Association of genetic variation in the natriuretic peptide system and left ventricular mass and blood pressure in newborns

Iwona Gorący1, Grażyna Dawid2, Karolina Skonieczna-Żydecka1, Mariusz Kaczmarczyk1, Beata Łoniewska3, Jarosław Gorący4

1Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland
2Department of Paediatrics, Pomeranian Medical University, Szczecin, Poland
3Department of Neonatal Diseases, Pomeranian Medical University, Szczecin, Poland
4Department of Cardiology, Pomeranian Medical University, Szczecin, Poland

Address for correspondence:
Iwona Barbara Gorący, MD, PhD, Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, ul. Powstańców Wlkp. 72, 70–111 Szczecin, Poland, e-mail: igor@pum.edu.pl
Received: 05.08.2014 Accepted: 09.10.2014 Available as AoP: 28.10.2014

INTRODUCTION

Left ventricular hypertrophy is a risk factor for cardiovascular events [1]. Left ventricular mass (LVM) is influenced by factors including blood pressure (BP), age, gender, obesity, and/or dietary habits. However, genetic factors play an important role in modulating LVM in humans. Identification of genes involved in the modulation of LVM could contribute to an understanding of the etiopathogenesis of left ventricular hypertrophy development.

The natriuretic peptide (NP) family consisting of the three NPs, the genes encoding A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), are named from NP precursors A, B, and C, i.e. NPPA (natriuretic peptide precursor A gene), NPPB (natriuretic peptide precursor B gene), and NPPC (natriuretic peptide precursor C gene), respectively. The actions of the NP include vasodilatation, natriuresis, suppression of the rennin–angiotensin system (RAS), and inhibition of both cardiomyocyte hypertrophy and cardiac fibroblast activation [2]. NP indicates the impact on the development of cardiovascular diseases [3, 4]. Published papers have shown that BNP is a good marker in identifying heart failure and left ventricular dysfunction in children [5]. However, the measurements NP are not generally part of the routine testing performed in children with cardiac disease because relatively little is known about their function and accuracy as a diagnostic test in children [6]. Furthermore, it should be emphasised that BNP levels were higher in the umbilical artery from birth and not related to maternal levels [7].

The NP might also be important during development. This hypothesis has been based on the findings that NP can act as a modulator of growth in cells of the heart. Experimental studies exhibit dynamic expression of cardiac NP during cardiogenesis (modulating growth of vascular cells, fibroblast proliferation, collagen production) and BP regulation in foetal life [8, 9]. Polymorphisms of the gene through variation of the concentration of the peptide or modification its structure can influence the pathogenesis of cardiovascular diseases. Recently Newton-Cheh et al. [10] reported that genetic variants of NPPA and NPPB genes are associated with increased circulating levels of BNP and ANP.

Therefore, we hypothesised that NPPA, NPPB, and NPPC are candidate genes possibly involved in the development or modulation of LVM in foetal life. In this study we evaluated the effects of the T2238C NPPA (rs5065), T(-381)C NPPB (rs198389), and G2628A NPPC (rs5268 ) gene polymorphisms in the development of LVM and regulation of BP in Polish newborns.

METHODS

Study subjects

The population included 206 healthy newborns born after the end of the 37th week of gestation (37 to 40 weeks). The mothers in this study were healthy without any complications. Body surface area (BSA) was calculated using the following equation: BSA = (BL [cm] × BW [kg]/3600)1/2, where BL is body length and BW is body weight [11].

Echocardiographic and blood pressure measurements

Echocardiographic and BP measurements in neonates were made on the 3rd day after delivery. Two-dimensional M-mode echocardiography was performed using an Acuson Sequoia 512 unit (USA) equipped with a 2–4 MHz imaging transducer. Measurement techniques were consistent with the American Society of Echocardiography conventions. The LVM were calculated from the echocardiographic left ventricular dimension measurements using the Penn convention with the equation modified by Huwez et al. [12].

To standardise the LVM, it was indexed with respect to BL (LVM/BL), BW (LVM/BW), and BSA (LVM/BSA), respectively. A Diascope oscillometer (Artema) was used to determine systolic BP (SBP) and diastolic BP (DBP), and one of the investigators performed all of the BP measurements using a standardised protocol.

Genetic analysis

Genomic DNA from cord blood was isolated using the QIAamp Blood DNA Mini Kit (QIAGEN, Germany). For the analysis of the C(-381)T NPPB (rs198389) and the C(-381 T NPPB (rs198389) gene polymorphisms, a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) method was designed. For the analysis of the T2238C NPPA (rs5065) gene polymorphism, the following primer pairs were used: forward 5’-gCCggggCTgTTTTCgCTgTgAgT-3’ and reverse 5’-CggAggCTgCTgCTgCTgCTTCTg-3’. The NPPA amplicons were subsequently digested with the enzyme ScaI enzyme (MBI Fermentas, Vilnius, Lithuania). DNA fragments that contained the C(-381)T NPPB (rs198389) gene polymorphism were amplified using PCR with forward 5’-gCC- ggggCTgTTTTCgCTgTgAgT-3’ and reverse 5’-CggAggCTgCTgCTgCTgCTTCTg-3’ primers. The reaction was run with the addition of betaine (20% of the final volume). Afterwards the amplicons were purified with Gene Elute PCR Clean Up Kit (Sigma Aldrich) and PCR-RFLP with the EcoRII restriction enzyme was performed. For analysis of the G2628A NPPC (rs5268) gene, PCR with forward 5’-gCTgAGCCgTTTCTgACCTT-3’ and reverse 5’-ATgAGCggCCTgggATGTTAgT-3’ primers were used. The amplification was run with the addition of Dimethyl sulfoxidate PCR reagent (10% of the final volume) and the amplicons were subsequently digested with Taq restriction enzyme (MBI Fermentas, Vilnius, Lithuania).

Statistical analysis

The divergence of NPPA, NPPB, and NPPC genotype frequencies from Hardy-Weinberg equilibrium was assessed using χ2 tests, and the distribution of each quantitative variable was tested for skewness. Quantitative data were presented as means ± standard deviation and analysed either by Student`s t-test or by one-way ANOVA. LVM index (LVMI) was tested for association with genotype in multivariate analysis (ANCOVA) in order to adjust for possible confounding factors: neonatal (gestational age, gender, APGAR at 3 min) and maternal (age, body mass index [BMI] at beginning and end of the pregnancy, smoking status, and hypertension status). Dominant, recessive, and additive modes of inheritance were tested. Statistical significance was defined as p < 0.05. All data were analysed with STATISTICA software (data analysis software system, version 8.0, StatSoft, Inc. 2007, www.statsoft.com).

RESULTS

The characteristics of the newborn cohort (n = 206) are shown in Table 1. The distribution of these characteristics in our cohort approached normality (skewness < 2 for all variables). There were no significant differences in NPPA rs5065, NPPB rs198389, and NPPC rs5268 genotypes or allele distributions between boys and girls, and the genotype distributions conformed to the expected Hardy-Weinberg equilibria (Table 1).

Table 1. Clinical and echocardiographic characteristics of the newborns with regard to gender

LVMI were tested for association with genotype in multivariate analysis (ANCOVA) in order to adjust for possible confounding factors. After adjusting for neonatal (gestational age, gender, APGAR at 3 min) and maternal (age, BMI at beginning and end of the pregnancy, smoking status, and hypertension status) parameters, we revealed a significant association between LVMIs (LVM/BSA and LVM/BW) and the NPPB polymorphism. The carriers of the C allele of the NPPB polymorphism had significantly lower LVM/BSA and LVM/BW values when compared with newborns homozygous for the T allele (41.76 g/m2 vs. 48.31 g/m2, padjusted = 0.044 and 2.78 g/kg vs. 3.26 g/kg, padjusted = 0.031, respectively) (Fig. 1).

Figure 1. Left ventricular mass indexes according to B-type natriuretic peptide (BNP) genotype. Mean and standard error of mean are shown; ap = 0.044; bp = 0.080; cp = 0.031; a, b, cCC+CT vs. TT; BSA — body surface area; BL — body length; BW — body weight

There was no independent association between NPPA, NPPC genetic variation, and LVMI in newborns. The frequency of homozygous TT NPPA gene was low (only 3 homozygous), and therefore no effect of the TT homozygous NPPA gene on the LVM in this study was considered. Owing to the low number of CC homozygotes for the NPPA polymorphism, only the dominant mode was considered (date not shown).

An association was observed between NPPA genotype and SBP, DBP, and mean arterial pressure (MAP) ≥ 90th percentile (p = 0.029, p = 0.0048, p = 0.004, respectively). The frequency of newborns with SBP ≥ 90th percentile was greater in the carriers of C allele (CC+TC vs. TT, p = 0.013), while the frequencies of newborns of DBP ≥ 90th percentile, MAP ≥ 90th percentile were greater in the homozygotes CC (p = 0.001 and p = 0.002, respectively). An association of SBP ≥ 90th percentile for NPPB (p = 0.016) was additionally observed. The homozygotes CC exhibited higher frequency of SBP ≥ 90th percentile, compared to the carriers of T allele (23% vs. 7%, p = 0.004). The NPPC gene variant was unrelated to BP in newborns. Additionally, NPPB and NPPC polymorphisms were significantly correlated with maternal history of smoking habits and NPPA, NPPB, and NPPC genotypes were significantly correlated with parity (Table 2).

Table 2. Overview of results depending on foetal genotypes

DISCUSSION

The present study demonstrates an association between NPPA, NPPB, and NPPC gene polymorphisms and LVM and BP in newborns. It is known that the role of genetic factors in the development or heart parameters may be particularly relevant. Recently we presented a study in which is was suggested that RAS or bone morphogenetic protein (BMP4) and bone morphogenetic protein type 1 receptor (BMPR1A) and calmodulin (CALM2) genetic variation may partially account for subtle variations in LVM or heart parameters in newborns, when external environmental factors have not yet had a marked impact [13, 14].

The present study demonstrates a significant association between variants of NPPB and a decrease in LVMIs in newborns. We used the suggested methods of determining the LVM in relation to BSA, BL, and BW to accurately transform and index the determined LVM. In carriers of the C allele of the NPPB gene, we observed a significant decrease in LVM/BSA and LVM/BW. This finding may be connected with varying levels of NP plasma concentrations. The T(-381)C rs198389 is located in the promoter region of NPPB. A previous study has demonstrated that the rs198389 C allele may be associated with higher BNP promoter activity [15], and that the rs198389 is correlated with levels of plasma BNP. As Lanfear [4] suggested, the NPPB promoter variants are the key source of inter-individual variation in BNP levels and may also impact the clinical interpretation of BNP testing. NP levels were not measured in the present study, which is its main limitation.

In the current study we investigated healthy newborns born full term, when external environmental factors had not yet had a marked impact and the influence of genes on LVM could be expressed. The strength of our study is determined by the well-documented cohort of healthy newborns born full term without coexisting heart defects. It is known that BNP is an important biomarker in cardiology in adults, and it is correlated with poor ventricular function. Genetic variation in the NPPB has potential implication in the BNP pathway and is associated with alterations in BNP levels. We hypothesised that genetic variation in the NP system may also cause minor changes in the development or modulation of LVM in healthy newborns. Our data on the genetic variability in NPPB shows the possible importance in the subtle modulating of LVM in foetal life and in the first days of life in healthy newborns. La Pointe [16] expressed a theory that BNP may work more as an autocrine/paracrine inhibitor of cell growth in the heart, while ANP is potent diuretic vasorelaxant hormone. Some studies indicate that BNP inhibits growth of vascular cells, as well as fibroblast proliferation and collagen production. We continue observing our population and we are considering conducting a follow-up, which will show in later years whether the variation of NPPB has a predisposition to modulate LVM and several echocardiographic variables. However, our results require confirmation in further independent large studies.

Another finding in our study is the influence of genetic variation of the NPPA and NPPB on the BP. NPPA rs5065, which introduces the stop codon and extends ANP by two amino acids, is located in the 3’UTR. Most of the mechanisms that this regulation affects are trough vascular volume (mainly natriuresis) and tone, resulting in lower BP [17]. In addition, the NP system has haemodynamic effects via antagonisation of the RAS and the adrenergic system, which also contribute to lowering of BP and vascular tone [4]. Genetic variation in the NP system is well documented. For blood, BNP levels have been shown to be heritable, supporting the notion that genetic variation in the system impacts its function [18]. However, the observed association with hypertension may be explained not only by increased levels of NP, but also by an increase in ANP receptor binding or activity in individuals carrying the rs5065 C allele. It is worth mentioning that the predominant phenotypes of NPPA and NPR1 (natriuretic peptide receptor type 1) knockout mice are arterial hypertension and myocardial hypertrophy [19]. In the current study an association was observed between NPPA rs5065 and SBP, DBP, and MAP ≥ 90th percentile, and also for NPPB rs198389 and SBP ≥ 90th percentile. In addition to its role in cardiovascular homeostasis in adults, the NP might also be important during development. In newborns this may not only be related to different levels of plasma concentrations of NP associated with different genetic variants, but also to diminished water and electrolyte distribution in the perinatal period. Therefore, these results should be interpreted with caution. Interestingly, Hammerer-Lerchel et al. [20] showed that umbilical cord N-terminal-proBNP levels were higher than maternal levels. The reason for this neonatal surge is not clear. Our suggested hypothesis is that genetic variation of NPPA and NPPB may be partly involved in modulating the levels of NP as early as in foetal life.

CONCLUSIONS

In conclusion, LVM is a continuous trait influenced by interaction between genetic, environmental, and lifestyle factors. The present study shows an association between NPPB gene polymorphism and LVM in newborns. Our data confirm a role of NPPB in LVM development in newborns and provide the first evidence suggesting that the NPPB polymorphism might be considered as one of the factors important in the development and/or modulation of LVM in foetal life. Additionally, an association between NPPA, NPPB, and BP was found. Further studies are needed to define the role of genetic variation in the NP system in the development or modulation of LVM and BP in newborns.

Conflict of interest: none declared

References

1. 1. Levy D, Garrison RJ, Savage DD et al. Prognostic implication of echocardiographically determined left ventricular mass in the Framingham Study. N Engl J Med, 1990; 322: 1561–1566.
2. 2. Garbers DL, Chrisman TD, Wiegen P et al. Membrane guanylyl cyclase receptors: an uptade. Trends Endocrinol Metab, 2006; 17: 251–258.
3. 3. Rubbatu S, Ridker P, Meir J et al. The gene encoding atrial natriuretic peptide and the risk of human stroke. Circulation, 1999; 100: 1722–1726.
4. 4. Lanfear DE. Genetic variation in natriuretic peptide system and heart failure. Heart Fail Rev, 2010; 15: 219–228.
5. 5. Maher KO, Reed H, Cuadrado A et al. B-type natriuretic peptide in the emergency diagnosis of critical heart disease in children. Pediatrics, 2008; 121: e1484-8.
6. 6. Low YM, Hayer AW, Reller MD, Silberbach M. Accuracy of plasma B-type natriuretic peptide to diagnose significant cardiovascular disease in children: the Nat Pout Children Study. J Am Coll Cardiol, 2009; 54: 1467–1475.
7. 7. Itoh H, Sagawa N, Hasegawa M et al. Brain natriuretic peptide levels in the umbilical venous plasma are elevted in fetal distress. Bio Neonate, 1993; 64: 18–25.
8. 8. Auman HJ, Coleman H, Riley E et al. Functional modulation of cardiac form through regionally confined cellshape changes. PLoS Biol, 2007; 5: e53.
9. 9. Knowlton KL, Rockman HA, Itani M et al. Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventriculae myocardium. J Clin Invest, 1995; 96: 1311–1318.
10. 10. Newton-Cheh C, Larson MG, Vasan RS et al. Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure. Nat Genet, 2009; 41: 348–353.
11. 11. Mosteller RD. Simplified calculation of body surface area [letter]. N Eng J Med, 1987; 317: 1098.
12. 12. Huwez FU, Houston AB, Watsona J et al. Age and body surface area related normal upper and lower limits of M mode echocardiographic measurements and left ventricular volume and mass from infancy to early adulthood. Br Heart J, 1994; 72: 276–280.
13. 13. Gorący I, Dawid G, Łoniewska B et al. Genetics of the rennin-angitensin system with respect to cardiac and blood pressure phenotypes in healthy newborns infants. J Renin Angiotensin Aldosterone Syst, 2013; 14: 337–347.
14. 14. Gorący I, Safranow K, Dawid G et al. Common genetic variants of BMP4, BMPR1A, BMPR1B and ACVR1 genes, left ventricular mass and other parameters of heart in newborns. Gen Test Mol Bioma, 2012; 16: 1309–1316.
15. 15. Takeishi Y, Toriyama S, Takabatake N et al. Linkage disequilibrium analyses of natriuretic peptide precursor B locus reveal risk haplotype conffering high plasma BNP levels. Biochem Biophys Res Commun, 2007; 362: 480–484.
16. 16. La Pointe MC. Molecular regulation of the brain natriuretic peptide gene. Peptides, 2005; 26: 944–956.
17. 17. Sabrane K, Kruse MN, Fabritz L et al. Vascular endothelium is critically involved in the hypotensive and hypovolemic actions of atrial natriuretic peptide. J Clin Invest, 2005; 115: 1666–1674.
18. 18. Wang TJ, Larson MG, Levy D et al. Heritability and genetic linkage of plasma natriuretic peptide levels. Circulation, 2013; 108: 13–16.
19. 19. John SW, Veress AT, Honrath U et al. Blood pressure and fluid–electrolyte balance in mice with reduced or absent ANP. Am J Physiol, 1996; 271: R109–R114.
20. 20. Hammere-Lercher A, Mair J, Puschendorf B, Sommer R. N-terminal pro B-type natriuretic concentration in the umbilical cord blood of newborns exceeds the concentration of their mothers [abstract]. Clin Chem, 2004; 50 (Suppl.): A124.