Journal of Human Hypertension (2004) 18, 823–828 & 2004 Nature Publishing Group All rights reserved 0950-9240/04 $30.00 www.nature.com/jhh ORIGINAL ARTICLE The microsatellite alleles on chromosome 1 associated with essential hypertension and blood pressure levels T Nakayama1, M Soma2, K Kanmatsuse2 and S Kokubun1 1Division of Receptor Biology, Advanced Medical Research Center, Nihon University School of Medicine, Ooyaguchi-kamimachi, Tokyo, Japan; 2Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan Essential hypertension (EH) is thought to be a polygenic contributions in D1S507, D1S2713 and D1S2842 were disease. Several candidate genes of this disease have 0.0008, 0.0062 and 0.0084, respectively. All these values been investigated in studies using polymorphic genetic were significant after Bonferroni correction. Further- markers, but some studies have failed to show any more, we found that the three microsatellite alleles were association of EH with these genes. In this experiment, associated with the levels of systolic blood pressure. we used microsatellite markers on chromosome 1, and These data suggest that there are at least the three performed an association study between EH and control susceptibility loci for EH on chromosome 1, and that a subjects. Genomic DNA was amplified with fluores- case–control study using microsatellite markers on cently labelled primers from the Applied Biosystems genomewide basis is a useful method for isolating the PRISM linkage mapping set HD-5 comprising 63 highly susceptibility loci of multifactorial disorders. polymorphic microsatellite markers with an average Journal of Human Hypertension (2004) 18, 823–828. spacing of 4.5 cM. We isolated three loci showing doi:10.1038/sj.jhh.1001740 significant differences: D1S507, D1S2713 and D1S2842. Published online 10 June 2004 The P-values of the allele with the greatest post hoc Keywords: essential; case–control study; microsatellite; polymorphism Introduction During recent years, several scans of the human genome have been performed on families with Essential hypertension (EH) is likely to be a multiple cases of EH. Many potential susceptibility polygenic disorder that results from the inheritance loci have been identified.2 Some genomewide scans of a number of susceptibility genes. Although data have provided suggestive evidence of linkages on from rodent models and human twin-based and chromosome 1,3–7 prompting detailed analysis of population-based epidemiology studies suggest that this region. Chromosome 1 includes genes of the inherited factors contribute 50% or less to the natriuretic peptide system such as atrial natriuretic determination of an individual’s eventual blood 8 1 peptide, brain natriuretic peptide, A-type natriure- pressure (BP), the number of contributing genes or tic peptide receptor,9–11 and genes of the renin– their individual attributable risk remains unknown. angiotensin system such as angiotensinogen12 and Indeed, whether there are one or two major renin.13 hypertension susceptibility-causing genes with sev- Genetic markers that are sufficiently polymorphic eral more minor loci or many genes, each with small (as measured by their heterozygosities) can be used attributable risks, is an important question that has in linkage and association analyses to detect not previously been possible to tackle. The affected Mendelian segregation underlying disease pheno- sibling pair model has been used to identify loci in types. Each type of analysis can either be based on a various complex traits by determining how often specific genetic model, or need not make any affected siblings share alleles. assumptions about the mode of inheritance of the disease. Association analyses are more powerful, Correspondence: Dr T Nakayama, Division of Receptor Biology, provided there is linkage disequilibrium between Advanced Medical Research Center, Nihon University School of the marker and the disease loci. Recently, it had Medicine, Ooyaguchi-kamimachi, 30-1 Itabashi-ku, Tokyo been pointed out that when gametic disequilibrium 173-8610, Japan. E-mail: [email protected] Received 22 December 2003; revised 22 February 2003; accepted is suspected, methods testing for both linkage and 14–15 24 February 2004; published online 10 June 2004 association might be more powerful. Susceptibility loci of hypertension on chromosome 1 T Nakayama et al 824 The aim of the present study is to identify 551C for 15 s, extension at 721C for 30 s; followed by susceptibility loci of EH on chromosome 1 using 20 cycles of denaturation at 891C for 15 s, annealing microsatellite markers by a case–control study in at 551C for 15 s and extension at 721C for 30 s; and Japanese subjects. ended with a final extension at 721C for 10 min. Amplifications were run in a Gene Amp PCR 9600 thermocycler (Perkin Elmer, Foster City, CA, USA). Materials and methods Products from up to eight different PCR reactions Subjects were pooled and diluted 1:10 for products labelled with FAM (1:5 for products labelled with HEX and The EH group consisted of 144 patients with EH NED). The pooled reactions (1 ml) were then mixed diagnosed according to the following criteria: sitting with 0.5 ml LIZ internal size standard GS-500 and systolic blood pressure (SBP) greater than diluted 1:6 with HI-DI formamide (Applied Biosys- 160 mmHg and/or diastolic blood pressure (DBP) tems). The pooled reactions were denaturated at greater than 100 mmHg on three occasions within 2 951C for 3 min and were loaded on an ABI3700 DNA months after the first medical examination.10 Parti- analyzer (Applied Biosystems). The fluorescent cipants were not using antihypertensive medication. signal was recorded and analysed using the GeneS- Subjects diagnosed with secondary hypertension can version 2.1 software. Fragments from reactions were excluded. We also included 144 age-matched using each of the different fluorescent dyes were normotensive (NT) healthy subjects as controls. plotted separately, and the sizes of fluorescent peaks None of the NT subjects had a family history of were estimated for the base pairs by referencing the hypertension, and they all had SBP less than in-lane size standard. Marker alleles were classified 130 mmHg and DBP less than 85 mmHg. A family according to their size using the Genotyper version 2 history of hypertension was defined as prior software. In addition to the automated allele calling, diagnosis of hypertension in grandparents, uncles, we performed manual surveillance of all genotypes. aunts, parents or siblings. Both the EH patients and the NT control subjects were recruited from the northern part of Tokyo, and informed consent was Statistical analysis obtained from each individual according to a protocol approved by the Human Studies Committee Data represent mean7s.d. Clinical data were tested of Nihon University. using ANOVA followed by Fisher’s protected least significant difference test, and P-values less than 0.05 indicated significant differences. Biochemical analysis Hardy–Weinberg equilibrium was assessed by w2 analysis. The overall distribution of alleles between Plasma concentrations of total cholesterol and HDL- EH patients and NT subjects was analysed by 2 Â n cholesterol, and serum concentrations of creatinine contingency tables19 and P-values less than 0.05 and uric acid were measured as previously were considered significant. The calculations of described.16 expected values and post hoc cell contributions were also performed. Cells with expected values Polymerase chain reaction (PCR) and genotyping below 1 were omitted, and the frequency of expected values below 4 was set to below 20%.20 Blood samples were collected from all participants, Individual differences in allele frequencies were and genomic DNA was extracted from the peripheral tested using 2 Â 2 contingency tables for each allele blood mononuclear cells by standard procedures.17 and combined the remaining alleles, and a P-value PCR was performed with fluorescently labelled of less than 0.05/n was considered significant to primers from the Applied Biosystems PRISM link- correct for the number of comparisons made age mapping set HD-5 comprising 63 highly poly- (Bonferroni correction).19 morphic markers arranged in panels of compatible markers with an average spacing of 4.5 cM and average heterozygosity of 0.76. The primers were Results dye-conjugated with FAM, HEX or NED (Applied Biosystems). The loci were selected from the We successfully identified the genotypes of more Genethon linkage map,18 based on location on than 92% of the total 18 144 genotypes found in 63 chromosome and heterozygosity. PCR amplification microsatellites from the 288 study subjects. The was carried out using 15 ng of genomic DNA, 3 ml observed and expected frequencies of each genotype True Allele PCR Premix (containing PCR buffer, for all 63 microsatellites in the NT group were in MgCl2, dNTP, Amplitaq Gold, Applied Biosystems) Hardy–Weinberg equilibrium (data not shown). We and 0.33 ml primer mix in a total reaction volume of identified three loci showing significant (Pp0.05) 5 ml. Amplifications were carried out at 951C for among the 63 loci (Table 1). The P-values of D1S507, 12 min to activate the Amplitaq Gold; followed by 10 D1S2713 and D1S2842 were 0.037, 0.035 and 0.018, cycles of denaturation at 941C for 15 s, annealing at respectively. Journal of Human Hypertension Susceptibility loci of hypertension on chromosome 1 T Nakayama et al 825 Table 1 Association study of microsatellites Allelic distributions of the three loci with the post hoc cell contributions for each cell are shown in 2 Microsatellite w value P-value Table 2. The P-values of the allele showing the highest post hoc cell contributions for D1S507, D1S468 11.2 0.189 D1S2660 5.8 0.218 D1S2713 and D1S2842 were 0.0008, 0.0062 and D1S214 6.0 0.640 0.0084, respectively. All these values were signifi- D1S450 12.9 0.115 cant after Bonferroni correction. In the tree micro- D1S2667 7.7 0.263 D1S434 3.7 0.595 satellite markers, five alleles showing significant D1S507 17.8 0.037* differences of frequencies between the two groups D1S2697 1.0 0.915 were discriminated (Table 2).