Genetic Variants on Chromosome 1q41 Influence Ocular
Axial Length and High Myopia
Qiao Fan1, Veluchamy A. Barathi2,3, Ching-Yu Cheng1,2,3, Xin Zhou1, Akira Meguro4, Isao Nakata5,6,
Chiea-Chuen Khor2,7,8,9, Liang-Kee Goh1,10,11, Yi-Ju Li12,13, Wan’e Lim2, Candice E. H. Ho2,
Felicia Hawthorne13, Yingfeng Zheng2, Daniel Chua2, Hidetoshi Inoko14, Kenji Yamashiro5, Kyoko Ohno-
Matsui15, Keitaro Matsuo16, Fumihiko Matsuda6, Eranga Vithana2,3, Mark Seielstad17, Nobuhisa Mizuki4,
Roger W. Beuerman2,3,10, E.-Shyong Tai1,18, Nagahisa Yoshimura5, Tin Aung2,3, Terri L. Young10,13,
Tien-Yin Wong1,2,3,19, Yik-Ying Teo1,7,20,21.*, Seang-Mei Saw1,2,3,10,20.*
1Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore, 2Singapore Eye Research Institute, Singapore National Eye Centre,
Singapore, Singapore, 3Department of Ophthalmology, National University of Singapore, Singapore, Singapore, 4Department of Ophthalmology, Yokohama City
University School of Medicine, Yokohama, Japan, 5Department of Ophthalmology, Kyoto University Graduate School of Medicine, Kyoto, Japan, 6Center for Genomic
Medicine and Inserm U.852, Kyoto University Graduate School of Medicine, Kyoto, Japan, 7Genome Institute of Singapore, Agency for Science, Technology, and Research,
Singapore, Singapore, 8Centre for Molecular Epidemiology, National University of Singapore, Singapore, Singapore, 9Department of Pediatrics, National University of
Singapore, Singapore, Singapore, 10Duke–National University of Singapore Graduate Medical School, Singapore, Singapore, 11Department of Medical Oncology,
National Cancer Centre Singapore, Singapore, Singapore, 12Department of Biostatistics and Bioinformatics, Duke University Medical School, Durham, North Carolina,
United States of America, 13Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, United States of America, 14Department of Molecular
Life Science, Division of Molecular Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Japan, 15Department of Ophthalmology and
Visual Science, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan, 16Division of Epidemiology and Prevention, Aichi Cancer Center
Research Institute, Nagoya, Japan, 17Institute for Human Genetics and Department of Laboratory Medicine, University of California San Francisco, San Francisco,
California, United States of America, 18Department of Medicine, National University of Singapore, Singapore, Singapore, 19Centre for Eye Research Australia, University
of Melbourne, Melbourne, Australia, 20Graduate School for Integrative Science and Engineering, National University of Singapore, Singapore, Singapore, 21Department
of Statistics and Applied Probability, National University of Singapore, Singapore, Singapore
As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia.
The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three
genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults.
We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-
wide significant association with AL variation (rs4373767, b=20.16 mm per minor allele, Pmeta=2.69610210). The minor C
allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds
ratio (OR)=0.75, 95% CI: 0.68–0.84, Pmeta=4.3861027) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two
neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and
sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the
retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This
supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.
Citation: Fan Q, Barathi VA, Cheng C-Y, Zhou X, Meguro A, et al. (2012) Genetic Variants on Chromosome 1q41 Influence Ocular Axial Length and High
Myopia. PLoS Genet 8(6): e1002753. doi:10.1371/journal.pgen.1002753
Editor: Janey L. Wiggs, Harvard University, United States of America
Received February 10, 2012; Accepted April 20, 2012; Published June 7, 2012
Copyright: ? 2012 Fan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study is supported by the Singapore Bio-Medical Research Council (BMRC 06/1/21/19/466), the National Medical Research Council of Singapore
(NMRC 0796/2003, NMRC 1176/2008, NMRC/IRG/1117/2008, and NMRC/CG/T1/2010), and the National Research Foundation (NRF-RF-2010-05). KO-M
acknowledges funding from the Japan Society for the Promotion of Science (JSPS 22390322 and 23659808). TLY acknowledges funding from the National
Institutes of Health, National Eye Institute, R01EY014685, and an internal grant from the Duke-National University of Singapore Graduate Medical School. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (S-MS); firstname.lastname@example.org (Y-YT)
. These authors contributed equally to this work.
Myopia increases the risk of visual morbidity and poses a
considerable public health and economic burden globally,
especially in Asia, where the prevalence is significantly higher
than other parts of the world . Human myopia primarily results
from an abnormal increase in ocular axial length (AL), the distance
between the anterior and posterior poles of the eye globe, whereas
the role of corneal curvature and lens thickness is minimal . A
1 millimeter (mm) increase in AL is equivalent to a myopic shift of
22.00 to 23.00 diopters (D) with no corresponding changes in the
optical power of the cornea and lens. High myopia, often defined
as ocular spherical equivalent (SE) refraction below 26.00 D, is
associated with an abnormally long AL, and this affects between
PLoS Genetics | www.plosgenetics.org1June 2012 | Volume 8 | Issue 6 | e1002753
1% to 10% of the general population . The degenerative
changes in the retina and the choroid due to the excessive
elongation of the globe are not prevented by optical correction and
this subsequently increases the risk of visual morbidity through
myopic maculopathy, choroidal neovascularization, retinal de-
tachment and macular holes . The active remodeling of the
sclera, mediated by the signaling cascade initiated in the retina
under visual input, has also been found to be critical in
determining axial growth, and thus the refractive state of the eye
Environmental factors such as the extent of near work, level of
educational attainment and amount of outdoor activities have
been documented to affect myopia development . Evidence
from family and twin studies has also supported a substantial
genetic component in spherical refractive error and AL [7–9]. The
heritability of the quantitative trait AL has been estimated to be as
high as 94% comparable to that for SE (for a review, see ).
Although linkage scans on pedigrees (myopia loci MYP1 to
MYP18; see http://www.omim.org) and genome-wide association
studies (GWAS) [11–16] have implicated several regions in the
human genome as being significant for refractive error and
myopia, no myopia genes have been consistently identified within
or across different population groups. This scenario reflects the
complexity in the disease architecture of myopia pathogenesis.
Genetic factors influencing AL and refraction appear to be at
least partly shared, given previous literature from twin studies
illustrating that at least half of the covariance between AL and
refraction are due to common genetic factors . The
measurement of AL is more precise and less prone to errors
compared to cycloplegic or non-cycloplegic assessments of
refraction. As AL is an endophenotype for spherical refractive
error, identifying genes that are responsible for AL variation
provides insight into myopia predisposition and development.
Presently there are only two genome-wide linkage studies
performed in European descent populations that suggest the
presence of AL quantitative trait loci (QTLs) on chromosomes
2p24  and 5q (at 98 centimorgans) along with two classical
myopia loci (MYP3 at 12q21 and MYP9 at 4q12) , and there
are no reports of any genes that are indisputably confirmed to be
associated with AL.
We thus performed a meta-analysis of three genome-wide
surveys of AL in a total of 4,944 individuals in Asian populations
comprising (i) Chinese adults from the Singapore Chinese Eye
Study (SCES); (ii) Chinese children from the Singapore cohort
Study of the Risk factors for Myopia (SCORM); and (iii) Malay
adults from the Singapore Malay Eye Study (SiMES). SNPs that
have been identified from this meta-analysis to be significantly
associated with AL were further assessed for association with high
myopia in an additional two independent case-control studies from
Japan. We also examined the expression patterns of the candidate
genes located in the vicinity of the identified SNPs in human
ocular tissues and in the eyes of myopic mice.
A genome-wide meta-analysis of three GWAS on AL was
performed in the post quality control samples from SCES
(n=1,860), SCORM (n=929) and SiMES (n=2,155). Principal
component analysis (PCA) of these samples with reference to the
HapMap Phase 2 individuals showed that the two Chinese cohorts
(SCES and SCORM) are indistinguishable with respect to samples
of Han Chinese descent, and the differentiation from samples of
Japanese descent is evident only on the fourth principal
component (Figure S1). The SiMES Malays are genetically similar
to the Chinese-descent samples relative to individuals with
European or African ancestries. The distributions of AL measure-
ments in the three cohorts were approximately Gaussian and the
baseline characteristics are summarized in Table 1. The mean AL
were 23.98 mm (SD=1.39 mm), 24.10 mm (SD=1.18 mm) and
23.57 mm (SD=1.04 mm) for SCES, SCORM and SiMES
respectively. Moderate to high correlations between AL and SE
were observed (SCES/SCORM/SiMES; Pearson correlation
coefficient r=20.75, 20.76 and 20.62 respectively). The meta-
analysis was performed on 456,634 SNPs present in all three
studies, and the quantile-quantile (QQ) plots of the P-values
showed only modest inflation of the test statistics in SCES and in
the meta-analysis (genomic control inflation factor: lmeta=1.03;
lSCES=1.05; lSCORM=1.00; lSiMES=1.00, Figure S2).
A cluster of four SNPs on chromosome 1q41 (rs4373767,
rs10779363, rs7544369 and rs4428898) attained genome-wide
significance on meta-analysis for AL, adjusting for age, gender,
height and education level (Figure 1). Analyses conducted without
adjustment for height or education level yielded the same pattern of
results. The most significant SNP rs4373767 (Pmeta=2.69610210)
explained 0.98% of AL variance in SCES, 0.86% in SCORM and
0.73% in SiMES, and each copy of the minor allele (cytosine)
decreased AL by 0.16 mm on average (Table 2). These top
associated SNPs at chromosome 1q41 remained significant after
adjustment for genomic control (Pmeta#1.8561028). Table 2 also
lists three genetic loci at chromosome 2p13.1 (SEMA4F), 2p21
(SPTBN1) and 5q11.1 (PARP8) exhibiting suggestive evidence of
association with AL that were seen in at least one SNP with P-
To assess whether these four SNPs at chromosome 1q41 have
any role in high myopia predisposition, we performed association
testing of these SNPs with high myopia in two independent case-
control studies from Japan consisting of 987 high myopes and
1,744 controls. High myopes were defined as individuals with
SE#29.00 D or AL$28 mm (see Materials and Methods). All
four SNPs exhibited consistent evidence of association (P,0.05) in
both Japanese studies, suggesting a potential role of these SNPs for
high myopia (Table 3).
We further dichotomized the quantitative refraction from our
three population-based studies (SCORM, SCES, and SIMES) to
define samples as high myopes and controls according to similar
criteria from the Japanese datasets. High myopes in SCES and
Myopic individuals exhibit an increase in ocular axial
length (AL). As a highly heritable ocular biometry of
refractive error, identification of quantitative trait loci
influencing AL variation would be valuable in informing
the biological etiology of myopia. We have determined
that a genetic locus on chromosome 1q41 containing zinc-
finger pseudogene ZC3H11B is associated with AL and
high myopia through a meta-analysis of three genome-
wide association scans on AL in Chinese and Malays, with
validation for high myopia association in two additional
Japanese cohorts. In addition, variations in the expression
of murine gene ZC3H11A and two neighboring genes
SLC30A10 and LYPLAL1 in the retina and sclera in a myopic
mouse model implicate the role of these genes in myopia
onset. To our knowledge, this is the first genome-wide
survey of single nucleotide polymorphism (SNP) variation
of AL in Asians. Our results suggest that genetic variants at
chromosome 1q41 have potential roles in both common
and high myopia.
Chromosome 1q41 Associated with Myopia
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SiMES were younger and more highly educated than controls
(Table S1). While the case-control associations of these 4 SNPs
with high myopia did not achieve statistical significance in SCES
and SiMES, this is likely a consequence of the small sample sizes
since the direction and magnitude of the odds ratios were highly
similar across all cohorts. The meta-analysis of 1,118 high myopia
cases and 5,433 controls from all the five cohorts yielded strong
evidence of association with high myopia at these SNPs (Pmeta
between 1.4561026to 7.8661028, Table 3), with no evidence of
inter-study heterogeneity (P$0.75 for heterogeneity). The minor
allele cytosine at rs4373767 lowered the odds of high myopia by
25% with respect to the thymidine allele (ORmeta=0.75, 95% CI:
Table 1. Characteristics of study participants in the five Asian cohorts.
Japan Dataset 1d
Japan Dataset 2e
High myopiaControls High myopiaControls
Individuals (n)1,8609292,155 4831,194 504 550
Male (%)51.551.7 49.333.741.3 43.349.5
Agea(yrs)58.4 (9.5)10.8 (0.8) 57.7 (13.9) 58.8 (13.2) 50.3 (15.9)37.8 (11.9)39.7 (12.6)
Range of age [44,85] [10,12][40,80][14,91] [20,79][12,76] [21,75]
Male 168.5 (6.3)144.8 (8.7)165.5 (6.4)NAf
Female156.7 (5.5) 145.5 (8.9)152.3 (6.2)NA NANA NA
No formal education21.2 3.1 18.0 NANA NA NA
Primary education 33.519.1 8.5NANA NA NA
Secondary education24.9 39.746.6NA NANA NA
Polytechnic13.1 18.1 19.7NA NANANA
University7.320.0 7.2NANANA NA
Average ALa(mm) 23.97 (1.39)24.13 (1.18)23.57 (1.04)30.08 (1.38) NA27.83 (1.28) NA
Range of AL[20.64, 33.36][21.05,28.20][20.48, 31.11][28.00, 38.03]NA [24.25, 34.74] NA
214.86 (4.28) NA
211.61 (2.22) NA
Range of SE[215.40, 6.25][211.09, 3.78][217.46, 8.56][242.00, 22.50]NA[223.00, 29.25]
aData presented are means (standard deviation). AL, ocular axial length; SE, spherical equivalent.
bThe education levels of the children in SOCRM was presented by the level of educational attainment of the father, as.
cGWAS cohorts. SCES, Singapore Chinese Eye Study; SCORM, Singapore Cohort study of the Risk factors for Myopia; SiMES, Singapore Malay Eye Study.
dFor the Japan dataset 1, high myopia, AL$28 mm for both eyes; controls, general healthy population.
eFor the Japan dataset 2, high myopia, SE#29.0 D for either eye; controls, SE$23.0 D for both eyes.
fNA, data not available.
Figure 1. Manhattan plot of -log10(P) for the association on axial length from the meta-analysis in the combined cohorts of SCES,
SCORM, and SiMES. The red horizontal line denotes genome-wide significance (P=561028).
Chromosome 1q41 Associated with Myopia
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0.68–0.84, Pmeta=4.3861027). The stringent definition of high
myopia (SE#29.00D) used here only considered between 1.0% to
2.4% of our samples as cases, and relaxing this criterion to the
commonly adopted threshold of SE#26.00D identified more
myopia cases and increased the statistical support of all four SNPs
(Pmetabetween 1.4761027to 9.1361029, Table S2).
This associated interval spans approximately 70 kb in the
extended linkage disequilibrium (LD) block within an intergenic
region on chromosome 1q41 (pairwise r2.0.5 with the most
significant SNP rs4373767, Figure 2A). Zinc finger family CCCH-
type 11B pseudogene ZC3H11B (RefSeq NG_007367.2) is
embedded between the associated top SNPs rs4373767 and
rs10779363 (Figure 2B). The most significant SNP rs4373767 is
located 223 kbdownstream
NM_018713.2), which is a member of solute carrier family 30,
and 354 kb downstream of LYPLAL1 (RefSeq NM_138794.3),
encoding a lysophospholipase-like protein.
The mRNA expression levels of ZC3H11B, SLC30A10 and
LYPLAL1 were surveyed in 24-week human fetal and adult tissues
using reverse-transcriptase polymerase chain reaction (RT-PCR).
Whilst ZC3H11B and LYPLAL1 were found to be expressed across
all the tissues including brain, placenta, neural retina, retina
pigment epithelium (RPE) and sclera, the expression of ZC3H11B
was more abundant compared to LYPLAL1 (Figure 3). SLC30A10
was expressed in all tissues but the adult sclera, analogous to
observations made in other zinc transporters .
Gene expressions for ZC3H11A, SLC30A10 and LYPLAL1 from
the tissues of myopic (with SE,25.0 D) and fellow non-occluded
eyes of the experimental mice were compared with age-matched
control tissues (Figure 4). The mRNA levels of ZC3H11A, a gene
that is conserved with respect to ZC3H11B in human, were
significantly down-regulated in myopic eyes compared to naive
controls (retina/RPE/sclera, Fold change=22.88, 23.24 and
22.07; P=2.6061025, 2.6261026and 1.0861024, respectively).
At the neighboring gene SLC30A10, there was a similarly
significant reduction in the expression of mRNA in the retina
tissue of myopic eyes in contrast to independent controls (retina/
RPE, Fold change=22.02, 22.69; P=2.0061024, 2.0061024,
respectively), with elevated expression in the sclera (Fold
change=4.58; P=4.0261024). Another neighboring gene LY-
PLAL1 exhibited up-regulation of transcription levels in retina
tissue but was down-regulated in the sclera (retina/RPE/sclera,
Fold change=2.71, 3.45 and 22.36; P=1.5061024, 1.5061024
and 1.5461024, respectively).
Immunohistochemical results confirmed the localization of
ZC3H11A, SLC30A10 and LYPLAL1 proteins in the neural retina,
Table 2. Top SNPs (Pmeta-value#161025) associated with AL from the meta-analysis in the three Asian cohorts.
Gene CHR BPMAaMAFb
b (s.e.)P MAF
b (s.e.) P
aMA, minor allele.
bMAF, minor allele frequency in each cohort.
cGWAS cohorts. SCES - Singapore Chinese Eye Study; SCORM - Singapore Cohort study of the Risk factors for Myopia; SiMES - Singapore Malay Eye Study.
db, coefficient of linear regression; s.e., standard error for coefficient b. Association between each genetic marker and AL was examined using linear regression, adjusted
for age, gender, height and level of education. The effect sizes denote changes in millimeter of AL per each additional copy of the minor allele.
ePhet, P-value for heterogeneity by Cochran’s Q test across three study cohorts.
Chromosome 1q41 Associated with Myopia
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Table 3. Association between genetic variants at chromosome 1q41 and high myopia in the five Asian cohorts.
Japan Dataset 1 (483/
Japan Dataset 2 (504/550) SCESb(44/1,305)
OR (95% CI) P
OR (95% CI) P
0.74 (0.64, 0.87)
0.76 (0.64, 0.91)2.1561023
0.73 (0.45, 1.19)2.0661021
0.74 (0.63, 0.86)
0.81 (0.68, 0.96)1.8061022
0.73 (0.44, 1.18)1.9961021
0.74 (0.63, 0.87)
0.81 (0.68, 0.96)1.4161022
0.68 (0.41, 1.13)1.3561021
0.75 (0.64, 0.88)
0.82 (0.69, 0.97)2.3261022
0.67 (0.39, 1.15)1.4561021
aMA, minor allele.
bGWAS cohorts; SCES - Singapore Chinese Eye Study; SCORM - Singapore Cohort study of the Risk factors for Myopia; SiMES - Singapore Malay Eye Study.
cThe sample sizes for each study denote the number of high-myopia cases versus controls. For the Japan dataset 1, high myopia, AL$28mm for both eyes; controls, general healthy population; For the Japan dataset 2, SCES, and
SiMES, high myopia, SE#29.0 D for either eye; controls, SE$23.0 D for both eyes; For SCORM children, high myopia, SE#26.0 D for either eye; controls, SE$21.0 D for both eyes.
dOR, odds ratio per copy of minor allele.
ePhet, P-value for heterogeneity by Cochran’s Q test across five study cohorts.
Chromosome 1q41 Associated with Myopia
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Figure 2. The chromosome 1q41 region and its association with axial length in the Asian cohorts. A) Regional plots for AL from the
meta-analysis of three Asian GWAS cohorts: SCES, SCORM and SiMES. The association signals in a 1 megabase (Mb) region at chromosome 1q41 from
217,400 kb to 218,400 kb around the top SNP rs4373767 (red diamond) are plotted. The degree of pair-wise LD between the rs4373767 and any
genotyped SNPs in this region is indicated by red shading, measured by r2. Superimposed on the plots are gene locations and recombination rates in
HapMap Chinese and Japanese populations (blue lines). B) LD plot showing pair-wise r2for all the SNPs genotyped in HapMap database residing
between rs4428898 and rs7544369, inclusively, at chromosome 1q41. The four identified top SNPs are in red rectangles. The LD plot is generated by
Haploview using SNPs (MAF.1%) genotyped on Han Chinese and Japanese samples in the HapMap database. All coordinates are in Build hg18.
Chromosome 1q41 Associated with Myopia
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RPE and sclera (Figure 5). For ZC3H11A, positive immunostaining
intensity was reduced significantly in the myopic tissues of
experimental mice compared to the non-myopic independent
controls (Figure 5A). This is consistent with the differential
expression patterns at the transcription level. For SLC30A10 and
LYPLAL1, there were also similarly noticeable changes in the
expression of proteins to that of their mRNA levels (Figure 5B and
We report that the chromosome 1q41 locus (most significant
SNP rs4373767) is associated with AL in a meta-analysis of three
GWAS performed in the study cohorts consisting of Chinese
adults, Chinese children, and Malay adults. The discovery of
chromosome 1q41 as a locus for high myopia in our data is further
supported by validation in two independent Japanese cohorts, and
the observed genetic effects are highly consistent across all five
studies. The pseudogene ZC3H11B and two nearby genes
SLC30A10 and LYPLAL1 were found to be expressed in the
human retina and sclera. The potential roles in regulating myopia
at three candidate genes were further implicated by the
concordant changes in the pattern of transcription and protein
expression in the mouse model.
The ZC3H11B pseudogene belongs to the CCCH-type zinc
finger family, whereas such type of zinc finger protein has been
shown as a RNA-binding motif to facilitate the mRNA processing
at transcription . Emerging evidence suggests that pseudo-
genes, resembling known genes but not producing proteins, play a
significant role in pathological conditions by competing for
binding sites to regulate the transcription of its protein-coding
counterpart [23–25]. Although the function of the ZC3H11B in
humans is presently unknown, the implicated role of the murine
gene ZC3H11A (conserved gene of ZC3H11B in mouse) in myopia
development is in keeping with previous findings that several zinc
finger proteins are involved in myopia [26,27]. Given their role as
transcription factors , zinc finger protein ZENK has been
proposed to function as a messenger in modulating the visual
signaling cascade in the chicken retina, where the expression of the
ZENK was suppressed by the condition of minus defocus (induced
myopic eye growth) and enhanced by positive defocus (induced
hyperopic eye growth) [29–31]. Similarly, it has been reported that
ZENK knockout mice had elongated AL and a myopic shift in
refraction . Moreover, early growth response gene type1 EGR-
1 (the human homologue of ZENK) has been shown to activate
transforming growth factor beta 1 gene TGFB1 by binding its
promoter [32,33], a gene that is implicated to be associated with
myopia [34,35]. Another zinc protein finger protein 644 isoform
ZNF644 has recently been identified to be responsible for high
myopia using whole genome exome sequencing in a Han Chinese
family , whereas its influence on ‘‘myopia genes’’ remains to
be elucidated. In light of this, the observation that ZC3H11B is
abundantly expressed in retina and sclera, together with the
significant down-regulation of the coding counterpart ZC3H11A in
Figure 3. mRNA expression of ZC3H11B, SLC30A10, and LYPLAL1 in human tissues. Expression of mRNA for the three genes was examined in
human brain, placenta, neural retina (retina), retinal pigment epithelium (RPE) and sclera from adult tissues, and retina/RPE and sclera from 24-week
gestation fetal tissues using reverse transcription polymerase chain reaction (RT-PCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a
housekeeping gene and was used as an internal control for the quantification of mRNA expression. NTC (No template control) served as a negative
control with the use of water rather than cDNA during PCR.
Chromosome 1q41 Associated with Myopia
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myopic mice eyes, suggests it may promote or inhibit the
transcription of ocular growth genes vital in myopia development.
One of the two neighboring genes SLC30A10 is an efflux
transporter that reduces cytoplasmic zinc concentrations . The
SLC30 zinc transporters are expressed abundantly in human RPE
cells, and the retina has been observed to possess the highest
concentration of zinc in the human body . Zinc deficiency in
the intracellular retina has thus been implicated in the pathogen-
esis of age-related macular degeneration (AMD) [37,38], and in
RPE-photoreceptor complex deficits, which can affect visual signal
transduction from retina to sclera and lead to visual impairment
. LYPLAL1 functions as a triglyceride lipase and this gene has
been shown to be up-regulated in subcutaneous adipose tissue in
obese individuals [40–42]. While the relationship between
LYPLAL1 and myopia is unknown, elevated saturated-fat intake
has been proposed to influence myopia development through the
retinoid receptor pathway [43–45]. Interestingly, the SNPs
pinpointing chromosome 1q41 in our study are 1 Mb away from
the transforming growth factor beta 2 gene (TGFb2) which has
been implicated in the down-regulation of mRNA levels in myopia
progression of an induced tree shrew myopia model . None of
these nearby genes, however, are within the LD block containing
our identified SNPs.
Chromosome 1q41 is a previously reported locus for refraction
from a linkage analysis of 486 pedigrees in the Beaver Dam Eye
Study, US . Using microsatellite markers, Klein et al identified
novel regions of linkage to SE on chromosome 1q41, whereas the
peak spanned a broad region near Marker D1S2141 (multipoint
P,1.961024). This result however was not replicated in a
subsequent genome-wide linkage scan for SE with denser SNP
markers, partially due to varying information of linkage conveyed
by SNPs versus microsatellites . The identified variants at
chromosome 1q41 in our study were noted to exhibit weaker,
albeit still significant, association with SE in SCES and SCORM
(rs4373767, SCES/SCORM: P=3.5461023, 3.4961022, respec-
tively; Table S3), but not in SiMES (3.5161021), which is
consistent with the lower correlation of AL and SE seen in the
SiMES data, partially from increasing lens opalescence in the
Malay population [49,50].
Our data have shown that genetic variants on chromosome
1q41 influence the physiological attribute of AL and are also
associated with high myopia. Elongation of AL is the major
underlying structural determinant of high myopia, mostly accom-
panied with prolate eyeballs and thinning of the sclera, macula
and retina . Thus, high myopia is also defined as AL of
.26 mm in some studies [13,51]. It is possible that genes involved
in a quantitative trait (refraction or underlying AL) also play a role
in the extreme forms of the trait (high myopia) . Two recent
GWAS performed in general Caucasians population have
identified genetic variants for quantitative refraction at chromo-
some 15q14  and 15q25 , of which the locus on 15q14 was
subsequently confirmed to be associated with high myopia in the
Japanese . Our GWAS results herein highlight AL QTLs
relevant for high myopia predisposition, which advances our
understanding of the genetic etiology of myopia at different levels
The meta-analysis of three GWAS in our discovery suggests that
the quantitative trait locus at chromosome 1q41 accounts for
variation in AL in both school children and adults, regardless of
age differences. Notably, the early-onset of myopia in childhood
may continuously progress toward high myopia in later life, while
adult-onset of myopia is usually in the low or moderate form
[54,55]. The significant association on chromosome 1q41 for high
myopia in adults and children thus also implicates this locus
identified for AL is likely to be associated with early-onset myopia.
Figure 4. Transcription quantification of ZC3H11A, SLC30A10, and LYPLAL1 in mouse retina, retinal pigment epithelium, and sclera in
induced myopic eyes, fellow eyes, and independent control eyes. Myopia was induced using 215 diopter negative lenses in the right eye of
mice for 6 weeks. Uncovered left eyes were served as fellow eyes and age-matched naive mice eyes were controls. Quantification of mRNA expression
in mice neural retina (retina), retinal pigment epithelium (RPE) and sclera using quantitative real-time PCR. The bar represents the fold changes of
mRNA for each gene after normalization using GAPDH as reference. The mRNA levels of murine ZC3H11A, a gene that is conserved with respect to
ZC3H11B in human, SLC30A10 and LYPLAL1 in myopic and fellow retina, RPE and sclera are compared with independent controls with P-values as
follows: ZC3H11A (retina/RPE/sclera, P=2.6061025, 2.6261026and 1.0861024respectively), SLC30A10 (P=2.0061024, 2.0061024and 4.0261024
respectively) and LYPLAL1 (P=1.5061024, 1.5061024, 1.5461024respectively). *P,0.0001.
Chromosome 1q41 Associated with Myopia
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The prevalence of myopia among Asian population is consid-
erably higher than in Caucasians . Although distinct genetic
mechanisms governing myopia may exist for populations with
different genetic backgrounds, we believe there are polymorphisms
involved in refractive variation that are shared across populations.
However, the allele frequencies of these identified SNPs vary
across populations. For instance, the minor C allele of rs4373767
was a major allele in the HapMap Africans and Europeans with
frequency of 0.92 and 0.62 respectively. Four distinct linkage
disequilibrium (LD) blocks existed in 50 kb region encapsulating
our top SNPs in the HapMap Africans, whereas high LD was
observed for the Chinese, Malays and Japanese populations. Such
heterogeneity may confer different statistical power and confound
the transferability of the same variants across populations [56,57].
In addition, we note that the variability in refraction attributed to
AL may vary in different ethnic groups. For example, AL has been
reported to account for a larger proportion of the variation in
refraction in East-Asian children compared to their Caucasian
counterparts , therefore the increased power of refraction may
reflect more variation in factors other than pure elongation of AL
in certain ethnic groups.
In conclusion, our findings suggest that common variants at
chromosome 1q41 are associated with AL and high myopia in a
pediatric and an adult cohort, the latter incorporating Chinese,
Figure 5. Immunofluorescent labeling. Immunofluorescent labeling of (A) ZC3H11A (B) SLC30A10 and (C) LYPLAL1 in mouse retina, retinal
pigment epithelium and sclera in induced myopic eyes, fellow eyes and independent control eyes. The neural retina (retina), retinal pigment
epithelium (PRE) and scleral cells were immunolabeled with the polycolonal antibodies against ZC3H11A, SLC30A10 and LYPLAL1 and were co-labeled
with 49,6-diamidino-2-phenylindole (DAPI). Negative controls were devoid of a fluorescence signal, treated with the secondary antibody alone and
DAPI. No immunostaining was observed in the negative controls. Scale bar represents 50 mM and magnification is 2006. The florescence intensity
labeled of the green color shows the localization of proteins and blue color indicates the nuclei that were stained with DAPI. Expression of the
proteins had a trend in abundance similarly to that of their mRNA levels as depicted in Figure 4. Lower level of expression was determined for
ZC3H11A in all tissues for myopic mice. Similarly significant reduction was shown in the expression of SLC30A10 in retina and RPE while higher level of
expression was found in myopic sclera. LYPLAL1 showed higher level of expression in the retina and RPE tissue but reduced expression in the sclera in
myopic mice. The following abbreviations represent the retinal layers: nerve fibre layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL),
inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photo receptor layer (PRL) and retinal pigment epithelium (RPE).
Chromosome 1q41 Associated with Myopia
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Malay and Japanese populations. Further evaluation of causal
variants and underlying pathway mechanisms may contribute to
early identification of children at highest risk of developing
myopia, and eventually lead to appropriate interventions to retard
the progression of myopia.
Materials and Methods
Singapore Chinese Eye Study (SCES).
population-based cross-sectional survey of eye diseases in Chinese
adults aged 40 to 80 years residing in the Southwestern part of
Singapore. The study began in 2007 and a detailed description
was published elsewhere . In brief, a total of 2,226 residents in
the Southwestern area of Singapore completed comprehensive
ophthalmologic examinations, including visual acuity assessments,
refraction, lens and retinal imaging, and slit lamp examinations.
Genome-wide genotyping was performed in 1,952 individuals.
Completed post quality control (QC) data for GWAS were
available for 1,860 adults with AL measurements.
Singapore Cohort study of the Risk factors for Myopia
A total of 1,979 children in grades 1, 2, and 3 from
three schools in Singapore were recruited from 1999 to 2001 .
The children were examined on their respective school premises
annually by a team of eye care professionals. The GWAS was
conducted in a subset of 1,116 Chinese children [14,60]. The
phenotype used in this study was based on the AL measured on the
4thannual examination of the study (children at age 10 to 12
years). Complete post-filtering data on AL measurements and SNP
data were available in 929 children.
Singapore Malay Eye Study (SiMES).
tion-based cross-sectional survey of eye diseases in Malay adults
aged 40 to 80 years living in Singapore. It was conducted between
August of 2004 and June of 2006 . A total of 4,168 Malay
residents in the Southwestern area of Singapore were identified
and invited for a detailed ocular examination where 3,280 (78.7%)
participated. Genome-wide genotyping was performed in 3,072
individuals [62,63]. Complete post-filtering data for GWAS with
AL measurements were available for 2,155 subjects.
SCES is an ongoing
SiMES is a popula-
Validation cohorts for high myopia
Japan dataset 1.
myopia cases and 1,194 general healthy population controls. High
myopia status was determined primarily on the basis of
AL$28 mm for both eyes, which corresponded to the spherical
equivalent (SE) cut-off of at least 29.00 D . Cases were
recruited at the Center for Macular Disease of Kyoto University
Hospital, the High Myopia Clinic of Tokyo Medical and Dental
University, and the Fukushima Medical University Hospital.
Details of the data have been reported elsewhere . The
population controls were recruited at the Aichi Cancer Center
Japan dataset 2.
The Japan dataset 2 was comprised of 504
high myopia cases (SE#29.00 D in either eye) and 550 non-
highly myopic controls (SE$23.00 D in both eyes). Less stringent
thresholds were adopted for controls for the purpose of ease of
recruitment from the clinics. Given the large phenotypic
separation between the cases and controls, and assumption of
homoscedasticity across genotype categories, such a study design
using the extreme on one end (i.e. SE#29.00 D) but sampling less
extreme controls (i.e. SE$23.00 D) still provides sufficient
statistical power to detect the true positive signals in the association
study . Cases were recruited at the Yokohama City University
The Japan dataset 1 consisted of 483 high
and Okada Eye Clinic. Controls were obtained from the
Yokohama City University and Tokai University Hospital.
Measurements of AL, refractive error, and covariates
All the studies used a similar protocol for ocular phenotype
measurements. For subjects in SCES and SiMES, AL for both eyes
were measured using optical laser interferometry (IOLMaster
V3.01, Carl Zeiss; Meditec AG Jena, Germany) [59,61]. Children
in the SCORM study underwent AL measurements using the A-
scan ultrasound biometry machine (Echoscan US-800; Nidek Co,
Tokyo, Japan) . For subjects in the Japan dataset 1,
applanation A-scan ultrasongraphy (UD-6000, Tomey, Nagoya,
Japan) or partial coherence interferometry (IOLMaster, Carl Zeiss
Meditec, Dublin, CA) were used to measure AL. AL was assessed
using a portable A-scan Biometer/pachymeter (AL-2000, Tomey,
Negoya, Japan) for the participants in the Japan dataset 2.
Non-cycloplegic refraction in SCES and SiMES as well as
cycloplegic refraction in SCORM (three drops of 1% cyclopen-
tolate at 5 minutes apart) were measured by autorefractor (Canon
RK-5, Tokyo, Japan) . For subjects in the Japan dataset 2,
refraction was measured using auto-refraction ARK-730A (NI-
DEK), ARK-700A (NIDEK) and KR-8100P (TOPCON). SE was
calculated as the sphere power plus half of the cylinder power for
To perform the genetic association of high myopia in SCES and
SiMES, we used the definition adopted by the Japan case-control
studies and defined high myopia cases as subjects having
SE#29.0 D in at least one eye, and non high-myopia controls
as samples with SE$23.0 D in both eyes. For children from
SCORM aged 10 to 12 years, cases were defined as SE#26.0 D
for at least one eye, while controls were defined as SE$21.0 D for
both eyes; this is approximately equivalent to the projected SE of
29.0 and 23.0 respectively at university age based on the
estimated annual progression rate in SE of 20.6 D for Chinese
myopic children and 20.3 D in the controls . Given the small
sample sizes of high myopia cases identified in our population-
based cohorts, in the supplementary analysis, we further applied
the commonly adopted criteria of SE#26.0 D in either eye as
cases. Controls were defined as SE$21.0 D in both eyes. For
SCORM children, we retained the same criteria in both analyses.
The detailed definitions of cases and controls are described in
Age, gender, height and level of education were obtained from
all Singapore participants who underwent ophthalmologic exam-
ination. Education was measured on an ordinal scale from no
formal education to the highest educational level. For participants
in SCORM, the education of the child was defined by the level of
educational attainment of the father, as a marker of socioeconomic
All studies followed the principle of the Declaration of Helsinki.
Study procedures and protocols were approved by the Institutional
Review Board of each local institution involved in the study. In all
cohorts, participants provided written, informed consent at the
recruitment into the studies. Informed written consent was
obtained from adult participants, and from the parents of the
Animal study approval was obtained from the SingHealth
IACUC (AAALAC accredited). All procedures performed in this
study complied with the Association of Research in Vision and
Ophthalmology (ARVO) Statement for the Use of Animals in
Ophthalmology and Vision Research.
Chromosome 1q41 Associated with Myopia
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Genotyping and data quality control in discovery cohorts
For SCES, a total of 1,952 venous blood-derived samples were
genotyped using Illumina Human 610 Quad Beadchips (Illumina
Inc., San Diego, US) according to the manufacturer’s protocols.
Samples which failed genotyping or with low call rate (,95%,
n=11), with excessive heterozygosity (defined as sample heterozy-
gosity exceeding 3 standard deviations from the mean sample
heterogzygosity; n=3), with gender discrepancies (n=2) were
excluded, as were cryptically related samples identified by the
identity-by-state (IBS) (n=41) and population structure in the
principal components analyses (PCA) (n=6). The criteria to define
cryptically related samples and outliers with population structure in
the discovery cohorts are described in the following paragraph. After
the removal of the samples, SNP QC was then applied on a total of
579,999 autosomal SNPs for the 1,889 post-QC samples. SNPs were
excluded based on (i) high rates of missingness (.5%) (n=26,437); (ii)
monomorphism or minor allele frequency (MAF),1% (n=59,633);
or (iii) genotype frequencies deviating from Hardy-Weinberg
Equilibrium (HWE) defined as HWE P-value,1026(n=1,821).
This yielded 492,108 autosomal SNPs. Those individuals with
missing data on phenotypes were further removed (n=29). Finally,
492,108 SNPs in 1,860 samples were available for analyses.
For SCORM, 1,116 DNA samples (1,037 from buccal swab and
79 from saliva) were genotyped on the Illumina HumanHap 550
Beadchips and 550 Duo Beadarrays. A total of 108 samples were
excluded, comprising (i) 70 samples with call rates below 98%; (ii)
6 with poor genotyping quality; (iii) 11 samples identified from sib-
ships; (iv) 18 with inconsistent gender information; and (v) 3 due to
population structure. This left a total of 1,008 samples for further
SNP QC. Based on 514,849 autosomal SNPs, we excluded 32,669
markers if they had missing genotype calls .5%, MAF,1%, or
significantly deviated from HWE (P,1026) . A final set of 929
samples with 482,180 post-QC SNPs and completed AL
measurement were included in analyses.
For SiMES, 3,072 DNA samples were genotyped using the
Illumina Human 610 Quad Beadchips. The detailed QC
procedures were provided elsewhere . In brief, we omitted a
total of 530 individuals due to: (i) subpopulation structure
(n=170); (ii) cryptic relatedness (n=279); (iii) excessive heterozy-
gosity or high missingness rate .5% (n=37); and (iv) gender
discrepancy (n=44). After the removal of the samples, SNP QC
was then applied on a total of 579,999 autosomal SNPs for the
2,542 post-QC samples. SNPs were excluded based on: (i) high
rates of missingness (.5%) (n=26,343); (ii) monomorphism or
MAF,1% (n=34,891); or (iii) genotype frequencies deviating
from HWE (P,1026) (n=3,645). This yielded 515,120 SNPs after
the same SNP QC criteria. Individuals without valid measure-
ments for AL were further removed (n=387). After the above
filtering criteria, 515,120 SNPs in 2,155 samples were available for
In our discovery cohorts, IBS was estimated with the genome-
wide SNP data using PLINK software to assess the degree of
recent shared ancestry for a pair of individuals . For a pair of
putatively-related samples defined as an identity by descent (IBD)
value greater than 0.185 , we removed one individual from
each pair of monzygotic twins/duplicates, parent-offspring or full-
siblings etc. Population structure was ascertained using PCA with
the EIGENSTRAT program and genetic outliers were defined as
individuals whose ancestry was at least 6 standard deviations from
the mean on one of the top ten inferred axes of variation .
For SiMES Malays, we also excluded the samples falling in the
main clusters of PCA plots of the Chinese and Indians ethnic groups,
as described in the previous study . In SiMES, we noticed some
degree of admixture in genetic ancestry of Malays and thus adjusted
for ancestry along the top five axes of variation, as the spread of
principal component scores was greater for the top five eigenvectors
in the bivariate plots of PCA (Figure S3), The top ten principal
components explained a small percentage of the global genetic
variability of 1.3% while top five explained 1.0%, suggesting, all
together, they had minimal effects on our association analyses.
Validation cohorts for high myopia
High myopia cases in the Japan dataset 1 were genotyped using
Illumina Human-Hap550 and 660 chips , while controls in the
Japan dataset 1 weregenotyped on Illumina Human-Hap610 chips.
Subjects in the Japan dataset 2 were genotyped on the Affymetrix
GeneChip Human Mapping 500 K Array Set (Affymetrix Inc.,
Santa Clara, US). For SNPs not available on the Affymetric chips
(rs43737678, rs10779363 and rs7544369), genotyping was per-
formed with TaqMan 59 exonuclease assays using primers supplied
by Applied Biosystems (Foster City, US). The probe fluorescence
signal was detected using the TaqMan Assay for Real-Time PCR
(7500 Fast Real-Time PCR System, Applied Biosystems).
Gene expression in a mouse model of myopia
Experimental myopia was induced in B6 wild-type (WT) mice
(n=36) by applying a 215.00 D spectacle lens on the right eye
(experimental eye) for 6 weeks since post-natal day 10. The left eyes
were uncovered and served as contra-lateral fellow eyes. Age
matched naive mice eyes were used as independent control eyes
(n=36). Each eye was refracted weekly using theautomated infrared
photorefractor as described previously . AL was measured by
AC- Master, Optic low coherence interferometry (Carl-Zeiss), in-
vivo at 2, 4 and 6 weeks after the induction of myopia . The
minus-lens-induced eyes after six weeks were significantly associated
with increased AL and myopic shift in refraction of ,25.00 D as
compared to independent control eyes (n=36, P=3.0061026for
AL, and 2.0561024for refraction). Eye tissues were collected at 6
weeks post myopia induction for further analyses.
neural retina (retina), retinal pigment epithelium (RPE) and sclera
for three batches using TRIzol Reagent (Invitrogen, Carlsbad, CA)
with each batch (n=6) comprising the myopic eye, fellow eye and
control eye. RNA concentration and quality were assessed by the
absorbance at 260 nm and the ratio of absorbance ratio at 260 and
280 nmrespectively,usingNanodrop ND-1000Spectrophotometer
(Nanodrop Technologies, Wilmington, DE). RNA was purified
using the RNeasy Mini kit (Qiagen, GmbH).
500 ng of purifed RNA was reverse-transcribed into cDNA
using random primers and reagents from iScriptTM select cDNA
synthesis kit (Bio-rad Laboratories, Hercules, CA). The pseudo-
gene ZC3H11B (zinc finger CCCH type containing 11B) is not
characterized in the mouse genome, therefore we examined a
similar gene ZC3H11A (zinc finger CCCH type containing 11A) in
mice. ZC3H11A in mice and ZC3H11B in humans are highly
conserved with 79% nucleotide similarity by BLAST alignment
analysis (http://blast.ncbi.nlm.nih.gov). We used quantitative
Real-Time PCR (qRT-PCR) to validate the gene expression.
qRT-PCR primers (Table S5) were designed using ProbeFinder
2.45 (Roche Applied Science, Indianapolis, IN) and this was
performed using a Lightcycler 480 Probe Master (Roche Applied
Science, Indianapolis, IN). The reaction was run in a Lightcycler
480 for 45 cycles under the following conditions: 95uC for 10 s,
56uC for 10 s and 72uC for 30 s. Gene expressions in the retina,
RPE and sclera after six weeks of myopic eyes and the fellow eyes
were compared to the control eyes. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as an endogenous internal
Chromosome 1q41 Associated with Myopia
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Whole mouse eyes (6 weeks minus lens treated myopic, contra-
lateral fellow and independent control eyes, n=6 per type) were
embedded in frozen tissue matrix compound at 220uC for 1 hour.
Prepared tissue blocks were sectioned with a cryostat at 6 microns
thicknesses and collected on clean polysineTMglass slides. Slides
with the sections were air dried at room temperature (RT) for
1 hour and fixed with 4% para-formaldehyde for 10 min. After
washing 3X with 1x PBS for 5 minutes, 4% bovine serum albumin
(BSA) diluted with 1x PBS was added as a blocking buffer. The
slideswerethen covered andincubated for 1 hour at RTina humid
chamber. After rinsing with 1x PBS, a specific primary antibody
raised in rabbit against ZC3H11A, SLC30A10 and raised in goat
against LYPLAL1 (Abcam,Cambridge, UK) diluted (1:200) with 4%
BSA was added and incubated further at 4uC in a humid chamber
overnight. After washing 3X with 1x PBS for 10 min, fluorescein-
labeled goat anti-rabbit secondary antibody (1:800, Invitrogen-
Molecular Probes, Eugene, OR) and fluorescein-labeled rabbit anti-
goat secondary antibody (1:800, Santa Cruz Biotechnology, Inc.
CA, USA) was applied respectively and incubated for 90 min at
RT. After washing and air-drying, slides were mounted with
antifade medium containing DAPI (4,6-diamidino-2-phenylindole;
Vectashield, Vector Laboratories, Burlingame, CA) to visualize the
cell nuclei. Sections incubated with 4% BSA and omitted primary
(Axioplan 2; Carl Zeiss Meditec GmbH, Oberkochen, Germany)
was used to examine the slides and capture images. Experiments
were repeated in duplicates from three different samples.
Gene expression in human tissues
GAPDH, ZC3H11B, SLC30A10, and LYLPLAL1 were run using
10 ul reactions with Qiagen’s PCR products consisting of 1.26 ul
H2O, 1.0 ul 10X buffer, 1.0 ul dNTPs, 0.3 ul MgCl, 2.0 ul Q-
Solution, 0.06 ul taq polymerase, 1.0 ul forward primer, 1.0 ul
reverse primer and 1.5.0 ul cDNA. The reactions were run on a
Eppendorf Mastercycler Pro S thermocycler with touchdown PCR
ramping down 1uC per cycle from 72uC to 55uC followed by 50
cycles of 94uC for 0:30, 55uC for 0:30 and 72uC for 0:30 with a
final elongation of 7:00 at 72uC. All primer sets were designed
using Primer3 . The gel electrophoresis was run on a 2%
agarose gel at 70 volts for 35 minutes. The primers were run on a
custom tissue panel including Clontech’s Human MTC Panel I,
Fetal MTC Panel I and an ocular tissue panel. The adult ocular
samples were obtained from normal eyes of an 82-year-old
Caucasian female from the North Carolina Eye Bank, Winston-
Salem, North Carolina, USA. The fetal ocular samples were from
24-week fetal eyes obtained by Advanced Bioscience Resources
Inc., Alameda, California, USA. All adult ocular samples were
stored in Qiagen’s RNAlater within 6.5 hours of collection and
shipped on ice overnight to the lab. Fetal eyes were preserved in
RNAlater within minutes of harvesting and shipped over night on
ice. Whole globes were dissected on the arrival day. Isolated tissues
were snap-frozen and stored at 280uC until RNA extraction.
RNA was extracted from each tissue sample independently using
the Ambion mirVana total RNA extraction kit. The tissue samples
were homogenized in Ambion lysis buffer using an Omni Bead
Ruptor Tissue Homogenizer per protocol. Reverse transcription
reactions were performed with Invitrogen SuperScript III First-
Strand Synthesis kit.
The primary analysis was performed on the AL quantitative
trait. As a strong correlation exists in AL measurements from both
eyes (r.0.9), we used the mean AL across both eyes in the GWAS
analysis, as was recommended in a review . Linear regression
was used to interrogate the association of each SNP with AL after
adjusting for age, gender, height and level of education, under the
assumption of an additive genetic effect where the genotypes of
each SNP are coded numerically as 0, 1 and 2 for the number of
minor alleles carried. In addition, for SiMES, the top five principal
components of genetic ancestry from the EIGENSTRAT PCA
were also included as covariates to account for the effects of
population substructure as described in genotype QC section .
Association tests between each genetic marker and phenotype
were carried out using PLINK software  (version 1.07).
Analyses were also repeated without adjustment for education
level or height for the purpose of comparison.
In the discovery phase, we conducted a meta-analysis of GWAS
results from 3 cohorts for AL using a weighted-inverse variance
approach by fixed-effect modeling in METAL (http://www.sph.
umich.edu/csg/abecasis/metal). In the secondary analyses, SNPs
that have been identified from the primary analyses were tested for
association with high myopia onset (as a binary trait) and SE (as a
quantitative trait). For Singapore cohorts, the association analyses
adjusted for the same covariates as the primary analyses within a
linear regression and logistic regression framework respectively.
For Japan case-control datasets, only age and gender were
included as covariates in the model for high myopia, as the other
covariates were not available.
The regional association plots were constructed by SNAP
(http://www.broad.mit.edu/mpg/haploview) was used to visual-
ize the LD of the genomic regions. Genotyping quality of all
reported SNPs has been visually evaluated by the intensity
clusterplots. The coordinates reported in this paper are on
For functional studies in the myopic mouse model, gene
expression of all three identified genes in control and experimental
groups was quantified using the 22DDCtmethod . The
standard student’s t-test was performed to determine the
significance of the relative fold change of mRNA between the
myopic eyes of the experimental mice with the independent age-
cohorts SCES, SCORM and SiMES with respect to the four
population panels in phase 2 of the HapMap samples (CEU -
European,YRI – African,CHB–Chinese,JPT–Japanese)(A),and
with respect to two reference population panels CHB and JPT (B–
D). (A) Principal components 1 versus 2; the principal components
(PCs) were calculated with SCES, SCORM, SiMES and four
HapMap panels on the thinned set of 102,122 SNPs (r2,0.2). (B)
Principal components 1 versus 2; (C) Principal components 1 versus
3; (D) Principal components 1 versus 4. For (B–D), the PCs were
calculated with SCES, SCORM, SiMES and HapMap Asian
population panels on the thinned set of 86,516 SNPs (r2,0.2).
Principal Component Analysis (PCA) of discovery
association between all SNPs and AL in the individual cohort
(A) SCES, (B) SCORM, (C) SiMES, and combined meta-analysis
of the discovery cohorts (D) SCES+SCORM+SiMES.
Quantile-Quantile (Q-Q) plots of P-values for
in SiMES to assess the extent of population structure. Each figure
represents a bivariate plot of two principal components from the
Principal Component Analysis (PCA) was performed
Chromosome 1q41 Associated with Myopia
PLoS Genetics | www.plosgenetics.org12 June 2012 | Volume 8 | Issue 6 | e1002753
PCA of genetic diversity within SiMES on the thinned set of
83,585 SNPs (r2,0.2). The first 5 principal components were used
as covariates to account for population structure.
three Singapore cohorts.
Characteristics of high myopia cases and controls in
1q41 and high myopia in the meta-analysis of five cohorts.
Association between genetic variants at chromosome
1q41 and spherical equivalent (SE) in the meta-analysis of three
Association between genetic variants at chromosome
controls used in the main and supplementary association analyses
for high myopia.
Definitions and numbers of high-myopia cases and
database (NCBI), and qRT-PCR primer sequences in mice
Gene accession number in the nucleotide sequence
We wish to express our gratitude to all the normal subjects and patients
who volunteered to take part in this study. We acknowledge the Genome
Institute of Singapore for genotyping all the samples collected from the
cohort studies in Singapore and the research team of the Singapore Eye
Research Institute who phenotyped the subjects.
Conceived and designed the experiments: TLY T-YW Y-YT S-MS.
Performed the experiments: VAB AM IN WL CEHH FH YZ DC HI
KY. Analyzed the data: QF VAB XZ C-YC. Contributed reagents/
materials/analysis tools: C-CK L-KG Y-JL KO-M KM FM EV MS NM
RWB E-ST NY TA TLY T-YW Y-YT S-MS. Wrote the paper: QF VAB
Y-YT S-MS. Critically reviewed the manuscript: C-YC C-CK L-KG E-
ST TLY T-YW.
1. Pan CW, Ramamurthy D, Saw SM (2012) Worldwide prevalence and risk
factors for myopia. Ophthalmic Physiol Opt 32: 3–16.
Saw SM, Chua WH, Gazzard G, Koh D, Tan DT, et al. (2005) Eye growth
changes in myopic children in Singapore. Br J Ophthalmol 89: 1489–1494.
Wong TY, Foster PJ, Hee J, Ng TP, Tielsch JM, et al. (2000) Prevalence and risk
factors for refractive errors in adult Chinese in Singapore. Invest Ophthalmol
Vis Sci 41: 2486–2494.
Saw SM, Gazzard G, Shih-Yen EC, Chua WH (2005) Myopia and associated
pathological complications. Ophthalmic Physiol Opt 25: 381–391.
McBrien NA, Gentle A (2003) Role of the sclera in the development and
pathological complications of myopia. Prog Retin Eye Res 22: 307–338.
Saw SM, Katz J, Schein OD, Chew SJ, Chan TK (1996) Epidemiology of
myopia. Epidemiol Rev 18: 175–187.
Hammond CJ, Snieder H, Gilbert CE, Spector TD (2001) Genes and
environment in refractive error: the twin eye study. Invest Ophthalmol Vis Sci
Klein AP, Suktitipat B, Duggal P, Lee KE, Klein R, et al. (2009) Heritability
analysis of spherical equivalent, axial length, corneal curvature, and anterior
chamber depth in the Beaver Dam Eye Study. Arch Ophthalmol 127:
Lyhne N, Sjolie AK, Kyvik KO, Green A (2001) The importance of genes and
environment for ocular refraction and its determiners: a population based study
among 20–45 year old twins. Br J Ophthalmol 85: 1470–1476.
10. Sanfilippo PG, Hewitt AW, Hammond CJ, Mackey DA (2010) The heritability
of ocular traits. Surv Ophthalmol 55: 561–583.
11. Solouki AM, Verhoeven VJ, van Duijn CM, Verkerk AJ, Ikram MK, et al.
(2010) A genome-wide association study identifies a susceptibility locus for
refractive errors and myopia at 15q14. Nat Genet 42: 897–901.
12. Hysi PG, Young TL, Mackey DA, Andrew T, Fernandez-Medarde A, et al.
(2010) A genome-wide association study for myopia and refractive error
identifies a susceptibility locus at 15q25. Nat Genet 42: 902–905.
13. Nakanishi H, Yamada R, Gotoh N, Hayashi H, Yamashiro K, et al. (2009) A
genome-wide association analysis identified a novel susceptible locus for
pathological myopia at 11q24.1. PLoS Genet 5: e1000660. doi:10.1371/
14. Li YJ, Goh L, Khor CC, Fan Q, Yu M, et al. (2011) Genome-wide association
studies reveal genetic variants in CTNND2 for high myopia in Singapore
Chinese. Ophthalmology 118: 368–375.
15. Li Z, Qu J, Xu X, Zhou X, Zou H, et al. (2011) A genome-wide association
study reveals association between common variants in an intergenic region of
4q25 and high-grade myopia in the Chinese Han population. Hum Mol Genet
16. Shi Y, Qu J, Zhang D, Zhao P, Zhang Q, et al. (2011) Genetic variants at
13q12.12 are associated with high myopia in the han chinese population.
Am J Hum Genet 88: 805–813.
17. Saw SM, Shankar A, Tan SB, Taylor H, Tan DT, et al. (2006) A cohort study of
incident myopia in Singaporean children. Invest Ophthalmol Vis Sci 47:
18. Dirani M, Shekar SN, Baird PN (2008) Evidence of shared genes in refraction
and axial length: the Genes in Myopia (GEM) twin study. Invest Ophthalmol
Vis Sci 49: 4336–4339.
19. Biino G, Palmas MA, Corona C, Prodi D, Fanciulli M, et al. (2005) Ocular
refraction: heritability and genome-wide search for eye morphometry traits in an
isolated Sardinian population. Hum Genet 116: 152–159.
20. Zhu G, Hewitt AW, Ruddle JB, Kearns LS, Brown SA, et al. (2008) Genetic
dissection of myopia: evidence for linkage of ocular axial length to chromosome
5q. Ophthalmology 115: 1053–1057 e1052.
21. Leung KW, Liu M, Xu X, Seiler MJ, Barnstable CJ, et al. (2008) Expression of
ZnT and ZIP zinc transporters in the human RPE and their regulation by
neurotrophic factors. Invest Ophthalmol Vis Sci 49: 1221–1231.
22. Liang J, Song W, Tromp G, Kolattukudy PE, Fu M (2008) Genome-wide survey
and expression profiling of CCCH-zinc finger family reveals a functional module
in macrophage activation. PLoS ONE 3: e2880. doi:10.1371/journal.
23. Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, et al. (2010) A coding-
independent function of gene and pseudogene mRNAs regulates tumour
biology. Nature 465: 1033–1038.
24. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP (2011) A ceRNA Hypothesis:
The Rosetta Stone of a Hidden RNA Language? Cell 146: 353–358.
25. D’Errico I, Gadaleta G, Saccone C (2004) Pseudogenes in metazoa: origin and
features. Brief Funct Genomic Proteomic 3: 157–167.
26. Shi Y, Li Y, Zhang D, Zhang H, Lu F, et al. (2011) Exome sequencing identifies
ZNF644 mutations in high myopia. PLoS Genet 7: e1002084. doi:10.1371/
27. Schippert R, Burkhardt E, Feldkaemper M, Schaeffel F (2007) Relative axial
myopia in Egr-1 (ZENK) knockout mice. Invest Ophthalmol Vis Sci 48: 11–17.
28. Laity JH, Lee BM, Wright PE (2001) Zinc finger proteins: new insights into
structural and functional diversity. Curr Opin Struct Biol 11: 39–46.
29. Fischer AJ, McGuire JJ, Schaeffel F, Stell WK (1999) Light- and focus-
dependent expression of the transcription factor ZENK in the chick retina. Nat
Neurosci 2: 706–712.
30. Bitzer M, Schaeffel F (2002) Defocus-induced changes in ZENK expression in
the chicken retina. Invest Ophthalmol Vis Sci 43: 246–252.
31. Simon P, Feldkaemper M, Bitzer M, Ohngemach S, Schaeffel F (2004) Early
transcriptional changes of retinal and choroidal TGFbeta-2, RALDH-2, and
ZENK following imposed positive and negative defocus in chickens. Mol Vis 10:
32. Liu C, Adamson E, Mercola D (1996) Transcription factor EGR-1 suppresses
the growth and transformation of human HT-1080 fibrosarcoma cells by
induction of transforming growth factor beta 1. Proc Natl Acad Sci U S A 93:
33. Baron V, Adamson ED, Calogero A, Ragona G, Mercola D (2006) The
transcription factor Egr1 is a direct regulator of multiple tumor suppressors
including TGFbeta1, PTEN, p53, and fibronectin. Cancer Gene Ther 13:
34. Khor CC, Fan Q, Goh L, Tan D, Young TL, et al. (2010) Support for TGFB1
as a susceptibility gene for high myopia in individuals of Chinese descent. Arch
Ophthalmol 128: 1081–1084.
35. Zha Y, Leung KH, Lo KK, Fung WY, Ng PW, et al. (2009) TGFB1 as a
susceptibility gene for high myopia: a replication study with new findings. Arch
Ophthalmol 127: 541–548.
36. Seve M, Chimienti F, Devergnas S, Favier A (2004) In silico identification and
expression of SLC30 family genes: an expressed sequence tag data mining
strategy for the characterization of zinc transporters’ tissue expression. BMC
Genomics 5: 32.
37. van Leeuwen R, Boekhoorn S, Vingerling JR, Witteman JC, Klaver CC, et al.
(2005) Dietary intake of antioxidants and risk of age-related macular
degeneration. JAMA 294: 3101–3107.
38. Ugarte M, Osborne NN (2001) Zinc in the retina. Prog Neurobiol 64: 219–249.
Chromosome 1q41 Associated with Myopia
PLoS Genetics | www.plosgenetics.org13 June 2012 | Volume 8 | Issue 6 | e1002753
39. Huibi X, Kaixun H, Qiuhua G, Yushan Z, Xiuxian H (2001) Prevention of axial Download full-text
elongation in myopia by the trace element zinc. Biol Trace Elem Res 79: 39–47.
40. Steinberg GR, Kemp BE, Watt MJ (2007) Adipocyte triglyceride lipase
expression in human obesity. Am J Physiol Endocrinol Metab 293: E958–964.
41. Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, et al. (2010) Meta-
analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual
dimorphism in the genetic basis of fat distribution. Nat Genet 42: 949–960.
42. Lindgren CM, Heid IM, Randall JC, Lamina C, Steinthorsdottir V, et al. (2009)
Genome-wide association scan meta-analysis identifies three Loci influencing
adiposity and fat distribution. PLoS Genet 5: e1000508. doi:10.1371/
43. Cordain L, Eaton SB, Brand Miller J, Lindeberg S, Jensen C (2002) An
evolutionary analysis of the aetiology and pathogenesis of juvenile-onset myopia.
Acta Ophthalmol Scand 80: 125–135.
44. Cordain L, Eades MR, Eades MD (2003) Hyperinsulinemic diseases of
civilization: more than just Syndrome X. Comp Biochem Physiol A Mol Integr
Physiol 136: 95–112.
45. Lim LS, Gazzard G, Low YL, Choo R, Tan DT, et al. (2010) Dietary factors,
myopia, and axial dimensions in children. Ophthalmology 117: 993–997 e994.
46. Gao H, Frost MR, Siegwart JT, Jr., Norton TT (2011) Patterns of mRNA and
protein expression during minus-lens compensation and recovery in tree shrew
sclera. Mol Vis 17: 903–919.
47. Klein AP, Duggal P, Lee KE, Klein R, Bailey-Wilson JE, et al. (2007)
Confirmation of linkage to ocular refraction on chromosome 22q and
identification of a novel linkage region on 1q. Arch Ophthalmol 125: 80–85.
48. Klein AP, Duggal P, Lee KE, Cheng CY, Klein R, et al. (2011) Linkage analysis
of quantitative refraction and refractive errors in the beaver dam eye study.
Invest Ophthalmol Vis Sci 52: 5220–5225.
49. Wong TY, Foster PJ, Johnson GJ, Seah SK (2003) Refractive errors, axial ocular
dimensions, and age-related cataracts: the Tanjong Pagar survey. Invest
Ophthalmol Vis Sci 44: 1479–1485.
50. Wu R, Wang JJ, Mitchell P, Lamoureux EL, Zheng Y, et al. (2010) Smoking,
socioeconomic factors, and age-related cataract: The Singapore Malay Eye
study. Arch Ophthalmol 128: 1029–1035.
51. Tokoro T (1988) On the definition of pathologic myopia in group studies. Acta
Ophthalmol Suppl 185: 107–108.
52. Plomin R, Haworth CM, Davis OS (2009) Common disorders are quantitative
traits. Nat Rev Genet 10: 872–878.
53. Hayashi H, Yamashiro K, Nakanishi H, Nakata I, Kurashige Y, et al. (2011)
Association of 15q14 and 15q25 with High Myopia in Japanese. Invest
Ophthalmol Vis Sci.
54. Dirani M, Shekar SN, Baird PN (2008) Adult-onset myopia: the Genes in
Myopia (GEM) twin study. Invest Ophthalmol Vis Sci 49: 3324–3327.
55. Jensen H (1995) Myopia in teenagers. An eight-year follow-up study on myopia
progression and risk factors. Acta Ophthalmol Scand 73: 389–393.
56. McCarthy MI, Hirschhorn JN (2008) Genome-wide association studies:
potential next steps on a genetic journey. Hum Mol Genet 17: R156–165.
57. Teo YY, Small KS, Fry AE, Wu Y, Kwiatkowski DP, et al. (2009) Power
consequences of linkage disequilibrium variation between populations. Genet
Epidemiol 33: 128–135.
58. Ip JM, Huynh SC, Kifley A, Rose KA, Morgan IG, et al. (2007) Variation of the
contribution from axial length and other oculometric parameters to refraction by
age and ethnicity. Invest Ophthalmol Vis Sci 48: 4846–4853.
59. Lavanya R, Jeganathan VS, Zheng Y, Raju P, Cheung N, et al. (2009)
Methodology of the Singapore Indian Chinese Cohort (SICC) eye study:
quantifying ethnic variations in the epidemiology of eye diseases in Asians.
Ophthalmic Epidemiol 16: 325–336.
60. Fan Q, Zhou X, Khor CC, Cheng CY, Goh LK, et al. (2011) Genome-wide
meta-analysis of five Asian cohorts identifies PDGFRA as a susceptibility locus
for corneal astigmatism. PLoS Genet 7: e1002402. doi:10.1371/journal.
61. Foong AW, Saw SM, Loo JL, Shen S, Loon SC, et al. (2007) Rationale and
methodology for a population-based study of eye diseases in Malay people: The
Singapore Malay eye study (SiMES). Ophthalmic Epidemiol 14: 25–35.
62. Vithana EN, Aung T, Khor CC, Cornes BK, Tay WT, et al. (2011) Collagen-
related genes influence the glaucoma risk factor, central corneal thickness. Hum
Mol Genet 20: 649–658.
63. Khor CC, Ramdas WD, Vithana EN, Cornes BK, Sim X, et al. (2011) Genome-
wide association studies in Asians confirm the involvement of ATOH7 and
TGFBR3, and further identify CARD10 as a novel locus influencing optic disc
area. Hum Mol Genet 20: 1864–1872.
64. Grosvenor TP (2007) Primary care optometry. St. Louis, Mo: Butterworth-
Heinemann/Elsevier. xiii, 510 p.
65. Schork NJ, Nath SK, Fallin D, Chakravarti A (2000) Linkage disequilibrium
analysis of biallelic DNA markers, human quantitative trait loci, and threshold-
defined case and control subjects. Am J Hum Genet 67: 1208–1218.
66. Saw SM, Chan YH, Wong WL, Shankar A, Sandar M, et al. (2008) Prevalence
and risk factors for refractive errors in the Singapore Malay Eye Survey.
Ophthalmology 115: 1713–1719.
67. Fan DS, Lam DS, Lam RF, Lau JT, Chong KS, et al. (2004) Prevalence,
incidence, and progression of myopia of school children in Hong Kong. Invest
Ophthalmol Vis Sci 45: 1071–1075.
68. Sim X, Ong RT, Suo C, Tay WT, Liu J, et al. (2011) Transferability of type 2
diabetes implicated loci in multi-ethnic cohorts from Southeast Asia. PLoS
Genet 7: e1001363. doi:10.1371/journal.pgen.1001363.
69. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007)
PLINK: a tool set for whole-genome association and population-based linkage
analyses. Am J Hum Genet 81: 559–575.
70. Anderson CA, Pettersson FH, Clarke GM, Cardon LR, Morris AP, et al. (2010)
Data quality control in genetic case-control association studies. Nat Protoc 5:
71. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, et al. (2006)
Principal components analysis corrects for stratification in genome-wide
association studies. Nat Genet 38: 904–909.
72. Schaeffel F, Burkhardt E, Howland HC, Williams RW (2004) Measurement of
refractive state and deprivation myopia in two strains of mice. Optom Vis Sci 81:
73. Barathi VA, Boopathi VG, Yap EP, Beuerman RW (2008) Two models of
experimental myopia in the mouse. Vision Res 48: 904–916.
74. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 132: 365–386.
75. Fan Q, Teo YY, Saw SM (2011) Application of advanced statistics in
ophthalmology. Invest Ophthalmol Vis Sci 52: 6059–6065.
76. Brink N, Szamel M, Young AR, Wittern KP, Bergemann J (2000) Comparative
quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in
UV-irradiated human skin in vivo. Inflamm Res 49: 290–296.
Chromosome 1q41 Associated with Myopia
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