AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia

Abstract

The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls.
High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities.
No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths.
Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.

Full text

AC and AG dinucleotide repeats in the PAX6 P1 promoter are
associated with high myopia
Tsz Kin Ng,1 Ching Yan Lam,1 Dennis Shun Chiu Lam,1 Sylvia Wai Yee Chiang,1 Pancy Oi Sin Tam,1
Dan Yi Wang,1,2 Bao Jian Fan,1,2 Gary Hin-Fai Yam,1 Dorothy Shu Ping Fan,1 Chi Pui Pang1
(The first two authors contributed equally to this work.)
1Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong S.A.R.; 2Present affiliation:
Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA
Purpose: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be
associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high
myopia patients and 349 controls.
Methods: High myopia patients had refractive errors of –6.00 diopters or greater and axial length longer than 26 mm.
Control subjects had refractive errors less than –1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all
coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase
chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths
were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities.
Results: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two
dinucleotide repeats, (AC)m and (AG)n, in the P1 promoter region were found to be highly polymorphic and significantly
associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)m (empirical
p = 0.013) and (AG)n (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with
the (AG)n repeat (empirical p = 0.016; 95% confidence interval: 1.059–1.663). Luciferase-reporter analysis showed
elevated transcription activity with increasing individual (AC)m and (AG)n and combined (AC)m(AG)n repeat lengths.
Conclusions: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in
the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.
Myopia, one of the most common refractive errors of the
eye worldwide, is an important public health issue, especially
in Asia, because of its higher prevalence in Asians than in
other populations [1]. The progression of myopia in Chinese
children in Hong Kong and Singapore is also much higher than
in Caucasians [2,3]. In Hong Kong, the prevalence of myopia
in Chinese schoolchildren aged 11–16 was 36.7%, according
to a 2004 report, which is several times higher than among
Caucasian children of similar ages [4]. The prevalence of high
myopia, defined as a refractive error equal to or greater than
–6.00 diopters (D), is also higher in Chinese than in
Caucasians [5,6]. Individuals with high myopia are more
prone to develop serious ocular complications, such as retinal
detachment, glaucoma, premature cataracts, and macular
degeneration, which may lead to visual impairment or even
blindness [7-10].
Myopia is a complex disorder. Multiple interacting
environmental and genetic causes are implicated. Myopia
Correspondence to: Prof. C.P. Pang, Department of Ophthalmology
& Visual Sciences, The Chinese University of Hong Kong,
University Eye Center, Hong Kong Eye Hospital, 147K Argyle
Street, Kowloon, Hong Kong; Phone: +852 27623129; FAX: +852
27159490; email: cppang@cuhk.edu.hk
development in schoolchildren has been attributed to
environmental factors, such as near work, reading habits, and
school achievement [3,11,12]. In addition, high heritability of
refractive errors has been observed in dizygotic and
monozygotic twin studies [13-17]. Family and sibling studies
have shown that children of myopic parents have greater
chances of developing myopia than those with nonmyopic
parents [11,18]. Twenty-four chromosomal loci have been
identified for myopia: Xq28 (MYP1) [19], 18p11.31 (MYP2)
[20,21], 12q21-31 (MYP3) [22], 7q36 (MYP4) [23], 17q21-22
(MYP5) [24], 22q37.1 (MYP6) [25], 11p13 (MYP7) [26], 3q26
(MYP8) [26], 4q12 (MYP9) [26], 8p23 (MYP10) [26], 4q22-
q27 (MYP11) [27], 2q37.1 (MYP12) [28], Xq23 (MYP13)
[29], 1p36 (MYP14) [30], 10q21.2 (MYP15) [31], 5p15.33-
p15.2 (MYP16) [32], 7p15 (MYP17) [33,34], 14q22.1-q24.2
(MYP18) [35], 15q12-13 [36], 21q22.3 [37], 12q24 [38], 4q21
[38], 9q34.11 [39] and 2q37 [40]. Among them, MYP1–5,11–
13,16, and 18 are linked to high myopia, and MYP2,11,13,
16, and 18 are found in the Chinese population. Some
candidate genes have been postulated for myopia, such as
TGIF [41], HGF [42], MMP3 [43], MMP9 [43], COL1A1
[44], COL2A1 [45], TGFB1 [46], TGFB2 [47], LUM [48], and
CMET [49].
Molecular Vision 2009; 15:2239-2248 <http://www.molvis.org/molvis/v15/a241>
Received 10 August 2009 | Accepted 27 October 2009 | Published 5 November 2009
© 2009 Molecular Vision
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A genome-wide scan in dizygotic twins revealed a
susceptibility locus for myopia on chromosome 11p13 [26].
The PAX6 gene at this locus, a member of the paired-domain
PAX family, has been postulated as a candidate gene for
myopia. PAX6 is expressed in the human eye [50] and plays
an evolutionarily conserved role in ocular development
[51-53]. PAX6 mutations are associated with ocular disorders,
such as aniridia (OMIM 106210), cataracts (OMIM 604219),
Peters anomaly (OMIM 604229), and optic nerve hypoplasia
(OMIM 16550). PAX6 encodes a transcriptional regulator
containing the DNA-binding paired domain, paired-type
homeodomain, and COOH-terminal transactivation domain.
The Pax6 protein regulates cell adhesion molecules, cell-to-
cell signaling molecules, hormones, and structural proteins
[54] through interactions with transcription factors such as
Mitf [55] and Sox2 [56]. Transcription of PAX6 is regulated
by at least two promoters, P0 and P1 [57-60]. Within the P1
promoter (promoter B in Okladnova et al. [59]), two
dinucleotide repeats, (AC)m and (AG)n, are located about 1 kb
from the transcription start site [58] and are highly
polymorphic in Caucasians. The poly AC and poly AG repeats
are independently polymorphic [60]. Luciferase analysis in
Cos-7 cells has shown that the longer the combined length of
the AC and AG repeats, the higher the transcriptional activity,
implying that the length of this dinucleotide repeat might
influence the transcriptional activity of promoter B, or P1, and
subsequently the transcription of PAX6.
Pax6 levels are tightly controlled. Both overexpression
and haploinsufficiency lead to abnormal phenotypes [61-63].
Polymorphisms or mutations in the PAX6 promoter could
influence PAX6 expressions that ultimately lead to a disease
phenotype. However, although PAX6 has been postulated to
be a candidate gene for myopia, several studies in Caucasian
populations could not find an association between PAX6 and
myopia [26,45,64]. Still, an Australian study suggested
PAX6 mutations might be associated with high myopia [65].
Intronic sequence alterations (SNPs) in PAX6 have been
reported to associate with high myopia in Han Chinese nuclear
families [66] and with extreme myopia in a Taiwan Chinese
population [67], but not in Caucasians. To attest the
association between PAX6 and high myopia, we should look
for mutations that may affect PAX6 expressions. We therefore
screened for sequence alterations in the P1 promoter, coding
exons, and adjacent splice-site regions of PAX6 in unrelated
high myopia patients and control subjects. We also examined
transcriptional effects of dinucleotide repeats within the P1
promoter in cultured human APRE-19 cells by a luciferase-
reporter assay and predicted the presence of transcription
factor binding sites within the repeats.
METHODS
Study subjects: We recruited 379 unrelated Han Chinese
patients with high myopia at the Hong Kong Eye Hospital.
They were given complete ophthalmoscopic examinations.
None of them had known diseases predisposing them to
myopia, such as Stickler or Marfan syndromes. Their
refractive errors were equal to or greater than –6.00 D, and
their axial length was longer than 26 mm. We also recruited
349 unrelated Chinese control subjects who visited the
hospital for ophthalmic examinations. They had no eye
diseases except senile cataracts and slight floaters. All of them
had refractive errors of less than –1.00 D and axial length
shorter than 24 mm. The study protocol was approved by the
Ethics Committee for Human Research at the Chinese
University of Hong Kong and was in accordance with the
tenets of the Declaration of Helsinki. Informed consent was
obtained from the study subjects after explanation of the
nature and possible consequences of the study.
Construction of PAX6 P1 promoter-luciferase constructs: A
1,851 bp genomic fragment (from –1278 to +573) containing
the PAX6 P1 promoter was cloned into an empty pGL3-Basic
vector, pGL3 (Promega, Madison, WI) between the SacI and
BglII sites (OriGene Technologies, Rockville, MD).
Constructs with different repeat lengths were generated.
Genomic DNA from the study subjects was amplified by PCR
(forward primer 5'-ACA CAC AGA TGA CCG GTG G-3';
reverse primer 5'-AAG CCT AGG CCG AGA GGA-3'). AgeI
and AvrII digested products were ligated into a linearized
pGL3-Basic vector containing the P1 promoter (pGL3-
Pax6p). A positive control construct was made by cloning a
pCMV5 promoter [68] into the pGL3-Basic vector (pGL3-
pCMV). All constructs were verified by direct sequencing.
Cell culture and transfection: The human retinal pigment
epithelial cell line ARPE-19 (American Type Culture
Collection, Manassas, VA) [69] was cultured in Dulbecco’s
modified Eagle’s medium and F-12 nutrient mixture
Molecular Vision 2009; 15:2239-2248 <http://www.molvis.org/molvis/v15/a241> © 2009 Molecular Vision
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PAX6 genotyping: The whole blood specimens (5 ml) from
all the patients and controls were collected in EDTA tube and
stored at -80 °C for fewer than two months. Genomic DNA
was extracted (QIAamp DNA kit; Qiagen, Hiden, Germeny)
according to the supplier’s instructions. All samples were
screened for sequence alterations in the P1 promoter region
flanking –3,433 to –118, coding exons, and intron-exon
boundaries of PAX6 (ENSG00000007372 and
ENST00000241001; Ensembl genome browser) by
polymerase chain reaction (PCR) with primer sets [61]. PCR
was performed in a final volume of 25 μl containing 1X PCR
buffer (Invitrogen™ Life Technology, Carlsbad, CA), 1.5
mM MgCl2, 0.2 mM of dNTP (Roche, Indianapolis, IN), 0.2
mM of each primers, 0.5 U of Platinum® Taq DNA
polymerase (Invitrogen). After the initial denaturation at 95
°C for 2 min, 40 PCR cycles were conducted: 95 °C for 45 s,
57 °C for 45 s and 72 °C for 45 s. The final extension lasted
for 5 min at 72 °C. Direct sequencing was performed using a
BigDye Terminator Cycle Sequencing Reaction Kit (v3.1,
Applied Biosystems, Foster City, CA) on an ABI 3130XL
capillary DNA sequencer (Applied Biosystems).

supplemented with 10% fetal bovine serum (Gibco BRL,
Rockville, MD). Cells were plated in 60 mm tissue culture
dishes at a density of 2–3×105 cells/dish one day before
transfection. At 60–80% confluence, cells were transfected
with 2 μg luciferase constructs in 6 μl FuGene HD (Roche)
transfection reagent per dish. Empty pGL3 and pGL3-pCMV
were used as negative and positive controls, respectively. At
36 h after transfection, cell lysates were extracted using Cell
Culture Lysis Reagent (Promega, Madison, WI) for
immunoblotting.
Immunoblotting: The denatured cell lysates of the transfected
cells were resolved on 10% SDS-polyacrylamide gel and
electro-transferred to nitrocellulose membranes for probing
with a rabbit polyclonal primary antibody against firefly
luciferase (Sigma-Aldrich, St. Louis, MO) and a secondary
antibody against rabbit IgG conjugated with horseradish
peroxidase (Jackson Immuno Res., West Grove, PA). The
chemiluminescence was detected by an enhanced
chemiluminescence system (Amersham Pharmacia,
Cleveland, OH) and quantified by ChemiDoc (BioRad,
Hercules, CA). Normalized luciferase intensities were
calculated by dividing the quantified luciferase intensities by
the housekeeping β-actin intensities. Triplicates were
performed.
Statistical analysis: The χ2 test or Fisher exact test was used
to compare the allele and genotype frequencies of SNPs in
patients and control subjects. For the comparison of (AC)m
and (AG)n repeat alleles and genotypes between high myopia
patients and control subjects, the χ2 test was performed using
the CLUMP program (version 2.3) [70]. For multiple testing
corrections, 10,000 Monte Carlo permutations were chosen to
simulate the empirical significance levels of the statistics
produced by the program, resulting in an empirical p-value.
Due to low frequencies of some alleles, and in order to
determine whether the transcriptional activities were affected
by the thresholds, (AC)m and (AG)n repeats were collapsed
into groups for association study and immunoblotting analysis
[71,72]. The risk of high myopia was also determined by odds
ratio using the χ2 test. Activity of each allelic construct was
expressed relative to (AC)20(AG)6. One-way ANOVA and
independent T-testing were used to compare the means among
(AC)m groups and between (AG)n repeats, respectively. SNP-
trait association, odds ratio calculation, and immunoblotting
analysis were performed on SPSS version 16.0 (SPSS
Science, Chicago, IL). Significance was defined as p < 0.05.
Transcription factor binding site prediction: The DNA
sequence of the cloned PAX6 P1 promoter was used to predict
transcription factor binding sites. The Transcription Element
Search System (TESS: University of Pennsylvania,
Philadelphia, PA) [73,74] was used to predict the transcription
factors that would bind to the region of the dinucleotide
repeats in the PAX6 P1 promoter. Predictions for different
lengths of dinucleotide repeats were also performed. As in the
statistical analysis for immunoblotting, (AC)20(AG)6 was set
Within the PAX6 P1 promoter, two dinucleotide repeats,
(AC)m and (AG)n, were observed about 1 kb from the
transcription start site, both highly polymorphic (Table 1).
The AC repeats ranged from 16 to 26 in high myopia patients
and from 7 to 26 in control subjects, while 5 to 8 AG repeats
were observed in patients and 4 to 8 in controls. The median
numbers of AC and AG repeats were 20 and 6, respectively,
in both patients and controls. Distribution of the allele
frequencies was slightly skewed in patients for both AC and
AG repeats. Allele frequencies of the AC and AG repeats were
significantly different between patients and controls
(empirical p = 0.013 and 0.012, respectively; Table 1).
Because the frequencies of some of the alleles were low, the
AC and AG repeats were collapsed into groups. The grouped
repeat lengths were longer in patients than in controls
(empirical p = 0.016 for (AC)m and empirical p = 0.016 for
(AG)n; Table 2). In terms of risk analysis, individuals with
(AG)7-8 repeats had a 1.327-fold increased risk of developing
high myopia compared with the those with (AG)4-6 repeats
(empirical p = 0.016; 95% confidence interval = 1.059–
1.663). Both grouped AC and grouped AG genotypes were
significantly different between high myopia patients and
control (empirical p = 0.004 and 0.039, respectively; Table 3).
We found that the dinucleotide repeats affected the
transcriptional activity of the PAX6 P1 promoter (Figure 1).
For a given (AG)n repeat length, elevated transcriptional
Molecular Vision 2009; 15:2239-2248 <http://www.molvis.org/molvis/v15/a241> © 2009 Molecular Vision
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RESULTS
In our study cohort, high myopia patients had a mean age of
39.52±14.96 years and a male-to-female ratio of 1.2:1.
Refractive errors ranged from –6.00 to –30.00 D. For the
controls, the mean age was 64.85±14.85 years, with a male-
to-female ratio of 1.6:1. There was no significant difference
in the sex ratio between high myopia patients and controls.
Two sequence changes were identified in coding exons
with the intron-exon boundary of PAX6. One novel
heterozygous silent variant, 678A>G (R67R), was found in
one high myopia patient, and a noncoding sequence change,
rs667773, was found in both patients and controls. Allelic and
genotypic frequencies of both polymorphisms showed no
significant difference (p > 0.05) between patients and controls
(data not shown). Within the P1 promoter region, 20
polymorphisms were identified, with no significant difference
in frequencies between patients and controls: –186C>T, –
215G>A, –242G>A, –263A>G, –292A>G, –331A>G, –
337A>T, –354A>G, –382G>A, –407G>A, –409G>A, –
692A>G, –758C>T, –782A>G, –933C>G, –3050C>A, –
3070C>A, –3078A>G, –3090C>T, and –3282T>C (data not
shown). For -186C>T, -292A>G, -331A>G, -933C>G, and
-3282T>C, each SNP was only found in 1 high myopia patient.
Therefore, they were statistically not significant under
Pearson’s χ2 test (p > 0.05).

activity was observed with increasing length of (AC)m repeats
(p = 0.004, one-way ANOVA; post-hoc tests adjusted by
Tukey HSD: (AC)Below20–22 versus (AC)20–22, p = 0.033; and
(AC)Below20–22 versus (AC)Above20–22, p = 0.004; Figure 1A,B).
Similarly, at a given (AC)m repeat length, transcriptional
activity of (AG)8 was increased when compared with (AG)6,
although the increase was not significant, likely due to the
substantial standard deviation (p = 0.205, independent T-test;
Figure 1C,D). For combined repeats of the same length,
transcriptional activity of (AC)23(AG)6 was similar to that of
(AC)21(AG)8 (p = 0.627, independent T-test; Figure 1E,F).
Thus, both AC and AG repeats contributed to the
transcriptional activity of the PAX6 P1 promoter.
Our luciferase-reporter analysis showed that
transcription activity increased with AC and AG repeat length.
This phenomenon may be due to influences of transcription
factor binding sites within this region. Thus, we used
(AC)20(AG)6 as a reference and predicted one binding site for
T-cell factor/Lymphoid enhancer factor family transcription
factors, one glucocorticoid receptor binding site, and four
transcription factor (TF) II-I binding sites (Figure 2B). With
decreasing AG repeat lengths, the T-cell factor/Lymphoid
enhancer factor and glucocorticoid receptor sites were
unchanged, but the TFII-I sites were reduced. Only two
predicted TFII-I sites were observed in (AC)15(AG)4 (Figure
2A). No alteration was observed with a decrease in AC repeat
TABLE 1. ALLELIC FREQUENCIES OF PAX6 P1 PROMOTER DINUCLEOTIDE REPEATS IN HIGH MYOPIA (HM) AND CONTROL SUBJECTS.
(AC)m repeat
Allelic count (%) Empirical
p-value (AG)n repeat
Allelic count (%) Empirical
p-value
HM n=750 Control n=678 HM n=758 Control n=698
(AC)70 (0.0) 1 (0.1) 0.013 (AG)40 (0.0) 1 (0.1) 0.012
(AC)15 0 (0.0) 2 (0.3) (AG)545 (5.9) 51 (7.3)
(AC)16 10 (1.3) 9 (1.3) (AG)6464 (61.2) 458 (65.6)
(AC)17 43 (5.7) 41 (6.0) (AG)7218 (28.8) 176 (25.2)
(AC)18 80 (10.7) 67 (9.9) (AG)831 (4.1) 12 (1.7)
(AC)19 100 (13.3) 138 (20.4)
(AC)20 155 (20.7) 134 (19.8)
(AC)21 149 (19.9) 99 (14.6)
(AC)22 161 (21.5) 138 (20.4)
(AC)23 29 (3.9) 33 (4.9)
(AC)24 13 (1.7) 13 (1.9)
(AC)25 6 (0.8) 2 (0.3)
(AC)26 4 (0.5) 1 (0.1)
TABLE 2. ALLELIC FREQUENCIES OF PAX6 P1 PROMOTER GROUPED DINUCLEOTIDE REPEATS, (AC)m AND (AG)n, IN HIGH MYOPIA (HM) AND CONTROL SUBJECTS.
Grouped
(AC) m repeat
Allelic count (%) Empirical
p-value
Grouped
(AG)n repeat
Allelic count (%) Empirical
p-value
HM n=750 Control n=678 HM n=758 Control n=698
(AC)Below 20-22 233 (31.1) 258 (38.1) 0.016 (AG)4-6 509 (67.2) 510 (73.1) 0.016
(AC)20-22 465 (62.0) 371 (54.7) (AG)7-8 249 (32.8) 188 (26.9)
(AC)Above 20-22 52 (6.9) 49 (7.2)
TABLE 3. GENOTYPIC FREQUENCIES OF PAX6 P1 PROMOTER GROUPED DINUCLEOTIDE REPEATS IN HIGH MYOPIA (HM) AND CONTROL SUBJECTS.
Grouped
(AC) m
genotype
Genotypic count (%) Empirical
p-value
Grouped
(AG)n
genotype
Genotypic count (%) Empirical
p-value
HM n=375 Control n=339 HM n=379 Control n=349
(AC)Below 20-22 /
(AC)Below 20-22
16 (4.3%) 40 (11.8%) 0.004 (AG)4-6 /
(AG)
173 (45.6%) 192 (55.0%) 0.039
(AC)Below 20-22 /
(AC)20-22
178 (47.5%) 149 (44.0%) (AG )4-6 /
(AG) 7-8
163 (43.0%) 126 (36.1%)
(AC)Below 20-22 /
(AC)Above 20-22
24 (6.4%) 29 (8.6%) (AG) 7-8 /
(AG) 7-8
43 (11.3%) 31 (8.9%)
(AC)20-22 / (AC)20-22 130 (34.7%) 103 (34.7%)
(AC)20-22 / (AC)Above
20-22
26 (6.9%) 16 (4.7%)
(AC)Above 20-22 /
(AC)Above 20-22
1 (0.3%) 2 (0.6%)
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lengths. Accordingly, more TFII-I sites were predicted with
increasing AG repeat lengths. Multiple sites for Wilms’ tumor
transcription factor without lysine-threonine-serine [Wt1(–
KTS)] were observed with an increase in AC repeat length,
and one GAGA factor binding site appeared with an increase
in AG repeat length. In (AC)26(AG)8, six TFII-I sites, six
Wt1(–KTS) sites, and one GAGA factor site were predicted
(Figure 2C).
DISCUSSION
We found no myopia mutations in the coding regions and
splice sites in PAX6 in our cohort of Chinese high myopia
patients. Some SNPs were detected in the P1 promoter, exon
7, and intron 10, but these were not statistically significant
(data not shown). In a recent report, two intronic SNPs
(rs3026390 and rs3026393, located in introns 12 and 13,
respectively) have been shown to be associated with high
myopia in Han Chinese nuclear families [66]. SNP
rs667773, located in intron 10, is in the same linkage
disequilibrium block with rs3026390 and rs3026393 [66].
However, in our study, no significant association was found
for rs667773 between high myopia patients and controls,
which was consistent with a previous case-control association
study in a Taiwan Chinese population [67]. The discrepancy
might be due to the much lower minor allele frequency of
rs667773 (0.137) than of rs3026390 and rs3026393 (0.472
and 0.493, respectively) [66]. Other studies have suggested
that rs667773, as a neural polymorphism, is an unlikely cause
of overt phenotypes such as aniridia [75,76].
The (AC)m(AG)n dinucleotide repeat sequence, located
about 1 kb from the transcription start site of the PAX6 P1
promoter, is highly polymorphic. The AC dinucleotide
polymorphism ranged from 18 to 31 repeats and AG ranged
from 5 to 7 repeats in a Caucasian population [60]. In our
Chinese cohort, the AC repeats ranged from 7 to 26 and the
AG repeats from 4 to 8 (Table 1). The allele size of the AG
repeats was similar in Caucasians and Chinese, but the AC
repeat length was longer in Caucasians. Notably, one (AC)7
allele was found in a control subject, far from the common
range of repeats between 15 and 26. In addition, many of the
dinucleotide repeats were heterozygous in both poly AC and
poly AG repeats (AC: 55.3% in controls and 75.9% in
patients; AG: 42.9% in controls and 53.4% in patients). The
observed heterozygosity rate was 65% in a Caucasian
population [60]. Although the allele number in that study was
defined as combined units of AC and AG repeats instead of
independent AC and AG alleles, the trend of heterozygosity
was similar to that in our work. These two dinucleotide repeats
Figure 1. Transcriptional activity of
dinucleotide repeats in the PAX6 P1
promoter. A 1,851 bp genomic fragment
(from –1278 to +573) containing the
PAX6 P1 promoter with different
dinucleotide repeats was cloned into an
empty pGL3-Basic vector (pGL3) and
transfected into ARPE-19 cells. The
activity of each allelic construct is
expressed relative to the construct
(AC)20(AG)6. Data are represented as
mean±SD for five independent
experiments. A and B: Immunoblotting
results and a bar chart show relative
luciferase activity for grouped (AC)m
repeats with a stable (AG)6. C and D:
Immunoblotting results and a bar chart
show relative luciferase activity for
(AG) repeats with (AC) . E and F:
Immunoblotting results and a bar chart
show relative luciferase activity for
combined (AC) (AG) repeats.
Molecular Vision 2009; 15:2239-2248 <http://www.molvis.org/molvis/v15/a241> © 2009 Molecular Vision
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21
n
mn

are, therefore, highly polymorphic both in Caucasians and in
Chinese.
The PAX6 P1, containing CCAAT boxes and a TATA-
like box, is likely a real promoter [58-60]. We evaluated the
influence of (AC)m(AG)n dinucleotide repeats on PAX6 P1
promoter activity by a luciferase-reporter assay and examined
the effects of repeat lengths as obtained from our high myopia
patients and controls. Since retinal pigment epithelium (RPE)
has been shown to have PAX6 P1 promoter activity [57], we
used an RPE cell line, ARPE-19, for transfection.
Immunoblotting showed that longer lengths of (AC)m have a
significant trend of increasing luciferase expression compared
with shorter lengths (Figure 1A,B), although this was not
observed for (AG)n, likely due to the substantial standard
deviation (Figure 1C,D).
We confirmed that transcriptional activity of
(AC)23(AG)6 was similar to that of (AC)21(AG)8 (Figure 1E,F),
suggesting that both (AC)m and (AG)n dinucleotide repeats
within the PAX6 P1 promoter contribute to transcriptional
activity and might work cooperatively as an unit. Previous
studies on luciferase-reporter assays assessed the promoter
activity invisibly by a luminometer [60,77]. In our study, we
monitored the luciferase-reporter assay by immunoblotting
using a commercially available antibody against firefly
luciferase and luciferase overexpression by pGL3-pCMV as
a positive control. There are technical advantages to this
method. The promoter activity could be visualized, and co-
transfection with another normalizing vector was not required,
as the luciferase intensity could be directly normalized with
the housekeeping protein, assuming the same transfection
efficiency among the constructs. The limitation of the
luciferase-reporter assay is that the effect of the dinucleotide
repeats on the transcriptional activity was performed using
RPE cells from normal controls, which might not truly reflect
the situation in high myopia unless the experiment were
performed using cells from a highly myopic individual.
Since levels of Pax6 are tightly controlled, small and
seemingly insignificant changes in the levels of Pax6 may lead
to significant phenotypic consequences [78]. Moreover, the
Pax6 protein could upregulate PAX6 P1 promoter activity
Figure 2. Transcription factor binding
site prediction for dinucleotide repeats
in the PAX6 P1 promoter. The cloned
PAX6 P1 promoter DNA sequence was
used to predict transcription factor
binding sites. Predicted transcription
factor binding sites around the region of
the dinucleotide repeats are shown, and
different lengths of AC and AG repeats
are assessed. As in the immunoblotting
analysis, [(AC)20(AG)6] was set as a
reference. A: Predicted transcription
factor binding sites for (AC) (AG)
are shown. B: Predicted transcription
factor binding sites for (AC) (AG)
are shown. C: Predicted transcription
factor binding sites for (AC) (AG)
are shown.
Molecular Vision 2009; 15:2239-2248 <http://www.molvis.org/molvis/v15/a241> © 2009 Molecular Vision
2244
15 4
20 6
26 8

[77]. Results of our genotyping and promoter activity analyses
indicate that longer lengths of dinucleotide repeats increase
the expression of PAX6, which increases the risk of high
myopia. This postulation may be supported by several
assertions: (1) PAX6 gene expression has been shown to be
significantly higher in the retinas of optical defocused eyes
than in contralateral eyes in the rhesus monkey [79], and
expression of PAX6 was also increased in posthatch chicken
eyes with form-deprivation myopia [78]. (2) In another study,
the number of dividing retinal progenitor cells, of which
PAX6 is a marker, was highly correlated with axial elongation
of the eye, resulting in myopic refractive errors in primates
with form-deprivation myopia [80]. (3) Pax6 has been shown
to transactivate insulin promoters [81] and promote proinsulin
processing [82]. As insulin is a strong stimulator of axial
myopia in chicks [83], elevated PAX6 expression may
increase the risk of developing myopia through increased
expression of insulin. Chronic hyperinsulinemia has been
proposed as a key player in the pathogenesis of juvenile-onset
myopia [84]. Although Pax6 also transactivates the glucagon
promoter [81], which is a “stop” for myopia [85], insulin
might overcome the effects of glucagon in the development
of myopia [86].
The transcription factor binding site prediction (Figure 2)
showed that an increase in AC repeat length created additional
Wt1(–KTS) binding sites, while an increase in AG repeat
length created TFII-I and GAGA factor binding sites. If the
AG repeat length was reduced, TFII-I sites were also reduced.
Wt1(–KTS) is necessary for normal retina formation in mice
[87], while TFII-I is a signal-induced multifunctional
transcription factor that plays a key role in the regulation of
cell proliferation [88]. Moreover, the GAGA factor, a
transcription activator, is activated by epidermal growth
factors, platelet-derived growth factors, and insulin [89].
These growth factors could regulate PAX6 transcription
through the GAGA factor binding site.
In summary, we found no association between
polymorphisms in the PAX6 coding region and high myopia
in our Hong Kong Chinese cohort. Two dinucleotide repeats,
AC and AG, in the PAX6 P1 promoter were associated with
high myopia. These two repeats were also associated with the
elevation of PAX6 P1 promoter activity, and hence an increase
in the transcriptional activity of PAX6. Our results provide
evidence for the role of PAX6 in the pathogenesis of high
myopia.
ACKNOWLEDGMENTS
We express our greatest appreciation to all the participants in
the study. This study was supported by a block Grant, The
Chinese University of Hong Kong and the Lim Por Yen Eye
Foundation Endowment Fund.
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