This article has Open Peer Review reports available.
A genetic variant of the atrial natriuretic peptide gene is associated with left ventricular hypertrophy in a non-diabetic population – the Malmö preventive project study
- Amra Jujić1, 2, 3Email author,
- Margret Leosdottir†2,
- Gerd Östling†3,
- Petri Gudmundsson†1,
- Peter M Nilsson†3, 4,
- Olle Melander†3, 4 and
- Martin Magnusson†2, 3
© Jujić et al.; licensee BioMed Central Ltd. 2013
Received: 1 July 2012
Accepted: 20 June 2013
Published: 24 June 2013
Epidemiological studies have shown considerable heritability of blood pressure, thus suggesting a role for genetic factors. Previous studies have shown an association of a single nucleotide polymorphism rs5068 in the NPPA locus gene with higher levels of circulating atrial natriuretic peptide as well as with lower intra individual blood pressure, but up to date, no association between rs5068 and cardiac organ damage, i.e. left ventricular hypertrophy, has been accounted for in humans. We sought to explore if rs5068 is associated with left ventricular hypertrophy as measured by echocardiographic examination in a non-diabetic population.
968 non-diabetic individuals from the Malmö Preventive Project (mean age 67 years; 31% women) were genotyped and examined with echocardiography. Logistic regression was used to adjust for covariates.
The minor allele of rs5068 was associated with decreased prevalence of left ventricular hypertrophy (p = 0.021) after adjustment for sex and age. In the multivariate logistic analysis including; age, sex, systolic blood pressure, antihypertensive and/or cardioprotective treatment, body mass index and fasting plasma glucose, the association of rs5068 with left ventricular hypertrophy was, as expected, attenuated (p = 0.061).
In a non-diabetic population, the minor allele of rs5068 was associated with lower left ventricular mass. These findings suggest that rs5068, or genetic variants in linkage disequilibrium, might affect susceptibility to left ventricular hypertrophy and support the possible protective role of natriuretic peptides.
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are secreted from cardiomyocytes in response to cardiac wall stress and have important roles in the regulation of extracellular fluid volumes and blood pressure (BP) [1–3]. Animal studies have shown that over expression of natriuretic peptide genes is associated with low BP, whereas under expression is associated with high BP and left ventricular hypertrophy (LVH) [4, 5], and studies on mice with inactivated type A natriuretic peptide receptors (NPRA) have even shown hypertrophic growth of the heart independently of BP levels . The suppressive effect of ANP on hypertrophy has also been demonstrated in cardiomyocytes in culture  and a recent study showed salt-sensitive hypertension in natriuretic peptide precursor A (NPPA) knockout mice, demonstrating that genetically reduced production of ANP can lead to salt-sensitive hypertension . Evidence of substantial heritability of plasma natriuretic peptide (NP) levels has also been accounted for  and low circulating NP levels have been associated with obesity  and new onset diabetes , which may contribute to their predisposition to hypertension.
The NPPA and natriuretic peptide precursor B (NPPB) genes lie in tandem 9.7 kb apart on chromosome 1. Associations between common variations (rs5068, rs198358 and rs632793) in NPPA-NPPB locus genes and higher levels of circulating ANP were found in a recent study as well as association with lower interindividual BP, with the strongest effect for rs5068, findings that have been replicated in larger cohorts . The minor allele of rs5068 has also been associated with favorable cardiometabolic profile , and there is evidence for an important protective role of the natriuretic peptides in individuals with coronary artery disease , but surprisingly no association with echocardiographic evidence of cardiac organ damage, such as LVH, for this particular SNP. However, since LVH is well known to be overrepresented in patients with diabetes mellitus [11, 16–19] the inclusion of subjects with diabetes, 17% and 15% respectively) might explain the lack of association of rs5068 and LVH [14, 15].
Here we tested the hypothesis that rs5068 suppressively regulates the development of LVH in a non-diabetic population.
Between 1974–1992, birth cohorts (men born in 1921, 1926–1942, 1944, 1946 and 1948–9; women born in 1926, 1928, 1930–6, 1938, 1941–2 and 1949) of inhabitants in Malmö, Sweden, were invited to participate in a large cohort study The Malmö Preventive Project (MPP) with a total of 33,346 individuals attending (67% men, attendance rate 71%, mean age 46 ± 7 years). Re-examination of 18,240 surviving MPP participants, the MPP-Re-examination Study (MPP-RES) was conducted during 2002–2006 (63% men, 72% attendance rate, mean age 69 ± 6 years). All participants answered a self-administered questionnaire on lifestyle and medical history. Height and weight were measured in light in-door clothing, waist circumference was measured and body mass index (BMI) was calculated (kg/m2). Patient BP and pulse rate were measured twice in the supine position after 10 minutes of rest and blood samples were drawn after an overnight fast. In a sample of 1,792 participants, echocardiography (UCG) and electrocardiography (ECG) recordings were carried out. These subjects were randomly selected from groups defined by glucometabolic status: normal fasting plasma glucose (FPG) (≤6.0 mmol/l); impaired FPG (IFG); new-onset type 2 diabetes mellitus; and prevalent diabetes mellitus; with oversampling in groups of subjects with glucometabolic disturbances to ensure sufficient numbers of subjects studied from each group. A full description of this study population has been presented elsewhere . Subjects with prevalent diabetes mellitus type 1 or 2 (n = 677) were excluded from our analysis due to previously shown overrepresentation of diastolic dysfunction and LVH within the diabetic phenotype [11, 16, 17], resulting in a total of 1,115 subjects. Of these 1,115 subjects, 147 had missing values of one or more covariates used in the multiple regression analysis, resulting in 968 eligible subjects.
All participants signed a written informed consent form before entering the MPP-RES. The study was approved by The Ethics committee at Lund University, Sweden.
Conventional echocardiograms were obtained with an Acuson Sequoia, Mountain View, CA, USA (3V2c transducer) or Sonos 5500 Philips, Andover, MA, USA (S3 transducer). All echocardiograms were carried out by experienced sonographers. Internal left ventricular dimensions were evaluated from parasternal long axis view according to the recommendations of the European Association of Echocardiography. Measurements of wall thickness were obtained in two-dimensional end-diastolic parasternal long axis view, where left ventricular mass (LVM) was calculated during a single heart cycle if recordings were homogeneous, otherwise a mean of three to five measurement of different heart cycles was used. LVM was calculated according to clinical standards and indexed for height2.7 to minimize the effect of obesity on LVM calculations .
Two sonographers analysed images independently from a random sub-sample of subject to test inter- and intra-observer variability which was 13.0% and 10.5% for intraventricular end-diastolic septum diameter, and 4.1% and 3.3% for left ventricular end-diastolic diameter. Both sonographers were experienced and blinded to clinical data. All analyses were performed offline using Xcelera software (Philips).
Variants in NPPA-NPPB locus genes were analysed by direct genotyping as described previously using TaqMan (Applied Biosystems) with primers and conditions as recommended by the manufacturer . Twenty five percent of the samples were run in duplicates without discrepancies.
All FPG analyses were performed by Department of Clinical Chemistry, Malmö University Hospital which is attached to a national standardization and quality control system (Beckman Coulter LX20, Beckman Coulter Inc, Brea, USA).
If LVM was >51 g/m2.7, LVH was considered prevalent [21, 22]. Antihypertensive and/or cardioprotective treatment was defined as use of one or more of the following; β-receptor blockers, calcium channel blockers, lipid lowering drugs, angiotensin converting enzyme inhibitors, long-acting nitrates, angiotensin receptor blockers, digitalis and diuretics. Prevalent hypertension was defined as the presence of either one or more of the following; systolic BP (SBP) > 140 mmHg and/or diastolic BP >90 mmHg and/or antihypertensive and/or treatment by the time of the third visit. Diabetes was defined as previously known diabetes mellitus type 1 or 2, whereas new-onset diabetes mellitus type 2 was defined by either two separate measurements of FPG ≥7.0 mmol/l, or one single measurement of ≥11.1 mmol/l.
Continuous variables were expressed as mean ± standard deviation (SD). FPG was not normally distributed and was log transformed prior to analysis. LVM was modeled as the dependent variable in linear regression and the presence of LVH via logistic regression with rs5068 genotype as independent variable using additive models with the minor allele (G) coded. All modeling was performed in models adjusted for age and sex (model 1) and for age, sex, BMI, SBP, antihypertensive and/or cardioprotective treatment, and FPG (model 2). A two-tailed significance level of p < 0.05 was considered statistically significant. All calculations were done in SPSS 19.0 (SPSS Inc, Illinois, USA).
Baseline characteristics of the study population
Subjects without LVH
Subjects with LVH
Sex (% women)
Systolic BP (mmHg)
Diastolic BP (mmHg)
Antihypertensive and/or cardioprotective treatment (%)
Genotype frequencies of rs5068
Subjects without LVH
Subjects with LVH
Left ventricular mass index (g/m 2.7 )
LVM index (g/m2.7)
Subjects without LVH
Subjects with LVH
Association of rs5068 with LVH in a non diabetic population adjusted for age and gender*
OR (95% CI)
Association of rs5068 with LVH in a non diabetic population in a fully adjusted model*
OR (95% CI)
Hosmer-Lemeshow goodness of fit test, sensitivity, specificity and percent of overall correct for association of rs5068 and LVH in a fully adjusted model*
Number of observations
Number of groups
Prob > chi2
Association of rs5068 with LVM Indexed for Height 2.7 in a non Diabetic Population adjusted for age and sex*
Adjusted R Square
Association of rs5068 with LVM Indexed for Height 2.7 in a non diabetic population in a fully adjusted model*
Adjusted R Square
The key finding of our study is that the minor allele of rs5068 is associated with lower LVM as well as lower prevalence of LVH in a non-diabetic population after adjustments for age and sex.
These associations were attenuated in the multivariate analysis in which SBP and antihypertensive and/or cardioprotective treatment were included. One possible explanation for this could be that the observed association between the minor allele of rs5068 and lower likelihood of LVH is explained by lower BP mediated by the same polymorphism . On the other hand, the attenuation of the association between rs5068 and LVH after full adjustment was rather modest, suggesting that the ANP elevating effects of the rs5068 G-allele also may contribute with BP independent effects on LVH. We acknowledge that the effect size is small, as commonly the case is in polygenic situations. However, the effect size for most variants is small and a single SNP can only explain a small proportion of the expected heritability.
In contrast to the results of Cannone and co-workers , but also Ellis and co-workers , we were able to show a significant association between the minor allele of rs5068 and reduced prevalence of LVH, which can be explained by our exclusion of subjects with diabetes. LVH is heavily overrepresented in patients with diabetes [19, 23] and the existence of a specific diabetic cardiomyopathy has been an ongoing debate over the past decades . It is likely to presume that the “harmful” diabetic phenotype overrides the cardioprotective and/or the blood pressure lowering effects of the minor allele of rs5068. Also, in comparison of clinical studies of LVH, adequate correction is of critical importance, since both body habitus and size are associated with LV mass and dimensions. Normalization of LV mass is still a subject for debate, since different criteria for body size adjustment result in different prevalence of patients with LVH. The body surface area correction has been shown to underestimate LVM in the upper range of body surface area distribution, and a height-based adjustment has been proposed to correct for obesity. The most accurate estimation of LVH appears to be derived from indexing for height2.7, where 51 g/m2.7 appears to be a reliable cut off value to define LVH, as used in our study.  As both Cannone and Ellis corrected for body surface area, our results may not be fully comparable.
Echocardiography is the most commonly used imaging modality in assessing LVM, left ventricular size and performance. However, it is subjective and operator dependent and contends with inter- and intraobserver variability, particularly when using harmonic imaging in assessing LVM. Even though harmonic imaging tends to overestimate wall thickness, it has also improved delineation of the left ventricle, especially in subjects with bad image quality . All methods for quantitation of LVM using two-dimensional imaging have been validated previously [26, 27]. We sought to minimize the effects of subjectivity on our data by testing if inter- and intraobserver variability between two independent readers analysing images was within acceptable levels.
As our sample size was relatively small, the results warrant replication in a larger cohort.
As this is a cross-sectional study, it shares the inherent limitations about causality and control as all cross-sectional studies. Still, the genetic effect is not expected to be hampered by such confounding, as the genome is constant throughout life.
Our data was collected at a single regional centre which limits the applicability to other populations and allows the influence of community characteristics. On the other hand, inter-observer bias regarding the UCG measurement was less than would be expected in a multi-centre study. Also, although the subjects with congestive heart failure were overrepresented in the original population, excluding subjects with diabetes resulted in low number of subjects with congestive heart failure (n = 4). In order to prove that inclusion of those subjects does not have any impact on our data, we performed a sensitivity analysis by excluding patients with prevalent heart failure and received very similar data (not shown). Hence, inclusion of patients with congestive heart failure is not considered to have any impact on our results.
Finally, our study results may not be generalizable to all ancestries since the study was undertaken in mainly individuals of Swedish descent.
In a non-diabetic population, the minor allele of rs5068 was associated with lower LVM and lower prevalence of LVH. Whether the association is mediated by the BP lowering effect of rs5068 or is independent of BP is uncertain. However, our findings suggest that rs5068 or genetic variants in linkage disequilibrium might affect susceptibility for LVH and support the possible protective role of NP.
Funding support was obtained by grants from the Swedish Medical Research Council (OM grant K2009-64X-20129-04-3), the European Research Council (OM grant StG-282255), the Swedish Heart and Lung Foundation (OM), the Medical Faculty of Lund University (OM), Skåne University Hospital (OM, MM), the Albert Påhlsson Research Foundation (OM), the Crafoord Foundation (OM), the Ernhold Lundströms Research Foundation (OM, MM), the Region Skane (OM, MM), the Hulda and Conrad Mossfelt Foundation (OM, MM), the King Gustaf V and Queen Victoria Foundation (OM), the Lennart Hanssons Memorial Fund (OM), Knut and Alice Wallenberg Foundation and the Marianne and Marcus Wallenberg Foundation, the Southwest Skanes Diabetes foundation (OM, MM). The Swedish Heart- and Lung Foundation grant 20060666 (PN, ML). The funding organizations had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; or the preparation or approval of the manuscript.
- Inagami T: Atrial natriuretic factor as a volume regulator. J Clin Pharmacol. 1994, 34: 424-426. 10.1002/j.1552-4604.1994.tb04982.x.View ArticlePubMedGoogle Scholar
- Clerico A, Emdin M: Diagnostic accuracy and prognostic relevance of the measurement of cardiac natriuretic peptides: a review. Clin Chem. 2004, 50: 33-50. 10.1373/clinchem.2003.024760.View ArticlePubMedGoogle Scholar
- Levin ER, Gardner DG, Samson WK: Natriuretic peptides. N Engl J Med. 1998, 339: 321-328. 10.1056/NEJM199807303390507.View ArticlePubMedGoogle Scholar
- John SW, Krege JH, Oliver PM, Hagaman JR, Hodgin JB, Pang SC, Flynn TG, Smithies O: Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science. 1995, 267: 679-681. 10.1126/science.7839143.View ArticlePubMedGoogle Scholar
- Melo LG, Steinhelper ME, Pang SC, Tse Y, Ackermann U: Anp in regulation of arterial pressure and fluid-electrolyte balance: lessons from genetic mouse models. Physiol Genomics. 2000, 3: 45-58.PubMedGoogle Scholar
- Oliver PM, Fox JE, Kim R, Rockman HA, Kim HS, Reddick RL, Pandey KN, Milgram SL, Smithies O, Maeda N: Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor a. Proc Natl Acad Sci USA. 1997, 94: 14730-14735. 10.1073/pnas.94.26.14730.View ArticlePubMedPubMed CentralGoogle Scholar
- Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K: Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes. Hypertension. 2000, 35: 19-24. 10.1161/01.HYP.35.1.19.View ArticlePubMedGoogle Scholar
- Melo LG, Veress AT, Chong CK, Pang SC, Flynn TG, Sonnenberg H: Salt-sensitive hypertension in anp knockout mice: potential role of abnormal plasma renin activity. Am J Physiol. 1998, 274: R255-R261.PubMedGoogle Scholar
- Wang TJ, Larson MG, Levy D, Benjamin EJ, Corey D, Leip EP, Vasan RS: Heritability and genetic linkage of plasma natriuretic peptide levels. Circulation. 2003, 108: 13-16. 10.1161/01.CIR.0000081657.83724.A7.View ArticlePubMedGoogle Scholar
- Wang TJ, Larson MG, Levy D, Benjamin EJ, Leip EP, Wilson PW, Vasan RS: Impact of obesity on plasma natriuretic peptide levels. Circulation. 2004, 109: 594-600. 10.1161/01.CIR.0000112582.16683.EA.View ArticlePubMedGoogle Scholar
- Magnusson M, Jujic A, Hedblad B, Engstrom G, Persson M, Struck J, Morgenthaler NG, Nilsson P, Newton-Cheh C, Wang TJ, Melander O: Low plasma level of atrial natriuretic peptide predicts development of diabetes: the prospective malmo diet and cancer study. J Clin Endocrinol Metab. 2012, 97: 638-645. 10.1210/jc.2011-2425.View ArticlePubMedGoogle Scholar
- Newton-Cheh C, Larson MG, Vasan RS, Levy D, Bloch KD, Surti A, Guiducci C, Kathiresan S, Benjamin EJ, Struck J, Morgenthaler NG, Bergmann A, Blankenberg S, Kee F, Nilsson P, Yin X, Peltonen L, Vartiainen E, Salomaa V, Hirschhorn JN, Melander O, Wang TJ: Association of common variants in nppa and nppb with circulating natriuretic peptides and blood pressure. Nat Genet. 2009, 41: 348-353. 10.1038/ng.328.View ArticlePubMedPubMed CentralGoogle Scholar
- Newton-Cheh C, Johnson T, Gateva V, Tobin MD, Bochud M, Coin L, Najjar SS, Zhao JH, Heath SC, Eyheramendy S, Papadakis K, Voight BF, Scott LJ, Zhang F, Farrall M, Tanaka T, Wallace C, Chambers JC, Khaw KT, Nilsson P, van der Harst P, Polidoro S, Grobbee DE, Onland-Moret NC, Bots ML, Wain LV, Elliott KS, Teumer A, Luan J, Lucas G, et al: Genome-wide association study identifies eight loci associated with blood pressure. Nat Genet. 2009, 41: 666-676. 10.1038/ng.361.View ArticlePubMedPubMed CentralGoogle Scholar
- Cannone V, Boerrigter G, Cataliotti A, Costello-Boerrigter LC, Olson TM, McKie PM, Heublein DM, Lahr BD, Bailey KR, Averna M, Redfield MM, Rodeheffer RJ, Burnett JC: A genetic variant of the atrial natriuretic peptide gene is associated with cardiometabolic protection in the general community. J Am Coll Cardiol. 2011, 58: 629-636. 10.1016/j.jacc.2011.05.011.View ArticlePubMedPubMed CentralGoogle Scholar
- Ellis KL, Newton-Cheh C, Wang TJ, Frampton CM, Doughty RN, Whalley GA, Ellis CJ, Skelton L, Davis N, Yandle TG, Troughton RW, Richards AM, Cameron VA: Association of genetic variation in the natriuretic peptide system with cardiovascular outcomes. J Mol Cell Cardiol. 2011, 50: 695-701. 10.1016/j.yjmcc.2011.01.010.View ArticlePubMedGoogle Scholar
- Ryden L, Standl E, Bartnik M, Van den Berghe G, Betteridge J, de Boer MJ, Cosentino F, Jonsson B, Laakso M, Malmberg K, Priori S, Ostergren J, Tuomilehto J, Thrainsdottir I, Vanhorebeek I, Stramba-Badiale M, Lindgren P, Qiao Q, Priori SG, Blanc JJ, Budaj A, Camm J, Dean V, Deckers J, Dickstein K, Lekakis J, McGregor K, Metra M, Morais J, Osterspey A, et al: Guidelines on diabetes, pre-diabetes, and cardiovascular diseases: executive summary. The task force on diabetes and cardiovascular diseases of the european society of cardiology (esc) and of the european association for the study of diabetes (easd). Eur Heart J. 2007, 28: 88-136.View ArticlePubMedGoogle Scholar
- Shehadeh A, Regan TJ: Cardiac consequences of diabetes mellitus. Clin Cardiol. 1995, 18: 301-305. 10.1002/clc.4960180604.View ArticlePubMedGoogle Scholar
- Fang ZY, Prins JB, Marwick TH: Diabetic cardiomyopathy: evidence, mechanisms, and therapeutic implications. Endocr Rev. 2004, 25: 543-567. 10.1210/er.2003-0012.View ArticlePubMedGoogle Scholar
- Palmieri V, Bella JN, Arnett DK, Liu JE, Oberman A, Schuck MY, Kitzman DW, Hopkins PN, Morgan D, Rao DC, Devereux RB: Effect of type 2 diabetes mellitus on left ventricular geometry and systolic function in hypertensive subjects: hypertension genetic epidemiology network (hypergen) study. Circulation. 2001, 103: 102-107. 10.1161/01.CIR.103.1.102.View ArticlePubMedGoogle Scholar
- Leosdottir M, Willenheimer R, Plehn J, Borgquist R, Gudmundsson P, Harris TB, Launer LJ, Bjornsdottir H, Nilsson PM, Gudnason V: Myocardial structure and function by echocardiography in relation to glucometabolic status in elderly subjects from 2 population-based cohorts: a cross-sectional study. Am Heart J. 2010, 159: 414-420. 10.1016/j.ahj.2009.12.028. e414View ArticlePubMedPubMed CentralGoogle Scholar
- Foppa M, Duncan BB, Rohde LE: Echocardiography-based left ventricular mass estimation. How should we define hypertrophy?. Cardiovasc Ultrasound. 2005, 3: 17-10.1186/1476-7120-3-17.View ArticlePubMedPubMed CentralGoogle Scholar
- Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ: Recommendations for chamber quantification: a report from the American society of echocardiography's guidelines and standards committee and the chamber quantification writing group, developed in conjunction with the European association of echocardiography, a branch of the European society of cardiology. J Am Soc Echocardiogr. 2005, 18: 1440-1463. 10.1016/j.echo.2005.10.005.View ArticlePubMedGoogle Scholar
- Santra S, Basu AK, Roychowdhury P, Banerjee R, Singhania P, Singh S, Datta UK: Comparison of left ventricular mass in normotensive type 2 diabetes mellitus patients with that in the nondiabetic population. J Cardiovasc Dis Res. 2011, 2: 50-56. 10.4103/0975-3583.78597.View ArticlePubMedPubMed CentralGoogle Scholar
- Poornima IG, Parikh P, Shannon RP: Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res. 2006, 98: 596-605. 10.1161/01.RES.0000207406.94146.c2.View ArticlePubMedGoogle Scholar
- Hirata K, Watanabe H, Beppu S, Muro T, Teragaki M, Yoshiyama M, Takeuchi K, Yoshikawa J: Pitfalls of echocardiographic measurement in tissue harmonic imaging: in vitro and in vivo study. J Am Soc Echocardiogr. 2002, 15: 1038-1044. 10.1067/mje.2002.121537.View ArticlePubMedGoogle Scholar
- Helak JW, Reichek N: Quantitation of human left ventricular mass and volume by two-dimensional echocardiography: in vitro anatomic validation. Circulation. 1981, 63: 1398-1407. 10.1161/01.CIR.63.6.1398.View ArticlePubMedGoogle Scholar
- Reichek N, Helak J, Plappert T, Sutton MS, Weber KT: Anatomic validation of left ventricular mass estimates from clinical two-dimensional echocardiography: initial results. Circulation. 1983, 67: 348-352. 10.1161/01.CIR.67.2.348.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2350/14/64/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.