Association of CT Dinucleotide Repeat Polymorphism in the 5′-Flanking Region of the Guanylyl Cyclase (GC)-A Gene with Essential Hypertension in the Japanese

89 Hypertens Res Vol.31 (2008) No.1 p.89-96 Original Article Association of CT Dinucleotide Repeat Polymorphism in the 5′-Flanking Region of the Guanylyl Cyclase (GC)-A Gene with Essential Hypertension in the Japanese Satoru USAMI1), Ichiro KISHIMOTO2), Yoshihiko SAITO3), Masaki HARADA1), Koichiro KUWAHARA1), Yasuaki NAKAGAWA1), Michio NAKANISHI1), Shinji YASUNO1), Kenji KANGAWA2), and Kazuwa NAKAO1) Guanylyl cyclase (GC)-A (natriuretic peptide receptor [NPR]-1), the receptor for atrial and brain natriuretic peptide, is important in the regulation of blood pressure in animal models and, possibly, in humans. In this study, we examined the association between dinucleotide repeat polymorphism within the 5′-flanking region of the GC-A gene and essential hypertension in a group of Japanese subjects. By genotyping 177 hypertensive and 170 normotensive subjects, we identified 5 allele types with 6, 9, 10, 11 and 12 CT dinucleotide repeats, respectively, around position – 293, upstream of the ATG codon in the human GC-A gene. The frequency of the (CT)n = 6 allele was significantly higher among hypertensive than normotensive subjects, while the frequencies of the other allele types did not differ between the two groups. We also examined the linkage between G/A polymorphism at position – 77 (rs13306004), downstream of the (CT)n polymorphism, and found that the (CT)n = 6 allele was tightly linked to an A at position – 77, while all other (CT)n alleles were linked to G. Promoter-reporter analyses carried out in cultured human aortic smooth muscle cells using a luciferase gene fused to the 5′-flanking region of the GC-A gene revealed that the promoter containing (CT)n = 6 drove less transcriptional activity than that containing (CT)n = 10. Finally, site-directed mutation showed that the (CT)n and G/A polymorphisms act synergistically to affect GC-A promoter activity. Our results thus define the (CT)n polymorphism in the 5′-flanking region of the GC-A gene as a potent and novel susceptibility marker for hypertension. (Hypertens Res 2008; 31: 89–96) Key Words: guanylyl cyclase (GC)-A, polymorphism, hypertension, transcriptional activity Introduction Atrial and brain natriuretic peptide (ANP and BNP, respectively) bind to and activate guanylyl cyclase (GC)-A (also termed natriuretic peptide receptor [NPR]-1) (1–6), thereby inducing relaxation of the vasculature and inhibition of vascular smooth muscle cell proliferation through the action of the second messenger cGMP (7). The important physiological role played by the natriuretic peptide/GC-A system in the regulation of arterial blood pressure (BP) and blood volume is now well documented in studies carried out in a variety of From the 1)Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2)Department of Biochemistry, National Cardiovascular Center, Research Institute, Suita, Japan; and 3)First Department of Internal Medicine, Nara Medical University, Nara, Japan. This work was supported by research grants from the Japanese Ministry of Education, Science and Culture and the Japanese Ministry of Health and Welfare. Address for Reprints: Ichiro Kishimoto, M.D., Ph.D., Department of Biochemistry, National Cardiovascular Center, Research Institute, 5–7–1 Fujishirodai, Suita 565–8565, Japan. E-mail: Received March 29, 2007; Accepted in revised form August 13, 2007. 90 Hypertens Res Vol. 31, No. 1 (2008) Table 1. Characteristics of the Normotensive and Hypertensive Groups Subject (n) Gender (male/female) Age (years) SBP (mmHg) DBP (mmHg) HR (beat/min) BMI (kg/m2) T-cho (mg/dL) HDL-cho (mg/dL) Cre (mg/dL) UA (mg/dL) FBS (mg/dL) PRA (ng/dL) PAC (ng/dL) ANP (pg/mL) BNP (pg/mL) UCG IVST (mm) PWT (mm) Normotensive Hypertensive p 170 113/57 54.9± 14.1 125.2± 10.9 74.4± 9.5 70.6± 12.3 23.1± 2.6 198± 36.6 47± 17 0.96± 0.3 5.3± 1.4 123.9± 42.0 1.02± 0.98 61.1± 39.4 28.8± 22.4 20.9± 35.0 177 91/86 57.3± 14.1 161.1± 18.8 92.9± 13.7 72.9± 13.1 24.1± 3.5 202.2± 34.9 49± 17 0.96± 0.99 6.0± 1.3 114± 31.3 0.99± 1.44 67.0± 42.5 38.3± 32.9 39.4± 32.9 <0.05 n.s. <0.001 <0.001 n.s. 0.01 n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.006 0.002 9.86± 1.37 9.88± 1.38 10.7± 2.25 10.7± 2.27 0.001 0.001 SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; BMI, body mass index; T-cho, serum total cholesterol; HDL-cho, serum high-density lipoprotein cholesterol; Cre, serum creatinine; UA, uric acid; FBS, fasting blood glucose; PRA, plasma renin activity; PAC, plasma aldosterone concentration; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; UCG, ultrasonic echocardiography; IVST, interventricular septal thickness; PWT, posterior wall thickness; n.s., not significant. Data are means±SD. genetically engineered mouse models. For instance, targeted deletion of GC-A leads to chronic hypertension (8–10), whereas its overexpression leads to a “dose-dependent” fall in BP (11). Although hypertension is a multifactorial disease controlled by multiple genes and environmental factors, these results demonstrating GC-A’s critical role in the regulation of BP suggest that variations in GC-A gene expression could contribute significantly to the pathogenesis of essential hypertension in humans. The GC-A gene is located on chromosome 1q21-22 in humans and is comprised of 22 exons spanning 16 kb (12). Recently, several polymorphisms were identified within the 5′-flanking region of the GC-A gene. Nakayama et al. identified an insertion/deletion polymorphism (8 bp deletion) at position −60 and a (CT)n dinucleotide repeat polymorphism at position −293 of the gene in a Japanese population (13, 14). In addition, Knowles et al. also identified the (CT)n dinucleotide repeat site as well as nine other polymorphic sites in the noncoding region of the gene (15). These polymorphisms in the 5′-flanking region and other noncoding regions could affect the transcriptional activity of the GC-A gene and are thus potentially involved in the pathogenesis of essential hypertension. Indeed, Nakayama et al. showed that there is an association between the insertion/deletion polymorphism at position −60 and essential hypertension (13). With respect to variations in the (CT)n dinucleotide repeat at position −293, Nakayama et al. reported 3 allele types containing 10, 11 and 12 CT repeats, respectively, and found no association between these variations and essential hypertension (14). On the other hand, Knowles et al. reported alleles with 6, 10 or 11 CT repeats at the same position, but did not test for an association between this variation and any diseases. Thus, the association between the (CT)n= 6 allele at position −293 of the GC-A gene and essential hypertension has not yet been evaluated. In the present study, therefore, we examined the association between (CT)n repeat polymorphism at position −293 of the GC-A gene and essential hypertension in our Japanese subjects. We identified 5 allele types havin (...truncated)