the scn5a gene , encoding a voltage - gated na channel , is predominately expressed in the heart , where it has a key role in the generation and propagation of the cardiac impulse . voltage - gated na channels are transmembrane proteins that produce the fast inward na current responsible for the depolarization phase of the cardiac action potential . inherited variations in scn5a , the gene encoding the pore - forming -subunit of the cardiac - type na channel ( nav1.5 ) , result in a spectrum of disease entities , termed na channelopathies . in many previous studies , lots of variations of the scn5a gene have been observed in various cardiac diseases , such as long - qt syndrome ( lqt ) , brugada syndrome ( brs ) , progressive cardiac conduction defect , atrial fibrillation ( af ) , dilated cardiomyopathy , and overlapping syndromes [ 2 , 3 ] . recently , cardiac conduction system disease , manifesting as slower intramyocardial conduction and , in some cases , atrioventricular conduction block ( avb ) , has been related to scn5a mutation . familial avb , characterized by progressive " degree of block " in association with variable apparent " site of block , " may be transmitted as an autosomal dominant trait . established a genetic link between avb and a genetic locus at chromosome 19q13 , and schott et al . such genetic variations in avb patients have been widely studied in caucasians , han chinese , and japanese , but no study has yet been published in koreans as far we know . therefore , we carried out a complete sequencing of coding regions of the scn5a gene , except the untranslated region , in korean avb patients to investigate the scn5a variations associated with avb and compared them with normal control subjects . this is the first study of scn5a genetic variations that examined all coding regions in korean patients with avb . we enrolled 113 korean avb patients who were diagnosed with electrocardiograms and who had inserted permanent pacemakers and 80 normal controls with no cardiac symptoms . all patients and control subjects were recruited from four medical centers - keimyung university , yeungnam university , catholic university , and fatima hospital ( daegu , korea ) - from august 2009 to september 2011 . this study was approved by the ethical committee at each hospital ; consent was obtained from all individuals before enrollment into the study . in this study , peripheral blood was collected into ethylenediamine tetraacetic acid - containing tubes , and genomic deoxyribonucleic acid ( dna ) was extracted from whole blood samples using the qiaamp dna blood mini kit ( qiagen , gmbh , hilden , germany ) . dna concentration was determined using a nanodrop nd1000 spectrophotometer ( thermo fisher scientific inc . , wilmington , de , usa ) , and purity of the dna was assessed based on the 260/280 nm absorbance ratio . the scn5a gene is located on human chromosome 3p21 , and the gene consists of 28 exons ( fig . 1 ) and encodes a protein of 2,016 amino acids with a molecular mass of 227 kda . entire coding regions , including exon - intron boundaries of the scn5a gene , were amplified by multiplex pcr - based direct sequencing . the primers for pcr amplification were designed based on the genbank reference sequence ; primers are shown in table 1 . pcr conditions were as follows : an initial denaturation at 95 for 15 min ; and denaturation at 94 for 30 s , annealing at 68 - 70 for 30 - 60 s , and extension at 72 for 60 - 90 s , repeated for 30 cycles ( table 1 ) . after multiplex pcr , the reaction mixture was electrophoresed in a 2% agarose gel and stained with ethidium bromide ( fig . then , amplified pcr products were purified using the qiaquick pcr purification kit ( qiagen ) and directly sequenced using the bigdye terminator ver 3.1 cycle sequencing kit on an abi prism 3100 genetic analyzer ( applied biosystems , foster city , ca , usa ) . sequencing results were compared with the reference sequences ( scn5a / nm_198056.2/ensg00000183873/enst00000333535 ) using the alignment program blast 2.0 of ncbi , and we determined the portion of variation that occurred . when a variation was discovered in genomic dna from patients , we confirmed whether the same variation existed in the control genomic dna . p - values and odds ratios with 95% confidence intervals for each genotype and allele were evaluated by  analysis . a p - value of < 0.05 was considered to indicate a statistically significant difference between avb patients and normal controls of the study . we enrolled 113 korean avb patients who were diagnosed with electrocardiograms and who had inserted permanent pacemakers and 80 normal controls with no cardiac symptoms . all patients and control subjects were recruited from four medical centers - keimyung university , yeungnam university , catholic university , and fatima hospital ( daegu , korea ) - from august 2009 to september 2011 . this study was approved by the ethical committee at each hospital ; consent was obtained from all individuals before enrollment into the study . in this study , peripheral blood was collected into ethylenediamine tetraacetic acid - containing tubes , and genomic deoxyribonucleic acid ( dna ) was extracted from whole blood samples using the qiaamp dna blood mini kit ( qiagen , gmbh , hilden , germany ) . dna concentration was determined using a nanodrop nd1000 spectrophotometer ( thermo fisher scientific inc . , wilmington , de , usa ) , and purity of the dna was assessed based on the 260/280 nm absorbance ratio . the scn5a gene is located on human chromosome 3p21 , and the gene consists of 28 exons ( fig . 1 ) and encodes a protein of 2,016 amino acids with a molecular mass of 227 kda . entire coding regions , including exon - intron boundaries of the scn5a gene , were amplified by multiplex pcr - based direct sequencing . the primers for pcr amplification were designed based on the genbank reference sequence ; primers are shown in table 1 . pcr conditions were as follows : an initial denaturation at 95 for 15 min ; and denaturation at 94 for 30 s , annealing at 68 - 70 for 30 - 60 s , and extension at 72 for 60 - 90 s , repeated for 30 cycles ( table 1 ) . after multiplex pcr , the reaction mixture was electrophoresed in a 2% agarose gel and stained with ethidium bromide ( fig . then , amplified pcr products were purified using the qiaquick pcr purification kit ( qiagen ) and directly sequenced using the bigdye terminator ver 3.1 cycle sequencing kit on an abi prism 3100 genetic analyzer ( applied biosystems , foster city , ca , usa ) . sequencing results were compared with the reference sequences ( scn5a / nm_198056.2/ensg00000183873/enst00000333535 ) using the alignment program blast 2.0 of ncbi , and we determined the portion of variation that occurred . when a variation was discovered in genomic dna from patients , we confirmed whether the same variation existed in the control genomic dna . p - values and odds ratios with 95% confidence intervals for each genotype and allele were evaluated by  analysis . a p - value of < 0.05 was considered to indicate a statistically significant difference between avb patients and normal controls of the study . in the present study , we sequenced all translated exons and exon - intron boundaries of the scn5a gene from 113 korean avb patients . as a result , we identified 7 genetic variations ( of these , 5 were known variations and 2 were novel variations ) , consisting of 3 synonymous ( g87a - a29a , g3048a - t1016 t , and t5457c - d1819d ) , 3 non - synonymous ( c48g - f16l , a1673g - h558r , and g3578a - r1193q ) , and 1 splicing donor site of the intron 9-exon 10 boundary ( intervening sequence [ ivs ] 9 - 3c > a ) ( table 2 ) . have been reported by american and japanese researchers : non - synonymous snp in exon 12 ( rs1805124 , a1673g - h558r ) and synonymous snp in exon 28 ( rs1805126 , c5457t - d1819d ) . a1673g - h558r was identified in 18% of the american populations ( n = 71 ) and 8% of the japanese populations ( n = 100 ) [ 8 , 9 ] , and c5457t - d1819d was observed in 12.3% of the american populations ( n = 130 ) and 46% of the japanese populations ( n = 100 ) [ 9 , 10 ] . we also discovered a synonymous snp ( rs6599230 : g87a - a29a , in exon 2 ) and non - synonymous snp ( rs41261344 , g3578a - r1193q , in exon 20 ) that are very common polymorphisms in the han chinese population . one of these variations was heterozygous synonymous ( g3048a - t1016 t ) , and the other was heterozygous non - synonymous ( c48g - f16l ) . the genotype and allele frequencies of the identified genetic variations are summarized in table 2 . there were significant differences in the allele frequencies of a1673g - h558r and g3048a ( t1016 t ) between the two groups ( p < 0.05 ) . there were no statistically significant differences in allele frequencies of the other genetic variations between the two groups ( all p > 0.05 ) . however , g298s , g514c , p1008s , g1406r , and d1595n , identified in other ethnic populations , were not detected , and a3075t - e1025d and t4847a - f1616y , discovered in 105 korean sick sinus syndrome patients in our previous study , were also not detected in this study . next , to compare with normal controls , we analyzed normal samples by the same methods whether the same variations existed in their genes . just as in the patient group , 4 snps ( rs6599230 , g87a - a29a ; rs41312434 , ivs9 - 3c > a ; rs1805124 , a1673g - h558r ; rs1805126 , c5457t - d1819d ) were detected in the normal control group , but 1 non - synonymous snp ( rs41261344 , g3578a - r1193q ) and 2 novel variations ( c48g - f16l and g3048a - t1016 t ) were not detected in the normal control group . this is the first case report with genetic variations of scn5a in korean patients with avb . to date , a number of scn5a variations associated with various cardiac diseases , such as lqt , brs , progressive cardiac conduction defect , af , and overlapping syndromes , have been reported . although scn5a genetic variations associated with avb have been well studied in caucasians , han chinese , and japanese , no study has yet been published in koreans as far we know . in previous studies , it has been shown that in some cases , progressive avb has been linked to scn5a gene variations . furthermore , in our prior study , 2 novel variations were detected in korean sick sinus syndrome ( sss ) patients , which , to our knowledge , has not been previously reported in any ethnic group . based on these results , we searched for scn5a genetic variations in korean avb and compared them with those of control subjects . the scn5a gene consists of 28 exons and encodes a protein of 2016 amino acids with a molecular mass of 227 kda . in our patients with avb , there were 7 sites of nucleotide change from exon 2 to exon 28 of the scn5a gene . among of them , 2 sites ( g87a - a29a , ivs9 - 3c > a ) were reported as genetic variations in a western study , and t5457c - d1819d has already been reported as a variation or polymorphism without functional effects on the channel in asian studies [ 7 , 15 , 16 ] . a1673g - h558r is located in the na channel i , ii interdomain linker , and previous functional studies have shown that the h558r - encoding minor allele can alter the phenotype of true disease - causing scn5a mutations . it has been suggested that it modulates na channel functional changes caused by other variations and plays a role in intragenic complementation [ 18 , 19 ] . previously , the h558r g allele has been associated with occurrence of early - onset af and lqt interval in healthy populations . the h558r g allele frequency has been reported to be 20.4 - 32% in the white population , 29% in the black population , and 10.4% in the chinese population . in our study , the g allele frequency of scn5a mutation carriers was 8% and 13.1% in the normal control group . g3578a - r1193q , which can cause a gain of the sodium ion channel function and cause lqt3 and brs , was also found . it has been reported that g3578a - r1193q confers a risk of lqt3 or brs in the general population , although it seems to be a common polymorphism in the han chinese population . electrophysiological studies showed that r1193q destabilizes inactivation gating and generates a persistent , non - inactivating current , which results in a gain of the sodium channel function , as in lqt3 syndrome [ 20 , 21 ] . in addition to previously reported variations , we identified 2 novel variations that were not reported in the case of japanese and chinese populations . one of these variations was heterozygous non - synonymous ( c48g - f16l ) , and the other was heterozygous synonymous ( g3048a - t1016 t ) . since a non - synonymous nucleotide change in the scn5a gene is likely to induce a functional change in the sodium channel , a non - synonymous substitution was considered a candidate for the variation associated with this disorder . in this study , a non - synonymous variation , c48g - f16l , was not found in the normal control group but was detected only in the patient group , suggesting that it may effect avb . however , g298s , g514c , p1008s , g1406r , and d1595n , identified in other ethnic populations , were not detected , thought to be due to very low frequency or no variation in the korean population . in summary , our data may provide useful information for the identification of novel variations related to avb , and the novel variants may provide useful bio - markers to study korean cardiac disease . in a future study , we will conduct a functional analysis of the novel variation ( c48g - f16l ) and propose a mechanism for its contribution to the avb phenotype in patients . also , we will try to find out genetic variation sites in other genes that might be responsible for avb .
recent several studies have shown that the genetic variation of scn5a is related with atrioventricular conduction block ( avb ) ; no study has yet been published in koreans . 
 therefore , to determine the avb - associated genetic variation in korean patients , we investigated the genetic variation of scn5a in korean patients with avb and compared with normal control subjects . 
 we enrolled 113 patients with avb and 80 normal controls with no cardiac symptoms . 
 dna was isolated from the peripheral blood , and all exons ( exon 2-exon 28 ) except the untranslated region and exon - intron boundaries of the scn5a gene were amplified by multiplex pcr and directly sequenced using an abi prism 3100 genetic analyzer . when a variation was discovered in genomic dna from avb patients 
 , we confirmed whether the same variation existed in the control genomic dna . in the present study , 
 a total of 7 genetic variations were detected in 113 avb patients . 
 of the 7 variations , 5 ( g87a - a29a , intervening sequence 9 - 3c > a , a1673g - h558r , g3578a - r1193q , and t5457c - d1819d ) have been reported in previous studies , and 2 ( c48g - f16l and g3048a - t1016 t ) were novel variations that have not been reported . 
 the 2 newly discovered variations were not found in the 80 normal controls . 
 in addition , g298s , g514c , p1008s , g1406r , and d1595n , identified in other ethnic populations , were not detected in this study . 
 we found 2 novel genetic variations in the scn5a gene in korean patients with avb . 
 however , further functional study might be needed .