207 Y
GOSR2 17 45000486 45018733 44900486 45118733 GOSR2 128 Y
SIX5 19 46268044 46272312 46168044 46372312 SIX5 173
PAX1 20 21686297 21696620 21586297 21796620 PAX1 151 Y
EYA2 20 45523509 45817492 45423509 45917492 EYA2 413 Y
Total 38 35 9817 15 16
To determine gene positions we used NCBI build version 36.3 (ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/ARCHIVE/BUILD.36.3/mapview/seq_gene.md.gz). We extracted SNPs within a region of interest comprising 100 kb on each side of the 38 candidate genes to be certain we included the promoter regions. Only SNPs with P ≥ 0.0001 for Hardy-Weinberg equilibrium test and with genotype call rate ≥ 95% were included. A total of 9611 SNPs met these criteria and were selected for the association test. 
Statistical Analysis
All analyses were performed using R statistical package (version 2.10.0 for Linux; www.r-project.org). Allele-based linear regression methods were used to test for association between a single SNP and the traits using ProbABEL (version 0.2.0-beta). 13 Betas and their standard errors for each SNP were derived adjusting for age and sex in the analyses of DA, and additionally adjusted for DA in the analyses of VCDR. All genes with nominal significant results in RS-I were followed up in the RS-II and RS-III cohorts in a meta-analysis using MetABEL R package (version 0.0-3). 14 For each candidate gene we calculated the P value significance threshold (p-sig) according to Bonferroni by adjusting for the number of SNPs tested within the respective region. After the meta-analysis in the three Rotterdam Study cohorts, the top SNPs were then followed up in another independent cohort, ERF. Finally we performed a meta-analysis in all four cohorts using the MetABEL R package. We used two different cutoffs for significance: Bonferroni adjustment for the number of SNPs sent for replication (P ≤ 0.05/6 = 8.33 × 10−3); and the more conservative Bonferroni adjustment on all SNPs tested in the discovery cohort (P ≤ 0.05/9611 = 5.20 × 10−6). 
Results
The general characteristics of the study populations are shown in Table 2. Participants of RS-III and ERF were younger compared with the other two cohorts. Slightly > 50% of participants were female in all populations. Of note is that in RS-III and ERF the mean DA and VCDR were smaller than in the other two populations. This is due to the difference in equipment 11 and does not affect the results 3 because all cohorts were analyzed separately and later meta-analyzed. 
Table 2.
 
General Characteristics of the Four Study Populations
Table 2.
 
General Characteristics of the Four Study Populations
RS-I RS-II RS-III ERF
Total sample size (N) 5312 2048 1966 1919
Age, y 68.0 ± 8.4 (55–99) 64.3 ± 7.8 (55–98) 55.6 ± 5.5 (45–89) 47.0 ± 14.0 (18–85)
Gender, female, n (%) 3099 (58.3) 1109 (54.2) 1102 (56.1) 1081 (56.3)
Disc area, mm2 * 2.42 ± 0.48 (0.58–5.44) 2.32 ± 0.48 (1.06–6.20) 1.92 ± 0.45 (0.07–7.20) 1.91 ± 0.35 (1.07–4.33)
VCDR* 0.50 ± 0.14 (0.00–0.89) 0.50 ± 0.14 (0.00–0.87) 0.42 ± 0.17 (0.00–1.00) 0.46 ± 0.15 (0.00–0.84)
Of the 38 investigated genes in our initial analysis in RS-I we found 15 regions to be nominally significant (P < 0.05) for DA and 16 for VCDR (Table 1). Next we performed a meta-analysis for these candidate genes including all three Rotterdam Study cohorts. Four genes reached statistical significance for DA after adjusting for multiple testing (based on the number of SNPs in the gene). These were paired box 6 (PAX6), paired box 1 (PAX1), ephrin receptor tyrosine kinase A3 (EPHA3), and sine oculis homeobox homolog 2 (SIX2) (data not shown). A single SNP in the gene paired box 2 (PAX2) was significant for VCDR (rs11190730). However, the SNP was discarded due to its low imputation quality (R 2 = 0.50). 
To corroborate the association with DA found in the Rotterdam Study cohorts, we tested the six top SNPs—three from PAX6 and one each from PAX1, EPHA3, and SIX2 each in a fourth cohort, ERF. These data were combined with those of the other three cohorts in a final meta-analysis (Table 3). When adjusting for the number of SNPs selected for replication, five of the six SNPs remained significant (rs953476 from gene EPHA3 with a P-value of 0.02 was discarded), but when performing a more stringent multiple testing adjustment only the PAX6 region remains significant (p-sig < 5.20 × 10−6). Though the SNPs for PAX6 were not significant in ERF, the direction of the effect was the same as in the other three cohorts. 
Table 3.
 
Results of SNPs Sent for Replication on Disc Area in the Four Meta-analyzed Cohorts
Table 3.
 
Results of SNPs Sent for Replication on Disc Area in the Four Meta-analyzed Cohorts
Gene SNP Chr Position Distance Effallele EAF RS-I RS-II RS-III ERF Meta-analysis
β SE P β SE P β SE P β SE P β SE P P-sig
PAX6 rs7126851 11 31710522 96 C 0.75 −0.04 0.01 7.27 × 10−4 −0.02 0.02 0.21 −0.07 0.02 1.68 × 10−4 −0.01 0.01 0.46 −0.03 0.01 4.79 × 10−6 0.0083
PAX6 rs7104512 11 31725460 81 C 0.24 0.04 0.01 4.94 × 10−4 0.02 0.02 0.24 0.07 0.02 1.39 × 10−4 0.01 0.01 0.50 0.03 0.01 3.92 × 10−6 0.0083
PAX6 rs10835818 11 31749627 57 G 0.24 0.04 0.01 7.19 × 10−4 0.03 0.02 0.14 0.06 0.02 2.74 × 10−4 0.01 0.01 0.47 0.03 0.01 4.88 × 10−6 0.0083
PAX1 rs2064773 20 21781737 85 G 0.67 −0.03 0.01 1.65 × 10−3 −0.04 0.02 0.01 −0.02 0.02 0.16 −0.02 0.01 0.21 −0.03 0.01 1.58 × 10−5 0.0083
EPHA3 rs953476 3 89095996 61 G 0.63 0.03 0.01 8.84 × 10−3 0.04 0.02 0.01 0.02 0.01 0.24 −0.02 0.01 0.10 0.01 0.01 0.02 0.0083
SIX2 rs2280220 2 45245405 13 G 0.17 0.04 0.01 2.84 × 10−3 0.02 0.02 0.34 0.01 0.02 0.44 0.00 0.02 0.97 0.02 0.01 4.70 × 10−3 0.0083
Figure 1 shows regional plots of the three candidate genes using the P values of RS-I only, as this cohort is the only one in which we investigated all SNPs. The three top SNPs for PAX6 (rs7126851, rs7104512, and rs10835818) were located 57 kb to 96 kb 3′ of PAX6 in its neighboring gene ELP4 (Fig. 1A). The top SNP (rs2064773) for PAX1 was located 85 kb from the 3′ end of the gene with no genes in between the top SNP and the gene of interest (Fig. 1B). Also for SIX2 (Fig. 1C) no genes could be found between the top SNP and the gene of interest. The top SNP (rs2280220) for SIX2 was located only 13 kb from the 5′ end of the gene. 
Figure 1.
 
Regional plots of the three loci found to be associated with optic disc area in the meta-analysis with our four cohorts for PAX6 (A), PAX1 (B) and SIX2 (C).
Figure 1.
 
Regional plots of the three loci found to be associated with optic disc area in the meta-analysis with our four cohorts for PAX6 (A), PAX1 (B) and SIX2 (C).
Discussion
We found three genes to be associated with DA: PAX6, PAX1, and SIX2. When adjusting for the number of SNPs sent for replication, one gene (PAX6) survived the most stringent Bonferroni correction on all SNPs tested in this study. None of the selected candidate genes could be associated with VCDR. 
The protein products of PAX6 and PAX1 are among 25 proteins listed in the internet databases (GeneCards; www.genecards.org) which interact with SIX1. The PAX family of genes is a group of highly conserved developmental control genes encoding nuclear transcription factors, regulating the expression of other genes, which have been shown to play a role in organogenesis. Our only significant finding after adjustment for multiple testing concerns PAX6 (three SNPs with P values to the order of 10−6). Pax6 has been shown to function in the genetic control of eye development in organisms ranging from planarians to humans. 15 In both insects and vertebrates, Pax6 is expressed in the embryo just before and during formation of the eye in the region of its development. 16 For eye morphogenesis in insects and vertebrates, the Pax6 paired domain seems to be paramount. 17 The Drosophila Pax6 homologue is the eyeless gene (ey). 18 In mice Pax6 causes Small eye (sey); in humans PAX6 is associated with aniridia, Peter's anomaly and other types of anterior segment dysgenesis. 19 21 Clinically, aniridia has frequently been associated with glaucoma; more than half of aniridic patients will develop glaucoma. 22,23 A mutation in one allele is sufficient to cause ocular defects through haploinsufficiency, while compound heterozygosity is usually lethal and includes defects in the brain and other organs. 21,24 PAX6 has tissue maintenance functions and continues to be expressed in the adult eye, leading investigators to speculate that PAX6 may possibly play a role in adult-onset POAG and/or age-related macular degeneration. 5,24 The three SNPs that were found to be significantly associated with DA in this study were located downstream of the PAX6 gene within an intronic region of neighboring ELP4. PAX6 is one of many genes listed in the internet databases (GeneCards; www.genecards.org) which interact with ELP4. Possibly ELP4 has a role in regulating PAX6. 25,26 The ELP4 gene encodes a component of the six subunit elongator complex, a histone acetyltransferase complex that associates directly with the RNA polymerase II (Pol II) holoenzyme, and is involved in transcriptional elongation. Elongator may play a role in chromatin remodeling and is involved in acetylation of histones H3 and probably H4. Some familial aniridia cases have an undamaged PAX6 gene but a deletion in the region 3′ to it at the level of the ELP4 gene; microdeletions 3′ of PAX6 have been known to suppress expression of PAX6 and cause aniridia. 25 27 Perhaps new techniques like Next Generation Sequencing will be able to discover which of the two genes is truly associated with DA. 
Although not significant after adjusting for multiple testing like PAX6, PAX1 also plays a role in the development of the central nervous system. In mice Pax1 is known to be expressed in segmented structures during embryogenesis. The mouse Pax1 mutant phenotype undulated, caused by a missense mutation in the paired box of Pax1, shows segmentation anomalies along the entire axial skeleton and defects in the pectoral girdle. 28 By phenotypic similarity, human PAX1 has been postulated as a candidate gene for Klippel-Feil syndrome, characterized by failed segmentation of the cervical vertebrae and other vertebral anomalies. 28 However, this association is inconclusive. 28,29 Interesting to note is that in human PAX1 the protein product contains additional amino acids upstream of the paired box, while in PAX6 the protein sequences are identical between mice and humans. 24,28 As of yet, the literature on PAX1 is limited. As pointed out by several authors, many members of the eye developmental cascade of transcription factors, including PAX6, also play a role in the genesis of other tissues. 17,24 Further research may reveal more about the nature of the association between PAX1 and DA. 
SIX2, which interacts with SALL1, is a member of the Drosophila sine oculis homeobox homologues and encodes a transcription factor associated with organ development. Also this finding is not significant after adjusting for multiple testing but is of interest. The Six proteins are part of the Pax/Eya/Six/Dach retinal determination cascade involved in embryonic cell fate determination. 30 As are a number of other genes in the Pax/Eya/Six network, Six2 is expressed in the mesenchyme and plays an important role in early kidney development as well as in eye and muscle formation in mammals. 31 In mammals and humans, Six2 has been associated with renal hypoplasia, 32 suggesting a role in regulating mesenchymal progenitor cells, 33 frontonasal dysplasia 34 and anterior cranial base defects. 35 In mouse studies, Six2 transcripts have been found in a variety of nonneuronal connective tissues, including the eye, during organogenesis. 36  
Other candidate genes in the network, for example SIX3 and PAX2, did not interact with PAX6 17 . This may in part be explained by a lack of statistical power. Larger numbers will be needed to determine whether these genes are associated with DA or not. More generally, one would expect that more of the genes that interact in the pathways of the PAX regulatory network, such as the SIX and EYA (eyes absent homolog) families, known to play a role in oculogenesis in Drosophila, would be significantly associated with either DA or VCDR. Obviously, these transcription factors play multiple roles in complex pathways involved not specifically or exclusively in the eye but more broadly in the central nervous system and in sensory neurogenesis, affecting the development of multiple organ systems. Many of these genes are highly conserved and serve multiple functions within a complex network of interdependencies and feedback loops. The individual effect of a gene on human optic nerve tissue may be subtle, absent, or subject to dosage effects. We may not have sufficient statistical power to detect these by association with either DA or VCDR in our populations. Further research is needed to tease out any of the possible roles these genes may play in the formation of the optic nerve head or optic disc cupping, or more generally in ocular pathology; at present these roles remain somewhat speculative. 
While none of our SNPs reached genome-wide significance, the PAX6/ELP4 region remained significant after adjusting for multiple testing. When we attempted to corroborate the associations we found with DA in the ERF cohort we found that, although not significant, the direction of the effect for the SNPs in PAX6 was the same as in the other three cohorts. Further research in a larger sample is required to validate the association between PAX6/ELP4 and DA. 
Footnotes
 Supported by Stichting Lijf en Leven, Krimpen aan de Lek; MD Fonds, Utrecht; Rotterdamse Vereniging Blindenbelangen, Rotterdam; Stichting Oogfonds Nederland, Utrecht; Blindenpenning, Amsterdam; Blindenhulp, The Hague; Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB), Doorn; Landelijke Stichting voor Blinden en Slechtzienden, Utrecht; Swart van Essen, Rotterdam; Stichting Winckel-Sweep, Utrecht; Henkes Stichting, Rotterdam; Laméris Ootech BV, Nieuwegein; Medical Workshop, de Meern; Topcon Europe BV, Capelle aan de IJssel, all in the Netherlands; and Heidelberg Engineering, Dossenheim, Germany.
Footnotes
 Disclosure: A.C. Gasten, None; W.D. Ramdas, None; L. Broer, None; L.M.E. van Koolwijk, None; M.K. Ikram, None; P.T.V.M. de Jong, None; Y.S. Aulchenko, None; R.C. Wolfs, None; A. Hofman, None; F. Rivadeneira, None; A.G. Uitterlinden, None; B.A. Oostra, None; H.G. Lemij, None; C.C.W. Klaver, None; N.M. Jansonius, None; J.R. Vingerling, None; C.M. van Duijn, None
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Figure 1.
 
Regional plots of the three loci found to be associated with optic disc area in the meta-analysis with our four cohorts for PAX6 (A), PAX1 (B) and SIX2 (C).
Figure 1.
 
Regional plots of the three loci found to be associated with optic disc area in the meta-analysis with our four cohorts for PAX6 (A), PAX1 (B) and SIX2 (C).
Table 1.
 
The 38 Genes/35 ROIs for Association with the Traits DA and VCDR
Table 1.
 
The 38 Genes/35 ROIs for Association with the Traits DA and VCDR
Gene Chr Start Gene* End Gene* Start Region End Region ROI SNPs (n) DA† VCDR†
EYA3 1 28300819 28415131 28200819 28515131 EYA3 150
CTBS 1 85018804 85040163 84918804 85140163 CTBS 273 Y Y
MYOG 1 203052257 203055377 202952257 203155377 MYOG 230 Y
SIX3 2 45169037 45172390 45069037 45272390 SIX32 358 Y
SIX2 2 45232324 45236542 45132324 45336542 SIX32
HOXD13 2 176957532 176960666 176857532 177060666 HOXD13 134
EPHA4 2 222282747 222437010 222182747 222537010 EPHA4 281
PAX3 2 223064607 223163700 222964607 223263700 PAX3 282 Y
EPHA3 3 89156674 89531284 89056674 89631284 EPHA3 457 Y
MDFI 6 41606195 41621982 41506195 41721982 MDFI 150 Y
TAF8 6 42018251 42048644 41918251 42148644 TAF8 138 Y
EYA4 6 133562495 133853258 133462495 133953258 EYA4 586 Y
EZR 6 159186773 159240456 159086773 159340456 EZR 262
HOXA13 7 27236499 27239725 27136499 27339725 HOXA13 326
SHH 7 155595558 155604967 155495558 155704967 SHH 299 Y
MNX1 7 156797547 156803347 156697547 156903347 MNX1 151 Y
EYA1 8 72109668 72274467 72009668 72374467 EYA1 500 Y Y
TERF1 8 73921097 73959987 73821097 74059987 TERF1 382
TG 8 133879205 134147143 133779205 134247143 TG 746 Y Y
PAX2 10 102505468 102589695 102405468 102689695 PAX2 231 Y
LBX1 10 102986733 102988717 102886733 103088717 LBX1 169
FGF8 10 103529887 103535827 103429887 103635827 FGF8 43
MYOD1 11 17741110 17743678 17641110 17843678 MYOD1 174
PAX6 11 31806340 31839509 31706340 31939509 PAX6 151 Y
MYF5 12 81110708 81113447 81010708 81213447 MYF5 135
TBX5 12 114791735 114846247 114691735 114946247 TBX5 281 Y Y
DACH1 13 72012098 72441330 71912098 72541330 DACH1 641 Y Y
SLC22A17 14 23815527 23822080 23715527 23922080 SLC22A17 182 Y Y
COCH 14 31343741 31359822 31243741 31459822 COCH 256 Y Y
OTX2 14 57267425 57277184 57167425 57377184 OTX2 246 Y
SIX6 14 60975938 60978525 60875938 61078525 SIX614 531 Y
SIX1 14 61111417 61116155 61011417 61216155 SIX614
SIX4 14 61176256 61190792 61076256 61290792 SIX614
SALL1 16 51169886 51185183 51069886 51285183 SALL1 207 Y
GOSR2 17 45000486 45018733 44900486 45118733 GOSR2 128 Y
SIX5 19 46268044 46272312 46168044 46372312 SIX5 173
PAX1 20 21686297 21696620 21586297 21796620 PAX1 151 Y
EYA2 20 45523509 45817492 45423509 45917492 EYA2 413 Y
Total 38 35 9817 15 16
Table 2.
 
General Characteristics of the Four Study Populations
Table 2.
 
General Characteristics of the Four Study Populations
RS-I RS-II RS-III ERF
Total sample size (N) 5312 2048 1966 1919
Age, y 68.0 ± 8.4 (55–99) 64.3 ± 7.8 (55–98) 55.6 ± 5.5 (45–89) 47.0 ± 14.0 (18–85)
Gender, female, n (%) 3099 (58.3) 1109 (54.2) 1102 (56.1) 1081 (56.3)
Disc area, mm2 * 2.42 ± 0.48 (0.58–5.44) 2.32 ± 0.48 (1.06–6.20) 1.92 ± 0.45 (0.07–7.20) 1.91 ± 0.35 (1.07–4.33)
VCDR* 0.50 ± 0.14 (0.00–0.89) 0.50 ± 0.14 (0.00–0.87) 0.42 ± 0.17 (0.00–1.00) 0.46 ± 0.15 (0.00–0.84)
Table 3.
 
Results of SNPs Sent for Replication on Disc Area in the Four Meta-analyzed Cohorts
Table 3.
 
Results of SNPs Sent for Replication on Disc Area in the Four Meta-analyzed Cohorts
Gene SNP Chr Position Distance Effallele EAF RS-I RS-II RS-III ERF Meta-analysis
β SE P β SE P β SE P β SE P β SE P P-sig
PAX6 rs7126851 11 31710522 96 C 0.75 −0.04 0.01 7.27 × 10−4 −0.02 0.02 0.21 −0.07 0.02 1.68 × 10−4 −0.01 0.01 0.46 −0.03 0.01 4.79 × 10−6 0.0083
PAX6 rs7104512 11 31725460 81 C 0.24 0.04 0.01 4.94 × 10−4 0.02 0.02 0.24 0.07 0.02 1.39 × 10−4 0.01 0.01 0.50 0.03 0.01 3.92 × 10−6 0.0083
PAX6 rs10835818 11 31749627 57 G 0.24 0.04 0.01 7.19 × 10−4 0.03 0.02 0.14 0.06 0.02 2.74 × 10−4 0.01 0.01 0.47 0.03 0.01 4.88 × 10−6 0.0083
PAX1 rs2064773 20 21781737 85 G 0.67 −0.03 0.01 1.65 × 10−3 −0.04 0.02 0.01 −0.02 0.02 0.16 −0.02 0.01 0.21 −0.03 0.01 1.58 × 10−5 0.0083
EPHA3 rs953476 3 89095996 61 G 0.63 0.03 0.01 8.84 × 10−3 0.04 0.02 0.01 0.02 0.01 0.24 −0.02 0.01 0.10 0.01 0.01 0.02 0.0083
SIX2 rs2280220 2 45245405 13 G 0.17 0.04 0.01 2.84 × 10−3 0.02 0.02 0.34 0.01 0.02 0.44 0.00 0.02 0.97 0.02 0.01 4.70 × 10−3 0.0083
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