Research ArticleLack of Association between Variant rs7916697 in ATOH7 andPrimary Open Angle Glaucoma in a Saudi Cohort
Altaf A Kondkar 1 Taif A Azad 1 Faisal A Almobarak 1 IbrahimM Bahabri2
Hatem Kalantan1 Khaled K Abu-Amero 1 and Saleh A Al-Obeidan 1
1Glaucoma Research Chair Department of Ophthalmology College of Medicine King Saud University Riyadh Saudi Arabia2King Khaled University Hospital King Saud University Riyadh Saudi Arabia
Correspondence should be addressed to Altaf A Kondkar akondkargmailcom
Received 29 August 2018 Revised 2 October 2018 Accepted 22 October 2018 Published 1 November 2018
Academic Editor Francine Durocher
Copyright copy 2018 AltafAKondkar et alThis is an open access article distributedunder theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
A case-control genetic association study was performed to investigate whether variant rs7916697 in atonal bHLH transcriptionfactor 7 (ATOH7) which has been previously reported to be associated with optic disc parameters and primary open angleglaucoma (POAG) in different ethnic groups is a risk factor for POAG or any of its clinical phenotypes in a Saudi cohortGenotyping of rs7916697 (GgtA) variant was performed in 186 unrelated POAG cases and 171 unrelated nonglaucomatous controlsof Saudi origin using real-time Taq-Man assay Genotypic and allelic association with POAG and its related clinical indices wereevaluated Demographic and systemic disease status did not differ significantly between POAG cases and controls Associationanalysis between POAG cases and controls showed no significant genotype effect under additive (p=0707) dominant (p=0458)and recessive (p=0554) models Besides the minor lsquoArsquo allele frequency was 039 in POAG cases and 036 in controls with nosignificant distribution (p=0406) In addition there was no significant difference between genotypes and clinical phenotypes suchas intraocular pressure and cupdisc ratio within the POAG group or any age and sex adjusted genotype effect on the diseaseoutcome in regression analysis Variant rs7916697 in ATOH7 is not associated with POAG or its clinical indices such as IOP andcupdisc ratio in a Saudi cohort
1 Introduction
With an estimated heritability of 081 [1] primary open angleglaucoma (POAG) follows a complex multi-factorial inher-itance pattern involving both genetics and environmentalfactors [2] Atonal bHLH transcription factor 7 (ATOH7)located on cytogenic band 10q213-221 is a single exon genethat encodes transcription factor known to play a centralrole in differentiation of retinal ganglion cells (RGCs) andoptic nerve formation [3] Studies in animal model of glau-coma have demonstrated that abnormal ATOH7 expressionresults in an increased number of differentiated RGCs [45] Mice retinal progenitor cells have been demonstrated toexpress Atoh7 [6] and its regulation by Pax6 in embryonicretina [7] is clinically relevant because PAX6 gene mutationshave been associated with various optic nerve abnormalitiesin humans [8] Genome-wide association studies (GWAS)
have previously reported strong association between opticdisk parameters and variant rs7916697 near ATOH7 in anAustralian twin cohort [6] the Rotterdam study [9] andSingapore Asians [10] and very recently in Latino popu-lation [11] A recent meta-analysis of GWAS within theInternational Glaucoma Genetics Consortium revealed thatrs7916697 among others significantly affected at least oneof the optic disc parameters [12] In addition a suggestiveprotective association was also noted for rs7916697 in Afro-Caribbean Barbados population in POAG [13] indicatingATOH7 as an important susceptibility gene associated withglaucoma and optic disc parameters Despite strong evi-dence for involvement of ATOH7 in pathogenesis of POAGthe exact molecular mechanism(s) leading to glaucoma-tous damage of the optic nerve and the possibility of anyinteraction of ATOH7 with other risk factors is still largelyunknown
HindawiGenetics Research InternationalVolume 2018 Article ID 2148056 6 pageshttpsdoiorg10115520182148056
2 Genetics Research International
Our previous study has shown that another polymor-phism rs1900004 in ATOH7 also reported to be stronglyassociated with optic disk parameters and POAG [6 9]was not associated with POAG in a small number of Saudipatients that we investigated [14] The present study wasperformed in a different and almost double the number ofsample cohorts of Saudi origin to investigate any associationbetween variant rs7916697 in ATOH7 and POAG or any of itsclinical indices
2 Material and Methods
21 Study Population A case-control study was performedaccording to the principles of the Declaration of Helsinki forhuman research and all of the study participants provided awritten informed consent The study received ethical clear-ance from College of Medicine institutional review boardcommittee at King Saud University (approval number 08-657) Saudi patients with clinically confirmed diagnosisof POAG (n=186) and nonglaucomatous healthy controls(n=171) of the same ethnicity were recruited into the studyat King Abdulaziz University Hospital King Saud Uni-versity Riyadh Saudi Arabia The criteria for selection ofpatients and controls have been detailed elsewhere [15 16]A standardized ophthalmic examination was performed inall the participating patients that included measurementof intraocular pressure (IOP) by Goldmann applanationtonometry mounted at the slit lamp examination of anteriorchamber angles by gonioscopy dilated pupil examination ofthe lens and fundus and visual field testing by Humphreyautomated field analyzer POAG patients were diagnosedby glaucoma specialist ophthalmologist and satisfied thefollowing diagnostic criteria (1) presence of progressiveglaucomatous damage at the optic disk or retinal nerve fiberlayer changes such as narrowing of the neuroretinal rimdiffuse thinning of retinal nerve fibre layer or localizeddefects (2) presence of visual field defects which are typicalof glaucoma such as nasal step defect arcuate or paracentralscotomata or generalized tunnel vision (3) bilaterally openanterior chamber angles as examined by gonioscopy and (4)adult onset Any secondary form of glaucoma cases such aspigmentary glaucoma uveitic pseudoexfoliation and historyof steroid use or ocular trauma was excluded Ethicallymatched control subjects were selected from ophthalmologyscreening clinics and subjects with normal IOP (withoutanti-glaucoma medicine) open anterior chamber angles andhealthy optic disk on examination with no previous historyof ocular disease(s) or ophthalmic surgeries Patient orcontrol subjects refusing to participate were excluded Otherdetails on history of systemic diseases health awareness andsmoking habitswere procured throughmedical records of thepatients or personal interviews for controls
22 Genotyping of rs7916697 in ATOH7 Gene DNA sampleswere obtained from peripheral EDTA blood using the illustrablood genomicPrep Mini Spin kit (GE Healthcare Bucking-hamshire UK) and genotyped for rs7916697 GgtA polymor-phism near ATOH7 gene (NG 0319341) using the TaqMan
SNP Genotyping Assay (assay ID C 27850155 10 AppliedBiosystems Inc Foster City CA USA) on ABI 7500 Real-Time PCR System (Applied Biosystems) using recommendedcycling conditions as described previously [17] Fluorescencewas measured at annealing step and genotype calling wasperformed using the automated 2-color allele discriminationsoftware on ABI 7500
23 Statistical Analyses SPSS version 22 (IBM Inc ChicagoIllinois USA) was used to perform the analyses Chi-square analysis was used to test Hardy-Weinberg Equilibrium(HWE) and allelicgenotype association Independent sam-ples t-test one-way ANOVA and Kruskal-Wallis tests wereused to detect the mean difference across genotypesgroupsRegression analysis was done to determine the effect of agesex and genotype on the disease (POAG) outcome Oddsratios (OR) were calculated confidence interval (CI) levelwas set to 95 and a p value less than 005 was consideredstatistically significant
3 Results
31 Demographic and Clinical Characteristics of the Partici-pants A total of 357 participants consisting of 186 POAGcases and 171 nonglaucomatous controls were included in thisstudy Except for family history of glaucoma (p=0039) themean age gender distribution smoking habits and status ofsystemic diseases such as diabetes hypertension coronaryartery disease and hypercholesterolemia showed no signif-icant difference between the two study groups (Table 1)
32 Genotype and Allelic Association with POAG The geno-types did not deviate significantly from the HWE (pgt005)Both the genotype and allele frequency of rs7916697 inATOH7 did not differ significantly between POAG casesand controls (Table 2) The minor lsquoArsquo allele frequency was039 in POAG cases and 036 in controls with no significantdistribution between cases and controls (p=0406)There wasno significant genotype association with POAG as assessedby additive (p=0707) dominant (p=0458) and recessivemodels (p=0554) In addition there was no significantdifference between genotypes and clinical phenotypes suchas intraocular pressure and cupdisc ratio within the POAGgroup (Table 3) or any age and sex adjusted genotype effecton the disease outcome in regression analysis (Table 4)
4 Discussion
Variant rs7916697 is located in the 51015840 untranslated region ofATOH7 an important candidate for human optic nerve apla-sia and related clinical syndromes [3] A genetic associationbetween rs7916697 and risk of POAG in a Saudi cohort wasinvestigated
The minor allele frequency (MAF) of rs7916697rsquoArsquo inATOH7 varies across different population ranging from 025to 082 with an overall MAF of 044 (1000 Genomes ProjectEnsembl database) The MAF in our Saudi cohort was 036in controls and 039 in POAG cases The observed MAF was
Genetics Research International 3
Table 1 Demographic and clinical characteristics of POAG cases and controls genotyped for SNP rs7916697
VariablesControls(n = 171)No ()
Cases(n = 186)No ()
p valuea
Demographic CharacteristicsAge in years mean (plusmnSD) 609 (106) 589 (115) 0096lowast
Dominant - - 085 055 ndash 130 0458Recessive - - 083 045 ndash 152 0554aPearson Chi2 test lowastRisk variant HWE P Hardy-Weinberg equilibrium p value sectFisher exact test
higher than 027 reported in US Caucasians [18] similar to038 reported in Latino study [11] and much lower than 076observed in Afro-Caribbeans where lsquoGrsquo was reported to bethe minor allele [13] highlighting an ethnic specific complexgenetic etiology in POAG
Genetic studies have reported both positive and negativeassociation of variants in ATOH7 and risk of POAG indifferent ethnic groups Polymorphism rs7916697 in ATOH7was reported to be an important genetic determinant ofoptic disc size in a meta-analysis consisting of UK andAustralian cohort (p=13x10minus10) that explained 17 variationin the optic disc area in the UK cohort [6] implicatingits pathophysiological role in POAG Studies in Rotterdamand Singapore had identified significant association betweenrs7916697 and optic disc area (p=2x10minus15) in Asians [10]
The Blue Mountains Eye Study and the Twins study alsoreplicated a strong association at rs7916697 for optic disc sizethat was dependent on vertical cupdisc ratio (VCDR) [19]Similarly consistent with other findings rs7916697 showed aborderline genome-wide significance (p=544x10minus8) and wasassociated with decrease in VCDR in the Latino population[11] In contrast we did not find any significant associationbetween rs7916697 and POAG or any of its clinical indicessuch as IOP cupdisc ratio and number of anti-glaucomamedications that serve as clinical markers of disease sever-ityprogression Consistent with our findings rs7916697 wasalso not found to be significantly associated with POAGand other clinical indices in US Caucasians where anothervariant rs1900004 in ATOH7 was reported to influence opticdisc area [18] Similarly a modest protective effect was
4 Genetics Research International
Table 3 Analysis of genotype effect on demographic and clinical characteristics within PAOG group
Characteristics
Genotypes
p valueaGG(n= 69)No ()
GA(n= 89)No ()
AA(n= 28)No ()
DEMOGRAPHICAge in years mean (SD) 622 (97) 600 (110) 604 (114) 0413lowast
Cupdisc ratio 084 (06) 075 (02) 082 (01) 0259lowastlowast
Number of anti-glaucomamedications 17 (11) 20 (10) 19 (11) 0325lowastlowastaPearson Chi2 test lowastOne-way ANOVA lowastlowastKruskal-Wallis test
Table 4 Binary logistic regression analysis to assess the effect of age sex and genotype on disease outcome
Variables Odds ratio 95 confidence interval p valueAge 101 099 ndash 103 0096Sexa 091 059 ndash 139 0676Genotypeb - - 0627GA 117 074 ndash 183 0499AA 134 070 ndash 259 0371aFemale as reference bGG as reference
reported for rs7916697 (allelic p=00096 genotypic p=001)for the lsquoGrsquo allele (OR=067 95 CI=050minus091) in Afro-Caribbean subjects Though this finding failed to withstandcorrection for multiple testing there was significant evidencefor an interactive effect with rs1063192 (near CDKN2BAS1 onchromosome 9) [13]
The molecular mechanisms leading to the developmentand progression of optic nerve defect in POAG could be IOP-andor non-IOP-dependent (RGCoptic nerve vulnerability-related) Genes affecting optic nerve quantitative traits suchas optic disc area and VCDR may have a plausible role inpathogenesis of POAG by affecting developmental-relatedpathways [20] ATOH7 gene encodes Math5 a protein thatplays a central role in RGC differentiation [4] and thus maynot be a major risk modulator or causal factor in IOP-relatedpathogenesis of late-onset POAG A complex disease such asglaucoma can result from interactions between several genesAn additive effect of genetic variants associated with IOPVCDR and high or normal tension glaucoma [13 21] cannotbe ruled out in this study In addition we have previouslyshown that polymorphism rs1900004 in ATOH7 was also not
associated with POAG [14] indicating that specific geneticvariants may bemore enriched in one ethnic population thananother highlighting racial differences and that ATOH7maynot have a major role in POAG developmentprogressionin this population Nonetheless the findings of our studyrequire cautious interpretation Considering the global MAF(from 1000 Genomes database) or the MAF observed in ourSaudi cohort the current sample size of the study has anestimated power of gt80 (with alpha risk of 5) to detect anodds ratio of 20 However it will certainly require a muchlarger sample size to detect a 15-fold relative risk As withother complex diseases large sample sizes are required toensure sufficient power to fully define the underlying geneticcausal effect which may be a major limitation in this studyBesides the role of other variants and gene-gene interactionalso cannot be ruled out
5 Conclusion
In an attempt to link this polymorphism with POAG amongthe Saudi cohort our study shows that variant rs7916697 in
Genetics Research International 5
the ATOH7 gene lacks significant association with POAGor related phenotypes such as IOP and cupdisc ratioThis observation needs further validation in a much largersample population of clinically well-defined POAG patientspotentially with age gender and ethnicity matched controlsto assess the risk it may contribute to the development orprogression of the disease in this population
Data Availability
The data supporting the conclusions of this article are allpresented within the article
Ethical Approval
The study adhered to the tenets of the Declaration of Helsinkiand had received approval from the Institutional ReviewBoard and Research Ethics Committee (approval 08-657)
Consent
Written informed consent was obtained from all participantsprior to their inclusion in this study
Disclosure
The funder had no role in the design of the study andcollection analysis and interpretation of data and in writingthe manuscript
Conflicts of Interest
The authors report no conflicts of interest in this work
Acknowledgments
The authors would like to thank the Deanship of ScientificResearch and Glaucoma Research Chair of Department ofOphthalmology College of Medicine King Saud Universityfor their support and use of laboratory facilities
References
[1] J Charlesworth P L Kramer T Dyer et al ldquoThe path toopen-angle glaucoma gene discovery Endophenotypic statusof intraocular pressure cup-to-disc ratio and central cornealthicknessrdquo Investigative Ophthalmology ampVisual Science vol 51no 7 pp 3509ndash3514 2010
[2] K Abu-Amero A A Kondkar and K V Chalam ldquoAn updatedreview on the genetics of primary open angle glaucomardquoInternational Journal of Molecular Sciences vol 16 no 12 pp28886ndash28911 2015
[3] N L Brown S L Dagenais C-M Chen and T GlaserldquoMolecular characterization and mapping of ATOH7 a humanatonal homolog with a predicted role in retinal ganglion celldevelopmentrdquo Mammalian Genome vol 13 no 2 pp 95ndash1012002
[4] Z Yang K Ding L Pan M Deng and L Gan ldquoMath5 deter-mines the competence state of retinal ganglion cell progenitorsrdquoDevelopmental Biology vol 264 no 1 pp 240ndash254 2003
[5] W-T Song X-Y Zhang and X-B Xia ldquoAtoh7 promotes thedifferentiation of Muller cells-derived retinal stem cells intoretinal ganglion cells in a rat model of glaucomardquo ExperimentalBiology and Medicine vol 240 no 5 pp 682ndash690 2015
[6] S Macgregor A W Hewitt P G Hysi et al ldquoGenome-wideassociation identifies ATOH7 as a major gene determininghuman optic disc sizerdquo Human Molecular Genetics vol 19 no13 pp 2716ndash2724 2010
[7] A N Riesenberg T T LeM IWillardsen D C BlackburnML Vetter and N L Brown ldquoPax6 regulation of Math5 duringmouse retinal neurogenesisrdquo Genesis vol 47 no 3 pp 175ndash1872009
[8] N Azuma Y Yamaguchi H Handa et al ldquoMutations of thePAX6 gene detected in patients with a variety of optic-nervemalformationsrdquo American Journal of Human Genetics vol 72no 6 pp 1565ndash1570 2003
[9] W D Ramdas L M van Koolwijk M K Ikram et al ldquoAgenome-wide association study of optic disc parametersrdquo PLoSGenetics vol 6 no 6 2010
[10] A BroeksM K Schmidt andM E Sherman ldquoLow penetrancebreast cancer susceptibility loci are associated with specificbreast tumor subtypes findings from the Breast Cancer Associ-ation Consortiumrdquo Human Molecular Genetics vol 20 no 16pp 3289ndash3303 2011
[11] D R Nannini M Torres Y I Chen et al ldquoA genome-wide association study of vertical cup-disc ratio in a latinopopulationrdquo Investigative Opthalmology amp Visual Science vol58 no 1 pp 87ndash95 2017
[12] H Springelkamp A I Iglesias and A Mishra ldquoNew insightsinto the genetics of primary open-angle glaucoma based onmeta-analyses of intraocular pressure and optic disc character-isticsrdquo Human Molecular Genetics vol 26 no 2 pp 438ndash4532017
[13] D Cao X Jiao X Liu et al ldquoCDKN2B polymorphism isassociated with primary open-angle glaucoma (POAG) in theAfro-Caribbean population of Barbados West Indiesrdquo PLoSONE vol 7 no 6 2012
[14] A A Kondkar A Mousa T A Azad et al ldquoAnalysis ofPolymorphism rs1900004 inAtonal bHLHTranscription Factor7 in Saudi Patients with Primary Open Angle GlaucomardquoGenetic Testing and Molecular Biomarkers vol 20 no 11 pp715ndash718 2016
[15] K Abu-Amero T Sultan S Al-Obeidan and A KondkarldquoAnalysis of CYP1B1 sequence alterations in patients withprimary open-angle glaucoma of Saudi originrdquo Clinical Oph-thalmology vol 12 pp 1413ndash1416 2018
[16] A A Kondkar N B Edward H Kalantan et al ldquoLack ofassociation between polymorphism rs540782 andprimary openangle glaucoma in Saudi patientsrdquo Journal of Negative Results inBioMedicine vol 16 no 1 2017
[17] A A Kondkar T A Azad F A Almobarak et al ldquoPolymor-phism rs10483727 in the SIX1SIX6 gene locus is a risk factor forprimary open angle glaucoma in a saudi cohortrdquoGenetic Testingand Molecular Biomarkers vol 22 no 1 pp 74ndash78 2018
[18] B J FanD YWang L R Pasquale J L Haines and J LWiggsldquoGenetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucomain a US Caucasian populationrdquo Investigative Ophthalmology ampVisual Science vol 52 no 3 pp 1788ndash1792 2011
6 Genetics Research International
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
Our previous study has shown that another polymor-phism rs1900004 in ATOH7 also reported to be stronglyassociated with optic disk parameters and POAG [6 9]was not associated with POAG in a small number of Saudipatients that we investigated [14] The present study wasperformed in a different and almost double the number ofsample cohorts of Saudi origin to investigate any associationbetween variant rs7916697 in ATOH7 and POAG or any of itsclinical indices
2 Material and Methods
21 Study Population A case-control study was performedaccording to the principles of the Declaration of Helsinki forhuman research and all of the study participants provided awritten informed consent The study received ethical clear-ance from College of Medicine institutional review boardcommittee at King Saud University (approval number 08-657) Saudi patients with clinically confirmed diagnosisof POAG (n=186) and nonglaucomatous healthy controls(n=171) of the same ethnicity were recruited into the studyat King Abdulaziz University Hospital King Saud Uni-versity Riyadh Saudi Arabia The criteria for selection ofpatients and controls have been detailed elsewhere [15 16]A standardized ophthalmic examination was performed inall the participating patients that included measurementof intraocular pressure (IOP) by Goldmann applanationtonometry mounted at the slit lamp examination of anteriorchamber angles by gonioscopy dilated pupil examination ofthe lens and fundus and visual field testing by Humphreyautomated field analyzer POAG patients were diagnosedby glaucoma specialist ophthalmologist and satisfied thefollowing diagnostic criteria (1) presence of progressiveglaucomatous damage at the optic disk or retinal nerve fiberlayer changes such as narrowing of the neuroretinal rimdiffuse thinning of retinal nerve fibre layer or localizeddefects (2) presence of visual field defects which are typicalof glaucoma such as nasal step defect arcuate or paracentralscotomata or generalized tunnel vision (3) bilaterally openanterior chamber angles as examined by gonioscopy and (4)adult onset Any secondary form of glaucoma cases such aspigmentary glaucoma uveitic pseudoexfoliation and historyof steroid use or ocular trauma was excluded Ethicallymatched control subjects were selected from ophthalmologyscreening clinics and subjects with normal IOP (withoutanti-glaucoma medicine) open anterior chamber angles andhealthy optic disk on examination with no previous historyof ocular disease(s) or ophthalmic surgeries Patient orcontrol subjects refusing to participate were excluded Otherdetails on history of systemic diseases health awareness andsmoking habitswere procured throughmedical records of thepatients or personal interviews for controls
22 Genotyping of rs7916697 in ATOH7 Gene DNA sampleswere obtained from peripheral EDTA blood using the illustrablood genomicPrep Mini Spin kit (GE Healthcare Bucking-hamshire UK) and genotyped for rs7916697 GgtA polymor-phism near ATOH7 gene (NG 0319341) using the TaqMan
SNP Genotyping Assay (assay ID C 27850155 10 AppliedBiosystems Inc Foster City CA USA) on ABI 7500 Real-Time PCR System (Applied Biosystems) using recommendedcycling conditions as described previously [17] Fluorescencewas measured at annealing step and genotype calling wasperformed using the automated 2-color allele discriminationsoftware on ABI 7500
23 Statistical Analyses SPSS version 22 (IBM Inc ChicagoIllinois USA) was used to perform the analyses Chi-square analysis was used to test Hardy-Weinberg Equilibrium(HWE) and allelicgenotype association Independent sam-ples t-test one-way ANOVA and Kruskal-Wallis tests wereused to detect the mean difference across genotypesgroupsRegression analysis was done to determine the effect of agesex and genotype on the disease (POAG) outcome Oddsratios (OR) were calculated confidence interval (CI) levelwas set to 95 and a p value less than 005 was consideredstatistically significant
3 Results
31 Demographic and Clinical Characteristics of the Partici-pants A total of 357 participants consisting of 186 POAGcases and 171 nonglaucomatous controls were included in thisstudy Except for family history of glaucoma (p=0039) themean age gender distribution smoking habits and status ofsystemic diseases such as diabetes hypertension coronaryartery disease and hypercholesterolemia showed no signif-icant difference between the two study groups (Table 1)
32 Genotype and Allelic Association with POAG The geno-types did not deviate significantly from the HWE (pgt005)Both the genotype and allele frequency of rs7916697 inATOH7 did not differ significantly between POAG casesand controls (Table 2) The minor lsquoArsquo allele frequency was039 in POAG cases and 036 in controls with no significantdistribution between cases and controls (p=0406)There wasno significant genotype association with POAG as assessedby additive (p=0707) dominant (p=0458) and recessivemodels (p=0554) In addition there was no significantdifference between genotypes and clinical phenotypes suchas intraocular pressure and cupdisc ratio within the POAGgroup (Table 3) or any age and sex adjusted genotype effecton the disease outcome in regression analysis (Table 4)
4 Discussion
Variant rs7916697 is located in the 51015840 untranslated region ofATOH7 an important candidate for human optic nerve apla-sia and related clinical syndromes [3] A genetic associationbetween rs7916697 and risk of POAG in a Saudi cohort wasinvestigated
The minor allele frequency (MAF) of rs7916697rsquoArsquo inATOH7 varies across different population ranging from 025to 082 with an overall MAF of 044 (1000 Genomes ProjectEnsembl database) The MAF in our Saudi cohort was 036in controls and 039 in POAG cases The observed MAF was
Genetics Research International 3
Table 1 Demographic and clinical characteristics of POAG cases and controls genotyped for SNP rs7916697
VariablesControls(n = 171)No ()
Cases(n = 186)No ()
p valuea
Demographic CharacteristicsAge in years mean (plusmnSD) 609 (106) 589 (115) 0096lowast
Dominant - - 085 055 ndash 130 0458Recessive - - 083 045 ndash 152 0554aPearson Chi2 test lowastRisk variant HWE P Hardy-Weinberg equilibrium p value sectFisher exact test
higher than 027 reported in US Caucasians [18] similar to038 reported in Latino study [11] and much lower than 076observed in Afro-Caribbeans where lsquoGrsquo was reported to bethe minor allele [13] highlighting an ethnic specific complexgenetic etiology in POAG
Genetic studies have reported both positive and negativeassociation of variants in ATOH7 and risk of POAG indifferent ethnic groups Polymorphism rs7916697 in ATOH7was reported to be an important genetic determinant ofoptic disc size in a meta-analysis consisting of UK andAustralian cohort (p=13x10minus10) that explained 17 variationin the optic disc area in the UK cohort [6] implicatingits pathophysiological role in POAG Studies in Rotterdamand Singapore had identified significant association betweenrs7916697 and optic disc area (p=2x10minus15) in Asians [10]
The Blue Mountains Eye Study and the Twins study alsoreplicated a strong association at rs7916697 for optic disc sizethat was dependent on vertical cupdisc ratio (VCDR) [19]Similarly consistent with other findings rs7916697 showed aborderline genome-wide significance (p=544x10minus8) and wasassociated with decrease in VCDR in the Latino population[11] In contrast we did not find any significant associationbetween rs7916697 and POAG or any of its clinical indicessuch as IOP cupdisc ratio and number of anti-glaucomamedications that serve as clinical markers of disease sever-ityprogression Consistent with our findings rs7916697 wasalso not found to be significantly associated with POAGand other clinical indices in US Caucasians where anothervariant rs1900004 in ATOH7 was reported to influence opticdisc area [18] Similarly a modest protective effect was
4 Genetics Research International
Table 3 Analysis of genotype effect on demographic and clinical characteristics within PAOG group
Characteristics
Genotypes
p valueaGG(n= 69)No ()
GA(n= 89)No ()
AA(n= 28)No ()
DEMOGRAPHICAge in years mean (SD) 622 (97) 600 (110) 604 (114) 0413lowast
Cupdisc ratio 084 (06) 075 (02) 082 (01) 0259lowastlowast
Number of anti-glaucomamedications 17 (11) 20 (10) 19 (11) 0325lowastlowastaPearson Chi2 test lowastOne-way ANOVA lowastlowastKruskal-Wallis test
Table 4 Binary logistic regression analysis to assess the effect of age sex and genotype on disease outcome
Variables Odds ratio 95 confidence interval p valueAge 101 099 ndash 103 0096Sexa 091 059 ndash 139 0676Genotypeb - - 0627GA 117 074 ndash 183 0499AA 134 070 ndash 259 0371aFemale as reference bGG as reference
reported for rs7916697 (allelic p=00096 genotypic p=001)for the lsquoGrsquo allele (OR=067 95 CI=050minus091) in Afro-Caribbean subjects Though this finding failed to withstandcorrection for multiple testing there was significant evidencefor an interactive effect with rs1063192 (near CDKN2BAS1 onchromosome 9) [13]
The molecular mechanisms leading to the developmentand progression of optic nerve defect in POAG could be IOP-andor non-IOP-dependent (RGCoptic nerve vulnerability-related) Genes affecting optic nerve quantitative traits suchas optic disc area and VCDR may have a plausible role inpathogenesis of POAG by affecting developmental-relatedpathways [20] ATOH7 gene encodes Math5 a protein thatplays a central role in RGC differentiation [4] and thus maynot be a major risk modulator or causal factor in IOP-relatedpathogenesis of late-onset POAG A complex disease such asglaucoma can result from interactions between several genesAn additive effect of genetic variants associated with IOPVCDR and high or normal tension glaucoma [13 21] cannotbe ruled out in this study In addition we have previouslyshown that polymorphism rs1900004 in ATOH7 was also not
associated with POAG [14] indicating that specific geneticvariants may bemore enriched in one ethnic population thananother highlighting racial differences and that ATOH7maynot have a major role in POAG developmentprogressionin this population Nonetheless the findings of our studyrequire cautious interpretation Considering the global MAF(from 1000 Genomes database) or the MAF observed in ourSaudi cohort the current sample size of the study has anestimated power of gt80 (with alpha risk of 5) to detect anodds ratio of 20 However it will certainly require a muchlarger sample size to detect a 15-fold relative risk As withother complex diseases large sample sizes are required toensure sufficient power to fully define the underlying geneticcausal effect which may be a major limitation in this studyBesides the role of other variants and gene-gene interactionalso cannot be ruled out
5 Conclusion
In an attempt to link this polymorphism with POAG amongthe Saudi cohort our study shows that variant rs7916697 in
Genetics Research International 5
the ATOH7 gene lacks significant association with POAGor related phenotypes such as IOP and cupdisc ratioThis observation needs further validation in a much largersample population of clinically well-defined POAG patientspotentially with age gender and ethnicity matched controlsto assess the risk it may contribute to the development orprogression of the disease in this population
Data Availability
The data supporting the conclusions of this article are allpresented within the article
Ethical Approval
The study adhered to the tenets of the Declaration of Helsinkiand had received approval from the Institutional ReviewBoard and Research Ethics Committee (approval 08-657)
Consent
Written informed consent was obtained from all participantsprior to their inclusion in this study
Disclosure
The funder had no role in the design of the study andcollection analysis and interpretation of data and in writingthe manuscript
Conflicts of Interest
The authors report no conflicts of interest in this work
Acknowledgments
The authors would like to thank the Deanship of ScientificResearch and Glaucoma Research Chair of Department ofOphthalmology College of Medicine King Saud Universityfor their support and use of laboratory facilities
References
[1] J Charlesworth P L Kramer T Dyer et al ldquoThe path toopen-angle glaucoma gene discovery Endophenotypic statusof intraocular pressure cup-to-disc ratio and central cornealthicknessrdquo Investigative Ophthalmology ampVisual Science vol 51no 7 pp 3509ndash3514 2010
[2] K Abu-Amero A A Kondkar and K V Chalam ldquoAn updatedreview on the genetics of primary open angle glaucomardquoInternational Journal of Molecular Sciences vol 16 no 12 pp28886ndash28911 2015
[3] N L Brown S L Dagenais C-M Chen and T GlaserldquoMolecular characterization and mapping of ATOH7 a humanatonal homolog with a predicted role in retinal ganglion celldevelopmentrdquo Mammalian Genome vol 13 no 2 pp 95ndash1012002
[4] Z Yang K Ding L Pan M Deng and L Gan ldquoMath5 deter-mines the competence state of retinal ganglion cell progenitorsrdquoDevelopmental Biology vol 264 no 1 pp 240ndash254 2003
[5] W-T Song X-Y Zhang and X-B Xia ldquoAtoh7 promotes thedifferentiation of Muller cells-derived retinal stem cells intoretinal ganglion cells in a rat model of glaucomardquo ExperimentalBiology and Medicine vol 240 no 5 pp 682ndash690 2015
[6] S Macgregor A W Hewitt P G Hysi et al ldquoGenome-wideassociation identifies ATOH7 as a major gene determininghuman optic disc sizerdquo Human Molecular Genetics vol 19 no13 pp 2716ndash2724 2010
[7] A N Riesenberg T T LeM IWillardsen D C BlackburnML Vetter and N L Brown ldquoPax6 regulation of Math5 duringmouse retinal neurogenesisrdquo Genesis vol 47 no 3 pp 175ndash1872009
[8] N Azuma Y Yamaguchi H Handa et al ldquoMutations of thePAX6 gene detected in patients with a variety of optic-nervemalformationsrdquo American Journal of Human Genetics vol 72no 6 pp 1565ndash1570 2003
[9] W D Ramdas L M van Koolwijk M K Ikram et al ldquoAgenome-wide association study of optic disc parametersrdquo PLoSGenetics vol 6 no 6 2010
[10] A BroeksM K Schmidt andM E Sherman ldquoLow penetrancebreast cancer susceptibility loci are associated with specificbreast tumor subtypes findings from the Breast Cancer Associ-ation Consortiumrdquo Human Molecular Genetics vol 20 no 16pp 3289ndash3303 2011
[11] D R Nannini M Torres Y I Chen et al ldquoA genome-wide association study of vertical cup-disc ratio in a latinopopulationrdquo Investigative Opthalmology amp Visual Science vol58 no 1 pp 87ndash95 2017
[12] H Springelkamp A I Iglesias and A Mishra ldquoNew insightsinto the genetics of primary open-angle glaucoma based onmeta-analyses of intraocular pressure and optic disc character-isticsrdquo Human Molecular Genetics vol 26 no 2 pp 438ndash4532017
[13] D Cao X Jiao X Liu et al ldquoCDKN2B polymorphism isassociated with primary open-angle glaucoma (POAG) in theAfro-Caribbean population of Barbados West Indiesrdquo PLoSONE vol 7 no 6 2012
[14] A A Kondkar A Mousa T A Azad et al ldquoAnalysis ofPolymorphism rs1900004 inAtonal bHLHTranscription Factor7 in Saudi Patients with Primary Open Angle GlaucomardquoGenetic Testing and Molecular Biomarkers vol 20 no 11 pp715ndash718 2016
[15] K Abu-Amero T Sultan S Al-Obeidan and A KondkarldquoAnalysis of CYP1B1 sequence alterations in patients withprimary open-angle glaucoma of Saudi originrdquo Clinical Oph-thalmology vol 12 pp 1413ndash1416 2018
[16] A A Kondkar N B Edward H Kalantan et al ldquoLack ofassociation between polymorphism rs540782 andprimary openangle glaucoma in Saudi patientsrdquo Journal of Negative Results inBioMedicine vol 16 no 1 2017
[17] A A Kondkar T A Azad F A Almobarak et al ldquoPolymor-phism rs10483727 in the SIX1SIX6 gene locus is a risk factor forprimary open angle glaucoma in a saudi cohortrdquoGenetic Testingand Molecular Biomarkers vol 22 no 1 pp 74ndash78 2018
[18] B J FanD YWang L R Pasquale J L Haines and J LWiggsldquoGenetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucomain a US Caucasian populationrdquo Investigative Ophthalmology ampVisual Science vol 52 no 3 pp 1788ndash1792 2011
6 Genetics Research International
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
Dominant - - 085 055 ndash 130 0458Recessive - - 083 045 ndash 152 0554aPearson Chi2 test lowastRisk variant HWE P Hardy-Weinberg equilibrium p value sectFisher exact test
higher than 027 reported in US Caucasians [18] similar to038 reported in Latino study [11] and much lower than 076observed in Afro-Caribbeans where lsquoGrsquo was reported to bethe minor allele [13] highlighting an ethnic specific complexgenetic etiology in POAG
Genetic studies have reported both positive and negativeassociation of variants in ATOH7 and risk of POAG indifferent ethnic groups Polymorphism rs7916697 in ATOH7was reported to be an important genetic determinant ofoptic disc size in a meta-analysis consisting of UK andAustralian cohort (p=13x10minus10) that explained 17 variationin the optic disc area in the UK cohort [6] implicatingits pathophysiological role in POAG Studies in Rotterdamand Singapore had identified significant association betweenrs7916697 and optic disc area (p=2x10minus15) in Asians [10]
The Blue Mountains Eye Study and the Twins study alsoreplicated a strong association at rs7916697 for optic disc sizethat was dependent on vertical cupdisc ratio (VCDR) [19]Similarly consistent with other findings rs7916697 showed aborderline genome-wide significance (p=544x10minus8) and wasassociated with decrease in VCDR in the Latino population[11] In contrast we did not find any significant associationbetween rs7916697 and POAG or any of its clinical indicessuch as IOP cupdisc ratio and number of anti-glaucomamedications that serve as clinical markers of disease sever-ityprogression Consistent with our findings rs7916697 wasalso not found to be significantly associated with POAGand other clinical indices in US Caucasians where anothervariant rs1900004 in ATOH7 was reported to influence opticdisc area [18] Similarly a modest protective effect was
4 Genetics Research International
Table 3 Analysis of genotype effect on demographic and clinical characteristics within PAOG group
Characteristics
Genotypes
p valueaGG(n= 69)No ()
GA(n= 89)No ()
AA(n= 28)No ()
DEMOGRAPHICAge in years mean (SD) 622 (97) 600 (110) 604 (114) 0413lowast
Cupdisc ratio 084 (06) 075 (02) 082 (01) 0259lowastlowast
Number of anti-glaucomamedications 17 (11) 20 (10) 19 (11) 0325lowastlowastaPearson Chi2 test lowastOne-way ANOVA lowastlowastKruskal-Wallis test
Table 4 Binary logistic regression analysis to assess the effect of age sex and genotype on disease outcome
Variables Odds ratio 95 confidence interval p valueAge 101 099 ndash 103 0096Sexa 091 059 ndash 139 0676Genotypeb - - 0627GA 117 074 ndash 183 0499AA 134 070 ndash 259 0371aFemale as reference bGG as reference
reported for rs7916697 (allelic p=00096 genotypic p=001)for the lsquoGrsquo allele (OR=067 95 CI=050minus091) in Afro-Caribbean subjects Though this finding failed to withstandcorrection for multiple testing there was significant evidencefor an interactive effect with rs1063192 (near CDKN2BAS1 onchromosome 9) [13]
The molecular mechanisms leading to the developmentand progression of optic nerve defect in POAG could be IOP-andor non-IOP-dependent (RGCoptic nerve vulnerability-related) Genes affecting optic nerve quantitative traits suchas optic disc area and VCDR may have a plausible role inpathogenesis of POAG by affecting developmental-relatedpathways [20] ATOH7 gene encodes Math5 a protein thatplays a central role in RGC differentiation [4] and thus maynot be a major risk modulator or causal factor in IOP-relatedpathogenesis of late-onset POAG A complex disease such asglaucoma can result from interactions between several genesAn additive effect of genetic variants associated with IOPVCDR and high or normal tension glaucoma [13 21] cannotbe ruled out in this study In addition we have previouslyshown that polymorphism rs1900004 in ATOH7 was also not
associated with POAG [14] indicating that specific geneticvariants may bemore enriched in one ethnic population thananother highlighting racial differences and that ATOH7maynot have a major role in POAG developmentprogressionin this population Nonetheless the findings of our studyrequire cautious interpretation Considering the global MAF(from 1000 Genomes database) or the MAF observed in ourSaudi cohort the current sample size of the study has anestimated power of gt80 (with alpha risk of 5) to detect anodds ratio of 20 However it will certainly require a muchlarger sample size to detect a 15-fold relative risk As withother complex diseases large sample sizes are required toensure sufficient power to fully define the underlying geneticcausal effect which may be a major limitation in this studyBesides the role of other variants and gene-gene interactionalso cannot be ruled out
5 Conclusion
In an attempt to link this polymorphism with POAG amongthe Saudi cohort our study shows that variant rs7916697 in
Genetics Research International 5
the ATOH7 gene lacks significant association with POAGor related phenotypes such as IOP and cupdisc ratioThis observation needs further validation in a much largersample population of clinically well-defined POAG patientspotentially with age gender and ethnicity matched controlsto assess the risk it may contribute to the development orprogression of the disease in this population
Data Availability
The data supporting the conclusions of this article are allpresented within the article
Ethical Approval
The study adhered to the tenets of the Declaration of Helsinkiand had received approval from the Institutional ReviewBoard and Research Ethics Committee (approval 08-657)
Consent
Written informed consent was obtained from all participantsprior to their inclusion in this study
Disclosure
The funder had no role in the design of the study andcollection analysis and interpretation of data and in writingthe manuscript
Conflicts of Interest
The authors report no conflicts of interest in this work
Acknowledgments
The authors would like to thank the Deanship of ScientificResearch and Glaucoma Research Chair of Department ofOphthalmology College of Medicine King Saud Universityfor their support and use of laboratory facilities
References
[1] J Charlesworth P L Kramer T Dyer et al ldquoThe path toopen-angle glaucoma gene discovery Endophenotypic statusof intraocular pressure cup-to-disc ratio and central cornealthicknessrdquo Investigative Ophthalmology ampVisual Science vol 51no 7 pp 3509ndash3514 2010
[2] K Abu-Amero A A Kondkar and K V Chalam ldquoAn updatedreview on the genetics of primary open angle glaucomardquoInternational Journal of Molecular Sciences vol 16 no 12 pp28886ndash28911 2015
[3] N L Brown S L Dagenais C-M Chen and T GlaserldquoMolecular characterization and mapping of ATOH7 a humanatonal homolog with a predicted role in retinal ganglion celldevelopmentrdquo Mammalian Genome vol 13 no 2 pp 95ndash1012002
[4] Z Yang K Ding L Pan M Deng and L Gan ldquoMath5 deter-mines the competence state of retinal ganglion cell progenitorsrdquoDevelopmental Biology vol 264 no 1 pp 240ndash254 2003
[5] W-T Song X-Y Zhang and X-B Xia ldquoAtoh7 promotes thedifferentiation of Muller cells-derived retinal stem cells intoretinal ganglion cells in a rat model of glaucomardquo ExperimentalBiology and Medicine vol 240 no 5 pp 682ndash690 2015
[6] S Macgregor A W Hewitt P G Hysi et al ldquoGenome-wideassociation identifies ATOH7 as a major gene determininghuman optic disc sizerdquo Human Molecular Genetics vol 19 no13 pp 2716ndash2724 2010
[7] A N Riesenberg T T LeM IWillardsen D C BlackburnML Vetter and N L Brown ldquoPax6 regulation of Math5 duringmouse retinal neurogenesisrdquo Genesis vol 47 no 3 pp 175ndash1872009
[8] N Azuma Y Yamaguchi H Handa et al ldquoMutations of thePAX6 gene detected in patients with a variety of optic-nervemalformationsrdquo American Journal of Human Genetics vol 72no 6 pp 1565ndash1570 2003
[9] W D Ramdas L M van Koolwijk M K Ikram et al ldquoAgenome-wide association study of optic disc parametersrdquo PLoSGenetics vol 6 no 6 2010
[10] A BroeksM K Schmidt andM E Sherman ldquoLow penetrancebreast cancer susceptibility loci are associated with specificbreast tumor subtypes findings from the Breast Cancer Associ-ation Consortiumrdquo Human Molecular Genetics vol 20 no 16pp 3289ndash3303 2011
[11] D R Nannini M Torres Y I Chen et al ldquoA genome-wide association study of vertical cup-disc ratio in a latinopopulationrdquo Investigative Opthalmology amp Visual Science vol58 no 1 pp 87ndash95 2017
[12] H Springelkamp A I Iglesias and A Mishra ldquoNew insightsinto the genetics of primary open-angle glaucoma based onmeta-analyses of intraocular pressure and optic disc character-isticsrdquo Human Molecular Genetics vol 26 no 2 pp 438ndash4532017
[13] D Cao X Jiao X Liu et al ldquoCDKN2B polymorphism isassociated with primary open-angle glaucoma (POAG) in theAfro-Caribbean population of Barbados West Indiesrdquo PLoSONE vol 7 no 6 2012
[14] A A Kondkar A Mousa T A Azad et al ldquoAnalysis ofPolymorphism rs1900004 inAtonal bHLHTranscription Factor7 in Saudi Patients with Primary Open Angle GlaucomardquoGenetic Testing and Molecular Biomarkers vol 20 no 11 pp715ndash718 2016
[15] K Abu-Amero T Sultan S Al-Obeidan and A KondkarldquoAnalysis of CYP1B1 sequence alterations in patients withprimary open-angle glaucoma of Saudi originrdquo Clinical Oph-thalmology vol 12 pp 1413ndash1416 2018
[16] A A Kondkar N B Edward H Kalantan et al ldquoLack ofassociation between polymorphism rs540782 andprimary openangle glaucoma in Saudi patientsrdquo Journal of Negative Results inBioMedicine vol 16 no 1 2017
[17] A A Kondkar T A Azad F A Almobarak et al ldquoPolymor-phism rs10483727 in the SIX1SIX6 gene locus is a risk factor forprimary open angle glaucoma in a saudi cohortrdquoGenetic Testingand Molecular Biomarkers vol 22 no 1 pp 74ndash78 2018
[18] B J FanD YWang L R Pasquale J L Haines and J LWiggsldquoGenetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucomain a US Caucasian populationrdquo Investigative Ophthalmology ampVisual Science vol 52 no 3 pp 1788ndash1792 2011
6 Genetics Research International
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
Cupdisc ratio 084 (06) 075 (02) 082 (01) 0259lowastlowast
Number of anti-glaucomamedications 17 (11) 20 (10) 19 (11) 0325lowastlowastaPearson Chi2 test lowastOne-way ANOVA lowastlowastKruskal-Wallis test
Table 4 Binary logistic regression analysis to assess the effect of age sex and genotype on disease outcome
Variables Odds ratio 95 confidence interval p valueAge 101 099 ndash 103 0096Sexa 091 059 ndash 139 0676Genotypeb - - 0627GA 117 074 ndash 183 0499AA 134 070 ndash 259 0371aFemale as reference bGG as reference
reported for rs7916697 (allelic p=00096 genotypic p=001)for the lsquoGrsquo allele (OR=067 95 CI=050minus091) in Afro-Caribbean subjects Though this finding failed to withstandcorrection for multiple testing there was significant evidencefor an interactive effect with rs1063192 (near CDKN2BAS1 onchromosome 9) [13]
The molecular mechanisms leading to the developmentand progression of optic nerve defect in POAG could be IOP-andor non-IOP-dependent (RGCoptic nerve vulnerability-related) Genes affecting optic nerve quantitative traits suchas optic disc area and VCDR may have a plausible role inpathogenesis of POAG by affecting developmental-relatedpathways [20] ATOH7 gene encodes Math5 a protein thatplays a central role in RGC differentiation [4] and thus maynot be a major risk modulator or causal factor in IOP-relatedpathogenesis of late-onset POAG A complex disease such asglaucoma can result from interactions between several genesAn additive effect of genetic variants associated with IOPVCDR and high or normal tension glaucoma [13 21] cannotbe ruled out in this study In addition we have previouslyshown that polymorphism rs1900004 in ATOH7 was also not
associated with POAG [14] indicating that specific geneticvariants may bemore enriched in one ethnic population thananother highlighting racial differences and that ATOH7maynot have a major role in POAG developmentprogressionin this population Nonetheless the findings of our studyrequire cautious interpretation Considering the global MAF(from 1000 Genomes database) or the MAF observed in ourSaudi cohort the current sample size of the study has anestimated power of gt80 (with alpha risk of 5) to detect anodds ratio of 20 However it will certainly require a muchlarger sample size to detect a 15-fold relative risk As withother complex diseases large sample sizes are required toensure sufficient power to fully define the underlying geneticcausal effect which may be a major limitation in this studyBesides the role of other variants and gene-gene interactionalso cannot be ruled out
5 Conclusion
In an attempt to link this polymorphism with POAG amongthe Saudi cohort our study shows that variant rs7916697 in
Genetics Research International 5
the ATOH7 gene lacks significant association with POAGor related phenotypes such as IOP and cupdisc ratioThis observation needs further validation in a much largersample population of clinically well-defined POAG patientspotentially with age gender and ethnicity matched controlsto assess the risk it may contribute to the development orprogression of the disease in this population
Data Availability
The data supporting the conclusions of this article are allpresented within the article
Ethical Approval
The study adhered to the tenets of the Declaration of Helsinkiand had received approval from the Institutional ReviewBoard and Research Ethics Committee (approval 08-657)
Consent
Written informed consent was obtained from all participantsprior to their inclusion in this study
Disclosure
The funder had no role in the design of the study andcollection analysis and interpretation of data and in writingthe manuscript
Conflicts of Interest
The authors report no conflicts of interest in this work
Acknowledgments
The authors would like to thank the Deanship of ScientificResearch and Glaucoma Research Chair of Department ofOphthalmology College of Medicine King Saud Universityfor their support and use of laboratory facilities
References
[1] J Charlesworth P L Kramer T Dyer et al ldquoThe path toopen-angle glaucoma gene discovery Endophenotypic statusof intraocular pressure cup-to-disc ratio and central cornealthicknessrdquo Investigative Ophthalmology ampVisual Science vol 51no 7 pp 3509ndash3514 2010
[2] K Abu-Amero A A Kondkar and K V Chalam ldquoAn updatedreview on the genetics of primary open angle glaucomardquoInternational Journal of Molecular Sciences vol 16 no 12 pp28886ndash28911 2015
[3] N L Brown S L Dagenais C-M Chen and T GlaserldquoMolecular characterization and mapping of ATOH7 a humanatonal homolog with a predicted role in retinal ganglion celldevelopmentrdquo Mammalian Genome vol 13 no 2 pp 95ndash1012002
[4] Z Yang K Ding L Pan M Deng and L Gan ldquoMath5 deter-mines the competence state of retinal ganglion cell progenitorsrdquoDevelopmental Biology vol 264 no 1 pp 240ndash254 2003
[5] W-T Song X-Y Zhang and X-B Xia ldquoAtoh7 promotes thedifferentiation of Muller cells-derived retinal stem cells intoretinal ganglion cells in a rat model of glaucomardquo ExperimentalBiology and Medicine vol 240 no 5 pp 682ndash690 2015
[6] S Macgregor A W Hewitt P G Hysi et al ldquoGenome-wideassociation identifies ATOH7 as a major gene determininghuman optic disc sizerdquo Human Molecular Genetics vol 19 no13 pp 2716ndash2724 2010
[7] A N Riesenberg T T LeM IWillardsen D C BlackburnML Vetter and N L Brown ldquoPax6 regulation of Math5 duringmouse retinal neurogenesisrdquo Genesis vol 47 no 3 pp 175ndash1872009
[8] N Azuma Y Yamaguchi H Handa et al ldquoMutations of thePAX6 gene detected in patients with a variety of optic-nervemalformationsrdquo American Journal of Human Genetics vol 72no 6 pp 1565ndash1570 2003
[9] W D Ramdas L M van Koolwijk M K Ikram et al ldquoAgenome-wide association study of optic disc parametersrdquo PLoSGenetics vol 6 no 6 2010
[10] A BroeksM K Schmidt andM E Sherman ldquoLow penetrancebreast cancer susceptibility loci are associated with specificbreast tumor subtypes findings from the Breast Cancer Associ-ation Consortiumrdquo Human Molecular Genetics vol 20 no 16pp 3289ndash3303 2011
[11] D R Nannini M Torres Y I Chen et al ldquoA genome-wide association study of vertical cup-disc ratio in a latinopopulationrdquo Investigative Opthalmology amp Visual Science vol58 no 1 pp 87ndash95 2017
[12] H Springelkamp A I Iglesias and A Mishra ldquoNew insightsinto the genetics of primary open-angle glaucoma based onmeta-analyses of intraocular pressure and optic disc character-isticsrdquo Human Molecular Genetics vol 26 no 2 pp 438ndash4532017
[13] D Cao X Jiao X Liu et al ldquoCDKN2B polymorphism isassociated with primary open-angle glaucoma (POAG) in theAfro-Caribbean population of Barbados West Indiesrdquo PLoSONE vol 7 no 6 2012
[14] A A Kondkar A Mousa T A Azad et al ldquoAnalysis ofPolymorphism rs1900004 inAtonal bHLHTranscription Factor7 in Saudi Patients with Primary Open Angle GlaucomardquoGenetic Testing and Molecular Biomarkers vol 20 no 11 pp715ndash718 2016
[15] K Abu-Amero T Sultan S Al-Obeidan and A KondkarldquoAnalysis of CYP1B1 sequence alterations in patients withprimary open-angle glaucoma of Saudi originrdquo Clinical Oph-thalmology vol 12 pp 1413ndash1416 2018
[16] A A Kondkar N B Edward H Kalantan et al ldquoLack ofassociation between polymorphism rs540782 andprimary openangle glaucoma in Saudi patientsrdquo Journal of Negative Results inBioMedicine vol 16 no 1 2017
[17] A A Kondkar T A Azad F A Almobarak et al ldquoPolymor-phism rs10483727 in the SIX1SIX6 gene locus is a risk factor forprimary open angle glaucoma in a saudi cohortrdquoGenetic Testingand Molecular Biomarkers vol 22 no 1 pp 74ndash78 2018
[18] B J FanD YWang L R Pasquale J L Haines and J LWiggsldquoGenetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucomain a US Caucasian populationrdquo Investigative Ophthalmology ampVisual Science vol 52 no 3 pp 1788ndash1792 2011
6 Genetics Research International
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
the ATOH7 gene lacks significant association with POAGor related phenotypes such as IOP and cupdisc ratioThis observation needs further validation in a much largersample population of clinically well-defined POAG patientspotentially with age gender and ethnicity matched controlsto assess the risk it may contribute to the development orprogression of the disease in this population
Data Availability
The data supporting the conclusions of this article are allpresented within the article
Ethical Approval
The study adhered to the tenets of the Declaration of Helsinkiand had received approval from the Institutional ReviewBoard and Research Ethics Committee (approval 08-657)
Consent
Written informed consent was obtained from all participantsprior to their inclusion in this study
Disclosure
The funder had no role in the design of the study andcollection analysis and interpretation of data and in writingthe manuscript
Conflicts of Interest
The authors report no conflicts of interest in this work
Acknowledgments
The authors would like to thank the Deanship of ScientificResearch and Glaucoma Research Chair of Department ofOphthalmology College of Medicine King Saud Universityfor their support and use of laboratory facilities
References
[1] J Charlesworth P L Kramer T Dyer et al ldquoThe path toopen-angle glaucoma gene discovery Endophenotypic statusof intraocular pressure cup-to-disc ratio and central cornealthicknessrdquo Investigative Ophthalmology ampVisual Science vol 51no 7 pp 3509ndash3514 2010
[2] K Abu-Amero A A Kondkar and K V Chalam ldquoAn updatedreview on the genetics of primary open angle glaucomardquoInternational Journal of Molecular Sciences vol 16 no 12 pp28886ndash28911 2015
[3] N L Brown S L Dagenais C-M Chen and T GlaserldquoMolecular characterization and mapping of ATOH7 a humanatonal homolog with a predicted role in retinal ganglion celldevelopmentrdquo Mammalian Genome vol 13 no 2 pp 95ndash1012002
[4] Z Yang K Ding L Pan M Deng and L Gan ldquoMath5 deter-mines the competence state of retinal ganglion cell progenitorsrdquoDevelopmental Biology vol 264 no 1 pp 240ndash254 2003
[5] W-T Song X-Y Zhang and X-B Xia ldquoAtoh7 promotes thedifferentiation of Muller cells-derived retinal stem cells intoretinal ganglion cells in a rat model of glaucomardquo ExperimentalBiology and Medicine vol 240 no 5 pp 682ndash690 2015
[6] S Macgregor A W Hewitt P G Hysi et al ldquoGenome-wideassociation identifies ATOH7 as a major gene determininghuman optic disc sizerdquo Human Molecular Genetics vol 19 no13 pp 2716ndash2724 2010
[7] A N Riesenberg T T LeM IWillardsen D C BlackburnML Vetter and N L Brown ldquoPax6 regulation of Math5 duringmouse retinal neurogenesisrdquo Genesis vol 47 no 3 pp 175ndash1872009
[8] N Azuma Y Yamaguchi H Handa et al ldquoMutations of thePAX6 gene detected in patients with a variety of optic-nervemalformationsrdquo American Journal of Human Genetics vol 72no 6 pp 1565ndash1570 2003
[9] W D Ramdas L M van Koolwijk M K Ikram et al ldquoAgenome-wide association study of optic disc parametersrdquo PLoSGenetics vol 6 no 6 2010
[10] A BroeksM K Schmidt andM E Sherman ldquoLow penetrancebreast cancer susceptibility loci are associated with specificbreast tumor subtypes findings from the Breast Cancer Associ-ation Consortiumrdquo Human Molecular Genetics vol 20 no 16pp 3289ndash3303 2011
[11] D R Nannini M Torres Y I Chen et al ldquoA genome-wide association study of vertical cup-disc ratio in a latinopopulationrdquo Investigative Opthalmology amp Visual Science vol58 no 1 pp 87ndash95 2017
[12] H Springelkamp A I Iglesias and A Mishra ldquoNew insightsinto the genetics of primary open-angle glaucoma based onmeta-analyses of intraocular pressure and optic disc character-isticsrdquo Human Molecular Genetics vol 26 no 2 pp 438ndash4532017
[13] D Cao X Jiao X Liu et al ldquoCDKN2B polymorphism isassociated with primary open-angle glaucoma (POAG) in theAfro-Caribbean population of Barbados West Indiesrdquo PLoSONE vol 7 no 6 2012
[14] A A Kondkar A Mousa T A Azad et al ldquoAnalysis ofPolymorphism rs1900004 inAtonal bHLHTranscription Factor7 in Saudi Patients with Primary Open Angle GlaucomardquoGenetic Testing and Molecular Biomarkers vol 20 no 11 pp715ndash718 2016
[15] K Abu-Amero T Sultan S Al-Obeidan and A KondkarldquoAnalysis of CYP1B1 sequence alterations in patients withprimary open-angle glaucoma of Saudi originrdquo Clinical Oph-thalmology vol 12 pp 1413ndash1416 2018
[16] A A Kondkar N B Edward H Kalantan et al ldquoLack ofassociation between polymorphism rs540782 andprimary openangle glaucoma in Saudi patientsrdquo Journal of Negative Results inBioMedicine vol 16 no 1 2017
[17] A A Kondkar T A Azad F A Almobarak et al ldquoPolymor-phism rs10483727 in the SIX1SIX6 gene locus is a risk factor forprimary open angle glaucoma in a saudi cohortrdquoGenetic Testingand Molecular Biomarkers vol 22 no 1 pp 74ndash78 2018
[18] B J FanD YWang L R Pasquale J L Haines and J LWiggsldquoGenetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucomain a US Caucasian populationrdquo Investigative Ophthalmology ampVisual Science vol 52 no 3 pp 1788ndash1792 2011
6 Genetics Research International
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
[19] C Venturini A Nag P G Hysi et al ldquoClarifying the roleof ATOH7 in glaucoma endophenotypesrdquo British Journal ofOphthalmology vol 98 no 4 pp 562ndash566 2014
[20] WD Ramdas L M van Koolwijk H G Lemij et al ldquoCommongenetic variants associated with open-angle glaucomardquoHumanMolecular Genetics vol 20 no 12 pp 2464ndash2471 2011
[21] F Mabuchi N Mabuchi Y Sakurada et al ldquoAdditive effects ofgenetic variants associatedwith intraocular pressure in primaryopen-angle glaucomardquo PLoS ONE vol 12 no 8 2017
