many reports have demonstrated that gene polymorphisms of the renin - angiotensin system ( ras ) are associated with hypertension .
there have been some reliable reports about susceptible genes for hypertension including the results from  the millennium genome project for hypertension in japan ( 2000~2005 ) [ 1 , 2 ] ; however , no convincing gene has yet been detected .
some of the genes regulating blood pressure might also be related to the response to antihypertensive medication [ 3 , 4 ] .
indeed , we and other collaborators have investigated several susceptible genes related to hypertension [ 515 ] , including genes of not only the renin - angiotensin system and sodium handling but also insulin resistance , oxidative stress , and sympathetic nervous system ( described in the section 2 ) ; however , the genes involved in the response to antihypertensive medication have not yet been identified .
in addition , exhaustive gene expression analysis ( transcriptome analysis ) for lifestyle - related diseases has not been performed thus far .
we performed a large collaboration with the study group led by professor katayama at saitama medical university to perform a randomized controlled trial called  azelnidipine ( a calcium ( ca ) channel blocker ) and temocapril ( an ace inhibitor ) in hypertensive patients with type 2 diabetes study ( attest )  , which included genetic analysis to evaluate the therapeutic effects of azelnidipine and temocapril .
the goals of this study were , first , to assess the association between polymorphisms of susceptible genes for hypertension ( 18 genes ) and each treatment or phenotype and , second , to assess the association between expression in peripheral blood of susceptible genes for hypertension and each treatment or phenotype .
attest study , a multicenter , randomized , open - label study , was originally performed to investigate the efficacy and safety of combination therapy using the calcium channel blocker ( ccb ) azelnidipine and angiotensin - converting enzyme ( ace ) inhibitor temocapril in hypertensive diabetics , led by professor katayama of saitama medical university .
all of the subjects fulfilled the following inclusion criteria : ( 1 ) age : 30~70 years , outpatients , ( 2 ) mean systolic bp ( bp ) : 140~180  mmhg and/or mean diastolic bp : 90~110  mmhg in washout period ( four weeks ) and stable bp without fluctuation in systolic bp of more than 30  mmhg or diastolic bp of more than 15  mmhg , and ( 3 ) fasting blood glucose : more than 126  mg / dl or hba1c : more than 6.5% at 6 months before entry .
these mild or moderate hypertensive subjects with diabetes were treated with the following medication : ccb : azelnidipine and ace inhibitor : temocapril in accordance with the protocol ( figure 1 ) .
each medication was started at a dose of 8  mg azelnidipine or 2  mg temocapril and increased to 16  mg azelnidipine or 4  mg temocapril until bp of less than 130/80  mmhg was achieved .
all subjects were measured for body height , body weight , systolic bp , diastolic bp , fasting blood glucose level , triglyceride level , low density lipoprotein ( ldl ) cholesterol level , and serum creatinine ( cr ) level .
informed consent for genetic analysis was obtained from all subjects , and finally a total of 44 subjects were recruited in this study .
the genotypes of the 22 polymorphisms of the 18 susceptible genes ( ace ins / del , add1 gly460trp ( rs4961 ) , adipoq ile164thr , adrb1 ser49gly ( rs1801252 ) and arg389gly ( rs1801253 ) , adrb2 gly16arg ( rs1042713 ) and glu27gln ( rs1042714 ) , adrb3 trp64arg ( rs4994 ) , agt met235trp ( rs699 ) , agtr1 a1166c ( rs17231380 ) , aldh2 glu487lys ( rs671 ) , arhgap8 arg338gly ( rs6007334 ) , bdkrb2 t-58c , fasl g-670a , grk4 ala486val ( rs1801058 ) and arg65leu ( rs2960306 ) , hogg1 ser326cys ( rs1052133 ) , mthfr c677 t ( rs1801133 ) , rgs2 a to g in promoter ( rs3767489 ) , rgs2 a-638 g ( rs2746071 ) , slc12a3 arg904gln ( rs11643718 ) , and tgfb1 leu10pro ( rs1982073 ) ) for which positive associations with hypertension were previously reported were successfully determined using taqman pcr method ( applied biosystems inc . , foster city , ca , usa ) . according to quantitative rt - pcr analysis using cdna extracted from peripheral blood ,
11 genes were expressed in peripheral blood : ace , add1 , adrb1 , adrb2 , arhgap8 , fasl , grk4 , mthfr , rgs2 , slc12a3 , and tgfb1 .
rna samples were extracted from the peripheral blood of the subjects , and cdna was made from rna using reverse transcriptase .
quantitative real - time pcr ( rt - pcr ) for all the genes was performed using taqman pcr method to determine their expression levels .
the associations between polymorphisms and clinical variables were analyzed using one - way analysis of variance ( anova ) .
odd ratios were calculated as an index of the association of each genotype with the prevalence of hypertension . to assess the contribution of confounding factors , we performed multiple logistic regression analysis using the computer software application , jmp 7.0 ( sas institute inc . , cary , nc , u.s.a . ) .
we focused on testing for snp medication and gene - expression medication interactions ( i.e. , whether the effects of snps on systolic bp or diastolic bp differed between hypertensives on azelnidipine and temocapril ) .
multivariate analysis of variance ( manova ) was performed to determine whether bp change showed a significant interaction of each antihypertensive medication with the genotypes of the susceptible genes .
baseline characteristics of the subjects treated with azelnidipine and temocapril are shown in table 2 .
there was no difference in sex , age , body mass index ( bmi ) , affected period of hypertension , systolic bp , triglyceride level , ldl cholesterol level , and serum creatinine level between the azelnidipine and temocapril groups ; however , fasting glucose level was higher in the azelnidipine group than in the temocapril group at the baseline . in the current study , the same level of bp reduction
was observed in both the azelnidipine and temocapril groups , and both groups achieved a mean bp of less than 130/80  mmhg at 52 weeks .
there was no difference in the dose of each treatment at 52 weeks ( group started with azelnidipine : bp at 52 weeks 121.9  11.4/74.7  7.1  mmhg , final dose of azelnidipine 15.0  2.7  mg , final dose of temocapril 3.8  0.6  mg ; group started with temocapril : bp at 52 weeks 125.6  10.3/74.7  6.1  mmhg , final dose of azelnidipine 14.9  2.8  mg , final dose of temocapril 3.7  0.7  mg , figure 2 ) .
the genotypes of 22 polymorphisms of the 18 genes were successfully determined at each time point ( 0 , 8 , 16 , and 52 weeks ) .
only the ile164thr polymorphism of the adiponectin gene has no mutant genotype , and the other genotype frequencies were not significantly different from the values of hardy - weinberg 's expectation ( figure 3 ) . according to the analysis of the association between bp , changes of bp , and all genotypes at each time point ( 0 , 8 , 16 , and 52 weeks ) , only the a-638 g polymorphism of the regulator of g protein signaling-2 gene ( rgs2 ) showed a significant association with changes in bp ( bp : bp at 8 weeks  bp at 0 week ) .
as shown in figure 3 , there was a significant relationship between the a-638 g polymorphism of the rgs2 gene and the changes in bp between 0 and 8 weeks in subjects with azelnidipine ( systolic bp : aa  28.0  10.1  mmhg , ag  15.5  12.6  mmhg , gg + 7.0  12.2  mmhg , p = .0013 ; diastolic bp : aa  17.2  9.8  mmhg , ag  8.1  9.0  mmhg ,
gg  4.0  11.0  mmhg , p = .067 ) , but not in subjects with temocapril ( systolic bp : aa  5.3  11.8  mmhg , ag  8.4  13.0  mmhg , gg  9.9  10.0  mmhg , p = .81 ; diastolic bp : aa  0.5  7.2  mmhg , ag  4.4  3.5  mmhg , gg  10.0  4.5  mmhg , p = .34 ) . at 8 weeks , changes in bp showed a significant interaction between a-638 g and treatment with azelnidipine and temocapril ( systolic bp , diastolic bp : p = .0014 , p = .036 , resp .
, by manova , after adjustment for age , sex , and bmi ) ( table 3 ) .  
there was no gene whose expression was associated with bp phenotypes or the polymorphisms of each gene through analysis of gene expression in peripheral blood . in terms of the rgs2 gene ,
the a-638 g polymorphism was not related to the change of rgs2 expression between 0 and 8 weeks either in subjects with azelnidipine ( rgs2/18srna with azelnidipine : aa  1.06  2.1 , ag + 0.31  0.85 , gg + 0.57  0.44 , p = .13 , figure 4 ) or in subjects with temocapril ( rgs2/18srna with temocapril : aa + 0.75  1.3 , ag  0.10  0.62 , gg + 0.38  0.44 , p = .24 , figure 4 ) .
this study demonstrated the importance of pharmacogenetic research . previously , lynch et al . reported that the nppa t2238c variant was associated with modification of antihypertensive medication effects on cardiovascular disease and bp , and tt allele carriers had more favorable outcomes when randomized to receive a ccb ( amlodipine ) .
beitelshees et al . also demonstrated that the kcnmb1 genotype influenced responsiveness to verapamil sr and risk of adverse cardiovascular outcomes .
these reports could support the possibility of the existence of genes influencing drug efficacy . signaling by g - protein - coupled neurotransmitter receptors in the autonomic nervous system and vasoregulatory factor receptors in the periphery governs both blood pressure , by controlling the constriction and dilatation of resistance arterioles , and electrolyte and fluid balance by the kidney [ 19 , 20 ] . the recently identified regulator of g - protein signaling ( rgs ) proteins is important in regulating signaling cascades initiated by g - protein - coupled receptors ( gpcrs ) activation .
rgs proteins facilitate the intrinsic inactivating guanosine triphosphatase reaction of g - protein a - subunits , and thereby serve as effector channel blockers .
rgs2 is unique among the rgs proteins in its apparent selectivity towards gpa , which mediates the action of mouse physiological vasoconstrictors , including norepinephrine , angiotensin ii , endothelin-1 , and thrombin .
rgs2 can also attenuate gi- and gs - mediated pathways [ 22 , 23 ] , which can also affect blood pressure via other physiologically important agonists such as serotonin , dopamine , and bradykinin .
it was recently reported that mice lacking rgs2 exhibit a strong hypertensive phenotype ( increase in sbp of 50  mmhg ) and resistance vasculature [ 24 , 25 ] . both heterozygous and homozygous
rgs2-null mice exhibited a similar level of marked hypertension , suggesting that a naturally occurring mutation that affects the level of rgs2 protein may have a significant impact on blood pressure regulation .
recently there have been two reports that genetic changes in rgs2 are associated with a hypertensive phenotype [ 26 , 27 ] .
a-638 g , t1026a , and 1891 - 1892 del tc polymorphisms were extracted on the condition the allele frequencies of these polymorphisms were > 0.1 , and the t1026a and 1891 - 1892 del tc polymorphisms of this gene were associated with hypertension in women .
these findings suggest that some functional variants of the rgs2 gene might be involved in regulating blood pressure in humans .  
this attest gene study revealed that one possible gene related to the effect of antihypertensive agents . in the current study ,
the a-638 g polymorphism of the rgs2 gene was a predictive factor for therapeutic performance of a ccb , azelnidipine .
other classical candidate genes including genes associated with the renin - angiotensin system , sodium handling , vasodilatation and vasoconstriction , and the sympathetic nervous system did not show significant association between their polymorphisms and the effect of a ca channel blocker or ace inhibitor .
in addition , there was no significant relationship between rgs2 polymorphisms and bp - related phenotypes in this study , unlike previous reports .
it has not yet been reported that the promoter variant a-638 g of the rgs2 gene may change rgs2 function ; however , this polymorphism has the possibility to change rgs2 protein production because this polymorphism is in the promoter region .
there has been no evidence supporting the idea that rgs2 could be involved in the effect of ccbs . in several previous reports ,
genes without a drug - metabolizing effect , such as ace   or agtr1 , showed a significant association between their polymorphisms and response to antihypertensive medication ; however , the mechanisms have not been clarified in any of these reports . in terms of the present study , rgs2 is suggested to function as a switch to turn on or off the g protein - associated pathway , and rgs2 can regulate blood pressure through smooth muscle cells . from this viewpoint , rgs2 might have the possibility of changing the effect of ccbs , because those mainly act through smc ; however , the detailed mechanism merits further investigation in the future . peripheral blood mainly contains white blood cells , red blood cells , platelets , and other circulating hormones , so the distribution of genes expressed in peripheral blood has to be investigated to clarify the significance of disease susceptibility gene expression in peripheral blood . in the present study ,
there was no significant relationship between gene expression including the rgs2 gene and the effect of antihypertensives and phenotypes . from this viewpoint ,
rgs2 expression in peripheral blood does not seem to do anything and might not be a marker for bp regulation .
however , further study is needed on its clinical application as a marker of drug efficacy . as the limitation of this study
, we could not exclude the possibility of the false positive association ( type i error ) due to the use of small number of subjects .
however , this study was carried out under the strict protocol of a clinical trial so that the reliability of the results obtained seems to be high . to confirm the effect of rgs2 gene in the tailored medicine of hypertension , further study using another panel of hypertensive subjects should be required . in conclusion , the a-638 g polymorphism of the rgs2 gene could be a predictive factor for therapeutic effectiveness of ccbs such as azelnidipine .
further research is needed to determine the optimal approach for personalizing antihypertensive medication treatment regimens according to genotype information and for achieving the best blood pressure control .
 we performed a prospective study to examine the genetic effect on the response to a calcium ( ca ) channel blocker , azelnidipine and an ace inhibitor , temocapril treatment in patients with hypertension , as a part of the prior clinical trial , the azelnidipine and temocapril in hypertensive patients with type 2 diabetes study ( attest ) . 
 
 methods and results . 
 all subjects who gave informed consent for genetic research were divided into two groups : the subjects treated with azelnidipine or temocapril , for 52 weeks . 
 we selected 18 susceptible genes for hypertension and determined their genotypes using taqman pcr method . 
 rna samples were extracted from peripheral blood , and quantitative real time pcr for all genes was performed using taqman method . 
 one of the polymorphisms of the rgs2 gene was extracted as being able to influence the effect of these treatments to reduce bp . at eight weeks 
 , bp change showed a significant interaction between the a-638 g polymorphism of regulator of g protein signaling-2 ( rgs2 ) gene and treatment with azelnidipine or temocapril . 
 there was no gene whose expression was associated with bp phenotypes or the polymorphisms of each gene . 
 
 conclusions . 
 a-638 g polymorphism of the rgs-2 gene could be a predictive factor for therapeutic performance of ca channel blockers . 
 
1. Introduction
2. Methods
3. Results
4. Discussion