Mariko Banno

Genetic variations of regulator of G-protein signaling 2 in hypertensive patients and in the general population.

Objectives Mice deficient in the regulator of G-protein signaling 2 (RGS2) exhibit a strong hypertensive phenotype. We studied whether genetic variations in RGS2 are implicated in hypertension or other phenotypes in Japanese hypertensive individuals and the general population. Methods We sequenced all exons of RGS2 and the promoter region in 953 and 48 hypertensive individuals, respectively. Genotyping by the TaqMan polymerase chain reaction method was performed for six missense or frameshift mutations and common single nucleotide polymorphisms in the general population, with a sample size of 1872 individuals (862 men and 1011 women). Results We identified five novel missense mutations (Q2L; n = 2, Q2R; n = 1, M5V; n = 1, R44H; n = 2, Q78H; n = 1) and one novel frameshift mutation (1925–1926insT; n = 2) in a heterozygous state, in addition to 33 variations including five common single nucleotide polymorphisms. Six missense/frameshift mutations and three common single nucleotide polymorphisms (−638A > G, 1026T > A, 1891–1892delTC) were successfully genotyped in the general population. Mutations Q2L (n = 2), M5V (n = 1), and 1925–1926insT (n = 2) were only identified in hypertensive subjects. Six out of seven individuals with the R44H mutation, which occurs in the amphipathic α-helical domain of RGS2, had hypertension. The results showed a significant association of two common single nucleotide polymorphisms, 1026T > A [TT versus TA + AA: odds ratio (OR) 1.33; 95% confidence interval (CI) 1.02–1.74; P = 0.035] and 1891–1892delTC (I: insertion allele, D: deletion allele, II versus ID + DD: OR 1.47; 95% CI 1.09–1.97; P = 0.012), with hypertension in women by multivariate logistic regression analysis. Conclusion Our results suggest that genetic variations in RGS2 contribute partly to the hypertensive phenotype.

Identification of 108 SNPs in TSC, WNK1, and WNK4 and their association with hypertension in a Japanese general population

AbstractThe deletion of thiazide-sensitive Na-Cl cotransporter (TSC, SLC12A3) causes Gitelman′s syndrome characterized by low blood pressure, while deletions of the WNK1 (PRKWNK1) and WNK4 (PRKWNK4) genes cause familial hypertension known as pseudohypoaldosteronism type II. Recent studies have revealed that cell surface expression of TSC is regulated by WNK1 and WNK4. We hypothesized that molecular variations in TSC, WNK1, and WNK4 could lead to an increased morbidity of hypertension. We identified 52, 35, and 21 polymorphisms in Japanese hypertensives by sequencing the entire coding regions of TSC, WNK1 and WNK4, respectively. Twenty-one representative polymorphisms were genotyped in 1,818 Japanese individuals (771 subjects with hypertension and 1,047 controls) randomly sampled in Suita city. The results indicated that the systolic blood pressure in men with the CT+TT genotype in WNK4 C14717T was 3.1 mmHg higher than those with the CC genotype (p=0.042) after adjustment with confounding factors such as age, BMI, hyperlipidemia, diabetes mellitus, antihypertensive drug use, smoking, and drinking. Multivariate logistic regression analysis (with adjustment for the same parameters) in men revealed that the odds ratio for the presence of hypertension of the CT+TT genotype in C14717T to the CC genotype was 1.62 (p=0.010, 95% confidence interval, 1.12-2.33). Association of TSC and WNK1 with hypertension was not observed. In conclusion, our study suggests the possible involvement of WNK4 in essential hypertension in a Japanese general population.

Association of Sixty-One Non-Synonymous Polymorphisms in Forty-One Hypertension Candidate Genes with Blood Pressure Variation and Hypertension

We previously selected a group of hypertension candidate genes by a key word search using the OMIM database of NCBI and validated 525 coding single nucleotide polymorphisms (SNPs) in 179 hypertension candidate genes by DNA sequencing in a Japanese population. In the present study, we examined the association between 61 non-synonymous SNPs and blood pressure variations and hypertension. We used DNA samples taken from 1,880 subjects in the Suita study, a population-based study using randomly selected subjects. Analyses of covariance adjusting for age, body mass index, hyperlipidemia, diabetes, smoking, drinking, and antihypertensive medication revealed that 17 polymorphisms in 16 genes (APOB, CAST, CLCNKB, CTNS, GHR, GYS1, HF1, IKBKAP, KCNJ11, LIPC, LPL, P2RY2, PON2, SLC4A1, TRH, VWF) were significantly associated with blood pressure variations. Multivariate logistic regression analysis with adjustment for the same factors revealed that 11 polymorphisms in 11 genes (CAST, CTLA4, F5, GC, GHR, LIPC, PLA2G7, SLC4A1, SLCI8A1, TRH, VWF) showed significant associations with hypertension. Five polymorphisms in five genes, CAST (calpastatin), LIPC (hepatic lipase), SLC4A1 (band 3 anion transporter), TRH (thyrotropin-releasing hormone), and VWF (von Willebrand factor), were significantly associated with both blood pressure variation and hypertension. Thus, our study suggests that these five genes were susceptibility genes for essential hypertension in this Japanese population.

Hypertension susceptibility genes on chromosome 2p24-p25 in a general Japanese population.

Background Genome-wide scans from Italy and China suggest a hypertension-susceptible locus between D2S2278 (nucleotides 11 245 080–11 245 358) and D2S168 (nucleotides 11 467 214–11 467 422) on chromosome 2. Methods We performed a large association study of polymorphisms in this region with blood pressure modulation in a Japanese general population. Forty-seven polymorphisms in 14 genes between nucleotide 8 845 292 and nucleotide 11 946 689, which contains D2S2278 and D2S168, were genotyped in 1880 individuals, 796 of whom were hypertensive and 1084 normotensive. Results Multivariate logistic regression analysis with adjustment for age, body mass index, presence of hyperlipidemia, diabetes mellitus, and current smoking and drinking revealed that one single nucleotide polymorphism (SNP), IMS-JST126186, in HPCAL1 (hippocalcin-like 1) in women and two SNPs, IMS-JST149391 and IMS-JST149390, in GREB1 (gene regulated by estrogen in breast cancer 1) in men were significantly associated with both prevalence of hypertension and blood pressure levels. To examine the role of GREB1 in more detail, we identified 38 additional genetic variations in GREB1 by direct sequencing, and eight polymorphisms were genotyped. One SNP, 45718A>G, was significantly associated with hypertension and blood pressure level in men, and this SNP was in linkage disequilibrium with a SNP present at the 3′ splice site of intron 11. Conclusion Our study suggests that GREB1 and HPCAL1 are candidate hypertension-susceptibility genes in the Japanese general population and supports previous studies that also identified hypertension-related loci in this narrow region.

Association of genetic polymorphisms of ACADSB and COMT with human hypertension.

Objectives Genetically hypertensive rats provide an excellent model to investigate the genetic mechanisms of hypertension. We previously identified three differentially expressed genes, Acadsb (short/branched chain acyl-CoA dehydrogenase), Comt (catecholamine-O-methyltransferase), and Pnpo (pyridoxine 5′-phosphate oxidase), in hypertensive and normotensive rat kidneys as potential susceptibility genes for rat hypertension. We examined the association of human homologues of these genes with human hypertension. Methods We sequenced three genes using samples from 48 or 96 hypertensive patients, identified single nucleotide polymorphisms, and genotyped them in a population-based sample of 1818 Japanese individuals (771 hypertensive individuals and 1047 controls). Results After adjustments for age, body mass index, present illness (hyperlipidaemia, diabetes mellitus), and lifestyle (smoking, alcohol consumption), multivariate logistic regression analysis revealed that −512A>G in ACADSB was associated with hypertension in women (AA vs AG + GG: odds ratio = 0.70, 95% confidence interval = 0.53–0.94). This single nucleotide polymorphism was in tight linkage disequilibrium with −254G>A. Furthermore, −1187G>C in COMT was associated with hypertension in men (GG vs CG + CC: odds ratio = 0.69, 95% confidence interval = 0.52–0.93) and was in tight linkage disequilibrium with 186C>T. After adjustments described above, −512 A>G and −254G>A in ACADSB were associated with variations in systolic blood pressure. ACADSB was in tight linkage disequilibrium with MGC35392 across a distance of 18.3 kb. COMT was not in linkage disequilibrium with any adjacent genes. Analysis indicated that two haplotypes of COMT were significantly associated with hypertension in men. Conclusion Our study suggests the possible involvement of genetic polymorphisms in ACADSB and COMT in essential hypertension in the Japanese population.

Genetic Variations of HSD11B2 in Hypertensive Patients and in the General Population, Six Rare Missense/Frameshift Mutations

Mutations in the gene encoding 11β-hydroxysteroid dehydrogenase type 2, HSD11B2, cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, defective HSD11B2 enzyme activity results in overstimulation of the mineralocorticoid receptor (MR) by cortisol, causing sodium retention, hypokalemia, and salt-dependent hypertension. Here, we have studied whether genetic variations in HDS11B2 are implicated in essential hypertension in Japanese hypertensives and the general population. By sequencing the entire coding region and the promoter region of HDS11B2 in 953 Japanese hypertensives, we identified five missense mutations in 11 patients (L14F, n=5; R74H, n=1; R147H, n=3; T156I, n=1; R335H, n=1) and one novel frameshift mutation (4884Gdel, n=1) in a heterozygous state, in addition to 19 genetic variations. All genetic variations identified were rare, with minor allele frequencies less than 0.005. Four of 12 patients with the missense/frameshift mutations showed renal failure. Four missense mutations, L14F, R74H, R147H, and R335H, were successfully genotyped in the general population, with a sample size of 3,655 individuals (2,175 normotensives and 1,480 hypertensives). Mutations L14F, R74H, R147H, and R335H were identified in hypertensives (n=6, 8, 3, and 0, respectively) and normotensives (n=8, 12, 5, and 0, respectively) with a similar frequency, suggesting that these missense mutations may not strongly affect the etiology of essential hypertension. Since the allele frequency of all of the genetic variations identified in this study was rare, an association study was not conducted. Taken together, our results indicate that missense mutations in HSD11B2 do not substantially contribute to essential hypertension in Japanese.

A Novel Missense Mutation, F826Y, in the Mineralocorticoid Receptor Gene in Japanese Hypertensives: Its Implications for Clinical Phenotypes

A gain-of-function mutation resulting in the S810L amino acid substitution in the hormone-binding domain of the mineralocorticoid receptor (MR, locus symbol NR3C2) is responsible for early-onset hypertension that is exacerbated in pregnancy. The objective of this study was to test whether other types of missense mutations in the hormone-binding domain could be implicated in hypertension in Japanese. Here, we screened 942 Japanese patients with hypertension for the S810L mutation in exon 6 in the MR. We did not identify the S810L mutation in our hypertensive population, indicating that S810L does not play a major role in the etiology of essential hypertension in Japanese. However, we identified a novel missense mutation, F826Y, in three patients in a heterozygous state, in addition to four single nucleotide polymorphisms, including one synonymous mutation (L809L). The F826Y mutation is present in the MR hormone-binding domain and might affect the ligand affinity. The F826Y mutation was also identified in 13 individuals (5 hypertensives and 8 normotensives) in a Japanese general population (n=3,655). The allele frequency was 0.00178. The frequencies of the F826Y mutation in the hypertensive population (3/942) and in the hypertensive group (5/1,480) and the normotensive group (8/2,175) in the general population were not significantly different, suggesting that this mutation does not greatly affect hypertension. Although it is unclear at present whether or not the F826Y mutation makes a substantial contribution to the mineralocorticoid receptor activity, this missense mutation may contribute, to some extent, to clinical phenotypes through its effects on MR.

Association of intima-media thickening of carotid artery with genetic polymorphisms of the regulator of G-protein signaling 2 gene in patients with hypertension and in the general population

Regulator of G-protein signaling 2 (RGS2) is a key molecule in signal pathways of vasoactive peptides, such as angiotensin II and endothelin 1, and is believed to have an important role in the pathophysiology of atherosclerosis. We have previously reported that common polymorphisms of RGS2 are associated with hypertension in Japanese. In this study, we studied whether the three previously identified common polymorphisms of RGS2 (−638A>G, 1026T>A and 1891–1892delTC) could be implicated in carotid atherosclerosis in Japanese patients with hypertension (459 men and 382 woman) and in a Japanese general population (814 men and 956 woman). We assessed two criteria for carotid atherosclerosis: maximal intima-media thickness (M-IMT) and mean-IMT. When subjects with atherosclerotic lesions were defined as having mean-IMT≥1.0 mm, multivariate logistic regression analysis performed after adjusting for confounding factors showed a significant association of the three common polymorphisms, −638A>G (AA versus AG+GG: odds ratio (OR), 1.55; 95% confidence interval (CI), 1.105–2.185; P=0.0113 only for the general population), 1026T>A (TT versus TA+AA: OR, 1.42; 95% CI, 1.027–1.972; P=0.034 for hypertensive subjects and OR, 1.56; 95% CI, 1.129–2.151; P=0.0071 for the general population), and 1891–1892delTC (II versus ID+DD: OR, 1.44; 95% CI, 1.043–2.008; P=0.028 for hypertensive subjects, OR, 1.32; 95% CI 1.002–1.742; P=0.048 for the total general population and OR 1.59; 95% CI 1.155–2.207; P=0.0047 for the general population), with carotid atherosclerosis. When atherosclerosis was defined as M-IMT ⩾1.0 mm, the values of M-IMT were also significantly different between the three genotypes in the three common polymorphisms. Taken together, these data suggest that genetic polymorphisms in RGS2 are associated with intima-media thickening of carotid artery in humans.

Association of PLA2G7 polymorphisms with carotid atherosclerosis in hypertensive Japanese

Although the plasma platelet-activating factor-acetylhydrolase (pPAF-AH) gene (PLA2G7) polymorphisms are reportedly associated with atherosclerotic diseases, their effects in hypertensive patients have not been well examined. Thus, we genotyped V279F, a loss-of-function mutation commonly seen in the Japanese, and I198T and A379V commonly seen in Caucasians, and investigated the (1) ethnic differences in the frequencies and (2) association of these variants with prevalence of carotid plaque in 733 treated hypertensive Japanese patients. The distribution of V279F (V allele 75.1% and F allele 24.9%) in hypertensive patients was similar to that previously reported in the healthy Japanese; however, allele frequencies of I198T (I allele 71.7% and T allele 28.3%) and A379V (A allele 84.7% and V allele 15.3%) were markedly different from those reported in Caucasians. In addition, V279F and I198T showed a strong linkage disequilibrium (D′=1.0, r2=0.89). The phenotypes showed no difference among genotypes for each polymorphism except for the blood pressure level in I198T in women. Carotid plaque was significantly more prevalent in subjects with 279F and 198T than in those with the wild type among men but not women, whereas A379V did not affect it. In multivariate logistic regression analyses, 279F and 198T were detected as an independent risk factor even after adjustments for other atherosclerotic risk factors in men. Taken together, our data suggest an ethnic difference and the possible involvement of genetic polymorphisms of PLA2G7 in the prevalence of carotid atherosclerosis in the hypertensive Japanese, especially in men.

OR-39: Susceptible gene polymorphisms to the antihypertensive effect of thiazide diuretics

SUSCEPTIBLE GENE POLYMORPHISMS TO THE ANTIHYPERTENSIVE EFFECT OF THIAZIDE DIURETICS Tetsutaro Matayoshi, Kei Kamide, Shin Takiuchi, Masayoshi Yoshii, Yoshikazu Miwa, Yoichi Takami, Chihiro Tanaka, Mariko Banno, Takeshi Horio, Toshiyuki Miyata, Yuhei Kawano. Hypertension and Nephrology, National Cardiovascular Center, Suita, Osaka, Japan; Research Institute, National Cardiovascular Center, Suita, Osaka, Japan.