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(Hypertension. 2001;38:9.)
© 2001 American Heart Association, Inc.

Rapid Communications

Microsatellite DNA Polymorphism of Human Adrenomedullin Gene in Normotensive Subjects and Patients With Essential Hypertension

Toshihiko Ishimitsu; Kazuyoshi Hosoya; Kohju Tsukada; Junichi Minami; Yasuo Futoh; Hidehiko Ono; Masami Ohrui; Jun Hino; Kenji Kangawa; Hiroaki Matsuoka

From the Department of Hypertension and Cardiorenal Medicine (T.I., K.H., K.T., J.M., Y.F., H.O., H.M.), and Department of Health Care (M.O.), Dokkyo University School of Medicine, Mibu, Tochigi, Japan; and the Department of Biochemistry, National Cardiovascular Center Research Institute (J.H., K.K.), Suita, Osaka, Japan.

Correspondence to Toshihiko Ishimitsu, MD, Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. E-mail isimitu{at}dokkyomed.ac.jp

Abstract

Abstract—— Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues. We have cloned and sequenced the genomic DNA encoding the human AM gene and have determined that the gene is located in the short arm of chromosome 11. The 3'-end of the gene is flanked by the microsatellite marker of cytosine adenine (CA) repeats. In this study, we investigated the association between DNA variations in AM gene and the predisposition to hypertension. Genomic DNA was obtained from 272 healthy normotensive subjects (NT) age 57±5 years and 266 patients with essential hypertension (EH) age 53±11 years. The DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined by poly-acrylamide gel electrophoresis. The averaged blood pressure was 117±13/73±9 mm Hg in NT and 170±23/104±12 mm Hg in EH. In Japanese, there existed 4 types of alleles with different CA-repeat numbers: 11, 13, 14, and 19. The frequencies of these alleles were significantly different between NT and EH (²=9.43, P=0.024). Namely, 13.5% of EH carried the 19-repeat allele, whereas the frequency was 6.2% in NT (²=7.62, P=0.007). In NT, plasma AM concentrations were not significantly different between the genotypes. In conclusion, microsatellite DNA polymorphism of AM gene may be associated with the genetic predisposition to EH, although the gene expression is not likely to be affected by the genotypes.

Key Words: adrenomedullin • polymorphism • hypertension, essential • microsatellite repeats • genetics

Adrenomedullin (AM) is a hypotensive peptide produced in cardiovascular tissues such as the heart, lung, kidney, and vascular wall.^1,2 Besides the potent vasodilator action, AM has also been shown to cause natriuresis in the kidney and to inhibit growth of cardiovascular cells. Moreover, a significant level of AM has been identified in human plasma, and AM is supposed to be a circulating hormone.^1,3 We have previously reported that plasma AM levels are increased in patients with cardiovascular diseases such as hypertension, renal failure, and heart failure.^4,5 These findings suggest that AM has implications in pathophysiology of the cardiovascular system.

We have cloned and sequenced the genomic DNA-encoding human AM gene and have determined that the gene is located in the short arm of chromosome 11.⁶ Nucleotide sequencing of genomic DNA adjacent to the AM gene revealed that the 3'-end of the gene is flanked by the microsatellite marker with a variable number of cytosine adenine (CA)-repeats. This microsatellite marker is located approximately 4 kb downstream of the AM gene. Considering the possible implications of AM in the cardiovascular system, it seems of interest to elucidate whether this gene variation has any relation to the etiology of cardiovascular diseases. In the present study, we investigated the relationship between the microsatellite polymorphism adjacent to the AM gene and genetic predisposition to essential hypertension.

Subjects and Methods

Study Subjects
A group of 266 case patients with essential hypertension (EH) (166 men and 100 women), who had hypertension before the age of 50 years was selected from the outpatient clinic of Dokkyo University School of Medicine Hospital. Hypertension was defined as systolic blood pressure (SBP) >140 mm Hg and/or diastolic blood pressure (DBP) >90 mm Hg on multiple visits. Secondary causes of hypertension were denied through a comprehensive checkup. The normotensive control group (NT) included 272 healthy subjects (175 men and 97 women) age 50 years. They were recruited from participants of the health-check program of Dokkyo University School of Medicine Hospital. All the control subjects had SBP <140 mm Hg and DBP <90 mm Hg on multiple occasions. Blood pressure was measured by a sphygmomanometer in a sitting position after a 10-minute rest. All subjects were Japanese and unrelated each other.

The study protocol was approved by the institutional review board, and informed consent was obtained from each subject.

Genotype Analysis
Genomic DNA was extracted from peripheral leukocytes using Easy DNA kit (Invitrogen). The microsatellite region containing the CA-repeats 4 kb downstream of the AM gene was amplified by PCR. The PCR was performed with 0.5 µg of genomic DNA and 25 pmol of each primer (sense, 5'-AAGAGGCTGAGTCAGAAGGATTGG-3'; antisense, 5'-GCAACATCATTTTAATATCCTGCACAG-3') in a final volume of 50 µL containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 10 µg/mL gelatin, 0.2 mmol/L of each dNTP, 1 U of Taq DNA polymerase (Perkin-Elmer). The 5'-end of sense primer was labeled with the blue fluorescent dye 6-FAM. The DNA was amplified for 30 cycles with denaturation at 94°C for 45 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes. Then, an aliquot of the PCR product was mixed with the internal DNA size standard labeled with the red fluorescent dye ROX. The mixture was electrophoresed on 6% urea-polyacrylamide gel using an ABI 373 DNA sequencer (Perkin-Elmer). The length of PCR product was calculated from the calibration curve of internal standard using GENESCAN 672 software (Perkin-Elmer).

Measurement of Plasma AM
Antecubital venous blood was collected into ice-chilled tube containing EDTA (1 mg/mL) and aprotinin (500 U/mL) in the morning after the subjects had fasted overnight and had 20 minutes of supine rest. Plasma was separated by centrifugation at 4°C and stored at -80°C until assayed. Plasma AM concentration was measured by immunoradiometric assay using an AM-RIA Shionogi kit (Shionogi & Co), which detects both amidated mature AM and glycine-extended AM.⁷

Statistical Analysis
Data are presented as mean±SE. Clinical characteristics between the 2 groups were compared by unpaired Student’s t test for parametric data and by ² test for categoric data. The allele and genotype frequencies in the 2 groups were compared by ² test. Differences in plasma AM among genotypes were analyzed by 1-way ANOVA followed by Dunnett’s multiple range test. A P value <0.05 was considered statistically significant.

An expanded Methods section can be found in an online data supplement available at http://www.hypertensionaha.org.

Results

Clinical Characteristics of Study Subjects
Table 1 lists the physical and laboratory findings of the study subjects. EH were slightly younger and had a higher body mass index compared with that of NT, whereas the gender ratio was comparable between the 2 groups. As a matter of course, SBP and DBP was far higher in EH than in NT. In laboratory findings, EH showed higher fasting blood glucose, higher serum creatinine, and higher serum uric acid compared with those of NT. With regard to the serum lipids, triglycerides were higher and HDL cholesterol was slightly lower in EH than in NT, although total cholesterol did not differ between EH and NT.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of the NT and EH patients

Genotype and Allele Frequencies
In the Japanese population in the microsatellite region adjacent to the AM gene, there were 4 types of alleles with different CA-repeat numbers: 11, 13, 14, and 19. The alleles with 11-, 13-, 14-, and 19-repeats yield DNA fragment lengths of 248, 252, 254, and 264 bp, respectively. Table 2 shows observed frequencies of the alleles and the genotypes of this microsatellite gene polymorphism in the study subjects. The frequencies of the 11-, 13- and 14-repeat alleles were approximately 30%, whereas the frequency of the 19-repeat allele was <10% in either NT or EH. Combinations of 4 alleles yield 10 possible genotypes for this gene polymorphism. The genotype frequencies were not significantly deviated from those expected from the Hardy-Weinberg’s equilibrium in either NT or EH. The allele frequencies were significantly different between NT and EH (²=9.43, P=0.024). Namely, 13.5% of EH patients carried the 19-repeat allele, whereas the frequency was 6.2% in NT (²=7.62, P=0.007).

View this table: [in this window] [in a new window]   Table 2. Genotype and Allele Frequencies of Microsatellite Repeat Adjacent to AM Gene in NT and EH groups

Plasma AM Concentration
To clarify the influence of allele types on plasma AM levels, plasma AM was measured in homozygotes of each allele type in NT. However, because there were no homozygotes of 19-repeat allele, plasma AM was also measured in heterozygotes that included this allele. Plasma concentrations of AM in 21 homozygotes of 11-repeat allele, 27 homozygotes of 13-repeat allele, 30 homozygotes of 14-repeat allele, and 17 heterozygotes carrying 19-repeat allele were 7.5±1.0, 7.0±1.3, 7.2±1.3, and 7.3±1.7 pmol/L, respectively, and the values were not significantly different between the genotypes.

Discussion

In the present study, we examined the microsatellite DNA polymorphism lying 3'-downstream of the AM gene in NT and EH. Among the 4 types of alleles with CA-repeat numbers of 11, 13, 14, and 19, the frequency of 19-repeat allele was higher in EH than in NT. The frequency of EH patients carrying the 19-repeat allele was more than twice that of NT subjects. This result suggests the association of microsatellite DNA polymorphism adjacent to the AM gene with the genetic predisposition to hypertension. Namely, the existence of 19-CA-repeat allele is supposed to be associated with the risk of developing hypertension. However, because the frequency of this 19-repeat allele was small, this gene variation alone is not thought to reflect the large part of genetic predisposition to essential hypertension. So far, a number of gene polymorphisms have been indicated to have relation with the predisposition to hypertension. Most of them are related to the genes of cardiovascular hormones and their signal transduction systems, such as angiotensinogen,⁸ ß₂-adrenergic receptor,⁹ and endothelial NO synthase.¹⁰ As proposed by Page’s mosaic theory,¹¹ multiple genes are supposed to contribute to the development of hypertension. And together with these gene variations, the DNA polymorphism examined in this study may participate, to some degree, in the genetic predisposition to essential hypertension.

Some gene polymorphisms of cardiovascular hormones have been shown to affect expression of the genes or activity of the gene products. For instance, the serum ACE activity is known to be increased in individuals carrying the deletion allele of the gene,¹² and it has been reported that the methionine-to-threonine substitution at amino acid residue 235 (M235T) of angiotensinogen is associated with an increase in the gene expression.⁸ Because the microsatellite polymorphism examined in this study is located 3' downstream of the AM gene, it is unlikely that the gene transcription is affected by this polymorphism. Indeed, the plasma AM levels were not significantly different among the genotypes of this polymorphism. Therefore, the association of 19-CA-repeat allele with hypertension is not likely mediated by the gene expression and the action of AM itself. It may be possible that this microsatellite polymorphism is associated with the expression of other genes. Near the location of AM gene in the short arm of chromosome 11, there exist such genes as sphingomyelinase, parathyroid hormone and lactate dehydrogenase.^13–15 With regard to the parathyroid hormone gene, it is known that a high serum level of parathyroid hormone is associated with hypertension.¹⁶ In this context, it may be worth examining the relation of the current microsatellite polymorphism with parathyroid hormone and calcium metabolism.

Microsatellite markers, like the one examined in this study, consist of variable number of repeats of short nucleotides, and more than hundreds of such repeat markers are scattered throughout the genomic DNA. These microsatellite markers can be used to locate the genomic region responsible for hereditary diseases or traits. Until now, several diseases have been shown to be associated with such microsatellite DNA polymorphism. For instance, it has been reported that the CA-repeat polymorphism lying upstream of aldose reductase gene affects the development of nephropathy and retinopathy in type 1 diabetes mellitus.¹⁷ With regard to essential hypertension, a certain number of TCAT-repeat in the intron 1 of tyrosine hydroxylase gene has been shown to have positive association with essential hypertension.¹⁸ The intron 2 of type B natriuretic peptide receptor gene was also found to contain the GT microsatellite–repeat associated with essential hypertension.¹⁸ Including these and our results, accumulation of information about association of essential hypertension with various microsatellite markers may serve to understand the whole figure of the genetic predisposition to essential hypertension which is attributed to multiple genomic regions.

The results of this study revealed the association of the microsatellite CA-repeat polymorphism adjacent to the AM gene with genetic predisposition to essential hypertension in Japanese population. The frequency of 19-repeat allele was significantly increased in patients with essential hypertension. Although this DNA polymorphism is not likely to affect the AM gene transcription, the existence of 19-CA-repeat allele is supposed to reflect a certain part of the genetic predisposition to essential hypertension

Acknowledgments

The authors thank Ms Yasuko Kawamura and Dr Kazumi Akimoto for technological assistance in executing the study. This study was supported in part by the grant-in-aid for scientific research (10218209) from the Ministry of Education, Science and Culture of Japan.

Received April 16, 2001; first decision April 23, 2001; accepted May 8, 2001.

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