Heritability and linkage study on heart rates in a Mongolian population

Elevated heart rate has been proposed as an independent risk factor for cardiovascular diseases, but their interrelationships are not well understood. In this study, we performed a genome-wide linkage scan in 1,026 individuals (mean age 30.6 years, 54.5% women) from 73 extended families of Mongolia and determined quantitative trait loci that influence heart rate. The DNA samples were genotyped using deCODE 1,039 microsatellite markers for 3 cM density genome-wide linkage scan. Correlation analysis was carried out to evaluate the correlation of the covariates and the heart rate. T-tests of the heart rate were also performed on sex, smoking and alcohol intake. Consequently, this model was used in a nonparametric genome-wide linkage analysis using variance component model to create a multipoint logarithm of odds (LOD) score and a corresponding P value. In the adjusted model, the heritability of heart rate was estimated as 0.32 (P < .0001) and a maximum multipoint LOD score of 2.03 was observed in 77 cM region at chromosome 18. The second largest LOD score of 1.52 was seen on chromosome 5 at 216 cM. Genes located on the specified locations in chromosomes 5 and 18 may be involved in the regulation of heart rate.


Epidemiological Evidence
Epidemiological and clinical studies suggest that elevated heart rate is a potential risk factor for a variety of cardiovascular diseases including atherosclerosis, coronary artery disease, myocardial infarction, arterial hypertension and heart failure. 2,5,9,12,14,25,29 The elevated heart rate is closely associated with age, high blood pressure, body mass index, smoking, alcohol consumption, high cholesterol level, and physical inactivity. But, even after adjusting for other potential risk factors, elevated heart rate remains significantly associated with cardiovascular disease, mortality and all cause mortality in patients with cardiovascular disease as well as general population. 7, 9-11, 13, 15 16, 26,28, 36 The normal weight individuals with lower resting heart rate have lower levels of cardiovascular disease risk factor and mortality. 42 In the latter study those 5,713 working men were participated and followed up for 23 years. The sudden death and total mortality was increased with high heart rate, and this association was still significant after adjusting for potential confounding factors such as blood pressure, age, and physical inactivity. 45 Moreover, in a study those 125, 000 men and 96, 000 women were participated, a high heart rate was related with cardiovascular mortality and the hazard ration was 1.59. 46 The prognostic power of high heart rate for cardiovascular disease was clear not only within the general population but also among patients with acute coronary syndrome, diabetes mellitus, high blood pressure, acute myocardial infarction and heart failure. In a recent survey of 10, 267 patients with acute coronary syndromes, a higher initial and delayed heart rate highly predictive of higher short-and long-term mortality in patients with acute coronary syndromes. 43 In the Coronary Artery Surgery Study which enrolled 24,913 subjects who suspected cardiovascular diseases and followed up for 14 years. In this study, a total mortality and cardiovascular mortality were significantly associated with high heart rate in patients with coronary artery disease. 7 Moreover, in the Framingham heart study which enrolled patients with high blood pressure and followed up for 36years. In this study, after adjusting for age, systolic blood pressure, smoking, body mass index and other potential confounding factors, odds ratios for all cause mortality was 2.18 and 2.14 respectively, in men and women. 11 Overall, these data provide that the significant association between high heart rate and cardiovascular disease, and mortality.

Genetic Evidence
A number of twin and family studies have reported that genetic factors influence the regulation of heart rate. 18-23, 34, 39, 40 There is a genetic component in heart rate generation and heart rate variability in monozygotic and dizygotic twin pairs. 37 A significant genetic regions contributing to heart rate variability has been identified on chromosome 15 at 62 cM and chromosome 2 at 153 cM. 35 In a twin study, a gender difference in heart rate variability between men and women is demonstrated. Women have greater heart rate variability than men even after controlling for a large number of potential confounders such as age, oral contraceptive use and menopausal status. 48 In the recent meta-analysis of genome-wide scans for study networks that enrolled Caucasians and African-Americans, the replication between various ethnic groups as well as the study networks with low heterogeneity has been identified on chromosome 5p13-14. 18 Moreover, a polymorphism in the B1 adrenergic receptor was determined to be significantly related to heart rate in a hypertensive cohort. 47 In the Hypertension Genetic Epidemiology Network The GENDISCAN study is committed to incorporating most of methodological issues of complex diseases using genetically homogeneous population and emphasizing the quantitative phenotypes. 49 The purpose of this study was to assess the heritability of heart rate and heart rate variability and to identify susceptible loci for heart rate variability in an Asian population. Recognition of the genetic determinants of heart rate and heart rate variability may provide additional insight into the pathophysiology of the cardiovascular disease and mortality.

Subjects
One thousand and two Mongolian individuals (54.5 % women) from 95 extended families from Dornod, Mongolia were genotyped.
Informed consents to participate in the study were obtained from all subjects. The protocol for the GENDISCAN study was approved by the institutional review board (IRB) of Seoul National University of Korea (approval number, H-0307-105-002).

Heart Rate and Covariates
Four consecutive measurements of the heart rate and blood pressure were made on both arms of each participant while seated, employing a standard electronic sphygmomanometer. When the four blood pressure measurements differed by more than 10 mmHg, a fifth measurement was made and the lowest four were used in the analyses.
The larger means of each set of measurements were accepted as heart rate and the blood pressure respectively. Measurements in children from age 10 to 16 were made using appropriate cuffs and a mercury sphygmomanometer. Data on potentially confounding factors for heart rate such as age, sex, smoking, and alcohol consumption were collected through interviews performed by trained interviewers. The standing height in centimeter (cm) and weight in kilograms (kg) were measured, and body mass index (BMI) was calculated in kg/m².

Genotyping
For those who agreed to be genotyped, genomic DNA was extracted from peripheral venous blood leukocytes using standard procedure and genotyped using deCODE 1,000 STR marker platform.

Correlation analysis
We carried out a correlation analysis to evaluate the correlation of covariates (age, sex, smoking, alcohol intake, brachial systolic pressure, brachial diastolic pressure, brachial mean arterial pressure and body mass index) and heart rate, and Pearson correlation coefficients were identified. T-tests of heart rate were also performed on the three categories, which are sex, smoking and alcohol intake, by using SAS statistical software version 9.1.

Identity by Descent (IBD) Calculation
For the family relationship, nonpaternity was examined using PEDCHECK. Relationships other than paternity were checked using average identity by descent (IBD)-based method by PREST. After correcting pedigree error and mendelian errors, non-mendelian errors were examined and corrected using SimWalk. After pedigree and genotype errors were corrected, IBD matrices between every relationship pair were calculated. IBD matrix for single marker was calculated by SOLAR, and multipoint IBD (MIBD) matrices were computed on every 1 cM distance using Markov chain Monte Carlo method by LOKI. We used Haldane's mapping function to convert map distances into recombination fractions.

Heritability Estimation
Genetic components of selected phenotypes were estimated in terms of heritability. Narrow sense heritability, defined as the proportion of total phenotypic variation due to additive genetic effects, was calculated. Age-sex adjusted phenotype (adjusted for age, sex, agesquare, product of age and sex, product of age-square and sex) was estimated by SOLAR for quantitative traits.

Heart rate (Genome-wide Linkage Analysis)
To reduce the type I error from deviated distribution, original values were normalized using Z-transformation. The genetic variance of heart rate was decomposed into specific additive genetic effects from specific markers (Quantitative trait loci, QTL) and non-QTL given by: Where Ω is the covariance matrix of the entire family, π is a matrix of the proportions of the specific QTL that the relative pairs share as IBD, Φ is a kinship matrix, I is an identity matrix, σ 2 QTL is specific QTL effects of the genetic markers, σ 2 QTL N − is residual genetic effect, and e 2 is random environmental effects and errors. The likelihood that QTL effects can be estimated were compared with the likelihood of null hypothesis that specific QTL effect equals zero. The logarithm of odds (LOD) score between likelihood of null and alternative hypothesis were used to test the significance of linkage results. The multipoint linkage analyses were performed using SOLAR. In the genome-wide scans, age, sex, age 2 and the interactions between them retained in the models as covariates at p <0.10. Because variance composition method is sensitive to outliers, multivariate residual kurtosis in each analysis retained less than 0.8 thereby avoiding type 1 error.

III. RESULTS
Demographic and pedigree characteristics of the data set and the covariates are presented in table 1.   In the adjusted model, the heritability of heart rate was estimated to be 0.32 (p <.0001) (Table3).

IV. DISCUSSION
It has long been known that heart rate is under the control of the parasympatic and sympathetic nervous system, and that heightened sympathetic tone increases the heart rate. 1,10,26,27 In more recent studies, they pointed out that genetic components may play an essential role in the regulation of heart rate variability. 34,39 The Framingham heart study also demonstrated that genetic factors are involved in heart rate variability. 35 In the GENDISCAN study, the degree of heritability of heart rate was 0.32. This value is somewhat higher than the figure (0. 21) reported for Framingham Heart Study participants, 34 but is lower than the figure (0.41) reported for participants of Netherlands Twin Register. 17 All the three studies provide evidence for a strong genetic component in heart rate variability.
The GENDISCAN project is the first largest family studies in Asian population in Asia. The unique property of this study is that subjects are members of large extended families in isolated rural area. 49 We showed a peak with a maximum LOD of 2.03 on chromosome 18 at 77 cM. The SLC14A2 (solute carrier family 14 urea transporter) gene which lies near this loci, encodes UT-A protein expressed in the heart. 8 The expression of UT-A protein in failing left ventricle is 1.4-4.3 fold to that in normal nonfailing ventricle. 8 The LIPG (endothelial lipase precursor) gene also lies on chromosome 18 at 77cM. This gene encodes the protein that process substantial phospholipase activity and plays an important role in lipid metabolism. More recently, it has been known that the significant association between 584C/T SNP of LIPG gene and an acute myocardial infarction independent of HDL-C levels in a Japanese population. 33 It is also of interest that chromosome 5 yielded the second largest linkage peak which corresponds to its 216 cM region. An analysis of the database indicated that the chromosomal region 216 cM on chromosome 5 contains NSD1 (nuclear receptor SET domain containing gene1) gene. The protein encoded by NSD1 gene enhances transactivation of androgen receptor. It has been reported that the intragenic mutation of this gene is associated with the high frequency of congenital heart defects or heart conduction. 3 The F12 (coagulation factor XII) gene also located at 216 cM on chromosome 5. This gene encodes coagulation factor XII which circulates in blood as a zymogen. The C46T polymorphism of F12 is associated with a reduction of plasma FXII levels and a development of myocardial infarction, particularly in hypercholesterolemic patients. 30 The homozygosity for the C46T polymorphism of the F12 gene is significantly associated with high risk of coronary artery disease in the Spanish population. 31 The ZC3H12A (zinc finger CCCH type containing 12A) gene was located at 62cM on chromosome 1, which is a monocyte chemotactic protein-1 (MCP1) induced protein. MCP1 mediated inflammation plays a critical role in the development of cardiovascular disease. 1,4,41 In a recent study, it has been reported that MCP1 causes cell death of cardiomyocytes and plays an important role in the development of ischemic heart disease. 41 The ET2 (endothelin 2) gene at 62 cM on chromosome 1 was founded. High circulating plasma levels of ET have been reported in essential hypertension. 32 The polymorphism in the ET2 gene influences the hypertension when blood pressure is assessed as a quantitative trait. 32 In addition, ET2 messenger transcript is known to be present in varying quantities in human heart and kidney, two of the main target organs of hypertensive complications in severe hypertensives. Thus, the variability of ET2 tissue expression and, in particular, the ET2 linkage data makes it an ideal candidate gene for human essential hypertension. 32 The E2F3 gene at 39 cM on chromosome 6 encodes a transcription factor of the E2F family. This family protein has an important role of controlling tumor suppressor proteins and cell cycle.
The region at 21 cM on chromosome 3 has includes CAV3 gene, which encodes caveolin-3 muscle-specific protein. This finding is interesting in the background of evidence that CAV3 null mice show perivascular fibrosis, cellular infiltration in cardiac tissue and cardiac myocyte hypertrophy, which exhibits inter-and intrafamilial variations ranging from benign to malignant forms with high risk of cardiac failure and sudden cardiac death. 4,6,40

V. CONCLUSION
We identified susceptible loci for heart rate variability in an Asian population. This study strongly indicates that heart rate is controlled by genes mapped to several loci.
We believe characterization of genes that affects heart rate variability can lead to unraveling of the pathogenetic mechanisms underlying heart rate variability and its association with cardiovascular disease and to rational therapeutic interventions.