Structure of the Type B Human Natriuretic Peptide Receptor Gene and Association of a Novel Microsatellite Polymorphism With Essential Hypertension
Abstract—The natriuretic peptide (NP) system may play a crucial role in development of essential hypertension (EH). C-type NP dilates arteries and lowers blood pressure and inhibits proliferation of vascular smooth muscle cells via the type B NP receptor (NPR-B). However, the association of the human NPR-B gene with EH has not been studied, because little is known about the genomic organization of this gene. We designed oligonucleotide primers based on the cDNA sequence of the human NPR-B gene, and long-range polymerase chain reaction (PCR) was performed. The amplified fragments were sequenced directly, and the exon/intron organization of the human NPR-B gene was determined. The gene, which spans ≈16.5 kbp, is composed of 22 exons, and the intron-exon junctions follow the GT-AG rule. Seven hundred fifty base pairs of the 5′-flanking region were sequenced using a thermal asymmetric interlaced–PCR (TAIL-PCR) method. This region contains 10 potential Sp1 binding sites and lacks a TATA box. Rapid amplification of cDNA ends (RACE) revealed the transcriptional start site at −14 bp. A CA/GT microsatellite repeat was identified with a hybridization-based method and was converted to a sequence-tagged site (STS). The GT microsatellite repeat was localized to intron 2 ≈150 bp downstream of the exon-intron junction. Two alleles, (GT)10 and (GT)11, were detected in both EH patients and age-matched normotensive (NT) controls. Multiple logistic linear regression analysis indicated that the NPR-B genotype is associated significantly with EH (odds ratio 1.55; 95% confidence interval, 1.02 to 2.35). The (GT)11 frequency was 0.316 (65/206) for the EH group and 0.218 (44/202) for the NT group and differed significantly between the EH and NT groups (χ2=4.97, P=0.026). The structural organization of the human NPR-B gene was determined, and a novel GT repeat polymorphism, which associated with EH, was identified. These results suggest that one cause of EH is a mutation in this gene or a closely related gene or region.
The family of natriuretic peptides (NPs) elicits a number of vascular, renal, and endocrine effects, resulting in regulation of extracellular fluid volume and blood pressure.1 2 3 The NP family consists of 3 peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Although ANP and BNP show high affinity for the type A NP receptor (NPR-A), CNP selectively binds to the type B NP receptor (NPR-B).4 CNP is secreted by endothelial cells5 and binds to NPR-B, which is abundant in the vascular smooth muscle cell membrane, and inhibits proliferation of vascular smooth muscle cells through the activation of the intracellular cGMP cascade.6 Intravenous administration of CNP led to reduced blood pressure in human subjects7 and increased forearm blood flow after intra-arterial injection.8 Abnormalities of NPR-B may therefore lead to hypertension. These receptor abnormalities were suggested by spontaneously hypertensive rats, which have plasma CNP concentrations similar to those in normotensive Wistar-Kyoto rats, but the vascular relaxing action of CNP was markedly attenuated.9
Genetic dissection is a powerful tool for determining the cause of a complex trait such as essential hypertension (EH)10 11 and for understanding the pathophysiological role of the protein encoded by the gene. We recently reported the association of the endothelial constitutive nitric oxide synthase gene and EH.12 13 As for human NPR-B, the gene was mapped to chromosome 9p21-p12.14 The cDNA and deduced amino acid sequences of human NPR-B have been described.15 The genomic structure, however, has not been determined.
In the present study, we determined the genomic structure of human NPR-B and found a novel GT repeat polymorphism that is associated with EH.
Materials and Methods
Examination of Genomic Sequences Including Protein Coding Region
Genomic DNA was extracted from peripheral blood leukocytes by standard methods. On the basis of the information for the human NPR-B cDNA, we designed oligonucleotide primers; specific primers were designed to amplify the intronic regions (Table 1⇓). Long and accurate polymerase chain reaction (LA-PCR)16 was performed using these primers, and a single PCR product was generated in each case. More than 25 primers were designed and synthesized (Sawaday Technology, Tokyo, Japan) to cover the genomic sequence from the start codon to the termination codon (Table 1⇓). Total human genomic DNA from several individuals was used as a template to confirm identity with the structure of the PCR products. After an initial denaturing step of 3 minutes at 94°C, the PCR conditions were 30 cycles of denaturation for 25 seconds at 98°C, annealing and extension for 20 seconds at 68°C, and a final extension at 72°C for 10 minutes. LA-PCR was performed according to the manufacturer’s instructions (LA-PCR kit version No. 2, Takara Syuzo, Tokyo, Japan). LA-PCR products were electrophoresed on 0.4% agarose gels. For sequencing, the bands were cut out of the gel and purified on a column (Microcon 100, Amicon Inc, Beverly, Mass).
Isolation of the 5′-Flanking Region
To isolate the 5′-flanking region of the NPR-B gene, thermal asymmetric interlaced (TAIL)–PCR was used. The PCR conditions were described previously,17 except that the annealing temperature was 68°C. Five hundred nanograms of genomic DNA was used as the template in the primary PCR reaction. Four arbitrary degenerate (AD) primers were used: AD1, AD2, AD3, and AD4. Three specific primers, which are complementary to the NPR-B gene, were synthesized (Table 2⇓), and the PCR products were electrophoresed on 1.5% agarose gels. For sequencing, bands were cut out of the gel and purified as described above.
PCR products were sequenced directly using a Thermo Sequenase kit (Amersham) after purification over a column. More than 40 nucleotides of the coding region of the NPR-B gene were sequenced together with the intron-exon boundaries.
Rapid Amplification of 5′-cDNA Ends (5′-RACE)
The 5′-RACE experiments were conducted to map the transcriptional start site. 5′-RACE was performed using the 5′-full RACE core set (Takara Syuzo Co Ltd, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthesized using avian myeloblastosis virus (AMV) reverse transcriptase XL and a 5′-phosphorylated 15-nucleotide antisense primer (base pair +266 to +252 with respect to the ATG codon) and having the sequence 5′-(P) AGC TTG AGG TCC ACA-3′. One microgram of human pituitary gland poly-(A) selected +RNA (Clontech) was reverse-transcribed using 5 U of AMV reverse transcriptase and 200 pmol of the antisense primer. After purification of the single-stranded cDNA, a ring structure was made (concatenating) using T4 RNA ligase. A primary PCR was performed using a sense primer F1 5′-TGG ACC TGC GGT TTG TCA GCT C (+185 to +206) and antisense primer R1 5′-TGC CAG GGC TGC CAC CAA CA (+40 to +23). An aliquot of the initial PCR reaction served as the template for a secondary PCR with sense primer F2 5′-AAC TGG AAG GCG CCT GCT CTG A (+209 to +230) and antisense primer R2 5′-AGC AGA AGT GAT GGC AGC GCC ATG G (+23 to −2), which yields a 98-bp product. PCR products were analyzed on a 1.5% agarose gel and sequenced directly.
Detection and Identification of a Microsatellite Polymorphism
A CA/TG repeat microsatellite was identified in LA-PCR products with a hybridization method described previously.18 This microsatellite was converted to a sequence-tagged site (STS) using the following primers: NPR-B-STS-F1 (GT strand) 5′-GGAGAGCTCTAAGTCTTAAGGCAC and NPR-B-STS-R1 (CA strand) 5′-GGTCTCCCCCTCTTCTAATCCTG. The PCR products were separated on a 6% polyacrylamide sequencing gel and visualized by autoradiography (Figure 1⇓). To establish a control for the number of dinucleotide repeats, amplified fragments were subcloned into a pCR II plasmid vector (Invitrogen, Carlsbad, Calif) and sequenced. Plasmid clones containing known numbers of GT repeats were used as standard templates in the PCR (Figures 1⇓ and 2⇓).
The study population included 103 patients with EH (EH group; 48.5±9.1 years, mean±SD) who were given a diagnosis according to the criteria of the World Health Organization. These criteria include a sitting systolic blood pressure (SBP) of >160 mm Hg or diastolic blood pressure (DBP) of >95 mm Hg on 3 occasions spanning 2 months from the first medical examination, without administration of antihypertensive drugs. Subjects given a diagnosis of secondary hypertension were excluded. As a control group, 101 normotensive healthy subjects (NT group; 48.2±7.1 years) were also studied. The NT subjects had no family history of hypertension, and, in all instances, their SBPs were <140 mm Hg and their DBPs were <85 mm Hg. A positive family history was defined as hypertension diagnosed in grandparents, parents, or siblings. Both the patients and the control group were recruited from the northern part of Tokyo, and informed consent was obtained from each individual according to a protocol approved by the Human Studies Committee at Nihon University.
The plasma concentration of total cholesterol and the serum concentrations of creatinine and uric acid were measured by standard methods in the clinical laboratory department of the Nihon University hospital.
Data are presented as mean±SD. Allele frequencies were calculated from the genotypes of all subjects. Hardy-Weinberg equilibrium was assessed by χ2 analysis. Significant differences between the total number of alleles on all chromosomes for the EH and NT groups were assessed by χ2 analysis with one degree of freedom. The association between genotypes and hypertension was evaluated by logistic linear regression analysis. EH was regarded as the dependent variable and genotype, sex, age, body mass index (BMI), and the concentrations of plasma total cholesterol, serum, creatinine, and uric acid were entered as independent variables. Differences in the clinical data between the EH and NT groups and between genotypes were assessed by ANOVA followed by the Fisher protected least significant difference (PLSD) test. A value of P<0.05 was considered significant.
Structure of the NPR-B Gene
The structural organization of the human NPR-B gene was determined by analysis of genomic clones and total genomic DNA by PCR amplification across introns, followed by sequencing of the intron-exon junction. In the present study, we determined the genomic structure of human NPR-B by direct sequencing of the long PCR products, not by screening a genomic library. The LA-PCR can amplify more than 20 kbp of DNA. Confirmation of whether the PCR products amplified the NPR-B gene was performed by nucleotide sequencing of more than 40 nucleotides within the cDNA region of the 5′-portion and 3′-portion of the exon. The sizes and positions of the exons, together with the codons and corresponding amino acids interrupted by introns, are listed in Table 3⇓. The genomic DNA contains 21 introns of variable size. The NPR-B gene is ≈16.5 kbp in length, which is more than 30 times longer than the cDNA. The size range for the exons is 69 bp (exon 18) to 667 bp (ATG-exon 1) and for the introns is 87 bp (intron 14) to 6.5 kbp (intron 3). All the intron-exon junction sequences follow the GT-AG rule shown in Table 4⇓ and conform to the consensus.19 A GT/CA microsatellite repeat was found in intron 2 ≈150 bp downstream of the exon-intron junction (Figure 3⇓).
A TAIL-PCR method using the AD3 primer detected a single clear band. This band was ≈1 kbp, and the sequence was determined after purification with a column. Seven hundred fifty base pairs of the 5′-flanking region were sequenced (Figure 4⇓). This region did not include a TATA box. The transcriptional start site was identified by sequencing the concatenation product and is shown in Figure 4⇓.
Association of a Microsatellite Polymorphism With Essential Hypertension
Clinical characteristics of the EH and NT subjects are shown in Table 5⇓. SBP, DBP, BMI, pulse rate, and plasma concentration of total cholesterol were significantly higher in the EH group than in the NT group. Age and serum concentrations of creatinine or uric acid were not different between the 2 groups.
We found a novel GT/CA microsatellite repeat in intron 2, ≈150 bp downstream of the exon-intron junction. Two alleles, (GT)10 and (GT)11, were detected in both the EH group and the NT group. The observed and expected heterozygosities were 0.16 and 0.15, respectively, which were in good agreement with Hardy-Weinberg equilibrium (χ2=0.19, P=0.98). The frequencies of the (GT)10/(GT)10, (GT)10/(GT)11, and (GT)11/(GT)11 genotypes were 0.624 (63/101), 0.317 (32/101), and 0.059 (6/101) for the NT group and 0.515 (53/103), 0.340 (35/103), and 0.146 (15/103) for the EH group, respectively. Logistic linear regression analysis adjusted by age and sex revealed that NPR-B genotype was associated significantly with EH (odds ratio 1.55; 95% confidence interval, 1.02 to 2.35).The frequencies of the (GT)10 and (GT)11 alleles were 0.782 (158/202) and 0.218 (44/202) for the NT group and 0.684 (141/206) and 0.316 (65/206) for the EH group, respectively, and differed significantly between the 2 groups (χ2=4.97, P=0.026). The genotype or allele frequency was not associated with BMI, pulse rate, or plasma levels of total cholesterol.
In the present study, the genomic structure of the human NPR-B gene was determined by analysis of genomic clones and total genomic DNA by PCR amplification across the intron-exon junction followed by sequencing. The long-range PCR and direct sequencing of PCR products were useful for determining the genomic structure as described previously.20 Advantages of this method are that radioisotope facilities and culture techniques for genomic library screening are not required. The difficulty in using this method is that even in long-range PCR, the genome cannot be amplified when there is an intron in the gene that is >20 to 30 kbp long. The identification of the human NPR-B PCR products was done by sequencing and gave more than 40 bp of identity to the human cDNA.15 The genomic structure is very similar to that of the rat21 and the human NPR-A gene,21A which contains 22 exons. The 5′-flanking region of the human NPR-B gene lacks a TATA box but contains 10 potential Sp1 binding sites.
The significance of NP family genes in hypertension in animals has been reported, and the targeted deletion of the pro-ANP gene or NPR-A gene yielded mice with high blood pressure.22 23 However, the genetic studies of human ANP gene are conflicting. A study of 2 ANP restriction fragment length polymorphisms in healthy Norwegian twins revealed no association with SBP, DBP, or blood pressure variability.24 Rutledge et al25 reported that the allele frequency of an Hpa II mutation in intron 2 was significantly higher in the hypertensive individuals compared with normotensive African Americans, but this observation was not confirmed in Australians.26 The Hpa II polymorphism was not associated with salt-sensitive hypertension in Caucasians.27 In contrast to the ANP gene, there is no available data on NP receptors. In the present study, we found a novel GT repeat in intron 2 of the NPR-B gene and demonstrated a significant association with EH. No differences in sex, age, pulse rate, BMI, or the plasma concentration of total cholesterol were apparent between subjects who were homozygous for the (GT)10 allele and those who had at least one (GT)11 allele. These results suggest that the GT repeat polymorphism may be in linkage disequilibrium with the actual cause of EH, which may be the NPR-B gene or a locus near this gene.
In conclusion, we determined the genomic structure of the human NPR-B gene and found a novel GT repeat polymorphism that is associated with EH. This allele may be in linkage disequilibrium with the actual causal mutation, suggesting that one cause of EH is a mutation in the NPR-B gene or a closely located gene. The genomic structure of the human NPR-B gene may contribute to our understanding of the actual cause of EH.
This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan. We wish to thank Hideko Tobe for her technical assistance and Drs. A. Kubo, M. Kunimoto, N. Fukuda, and Y. Watanabe for collecting samples.
- Received April 21, 1998.
- Accepted January 3, 1999.
- © 1999 American Heart Association, Inc.
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