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The Purinergic Receptor P2Y, G-Protein Coupled, 2 (P2RY2) Gene Associated with Essential Hypertension in Japanese Men

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The Purinergic Receptor P2Y, G-Protein Coupled, 2 (P2RY2) Gene Associated with Essential Hypertension in Japanese Men Journal of Human Hypertension (2010) 24, 327–335 & 2010 Macmillan Publishers Limited All rights reserved 0950-9240/10 $32.00 www.nature.com/jhh ORIGINAL ARTICLE The purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) gene associated with essential hypertension in Japanese men Z Wang1,2, T Nakayama1,3, N Sato1, Y Izumi3, Y Kasamaki4, M Ohta4, M Soma5, N Aoi1, Y Ozawa4 andYMa2 1Division of Laboratory Medicine, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan; 2Department of Cardiovascular Medicine, the First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, PR China; 3Division of Nephrology, Hypertension and Endocrinology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan; 4Division of Cardiovascular Medicine, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan and 5Division of General Medicine, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan P2RY2 has an important function in the regulation of respectively). Logistic regression showed that for blood pressure by activating adenosine triphosphate the total and men groups, the TG þ TT genotype of (ATP). The aim of this study was to investigate the asso- rs4944831 was more prevalent in EH patients than in the ciation between the human P2RY2 gene and essential controls (P ¼ 0.026 and 0.011, respectively). For men, the hypertension (EH) through a haplotype-based case– overall distribution of the haplotype (SNP2-SNP4-SNP5) control study that included two gender groups. The 273 was significantly different between the EH patients EH patients and 255 age-matched controls were geno- and the controls (P ¼ 0.006). As compared with controls, typed for five single-nucleotide polymorphisms (SNPs) the frequency of the T-A-G haplotype was significantly of the human P2RY2 gene (rs4944831, rs1783596, higher, whereas the T-C-G haplotype was significan- rs4944832, rs4382936 and rs10898909). Data were ana- tly lower for the EH patients (P ¼ 0.001 and 0.014, respec- lysed for men and women separately and then as a com- tively). In conclusion, the present results indicate bined total group. For the total and the men only groups, that rs4944831 and the T-A-G haplotype of the human the genotype distribution of the T allele of rs4944831 and P2RY2 gene might be genetic markers for EH in the recessive model (GG vs TG þ TT) of rs4944831 Japanese men. differed significantly between the EH patients and Journal of Human Hypertension (2010) 24, 327–335; controls (P ¼ 0.028 and 0.019; P ¼ 0.009 and 0.008, doi:10.1038/jhh.2009.67; published online 27 August 2009 Keywords: P2RY2; single-nucleotide polymorphism; haplotype; case–control study; adenosine triphosphate Introduction considerable help in understanding the genetics of hypertension.2 Development of extensive collec- Essential hypertension (EH) is most likely a polyge- tions of single-nucleotide polymorphisms (SNPs) nic disorder that results from the inheritance of a raises the possibility that these SNPs can be used as number of susceptibility genes and involves envir- markers in genome-wide association mapping stu- onmental, demographic, vascular and neuroendo- 1 dies to identify hypertension susceptibility loci. crine factors. More than 50 genes have been Purinergic receptors have been classified as P1 examined in association studies with hypertension, and P2 receptors. At least eight P2 receptor subtypes with this number continuing to grow. A number of (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 polymorphisms in the candidate genes have been and P2Y14) have been identified3 in pharmacologi- associated with differences in blood pressure (BP) cal and molecular cloning studies. The P2Y2 level in certain populations and have been of receptor, which is also referred to as P2RY2 (official name: purinergic receptor P2Y, G-protein coupled, Correspondence: Professor T Nakayama, Division of Laboratory 2), belongs to the family of G-protein coupled Medicine, Department of Pathology and Microbiology, Nihon receptors. G-proteins mediate the intracellular ef- University School of Medicine, 30-1 Ooyaguchi-kamimachi, fects of many vasoactive and proliferative stimuli. Itabashi-ku, Tokyo 173 8610, Japan. E-mail: [email protected] Recently, G-protein signalling was found to be Received 21 February 2009; revised 11 May 2009; accepted 28 enhanced in cultured cells of various hypertensive May 2009; published online 27 August 2009 subjects.4 Thus, the purinergic system may be Haplotype of P2RY2 and hypertension Z Wang et al 328 similar in importance as the sympathetic and renin– and a DBP o85 mm Hg. Informed consent was angiotensin–aldosterone (RAA) systems are for obtained from each subject in accordance with the cardiovascular regulation and pathophysiology. protocol approved by the Human Studies Committee P2RY2 is expressed in different regions of the of Nihon University.15 kidney, which includes the vasculature and the glomerulus, in addition to the tubular epithelia and collecting duct system.5 The epithelial sodium channel (ENaC) in the renal collecting duct is Genotyping critically involved in the regulation of renal Na þ The human P2RY2 gene is located at chromosome reabsorption and K þ excretion, as well as in the 11q13.5-q14.1, spans approximately 18.1 kilobase genetic forms of arterial hypertension.6 Lehrmann pairs and contains three exons. A total of 104 SNPs et al.7 found that P2PY2 might mediate the adeno- are listed for the human P2RY2 gene in the National sine triphosphate (ATP) responses involved in the Center for Biotechnology Information SNP database inhibition of the ENaC-mediated Na þ reabsorption. Build 129 (http://www.ncbi.nlm.nih.gov/SNP). We Furthermore, a variety of experimental approaches screened the data for Tag SNPs on the International have provided evidence that local ATP release and HapMap Project website (http://www.hapmap.org/ 2X P2RY2 stimulation may inhibit water reabsorption index.html.ja) using a cutoff level of r 0.5. For the in the collecting duct.8 minor allele frequencies, we used a cutoff level of Previous studies in mice have identified impor- p0.2. SNPs with relatively high minor allele tant functions for P2RY2 in a variety of processes, frequencies have been shown to be very useful as including nucleotide-regulated Ca2 þ signalling in genetic markers for genetic case–control studies. lung fibroblasts and airway epithelial cells,9 nucleo- According to the above criterion, we selected tide-stimulated ClÀ secretion in the trachea and rs4944831 (C-12034890-10, registration number by gallbladder,10 neuronal growth,11 stimulation of K þ Applied Biosystems Inc., Foster City, CA, USA), secretion in the colon12 and neutrophil chemo- rs1783596 (C-8893575-10), rs4944832 (C-27987464- taxis.13 In 2007, Rieg et al.14 reported that salt- 10), rs4382936 (C-1830488-20) and rs10898909 (C- resistant hypertension occurs in the P2Y2 receptor 1830487-10) for this gene (Figure 1). SNP1 was located (mice homologue of the human P2RY2 gene) knock- in intron 1, whereas SNP2 was located in the coding regions of exon 3 of this gene. SNP3, SNP4 and SNP5 out mice because the baroreceptor response to 0 variations in salt intake remains intact. Further- were located in the 3 -flanking region of the gene. more, the study showed that the P2Y2 receptor gene Blood samples were collected from all partici- has an important function in the regulation of both pants, and genomic DNA was extracted from the þ peripheral blood leucocytes by phenol and chloro- the BP and the renal reabsorption of Na and fluid 16 in the animal model. form extraction. To the best of our knowledge, there have been no Genotyping was performed using the TaqMan SNP previous studies on the association between the Genotyping Assay (Applied Biosystems Inc.). The TaqMan SNP Genotyping Assays were performed human P2RY2 gene and EH. Therefore, the aim of 17 0 this study was to investigate the association between using the method of Taq amplification. In the 5 the human P2RY2 gene and EH in Japanese subjects nuclease assay, discrimination occurs during the through a haplotype-based case–control study that polymerase chain reaction (PCR) because of allele- used SNPs. specific fluorogenic probes that, when hybridized to the template, are cleaved by the 50 nuclease activity Materials and methods SNP1 SNP3 SNP4 rs4944831 rs4944832 rs4382936 Subjects C-12034890-10 C-27987464-10 C-1830488-20 Subjects diagnosed with EH were recruited at Nihon University Itabashi Hospital and other neighbouring SNP2 SNP5 hospitals in Tokyo from 1993 to 2008. We enrolled rs1783596 rs10898909 273 EH patients in this study, with a male/female C-8893575-10 C-1830487-10 (m/f) ratio of 1.97. EH was diagnosed based on the 5′ 3′ following criteria: seated systolic BP (SBP)4 160 mm Hg or diastolic BP (DBP)4100 mm Hg on three occasions within 2 months after the first BP exon 1 exon 2 exon 3 reading. None of the EH patients were receiving antihypertensive medication. Patients diagnosed –20 –10 0 10 kbp with secondary hypertension were excluded. A total of 255 normotensive age-matched healthy indivi- Figure 1 Structure of the human P2RY2 gene. The gene consists of three exons (boxes) separated by two introns. The lines show duals (m/f ratio ¼ 1.66) were enrolled as controls. introns and intergenic regions. The filled box shows the coding None of the controls had a family history of region. The arrows indicate the locations of single-nucleotide hypertension, and all had an SBP o130 mm Hg polymorphisms (SNPs). kbp, kilo-base pairs. Journal of Human Hypertension Haplotype of P2RY2 and hypertension Z Wang et al 329 of the Taq polymerase. The probes contain a 30 Fisher’s exact test. Hardy–Weinberg equilibrium minor groove-binding group that hybridizes to was assessed by w2 analysis. Differences in distribu- single-stranded targets with greater sequence speci- tions of genotypes and alleles between EH patients ficity than ordinary DNA probes.
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