left ventricular hypertrophy ( lvh ) and increased left ventricular mass ( lvm ) are strong risk factors for cardiovascular disease and morbidity .
cardiac hypertrophy is characterized by increased cell size , cardiac remodeling of myofilaments , and increased expression of fetal genes .
lvm results from a complex of interaction between genetic , environmental , and lifestyle factors .
increased knowledge concerning genes involved in the modulation of lvm will lead to a better understanding of the etiopathogenesis of lvh .
calcium ( ca ) is arguably the most important messenger in cardiac muscle and plays a central role in regulating contractility , gene expression , hypertrophy , and apoptosis .
it has been well described that ca transient movements regulate the transcription and gene expression that characterize the hypertrophic response of cardiomyocytes [ 2 , 3 ] .
the levels of ca are precisely controlled . a major sensor and mediator of intracellular ca transient movements is calmodulin ( cam ) . the cacam complex binds and activates enzymes , including protein kinases , protein phosphatases , phospholipases , nitric oxide synthases , and endonucleases .
three ca calmodulin dependent enzymes have significant roles in cardiac function : ca calmodulin - dependent protein kinase ( camk ) , protein phosphatase 2b ( calcineurin , can ) , and myosin light - chain kinase ( mlck ) .
camk and can have been shown to play key and often synergistic roles in transcriptional regulation in cardiomyocytes .
it has been suggested that camk regulates gene expression via activation of several transcription factors [ 5 , 6 ] .
ca - cam - dependent kinase ii ( camkii ) , a major cam target protein , is a uniquely regulated multifunctional regulatory enzyme .
there are several studies indicating the major role of camkii involvement in cardiac hypertrophy and heart failure . in hypertrophic myocardium of animal models ,
experimental studies have demonstrated that transgenic mice overexpressing nuclear camkii have increased incidence of cardiac hypertrophy .
inhibition of nuclear camkii activity causes transgenic mice to have smaller hearts than their nontransgenic littermates .
thus , any genetic variants that directly affect cam gene expression or function are promising as candidates involved in modulating lvm .
cam is encoded by a multigene family consisting of three members : calm1 , calm2 , and calm3 .
discovered that 2622a > g and 3001g > a polymorphism , both located in intron 1 , may be associated with osteoarthritis in the japanese population .
liu et al .   indicated that calm2 is a candidate gene for primary open - angle glaucoma . to date , only vasan et al
.   have demonstrated , in meta - analysis , the correlation between calm2 polymorphism rs7565161 and echocardiographic diameter lvm in adults .
the guanine to adenine transition at nucleotide position 474955027 ( g.474955027 g > a , rs7565161 ) of human chromosome 2p21 is intergenic , adjacent to the calm2 gene .
however , there are no reports which have focused on the association of intergenic adjacent calm2 polymorphisms with left ventricular mass in newborns .
the factors influencing heart development during fetal life or first days of life , when external environmental factors such as diet , lifestyle , smoking or diseases have not yet had a marked impact , are still being sought .
we hypothesize that adjacent intergenic calm2 polymorphism could potentially modify lvm during fetal life and in the first period of life in newborns . in the present study
, the relationships between g.474955027 g > a ( rs7565161 ) being adjacent intergenic calm2 gene polymorphism and lvm in a population of polish newborns have been investigated .
the population included 206 consecutive healthy polish newborns ( 92 females and 114 males ) , born after the end of the 37th week of gestation ( from 37 to 40 weeks ) .
mothers in this study were healthy without any complications such as preeclampsia or eclampsia , and there was no fetal growth restriction .
newborns in this study were appropriately grown for their gestational age ( defined as birth mass above the 10th centile ) .
exclusion criteria were twins , intrauterine growth restriction , chromosomal aberrations and/or congenital malformations , or 
small for gestational age ,  that is , below the 10th centile body length ( bl ) , birth weight ( bw ) , or head circumference ( hc ) . at birth , cord blood ( 500  l ) 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 using the following equation : ( 1)bsa=[bl(cm)bw(kg)3600 ] . a diascope oscillometer ( artema )
was used to determine systolic and diastolic blood pressure ( sbp or dbp , resp . ) , and only one of the investigators performed all of the blood pressure ( bp ) measurements using a standardized protocol .
the smallest cuff size that covered at least two thirds of the right upper arm and encompassed the entire arm was selected .
newborn measurements were taken at least one and a half hours following their last feeding or medical intervention .
an appropriately sized cuff was applied to the right upper arm , and the newborn was then left undisturbed for at least 15 minutes or until the infant was sleeping or in a quiet awake state .
echocardiographic measurements in newborn on the 3rd day after delivery were made by one pediatric cardiologist .
two - dimensional m - mode echocardiography was performed using an acuson sequoia 512 unit ( usa ) , equipped with a 24  mhz imaging transducer .
measurement techniques were consistent with the american society of echocardiography conventions . in a parasternal long - axis view , lvidd - left ventricular internal diameter - diastolic ,
lvids - left ventricular internal diameter - systolic , lvpw - left ventricular posterior wall thickness at end diastole , ivs - thickness of interventricular septum at end diastole , lad - left atrial diameter , aod - aortic diameter , pad - pulmonary artery diameter , lvv - left ventricular volume , and lvef - left ventricular ejection fraction were measured ( using m - mode formulas ) .
the left ventricular masses ( lvm ) were calculated from the echocardiographic left ventricular dimension measurements , using the penn convention with the equation modified by huwez et al .  
( 1994 ) as follows:(2)lvm=1.04[(ivst+lvpwt+lvid)3lvid3 ] , 
 where ivst , lvpwt , and lvid denote interventricular septal thickness , left ventricular posterior wall thickness , and left ventricular internal dimension , respectively . to accurately determine and standardize the left ventricular mass ,
the lvm was indexed with respect to body length ( lvm / bl ( g / m ) ) , body weight ( lvm / bw ( g / kg ) ) , and body surface area ( lvm / bsa ( g / m ) ) , respectively .
genomic dna from cord blood was isolated using the qiaamp blood dna mini kit ( qiagen , germany ) , according to the manufacturer 's protocol . for the analysis of the intergenic g
> a calm2 ( rs7565161 ) polymorphism , a polymerase chain reaction - restriction fragment length polymorphism ( pcr / rflp ) method was designed with the following primer pair : forward 5-agggcctgcaatctaat-3 and reverse 5-atataatccccaccttcag-3 ( tib mol biol , pozna , poland ) .
the calm amplicons were subsequently digested with the acii restriction enzyme ( mbi fermentas , vilnius , lithuania ) .
the pcr product of 417 base pairs ( bp ) was cut into fragments of 258  bp , 137  bp , and 22  bp in the presence of the g allele and into fragments of 395  bp and 22  bp in the presence of the a allele .
restriction fragments in each case were electrophoretically separated and visualized in midori green - stained ( nippon genetics ) 3% agarose gels . to verify the results ,
each calm2 amplicon was cleaned with genelute pcr clean - up kit ( sigma ) .
sequencing was performed according to the dideoxy sanger method in a geneamp pcr system 9700 thermal cycler ( applied biosystems ) , using bigdye terminator v3.1 cycle sequencing kit ( applied biosystems ) .
afterwards , samples were purified ( bigdye xterminator purification kit , applied biosystems ) , and 20  l deionized formamide ( applied biosystems ) was added .
the sequencing results were read using sequencing analysis software v5.1 ( applied biosystems ) . in each case , the result obtained with pcr - rflp method was identical with that appropriate one from sequencing .
the divergence of calm2 genotypes frequencies from hardy - weinberg equilibrium was assessed using  tests , and the distribution of each quantitative variable was tested for skewness .
quantitative data were presented as means  sd and analyzed either by student 's t - test or by one - way anova . left ventricular mass indexes ( lvmis ) were tested for association with genotype using multivariate analysis ( ancova ) in order to adjust for possible confounding factors : neonatal ( gestational age , gender , sbp , and apgar at three minutes ) and maternal ( age , bmi at the beginning and the end of the pregnancy , smoking status , and hypertension status ) .
all data were analyzed with statistica ( data analysis software system , version 10.0 , statsoft , inc .
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 ) .
mean bw and bsa values in boy newborns were significantly higher than those in girls .
69 gg calm2 homozygotes ( 33.5% ) , 95 ga heterozygotes ( 46.1% ) , and 42 aa homozygotes ( 20.4% ) were identified .
there were no significant differences in calm2 genotype or allele distributions between boys and girls ( p = 0.273 , and p = 0.107 , resp . ) .
the calm genotype distributions conformed to the expected hardy - weinberg equilibrium ( p = 0.396 ) .
lvmi measurements were tested for association using multivariate analysis ( ancova ) in order to adjust for possible confounding factors , after adjusting for newborn ( gestational age , gender , sbp , and apgar at three minutes ) and maternal ( age , bmi at the beginning and the end of the pregnancy , smoking status , and hypertension status ) parameters .
we revealed a significant association between lvmis ( lvm / bw in recessive and additive modes and the calm2 polymorphism ) .
the carriers of the g allele of the calm2 polymorphism had significantly higher lvm / bw values , when compared with newborns homozygous for the a allele ( 3.1  g / m versus 2.5  g / m , padjusted = 0.036 , resp . ) .
the ag genotype of calm2 was associated with the highest values of lvm / bw , exhibiting a pattern of heterozygote advantage ( 2.9  g / kg versus 3.1  g / kg versus 2.5  g / kg , padjusted = 0.037 ) ( figures 2 and 3 ) .
carriers of the a allele did not differ in lvm indexes ( figure 1 ) .
an association was observed between genotype and dbp  90 percentile ( p = 0.027 ) .
carriers of the allele a of the calm2 gene had an increased incidence ( % ) sbp  90 percentile ( p = 0.027 , 76.2% versus 23.8% ) .
lastly , the calm2 polymorphism was significantly correlated with maternal history of gestational age ( p = 0.019 ) .
genetic factors are estimated to be responsible for between 30% and 70% of cardiac mass variance .
studies in twins [ 20 , 21 ] and populations [ 22 , 23 ] showed that lvm is under genetic control .
the present study in a cohort of newborns has demonstrated for the first time the significant association between variants of the intergenic adjacent calm2  polymorphism and increases in lvm indices in newborns .
therefore , to minimize the disparities , we carefully selected homogenous group of full - term newborns . to accurately determine lvm , we used lvm in relation to bsa , bl , and bw , which are reported to be more appropriate .
it should be emphasized that confounding factors such as especially gestational age may play a role in the development of lvm in fetus .
the fetal programming hypothesis states that , for example , birth mass in newborns may be partially related to maternal factors . in this study ,
the ag genotype of intergenic adjacent calm2 polymorphism was associated with the subtle higher values of lvmi , exhibiting a pattern of heterozygote advantage in results .
what is important , in our study , the carriers of the g allele have higher lvm than the carriers of the a allele . these results were similar to those of a large cohort of adults , who were studied by vasan et al . . in this meta - analysis of echocardiographic data associated with interindividual variation in cardiac dimension ,
it should be mentioned that total sample included those with coronary heart disease , peripheral vascular disease , valvular heart disease , stroke , and circulation heart failure .
current results exhibit a pattern of heterozygote advantage , as heterozygote newborns had significantly higher lvmi than the carriers of homozygote genotypes .
the heterozygote advantage hypothesis attributes heterosis to the superior fitness of heterozygous genotypes over homozygous genotypes at a single locus .
some studies suggest that heterozygote advantage is a favorable process , the positive selection over evolution , as a natural consequence of adaptation role of variation in gene [ 2628 ] .
however , in light of vasan 's study , the feature that may be potentially beneficial in early life may lead to predisposition to increase or hypertrophy left ventricular in adults .
williams suggested  antagonistic pleiotropy  theory , which assumes that some genes responsible for increased fitness in the children , fertile organism contribute to decreased fitness in adults . we conclude that this theory may be relevant here .
we hypothesized that genetic variation in the intergenic adjacent calm2 gene polymorphism , analogously to the other common polymorphisms in developmental genes , may cause minor changes in the development or modulation of lvm in newborns .  
we continue observing our population and consider conducting follow - up , which will show in later years whether the heterozygotes have a predisposition to develop left ventricular hypertrophy or not .
however , our results require confirmation in further independent large studies . the connection between calmodulin and modulating cardiac contractile function and growth is well documented [ 30 , 31 ] .
otherwise , in an experimental animal study , the protein level of cam was shown with a relatively high level of calmodulin appearing on gestational days 14 - 15 , followed by a steady but significant decrease at birth and during the first week of postnatal life .
it is reported that specific elevation of cam levels directly affects the rate of cell proliferation .
also , gillett et al . , in animal study ( fetal sheep ) , showed that increased calm2 mrna expression levels may reflect an important role for calmodulin in expansion - induced fetal lung growth . a study
performed in human showed that genes encoding calmodulin ( calm1 , calm2 , and calm3 ) are involved in increasing proliferation [ 35 , 36 ] .
although such knowledge indicates the important role of calmodulin - dependent protein kinases and phosphatases in regulating cardiac hypertrophy , the role of genetic variation in cam in the physiology of the development human heart has not been clarified .
our results suggest that genetic variation of calm2 may be partly involved in regulating myocardial cell proliferation and growth , during embryogenesis and in the first days of life .
it is possible that genetic variation in cam may have been involved in regulating the activity or / and levels in serum kinases and phosphorylases ( e.g. , camkii , calcineurin ) during fetal life . in the current study , we investigated healthy newborns born at full term .
our previous studies reported that ras ( renin - angiotensin system ) or bmp4 ( bone morphogenetic protein 4 ) and bmpr1a ( bone morphogenetic protein , receptor type 1a ) genetic variation may partially account for subtle variation in lvm or parameters or heart parameters in newborns [ 37 , 38 ] . to the best of our knowledge ,
the recent results have never been replicated , and therefore the replication of the study findings in different population is needed . additionally , an association between calm2 polymorphism , and dbp and map was found , but the mechanism by which this might act is not clear .
blood pressure is regulated by multiple neuronal , hormonal , renal , and vascular control mechanisms , as well as genetic and environmental factors .
it is also dependent , inter alia , on the force of contraction of the heart muscle which is connected indirectly to the left ventricular mass .
there are many known candidate genes that have huge influence on the blood pressure or development of hypertension [ 3941 ] .
however , the mechanisms of interaction intensifying effects of these genes are still researched . it is known that changes in signaling mechanisms in the endothelium of vascular smooth muscle ( vsm ) cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increase in vascular resistance .
vascular tone that is a component of regulating blood pressure can be controlled indirectly by different genes activity .
an experimental study demonstrated findings that expression levels of several cam - related proteins are changed in vascular tissues and suggested that cam - related proteins might be at least in part related to the pathogenesis of hypertensive vascular diseases .
a recent study reported that camkii inhibitor inhibited the ang ii - induced vascular smooth muscle cell hypertrophy .
however , the role of cam - related protein in vascular pathophysiology is not yet fully clarified .
, we have shown that the intergenic adjacent calm2 polymorphism is associated with left ventricular mass in newborns
. this might be the consequences of variation in cell proliferation and growth , and this finding may indicate an important role for genetic variation of calm2 in expansion - induced heart growth in fetal life .
 calmodulin ii ( calm2 ) gene polymorphism might be responsible for the variation in the left ventricular mass amongst healthy individuals . 
 the aim was to evaluate the correlation between left ventricular mass ( lvm ) and g.474955027g > a ( rs7565161 ) polymorphism adjacent to the calm2 gene . 
 
 healthy polish newborns ( n = 206 ) were recruited . 
 two - dimensional m - mode echocardiography was used to assess lvm . 
 polymorphisms were determined by polymerase chain reaction - restriction fragment length polymorphism and sequencing analyses . 
 
 the carriers of the g allele of the calm2 polymorphism had significantly higher left ventricular mass / weight ( lvm / bw ) values , when compared with newborns homozygous for the a allele ( 3.1  g / m2 versus 2.5  g / m2 , padjusted = 0.036 ) . 
 the ag genotype of calm2 was associated with the highest values of lvm / bw , exhibiting a pattern of overdominance ( 2.9  g / kg versus 3.1  g / kg versus 2.5  g / kg , padjusted = 0.037 ) . 
 
 the results of this study suggest that g > a calm2 polymorphism may account for subtle variation in lvm at birth . 
 