loss - of - function mutations in scn5a , that encodes the alpha - subunit of the cardiac na channel , lead to variable phenotypes , viz . brugada syndrome ( brs ) , progressive cardiac conduction disease , atrial fibrillation , and sick sinus syndrome ( sss ) . in young children carrying scn5a mutations , reportedly , cardiac conduction disturbance appears as the most common manifestation of disease , with both atrial and ventricular arrhythmia . the phenotype of the patients with the same scn5a mutations is variable because various modifiers might modulate it . we describe a proband , and his family members with a novel nonsense scn5a mutation , who displayed variable phenotypes , and searched for the genetic modifiers that might affect the phenotype . gene analysis was performed for the proband , his parents , and two siblings as summarized in the family tree ( fig . genomic dna was isolated from blood lymphocytes , and screened for the open reading frame of kcnq1 , kcnh2 , kcne1 - 3 , kcne5 , kcnj2 , and scn5a . genetic screening was performed using denaturing high - performance liquid chromatography ( dhplc wave system , transgenomic , omaha , ne , usa ) . for abnormal screening patterns , direct sequencing was performed using an automated sequencer ( abi prism , 3100x , applied biosystems , foster city , ca , usa ) . for detection of the genetic modifiers , we performed targeted panel sequencing for coding region of 46 genes ( supplementary information ) that were related to primary arrhythmia syndrome for proband and 4 family members , using a bench - top next generation sequencer , the miseq ( illumina , san diego , ca , usa ) . we then evaluated the detected variants by frequently referring to the ncbi dbsnp database ( http://www.ncbi.nlm.nih.gov/snp/ ) , human genetic variation browser that is a japanese snp database http://www.genome.med.kyoto-u.ac.jp/snpdb/ ) , and the 1000 genomes database ( http://www.1000genomes.org/home ) . the pathogenicity of the variants was estimated by three different types of prediction software : ( 1 ) combined annotation dependent depletion ( cadd ) ( http://cadd.gs.washington.edu/ ) , ( 2 ) polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) , and ( 3 ) sift ( http://sift.jcvi.org/ ) . index proband was a 13-year - old ( yo ) boy who was suspected to have a sino - atrial block because of an auscultation abnormality at the age of three ( family tree in fig . his pr interval was 179  ms , qrs was 107  ms , and qt interval was 394  ms ( qtcf 451  ms ) . the sinus node recovery time was significantly prolonged to 7,268  ms , and hv interval to 70  ms . the atrio - ventricular nodal effective refractory period was 360  ms . against a diagnosis of sss the electrocardiogram ( ecg ) monitored by an autonomic external defibrillator ( aed ) showed ventricular fibrillation ( vf ) ( fig . his resting ecg in v2 showed st elevation of a saddleback type at the 4th , and coved type at the 3 intercostal leads sites ( fig . 2b ) . during atrial pacing at 60  bpm , his pr interval prolonged to 262  ms , qrs interval was 125  ms , and qtc was 436 ms ( table 1 ) . 2c depicts temporal changes in the ecg ( v1-v3 ) recorded from the age 4 to 13 . at age 4 , we failed to find any brugada pattern , while the j point gradually elevated from the age of 9 . the signal averaged electrocardiogram ( saecg ) revealed positive late potentials , and microvolt - t wave alternans ( v - twa ) was negative . the proband had asymptomatic parents and two siblings as shown in the family tree in fig . his mother ( 46-yo ) had a coved type st elevation in the 3rd precordial leads ( fig . had sss , and a coved type brugada ecg during a pilsicainide ( class ic antiarrhythmia drug ) challenge test ( fig . 3b ) , and his elder brother ( 21-yo ) had a saddle back brugada ecg during pilsicainide administration ( fig . the prolongation of the pr interval was detected in his mother at rest , and in his sister and brother on pilsicainide administration ( table 1 ) . his maternal grandfather , who died of cancer , had received a pacemaker for sss . we obtained his ecg recording that showed sinus rhythm at 70  bpm , a pr interval of 190  ms , qrs duration of 106  ms , and no brugada ecg pattern ( data not shown ) . we identified a scn5a mutation in the proband , and the three family members except for his father . the mutation , a single - base substitution at nucleotide 2335 ( c.2335c > t ) , is located in the cytoplasmic loop of the na channel alpha - subunit and changed the codon cag ( glutamine : q ) to an in - frame stop codon , tag , at position 779 , q779x ( fig . a premature stop codon in this position results in nonsense mediated decay ( nmd ) , leading to decay of mrna . therefore , the expression level of mrna and protein of cardiac na channel is presumably low in scn5a - q779x carriers . to clarify the phenotypic variation among the mutant scn5a carriers in the index family in the analysis , we further detected four non - synonymous variants of arrhythmia - related genes in the proband , his mother and his sister , but not in his brother exhibiting a milder phenotype . the first one was desmoglein 2 ( dsg2)-r773k ( rs2278792 ) and the others were scn1b l210p ( rs55742440 , ) , s248r ( rs67701503 ) , and r250 t ( rs67486287 ) . scn1b encodes an ancillary beta - subunit of the cardiac na channels and affects the cardiac conduction , and was reported as a causative gene for brs . table 2 summarizes minor allele frequencies ( maf ) and prediction scores for the pathogenicity of each variant . none of them seemed to be the main cause of the disease because their mafs of them were greater than 0.1 , and were predicted to be of benign or mild pathogenicity . index proband was a 13-year - old ( yo ) boy who was suspected to have a sino - atrial block because of an auscultation abnormality at the age of three ( family tree in fig . his pr interval was 179  ms , qrs was 107  ms , and qt interval was 394  ms ( qtcf 451  ms ) . the sinus node recovery time was significantly prolonged to 7,268  ms , and hv interval to 70  ms . the atrio - ventricular nodal effective refractory period was 360  ms . against a diagnosis of sss the electrocardiogram ( ecg ) monitored by an autonomic external defibrillator ( aed ) showed ventricular fibrillation ( vf ) ( fig . his resting ecg in v2 showed st elevation of a saddleback type at the 4th , and coved type at the 3 intercostal leads sites ( fig . 2b ) . during atrial pacing at 60  bpm , his pr interval prolonged to 262  ms , qrs interval was 125  ms , and qtc was 436 ms ( table 1 ) . 2c depicts temporal changes in the ecg ( v1-v3 ) recorded from the age 4 to 13 . at age 4 , we failed to find any brugada pattern , while the j point gradually elevated from the age of 9 . the signal averaged electrocardiogram ( saecg ) revealed positive late potentials , and microvolt - t wave alternans ( v - twa ) was negative . the proband had asymptomatic parents and two siblings as shown in the family tree in fig . 1 . all except for his father had abnormal ecg . his mother ( 46-yo ) had a coved type st elevation in the 3rd precordial leads ( fig . had sss , and a coved type brugada ecg during a pilsicainide ( class ic antiarrhythmia drug ) challenge test ( fig . 3b ) , and his elder brother ( 21-yo ) had a saddle back brugada ecg during pilsicainide administration ( fig . the prolongation of the pr interval was detected in his mother at rest , and in his sister and brother on pilsicainide administration ( table 1 ) . his maternal grandfather , who died of cancer , had received a pacemaker for sss . we obtained his ecg recording that showed sinus rhythm at 70  bpm , a pr interval of 190  ms , qrs duration of 106  ms , and no brugada ecg pattern ( data not shown ) . we identified a scn5a mutation in the proband , and the three family members except for his father . the mutation , a single - base substitution at nucleotide 2335 ( c.2335c > t ) , is located in the cytoplasmic loop of the na channel alpha - subunit and changed the codon cag ( glutamine : q ) to an in - frame stop codon , tag , at position 779 , q779x ( fig . a premature stop codon in this position results in nonsense mediated decay ( nmd ) , leading to decay of mrna . therefore , the expression level of mrna and protein of cardiac na channel is presumably low in scn5a - q779x carriers . to clarify the phenotypic variation among the mutant scn5a carriers in the index family , we performed a targeted panel sequence using miseq , for the mutation carriers . in the analysis , we further detected four non - synonymous variants of arrhythmia - related genes in the proband , his mother and his sister , but not in his brother exhibiting a milder phenotype . the first one was desmoglein 2 ( dsg2)-r773k ( rs2278792 ) and the others were scn1b l210p ( rs55742440 , ) , s248r ( rs67701503 ) , and r250 t ( rs67486287 ) . scn1b encodes an ancillary beta - subunit of the cardiac na channels and affects the cardiac conduction , and was reported as a causative gene for brs . table 2 summarizes minor allele frequencies ( maf ) and prediction scores for the pathogenicity of each variant . none of them seemed to be the main cause of the disease because their mafs of them were greater than 0.1 , and were predicted to be of benign or mild pathogenicity . we identified a novel , heterozygous nonsense scn5a mutation , q779x , in a family with overlapped phenotype with progressive conduction defects and brs . the proband suffered from sss at the age of three , and subsequent ventricular fibrillation with a coved type brugada ecg pattern at age of 13 was detected . mild prolongation of the hv intervals had been observed at the age of four , indicating the presence of an extensive conduction delay . at 9 years of age , sss is complicated in children with a left isomerism heart , or on surgical intervention for congenital heart disease . it is also seen in channelopathies such as long qt syndrome , brs , or as a part of a progressive cardiac conduction disease . loss - of - function scn5a mutants have been shown to be related to brs as well as profound bradyarrhythmias . in children with these mutations , prolonged cardiac conduction intervals have been reported to emerge as the most common manifestation of the disease , and are accompanied with both atrial and ventricular arrhythmias . the intravenous challenge test with a na channel blocker was conducted in two of our genotype - positive members , and the drug produced pr prolongation ( table 1 and fig . the pr prolongation , which results from common loss - of - function scn5a mutation , was enhanced by a na channel blocker in our family members . the heterozygous nonsense scn5a mutation of the family was at amino acid 779 that lies in the middle of the cytoplasmic loop of domain 2 ( fig . a missense scn5a mutation at the same position , q779k , was previously reported for a japanese pedigree of long qt syndrome , which harbored a compound heterozygous mutation , e284k , in kcnq1 
 . a change in the residual charge from neutral ( q : glutamine ) to basic ( k : lysine ) may cause a gain - of - function outcome . in contrast , the nonsense mutation at the same codon leads to nmd , thereby exerting severe loss - of - function effects . the index boy presented progressive conduction defects and vf during earlier stage of his life . his mother and two siblings had similar ecg abnormalities at rest and when challenged with na - channel blockers , but remained asymptomatic in spite of the same nonsense scn5a mutation . this variation in phenotypic severity could be partially explained by the gender difference , because brs is predominantly noted in males as the disease has an autosomal - dominant mode of transmission . the reason for the variation in phenotypic severity between the index boy and his elder brother is unknown . both had the same scn5a mutation , however , the proband had additional dsg2 and scn1b variants . dsg2 is located on chromosome 18 , and encodes a calcium - binding transmembrane glycoprotein component of desmosomes , desmoglein 2 . few mutations in dsg2 have been shown to decrease the na current , leading to conduction disturbances , and are associated with arrhythmogenic right ventricular dysplasia . the scn1b gene encodes a beta-1 subunit of the sodium channel that is known to modulate the sodium channel through a non - covalent interaction with the alpha - subunit encoded by scn5a . maf for each variant was not low , indicating that they were not pathogenic mutations ; although , the pathogenicity estimation scores of scn1b - s248r and r250 t were relatively high ( table 2 ) . therefore , the phenotype variation among the scn5a - q779x carriers could be partially explained by modifier effects of these variants , in addition to the gender difference . we could only once record the ecgs of family members ; therefore , we might have failed to record coved type brugada pattern ecg , which might have been recorded during follow - up . we did not perform functional analysis of genetic modifiers to confirm the effect on the phenotype . additionally , we only screened 46 genes related with cardiac arrhythmias , and did not screen introns that might affect the transcription or expression . thus , we might have overlooked mutation in other genes or in the intronic regions that could be related to the phenotype . scn5a - positive proband and the female members of the family with overlap syndrome phenotype carried additional variants in scn1b and dsg2 . in contrast , these variants were absent in their scn5a - positive male family member who showed a milder phenotype . therefore , in addition to the gender difference , scn1b and dsg2 variants could be modifiers of brs and sss phenotypic expression , and our results may help explain the enigma of variable phenotypes for those with scn5a mutations .
backgroundphenotypes often differ even within family members carrying the same scn5a mutation . 
 we aimed to evaluate the genetic modifiers in a family with brugada syndrome ( brs ) and sick sinus syndrome ( sss ) with an scn5a mutation that causes the truncated alpha - subunit of cardiac na channel protein.methodsto detect the genetic modifiers , we performed targeted panel sequencing of the coding region of 46 genes that are related to primary arrhythmia syndrome , by using a bench - top , next generation sequencer . 
 phenotype 
 genotype relationships were evaluated among the family members.resultsindex proband was a 13-year old ( yo ) boy with cardiac conduction defect as well as brs . 
 genetic analysis revealed that he and his three asymptomatic family members carried a novel nonsense mutation : scn5a - q779x . 
 both genotype - positive mother and sister exhibited coved type st elevation and his sister had sss , whereas his elder brother exhibited saddleback type st elevation induced by pilsicainide administration . 
 we detected four non - synonymous variants ( dsg2-r773k , scn1b - l210p , -s248r , and -r250 t ) in the proband , his mother and his sister , but not in his brother.conclusionphenotypic differences between the proband and his brother carrying the same nonsense scn5a mutation could be explained by modifiers such as scn1b , and dsg2 gene variants .