glaucoma is the most common cause of visual impairment or visual loss worldwide , and primary open - angle glaucoma ( poag ) is the most prevalent type of glaucoma . an estimated 70 million people suffer from glaucoma , and asian ethnic minorities are more susceptible to this visual disease . poag is a chronic and aggressive optic neuropathy characterized by loss of optic nerve fibers , leading to elevated intraocular pressure ( iop ) , visual field abnormality , and occurrence of open anterior chamber angle ( http://www.lifesciencesite.com/lsj/life1101/003_21749life1101_18_26.pdf ) . the poag etiology is partly attributed to advanced age , ethnicity , and family history . iop is considered the major risk factor for poag , and an elevated iop ( > 21 mmhg ) influences both the incidence and the progression of poag ; therefore , lowering iop is the primary goal of current treatments in poag patients . timolol is a non - selective beta - adrenergic receptor antagonist that has been widely used for the treatment of elevated iop and glaucoma . timolol is actively metabolized by the drug - metabolizing enzyme cytochrome p450 2c19 ( cyp2c19 ) , improving the efficacy of medical treatment . the use of timolol in poag patients can improve retinal vascular autoregulatory function , including ocular blood flow and blood pressure , thereby reducing the loss of retinal ganglion cells and vision damage . inactive prodrugs require biotransformation into active forms , and drug metabolism is mediated by the hepatic cytochrome p450 ( cyp450 ) system . the hepatic cyp2c19 enzyme , belonging to the cyp450 superfamily , metabolizes several important drugs such as mephenytoin , antidepressants , benzodiazepines , clopidogrel , and timolol . the cyp2c191 , 2 and 3 alleles are the most common loss - of - function alleles that affect cyp2c19-mediated bioactivation of inactive drugs to active metabolites , consequently leading to poor drug pharmacodynamics and intestinal absorption . patients carrying the cyp2c191 variant have full drug - metabolizing capacity , and cyp2c192 and 3 carriers exhibit attenuation of cyp function and impaired drug response ; therefore , individual differences in drug treatment response may be attributed to cyp2c192 and 3 genetic polymorphisms ( http://www.davidpublishing.com/davidpublishing/upfile/8/14/2014/2014081408950324.pdf ) . the present study investigated the cyp2c192 and 3 genetic polymorphisms for their association with the susceptibility to poag and individual differences in drug treatment response . this case - control study was performed in accordance with the guidelines received from the local ethics committee of the affiliated hospital of beihua university . the enrolled patients provided written informed consent for the procedures and agreed to genetic analyses . the study consisted of 93 poag subjects ( male 64 , female 29 ; age range , 19~78 ; average age , 44.810.3 ; bmi , 17~31 ) and 125 controls ( male 73 , female 52 ; age range , 20~76 ; average age , 43.110.7 ; bmi , 17~31 ) , divided into 2 groups ( case and control groups ) . all patients in this study were recruited between march 2013 and may 2014 at the department of ophthalmology , the affiliated hospital of beihua university . the diagnosis of poag was based on diagnostic criteria published by the chinese medical association glaucoma branch in 2008 . the criteria for diagnosis of poag were as follows : 1 ) iop 21 mmhg ; 2 ) abnormal optic disc determined by optical coherence tomography ; 3 ) glaucomatous visual field deletion ( on the basis of mean deviation and corrected pattern standard deviation ) ; 4 ) retinal nerve fiber layer defect ; and 5 ) open anterior chamber angle . patients were excluded if they had histories of chronic obstructive pulmonary diseases , bradycardia ( heart rate < 60 beats per min ) ( bpm ) , atrioventricular heart - block , bronchial asthma , spontaneous hypoglycemia , and diabetics under medication . all patients did not use medication for 3 weeks before inclusion were excluded as well . after all subjects fasted overnight for a period of 1012 h , peripheral venous blood ( 5 ml ) was collected in ethylene diamine tetraacetic acid ( edta ) tubes and stored at 80 c . the whole - blood genomic dna isolation kit ( tiangen biotech ( beijing ) co. ltd ) an ultraviolet spectrophotometer was used to measure the concentration and purity of the dna . cyp2c192 and 3 single - nucleotide polymorphisms ( snps ) were analyzed by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rlfp ) method . the pcr primers were designed by primer premier 5.0 software ( sangon biotech , shanghai , china ) and were synthesized by shanghai sangon biological engineering technology and service co. , ltd . the 50 l - pcr system consisted of 5 l 10 pcr buffer , 1 l dntp ( 10 mmol / l ) , 2 l primer compound , 0.5 l taq enzyme ( 5 u/l ) ( takara ) , and 1 l genomic dna . the amplification conditions were as follows : an initial denaturation step ( 94c for 5 min ) and 35 cycles of denaturation ( 94c for 60 s ) , followed by annealing and extension steps ( 53c for 30 s and 70c for 1 min ) . the sam i restriction digestion reaction consisted of 1 l sam i , 2.0 l 10 t buffer , 2 l 0.1% bsa , 10 l dna , and 5 l double distilled water . the bamhi restriction digestion reaction was as follows : 1 l bamh i , 2.0 l 10 k buffer , 12 l dna , and 5 l double - distilled water . the digestion products were separated on 3% agarose gels containing ethidium bromide and sequenced directly on an abi 370 automated dna sequencer ( abi systems ) . the polymorphisms of cyp2c192 and 3 were divided into 3 phenotypes according to definition of metabolic phenotype : cyp2c191/1 ( extensive metabolizers ) ; cyp2c191/2 and cyp2c191/3 ( intermediate metabolizers ) ; and cyp2c19 2/ 2 , cyp2c19 2/ 3 , and cyp2c19 3/ 3 ( poor metabolizers ) . the case group received 1 drop of 0.5% timolol without punctal occlusion , twice daily at 8 am and 6 pm . after the instillation , iop was measured on days 1 , 3 , 7 , 14 , 21 , and 28 with a perkins hand - held applanation tonometer ( perkins ) , in the sitting position . the iop daily curve was taken after measurements , and hr of patients was also recorded . patients who showed hr lower than 60 bpm after timolol treatment or lower than 20 bpm for the first - time measurement were considered as the side effect group ( se ) , and the remaining patients were considered as the non - side effect group ( nse ) . if patients had hr lower than 45 bpm or symptoms of chest stuffiness , general fatigue , or palpitations at any time during the study , they were requested to immediately stop drug use and consult a doctor . based on the effects of drug treatment and degree of iop improvement , patients were divided into 3 groups : the excellent group ( ex ) ( iop 8 mm hg ) ; the utility group ( ut ) ( 5 < iop 8 mm hg ) ; and the ineffective group ( in ) ( iop 5 mm hg ) . data analysis was performed using spss software for windows ( version 18.0 ; spss , chicago , il ) . the different genotypes of cyp2c192 and 3 polymorphisms were estimated by odds ratios ( ors ) with 95% confidence intervals ( cis ) . all tests were 2-sided , and p values less than 0.05 were considered to be statistically significant . this case - control study was performed in accordance with the guidelines received from the local ethics committee of the affiliated hospital of beihua university . the enrolled patients provided written informed consent for the procedures and agreed to genetic analyses . the study consisted of 93 poag subjects ( male 64 , female 29 ; age range , 19~78 ; average age , 44.810.3 ; bmi , 17~31 ) and 125 controls ( male 73 , female 52 ; age range , 20~76 ; average age , 43.110.7 ; bmi , 17~31 ) , divided into 2 groups ( case and control groups ) . all patients in this study were recruited between march 2013 and may 2014 at the department of ophthalmology , the affiliated hospital of beihua university . the diagnosis of poag was based on diagnostic criteria published by the chinese medical association glaucoma branch in 2008 . the criteria for diagnosis of poag were as follows : 1 ) iop 21 mmhg ; 2 ) abnormal optic disc determined by optical coherence tomography ; 3 ) glaucomatous visual field deletion ( on the basis of mean deviation and corrected pattern standard deviation ) ; 4 ) retinal nerve fiber layer defect ; and 5 ) open anterior chamber angle . patients were excluded if they had histories of chronic obstructive pulmonary diseases , bradycardia ( heart rate < 60 beats per min ) ( bpm ) , atrioventricular heart - block , bronchial asthma , spontaneous hypoglycemia , and diabetics under medication . all patients did not use medication for 3 weeks before inclusion were excluded as well . after all subjects fasted overnight for a period of 1012 h , peripheral venous blood ( 5 ml ) was collected in ethylene diamine tetraacetic acid ( edta ) tubes and stored at 80 c . the whole - blood genomic dna isolation kit ( tiangen biotech ( beijing ) co. ltd ) cyp2c192 and 3 single - nucleotide polymorphisms ( snps ) were analyzed by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rlfp ) method . the pcr primers were designed by primer premier 5.0 software ( sangon biotech , shanghai , china ) and were synthesized by shanghai sangon biological engineering technology and service co. , ltd . the 50 l - pcr system consisted of 5 l 10 pcr buffer , 1 l dntp ( 10 mmol / l ) , 2 l primer compound , 0.5 l taq enzyme ( 5 u/l ) ( takara ) , and 1 l genomic dna . the amplification conditions were as follows : an initial denaturation step ( 94c for 5 min ) and 35 cycles of denaturation ( 94c for 60 s ) , followed by annealing and extension steps ( 53c for 30 s and 70c for 1 min ) . the sam i restriction digestion reaction consisted of 1 l sam i , 2.0 l 10 t buffer , 2 l 0.1% bsa , 10 l dna , and 5 l double distilled water . the bamhi restriction digestion reaction was as follows : 1 l bamh i , 2.0 l 10 k buffer , 12 l dna , and 5 l double - distilled water . the digestion products were separated on 3% agarose gels containing ethidium bromide and sequenced directly on an abi 370 automated dna sequencer ( abi systems ) . the polymorphisms of cyp2c192 and 3 were divided into 3 phenotypes according to definition of metabolic phenotype : cyp2c191/1 ( extensive metabolizers ) ; cyp2c191/2 and cyp2c191/3 ( intermediate metabolizers ) ; and cyp2c19 2/ 2 , cyp2c19 2/ 3 , and cyp2c19 3/ 3 ( poor metabolizers ) . the case group received 1 drop of 0.5% timolol without punctal occlusion , twice daily at 8 am and 6 pm . after the instillation , iop was measured on days 1 , 3 , 7 , 14 , 21 , and 28 with a perkins hand - held applanation tonometer ( perkins ) , in the sitting position . the iop daily curve was taken after measurements , and hr of patients was also recorded . patients who showed hr lower than 60 bpm after timolol treatment or lower than 20 bpm for the first - time measurement were considered as the side effect group ( se ) , and the remaining patients were considered as the non - side effect group ( nse ) . if patients had hr lower than 45 bpm or symptoms of chest stuffiness , general fatigue , or palpitations at any time during the study , they were requested to immediately stop drug use and consult a doctor . based on the effects of drug treatment and degree of iop improvement , patients were divided into 3 groups : the excellent group ( ex ) ( iop 8 mm hg ) ; the utility group ( ut ) ( 5 < iop 8 mm hg ) ; and the ineffective group ( in ) ( iop 5 mm hg ) . data analysis was performed using spss software for windows ( version 18.0 ; spss , chicago , il ) . the different genotypes of cyp2c192 and 3 polymorphisms were estimated by odds ratios ( ors ) with 95% confidence intervals ( cis ) . all tests were 2-sided , and p values less than 0.05 were considered to be statistically significant . the frequencies of cyp2c19 alleles and genotypes are shown in table 2 , and it showed that there were no statistically significant differences in frequencies of cyp2c19 alleles and genotypes between the case group and the control group ( both p>0.05 ) . the results suggest that cyp2c192 and 3 polymorphisms were not associated with susceptibility to poag . no differences in sex , age , or bmi existed between the se group and nse group ( all p>0.05 ) . however , statistically significant differences were observed in cyp2c19 metabolizer phenotypes between the se group and nse group . as seen in table 3 , the frequencies of extensive metabolizer phenotype and poor metabolizer phenotype or poor metabolizer phenotype and intermediate metabolizer phenotype were significantly different ( both p<0.05 ) , but the distributions of intermediate metabolizer phenotype and extensive metabolizer phenotype showed no statistically significant differences ( p>0.05 ) . these results suggest that the poor metabolizer phenotype slows the rate of timolol metabolism , thereby leading to ses . there were no differences in sex , age and bmi among ex group , ut group , and in group ( all p>0.05 ) . distributions of poor metabolizer phenotype and intermediate metabolizer phenotype among ex group , ut group , and in group did not show statistically significant differences ( all p>0.05 ) , while the distributions of intermediate metabolizer phenotype and extensive metabolizer phenotype and extensive metabolizer phenotype and poor metabolizer phenotype among ex group , ut group , and in group were significantly different ( all p<0.05 ) ( table 4 ) . these results revealed that extensive metabolizers rapidly metabolize timolol , contributing to low utilization ratio of the drug and poor treatment responses . the frequencies of cyp2c19 alleles and genotypes are shown in table 2 , and it showed that there were no statistically significant differences in frequencies of cyp2c19 alleles and genotypes between the case group and the control group ( both p>0.05 ) . the results suggest that cyp2c192 and 3 polymorphisms were not associated with susceptibility to poag . no differences in sex , age , or bmi existed between the se group and nse group ( all p>0.05 ) . however , statistically significant differences were observed in cyp2c19 metabolizer phenotypes between the se group and nse group . as seen in table 3 , the frequencies of extensive metabolizer phenotype and poor metabolizer phenotype or poor metabolizer phenotype and intermediate metabolizer phenotype were significantly different ( both p<0.05 ) , but the distributions of intermediate metabolizer phenotype and extensive metabolizer phenotype showed no statistically significant differences ( p>0.05 ) . these results suggest that the poor metabolizer phenotype slows the rate of timolol metabolism , thereby leading to ses . there were no differences in sex , age and bmi among ex group , ut group , and in group ( all p>0.05 ) . distributions of poor metabolizer phenotype and intermediate metabolizer phenotype among ex group , ut group , and in group did not show statistically significant differences ( all p>0.05 ) , while the distributions of intermediate metabolizer phenotype and extensive metabolizer phenotype and extensive metabolizer phenotype and poor metabolizer phenotype among ex group , ut group , and in group were significantly different ( all p<0.05 ) ( table 4 ) . these results revealed that extensive metabolizers rapidly metabolize timolol , contributing to low utilization ratio of the drug and poor treatment responses . cyp450 metabolizes almost 90% of prescribed drugs , and cyp2c19 is an important cyp450 that can also catalyze drugs by inducing oxidation reactions . our study found no association between cyp2c19 polymorphisms and susceptibility to poag , possibly because cyp2c19 affects drug metabolism , and thus is unlikely to be involved in the poag disease process . however , genetic polymorphisms play a critical role in drug responses and adverse drug reactions . a previous study suggested that drug - drug interactions are correlated with activities of cyp2c19 , but the development of poag was not linked with cyp2c19 polymorphism . we support the view that different cyp2c19 metabolizer phenotypes impact drug treatment response to timolol in patients with poag . timolol monotherapy , used for management of poag patients , lowers iop and prevents other complications and drug - related adverse events . a study conducted by frezzotti paolo observed that , despite the iop - lowering effect , 2 formulations of timolol caused varying degrees of adverse events . therefore , combination therapy for glaucoma aims to achieve minimum medication , reduced iop - lowering doses , and improved convenience and compliance for patients , leading to excellent drug effects . owing to the drug metabolic enzymes , treatment of poag using timolol is improved ; however , patients carrying different cyp2c19 phenotypes may differ in drug response . carriers of 1 or more loss - of - function cyp2c19 alleles ( so - called intermediate metabolizers ) prevent drug conversion to their active metabolites and enhance adverse outcomes ( http://circinterventions.ahajournals.org.proxy.its.virginia.edu/content/4/5/514.full ) . accordingly , we support the notion that cyp2c19 genetic variants influence clinical benefits from drug treatment by modulating availability of the active drug ; thus , treatment of poag with timolol must consider these cyp2c19 metabolizer phenotypes . according to cyp2c19 genotypes , there are 3 main phenotypes  extensive metabolizers , intermediate metabolizers , and poor metabolizers  that are closely correlated with drug utilization , individual response to drug treatment , and drug effects . cyp2c19 polymorphisms are chief pharmacogenetic contributors of individual differences in response to drug treatment because of the carrier status of the cyp2c19 loss - of - function allele . poor metabolizers have lower activities of cyp2c19 enzyme , and the concentration of drug significantly increases , consequently leading to drug toxicity ( http://cdn.intechopen.com/pdfs-wm/39693.pdf ) . in contrast , intermediate and extensive metabolizers exhibit higher drug metabolism and absorption , but the rapid metabolism lower drug effects in the clinic , causing administration of larger doses of the drug and severe side effects . our findings also suggest that different cyp2c19 metabolizer phenotypes , due to cyp2c192 and 3 polymorphisms , have important effects on individual responses to drug treatment and adverse drug reactions . also , we did not use different timolol dosing regimens , which may be a significant study limitation . third , the time of iop measurement and timolol use was not matched , resulting in ignoring the peak of efficacy of drug treatment . additionally , other factors like symptoms and ocular surface staining , which may provide useful information , were not analyzed in the study . finally , we only discussed cyp2c192 and 3 polymorphisms in this paper , but there are other genetic variants of cyp2c19 polymorphisms that could alter the response to timolol treatment , such as 3 , 5 , and 17 . however , we observed that different cyp2c19 metabolizer phenotypes , due to cyp2c192 and 3 polymorphisms , have important effects on individual responses to drug treatment and treatment - related side effects .
backgroundthe aim of this study was to investigate the roles of cytochrome p450 2c19 ( cyp2c19 ) polymorphisms in primary open - angle glaucoma ( poag ) susceptibility and individual responses to drug treatment.material/methodsthis case - control study consisted of 93 cases with poag and 125 controls . 
 polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) was used to analyze cyp2c19 single - nucleotide polymorphisms ( snps ) . 
 after timolol treatment , patients were classified into side effect ( se ) group and non - side effect ( nse ) group . according to drug treatment responses 
 , patients were divided into 3 groups : excellent group ( ex ) ( iop 8 mm hg ) ; utility group ( ut ) ( 5 < iop 8 mm hg ) , and ineffective group ( in ) ( iop 5 mm hg ) . 
 data analysis was performed using spss software.resultswe found no statistical differences in the alleles and genotypes frequencies of cyp2c19 between the case group and the control group ( both p>0.05 ) . 
 frequencies of extensive metabolizer phenotype and poor metabolizer phenotype or poor metabolizer phenotype and intermediate metabolizer phenotype were significantly different between the se group and nse group ( both p<0.05 ) . 
 the distribution of intermediate metabolizer phenotype and extensive metabolizer phenotype were significantly different among ex group , ut group , and in group ( all p<0.05).conclusionswe found no evidence that cyp2c19 polymorphisms are associated with susceptibility to poag . 
 however , different cyp2c19 metabolizer phenotypes were identified and observed to have important effects on the individual differences in drug treatment response .