729463.pdf

Research ArticleEvaluation of Four Genetic Variants in Han Chinese Subjectswith High Myopia

Zimeng Ye12 Huaichao Luo2 Bo Gong23 Ying Lin23 Ping Shuai2 Pu Wang2

Changning Ye3 Zhenglin Yang234 Wanjun Wang1 and Yi Shi234

1College of Life Science and Engineering Southwest Jiaotong University 111 First Section Northern Second Ring RoadChengdu Sichuan 610031 China2Sichuan Provincial Key Laboratory for Human Disease Gene Study Hospital of the University of Electronic Science andTechnology of China and Sichuan Provincial Peoplersquos Hospital 32 the First Ring Road West 2 Chengdu Sichuan 610072 China3Department of Laboratory Medicine Sichuan Provincial Peoplersquos Hospital 32 the First Ring Road West 2Chengdu Sichuan 610072 China4School of Medicine University of Electronic Science and Technology of China 32 the First Ring Road West 2Chengdu Sichuan 610072 China

Correspondence should be addressed to Wanjun Wang wanjunwanghomeswjtueducn and Yi Shi yshiuestceducn

Received 24 September 2014 Revised 19 December 2014 Accepted 19 December 2014

Academic Editor Edward Manche

Copyright copy 2015 Zimeng Ye et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

High myopia is one of the leading causes of blindness worldwide However the exact etiology of high myopia remains unraveleddespite numerous attempts of elucidation Previous genome-wide association study (GWAS) has revealed that four single nucleotidepolymorphisms (SNPs) including rs2969180 rs1652333 rs9307551 and rs7837791 were associated with highmyopia in CaucasiansThe present study was conducted to investigate whether these genetic variants were associated with high myopia in Han ChineseThese four SNPs were genotyped by SNaPshot method in a Han Chinese cohort composed of 827 patients with high myopia and988 healthy controls Among the SNPs genotyped only rs9307551 was found to be significantly associated with high myopia in thisstudy Carriers of rs9307551A allele AA and AC genotypes had an increased risk of highmyopia (OR = 133 95CI 114ndash154 OR =175 95 CI 128ndash238 OR = 159 95 CI 124ndash201 resp) Interestingly when split by gender the association between rs9307551and high myopia proved to be gender-specific with significance observed only in females but not males These findings suggestedthat the SNP of rs9307551 showed a gender-specific association with high myopia in the Han Chinese population In additionLOC100506035 a lincRNA gene might play a crucial role in the susceptibility to high myopia

1 Introduction

Myopia is a common cause of visual impairment worldwide[1] with a prevalence of 331 in the USA [2] 164in Australia and 266 in the Western Europe [3] Theprevalence of myopia in Asian populations is up to 40ndash70[4ndash6] In mainland China almost 439 of the populationsuffer frommyopia [7] Lowmyopia (with a refractive error ⩾minus30D) andmediummyopia (with a refractive error betweenminus60 and minus30D) are primarily physiological whereas highmyopia (with a refractive error ⩽ minus60D) is one of theleading causes of blindness in the world High myopia (HM)is associated with pathologic ocular changes such as myopic

macular degeneration glaucoma and retinal detachment [8ndash10] In China the prevalence of HM is 41 [7] and is muchhigher in high school and university students [11]

Previous studies have indicated the involvement ofgenetic and environmental factors in the pathogenesis ofmyopia genetic factors play a major role in the developmentof high myopia [12 13] Recently the application of genome-wide association study (GWAS) has revealed several novelsingle nucleotide polymorphisms (SNPs) susceptible to HMin theCaucasian populations including rs2969180 rs1652333rs9307551 and rs7837791 [14] Here in order to investigatewhether these SNPs were associated with highmyopia inHanChinese population we conducted a case-control study in

Hindawi Publishing CorporationJournal of OphthalmologyVolume 2015 Article ID 729463 6 pageshttpdxdoiorg1011552015729463

2 Journal of Ophthalmology

Table 1 Demographic and clinical characteristics of HM patients and controls in this study

Group Number Average age (years) Gender Refractive errors (diopter) Axial length (mm)Male Female OD OS OD OS

Patient 827 3530 plusmn 1655 318 509 minus1134 plusmn 465 minus1104 plusmn 449 2816 plusmn 224 2797 plusmn 241Control 988 4968 plusmn 1722 581 407plusmn standard deviation OD right eye OS left eye

a Han Chinese cohort composed of 827 unrelated individualswith high myopia and 988 unrelated normal controls

2 Materials and Methods

21 Study Population This studywas approved by the Institu-tional Review Board of Sichuan Provincial Peoplersquos HospitalAll participants gave their written informed consent toparticipate in the study The study was performed under theprinciples of the Declaration of Helsinki Patients with HMand normal matched controls were recruited at the Ophthal-mology Clinic of Sichuan Provincial Peoplersquos Hospital Allparticipants went through a standard examination protocolIn this study the diagnosis for high myopia required thespherical equivalent less than or equal tominus60DS in both eyesand the axial length of the eye globe greater than or equal to260mm Individuals were excluded from the study if theyhad undergone ocular procedures that might alter refractionor if they had other symptoms besides HM For the controlsthe criteria were no evidence of disease in either eye and aspherical equivalent from minus10 to +10DS The controls werefrom the same district as the cases All of the participantsin this study were Han Chinese In total 827HM patientsand 988 normal controls were recruited Demographic andclinical information of the patients and controls were listedin Table 1

22 Selection of SNPs In 2013 the Consortium of RefractiveError and Myopia (CREAM) performed a genome-widemeta-analysis of multiancestry cohorts and identified 16 newloci for refractive error in individuals of European ancestryof which 8 were shared with Asians Combined analysisidentified 8 additional associated loci of which 5 were sharedwith Asians [14] In this study we chose 4 of these 13 sharedSNPs and planned to investigate the other 9 in future studiesThe 4 SNPs we selected were rs2969180 (intronic SHISA6gene) rs1652333 (24 kb 51015840 of CD55 gene) rs9307551 (33 kb51015840 of LOC100506035 gene) and rs7837791 (147 kb 31015840 of TOXgene)

23 Genotyping Venous blood of each subject was drawnand collected in an EDTA-containing tube Genomic DNAwas extracted from the blood using a Gentra Puregene BloodDNA kit (Gentra Minneapolis MN) SNP genotyping wasconducted by the dye terminator-based SNaPshot methodwith success rate and accuracy greater than 99 as judgedby random regenotyping of 10 of the samples in the subjectgroup

24 Statistical Analysis The Hardy-Weinberg equilibrium(HWE) for each SNP was tested with a standard observed-expected chi-square test (1205942 test) The overall associationstudy was adjusted for age and gender with binary logisticregression whereas gender-stratified analysis was adjustedfor age The genetic association analysis was carried out byconstructing 2 times 3 tables of the genotype counts and 2 times2 tables of the allele counts for each SNP in both patientand control groups Subsequently Pearson 1205942 statistics werecalculated and 119875 values were computed by comparing thestatistic to a 1205942 distribution with 1 or 2 degrees of free-dom for the allelic and genotypic tests Odds ratio (OR)corresponding to 95 CI was used to assess the strengthof association between the SNPs and susceptibility to highmyopiaThe significance of the pooledORwas determined bythe 119885-test All statistical analyses were conducted using SPSS(version 170) software and all results were considered to bestatistically significant when 119875 lt 005

3 Results

All of the four SNPs were successfully genotyped and thegenotype distributions were within Hardy-Weinberg equi-librium in both patient and control groups (119875 gt 005Table 2) Among the SNPs genotyped rs9307551 was foundto be significantly associated with high myopia The minorallele (A) frequency (MAF) of rs9307551 was 0445 in controlsand 0505 in patients and the adjusted allelic 119875 valuewas 246 times 10minus4 with an adjusted odds ratio of 133 (95confidence interval 114ndash154) The 119875 value of rs9307551 stillshowed significant association with HM after the Bonferronicorrection (corrected 119875 = 984 times 10minus4) No significantassociation was observed between HM and the other threeSNPs (119875 gt 005 Table 2)

We further investigated the association between HM andrs9307551 by using different genetic models The genotypedistribution of rs9307551 is shown in Table 3 The frequencyof CC genotype was significantly higher in the controls thanthat in HMpatients (312 in controls and 236 in patients)The results suggested that subjects carrying rs9307551ACrs9307551AA and rs9307551AA + AC were more likely tosuffer from HM than those carrying rs9307551CC (additive1 model 119875 = 0012 adjusted OR [95 CI] = 149 [115ndash194]additive 2model119875 = 337times10minus4 adjustedOR [95CI] = 175[128ndash238] dominant model 119875 = 212 times 10minus4 adjusted OR[95 CI] = 159 [124ndash201]) In addition the recessive modelindicated that subjects bearing rs9307551AA had a 134-fold (95 CI 104ndash173) increased likelihood of HM com-pared to those who harbor rs9307551AC and rs9307551CC

Journal of Ophthalmology 3

Table2As

sociationbetweenHM

andfour

SNPs

(rs2969180rs1652333rs9307551and

rs7837791)in

aHan

Chinesep

opulation

SNP

Chromosom

ePo

sition

Gene

Minor

allele

MAF

119875HWE

Allelic119875lowast

Corrected119875amp

OR(95

CI)lowastlowast

Patie

ntCon

trol

Patie

ntCon

trol

rs2969180

17114

07900

SHISA6

G0472

0483

0208

0722

0581

110

4(090ndash

122)

rs1652333

1203858855

CD55

T0452

0420

0615

0707

0936

110

0(085ndash116)

rs9307551

480530670

LOC100506035

A0505

044

50302

0580

246times10minus4

984times10minus4

133(114

ndash154)

rs7837791

860179085

TOX

T0478

0473

0888

006

40605

110

4(090ndash

121)

lowastAllelic119875valueh

asbeen

adjuste

dby

agea

ndsex

lowastlowastORs

(95

CI)h

aveb

eenadjuste

dby

agea

ndsexandwered

etermined

bythe1205942test

patientsv

ersusc

ontro

lsampCorrected119875=allelic119875times4(th

enum

bero

fgenotyped

SNPs)

SNP

singlen

ucleotidep

olym

orph

ismM

AFminor

allelefre

quencyH

WE

Hardy-W

einb

ergequilib

riumO

Rod

dsratio


Table 3 The SNP of rs9307551 in the LOC100506035 gene was associated with HM in this study

Group Genotype (119899)119875 HWE Model Crude OR (95 CI) Adjusted OR (95 CI) Adjusted 119875

AA AC CCControl 200 (203) 479 (485) 308 (312) 0580 1 1Patient 206 (257) 408 (507) 190 (236) 0302 Allelic 124 (110ndash141) 133 (114ndash154) 246 times 10minus4

Additive 1 140 (113ndash173) 149 (115ndash194) 0012Additive 2 154 (120ndash198) 175 (128ndash238) 337 times 10minus4

Dominant 144 (118ndash176) 159 (124ndash201) 212 times 10minus4

Recessive 124 (100ndash152) 134 (104ndash173) 0025Crude ORs (95 CI) were determined by the 1205942 test patients versus controls Adjusted ORs (95 CI) and adjusted 119875 values were obtained by adjusting forage and sexGenotype (AAACCC) analyses were conducted for the allelic model (A compared with C) additive model 1 (AC compared with CC) additive model 2 (AAcompared with CC) dominant model (AA + AC compared with CC) and the recessive model (AA compared with AC + CC)

Table 4 The association between HM and rs9307551 was gender-specific

Gender Group Genotype (119899)119875 HWE Model Crude OR (95 CI) Adjusted OR (95 CI) Adjusted 119875

AA AC CC

Female

Control 82 (203) 191 (473) 131 (324) 0417 1 1Patient 133 (269) 259 (524) 102 (207) 0241 Allelic 145 (120ndash174) 147 (119ndash184) 286 times 10minus4



Recessive 145 (106ndash198) 141 (100ndash201) 0050

Male

Control 118 (202) 288 (494) 177 (304) 0966 1 1Patient 73 (235) 149 (481) 88 (284) 0521 Allelic 111 (091ndash135) 118 (079ndash175) 0411

Additive 1 124 (084ndash183) 118 (094ndash147) 0142Additive 2 104 (076ndash144) 139 (089ndash217) 0142Dominant 109 (081ndash149) 124 (088ndash175) 0223Recessive 121 (087ndash169) 125 (086ndash182) 0237

119875 value and ORs (95 CI) were adjusted by age

In the meantime different genetic models were also appliedto analyze the association between rs2969180 rs1652333 andrs7837791 and HM Still no significant association was found(data not shown)

In order to study whether rs9307551 showed significantassociation only with one gender we split the subjects bygender and investigated the association by using differentgenetic models (Table 4) Interestingly our data indicatedthat the association between rs9307551 and high myopiawas gender-specific The association was only observed infemales but not males We also found that the associationwas not only maintained but also strengthened in the femalegroup despite the reduced sample size The allelic modelshowed that rs9307551A was the risk allele of HM (OR[95 CI] = 147 [119ndash184] 119875 = 286 times 10minus4) Femaleswith rs9307551AC rs9307551AA and rs9307551AA + ACrespectively had a 166-fold (95 CI 128ndash245 119875 = 0012)212-fold (95 CI 139ndash324 119875 = 465 times 10minus4) and 195-fold(95 CI 140ndash272 119875 = 799 times 10minus5) increased likelihoodof HM compared to those females carrying rs9307551CCFurthermore the recessive model showed also that femalesubjects bearing rs9307551AA had a 141-fold (95 CI 100ndash201) increased likelihood of HM compared to those females

with rs9307551AC and rs9307551CC None of these effectshowever was observed in males

4 Discussion

In 2013 the Consortium of Refractive Error and Myopia(CREAM) presented the results from the largest internationalgenome-wide meta-analysis and reported 24 new loci asso-ciated with myopia in the Caucasian populations includingrs2969180 rs1652333 rs9307551 and rs7837791 [14] As shownin the results of the CREAM study [14] the ldquoArdquo allele ofrs9307551 showed decreasing effect with spherical equivalentIn this study our results demonstrated that the ldquoArdquo allele ofrs9307551 had an increased risk of high myopia (Tables 2 and3) And taking the results of these two studies together weconcluded that the direction of the effect was the same inCREAM as in our study However no significant associationwas found in the other three SNPs in this study The samplesize in this study was much smaller than that in CREAMstudy This might be one of the reasons why we cannotreplicate the results of CREAM And other reasons could beallele frequency differences different LD patterns betweenpopulations and ethnic differences


On the other hand gender-specific genetic effects havebeen observed in other traits [15 16] In this study ourresults suggested that the genetic variant located at 33 kb 51015840of LOC100506035 gene was associated with HM in a gender-specific way This association was only found in femalesbut not in males However there have been too few studiesaddressing the effect of gender on the risk of HM In 2002Saw et al pointed out that myopia was associated with beingmale in Singaporean Chinese children [17] But in 2012You et al noted that myopia in school children in GreaterBeijingwas associatedwith female gender [18] As a result thegender-specific association seen in our study could provideinsights into the understanding of the etiology of HM inthe future Gender-specific associations might result fromgene-gene or gene-environment interactions It is possiblethat women are exposed to an environment which interactswith the geneThese results underlie the potential importanceof analyzing HM data both with and without gender as astratifying factor

LOC100506035 gene has also been called long intergenicnonprotein coding RNA 989 (LINC00989) Large intergenicnoncoding RNAs (lincRNAs) have recently been identifiedas a kind of new RNA molecule in gene regulation [1920] The lincRNAs which proved functional showed diversebiological activities such as involvement in X chromosomeinactivation and regulation of gene expression in cancer cellsstem cells and development [21ndash24] More than 30 oflincRNAs were estimated to be associated with chromatin-modifying complexes such as co-REST and PRC2 and sub-sequently target those complexes to specific genomic regions[19] HOTAIR a lincRNA which is part of the Hox genecluster proved to be related to breast cancer progression [25]And ANRIL a lincRNA regulating CDKN2A and CDKN2Bwas reported to be associated with atherosclerosis [26]However despite the potentially broad functional impact oflincRNAs in regulation of gene expression no lincRNAs havebeen reported to be related to HM In this study the datashowed that a genetic variant of a lincRNA gene may atleast in part play a crucial role in the development of HMin Han Chinese population This may facilitate the studyof lincRNAs and provide a new direction to investigate thegenetic mechanism implicated in HM

In summary our data revealed the gender-specific associ-ation between rs9307551 andHM inHanChinese populationFemale carriers of rs9307551 risk allele (A) weremore likely tosuffer from HM Our results suggested the potential impor-tance of analyzing HM data both with and without genderas a stratifying factor In addition our results suggested that agenetic variant in a lincRNA gene (LOC100506035) increasedsusceptibility to high myopia in Han Chinese

DisclaimerAll authors had full access to the data and take full responsi-bility for its integrity

Conflict of InterestsThe authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank the patients and their families for theirparticipationThis work was supported by the National BasicResearch Program of China (973 Program 2011CB504604 toZhenglin Yang) the National Science Foundation of China[81170882 to Yi Shi 81170883 to Zhenglin Yang 81371048to Bo Gong] the Department of Science and Technologyof Sichuan Province China [2012JQ0023 and 2014JZ0004 toYi Shi] and the Department of Sichuan Provincial Health[130167 to Bo Gong]

References

[1] Y Tano ldquoPathologic myopia where are we nowrdquoTheAmericanJournal of Ophthalmology vol 134 no 5 pp 645ndash660 2002

[2] S Vitale L EllweinM F Cotch F L Ferris III andR SperdutoldquoPrevalence of refractive error in the United States 1999ndash2004rdquoArchives of Ophthalmology vol 126 no 8 pp 1111ndash1119 2008

[3] J H Kempen P Mitchell K E Lee et al ldquoThe prevalence ofrefractive errors among adults in the United States WesternEurope and Australiardquo Archives of Ophthalmology vol 122 no4 pp 495ndash505 2004

[4] A Sawada A Tomidokoro M Araie A Iwase and TYamamoto ldquoRefractive errors in an elderly Japanese populationthe Tajimi studyrdquo Ophthalmology vol 115 no 2 pp 363ndash3702008

[5] M He Y Zheng and F Xiang ldquoPrevalence of myopia in urbanand rural children in mainland chinardquo Optometry and VisionScience vol 86 no 1 pp 40ndash44 2009

[6] P J Foster J Hee J M Tielsch G J Johnson and S K LSeah ldquoPrevalence and risk factors for refractive errors in adultChinese in Singaporerdquo Investigative Ophthalmology and VisualScience vol 41 no 9 pp 2486ndash2494 2000

[7] L Xu J Li T Cui et al ldquoRefractive error in urban and ruraladult Chinese in Beijingrdquo Ophthalmology vol 112 no 10 pp1676ndash1683 2005

[8] B J Curtin and D B Karlin ldquoAxial length measurementsand fundus changes of the myopic eyerdquo American Journal ofOphthalmology vol 71 no 1 pp 42ndash53 1971

[9] N A McBrien and A Gentle ldquoRole of the sclera in the devel-opment and pathological complications of myopiardquo Progress inRetinal and Eye Research vol 22 no 3 pp 307ndash338 2003

[10] SM Saw G Gazzard E C Shin-Yen andWH Chua ldquoMyopiaand associated pathological complicationsrdquo Ophthalmic andPhysiological Optics vol 25 no 5 pp 381ndash391 2005

[11] M He J Zeng Y Liu J Xu G P Pokharel and L B EllweinldquoRefractive error and visual impairment in urban childrenin southern Chinardquo Investigative Ophthalmology and VisualScience vol 45 no 3 pp 793ndash799 2004

[12] M Dirani M Chamberlain S N Shekar et al ldquoHeritabilityof refractive error and ocular biometrics the genes in myopia(GEM) twin studyrdquo Investigative Ophthalmology amp Visual Sci-ence vol 47 no 11 pp 4756ndash4761 2006

[13] M C Lopes T Andrew F Carbonaro T D Spector and C JHammond ldquoEstimating heritability and shared environmentaleffects for refractive error in twin and family studiesrdquo Investiga-tive Ophthalmology andVisual Science vol 50 no 1 pp 126ndash1312009

[14] V J Verhoeven P G Hysi R Wojciechowski et al ldquoGenome-wide meta-analyses of multiancestry cohorts identify multiple


new susceptibility loci for refractive error and myopiardquo NatureGenetics vol 45 no 3 pp 314ndash318 2013

[15] R Caliz L M Canet C B Lupianez et al ldquoGender-specificeffects of genetic variants within Th1 and Th17 cell-mediatedimmune response genes on the risk of developing rheumatoidarthritisrdquo PLoS ONE vol 8 no 8 Article ID e72732 2013

[16] C Perez-Becerril AGMorris AMortimer P JMcKenna andJ de Belleroche ldquoAllelic variants in the zinc transporter-3 geneSLC30A3 a candidate gene identified from gene expressionstudies show gender-specific association with schizophreniardquoEuropean Psychiatry vol 29 no 3 pp 172ndash178 2014

[17] S M Saw A Carkeet K S Chia R A Stone and D T H TanldquoComponent dependent risk factors for ocular parameters inSingapore Chinese childrenrdquoOphthalmology vol 109 no 11 pp2065ndash2071 2002

[18] Q S You L J Wu J L Duan et al ldquoFactors associated withmyopia in school children in China the Beijing Childhood EyeStudyrdquo PLoS ONE vol 7 no 12 Article ID e52668 2012

[19] A M Khalil M Guttman M Huarte et al ldquoMany humanlarge intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expressionrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 28 pp 11667ndash11672 2009

[20] M Guttman I Amit M Garber et al ldquoChromatin signaturereveals over a thousand highly conserved large non-codingRNAs in mammalsrdquo Nature vol 458 no 7235 pp 223ndash2272009

[21] M E Talkowski C Ernst A Heilbut et al ldquoNext-generationsequencing strategies enable routine detection of balancedchromosome rearrangements for clinical diagnostics andgenetic researchrdquo American Journal of Human Genetics vol 88no 4 pp 469ndash481 2011

[22] M Huarte M Guttman D Feldser et al ldquoA large intergenicnoncoding RNA induced by p53 mediates global gene repres-sion in the p53 responserdquo Cell vol 142 no 3 pp 409ndash419 2010

[23] S Loewer M N Cabili M Guttman et al ldquoLarge intergenicnon-coding RNA-RoR modulates reprogramming of humaninduced pluripotent stem cellsrdquo Nature Genetics vol 42 no 12pp 1113ndash1117 2010

[24] K C Wang Y W Yang B Liu et al ldquoA long noncodingRNA maintains active chromatin to coordinate homeotic geneexpressionrdquo Nature vol 472 no 7341 pp 120ndash124 2011

[25] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010

[26] C E Burd W R Jeck Y Liu H K Sanoff Z Wang and NE Sharpless ldquoExpression of linear and novel circular formsof an INK4ARF-associated non-coding RNA correlates withatherosclerosis riskrdquo PLoS genetics vol 6 no 12 Article IDe1001233 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Demographic and clinical characteristics of HM patients and controls in this study

Group Number Average age (years) Gender Refractive errors (diopter) Axial length (mm)Male Female OD OS OD OS

Patient 827 3530 plusmn 1655 318 509 minus1134 plusmn 465 minus1104 plusmn 449 2816 plusmn 224 2797 plusmn 241Control 988 4968 plusmn 1722 581 407plusmn standard deviation OD right eye OS left eye

a Han Chinese cohort composed of 827 unrelated individualswith high myopia and 988 unrelated normal controls

2 Materials and Methods

21 Study Population This studywas approved by the Institu-tional Review Board of Sichuan Provincial Peoplersquos HospitalAll participants gave their written informed consent toparticipate in the study The study was performed under theprinciples of the Declaration of Helsinki Patients with HMand normal matched controls were recruited at the Ophthal-mology Clinic of Sichuan Provincial Peoplersquos Hospital Allparticipants went through a standard examination protocolIn this study the diagnosis for high myopia required thespherical equivalent less than or equal tominus60DS in both eyesand the axial length of the eye globe greater than or equal to260mm Individuals were excluded from the study if theyhad undergone ocular procedures that might alter refractionor if they had other symptoms besides HM For the controlsthe criteria were no evidence of disease in either eye and aspherical equivalent from minus10 to +10DS The controls werefrom the same district as the cases All of the participantsin this study were Han Chinese In total 827HM patientsand 988 normal controls were recruited Demographic andclinical information of the patients and controls were listedin Table 1

22 Selection of SNPs In 2013 the Consortium of RefractiveError and Myopia (CREAM) performed a genome-widemeta-analysis of multiancestry cohorts and identified 16 newloci for refractive error in individuals of European ancestryof which 8 were shared with Asians Combined analysisidentified 8 additional associated loci of which 5 were sharedwith Asians [14] In this study we chose 4 of these 13 sharedSNPs and planned to investigate the other 9 in future studiesThe 4 SNPs we selected were rs2969180 (intronic SHISA6gene) rs1652333 (24 kb 51015840 of CD55 gene) rs9307551 (33 kb51015840 of LOC100506035 gene) and rs7837791 (147 kb 31015840 of TOXgene)

23 Genotyping Venous blood of each subject was drawnand collected in an EDTA-containing tube Genomic DNAwas extracted from the blood using a Gentra Puregene BloodDNA kit (Gentra Minneapolis MN) SNP genotyping wasconducted by the dye terminator-based SNaPshot methodwith success rate and accuracy greater than 99 as judgedby random regenotyping of 10 of the samples in the subjectgroup

24 Statistical Analysis The Hardy-Weinberg equilibrium(HWE) for each SNP was tested with a standard observed-expected chi-square test (1205942 test) The overall associationstudy was adjusted for age and gender with binary logisticregression whereas gender-stratified analysis was adjustedfor age The genetic association analysis was carried out byconstructing 2 times 3 tables of the genotype counts and 2 times2 tables of the allele counts for each SNP in both patientand control groups Subsequently Pearson 1205942 statistics werecalculated and 119875 values were computed by comparing thestatistic to a 1205942 distribution with 1 or 2 degrees of free-dom for the allelic and genotypic tests Odds ratio (OR)corresponding to 95 CI was used to assess the strengthof association between the SNPs and susceptibility to highmyopiaThe significance of the pooledORwas determined bythe 119885-test All statistical analyses were conducted using SPSS(version 170) software and all results were considered to bestatistically significant when 119875 lt 005

3 Results

All of the four SNPs were successfully genotyped and thegenotype distributions were within Hardy-Weinberg equi-librium in both patient and control groups (119875 gt 005Table 2) Among the SNPs genotyped rs9307551 was foundto be significantly associated with high myopia The minorallele (A) frequency (MAF) of rs9307551 was 0445 in controlsand 0505 in patients and the adjusted allelic 119875 valuewas 246 times 10minus4 with an adjusted odds ratio of 133 (95confidence interval 114ndash154) The 119875 value of rs9307551 stillshowed significant association with HM after the Bonferronicorrection (corrected 119875 = 984 times 10minus4) No significantassociation was observed between HM and the other threeSNPs (119875 gt 005 Table 2)

We further investigated the association between HM andrs9307551 by using different genetic models The genotypedistribution of rs9307551 is shown in Table 3 The frequencyof CC genotype was significantly higher in the controls thanthat in HMpatients (312 in controls and 236 in patients)The results suggested that subjects carrying rs9307551ACrs9307551AA and rs9307551AA + AC were more likely tosuffer from HM than those carrying rs9307551CC (additive1 model 119875 = 0012 adjusted OR [95 CI] = 149 [115ndash194]additive 2model119875 = 337times10minus4 adjustedOR [95CI] = 175[128ndash238] dominant model 119875 = 212 times 10minus4 adjusted OR[95 CI] = 159 [124ndash201]) In addition the recessive modelindicated that subjects bearing rs9307551AA had a 134-fold (95 CI 104ndash173) increased likelihood of HM com-pared to those who harbor rs9307551AC and rs9307551CC


Table2As

sociationbetweenHM

andfour

SNPs

(rs2969180rs1652333rs9307551and

rs7837791)in

aHan

Chinesep

opulation

SNP

Chromosom

ePo

sition

Gene

Minor

allele

MAF

119875HWE

Allelic119875lowast

Corrected119875amp

OR(95

CI)lowastlowast

Patie

ntCon

trol

Patie

ntCon

trol

rs2969180

17114

07900

SHISA6

G0472

0483

0208

0722

0581

110

4(090ndash

122)

rs1652333

1203858855

CD55

T0452

0420

0615

0707

0936

110

0(085ndash116)

rs9307551

480530670

LOC100506035

A0505

044

50302

0580

246times10minus4

984times10minus4

133(114

ndash154)

rs7837791

860179085

TOX

T0478

0473

0888

006

40605

110

4(090ndash

121)


asbeen

adjuste

dby

agea

ndsex

lowastlowastORs

(95

CI)h

aveb

eenadjuste

dby

agea

ndsexandwered

etermined

bythe1205942test

patientsv

ersusc

ontro


enum

bero

fgenotyped

SNPs)

SNP

singlen

ucleotidep

olym

orph

ismM

AFminor

allelefre

quencyH

WE

Hardy-W

einb

ergequilib

riumO

Rod

dsratio










AA AC CC

Female





Male







4 Discussion








Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2As

sociationbetweenHM

andfour

SNPs

(rs2969180rs1652333rs9307551and

rs7837791)in

aHan

Chinesep

opulation

SNP

Chromosom

ePo

sition

Gene

Minor

allele

MAF

119875HWE

Allelic119875lowast

Corrected119875amp

OR(95

CI)lowastlowast

Patie

ntCon

trol

Patie

ntCon

trol

rs2969180

17114

07900

SHISA6

G0472

0483

0208

0722

0581

110

4(090ndash

122)

rs1652333

1203858855

CD55

T0452

0420

0615

0707

0936

110

0(085ndash116)

rs9307551

480530670

LOC100506035

A0505

044

50302

0580

246times10minus4

984times10minus4

133(114

ndash154)

rs7837791

860179085

TOX

T0478

0473

0888

006

40605

110

4(090ndash

121)


asbeen

adjuste

dby

agea

ndsex

lowastlowastORs

(95

CI)h

aveb

eenadjuste

dby

agea

ndsexandwered

etermined

bythe1205942test

patientsv

ersusc

ontro


enum

bero

fgenotyped

SNPs)

SNP

singlen

ucleotidep

olym

orph

ismM

AFminor

allelefre

quencyH

WE

Hardy-W

einb

ergequilib

riumO

Rod

dsratio










AA AC CC

Female





Male







4 Discussion








Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























AA AC CC

Female





Male







4 Discussion








Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















729463.pdf

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