Common Genetic Variants of the BMP4,BMPR1A,BMPR1B,and ACVR1 Genes,Left Ventricular Mass,and Other Parameters of the Heart in Newborns

Abstract

The members of the family of bone morphogenetic proteins (BMPs) are important regulators in cardiac development. The present study was designed to evaluate the effect of common genetic variants of BMP-4 and its receptors BMPR1A, BMPR1B, and ACVR1 on left ventricular mass (LVM) and other parameters of the heart and blood pressure in newborns. The study included 210 healthy newborns. Two-dimensional M-mode echocardiography was used to assess LVM between days 3 and 4 after birth. Polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism technique. We found lack of associations between LVM, values of blood pressure, and the BMP4, BMPR1A, BMPR1B, and ACVR1 genotypes. A significant association was observed between the 455C allele of BMP4 and increased left ventricular internal diameter systolic (p=0.004) and between 1650T allele of BMPR1B and lower left atrium diameter (p=0.038). Presence of the 455C allele of BMP4 and the 8474T allele of ACVR1 gene was significantly associated with decreased left ventricular ejection fraction (LVEF) (p=0.0004 and p=0.046, respectively). The 455C allele of BMP4 and the 8474T allele of ACVR1 may play a role as significant predictors for decreased LVEF in newborns.

Introduction

Left ventricular hypertrophy (LVH) is a strong independent risk factor for cardiovascular disease morbidity and mortality not only in adults with hypertension but also in the general population (Levy et al., 1990). Although much of this condition is explained by conventional risk factors, the role of genetic factors may be particularly relevant, but remains poorly understood. The effect of some growth factors on cardiac differentiation of embryonic stem cells has been clarified (Wu et al., 1999; Paquin et al., 2002; Dell'Era et al., 2003). Moreover, studies about the exact role of some growth factors such as bone morphogenetic proteins (BMPs) are still continuing.

BMPs are multifunctional cytokines belonging to the transforming growth factor-β superfamily. BMPs are multipotential proteins that regulate a plethora of cellular functions during development and adult life (van Wijk et al., 2007). Their signals play a central role in vertebral mesodermal induction (Zhang et al., 2008) as well as in heart development (Schultheiss et al., 1997; Brand, 2003). BMPs are involved in the differentiation of mesodermal cells into the cardiac lineage; the cardiac progenitor cells from the lateral plate mesoderm are transformed to the primary heart field and later form the myocardium and endocardium of the heart (Schultheiss et al., 1997; Tirosh-Finkel et al., 2006). It was suggested that BMP-4 is essential for gastrulation and mesoderm formation (Winnier et al., 1995). BMPs control the differentiation of embryonic stem cells into cardiomyocytes in four stages; BMP signaling stimulates relevant transcription factors, and thereby results in formation of precardioblasts initially, then cardioblasts, and finally, when the expression of sarcomeric proteins is induced, cardiomyocytes (van Wijk et al., 2007). BMP-4 knockout mice die during gastrulation and show little or no mesodermal differentiation (Winnier et al., 1995). BMP4 induces the expression of Nkx2.5 and GATA-4 in cardiac progenitor cells, which are indispensable for differentiation into the cardiac lineage (Brand, 2003; Sachinidis et al., 2003). BMP4 or BMP2 induces both the early regulators and also terminal differentiation of cardiac tissue (Schultheiss et al., 1997), but not only. BMPs are crucial in the regulation at the distal borders of the heart during the formation of the four-chambered heart and septovalvular development (van Wijk et al., 2007). Cellular responses to BMPs have been shown to be mediated by the formation of a complex of the type 1 and type 2 receptors. BMPs have a higher affinity for type 1 than type 2 receptors (van Wijk et al., 2007; Miyazono et al., 2010). Among different isoforms, the type 1 receptors BMPR1A (encoding ALK3), BMPR1B (encoding ALK6), and ACVR1 (encoding ALK2) mediate most of the effect of BMPs (Miyazono et al., 2005). A few studies demonstrated that genetic variations within BMP4 and its receptors were significantly associated with cutaneous melanoma (Capasso et al., 2009), human obesity (Böttcher et al., 2009), and increased LDH and uric acid plasma levels (Abhishek et al., 2010), but did not contribute to lumbar spine bone mineral density in postmenopausal women (Özkan et al., 2010). However, it is known that BMP4 and its receptors play an important role in cardiac induction (Winnier et al., 1995; Taha et al., 2007; van Wijk et al., 2007), but there are no studies demonstrating the impact of genetic variation in the development of the heart. Therefore, BMP4 and its receptors, BMPR1A, BMPR1B, and ACVR1, seem to be candidate genes possibly involved in the development of LVM and parameters of the human heart. In this study, we evaluated the effect of T455C BMP4 (rs17563), A5659T BMPR1A (rs7922846), C1650T BMPR1B (rs1434536), and A8474T ACVR1 (rs1220134) gene polymorphisms in development on anatomical and functional parameters and left ventricular mass (LVM) of the heart in Polish newborns. Additionally, we examined the possible impact of genetic variation of BMP4 and its receptors in regulation of blood pressure (BP) in newborns.

Materials and Methods

Study subjects

The study was approved by the Pomeranian Medical University ethics committee. The population in this study included 210 healthy aboriginal European 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 such as pre-eclampsia or eclampsia and fetal growth restriction, and the scientists identifying the RAS genotypes were blinded to the clinical characteristics of subjects. Infants in this study were appropriately grown for their gestational age (defined as birth weight [BW] above 10th centile). Exclusion criteria were twins, intra-uterine growth restriction, chromosomal aberrations and/or congenital malformations, or small for gestational age, that is, below the 10th centile body length (BL), BW, or head circumference (HC). At birth, cord blood (500 μL) of neonates was obtained for isolation of genomic DNA. The gender of the newborn, BL, BW, and HC were taken from standard hospital records. Body surface area (BSA) was calculated with the following equation (Mosteller, 1987): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document}\begin{align*}{ \rm BSA} = \surd [ { \rm BL} \ ( { \rm cm} ) \times { \rm BW}\ ( { \rm kg} ) / 3600 ]\end{align*}\end{document}

BP measurements

The Diascope oscillometer (Artema) was used to determine systolic and diastolic blood pressure (SBP or DBP, respectively), and the only one of the investigators performed all of the BP measurements using a standarized protocol. The smallest cuff size that covered at least two-thirds of the right upper arm and encompassed the entire arm was selected. BPs were measured in supine position on the 3rd day after delivery. Newborn infants were studied at least one and one-half hours after their last feeding or medical intervention. An appropriate-sized cuff was applied to the right upper arm, and the baby was then left undisturbed for at least 15 min or until the infant was sleeping or in a quiet awake state. Three successive BP recordings were taken at 3-min intervals.

Echocardiographic measurements

Echocardiographic measurements in neonates on the 3rd day after delivery were made by one pediatric cardiologist. Two-dimensional M-mode echocardiography was performed using Acuson Sequoia 512 unit equipped with 2-4-MHz imaging transducer. Measurement techniques were consistent with the American Society of Echocardiography conventions (Schiller et al., 1989). In a parasternal long-axis view, left ventricular internal diameter diastolic (LVIDd), left ventricular internal diameter systolic (LVIDs), left ventricular posterior wall (LVPW) thickness at end diastole, thickness of interventricular septum (IVS) at end diastole, left atrial diameter (LAD), aortic diameter, pulmonary artery diameter, left ventricular volume, and left ventricular ejection fraction (LVEF) were measured (using M-mode formulas). The LVMs were calculated from the echocardiographic left ventricular dimension measurements on the Penn convention with the equation modified by Huwez (Huwez et al., 1994) as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document}\begin{align*}{ \rm LVM} = 1.04 [ ( { \rm IVST} + { \rm LVPWT} + { \rm LVID} ) ^3 - { \rm LVID}^3 ]\end{align*}\end{document}

where IVST, LVPWT, and LVID denote interventricular septal thickness, LVPW thickness, and left ventricular internal dimension, respectively. The LVM was indexed for BL, BW, and BSA. For LVM indexation, we also calculated LVM/BL^k, LVM/BW^l, and LVM/BSA^m according to the method described by Hashimoto (Hashimoto et al., 1999). To determine the exponents by which BL, BW, and BSA should be raised, logarithmic transformations were used, and the following regression equations were solved: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document}\begin{align*}{ \rm log}\ ( { \rm LVM} ) = \alpha + { \rm k} \times { \rm log} \ ( { \rm BL} ) \\ { \rm log}\ ( { \rm LVM} ) = \beta + { \rm l} \times { \rm log}\ ( { \rm BW} ) \\ { \rm log} \ ( { \rm LVM} ) = \gamma + { \rm m} \times { \rm log}\ ( { \rm BSA} )\end{align*}\end{document}

Genetic analysis

Genomic DNA from cord blood was isolated with the QIAamp Blood DNA Mini Kit (QIAGEN). For the analysis of the T455C BMP4 gene polymorphism, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed with the following primer pair: forward: 5′-ACTCTgCTTTTCgTTTCC TCTTTA-3′ and reverse 5′-ggCCCAATTCCCACTCC-3′ primers (TIB MOL BIOL). The BMP4 amplicons were subsequently digested with HphI enzyme (MBI Fermentas). The PCR BMP4 product of 395 base pairs (bp) was cut into fragments of 244, 75, 62, and 14 bp in the presence of the T allele and 306 bp, 75 bp, and 14 bp in the presence of the C allele. DNA fragments that contained the A5659T BMPR1A polymorphism were amplified by PCR with forward 5′-TTTCAgCGCTCAATAgACAC-3′ and reverse 5′-TCCCTCCCCCTTTCATA-3′ primers. The PCR-RFLP with AccI restriction enzyme was performed. In case of T allele, the final product of 541 bp remained undigested, while the A variant gave restriction fragments of 352 and 189 bp. For analysis of the C1650T BMPR1B, PCR with forward 5′-CTAAggCAgATgggAAAACTCA-3′ and reverse 5′-AAAAAggCAATCACAAAATACAgg-3′ primers was run. The amplicons were subsequently digested with DdeI restriction enzyme (MBI Fermentas). The PCR product (417 bp) was cut into fragments of 263 bp, 120 bp, and 33 bp, 1 bp for C allele, and 383 bp, 33 bp, and 1 bp for T allele. The A8474T ACVR1 polymorphism was analyzed with following primers: forward 5′-gCCCCgggAATCTgTgTCTT-3′ and reverse 5′-gggTgTgATgTgCCTATTTC-3′. The amplicons were subsequently digested with PsuI restriction enzyme (MBI Fermentas). The PCR product (556 bp) was cut into fragments of 364 bp, 192 bp for T allele, and remained undigested for A allele. The restriction fragments in each case were electrophoretically separated and visualized in ethidium bromide-stained 3% agarose gels.

Statistical analysis

Quantitative variables were compared between genotype groups with the Kruskal-Wallis test, followed by the Mann-Whitney test, whereas the Chi-square test or Fisher's exact test was used for qualitative variables. The general linear model was used for multivariate analysis. p<0.05 was considered statistically significant. Statistical analyses were performed with Statistica 9.1.

Results

The BL, BW, and BSA exponents used for the LVM indexes were determined as k=1.506, l=0.669, and m=1.038, respectively. Characteristics of the newborn cohort (n=210) (newborns and maternal factor characteristics) are shown in Tables 1 and 2. Mean BL, BW, and BSA values in boy newborns were significantly higher (borderline significance for BL) as compared to girls. We have shown the significant differences in values of LVM between boys and girls (Table 1). We recorded no significant differences in values of LVM indexes between boys and girls. There were no significant differences in T455C BMP4, A5659T BMPR1A, C1650T BMPR1B, and A8474T ACVR1 genotype or allele distribution between boys and girls (p>0.09), and the genotype distributions conformed to the expected Hardy-Weinberg equilibria (p>0.07).

Table 1.

Clinical and Echocardiographic Characteristics of the Newborns in Regard to Gender

	Total	Males	Females	p
n (%) n=210	210	115 (54.8%)	95 (45.2%)
BL (m)	0.56±0.03	0.56±0.03	0.55±0.03	0.059
BW (kg)	3.46±0.5	3.55±0.5	3.34±0.4	0.001
BSA (m²)	0.23±0.02	0.23±0.02	0.22±0.02	0.003
SBP (mmHg)	69.1±9.2	69.3±10.2	68.9±7.8	0.840
DBP (mmHg)	40.0±8.0	39.6±8.3	40.4±7.5	0.425
MAP (mmHg)	52.0±7.7	51.8±8.0	52.1±7.2	0.615
LVIDd (mm)	18.5±1.7	18.7±1.7	18.4±1.6	0.247
LVIDs (mm)	11.8±1.4	11.7±1.4	11.9±1.4	0.330
IVS (mm)	3.7±0.7	3.8±0.6	3.7±0.7	0.297
LVPW (mm)	2.8±0.7	2.8±0.7	2.7±0.7	0.212
LVM (g)	9.8±2.9	10.1±2.9	9.5±2.8	0.043
LVV (mL)	10.63±2.5	10.8±2.5	10.4±2.5	0.247
LVM/BL (g/m)	17.6±4.8	18.0±4.6	17.1±4.9	0.066
LVM/BW (g/kg)	2.9±0.8	2.9±0.8	2.8±0.8	0.707
LVM/BSA (g/m²)	42.5±11.5	43.0±11.3	41.9±11.7	0.260
LVM/BL^1.506	25.7±7.0	26.1±6.7	25.1±7.2	0.117
LVM/BW^0.669	3.9±1.1	3.9±1.1	3.8±1.2	0.411
LVM/BSA^1.038	54.9±14.8	55.4±14.6	54.1±15.1	0.322

BL, body length; BW, birth weight; BSA, body surface area; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; LVIDd, left ventricular internal diameter diastolic; LVIDs, left ventricular internal diameter systolic; IVS, interventricular septum; LVPW, left ventricular posterior wall; LVM, left ventricular mass; LVV, left ventricular volume.

Table 2.

Descriptive Data of the Newborn Population and Maternal Factors

N	210
Age (years)	28.2±5.4
BMI at beginning of pregnancy (kg/m²)	22.3±3.3
BMI at the end of pregnancy (kg/m²)	27.9±3.8
Gestational age at delivery (weeks)	39.3±1.0
Apgar 3 min	9.6±1.0
Apgar 5 min	9.8±0.7
Hypertension before/during pregnancy (%)	8.1
Diabetes mellitus before/during pregnancy (%)	2.9
Smoking habit history during pregnancy (%)	11.4
Parity (%) 1/2/3/4/5	62.4/25.2/9.0/1.4/1.9

BMI, body mass index.

There were no significant differences in values of LVM and LVM indexes in regard to BMP4, BMPR1A, BMPR1B, and ACVR1 genotypes assessed by Kruskal-Wallis test and Mann-Whitney test for dominant and recessive models of inheritance of the minor allele (Table 3). We conducted a separate analysis to compare the polymorphism distributions between the subjects in the lower tertile and the subjects in the upper tertile for LVM indexes, and no significant associations were found (data not shown). No significant associations were found between values of SBP and DBP, in regard to BMP4, BMPR1A, BMPR1B, and ACVR1 genotypes (Table 2). Additionally, we assessed the influence of the tested polymorphisms on the anatomical and functional parameters of the heart. A significant association was found between the 455C allele of BMP4 and increased LVIDs (p=0.004), whereas lower LAD was significantly associated with presence of the 1650T allele of BMPR1B (p=0.038) and the 455T allele of BMP4 (p=0.069, borderline significance). The presence of the allele 455C of BMP4 and 8474T of ACVR1 was significantly associated with decreased LVEF (p=0.0004 and p=0.046, respectively) (Table 4). A multivariate analysis using the general linear model shows that female gender and presence of the 455C allele of BMP4 and the 8474T allele of ACVR1 were independent significant predictors for decreased LVEF in newborn (Table 5).

Table 3.

The Blood Pressure and Left Ventricular Mass Values in the Newborns in Regard to T455C BMP4, A5659T BMPR1A, C1650T BMPR1B, and A8474T ACVR1 Gene Polymorphisms

	BMP4					BMPR1A					BMPR1B					ACVR1
	CC (n=67)	CT (n=112)	TT (n=31)	p _DO	p _RE	AA (n=100)	AT (n=91)	TT (n=19)	p _DO	p _RE	CC (n=47)	CT (n=119)	TT (n=44)	p _DO	p _RE	TT (n=99)	AT (n=97)	AA (n=14)	p _DO	p _RE
SBP (mmHg)	68.0±8.2	69.8±9.7	69.0±9.3	0.236	0.974	69.4±9.2	68.4±9.2	69.9±9.2	0.705	0.580	67.7±9.1	69.6±8.7	69.3±10.5	0.757	0.304	69.3±10.3	68.3±7.8	73.0±9.6	0.791	0.109
DBP (mmHg)	39.8±7.9	40.0±7.8	40.2±8.9	0.748	0.743	40.7±7.6	38.9±8.4	40.5±7.5	0.726	0.132	40.1±8.2	40.3±8.0	38.4±7.5	0.148	0.917	39.7±8.3	40.1±7.8	40.0±7.7	0.436	0.740
MAP (mmHg)	51.4±7.2	52.0±7.9	52.9±8.0	0.426	0.397	52.4±7.6	51.3±8.1	52.2±6.1	0.778	0.493	51.8±7.7	52.0±7.6	51.1±8.1	0.247	0.940	51.8±7.8	51.8±7.1	53.1±10	0.616	0.329
LVM (g)	10.1±3.3	9.7±2.4	9.7±3.2	0.520	0.445	9.7±2.7	9.8±2.9	10.6±3.2	0.255	0.353	10.0±3.1	9.6±2.6	10.2±3.4	0.719	0.573	9.7±2.9	9.8±2.8	10.7±3.2	0.323	0.123
LVM/BL (g/m)	18.1±5.8	17.3±4.0	17.6±5.4	0.731	0.695	17.5±4.7	17.5±4.8	19.0±5.5	0.275	0.490	18.0±5.1	17.3±4.5	18.2±5.5	0.634	0.461	17.4±4.8	17.6±4.8	19.1±5.6	0.452	0.130
LVM/BW (g/kg)	3.0±1.0	2.8±0.8	2.8±0.9	0.565	0.396	2.8±0.7	2.8±0.8	3.1±0.9	0.356	0.554	2.9±0.8	2.8±0.7	2.9±0.9	0.697	0.727	2.8±0.8	2.8±0.8	3.0±0.7	0.631	0.298
LVM/BSA (g/m²)	43.9±14	41.8±9.2	42.1±13	0.643	0.491	42.0±11	42.3±12	46.0±13	0.326	0.540	43.0±11	41.8±2.6	43.9±3.4	0.705	0.591	42.2±11.8	42.5±11	45.2±12	0.535	0.170
LVM/BL^1.506	26.3±8.4	25.2±5.7	25.9±7.8	0.886	0.864	25.6±6.9	25.4±6.8	27.6±7.9	0.300	0.567	26.2±7.2	25.2±6.5	26.4±7.8	0.613	0.446	25.4±6.9	25.6±6.9	27.7±7.9	0.584	0.137
LVM/BW^0.669	4.0±1.3	3.8±0.8	3.8±1.2	0.590	0.409	3.8±1.0	3.9±1.1	4.2±1.2	0.332	0.448	3.9±1.1	3.8±1.0	4.0±1.3	0.737	0.681	3.9±1.1	3.9±1.0	4.1±1.0	0.520	0.191
LVM/BSA^1.038	56.7±18	53.9±12	54.5±17	0.635	0.503	54.3±14	54.6±15	59.3±17	0.322	0.592	55.4±14	54.0±14	56.6±17	0.697	0.601	54.5±15	54.8±15	58.1±153	0.523	0.189

BMP, bone morphogenetic protein; p_DO, pdominant; p_RE, precessive.

Table 4.

The Echocardiographic Parameters of Heart Values in the Newborns in Regard to T455C BMP4, A5659T BMPR1A, C1650T BMPR1B, and A8474T ACVR1 Gene Polymorphisms

	BMP4					BMPR1A					BMPR1B					ACVR1
	CC	CT	TT	p _DO	p _RE	AA	AT	TT	p _DO	p _RE	CC	CT	TT	p _DO	p _RE	TT	AT	AA	p _DO	p _RE
LVIDd (mm)	18.6±1.6	18.5±1.6	18.2±1.8	0.328	0.585	18.6±1.7	18.4±1.6	18.7±1.6	0.661	0.760	18.2±1.5	18.6±1.7	18.7±1.6	0.089	0.444	18.4±1.7	18.6±1.6	19.2±2.1	0.166	0.157
LVIDs (mm)	11.9±1.3	11.9±1.4	11.0±1.5	0.004	0.347	11.9±1.5	11.7±1.3	11.9±1.5	0.773	0.506	11.5±1.3	11.8±1.5	11.9±1.3	0.233	0.541	11.6±1.4	12.0±1.2	11.2±1.4	0.127	0.624
IVS (mm)	3.8±0.8	3.7±0.5	3.8±0.7	0.394	0.467	3.7±0.7	3.8±0.6	3.8±0.8	0.281	0.121	3.8±0.7	3.7±0.6	3.8±0.6	0.473	0.838	3.8±0.7	3.7±0.7	3.6±0.5	0.203	0.263
LVPW (mm)	2.8±0.8	2.8±0.6	2.7±0.7	0.574	0.781	2.7±0.7	2.8±0.7	2.9±0.6	0.176	0.277	2.9±0.8	2.7±0.7	2.8±0.6	0.160	0.342	2.7±0.7	2.8±0.7	3.1±1.0	0.360	0.227
LAD (mm)	9.8±1.1	9.6±1.2	9.4±1.2	0.086	0.069	9.8±1.2	9.5±1.2	9.8±1.2	0.517	0.192	9.9±1.0	9.4±1.2	9.9±1.3	0.038	0.148	9.5±1.2	9.7±1.2	9.7±1.0	0.331	0.681
AoD (mm)	8.3±1.1	8.2±1.0	8.0±1.0	0.132	0.483	8.1±1.2	8.3±0.9	8.0±1.0	0.445	0.192	8.4±1.1	8.1±0.9	8.3±1.3	0.175	0.641	8.2±1.0	8.2±1.0	8.5±0.9	0.896	0.292
PAD (mm)	8.4±1.3	8.3±1.0	8.1±1.1	0.314	0.933	8.4±1.3	8.2±0.9	8.2±1.2	0.932	0.771	8.5±1.0	8.2±1.1	8.4±1.2	0.077	0.575	8.2±0.9	8.4±1.3	8.4±1.3	0.393	0.965
LVV (mL)	10.7±2.5	10.6±2.5	10.3±2.6	0.328	0.585	10.7±2.6	10.4±2.4	10.6±2.4	0.661	0.543	10.1±2.3	10.8±2.6	10.8±2.3	0.089	0.444	10.4±2.5	10.7±2.4	11.6±3.2	0.166	0.157
LVEF (%)	73.1±6.3	73.1±6.1	77.3±6.5	0.0004	0.371	73.4±6.7	74.0±6.5	74.3±4.3	0.331	0.134	74.0±6.8	73.7±6.7	73.6±5.4	0.845	0.630	74.2±6.5	72.8±6.4	76.9±5.2	0.305	0.046

LAD, left atrial diameter; AoD, aortic diameter; PAD, pulmonary artery diameter; LVEF, left ventricular ejection fraction.

Table 5.

General Linear Model Analysis of Left Ventricular Ejection Fraction Values in the Newborns

Independent variables	Regression equation coefficient ^a	95% CI	p
Gender (male vs. female)	+2.36	+0.68 to +4.03	0.006
BMP4 (455 CC+CT vs. TT)	−4.48	−6.82 to −2.14	0.0002
ACVR1(8474 TT+AT vs. AA)	−3.37	−6.71 to −0.026	0.048

Change of LVEF (%) associated with each independent variable.

Discussion

To the best of our knowledge, this is the first study that examined the ability of BMP4 and its receptors BMPR1A and BMPR1B and ACVR1 gene polymorphisms to modulate LVM and other parameters of the heart in newborns. The BMP expression has been linked to several aspects of embryonic development, including the establishment of the basic embryonic body plan, morphogenesis of organs, and regulation of cell proliferation and differentiation (Wozney, 1998). Over the past decade, several studies examined the effect of BMPs in cardiac differentiation and cardiomyocyte induction (Takei et al., 2009; Vidarson et al., 2010). Nothing is known about association of the BMP4, BMPR1A, BMPR1B, ACVR1 polymorphisms in modulating LVM or parameters of the human heart.

In the current study, we describe genetic analysis of BMP-4, BMPR1A, BMPR1B, and ACVR1 and the LVM at beginning of life, in newborns. In our study, no differences in genotype or allele frequencies were found between boys and girls. None of the single-nucleotide polymorphisms (SNPs) tested was significantly associated with the LVM. Possibly, the impact of this genetic variation of BMP-4, BMPR1A, BMPR1B, and ACVR1 may reflect changes in level of BMP-4 during the fetal and first days of life.

Experimental studies concerned with the BMP-4 in the development of the heart are well documented (van Wijk et al., 2007; Takei et al., 2009; Miyazono et al., 2010; Vidarson et al., 2010); however, the existence and effects of this proteins in the systemic circulation are not well known. Takei et al. (2009) showed that BMP-4 in combination with fetal bovine serum at the appriopriate time and concentration significantly promotes cardiomyocyte induction from human embryonic stem cells. In an animal model, Taha et al. (2007) demonstrated an inhibitory effect of BMP-4 on cardiomyocyte differentiation; however, Laflamme et al. (2007) reported a more efficient effect induced to differentiate into cardiomyocytes with BMP-4 supplementation. Son et al. (2011) showed that serum BMP-4 levels were significantly increased in individuals with obesity or metabolic syndrome.

Moreveor, activities of BMPs are regulated by various agonists and antagonists that have recently been reviewed (Yamada et al., 2000; Rutenberg et al., 2006; Singh et al., 2009). The T-box-containing transcriptional factor TBX2 plays a central role in the developing heart, the atrioventricular canal (AVC), chamber formation, and the level of BMP4/2 signaling plays an important role in the regulation of distinct T-box gene (including Tbx2) expression (Verhoeven et al., 2011). However BMP-4 levels or receptor activity may vary in fetal life, first day of life, or in adults. Unfortunately BMP-4 levels were not measured in the present study. Several studies have suggested that genetic variation in the renin-angiotensin system or insulin-like growth factor (IGF-1) or other are plausible factors that can lead to LVH (promote growth and hypertrophy); moreover, reports of this association are conflicting (Buck et al., 2009; Schreiber et al., 2011). The heterogenous results obtained in several studies in adults could be caused by ethnic differences of the studied populations and the impact of environmental factors, which can affect the development of increased LVM and modify the genetic factors in various ways. LVH is a continuous trait influenced by interaction between genetic, environmental, and lifestyle factors.

There is a lack of research concerning the factors influencing heart development during embryogenesis or first days of life, when external environmental factors have not yet had a marked impact. A few studies demonstrated the role of BMP-4 in cardiac differentiation. The formation of endocardial-derived mesenchyme is induced at stage 13 in chicken and E9.5 in mouse in AVC and later in the outflow tract (OFT) (Moreno-Rodriguez et al., 1997; Camenisch et al., 2002). In mice, BMP-4 is expressed in the myocardium of the AVC and OFT (Lyons et al., 1990). BMPR1A, BMPR1B, and ACVR1 are specific BMP receptors that are essential for BMP signaling and are widely expressed in various tissues (Miyazono et al., 2010).

The genetic variation in BMPR1A (rs7922846) and BMPR1B (rs1434536) leading to differentially regulates expression levels of these receptors (Böttcher et al., 2009; Saetrom et al., 2009). Böttcher et al. (2009) reported that genetic variation in BMPR1A may play role in the pathophysiology of human obesity, possibly mediated through an effect of mRNA expression.

Previous studies of mouse embryos with targeted disruption of genes encoding BMP ligands or receptors have shown cardiac OFT cushion hypoplasia (Delot et al., 2003; Lin et al., 2004). The BMPR1A-null mutant mouse dies at E9.5. No mesodermal forms in the mutant embryos suggest important role of the BMPR1A receptor in formation of mesoderm during gastrulation (Mishina et al., 1995). Disruption of the ACVR1 gene causes embryonic lethality in mouse, although the phenotype of ACVR1-null embryos is less than that of BMPR1A-null embryos (Gu et al., 1999). Nomura-Kitabayashi et al. (2009) in their study suggest that BMP signaling to cardiac neural crest cells is required for growth of the OFT cushions, and hypoplasia of the OFT cushions was associated with reduction in cell proliferation in the PO-Cre-deleted BMPR1 mutants. This study provides definitive evidence that the outflow cushions perform a valve-like function for survival of the early mouse embryos. Kaartinen et al. (2004) reported that the type 1 BMP receptor and ALK2 play an essential cell autonomous role in the development of the cardiac OFT and aortic-arch derivatives.

We also evaluated echocardiographic parameters of the heart in newborns. We showed that LVEF was significantly associated with both the BMP4 and ACVR1 genotypes, which may indicate a link to the impact of genetic variation of BMP4 and ACVR1 in development of the heart. This finding may be associated with identified altered cardiac parameters, significantly increase LVIDs with the ACVR1 C allele, and borderline significance increase LAD with T allele of BMP4. In addition, we did not find any significant association between BP and genetic variation of BMP4 and its receptors BMPR1A, BMPR1B, and ACVR1. Capasso et al. showed functional effect of the nonsynonymous SNP 6007 C/T (rs 17563) resulting Val152Ala amino acid change. The 6007 C/T substitution leads to a different mRNA structure and function. Authors show that Epstein-Barr virus transformed lymphoblastoid cell lines from healthy controls carrying the T allele had higher mRNA levels of BMP-4 than C-allele carriers, and the T allele was predicted to affect mRNA stability. Additionally, it suggests that the predicted functional polymorphism of BMP4 affects the risk to development of cutaneous melanoma (Capasso et al., 2009). The 6007 C/T polymorphism has recently been associated with other phenotypes such as bone mineral density (Choi et al., 2006) and hip bone density in postmenopausal women (Ramesh Babu et al., 2005). However to the best of our knowledge, there has been no published study examining the association between BMP4 and its receptor polymorphisms and parameters and LVM of the heart.

The strength of our study is determined by the well-documented cohort of polish newborns, and the sonographic examination of the heart dimension was performed according to protocols on the literature that gives reproducible results, and the heart dimensions were similar to those of small group of Slovakia healthy newborns who were studied at the 5 days of life (Jurko, 2004). The potential weakness of the present study stems from the fact that our sample was not systematically tested for genetic heterogeneity. Berger et al. (2006) showed that population substructures can be detected even in an apparently homogenous population. The confounded association resulting from stratification or admixture within can be reduced by matching by the geographical region of ethnic origin (Cordell et al., 2005). All newborns in our study came from the West Pomeranian Region, and the ethnic admixture cannot be completely ruled out. However, we believe that it is very unlikely that a cohort of newborns is a biased sample. Also, the relatively small amount of studied newborns is a limitation, and our study should therefore be interpreted with caution the result, because the result can be accidental. However, the effect of genetic variation of the BMP4, and its receptors on the LVM and the dimension of the heart remains to be elucidated, and replication of findings studies in different homogenous populations are needed. The BMPs are involved in cardiac development and are one of the inducers of cardiac differentiation (van Wijk et al., 2007). It is possible that the effect of these SNPs associated with an impaired LVEF is more strongly expressed in the coexistence of heart defects. It seems that association of decreased LVEF with a BMP4 455C allele and an ACVR1 8474T allele is in line with previous knowledge of the heart development, BMPs specifically related to the development of OFT. Conwey et al. (2011) suggested an important role of BMP ligands in heart valve formation and valvular heart disease. In humans, the levels of BMP2 are significantly detectable in diseased valves, and BMP4 levels are highly abundant in adult cardiac valves (Conwey et al., 2011). This expression of BMP ligands in adult heart valves is in accordance with the report showing an in vivo genetic interaction between BMP2 and BMP4 as indicated by valve abnormalities in BMP2^+/− and BMP4^+/− adult mice (Uchimura et al., 2009). Smith et al. (2009) have screened genes known to be involved in improper atrioventricular septum (AVS) development and identified novel heterozygous missense mutations in ALK2 in patients with AVS defect, which associates with congenital heart defects. To date, it is not known that genetic variation in BMP4 and their receptors BMPR1A, BMPR1B, and ACVR1 may in fact contribute to parameters or LVM of heart. It is possible that a stronger interaction effect may be more apparent after many years in adult life, in relation to environmental and cardiovascular risk factors.

However, although the roles of the BMPs in differentiation of heart are well documented, the effects of genetic variations of BMP and its receptors on LVM and parameters are more complex and less defined. In the present study, our findings demonstrated lack of associations between LVM, values of BP, and BMP4, BMPR1A, BMPR1B, and ACVR1 genotypes. Although, we found that the 455C allele of BMP4 and 8474T allele of ACVR1 may play a role as significant predictors for decreased LVEF in newborns.

These are preliminary data, and further studies are needed in other populations, with different ethnicity and age to explain the effect on LVM and parameters of the heart.

Footnotes

Acknowledgments

This research received no specific grant from any funding agency, commercial, or not-for-profit sectors.

Author Disclosure Statement

No competing financial interests exist.

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