Published online before print December 30, 2002, doi: 10.1161/01.HYP.0000049762.77830.89
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(Hypertension. 2003;41:308.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Haplotype Analysis of the Human Renin Gene and Essential Hypertension
From the Division of Receptor Biology (B.H., T.N., S.K.), Advanced Medical Research Center; the Second Department of Internal Medicine (B.H., Y.I., M.S.,Y.O.); and the Division of Genetic and Genomic Medicine (Y.M., S.A.), Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan.
Correspondence to Tomohiro Nakayama, MD, Division of Receptor Biology, Advanced Medical Research Center, Nihon University School of Medicine, Ooyaguchi-kamimachi, 30-1 Itabashi-ku, Tokyo 173-8610, Japan. E-mail tnakayam{at}med.nihon-u.ac.jp
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Abstract |
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The human renin gene is an attractive candidate for involvement in the underlying cause of essential hypertension (EH). Despite extensive examination, the relation between the renin gene and hypertension remains unclear. The aims of the present study were to discover new genetic markers of EH and to investigate the relations between polymorphisms of the renin gene and EH in the Japanese. Using the polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) method, we isolated 3 novel variants of the renin gene; a single nucleotide polymorphism (SNP) in intron 4 (T+17int4G), a variable number of tandem repeats (VNTR) polymorphism in intron 7, and a missense mutation in exon 9 (G1051A). We performed an association study with these polymorphisms in 212 patients with EH and 209 age-matched normotensive (NT) subjects. The frequency of genotypes VNTR and T+17int4G did not differ significantly between the 2 groups, whereas the overall distribution of G1051A was significantly different between EH and NT. Haplotype analysis revealed that the overall distribution of haplotypes differed significantly between the EH and NT groups. PRA levels in patients with EH with the G/G genotype were significantly higher than in subjects with EH with G/A and A/A genotypes. These data suggest that the missense mutation in exon 9 may affect the enzymatic function of renin and consequently may be involved in the etiology of hypertension.
Key Words: hypertension, essential • renin • polymorphism • haplotypes • genetics
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Introduction |
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High blood pressure or hypertension affects 25% of most adult populations and is an important risk factor for death from stroke, myocardial infarction, and congestive heart failure.1 The renin-angiotensin system plays a role in the pathophysiology of vascular diseases, and genes controlling this system may be involved in the development of hypertension.2 Renin is a protease that cleaves angiotensinogen to release the decapeptide angiotensin I, which in turn is cleaved by converting enzyme to angiotensin II, a potent vasoconstrictor and stimulator of aldosterone release.2 Renin, the rate-limiting factor in this cascade, plays a crucial role in the regulation of blood pressure, and renin gene may be a candidate gene for hypertension.3,4 However, there are a few reports of the association study between the human renin gene and essential hypertension (EH) comparing with genes of angiotensinogen and ACE and EH. Therefore we focused on isolating the useful genetic markers in the human renin gene and estimating the association between this gene and EH.
The human renin gene is located on 1q32, is 12.5 kb in length, and includes 10 exons and 9 introns.5,6 Although hypertensive animal studies indicating that the renin gene is cosegregated with blood pressure used the mouse gene in the transgenic rat and visa versa,7,8 irrefutable findings have not been obtained in human hypertension. Similarly, genetic analyses of the renin gene have been performed in animals9–13 and humans14–16; however, distinct genetic variants of the renin gene have yet to be isolated in human hypertension. According to the web site (http//www.ncbi.nlm.nih. gov/) of the National Center of Biotechnology Information (NCBI), the genetic variants of the human renin gene include 7 single nucleotide polymorphisms (SNPs) in exons 1, 2, 3, 9, and 10. Of these SNPs, only one in exon 9 (G1051A) yields an amino acid change (V351I). To date, no SNPs have been identified in exons 4, 5, 6, 7, and 8.
The aim of the present study was to screen for possible mutations and polymorphisms in exons 4, 5, 6, 7, and 8 and to assess the association between the genotypes or haplotypes of the renin gene and EH through a case-control study.
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Methods |
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Subjects
The study group consisted of 212 Japanese patients with EH, and a total of 209 Japanese normotensive (NT) healthy subjects were selected by the criteria as previously described.17 The subjects in both groups lived in the Kanto district in Japan and were recruited from the patients and healthy volunteers coming to the Nihon University Hospital located in Tokyo Japan. Informed consent was obtained from each individual, according to a protocol approved by the Human Studies Committee of Nihon University, Japan.
Biochemical Analysis
Plasma concentrations of total cholesterol and HDL cholesterol and serum concentrations of creatinine and uric acid were measured as previously described.18
Single-Strand Conformation Polymorphism
DNA was extracted from the peripheral blood leukocytes by using the standard protocols.19,20 Genomic segments corresponding to exons 4, 5, 6, 7, 8, and 9 from each EH and NT subject were amplified by polymerase chain reaction (PCR). Seven pairs of oligonucleotide primers were designed from the published sequence of the human renin gene at chromosome 1q32 (GeneBank accession number NT004662, Table 1). PCR buffer and Taq DNA polymerase were used the Long and Accurate PCR System (TaKaRa Shyuzo Co). The condition of PCR and electrophoresis were used as previously described.21
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Nucleotide Sequencing
The PCR products were sequenced directly with the use of an autosequencer (ABI model 310, Perkin-Elmer Biosystems).22
Genotyping
PCR–restriction fragment length polymorphism (RFLP) was performed for genotyping of T+17G in intron 4 (T+17int4G) with EcoT14I and G1051A in exon 9 (the nucleotide A of the ATG start codon is numbered 1) with Tth111I. The primers for T+17int4G were the same as used for single-strand conformation polymorphism (SSCP). The primers for G1051A were as follows: RENexon9F1 (5'-CCCACCCCAGTATGTCGTGAAGTG-3') and RENexon9R1 (5'-GGCCGACTCGAACCTCACCTGAAAGA-3'; underline shows artificial mutagenesis). Genotyping for a variable number of tandem repeats (VNTR) in intron 7 was performed as previously described23 with the primers RENint7F1 (5'-GAGGTGCCC0CATC- AGCCTTCTGTCT-3') and RENint7R1 (5'-CCACCC-ACAGCACCTTCCCTCTT -3').
Haplotype Analysis
On the basis of the data of the genotypes of the 3 genetic variations, the expectation/maximization (EM) algorithm24 was applied to estimate the frequency of the haplotype. The Arlequin version 2 was available from the web site (http://lgb.unige.ch/arlequin/).
Plasma Renin Activity
Twenty-four patients in the EH group who had the each genotype of G1051A in exon 9 were selected randomly. Eight patients were selected in each genotype. Blood samples were collected in patients before treatment with antihypertensive drugs. Plasma renin activity (PRA) was measured as previously described.25
Statistical Analysis
Data represent mean±SD. Hardy-Weinberg equilibrium was assessed by 
2 analysis. The overall distribution of alleles was analyzed by 2x2 contingency tables, and the distributions of the genotypes between patients with EH and NT subjects were tested with a 2-sided Fisher exact test and multiple logistic regression analysis. The statistical significance was established at the P<0.05 level. Differences in clinical data and PRA between the EH and NT groups were assessed by ANOVA followed by the Fisher protected least significant difference test. Statistical significance was established at the P<0.05 level. The probability value of each haplotype was applied to 0.05/number of haplotypes.26
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Results |
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The clinical characteristics of the patients with EH and NT subjects are shown in Table 2. The systolic blood pressure, diastolic blood pressure, pulse rate, body mass index (BMI), and plasma concentrations of total cholesterol were significantly higher in the EH group than in the NT group. No significant differences in age, serum concentrations of creatinine, and uric acid were observed between the 2 groups.
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Using SSCP, we discovered a new SNP 17 bp downstream of the boundary of exon 4 (T+17int4G) and a VNTR in intron 7 at 18 bp upstream of the boundary of exon 8. We could not find abnormally migrating bands in the regions of exon 5, 6, and 8 by SSCP; thus there may be no polymorphism or mutation in these regions. Nucleotide sequencing revealed that T+17int4G is a substitution from T to G and that the VNTR is a tandem repeat consisting of 4 the nucleotides TCTG. Because only the one missense mutation was listed in the NCBI information, we performed an association study by using these 3 genetic variants. The distributions of the genotypes of these 3 variants are displayed in Table 3. No significant difference in overall distribution of either T+17int4G or VNTR was observed between the EH and the NT groups. On the other hand, the distribution of G1051A did differ significantly between the EH and NT groups (2=21.1, df=2, P=0.0001). The frequency of the G allele was significantly higher than that of the A allele (2=8.1, df=1, P=0.0045). Multiple logistic linear regression analysis adjusted for age, gender, and BMI showed a significant difference in distributions of genotypes between the control and EH groups for the total study population (odds ratio=2.41; 95% CI, 1.93 to 3.01).
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The haplotype frequencies are shown in Table 4. Seventeen haplotypes were observed and consequently denoted H1 to H17. H15 and H16 were observed only in EH and H17 only in NT. The overall distribution of haplotypes was significantly different between the EH and NT groups. The H1 haplotype was more frequent in patients with EH than NT subjects (P=0.019), whereas the incidence of the H2 haplotype was significantly lower in the EH group (P=0.010).
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PRA Levels in Patients With EH
PRA levels in patients with EH are shown in the Figure. PRA level was significantly different in each group in patients with EH (P=0.012). PRA levels were significantly higher in patients with EH with the G/G genotype than in EH subjects with G/A and A/A genotype (P=0.004).
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Discussion |
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Animal model studies have clearly demonstrated that variations in the renin gene affect blood pressure and that the renin gene is directly involved in the development of hypertension.9–13 Some human studies have revealed positive associations between the human renin gene and EH by using case-control studies, family history of EH, and intermediate phenotypes. Frossard et al27–29 discovered that variations in the REN or nearby genes that may be in linkage disequilibrium with RFLPs of the renin gene could play a role in EH and stroke. On the other hand, many studies have shown no association between the renin gene and EH. Morris et al14 reported no relation between EH and a Hind III RFLP in the renin gene. Naftilan et al15 discovered no absolute linkage with hypertension in a large Utah pedigree. Jeunemaitre et al16 demonstrated no role for the renin gene in the pathogenesis of EH, using the sib-pair method of linkage analysis. These apparent discrepancies between reports are thought to be due to not only racial differences and different methods of sample selection but also the strategy of investigation in the noncoding region. For instance, of the reports showing positive association, the MboI RFLP was reported to be associated with EH in different races, including the Japanese.27,28,30,31 However, the MboI RFLP is located in intron 9, and as such is probably not a functional genetic variation. In the present study, our search for SNPs in the information of the NCBI revealed only one SNP, with an amino acid change in the human renin gene. Although the linkage between this SNP in exon 9 and the MboI RFLP is unknown, the SNP is thought to be more preferable as a genetic marker for case-control studies than the RFLP in the intron, as the SNP can influence the enzymatic activity of renin.
We discovered an SNP of T+17G in intron 4 and a VNTR in intron 7. No significant differences in the distributions of T+17int4G and VNTR were observed between the EH and NT groups, whereas the distribution of G1051A did differ significantly between the EH and NT groups. Haplotype analysis is thought to be a useful method for the identification of not only rare disease genes but also common disease genes and is frequently more powerful than the analysis using one polymorphism.32,33 We estimated haplotypes by using these 3 polymorphisms because this combination of polymorphisms has not been estimated by a case-control study between the EH and NT groups. In general, exact haplotypes are determined by continuous sequencing of a one chromosome or linkage analysis of a pedigree. Recently, methods for estimating the frequencies of haplotypes from genotypic data in unrelated individuals have been developed on the basis of the EM algorithm.24 The EM algorithm allows determination of haplotype frequencies and maximizes the probability of obtaining the observed genotypes.34 We applied the EM algorithm to the haplotype analysis in the present study, and succeeded in estimating an approximate value of haplotype frequency using the entire data of genotypes. Of the evaluated haplotypes, the H1 haplotype was more frequent in the EH group than the NT group, suggesting that it has a hypertensive effect, whereas the H2 haplotype was significantly less frequent in the EH subjects, suggesting a hypotensive effect.
In the present study, PRA level was measured, and the levels in patients with EH with the G/G genotype was significantly higher than that in EH subjects with G/A and A/A genotype. This finding suggests that the G/G genotype affects the enzymatic activity of renin. Hypertension of the EH patient with G/G genotype may be caused by increased renin activity.
In conclusion, this is the first study to demonstrate that a missense mutation in the coding region of the human renin gene is associated with EH. The amino acid change caused by this mutation may affect the functioning of this enzyme in the mediation of blood pressure.
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Acknowledgments |
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This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan (High-Tech Research Center, Nihon University), a research grant from the alumni association of Nihon University School of Medicine, and the Tanabe Biomedical Conference, Japan. We thank Ms Hideko Tobe for technical assistance.
Received July 3, 2002; first decision July 30, 2002; accepted November 19, 2002.
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