From the Department of Laboratory Medicine (H.O., H.E., M.X., W.P.), the First Department of Internal Medicine (B.H., B.P.), and the Department of Neurology (B.I.), Landeskliniken, Salzburg; and the Department of Internal Medicine, Krankenhaus Hallein (F.K.), Austria.
Abstract
Peroxisome proliferator–activated receptor-{gamma}
coactivator-1 (PPARGC1/PGC-1) is a transcriptional coactivator of nuclear hormone receptors implicated in blood pressure regulation. We therefore ascertained whether the PPARGC1 gene locus is associated with hypertension. We studied associations of 3 polymorphisms in PPARGC1 transcripts with hypertension in 683 middle-aged men and 530 middle-aged women of a cross-sectional Austrian population. Hypertension was defined by average values of systolic or diastolic ambulatory blood pressure readings (taken between 7 AM and 10 PM) above 140 and/or 90 and/or use of antihypertensive medication. Among the 3 polymorphic sites, genotype distributions associated with Gly482Ser differed by hypertension status in men (P=0.0038), but not in women. The less common Ser482 allele was associated with a modest, but significant, reduction in the prevalence of hypertension in men. The distribution of 3 loci haplotypes also differed in men with and without hypertension (P=0.015). Despite its moderate effect, but because of its high frequency ({approx} 64%), the more common risk allele contributed to hypertension in 35% (95% CI 16% to 54%) of our male population. These results suggest, but do not prove, that PPARGC1 participates in blood pressure control, and sequence substitutions at its gene locus confer an increased risk of hypertension to a substantial proportion of men.
Key Words: genetics • genes • gender • haplotypes • polymorphism
Introduction
In the great majority of hypertensive patients, etiology and pathogenesis of the disorder are unknown,1 but it is well established that between 30% and 50% of blood pressure variation in populations is determined by genetic factors.2 Despite the identification of genes underlying rare Mendelian forms of hypertension and reports on linkage of marker loci and associations of candidate genes with blood pressure,3 common genetic variants identified to date account for a small fraction of the blood pressure variance in the general population.1,4
PPARGC1, the human homolog of mouse Pgc1, is a coactivator of peroxisome proliferator–activated receptors (PPARs) {alpha}
and {gamma}
and other nuclear receptors, including the mineralocorticoid and estrogen receptors (ER) {alpha}
and ß,5 which are involved in blood pressure control. PPARGC1 was mapped to chromosome 4p15.1.6 In a genome-wide scan in Dutch families with familial combined hyperlipemia, linkage of chromosome 4p15.1-2 with systolic blood pressure was observed.7 We therefore studied possible associations of the PPARGC1 gene locus with hypertension in a well-characterized cross-sectional Austrian sample.
Methods
Study Population
We studied 40- to 65-year-old male and 45- to 70-year-old female participants of Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR), a population-based prospective study that investigates genetic and environmental factors contributing to atherosclerotic vascular disease. Recruitment procedures have been detailed.8 All study subjects provided informed consent, and the study was approved by the local ethics committee. The population comprised only white Europeans, mainly of Bavarian and Austrian German descent. Body mass index (BMI) was calculated from measurements of weight and height. Diabetes was diagnosed by fasting plasma glucose concentrations of ">="
7.0 mmol/L and/or use of hypoglycemic medications. Between 7 AM and 10 PM, ambulatory blood pressure was measured in intervals of 15 minutes using the TM-2430 device (Boso). Means of systolic and diastolic blood pressure measurements (SBP-day and DBP-day) were used for analyses. Hypertension was defined by SBP-day >140 mm Hg and/or DBP-day >90 mm Hg and/or antihypertensive drug therapy.
Laboratory parameters and the Gly482Ser and the +2962A/G polymorphisms were determined as described.9 For typing of the silent A-to-G substitution at position +1704 (Genbank Accession No. ), polymerase chain reaction (PCR) conditions described for Gly482Ser, but substituting C for T at position 1706 in the antisense primer, and digestion with BstU I were used. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance [HOMA-IR=fasting insulin (µU/mL)xglucose (mmol/L)/22.5].10
Statistics
Differences of continuous variables between hypertensive and normotensive men and women as well as effects of genotypes on clinical parameters were ascertained by 2-way analysis of variance. Logarithmic transformations were made if the equal variance and normality assumptions of ANOVA were rejected. Measurements were adjusted for effects of age and BMI as indicated. For testing the significance of multiple comparisons between hypertensive and normotensive subjects, the Bonferroni correction was used and a value of <0.003 was employed as level of significance instead of a value of <0.05.11 Allele frequencies were estimated by gene counting. Agreement with Hardy-Weinberg expectations was tested using a {chi}
2 goodness-of-fit test. The standardized pair-wise linkage disequilibrium statistics (D’) and haplotype frequencies were estimated according to Terwilliger and Ott.12 Differences in genotype frequencies between hypertensive and normotensive subjects were determined using a {chi}
2 distribution with 2 degrees of freedom. Because genotype distributions for 3 polymorphic sites were compared, a significance level of 0.016 was used for comparison in the entire study population. For gender-specific comparison of genotype distributions, a significance level of 0.008 was employed. To estimate odds ratios with confidence intervals (95% CI) for each genotype, 2 "dummy" variables with the respective wild-type as the reference were used in univariate logistic regression analysis. Adjustments for the study subjects’ age and BMI and, for comparisons in females, menopause and/or hormone replacement therapy were made by including these covariates in a second set of multivariate logistic regression models. Differences in haplotype distributions and population-attributable risk with 95% CI were calculated as described.8
Results
As expected from the selection criteria, systolic and diastolic blood pressures were higher in subjects with hypertension (). Average values for age and BMI were greater and the prevalence of type 2 diabetes was higher in hypertensive subjects. Gender-specific differences were observed for age, systolic blood pressure, and BMI. More women used antihypertensive medication than men. Among laboratory parameters, average values for insulin and HOMA-IR were higher in hypertensive than in normotensive subjects. Women displayed lower average values for glucose, HOMA-IR, creatinine, uric acid, and triglycerides but higher average values for HDL cholesterol. A significant interaction between hypertension and gender was observed for insulin.
fig-ommitted
TABLE 1. Characteristics of Study Population
The polymorphisms studied fulfilled Hardy-Weinberg expectations in male and female subjects with and without hypertension and displayed highly significant positive standardized pairwise linkage disequilibria (all D/Dmax>0.95, P<0.001). Among the 3 polymorphic sites, genotype distributions associated with the Gly482Ser differed significantly between hypertensive and normotensive subjects (P=0.0139, see , in online supplement available at http://www. hypertensionaha.org.). Gender-specific analysis showed differences in males (P=0.0038, ), but not in females (). Neither adjustment for menopause or hormone replacement therapy (not shown) nor stratification by type 2 diabetes status and BMI ( in online supplement available at http://www.hypertensionaha.org.) altered the lack of association. Genotype distributions differed by hypertension status in both obese and nonobese men without type 2 diabetes, but reliable analyses in men with type 2 diabetes were precluded by the low diabetes prevalence ( in online supplement available at http://www.hypertensionaha. org.). No interactions between genotypes and hypertension status were observed for the parameters shown in in either sex group (Tables 4 and 5 in online supplement available at http://www.hypertensionaha.org.). The distribution of 3 loci haplotypes differed significantly in the male study population (P=0.0149). Frequency distributions of 2-loci haplotypes comprising the Gly482Ser and the A1704G polymorphisms differed by hypertension status in males and in the entire study population (P=0.0022 and P=0.0182, respectively), whereas frequency distributions of 2-loci haplotypes comprising the Gly482Ser and A2962G polymorphisms did not differ in any study group (Table 6 in online supplement available at http://www.hypertensionaha.org.). Gly482Ser genotype distributions also differed by hypertension when male subjects without antihypertensive medications were considered (P=0.0013). Using either additive or dominant models, the population-attributable risk in males was 35% (95% CI 16% to 54%).
fig-ommitted
TABLE 2. PPARGC1 Polymorphisms and Associated Risk of Hypertension in Males
fig-ommitted
TABLE 3. PPARGC1 Polymorphisms and Associated Risk of Hypertension in Females
Discussion
The association of the PPARGC1 gene locus with hypertension in our male study population is consistent with linkage studies showing an association of chromosome 4p15.1-2 with systolic blood pressure.7 The discriminatory Gly482Ser polymorphism is located within a PPARGC1 region that mediates Mads Box Transcription Enhancer Factor 2C–induced GLUT4 expression in myocytes,13 but transient transfection studies in immortalized human adipocytes showed similar magnitudes of PPAR{gamma}
-mediated transactivation of the UCP1 promoter for PPARGC1 constructs harboring Gly or Ser at amino acid position 482.9,14 Thus, whether the Gly482Ser polymorphism is the causal site or reflects the effect of another sequence substitution that is in linkage disequilibrium with it remains to be determined.
We have no clear explanation for the fact that the associations were observed only in men. Gender-specific differences in age-dependent hypertension prevalence may have contributed to our results.15 PPARGC1 is expressed in endothelial cells (Figure 1 in online supplement available at http://www.hypertensionaha.org.), interacts with ER{alpha}
and -ß, and enhances their transcriptional activity. Both receptors are expressed in vascular endothelial and smooth muscle cells.16 ERß-deficient mice develop sustained systolic and diastolic hypertension as they age, and their vascular cells show multiple abnormalities.17 Hence, ER signaling may play a role in the association of PPARGC1 with hypertension. However, PPARGC1 mRNA is expressed in other tissues important for blood pressure regulation such as brain, heart, and kidney,6 and other nuclear receptors that are coactivated by PPARGC1 may play a role as well. Hypertension has been observed in patients with dominant-negative mutations in PPAR{gamma}
,18 and a Ser810Leu mutation in the ligand binding domain of the mineralocorticoid receptor causes autosomal dominant hypertension.19 Clearly, identification of the causative site(s) in PPARGC1 and functional studies are necessary to delineate the downstream factors involved in the pathogenesis of hypertension.
We reported an association of 2-loci haplotype combinations comprising the Gly482Ser and +2962A/G sites with obesity indices in females of SAPHIR.9 Support for our results comes from a recent genome-wide search showing linkage of chromosome 4p15-p14 with high BMI in females.20 Interestingly, the associations with obesity in females and hypertension in males differed in that hypertension status in men was associated with the Gly482Ser/A1704G haplotype combination, whereas obesity indices in females were associated with the Gly482Ser/A2962G haplotype combination,9 and associations with the Gly482Ser/A1704G haplotype combination did not reach statistical significance (unpublished results). Thus, the molecular mechanisms underlying the gender-specific associations of PPARGC1 with obesity and hypertension are probably different. Associations of the Gly482Ser polymorphism, either alone or in a haplotype context, with type 2 diabetes have been reported in Danish and Japanese populations.21,22 PPARGC1 may therefore be involved in disturbances that are components of the metabolic syndrome.
Perspectives
Hypertension has multiple etiological components. Our study strongly suggests that sequence substitutions at the PPARGC1 gene locus contribute to hypertension in a significant number of men. Our results are supported by a genome-wide scan in an independent population showing linkage of chromosome 4p15.1-2 with hypertension. Nevertheless, confirmation of the gender-specific association of PPARGC1 with hypertension is required in other male and female populations. PPARGC1 integrates several transcriptional programs and acts as a cofactor for numerous transcription factors. Hence, identification of the causal PPARGC1 sequence substitution(s) that may affect its expression or alter the surface interacting with specific transcription factor(s) is needed to gain mechanistic insight. Further studies may focus on downstream targets functionally compromised by altered PPARGC1 coactivation to delineate the role of this gene locus in the pathogenesis of hypertension.
Acknowledgments
This study was supported by grants from the Bundesland Salzburg and the Medizinische Forschungsgesellschaft Salzburg and by Jubilaeumsfondsprojekt No. 9364 from the Oesterreichische Nationalbank. The technical assistance of Daniela Pichler and Simone Fellner is acknowledged.
Footnotes
H.O. and B.H. contributed equally to this work.
References
Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell. 2001; 104: 545–556.
Ward R. Familial aggregation and genetic epidemiology of blood pressure. In: Laragh J, Brenner B, eds. Hypertension: Pathophysiology, Diagnosis, and Management. New York, NY: Raven Press; 1995: 67–88.
Jeunemaitre X, Soubrier F, Kotelevtsev YV, Lifton RP, Williams CS, Charru A, Hunt SC, Hopkins PN, Williams RR, Lalouel JM. Molecular basis of human hypertension: role of angiotensinogen. Cell. 1992; 71: 169–180.
Turner ST, Boerwinkle E. Genetics of hypertension, target-organ complications, and response to therapy. Circulation. 2000; 102: IV40–IV45.
Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 1998; 92: 829–839.
Esterbauer H, Oberkofler H, Krempler F, Patsch W. Human peroxisome proliferator activated receptor gamma coactivator 1 (PPARGC1) gene: cDNA sequence, genomic organization, chromosomal localization, and tissue expression. Genomics. 1999; 62: 98–102.
Allayee H, de Bruin TW, Michelle DK, Cheng LS, Ipp E, Cantor RM, Krass KL, Keulen ET, Aouizerat BE, Lusis AJ, Rotter JI. Genome scan for blood pressure in Dutch dyslipidemic families reveals linkage to a locus on chromosome 4p. Hypertension. 2001; 38: 773–778.
Esterbauer H, Schneitler C, Oberkofler H, Ebenbichler C, Paulweber B, Sandhofer F, Ladurner G, Hell E, Strosberg AD, Patsch JR, Krempler F, Patsch W. A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans. Nat Genet. 2001; 28: 178–183.
Esterbauer H, Oberkofler H, Linnemayr V, Iglseder B, Hedegger M, Wolfsgruber P, Paulweber B, Fastner G, Krempler F, Patsch W. Peroxisome proliferator-activated receptor-gamma coactivator-1 gene locus: associations with obesity indices in middle-aged women. Diabetes. 2002; 51: 1281–1286.
Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985; 28: 412–419.
Rosner B. Fundamentals of Biostatistics. Boston, Mass: PWS-Kent Publishing Co; 1990: 1–647.
Terwilliger JD, Ott J. Handbook of Human Genetic Linkage. Baltimore, Md: Johns Hopkins University Press; 1994: 188–194.
Michael LF, Wu Z, Cheatham RB, Puigserver P, Adelmant G, Lehman JJ, Kelly DP, Spiegelman BM. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci U S A. 2001; 98: 3820–3825.
Oberkofler H, Esterbauer H, Linnemayr V, Strosberg AD, Krempler F, Patsch W. Peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 recruitment regulates PPAR subtype specificity. J Biol Chem. 2002; 277: 16750–16757.
Burt VL, Whelton P, Roccella EJ, Brown C, Cutler JA, Higgins M, Horan MJ, Labarthe D. Prevalence of hypertension in the US adult population. Results from the Third National Health and Nutrition Examination Survey, 1988–1991. Hypertension. 1995; 25: 305–313.
Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999; 340: 1801–1811.
Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW, Thoren P, Smithies O, Gustafsson JA, Mendelsohn ME. Abnormal vascular function and hypertension in mice deficient in estrogen receptor beta. Science. 2002; 295: 505–508.
Barroso I, Gurnell M, Crowley VE, Agostini M, Schwabe JW, Soos MA, Maslen GL, Williams TD, Lewis H, Schafer AJ, Chatterjee VK, O’Rahilly S. Dominant negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension. Nature. 1999; 402: 880–883.
Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai FT, Sigler PB, Lifton RP. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science. 2000; 289: 119–123.
Stone S, Abkevich V, Hunt SC, Gutin A, Russell DL, Neff CD, Riley R, Frech GC, Hensel CH, Jammulapati S, Potter J, Sexton D, Tran T, Gibbs D, Iliev D, Gress R, Bloomquist B, Amatruda J, Rae PM, Adams TD, Skolnick MH, Shattuck D. A major predisposition locus for severe obesity, at 4p15-p14. Am J Hum Genet. 2002; 70: 1459–1468.
Ek J, Andersen G, Urhammer SA, Gaede PH, Drivsholm T, Borch-Johnsen K, Hansen T, Pedersen O. Mutation analysis of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) and relationships of identified amino acid polymorphisms to Type II diabetes mellitus. Diabetologia. 2001; 44: 2220–2226.
Hara K, Tobel K, Okada T, Kadowaki H, Akanuma Y, Ito C, Kimura S, Kadowaki T. A genetic variation in the PGC-1 gene could confer insulin resistance and susceptibility to type II diabetes. Diabetologia. 2002; 45: 740–743.
(Hannes Oberkofler; Bertram Hölzl; Harald Esterbauer; Mingqiang Xie; Bernhard Iglseder; Franz Kr)
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