Genetics Common Polymorphisms in the SERPINI2 Are Associated with Refractive Error in the 1958 British Birth Cohort

Pirro G. Hysi,1,2,3 Claire L. Simpson,1,3 Yvonne K. Y. Fok,4 Dianne Gerrelli,4 Andrew R. Webster,5 Shomi S. Bhattacharya,5 Christopher J. Hammond,2 Pak C. Sham,6 and Jugnoo S. Rahi1

PURPOSE. To identify heritable genetic factors altering suscep- physiological pathways controlling eye growth and develop- tibility to refractive error in the general population. ment and those controlling glucose metabolism. The findings METHODS. This was a genetic association study of refractive indicate that SERPINI2 is a promising candidate for further error investigating genetic polymorphisms in regions previ- investigations of the genetic susceptibility to myopia. (Invest ously reported through linkage. Two study panels were drawn Ophthalmol Vis Sci. 2012;53:440–447) DOI:10.1167/iovs.10- from the British 1958 Birth Cohort, composed of 2211 persons 5640 44 years of age at the time of visit. Two main outcomes were considered: refractive error as a continuous outcome (spheri- major challenge for the 21st century is to find the genetic cal equivalent) and myopia as a diagnosis (defined as spherical Afactors that influence susceptibility to common complex equivalent equal to or worse thanϪ1.00 diopter). Genotyping diseases and traits. Refractive error is an archetypal complex was initially performed in 1188 subjects from the outer tertiles quantitative trait, with myopia at one end of the distribution of the population distribution, using customized arrays of sin- and hypermetropia at the other. Refractive error is the com- gle nucleotide polymorphisms (SNPs) saturating regions of monest cause of visual impairment globally and has major previously reported highly significant linkage. In a second socioeconomic costs for the affected person and for society as stage, SNPs most significantly associated were validated in a whole; for example North Americans are reported to spend at 1023 more persons. Findings were investigated further least $12 billion annually on glasses, contact lenses, and refrac- 1 through human fetal expression studies. tive surgery. Myopia affects at least 25% of white North Americans and RESULTS. Polymorphisms within the SERPINI2 gene were asso- Europeans2 and up to 60% of Asians but only 5% of Africans.3,4 ciated with refractive error in two different European sub- The prevalence and severity of myopia have been increasing in groups from the 1958 British Birth Cohort (meta-analysis P ϭ recent years, especially in Southeast Asia.3,4 This suggests that, 7.4E-05 for rs9810473). Association was also significant for against a background of genetic susceptibility, changing envi- myopia (best association: OR ϭ 0.80; 95% CI, 0.69–0.93; P ϭ ronmental factors, possibly related to rapid urbanization, play a 0.003 for rs10936538). Expression profiling of SERPINI2 re- role in the development of refractive error. vealed that the gene is expressed in the retina and in other eye Family aggregation studies have shown a correlation be- and CNS tissues. tween parental refractive error and that of their children,5 CONCLUSIONS. The novel association of SERPINI2 with refrac- and twin studies have reported high heritability of 60% to tive error and myopia is suggestive of a possible link between 90% in the general population.6 However, until very re- cently, genetic research has focused on linkage analysis of cohorts of families, often with “syndromic” or “pathologic” From the 1MRC Centre of Epidemiology for Child Health, Institute myopia, whose similarities with common, population-wide of Child Health, University College London, London, United Kingdom; myopia are uncertain. The first linkage study of refractive 2Department of Twin Research and Genetic Epidemiology, Kings Col- error based on dizygotic twins reported four linkage peaks 4 lege London, St. Thomas’ Hospital, London, United Kingdom; Neural on 11p13, 8p23, 3q26, and 4q12.7 We investigated candi- Development Unit, UCL Institute of Child Health, London, United date in the top three linkage peaks from that study in Kingdom; 5Institute of Ophthalmology, University College London, London, United Kingdom; and 6Genome Research Center, The Univer- a total of 2211 subjects from the 1958 British Birth Cohort sity of Hong Kong, Hong Kong, SAR, China. and report findings from this candidate gene study and 3These authors contributed equally to the work presented here subsequent expression studies. and should therefore be regarded as equivalent authors. Supported by the Medical Research Council Component Project Grant G0301069 and Health of the Public Project Grant G0000934. METHODS PGH is funded by the EU MyEuropia Marie Curie Research Training Network. Candidate Gene Association Study Submitted for publication April 3, 2010; revised July 13 and The 1958 British Birth Cohort originally consisted of 17,000 persons August 30, 2010, and March 10, July 29, September 30, and October who were born during one week in March 1958 and were subse- 30, 2011; accepted November 1, 2011. quently followed up at intervals8 with physical examination and Disclosure: P.G. Hysi, None; C.L. Simpson, None; Y.K.Y. Fok, collection of data on environmental, social, and lifestyle factors. In None; D. Gerrelli, None; A.R. Webster, None; S.S. Bhattacharya, None; C.J. Hammond, None; P.C. Sham, None; J.S. Rahi, None 2002 to 2003, when the participants were aged 44 to 45 years, a Corresponding author: Jugnoo S. Rahi, MRC Centre of Epidemiol- biomedical survey was undertaken of the 9377 individuals still ogy for Child Health, Institute of Child Health, UCL, 30 Guilford Street, actively participating in the study. Of all the subjects, 2485 (27%) London WC1N 1EH, UK; [email protected]. were selected randomly and underwent noncycloplegic autorefrac-

Investigative Ophthalmology & Visual Science, January 2012, Vol. 53, No. 1 440 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.

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tion with an autorefractor (Retinomax 2; Nikon, Tokyo, Japan)9 Ͼ80% calculated from loci genotyped at the first stage was interpreted (equipment costs precluded autorefraction of all subjects). After as proof of identity, and the 644 samples concerned were removed exclusion of 251 subjects for whom no biological material was from the replication panel. The subjects’ Caucasian ancestry in the available and 23 subjects with high anisometropia, this study was replication stage was confirmed by eigenvector analysis.14 The 1023 conducted in two stages. In 1188 subjects in a discovery stage, subjects ultimately included in the replication stage had been geno- fine-mapping was performed of the regions of interest through typed using several platforms, including some of very high density custom genotyping. In a replication stage of another 1023 subjects, (Affymetrix 5 and 6; Illumina Human1M-Duo and Omni 660W-Quad not overlapping with those participating in the first stage, associa- and Illumina HumanHap550 SNP chips). tions were examined in subjects using genomewide data. Autore- fractor measurements in this age group provided a reliable objective Statistical Analysis measure of refractive error at the point at which primary myopia became manifest but before myopia secondary to other ocular After genotyping, SNPs were included in subsequent analytical stages Ͼ disorders occurred.9 This avoided misclassification and ensured if they were in Hardy-Weinberg equilibrium (P 0.01), polymorphic phenotypic stability. Blood was taken for the creation of immortal- (minor allele frequency at least) in the population sample, and above a Ͼ ized cell lines to provide a continuous source of DNA.8 threshold genotyping rate across the sample ( 95%). Refractive error was quantified using the summary measure of We considered two phenotypic outcomes: the spherical equivalent spherical equivalent of refraction (SphE) in diopters (D). We used as transformed using rank-based inverse normal transformation to over- autorefraction readings to calculate SphE for each eye of every subject come nonnormality of phenotype distribution (Fig. 1) and myopia as a in the conventional way (SphE ϭ S ϩ C/2), where S is the sphere and categorical disease status using the criterion of a spherical equivalent of Ϫ1.00 D or more extreme for myopia, with all subjects with0Dor C is the cylinder). Subjects with highly discordant SphE for both eyes 3,15 (within the worst 5% of the sample) were excluded.10,11 Inclusion higher classified as nonmyopes, as in previously published studies. Association was tested using linear and logistic regression models; criteria for the first stage (linkage fine-mapping and SNP association 14 discovery stage) were the mean spherical equivalent within either of PLINK and Stata 10 (Stata Corp, College Station, TX) were used for the analysis. Correction for multiple testing was made either through the outer tertiles of the distribution for this trait (half the participants the Bonferroni method or through phenotype-swapping 1 million in each tertile). The lower tertile spanned the interval Ϫ12.875D to permutations to control for multiple testing where linkage disequilib- Ϫ1.3125D, whereas the upper tertile spanned the interval Ϫ0.3125D rium was so high that the information made available from multiple to ϩ6.50D. As a categorical trait, myopia was determined as a mean SNP loci became highly redundant. spherical equivalent of Ϫ1D or less and nonmyopia as 0D or above. All genotypes obtained at the various stages of the work were Subjects whose spherical equivalent was between 0 and Ϫ1D were not converted with their positive strand alleles as reference. Information included in the qualitative analysis to remove uncertainties of myopia on variation at loci that were not directly genotyped in our assays at classification inherent in any arbitrary dichotomization of quantitative both stages were imputed using the program MACH16 using the traits.12 All participants gave informed written consent to participate in HapMap Phase 2 CEU population as a reference. Four hundred Markov genetic association studies, and the study and biomedical examination Chain iterations accounting for the 300 most common haplotypes protocols were approved by the South East MultiCentre Research overlapping SERPINI2 were used to provide maximal quality of impu- Ethics Committee and the Oversight Committee for the 1958 British tation (error rate equaled 2e-04). Metal was used to calculate fixed- Birth Cohort. The research adhered to the tenets of the Declaration of effect inverse variance weighted meta-analysis. Helsinki. The discovery stage of the study was designed with two phases Expression in Fetal Tissues. Human embryonic and fetal of genotyping. The first phase of genotyping consisted of fine-scale material was provided by a licensed tissue bank, Human Developmen- genotyping of 1536 single nucleotide polymorphisms (SNPs) cover- tal Biology Resource (HDBR), UCL Institute of Child Health, with ing 3 of the 4 linkage peaks on 11p13, 8p23, and 3q26 reported by National Research Ethics approval. Hammond et al.7 on 590 persons of Caucasian ancestry from the Cloning. We designed genomic amplicons to cover the SERPINI2 1958 Birth Cohort, with autorefractor measures randomly selected coding regions (see Table 4). PCRs were performed in 96-well plates. from the outer tertiles of the population distribution. These SNPs Fetal total cDNA (50 ng, supplied by HDBR) was amplified using the were selected so that they could provide information on genetic Invitrogen Platinum® Taq DNA polymerase PCR Kit. PCR products variations from 111 genes. The fourth peak reported in the original were purified using a PCR purification kit (QIAquick; Qiagen, Valencia, study7 was the one with the weakest evidence for linkage and CA) and were ligated (pGEM T-Easy; Promega, Madison, WI). coincidentally was the widest one. Based on cost efficiency consid- Production of DIG-Labeled Probes. Antisense and sense erations, it was not analyzed as part of the present work. Linkage probes were prepared by linearizing plasmids for SERPINI2 A, B, and disequilibrium patterns from the HapMap Phase 2 CEU population C with SpeI and SacII, respectively. Digoxigenin-UTP was incorporated were used to inform the choice of these SNPs. Selection of the tag into riboprobes during in vitro transcription using the DIG RNA label- SNPs was made using Tagger.13 An r2 of at least 0.8 was required for ing mix (Roche, Indianapolis, IN) according to the manufacturer’s genomic regions inside the genes, and an r2 of 0.7 or above was instructions. Antisense and sense probes were generated using T7 and required for the genomic regions that were not known to code for SP6 polymerase, respectively. Probe concentration was measured any transcript. Genotyping was performed using a methylation assay (Nanodrop; Thermo Scientific, Waltham, MA). platform (GoldenGate; Illumina, San Diego, CA). Approximately Tissues Tested. Sections from seven human embryos and four one-third of the strongest associations obtained (503 SNPs) from the fetuses were tested. Samples ranged from 47 to 63 days of gestation. first phase were taken forward and genotyped in an additional 598 In Situ Hybridization. In situ hybridization was carried out as subjects from the 1958 British Birth Cohort, also drawn from the described by Wilkinson.17 Human embryonic and fetal tissue was outer tertiles of the refractive error distribution, in a second phase. dissected and fixed in 4% paraformaldehyde (PFA) in phosphate-buff- As in the previous phase, genotyping was carried out using the same ered saline (PBS) overnight at 4°C. After fixation, tissues were dehy- methylation assay platform (GoldenGate; Illumina). drated and embedded in paraffin wax. Sagittal sections were cut at 8 In the replication stage of the analysis, we initially obtained and ␮m using a standard microtome and attached to microscope slides used data from all (N ϭ 1667) Caucasian participants in the 1958 Birth (Superfrost Plus; VWR, Leicestershire, UK). Before hybridization, tissue Cohort with autorefractor data who had undergone genotyping using sections were dewaxed, hydrated, fixed in 4% PFA/PBS, and rinsed genomewide SNP chip arrays in the course of other research. Subjects twice with PBS. were removed by incubation with proteinase who had been included in the discovery stage of our study were K (20 mg/mL) in PBS. After washing with PBS, the sections were excluded in the second stage using a relatedness analysis. Relatedness refixed in the same PFA solution and treated with 0.1 M triethanol-

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FIGURE 1. Full distribution of spheri- cal equivalent refractive error in the 1958 British Birth Cohort. Vertical dashed lines: values Ϫ1.3125 D and 0.3125 D, used to define our tertiles.

amine containing 0.25% acetic anhydride. Slides were dehydrated RESULTS through an alcohol series and air dried. Hybridization solution contained riboprobe (300 ng DIG-labeled Of the original 1536 SNPs, 1472 (95.83%) were genotyped RNA probe), RNAguard (1 ␮L/mL), and tRNA (0.5 mg/mL) in hybrid- successfully in 590 subjects in phase 1 of the study. Of all the ization buffer (50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.5, 5 SNPs included in the analysis, the strongest association with mM EDTA, pH 8.0, 10% dextran sulfate, and 1ϫ Denhardt’s solution). spherical equivalent was observed for a genomic segment A 100-␮L aliquot of hybridization probe was added to each slide, which spanning 36 kb on 3 (P ϭ 0.000022 for was incubated in a sealed chamber moistened with 50% formamide/1ϫ rs9810473 and other SNPs in perfect LD with it; data not standard saline citrate (SSC) overnight at 65°C. Stringency washes were shown.), which overlaid the serine protease inhibitor clade I performed in the following order: 2ϫ SSC (twice at 65°C), 50% forma- member 2 (SERPINI2) genomic coding region. This association mide/2ϫ SSC (twice at 65°C), 2ϫ SSC (twice at 65°C), 0.2ϫ SSC was stronger than what would have been expected from sim- (65°C), and 0.2ϫ SSC (65°C cooled to room temperature). Slides were ple multiple testing (P ϭ 0.03 after Bonferroni multiple testing then incubated for 1 hour in 150 mM NaCl and 100 mM Tris-HCl, pH adjustment). SNPs in which the strongest association was ob- 7.5, containing 10% fetal calf serum (FCS). For antibody detection, served, including 20 SNPs overlapping the SERPINI2 gene and slides were incubated in anti-digoxigenin antibody conjugated with flanking adjacent regions, were genotyped in an additional 598 alkaline phosphatase (anti-Dig antibody diluted 1:1000, containing subjects from the British Birth Cohort (Table 1 and Supplemen- 2% FCS) overnight at 4°C. Expression patterns were visualized using tary Table S1, http://www.iovs.org/lookup/suppl/doi: the NBT/BCIP system, and sections were mounted in aqueous 10.1167/iovs.10-5640/-/DCSupplemental). Imputation from mounting medium (VectaMount; Vector Laboratories, Burlingame, genotypes of all subjects participating in phases 1 and 2 of the CA) and analyzed using an imaging system (Axioplan 2; Zeiss, discovery stage was performed as described to improve the Thornwood, NY). accuracy of the localization of the association signal.

TABLE 1. Best Association Results for the SNPs Located within the SERPINI2 Genomic Sequence or Its Immediate Vicinity from the Discovery Stage for the Spherical Equivalent

Regression Chromosome SNP Position on Build 36 N Slope SE P

3 rs6784768 168639049 1188 Ϫ0.1019 0.029 0.00043 3 rs9864094 168642525 1188 Ϫ0.1019 0.029 0.00043 3 rs9844202 168642528 1188 Ϫ0.1019 0.029 0.00043 3 rs9810473 168642674 1188 Ϫ0.1019 0.029 0.00043 3 rs9848929 168643017 1188 Ϫ0.1019 0.029 0.00043 3 rs9884112 168645652 1188 Ϫ0.1019 0.029 0.00043 3 rs2272138 168646881 1188 Ϫ0.1019 0.029 0.00043 3 rs13085856 168647355 1188 Ϫ0.1019 0.029 0.00043 3 rs6444326 168641564 1188 Ϫ0.1019 0.029 0.00043 3 rs35216909 168642415 1188 Ϫ0.1019 0.029 0.00043

Effect sizes and SEs are standardized. Results shown are uncorrected.

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TABLE 2. Summary of the Association with Refractive Error for Selected SNPs for the SERPINI2 Loci, in Each of the Phases of This Study, Directionality of Effect, and Meta-Analysis Association

SNP Bp Allele 1 Allele 2 Stage 1 P Stage 2 P Direction of Effect Standardized Effect Meta P

rs6784768 168639049 T C 0.0004 0.02 — Ϫ0.1112 0.00007 rs9810473 168642674 A G 0.0004 0.02 ϩϩ 0.1112 0.00007 rs9864094 168642525 T C 0.0004 0.02 ϩϩ 0.111 0.00008 rs9844202 168642528 T C 0.0004 0.02 — Ϫ0.1108 0.00009 rs9848929 168643017 A G 0.0004 0.02 — Ϫ0.1108 0.00009 rs9884112 168645652 T G 0.0004 0.02 ϩϩ 0.1108 0.00009 rs2272138 168646881 A G 0.0004 0.02 ϩϩ 0.1108 0.00009 rs13085856 168647355 T C 0.0004 0.02 — Ϫ0.1108 0.00009 rs9848915 168642985 A G 0.0006 0.02 — Ϫ0.1091 0.0001 rs10936539 168674811 C G 0.004 0.03 — Ϫ0.1019 0.0004 rs13074812 168638486 C G 0.06 0.05 — Ϫ0.0923 0.001 rs9860065 168645314 A G 0.09 0.02 — Ϫ0.0658 0.02

Stage 1 and stage 2 refer to the discover and replication stages, respectively. Values shown are uncorrected.

A summary of the probabilities for association with refrac- with refractive error, was equally associated with myopia tive error for polymorphisms, directly genotyped or imputed, (meta-analysis OR ϭ 1.44; 95% CI, 1.10–1.89 for allele T). within the SERPINI2 genomic sequence is shown in Table 2 Association with myopia was overall significant for a number of and Supplementary Table S2 (http://www.iovs.org/lookup/ other SNPs (Table 3). suppl/doi:10.1167/iovs.10-5640/-/DCSupplemental). Analysis Serine protease inhibitor clade I member 2 (SERPINI2) is a of the 1188 subjects genotyped revealed strong association member of the plasminogen activator inhibitor-1 family, a sub- for a number of SNPs adjacent to one another and located set of the superfamily, and these proteins act as tissue- within the 3Ј end of the SERPINI2 gene transcript (best specific plasminogen-activator (tPA) inhibitors.18 The limited association P ϭ 0.00043 for rs9810473 [standard linear literature on gene function indicates that SERPINI2 has a com- regression coefficient ␤ ϭϪ0.10] and a number of other plex role in the control of growth, with a knockout mouse SNPs in proximity with it). This SNP and others remained model having apoptosis of the acinar cells.19 SERPINI2 expres- significant after correcting for multiple testing using 1 mil- sion has been reported to be inversely correlated with tumor lion phenotype-swapping permutations (P ϭ 0.0006). growth in breast, pancreatic, and other types of cancer.20–22 We extracted SERPINI2 genotypes within the associated However, there was no published report of SERPINI2 expres- interval (168522964–168806403 on genomic build 36) from sion in ocular tissues at the time of our study. For this reason 1023 additional subjects participating in the 1958 British we investigated the presence of SERPINI2 in human eyes. Birth Cohort in the replication stage. We fitted the geno- Using in situ hybridization (Table 4), we sought to assess the types and their refractive error measurements and SERPINI2 expression of SERPINI2 transcript expression in the human genotypes in linear regression models and noticed that a fetal and embryonic eye and brain. number of SNPs identified in association with the phenotype Antisense probes to SERPINI2A and SERPINI2C showed in the discovery stage were also nominally associated, with similar patterns of expression in the eye and brain. the same directionality of association in the replication stage SERPINI2B antisense probe did not yield any reproducible (Supplementary Table S3, http://www.iovs.org/lookup/ results. suppl/doi:10.1167/iovs.10-5640/-/DCSupplemental). Evi- We found SERPINI2 to be expressed in the anterior epithe- dence for association remained strong for SNP rs9810473 lium of the lens and within the lens (Fig. 4A). The anterior (P ϭ 0.02). In the second stage, the strongest effects were surface of the lens is covered by an epithelial cell layer that observed for rs6784768; each copy of the rarer allele T in the extends to the equatorial zone. These cells proliferate and at replication stage lowered refraction by an average 0.26 SE units the equatorial zone migrate posteriorly, elongate, and differen- (corresponding to 0.52 D) versus 0.1 SD units in the first stage tiate to produce the lens fibers. SERPINI2 may therefore have (corresponding to 0.27 D). a role in the production of lens fibers. SERPINI2 expression Combining both results confirmed the association at that was also seen in discrete cells within the neural retina and (P ϭ 7.4 ϫ 10Ϫ5), as shown in Table 2 and Figure 2. The mesenchyme around the eye. Additional markers would be overall meta-analysis standardized effect was Ϫ0.11 (approxi- needed to identify which cell types express SERPINI2 in the mately Ϫ0.22 D change for each additional copy of suscepti- neural retina. bility allele) and SE. SE ϭ 0.03 for these SNPs. SERPINI2 was expressed in the cortical plate, hippocam- We further looked at associations between SERPINI2 and pus, mesencephalon, and thalamus (Fig. 4B). The mesen- clinical myopia. We compared allele frequency distributions of cephalon includes the superior colliculi, an area known to subjects with myopia (ϽϪ1 D) with those of nonmyopic sub- have a visual function. SERPINI2 was expressed in the fetal jects (Ն0 D) and found good evidence for associations be- lens and neural retina at 63 days of gestation (Fig. 4). tween SERPINI2 SNPs and myopia, with consistency in the SERPINI2 was also shown to be expressed at the site of effect for all loci studied (Supplementary Tables S4, S5, http:// fusion of the eyelids. www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5640/-/DC Supplemental). The main association originated from a group of tightly linked markers, as shown in Figure 2. As an example, DISCUSSION rs10936538 is associated with myopia on both stages. The minor T allele on that locus significantly decreased the odds of Based on a joint analysis of two subpopulations drawn from having myopia (meta-analysis odds ratio [OR] ϭ 0.80; 95% the 1958 British Birth Cohort, we report that polymor- confidence interval [CI], 0.69–0.93; uncorrected P ϭ 0.004) as phisms in a novel candidate gene, SERPINI2, are associated shown in Figure 3. The SNP rs6784768, previously associated with refractive error. According to our results, SERPINI2

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FIGURE 2. Association in and around the SERPINI2 gene. The blue diamond represents the SNP with the best association (shown in a logarithmic scale on the y-axis). The colors of the other diamonds represent the linkage disequilibrium with the lead SNP (red for a pairwise r2 0.8–1, orange for r2 0.6–08, and yellow for r2 0.4–0.6 between the SNP they represent and the lead SNP). The blue line represents the recombination rate as calculated in the HapMap CEU subjects. SERPINI2 is superimposed at the bottom of the figure.

polymorphisms increase susceptibility to refractive error; The Serpin gene family contains numerous members, and odds of having myopia were also significant in the presence SERPINI2 belongs to the serine protease inhibitor (serpin) of specific alleles (OR ϭ 0.80; 95% CI, 0.69–0.93 for allele family. In a mouse model of pancreatic insufficiency, dele- T of rs10936538). tion of the SERPINI2 gene of causes apoptosis of the acinar

FIGURE 3. ORs (horizontal axis) for SNP rs10936538 in the two pan- els used in this study and the meta- analysis ORs (vertical axis).

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TABLE 3. Association with Myopia for Selected SNPs in Each of the Stages of this Study and Meta-Analysis of their Effects

Discovery Stage Replication Stage

Lower Upper Lower Upper Heterogeneity SNP Allele 1 Allele 2 OR Bound Bound P OR Bound Bound P Meta P of Effect

rs9848915 A G 1.63 1.16 2.30 0.005 1.33 0.80 2.22 0.28 0.004 0.52 rs9884112 G T 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs9864094 C T 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs2272138 G A 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs9844202 T C 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs13085856 T C 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs9848929 A G 1.64 1.16 2.33 0.005 1.33 0.80 2.22 0.28 0.004 0.50 rs11926880 C A 0.84 0.70 1.00 0.06 0.73 0.56 0.95 0.02 0.004 0.39 rs10936538 T C 0.83 0.69 0.99 0.04 0.75 0.58 0.98 0.04 0.004 0.54 rs6784768 G A 1.64 1.16 2.33 0.005 1.20 0.79 1.83 0.40 0.007 0.26

All the values are uncorrected.

cells.19 Many of the symptoms in this model of pancreatic planation is that the distribution of the refractive error was insufficiency can be attributed to malnutrition, with the different in the two panels, though they were drawn from resultant effects on growth and immune function.19 the same cohort. Although the opposing tertiles were se- Our finding of an association of Serpini2 with refractive lected for the discovery stage, subjects used in the replica- error and myopia raises intriguing questions about patho- tion stage were drawn from the remainder of the same genesis. SERPINI2 is involved in acinar cells of the pancreas cohort, which meant the replication sample was not en- and may be important to control glucose metabolism. The riched for extreme cases of refractive error. Richness in latter has been hypothesized to be linked to the develop- extremes of phenotypes is reflected in the power of associ- ment of myopia.23,24 Experimental findings in animal models ation analyses. This was particularly true when the effect on indicate that insulin and its metabolic pathways are impli- myopia as a dichotomous trait was studied. Second, by cated in retinal signaling, which results in excessive eye comparing myopic with all nonmyopic subjects, we are growth and myopia.23,25 Signaling between the neural retina likely to be reporting effect sizes higher than would be and lens is known to be necessary for eye growth and obtained comparing myopic with emmetropic subjects. This development.26 Expression of SERPINI2 in the anterior ep- comparison was made necessary by the design adapted in ithelium of the lens, within the lens, and in discrete cells this study, which was aimed at ensuring the highest power within the retina is compatible with a role in eye develop- for refractive error association detection. Third, our panel ment. Thus it is possible that SERPINI2 is implicated in consisted of Caucasian subjects, and it is possible that they refractive error through more than one mechanism. Further were not fully generalizable to populations of different racial functional characterization is needed of the properties of or ethnic composition. Fourth, we cannot exclude the pos- the SERPINI2 and the physiologic pathways in sibility that variants other than those in SERPINI2, either in which it participates. short or long distances from this gene, account for at least Polymorphisms located on that we report part of the linkage signal and variance of spherical equiva- here to be in association with refractive error and myopia lent observed in the original linkage study. Many complex are unlikely to be alone in predisposing to these traits. The genetic traits are controlled by multiple loci of small ef- presence of other SNPs that were initially found in associa- fect.27 Although the power to detect effects such as the ones tion in the first genotyping group but were not followed up reported here, at a significance threshold of ␣ ϭ 0.001 in the second (such as those in 8 and 11), as would be 0.90 for N ϭ 1188 (our discovery sample), for well as variants that were not associated at all in the present smaller effect sizes the power would have been considerably work either because they lacked sufficient power or because smaller. they lay outside the linkage regions of interest to us, suggest Refractive error is increasingly relevant to public health that SERPINI2 polymorphisms could potentially be just a in both developed and developing countries. The present few of what is likely to be a complex constellation of genetic lack of knowledge about environmental risk factors makes it factors behind refractive error and myopia. difficult to develop effective strategies for prevention and There are some issues to be considered regarding the treatment. Identifying genetic variants could lead to better results of our study. First, although the effect size and understanding of the pathogenesis of refractive error. In this direction of effect were similar in discovery and replication study, we have observed the association of variants in SER- data sets, the replication data set achieved lesser statistical PINI2 with refractive error. Investigation of the function of significance than the discovery cohort. The most likely ex- this gene—only very recently recognized to be expressed in

TABLE 4. Primers Used for SERPINI2 Cloning

(Exons Primer Sequence (5؅33؅) Size of PCR Product (bp

2–3 Forward: TGG ACA CAA TCT TCT TGT GGA 438 Reverse: CAC AAG CCT TTG CAT CTT GA 5–7 Forward: TGG CTC TCT GAG ATG CAA GA 179 Reverse: TGC GTC ACT TGG GAA ACA TA 2–3 Forward: GAG CCA AAG GAA AAG CAC AG 245 Reverse: TGC ATC TTG AAA ATC CAC CA

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the human eye—in relation to ocular development could lead to a better understanding of the pathophysiology of this complex trait and thus ultimately to therapeutic or preven- tive strategies.

Acknowledgments

Human embryonic and fetal material was provided by the MRC/ Wellcome-funded Human Developmental Biology Resource, UCL Institute of Child Health.

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