Muscarinic acetylcholine receptor 3 is dominant in myopia
progression
Hui-Ju Lin,1, 2, 3, Lei Wan,2, 3, 4*
Wen-Chi Chen2, 5, Jane-Ming Lin1, 2, 3, Chao-Jen Lin6 Fuu-Jen Tsai 2, 3*
1Department of Ophthalmology, China Medical University Hospital, Taichung, Taiwan; 2Department of Medical Genetics, China Medical University Hospital, Taichung, Taiwan; 3School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan; 4Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan; 5Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan; 6Department of Pediatrics, Changhua Christian Hospital, Changhua, Taiwan
Running title: Muscarinic acetylcholine receptor and myopia
*Corresponding author: Lei Wan, PhD and Fuu-Jen Tsai, MD, PhD
Address: Department of Medical Genetics and Pediatrics, China Medical University Hospital, No. 2 Yuh Der Road, Taichung 404, Taiwan
TEL: 886-4-22052121 ext. 2041 FAX: 886-4-22033295
Abstract
Purpose: Numerous studies have proven that the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine prevents the axial elongation that leads to myopia. Five distinct receptor genes (CHRM1–CHRM5), each encoding a muscarinic receptor protein (M[1]–M[5]), have been cloned. Copy number variations (CNVs), which constitute a substantial portion of genetic variability and structural genetic variants, are increasingly being recognized as modulators of human diseases. In this study, CNVs of CHRMs were detected to determine the genes associated with myopia. Methods: Participants were divided into 3 groups: high myopia group (myopia of 6–10 diopter [D]), severe high myopia group (myopia ≧10 D), and control group (myopia≦0.5 D). The CNVs were detected, and the relative copy number was estimated using the comparative2--Ct method. Syrian hamsters with form-deprivation myopia (FDM) were used as animal models of myopia. Results: The CNVs of CHRM2, CHRM3, and CHRM4 were significantly different among the groups, and the variations were most dominant in the CHRM3. The CNVs of CHRM3 showed significant differences among all 3 groups (p=0.005). A replication cohort was collected to further confirm the association of CHRM3 CNV with myopia ( p =0.011) . The expression of M(3) on the sclera of the FDM Syrian hamsters was upregulated and then downregulated after atropine administration. Conclusion: CHRM3 and M(3) were suggested to play important roles in the pathogenesis of myopia and in the arrested progression of myopia by atropine.
Introduction
Clinically significant refractive errors are the most common visual disorders. Myopia affects more than half of young adults in the world.1-3 In Asian countries, the prevalence of myopia has approached epidemic proportions.4 Animal studies of myopia have shown that the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine effectively prevents the axial elongation that leads to myopia.5-7 Human clinical trials have also shown the effectiveness of daily atropine administration in reducing the progression of myopia. The mAChR family proteins are a group of neurotransmitter proteins that belong to the 7-transmembrane superfamily of receptors. Five distinct receptor genes (CHRM1–CHRM5), each encoding a muscarinic receptor protein (M[1]–M[5]) with specific pharmacological properties, have been cloned.8-10 Different studies have shown the diverse effects of M(1)–M(5) on myopia; moreover, M(1)–M(5) have been proposed to play various roles in the “stop” signal of myopic progression.8-10 In vitro studies have shown that mAChR antagonists inhibited scleral proliferation and matrix synthesis. 8-10
Many studies have suggested that myopia is a complex condition with multiple causes and develops as a result of interactions between multiple genes and environmental stimuli.11 This study detected CHRM1–CHRM5 polymorphisms by using the copy number variation (CNV) method. CNV is a common genomic variation in which variable number of copies of DNA segments (>1 kb) are present in comparison to a reference genome. CNV caused by genomic rearrangements such as deletions, duplications, inversions, or translocations may contribute to altered gene expression and subsequent phenotypic variation, resulting in disease susceptibility or resistance.12,13 CNV has also been associated with autism,14,15 schizophrenia,16,17
cancer,18-20 and autoimmune diseases.21-23 Because CNVs of different genes are related to many phenotypes, we identified CNVs of genes encoding mAChRs in an effort to determine a possible correlation with high myopia in the present study. Form-deprived myopic (FDM) animals were used to study the expression levels of mAChRs in myopic eyes before and after atropine treatment.
Materials and methods
Genomic DNA extraction and quantification of CHRM1-CHRM5 copy number
Relative copy number (CN) of the CHRM1-CHRM5 genes in each study participant was estimated using a relative real-time quantitative polymerase chain reaction (PCR) method. Specific primers and TaqMan® probes for CHRM1-CHRM5 and for reference gene RNase P were purchased from ABI Biosciences (Figure 1).24
The TaqMan® probes for CHRM1-CHRM5 and RNase P were labeled with FAMTM and VIC®, respectively. PCRs were run on an ABI 9700 machine. Amplification reactions (15 L) were carried out using genomic DNA (10 ng), TaqMan® Copy Number Reference Assay for CHRM1-CHRM5 and RNase P, and TaqMan® Master Mix. Thermal cycling was initiated with 10 min denaturation at 95°C, followed by 40 cycles each of 15 s at 95°C and of 1 min at 60°C. Dissociationprocedure generate a melting curve to confirmof amplification specificity. Relative CHRM1-CHRM5 CN
per individual was estimated using the2-Ct method (Ct is cycle threshold, –Ct = [Ctgene – Ctreference]) describedpreviously.25 PCR products were quantified in triplicate, standard deviation and coefficient of variation (CV) were calculated on the basis of three runs. To control for reaction quality, each reaction plate also included a calibrator, positive control, and a no-template control (NTC). Data from a plate were included if the calibrator CV was less than 5%, positive control CV and sample CVs all less than 10%, and the NTC negative. To include across-plate data, CVs of the positive control had to be similar and NTC negative. Copy caller software (ABI Biosciences) were used to estimate the copy number. A maximum likehood algorithm was used to estimate the mean Ct expected for copy number 1 (CN=1) based on the probability density distribution of all samples and used to determine the copy number for each sample. The relative copy number of each gene was normalized to RNase P,
a reference gene with CN=2.
Association analysis and risk calculation
Significance of differences between the distributions of CN for CHRM1-CHRM5 in cases and controls was estimated using the Chi-square test or Fisher’s exact test or linear regression analysis . Odds ratios and confidence intervals were estimated with logistic regression models using SPSS 12.0. Myopia risks were estimated with CN for CHRM1-CHRM5 categories by comparing with reference category (CN = 2) as median CN for CHRM1-CHRM5. To estimate odds ratios, bins of CN were grouped as 0-1 CN for CHRM1-CHRM5 (<2 category), 3-5 CN for CHRM1-CHRM5 (>2 category), and 0-1 plus 3-5 CN for CHRM1-CHRM5 (CN≠2 category). For each category, odds ratio >1 indicated effect of CNV on disease susceptibility, <1 protective.
Patients: In this study, the refractive errors of 4 ,000 volunteers were measured. All of the participants were medical students unrelated and Taiwan-born Han Chinese, aged 16–25 years (mean age, 18 ± 3.3 years), and the male-to-female ratio 1.48:1.0. All participants had a visual acuity with distance correction of 0.2 logMAR (20/32) or better. Refractive error was measured in diopters (D) and determined using the mean spherical equivalent (SE) of both eyes of each individual after administering one drop of cycloplegic drug (1% tropicamide; Mydriacyl; Alcon, Berlin, Germany). Persons with myopia ≥6.0 D (both eyes) were included in the myopia group, those with myopia ≥10.0 D (both eyes) were included in the high myopia group, and those with myopia <0.5 D and hyperopia <1.0 D (both eyes) were in the control group. Patients with astigmatism >0.75 D were excluded, since astigmatism alters spherical equilibrium (SE) results. The study was reviewed by the ethics committee of China Medical University Hospital at Taichung, Taiwan, performed in accordance with the
tenets of the Declaration of Helsinki for research involving human subjects. Informed consent was obtained from all participants, comprehensive ophthalmic examination and blood collection performed. No participants had ocular disease or insult such as retinopathy, prematurity, neonatal problems, history of genetic disease, or connective tissue disorders associated with myopia such as Strickler or Marfan syndromes. Clinical examinations included visual acuity, refraction error, slit-lamp examination, ocular movements, intraocular pressure, and fundoscopy. Patients with organic eye disease, a history or evidence of intraocular surgery were excluded. As with all data collection procedures, auto-refraction (autorefractor/autokeratometer [ARK 700A; Nikon, Tokyo, Japan]) was conducted on both eyes by experienced optometrists trained and certified in the study protocols. Refractive data, sphere, negative cylinder, and axis measurements were analyzed through the calculation of SE refractive error. Animals: Three-week-old Golden Syrian hamsters (80–90 g) were used. All procedures were approved by the Institutional Animal Care and Use Committee of China Medical University and are in adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Right eyes of all Syrian hamsters in both the control group and the study group were induced FDM. Animals were randomly assigned into a study group (n = 30; 1% atropine applied to both eyes) and control group (n = 30; no eye drop applied). Left eyes of all Syrian hamsters were left open to use as a control. Hamsters were raised with right eyelid fusion for 31 days, a
drop of 1% atropine administered to both eyes of the study group every day.
Biological measurements: Refractive errors were measured using a hand-held streak
retinoscope; all referred to spherical-component refractive error, defined as mean refractive error in the horizontal and vertical meridians. Axial dimensions of eyes were measured by ultrasonography with a 10-MHz transducer while the animals were
anesthetized with 10% ether in oxygen. Axial length (AXL) of the eye was defined as the distance from front of the cornea to the back of the sclera. Ocular refraction and axial ocular dimensions were collected at start and end of the experiment.
Tissue preparation: The animals were sacrificed with a lethal dose of chloral hydrate
at the end of the study, and the eyes were enucleated. Digital calipers were used to measure the equatorial diameters and AXLs immediately. Using a surgical microscope (Topcon, Tokyo, Japan) and a razor blade, eyes were cut perpendicular to the anterior-posterior axis and approximately 1 mm posterior to the ora serrata on an ice plate. The anterior segment of the eye was discarded except for the iris and the ciliary body. Posterior sclera was excised using a 7-mm diameter trephine, the head of the optic nerve was discarded.
Immunohistochemistry: This procedure was performed on frozen eye sections,
positively stained cells quantified by averaging three optical fields (>200 cells per field) under 400× magnification on six samples from six individual animals (n = 6).
Results
Volunteers were enrolled in this study based on the following data: age, 16–25 years (mean, 18 ± 3.3 years); male to female ratio, 1.48:1.0; mean axial length (AXL), 24.8 mm; and mean SE, –4.5 diopter (D). No significant differences were observed between the control and the myopia groups with respect to age, gender, cornea diopter, anterior chamber depth, or lens thickness. The myopia groups comprised 150 patients with high myopia (≥6 D) and 25 patients with severe high myopia (≥10 D); the control group comprised 95 individuals with normal eyes. The CNV distribution of CHRM1–CHRM5 was compared between the control and high myopia groups, between control and severe high myopia groups, and between high myopia and severe high myopia groups. Table 1 and Figure 2 depict the distribution of CHRM1–CHRM5 CN. Significant differences were noted in CHRM3 CNV distribution among the groups, which showed both CN ≠ 2 (compare CN = 2 and CN ≠ 2) and CN > 2 (compare CN = 2, CN > 2, and CN < 2) tests. CNV differences of CHRM3 among all 3 groups (in CN > 2, p < 0.001; in CN ≠ 2, p < 0.001) as well as between the control and the high and severe high myopia groups were significant (in CN > 2, p = 0.009 and 0.001, respectively; in CN ≠ 2, p = 0.002 and p = 0.019, respectively). These results indicate that in patients with high and severe high myopia, the CNVs of CHRM3 (CN > 2) may be related to the pathogenesis of myopia. A significant difference was also noted in the CNVs of CHRM2 (CN > 2) between the control and the severe high myopia groups (p = 0.006) (Table 1), indicating that CHRM2 (CN >
was observed in the CNV distribution of CHRM4 (CN ≠ 2 and CN > 2) among all the groups (p = 0.005 and 0.021) and between the control group and high and severe high (in CN ≠ 2, p = 0.002 and in CN > 2, p = 0.004). However, significant differences were not observed in CNV distribution of CHRM4 between the control and high myopia groups, indicating that the CNV of CHRM4 is a useful marker for differentiating between high myopia and severe high myopia. A univariate linear regression model was also used to test the association of CHRM1~5 CNV with myopia. The results revealed that CHRM2 ( p =0.035) and CHRM3 ( p =0.005) were associated with myopia which is consistent with chi square or fisher ’ s exact test.
As shown in the previous results, CHRM3 is the most significant gene segment that has been found to be associated with myopia. To identify whether CHRM3 CNV affects the entire gene, we selected another 2 probes located on CHRM3. A total of 3 probes were used to determine the CHRM3 CNV (Figure 1). We used the McNemar test to determine the differences in copy numbers among the 3 probes. The p values of the McNemare test for the control, high myopia group, and severe high myopia group were not significant (p=0.607, p=0.324, and p=0.362, respectively), indicating that the CHRM3 CNV affected the entire gene. A replication study was also performed on CHRM3 CNV to confirm the association with myopia. The demographic data of the individuals are: age, 16–25 years (mean, 17 ± 4.3 years); male to female ratio, 1.54:1.0; mean AXL , 25.1 mm; and mean SE, –5. 7 diopter (D). We collected 48 control individuals, 112 patients with high myopia (≥6 D) and 12 patients with severe high myopia (≥10 D) . No significant differences were observed between the control and the myopia groups with respect to age, gender, cornea diopter, anterior chamber depth, or lens thickness. We found a significant association of CHRM3 gene with myopia ( p =0.011) through univariate linear regression analysis (Table 2).
To understand the involvement of CHRM3 in the development of myopia, an FDM model was used to determine the expression level of M[3] in the sclera of myopic eyes and to determine the effect of atropine on CHRM3 expression. Table 3 lists the ocular component changes in FDM Syrian hamsters; the refractive power of the occluded eyes averaged 7.25 ± 0.5 D more myopia than that of the non-occluded eyes at the end of the experiment (Table 3). The AXL of occluded eyes was +1.06 ± 0.09 mm longer than that of the non-occluded eyes (Table 3). Refractive power and axial length of FDM Syrian hamsters after atropine administration also showed less myopic change than that in control eyes. After atropine administration, the average refractive power of occluded eyes was 7.5 ± 0.5 D and that of the non-occluded eyes was 12.0 ± 0.5 D at the end of the experiment.
Immunohistochemistry showed that M(3) was expressed in the sclera, and this expression was higher in FDM eyes (Figure 3B) than that in non-FDM eyes (Figure 3A). After atropine administration, M(3) expression decreased in both the FDM (Figure 3D) and non-FDM eyes (Fig. 3C). However, the expressions of M(1), M(2), M(4) and M(5) during the immunohistochemistry analysis did not reach significance among the groups (data not shown).
Discussion
Genetic studies have determined the polymorphisms of separate loci that are correlated to high myopia, e.g., those on chromosomes 18p and 12q, myocilin gene, 26-28 transforming growth factor (TGF) gene,29 paired box 6 (PAX6) gene,30 and collagen type I alpha 1 (COL1A1) gene.31 Such polymorphisms indicate a genetic predisposition to high myopia. Despite this, no single gene is responsible for the development of myopia, especially considering the wide variability in the prevalence
of myopia across ethnic groups.32-34 Mechanisms underlying myopia development are further obscured by the uncertainty regarding the roles of environmental factors. A higher prevalence of myopia is observed among individuals with higher levels of education, e.g., medical students participating in the present study, than in the members of the general population. However, because the high myopia groups as well as the control group comprised medical students, we believed that the bias associated with environmental influence would be minimal.
Clinical trials in school-aged patients have shown the effectiveness of daily atropine administration, which reduced the progression of myopia by at least 60% during the first year of treatment.22,23 The mechanism of action of atropine in myopia is suspected to include reduction of neuronal activity and increase in the general release of retinal neurotransmitters.20 It has also been proven that pirenzepine and himbacine inhibit myopia in a dose-dependent manner, suggesting that these drugs mediate their effects via a receptoral mechanism.14,15 In the present study, the CN differences among 5 CHRMs were determined to evaluate the potential mechanisms of myopia progression through genetic inheritance.
Different mAChR antagonists have been investigated for their individual effectiveness in reducing myopia. In this study, the CNVs of CHRM3, CHRM2, and CHRM4 were significantly different in patients with myopia, with the most notable difference observed in the CNV of CHRM3. The main gene near this location is FMN2,35 which is present on chromosome 1 at 1q43 and belongs to the family of actin-binding proteins that are homologous to formin. FMN2 is involved in multicellular organization, actin cytoskeleton organization, and biogenesis.35,36 Moreover, formin homology domain proteins are involved in cytoskeletal organization and the establishment of cell polarity. Cellular growth function of these
genes may involve remodeling of the sclera and increase in AXL.35 Immunohistochemical examinations of the sclera of myopic Syrian hamsters showed that M(3) was upregulated after FDM development, indicating that M3 itself is involved in the progression of myopia. M(3) was downregulated after 1% atropine administration, indicating that the M3 receptor is an important mAChR for controlling the progression of myopia.
Earlier studies have shown that M(3) receptors coupled with G proteins of class Gq upregulate phospholipase C, which alters the inositol trisphosphate and intracellular calcium levels.37 Increase in intracellular calcium typically results in muscle constriction, including the constriction of the ciliary body. Moreover, with respect to the vasculature, the activation of M(3) in vascular endothelial cells increases nitric oxide synthesis and eye accommodation.38 Nitric oxide formation and eye accommodation are important factors in the progression of myopia and AXL. During myopia progression, M(3) expression levels may be upregulated to stimulate several important signaling molecules that induce tissue remodeling and promote myopia.
In addition to CHRM3, CHRM2 and CHRM4 also showed significant differences in patients with myopia. CHRM2 is located at Chr7: 136553416-136705002 on chromosome 7q33.38-39 It has been reported that the presence of a 10-Mb deletion at 7q33–q35 involving several genes can induce neurological diseases. In addition to CHRM2, the locus-encoded genes CNTNAP2, HIPK2, DGKI, EPHB6, and PTN are particularly interesting in complex neurodevelopmental phenotypes including high myopia.39,40 CHRM4 is at Chr11: 46406640-46408107 on chromosome 11p11.2. This location also contains the genes MDK, CREB3L1, and AMBRA1. MDK is a retinoic acid-responsive, heparin-binding growth factor that is expressed in various
cell types during embryogenesis; it also promotes angiogenesis, cell growth, and cell migration. The protein encoded by CREB3L1 is involved in intramembrane proteolysis and endoplasmic reticulum stress.41,42 AMBRA1 regulates autophagy and nervous system development and is involved in controlling protein turnover during neuronal development and in regulating normal cell survival and proliferation.41,42
The exact role of CHRM1–CHRM5 and M(1)–M(5) in myopia development remains unclear because different studies have reported varied expression levels of these biological factors.8-10,43,44 All mAChRs expressed on the sclera have been proven; in fact, M(1)–M(5) might play distinct roles in the “stop” signal of myopic progression, as proposed by different studies. Several studies have shown differential expressions of CHRM1–CHRM5 among different models, e.g., Barathi et al. used quantitative real-time polymerase chain reaction (PCR) and showed that CHRM3 expression levels increased after atropine treatment in myopic sclera.8 It has been reported that the expression of CHRMs varies between species (BJ and B6 mice v.s. Syrian hamsters). As we know, real-time PCR detects mRNA expression levels, but these expression levels are not always positively correlated with protein levels. Moreover, isolation of the sclera may contaminate adjacent tissues, which may also influence CHRM expression levels. Moreover, the atropine concentration may account for the difference. In Barathi et al.’s report, 10 μL of atropine was administered daily as a subconjunctival injection, whereas in our study, we administered 10 μL of atropine directly to the cornea. Therefore, the concentration of atropine would be lower in our model system. This might be the reason for the different outcomes observed in the present study, in which the CNVs of CHRM3, CHRM2, and CHRM4 reached significant difference in patients with myopia. Nonetheless, the changes in M(3) levels in FDM Syrian hamsters were the most
dominant after atropine administration. Discordant views of different mAChR studies were discussed in this study and advanced investigations of mAChR are needed in the future.
Early success in identifying disease-associated CNVs as candidate genes allows the determination of structural genetic variations.45 The importance of structural genetic variants in modulating human diseases is being increasingly recognized.45 Although the manner in which CNVs affect entire genes remains unknown, this newly appreciated form of genetic variation is a valuable tool. In this study, 5 CHRMs were examined to verify the function of atropine in the progression of myopia. FDM animals were used as a model. CHRM3 and M(3) showed significant differences, and CHRM3 was determined as an important molecule in the regulation of myopia progression.
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Figure legend
Figure 1. Gene structure, chromosome location, and probe locations used in this study (based on GRCh37).
Figure 2. (a) The distribution of CHRM3 copy number among healthy controls (dark grey bars), high myopia (≥6 D) groups (pale grey bar), and severe high myopia (≥10 D) groups (open bar). Fisher’s exact test was used to assess the distribution of gene copy number for CHRM3 between healthy controls, high myopia group, and severe high myopia group. n = number of subjects in each group. (b) The distribution of CHRM3 copy number among healthy controls (grey bars) and patients with myopia (open bar). Fisher’s exact test was used to assess the distribution of gene copy number for CHRM3 between healthy controls and patients with myopia. n = number of subjects in each group.
Figure 3. Immunohistochemical analysis CHRM3 was performed on control (A) myopic eyes and form-deprived myopic eyes (B). Atropine (1%) was administered, and CHRM3 expression in the non-occluded eye (C) and occluded eye (D) was determined.
