Journal of Human Hypertension (2011) 25, 327–333 & 2011 Macmillan Publishers Limited All rights reserved 0950-9240/11 www.nature.com/jhh ORIGINAL ARTICLE Effects of coding polymorphisms in the 1 on baseline blood pressure and antihypertensive response to irbesartan in Chinese hypertensive patients

S Jiang1,2, Y-H Hsu3,4, SA Venners5, Y Zhang2, H Xing2, X Wang6 andXXu2,7 1School of Life Sciences, Anhui University, Hefei, China; 2Institute of Biomedicine, Anhui Medical University, Hefei, China; 3Institute for Aging Research, HSL and Harvard Medical School, Boston, MA, USA; 4Molecular and Integrative Physiological Sciences Program, Harvard School of Public Health, Boston, MA, USA; 5Simon Fraser University, Faculty of Health Sciences, Burnaby, British Columbia, Canada; 6The Mary Ann and J. Milburn Smith Child Health Research Program, Children’s Memorial Hospital and Children’s Memorial Research Center, Chicago, IL, USA and 7Division of Epidemiology and Biostatistics, School of Public Health, University of Illinois at Chicago, Chicago, IL, USA

The aim of this study was to determine the association SBP) and BP responses, or rs5516 with baseline DBP between coding variants in the human tissue kallikrein 1 and BP response were observed. In a haplotype-based (KLK1) gene and baseline blood pressure (BP) and association test for the KLK1 gene, the Haplo-special antihypertensive response to irbesartan treatment in score analyses identified that haplotype AG was margin- Chinese hypertensive patients. A total of 1061 hyper- ally associated with SBP response (specific score: 1.75 tensives were recruited and received daily oral dosage for P ¼ 0.08), but not with DBP response. We did not find of 150 mg irbesartan for 4 weeks. Predose BPs, BPs and any associations between haplotypes (GC and AC) blood irbesartan concentrations at postdose on the 28th and BP responses. The Haplo-GLM analyses showed day were all measured. Common functional single- that, compared with haplotype GC subjects, the subjects nucleotide polymorphisms (SNPs) in the KLK1 gene with haplotype AG had a marginally greater SBP were genotyped. On the basis of the HapMap data of Han response (adjusted b±s.e.: 1.81±0.97, P ¼ 0.06), but Chinese in the Beijing population, two non-synonymous DBP response did not differ. This study suggests polymorphisms with minor allele frequency40.1, SNP that rs5516 in the KLK1 gene may be involved in rs5517 (Glu162Lys) and rs5516 (Gln121Glu), were se- the development of essential hypertension and in the lected. Those with GG genotype in the rs5516 locus regulation of SBP-lowering response to irbesartan in had higher average baseline systolic BP (SBP) than CC Chinese hypertensives. subjects (b±s.e.: 5.0±2.3, P ¼ 0.033); and no associa- Journal of Human Hypertension (2011) 25, 327–333; tions of rs5517 with baseline BP (diastolic BP (DBP) and doi:10.1038/jhh.2010.70; published online 8 July 2010

Keywords: kallikrein 1; irbesartan; response; polymorphism; essential hypertension

Introduction irbesartan, and only 56% of irbesartan-treated patients have a favourable response.2–5 Multiple genetic variants Irbesartan, a non-peptide angiotensin II type 1 as well as environmental risk factors and their inter- receptor (AT1R)-specific antagonist, acts at the final actions may, in part, explain these variations.6,7 step of the renin-angiotensin system by selectively The kallikrein-kinin system counter-regulates the blocking the binding of angiotensin II to AT1R, 1 detrimental actions of the renin-angiotensin sys- thereby exhibiting its antihypertensive effect. tem.8 Beneficial effects of the angiotension-convert- Although the drug has been proven to have good ing enzyme (ACE) inhibition in hypertension and antihypertensive efficacy with a dose of 150 mg once cardiovascular disease are partially attributed to daily for 6–8 weeks, there are considerable inter- kallikrein increase and kinin accumulation. Many individual variations in the therapeutic response to lines of evidence have shown an abnormality of the kallikrein levels in the pathogenesis of hyperten- Correspondence: Professor S Jiang, School of Life Sciences, Anhui sion.8 Epidemiological studies have identified an University, 3 Feixi Road, PO Box 41, Hefei 230039, China. inverse relationship between blood pressure (BP) E-mail: [email protected] and urinary or renal kallikrein levels in patients Received 10 November 2009; revised 2 June 2010; accepted 5 June 9 2010; published online 8 July 2010 with essential hypertension. A study using family Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 328 pedigrees10 indicated that a dominant gene ex- these two KLK1 functional genetic variants and the pressed as renal or urinary kallikrein may be baseline BP level as well as BP-lowering response to associated with a reduced risk of hypertension. irbesartan treatment were investigated. Mice and rats overexpressing human tissue kallik- rein gene are permanently hypotensive throughout their lifetime.11 A single injection of human tissue Materials and methods kallikrein plasmid cDNA constructs into SHRs Study population causes a prolonged reduction of systolic BP (SBP). Patients with mild-to-moderate hypertension were The extent of BP reduction is dependent on the dose 12 enrolled from two adjacent counties, Taihu and of injected DNA. Dongzhi, in Anhui Province, China, from April 2003 Human tissue kallikrein 1 (KLK1) protein (EC to January 2004. In brief, hypertensive subjects 3.4.21.35), a key protease, is encoded by who met the following criteria were enrolled: KLK1 gene located on 19q13.2–13.4. (1) SBP, 140–200 mm Hg or diastolic BP (DBP), The KLK1 generates Lys-bradykinin by specific 13 14 90–115 mm Hg; (2) aged 35–65 years old; and (3) proteolysis of kininogen-1. Gan et al. state that not taking any antihypertensive medications within the deduced 262-amino acid KLK1 protein contains 4 weeks before the study. Subjects with a history of a putative signal peptide, followed by a short any of the following conditions were excluded: activating peptide and the protease domain. The secondary hypertension, pregnancy, hypercalcaemia, active form of the KLK1 is produced by intracellular chronic cardiovascular disease, chronic cerebrovascular proteolysis of a short amino-terminal peptide. disease, chronic liver or renal diseases, or body mass Measured by RT-PCR, the KLK1 gene highly ex- index 433 kg mÀ2. The study was approved by the presses in 35 human tissues such as kidney, Ethics Committee of Anhui Medical University, pancreas, salivary gland, thyroid, parotid gland Hefei, China. The purpose and procedures of the and blood vessels. The human KLK1 gene contains study were carefully explained to all participants, five exons. The sequence of its promoter is highly and a written informed consent was obtained. polymorphic and the variants in the promoter of the kallikrein gene are associated with hypertension. The kallikrein promoter polymorphism regulates Measurement of BPs and biometric variables and modifies BP in response to BPs were measured by well-trained nurses using a dietary salt intake.15 Analysis of recombinant kal- mercury-gravity manometer with appropriate sized likrein variants reveals a major decrease in enzyme cuffs after the subjects kept silent in a seated pose activity when arginine is replaced by histidine for half an hour without drinking tea or smoking. at protein codon 53, and a model of kallikrein Triplicate measurements were carried out on the left derived from crystallographic data suggest that arm with a 30-s interval between each reading. We codon 53 is involved in substrate binding. Two defined SBP as Korotkoff phase I (appearance of common KLK1 gene polymorphisms, single-nucleotide sound) and DBP as Korotkoff phase V (disappear- polymorphism (SNP) rs5515 (Arg53His) and rs5516 ance of sound). The mean of the three BP measure- (Gln121Glu)) have long been implicated in the ments was used in statistical analysis. If the pathogenesis of human essential hypertension.16–18 difference between measurements was 44 mm Hg, SNP rs5517 (Glu162Lys) in exon 4 of the KLK1 gene the patient was asked to rest for 5 min, and then the has attracted a lot of attention recently. A large-scale measurements were repeated. The subjects’ age, case–control study in a Chinese Han population gender, weight, height, smoking and drinking status with tag SNPs approach suggested that SNP rs5517 and other demographic factors were also measured. in the KLK1 gene was significantly associated with essential hypertension.19 The KLK1 has a critical role in the cardioprotec- Irbesartan treatment and blood sample collection tive effect of ACE inhibitors in acute myocardial All subjects were instructed to take a 150-mg tablet ischaemia.20 Genetically engineered mice deficient of irbesartan (manufactured by Sanofi-Synthelabo, in the KLK1 and kinin confirm the role of the KLK1 Hongzhou, China), once daily, fasting in the morning, in the cardioprotection elicited by AT1R blockade.21 for 28 consecutive days. The 1st and the 28th doses of These data suggest that the KLK1 may be involved in the subjects were administered in our study centre. the progression of cardiovascular diseases, and further Subjects were excluded from our study if they were alter ACE inhibition therapeutic response. To investigate unable to tolerate the drug therapy or took any other whether the KLK1 has a critical role in the development medications during the follow-up. Blood samples of essential hypertension and in the drug response to were collected in vacutainers containing heparin as Irbesartan, we recruited 1061 essential hypertensive an anticoagulant at the predose time point of the first patients with administration of daily oral dosage of day and at the 24-h time point after the 27th day 150 mg irbesartan for 28 consecutive days. The blood of drug administration. Plasma was obtained from samples of patients were collected to determine the blood samples after centrifuging (5417R Centrifuge, genotypes of the protein coding SNP rs5516 and Eppendorf AG, Hamburg, Germany) at 4 1Candstored rs5517 in KLK1 gene. The relationship between at À80 1Cbeforeanalysis.

Journal of Human Hypertension Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 329 Determination of plasma irbesartan by high Table 1 Clinical and demographic characteristics of hyper- performance liquid chromatography fluorescence tensive patients Plasma irbesartan concentrations were measured by Variables Subject high performance liquid chromatography fluore- (n ¼ 1061) scence. Standard curves of irbesartan were con- Mean±s.d. structed using a series of standard working solutions dissolved with methanol. The lower limit of detec- Age, years 52.8±7.1 tion was 5 ng mlÀ1, and the upper limit of quantifi- BMI, kg mÀ2 22.3±2.9 cation was 5000 ng mlÀ1. Mean accuracy ranged from Trough plasma concentration on 28th day, 73.1±49.4 ng mlÀ1 97 to 104%. Within- and between-run precision Natural logarithm-transformed concentration 4.1±0.7 (% RSD) were o8% at all concentration levels. Baseline DBP, mm Hg 92.3±10.1 Quantitation of irbesartan was measured by compar- Baseline SBP, mm Hg 164.9±16.4 ing high performance liquid chromatography peak DBP response on 28th day, mm Hg 6.3±8.2 areas with those of authentic standards, with SBP response on 28th day, mm Hg 18.6±17.2 Percent change in DBP, % 6.6±8.7 reference to an internal standard. Percent change in SBP, % 11.1±10.1 N (%) Male 464 (43.7) DNA extraction and genotyping Current smokers 377 (35.5) Venous blood samples were collected from all Current alcohol drinkers 259 (24.4) Occupation, farmers 898 (84.6) participants. Genomic DNA was extracted using Education, middle school or higher 150 (14.1) the QIAamp Blood Kit (Qiagen, Valencia, CA, USA) by standard techniques and stored at À20 1C Community until genotypic analysis was performed. SNP rs5516 Taihu 709 (66.8) and rs5517 in KLK1 gene were genotyped using the Dongzhi 352 (33.2) Taqman allelic discrimination method (Applied 22 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; Biosystems, Foster City, CA, USA). SBP, systolic blood pressure. Data are presented as mean±s.d. or number and percent.

Statistical analysis The SAS statistical program (Release 8.0, SAS Institute, Cary, NC, USA) was used for data analysis. respectively. The subjects’ mean age, body mass index, Descriptive statistical analyses were presented as baseline SBP and DBP, SBP and DBP reductions and percentages for categorical variables and mean trough irbesartan concentration are shown in Table 1. values with s.d. for continuous variables. BP Among hypertensive subjects, 43.7% are male; 35.5% response was defined as BP before treatment minus were current smokers; 24.4% were current alcohol BP on the 28th day. Multiple linear regression drinkers; and 84.6% were farmers. analyses were used to assess the influences of specific SNPs on the baseline BP and BP response, Single-locus association test with adjustment of potential confounders, including We used multiple linear regression models to age, sex, body mass index, smoking status, alcohol estimate the association of SNP rs5517 and rs5516 consumption, plasma concentration of irbesartan, with baseline BPs (Table 2) and BP responses educational level, occupation and county. (Table 3). The average baseline DBP (mean±s.d.) Haplotypes of SNPs rs5517 and rs5516 were in individuals carried GG, GA and AA genotypes of estimated by expectation-maximization algorithm. SNP rs5517 were 92.2±10.21, 92.1±9.86 92.7± Association between haplotypes and phenotypes 10.70, respectively; and the average baseline SBP (baseline BP and BP response) were examined ± ± ± 23 were 166.3 16.41, 163.4 15.94 166.4 17.34, re- by score test and generalized linear model test. spectively (Table 2). The average baseline DBP Both Haplo.score and Haplo.glm were implemented (mean±s.d.) were 91.9±10.44, 93.0±9.56 and in the Haplo.stats software developed using the R 92.5±9.37 in individuals carried CC, CG and GG language. genotypes of SNP rs5516, respectively; the average baseline SBP were 164.8±15.97, 164.4±17.20 and Results 169.5±15.85, respectively. Baseline SBP was found to be significantly associated with SNP rs5516 Demographic and clinical characteristics of the study (b±s.e.: 5.0±2.3, P ¼ 0.033), but not with SNP population rs5517. Baseline DBP was not associated with both Totally, 1061 subjects from two adjacent rural SNP rs5516 and rs5517. The average DBP responses communities in Anhui Province, China, with com- (mean±s.d.) for rs5517 genotype subgroups with plete genotype and phenotype information were GG, GA and AA were 6.1±8.14, 6.5±8.20 and analysed. The genotype distribution of SNP rs5516 6.4±8.21 mm Hg, respectively (Table 3). The mean and rs5517 were not deviated from Hardy–Weinberg SBP responses for rs5517 genotype subgroups with equilibriums (w2 ¼ 0.01, P ¼ 0.995; w2 ¼ 0.19, P ¼ 0.666), GG, GA and AA were 17.88±16.82, 18.61±16.89

Journal of Human Hypertension Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 330 Table 2 Multiple linear regression analyses of baseline blood pressure with KLK1 rs5517 and rs5516 polymorphisms

Variables rs5517 rs5516 N Mean±s.d. Unadjusted Adjusteda

b±s.e. P-value b±s.e. P-value

Baseline DBP GG 363 92.21±10.21 0.0 0.0 GA 521 92.14±9.86 À0.1±0.7 0.923 À0.4±0.6 0.530 AA 177 92.73±10.70 0.5±1.0 0.585 0.2±0.9 0.789 CC 665 91.87±10.44 0.0 0.0 CG 350 92.98±9.56 1.1±0.7 0.089 0.6±0.6 0.328 GG 46 92.48±9.37 0.6±1.4 0.668 0.7±1.3 0.575 Baseline SBP GG 363 166.32±16.41 0.0 0.0 GA 521 163.37±15.94 À2.9±1.1 0.008 À2.7±1.1 0.011 AA 177 166.41±17.34 0.1±1.6 0.954 0.5±1.5 0.752 CC 665 164.81±15.97 0.0 0.0 CG 350 164.42±17.20 À0.4±1.1 0.726 0.2±1.1 0.845 GG 46 169.48±15.85 4.7±2.4 0.052 5.0±2.3 0.033

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; KLK1, kallikrein 1; SBP, systolic blood pressure. aAdjusted for age, BMI, smoking and alcohol status, education levels, occupation, community and plasma concentration of irbesartan.

Table 3 Multiple linear regression analyses of blood pressure response on 28th day with KLK1 rs5517 and rs5516 polymorphisms

Variables rs5517 rs5516 N Mean±s.d. Unadjusted Adjusteda

b±s.e. P-value b±s.e. P-value

DBP responseb on 28th day GG 363 6.07±8.14 0.0 0.0 GA 521 6.51±8.20 0.4±0.6 0.423 0.3±0.5 0.515 AA 177 6.41±8.21 0.3±0.8 0.648 0.0±0.7 0.962 CC 665 6.17±8.07 0.0 0.0 CG 350 6.72±8.44 0.6±0.5 0.308 0.3±0.5 0.574 GG 46 6.04±7.77 À0.1±1.2 0.916 À0.4±1.1 0.703 SBP responseb on 28th day GG 363 17.88±16.82 0.0 0.0 GA 521 18.61±16.89 0.7±1.1 0.522 1.9±1.1 0.072 AA 177 20.16±18.90 2.3±1.7 0.173 1.9±1.5 0.217 CC 665 18.12±17.52 0.0 0.0 CG 350 18.95±16.23 0.8±1.1 0.455 1.0±1.0 0.348 GG 46 23.35±19.63 5.2±2.9 0.076 2.9±2.8 0.291

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; KLK1, kallikrein 1; SBP, systolic blood pressure. aAdjusted for baseline DBP, baseline SBP, age, BMI, smoking and alcohol status, education levels, occupation, community and plasma concentration of irbesartan. bBP response was defined as BP before treatment minus BP on the 28th day.

and 20.16±18.90 mm Hg, respectively. The mean ungenotyped variants of the KLK1 gene. Tables 4 DBP responses for rs5516 genotype subgroups with and 5 displayed the results of haplotype-specific CC, CG and GG were 6.17±8.07, 6.72±8.44 and (Haplo-score) and haplotype-global (Haplo-GLM) 6.04±7.77 mm Hg, respectively. The mean SBP analyses for the association between the SNP responses for rs5516 genotype subgroups with CC, rs5517–rs5516 haplotypes and baseline BPs and BP CG and GG were 18.12±17.52, 18.95±16.23 and responses. Only three haplotypes with a frequency 23.35±19.63 mm Hg, respectively. Compared with 40.01 (G-C, A-C and A-G) were observed. Haplo- individuals carried CC genotype of rs5516, indivi- type-based association tests were performed with duals with GG genotype had a slightly higher SBP adjustment for baseline BPs, age, sex, body mass response (b±s.e.: 5.2±2.9, P ¼ 0.076) without index, smoking status, alcohol consumption, plasma adjustment of covariates, whereas there were no concentration of irbesartan, education and occupation associations between rs5517 and BP responses, as with the global score test. In Table 4, no significant well as between rs5516 and DBP responses. associations were detected for SNP rs5517–rs5516 haplotype with baseline DBP (global score ¼ 1.36, df ¼ 2, P ¼ 0.51) and SBP (global score ¼ 2.30, df ¼ 2, Haplotype-based association test for KLK1 gene P ¼ 0.32). In the Haplo-special score analyses, we Previous study result19 supported that the optimal did not observe any haplotype (G-C, A-C and A-G) rs5516 and rs5517 set was selected as tSNPs that that was significantly associated with baseline DBP efficiently covered both common haplotype and (specific score: À0.43, À0.61 and 1.13 for P ¼ 0.67,

Journal of Human Hypertension Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 331 Table 4 Haplotype analyses of KLK1 gene with baseline BP in patients with hypertensiona

Haplotype Baseline DBP Baseline SBP

Haplotype score analysis rs5517 rs5516 Hap-Freq Specific score P-value Specific score P-value G C 0.59 À0.43 0.67 0.74 0.46 A C 0.20 À0.60 0.55 À1.50 0.13 A G 0.21 1.13 0.26 0.63 0.53 Global score P-value Global score P-value 1.36 0.51 2.30 0.32 Haplotype GLM analysis rs5517 rs5516 Hap-Freq b (s.e.) P-value b (s.e.) P-value G C 0.59 0 0 A C 0.20 À0.17 (0.57) 0.77 À1.28 (0.92) 0.17 A G 0.21 0.57 (0.57) 0.32 0.19 (0.93) 0.84

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; KLK1, kallikrein 1; SBP, systolic blood pressure. aBoth Haplo-score and Haplo-GLM analyses were adjusted for age, BMI, smoking and alcohol status, education levels, occupation, community and plasma concentration of irbesartan.

Table 5 Haplotype analyses of KLK1 gene with BP response to irbesartan in patients with hypertensiona

Haplotype DBP responseb SBP responseb

Haplotype score analysis rs5517 rs5516 Hap-Freq Specific score P-value Specific score P-value G C 0.59 À0.89 0.37 À1.56 0.12 A C 0.20 0.22 0.82 0.12 0.90 A G 0.21 0.85 0.40 1.75 0.08 Global score P-value Global score P-value 0.95 0.62 3.49 0.17 Haplotype GLM analysis rs5517 rs5516 Hap-Freq b (s.e.) P-value b (s.e.) P-value G C 0.59 0 0 A C 0.20 0.22 (0.46) 0.63 0.63 (0.96) 0.52 A G 0.21 0.44 (0.47) 0.34 1.81 (0.97) 0.06

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; KLK1, kallikrein 1; SBP, systolic blood pressure. Bold P-value is denoted as marginal significant level. aBoth Haplo-score and Haplo-GLM analyses were adjusted for baseline DBP, baseline SBP, age, BMI, smoking and alcohol status, education levels, occupation, community and plasma concentration of irbesartan. bBP response was defined as BP before treatment minus BP on the 28th day.

0.55 and 0.26, respectively) or with baseline SBP Discussion (specific score ¼ 0.74, À1.50 and 0.63 for P ¼ 0.46, 0.13 and 0.53, respectively). The Haplo-GLM analyses Our results suggest that the rs5516 polymorphism in showed that, compared with haplotype GC subjects, exon 3 of the KLK1 gene, and their reconstructed the subjects with haplotype AC and AG did not have haplotype AG, are associated with baseline SBP significantly greater baseline SBP and DBP. As shown and SBP reduction to antihypertensive treatment in Table 5, no marginally significant associations were with irbesartan. Consistent with earlier finding in detected for KLK1 haplotypes with DBP response Japanese,24 SNP rs5517 did not associate with either (global score ¼ 0.95, df ¼ 2, P ¼ 0.62) or SBP response baseline BPs or BP responses to irbesartan treatment (global score ¼ 3.49, df ¼ 2, P ¼ 0.17). In the Haplo- in Chinese hypertensives. special score analyses, we found that haplotype KLK1 belongs to a subgroup of serine proteinases AG was marginally associated with SBP response and processes low molecular weight kininogen sub- (specific score: 1.75 for P ¼ 0.08) but not with DBP strates to release vasoactive kinin peptides. Intact response. Moreover, haplotypes (GC and AC) were kinins bind to bradykinin B2 receptors, whereas kinin not associated with SBP response or DBP response. metabolites produced by enzymes such as kiniases I The Haplo-GLM analyses showed that, compared and II and neutral , bind to bradykinin with haplotype GC subjects, the subjects with haplo- B1 receptors. The binding of kinins to their respective type AG had a marginally greater SBP response receptors activates signalling pathways such as (adjusted b±s.e.: 1.81±0.97, P ¼ 0.06), but not DBP NO-cGMP and prostacyclin-cAMP, which trigger a response. The haplotype AC did not show signifi- broad spectrum of biological effects including en- cant associations with BP responses. dothelial function in arteries, vasodilation, smooth

Journal of Human Hypertension Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 332 muscle contraction and relaxation, inflammation and morphism and subjects with rs5516 GG genotype of pain. Previously reported evidences indicate that the the KLK1 gene may have decreased enzyme activity cardioprotective effects of ACE inhibitors in ischae- or in linkage disequilibrium with functional vari- mia-reperfusion injury are the result of the protection ants and subsequent increased BP level. of endogenous kinins from degradation rather than Recently, Slim et al.27 reported that there is an inhibition of Ang II formation.20,25 Genetically en- SNP Arg53-His in exon 3 of KLK1, and found gineered mice deficient in KLK1 ascertained the role association with significant decrease in urinary of KLK1 in the cardioprotection elicited by AT1R kallikrein activity. When arg53 is replaced by blockade in ischemia-reperfusion injury.21 Therefore, histidine, analysis of recombinant kallikrein var- it is likely that KLK1 may directly contribute to iants revealed a major decrease in enzyme activity. AT1R inhibition of irbesartan therapeutic efficacy. A model of kallikrein derived from crystallographic Our findings have found that the KLK1 rs5516 data suggest that arg53 is involved in substrate polymorphism and reconstructed haplotype AG binding, and individuals with R53H have on average marginally alter SBP response to irbesartan treatment. a 50–60% reduction in urinary kallikrein activity.30 The KLK1 rs5517 polymorphism is a G to A The HapMap project data available in Chinese transition, which replaces Glu (GAA) to Lys (AAA) population exhibit that there are no any frequencies at position 162. A large-scale case–control study19 of both rs5516 and rs5515 loci, which indicated it is identified that in a Chinese Han population likely that rs5516 is in strong linkage disequilibrium although the rs5517 polymorphism in the KLK1 with known rs5515 (Arg53His) functional variant. gene was significantly associated with essential YRI population data further support the rs5515 and hypertension, the two different studies consistently rs5516 are in complete LD (D 0 ¼ 1). found that the rs5516 was significantly associated A limitation of this study was that the SNPs in the with hypertension risk (P ¼ 0.0298)19 or higher promoter region were not included. In fact, several baseline SBP level in our hypertensive patients studies have provided evidence of an association of (P ¼ 0.033). Our present study reports that the regulatory polymorphisms of the KLK1 gene with frequencies of rs5517 GG, GA and AA in the hypertension and related disease.13–15 hypertensive group were 34.21, 49.11 and 16.68%, In conclusion, this study suggests that rs5516 in respectively, whereas the frequencies of CC, CG and the KLK1 gene may be involved in the development GG for rs5516 were 62.68, 32.99 and 4.33%, of essential hypertension and in the regulation of respectively; which were similar in distribution to SBP-lowering response to irbesartan in Chinese the hypertensive subgroups in the previous study.19 hypertensives. Functional investigations are needed Hong et al.26 showed that the KLKI Glu162Lys to elucidate their relationships. polymorphism functionally affects plasma creati- nine levels, and suggested that the genetic variant is a risk factor for a decline in renal plasma creatinine What is known about topic K Many lines of evidences identified that KLK1 is involved in clearance rate in hypertensive individuals. How- the progression of cardiovascular diseases, and further alter ever, we did not find the rs5517 polymorphism is ACE inhibition therapeutic response. associated with higher baseline BP levels. The K Previous study result supported that the optimal KLK1 different conclusions are largely explained by the rs5516 and rs5517 set is selected as tSNPs that efficiently two different study designs (a case–control study vs covered both common haplotype and ungenotyped variants of the KLK1 gene. a prospective cohort study). K A large-scale case–control study identified that in Chinese The SNP rs5516 is a C to G transition located in population, the rs5517 polymorphism in the KLK1 gene exon 3, which causes Gln to Glu substitution at was significantly associated with essential hypertension. position 121. Our results show that SNP rs5516 (Gln121Glu), but not SNP rs5517 (Glu162Lys), What this study adds K Our results suggest that the rs5516 polymorphism and their is significantly associated with baseline SBP in reconstructed haplotype A-G are associated with baseline Chinese hypertensives, which is different from the SBP and SBP reduction to antihypertensive treatment with results found in Caucasian populations.18,27 To date, irbesartan. Whereas SNP rs5517 did not associate with molecular evidence indicated that the locus at either baseline BPs or BP responses to irbesartan treatment in Chinese hypertensives. position 162 (rs5517) of the KLK1 gene is not in the catalytic triads (His41, Asp96 and Ser189) or any known essential binding pockets (S1, S2, S10,S20 and kallikrein loop) of the protein,28 and in vitro Conflict of interest synthesis of recombinant kallikrein variants of SNP The authors declare no conflict of interest. rs5516 (Q121E) shows it does not reduce enzyme activity.27 The result of an in vivo study does not support the hypothesis that the Q121E SNP in the Acknowledgements KLK gene affects either endothelium-dependent vasodilatation or endothelium-independent vaso- This study was supported by National Natural Science dilatation in humans.29 However, we speculate that Foundation of China (Grant No. 30700454), Anhui rs5516 is most likely a functional common poly- Medical University Biomedical Institute. We gratefully

Journal of Human Hypertension Effects of protein-coding polymorphisms in the KLK1 gene S Jiang et al 333 acknowledge the assistance and cooperation of the 15 Song Q, Chao J, Chao L. DNA polymorphisms in the faculty and staff of the Anhui Medical University 50-flanking region of the human tissue kallikrein gene. and thank all of the participants in our study, as well Hum Genet 1997; 99(6): 727–734. as Dr Zhong Guisheng (Cornell University) who helped 16 Yu H, Song Q, Freedman BI, Chao J, Chao L, Rich SS et al. edit this manuscript. This study was conducted in Association of the tissue kallikrein gene promoter with ESRD and hypertension. Kidney Int 2002; 61: 1030–1039. accordance with the current regulations of the People’s 17 Hua H, Zhou S, Liu Y, Wang Z, Wan C, Li H et al. Republic of China. Relationship between the regulatory region poly- morphism of human tissue kallikrein gene and essential hypertension. 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