pulse rate , also called heart rate , is the number of heartbeats per unit of time , commonly used as beats per minute ( bpm ) .
pulse rate is varied in accordance with the body 's need for oxygen changes , such as during exercise or sleep .
since various health - related phenotypes are closely correlated with pulse rate [ 1 - 3 ] , measuring pulse rate is commonly used for diagnosing and accessing medical conditions .
pulse rate is known to be positively correlated with triglycerides and negatively correlated with high - density lipoprotein cholesterol .
metabolic syndrome is associated with poor exercise capacity and poor heart rate recovery in patients who have coronary disease . moreover ,
high pulse rate is negatively correlated with lifespan in mammals , including humans . despite the importance of pulse rate as a risk factor for various diseases ,
however , few genes are known to be associated with pulse rate , and the underlying mechanism for pulse rate remains unraveled .
we previously reported pulse rate - associated loci from the korea association resource ( kare ) genomewide association study ( gwas ) , comprising 8,842 samples in the korean population . in this study
, we extended the previous study and reexamined the relevance of pulse rate - associated single - nucleotide polymorphisms ( snps ) from an additional gwas of 3,703 korean samples .
in the discovery stage , the association results for pulse rate were obtained from the kare study .
subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .
the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .
pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts , the first measurement of the participants , who rested for 5 min ,
the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,
genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,
santa clara , ca , usa ) . from 4,302 genotyped samples , we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .
association analyses were performed using plink 
 ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .
the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .
the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .
meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .
in the discovery stage , the association results for pulse rate were obtained from the kare study .
subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .
the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .
pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts ,
the first measurement of the participants , who rested for 5 min , was used .
the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,
genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,
, we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .
association analyses were performed using plink 
 ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .
the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .
the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .
meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .
the discovery gwas for pulse rate included 352,225 snps profiled with affymetrix genome - wide human snp array 5.0 in 8,842 individuals from the ansung and ansan cohorts as described previously .
characteristics of the study , including age , sex , and trait summaries , are presented in table 1 .
the association analysis identified several genomic locations as potentially associated with pulse rate . unlike the previous gwas
, we did not apply a threshold with a p < 0.01 in both ansung and ansan cohort in addition to a combined p < 1  10 for the snp selection for the replication study so that we could include more promising snps for their association with pulse rate .
we observed 61 significant snps by applying only a threshold with a combined p < 1  10 . after excluding snps by cluster plot inspection and removing singletons , 3 independent snps ( that is , with pairwise linkage disequilibrium statistics r < 0.2 and maf  0.05 within a 500-kb window of the genomic region )
were finally chosen to validate in the independent population - i.e. , the hexa cohort , comprising 3,703 individuals . in the replication study ,
all 3 snps were confirmed for their association with pulse rate at a p < 0.05 ( table 2 ) . the first pulse rate signal located to 1q32.2a ( rs12731740 , maf = 0.103 , poverall = 2.20  10 ) .
three genes , such as cd46 , clorf132 , and cd34 , appear near this locus .
the physiological relevance of the 3 genes in relation to pulse rate is not clear [ 8 , 9 ] .
thus , fine mapping surrounding this locus by targeted resequencing is highly recommended to discover the causal variants for pulse rate .
the second pulse rate snp is rs12110693 ( maf = 0.494 , poverall = 1.10  10 ) on 6q22.31c .
the only gene located near this snp is loc644502 , which has no known function .
the third locus associated with pulse rate is at 6q22.31c ( rs2892767 , maf = 0.261 , poverall = 2.50  10 ) .
this locus is near gja1 ( gap junction protein , alpha 1 , 43 kda ) , which is a member of the connexin gene family .
the protein encoded by gja1 is a component of gap junctions , which are intercellular channels that provide a route for the diffusion of low - molecular - weight materials from cell to cell .
in this study , we performed a genomewide scan for pulse rate and identified 3 loci accounting for the variation in pulse rate .
two of 3 loci were previously reported and replicated once again in an independent hexa - shared cohort .
the other one ( rs2892767 ) near gja1 was previously implicated for its association with pulse rate but was not chosen for the replication study , since the snp did not meet the selection criteria in the discovery stage . in the present study ,
snp rs2892767 near gja1 was taken forward to the replication study using the independent dataset from the hexa - shared cohort and was nicely confirmed for its association with pulse rate .
two snps , rs2892767 near gja1 and rs12110693 near loc644502 , are 408 kb apart from each other and are located in a separate ld , indicating that both are independent snps .
we performed multiple linear regression analyses , conditioning the snps to each other to find a significantly stronger proxy for pulse rate .
however , no dramatic decrease in statistical significance was observed , and no interaction was found between rs2892767 and rs12110693 ( table 3 ) .
gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development .
it is also known that gap junctions , clusters of cell - to - cell channels composed of connexins , mediate the orderly spread of electrical excitation throughout the heart .
the gja1-encoding protein has been reported to be involved in the development and function of the heart by allowing direct cell - to - cell exchange of molecules , which mediate multiple signaling events .
cho et al .   previously reported the same locus near gja1 to be responsible for pulse rate without evidence of replication . in the present study
as indicated from the functional role of gja1 , it is a strong candidate gene for pulse rate .
thus , further efforts , such as fine mapping or imputation based on large - scale sequencing data near rs2892767 on 6q22.31 , would reveal the causal variant of this locus .
in addition , functional studies for gja1 would be worthwhile to facilitate a full understanding of its biological role in pulse rate .
our study was relatively underpowered to discover additional novel loci and demonstrated that large - scale genomewide meta - analysis comprising hundreds of thousands samples is required for understanding the missing heritability of pulse rate .
 pulse rate is known to be related to diverse phenotypes , such as cardiovascular diseases , lifespan , arrhythmia , hypertension , lipids , diabetes , and menopause . 
 we have reported two genomewide significant genetic loci responsible for the variation in pulse rate as a part of the korea association resource ( kare ) project , the genomewide association study ( gwas ) that was conducted with 352,228 single nucleoride polymorphisms typed in 8,842 subjects in the korean population . 
 gja1 was implied as a functionally causal gene for pulse rate from the kare study , but lacked evidence of replication . 
 to re - evaluate the association of a locus near gja1 with pulse rate , we looked up this signal in another gwas conducted in a health examinee - shared cohort of 3,703 samples . 
 not only we were able to confirm two pulse rate loci ( 1q32.2a near cd46 and 6q22.13c near locl644502 ) identified in the kare gwas , we also replicated a locus ( 6q22.31c ) near gja1 by the lookup in the health examinee gwas . considering that the gja1-encoded protein is a major component of cardiac gap junctions , 
 a functional study might be necessary to validate its genuine molecular biological role in the synchronized contraction of the heart . 
Introduction
Methods
Subjects
The measurement of pulse rate
Genotyping and quality control
Statistical analysis
Results
Discussion