dilated cardiomyopathy ( dcm ) is the most common form of primary cardiomyopathy and is a leading cause for heart transplantations   approximately 30%50% of affected individuals have a familial form of dcm . to date
, at least 40 disease genes have been reported to be associated with familial dcm , in which ; mutations of scn5a gene which encodes the alpha subunit of the major sodium channel in the heart have been demonstrated to result in myocardial damage and dcm [ 4 , 5 ] .
previously , scn5a has been implicated in conduction disturbances and several electrophysiological phenotypes [ 6 , 7 ] , including atrioventricular block ( avb ) , long qt syndrome , sick sinus syndrome , sudden infant death syndrome , and atrial fibrillation [ 8 , 9 ] . meanwhile
, more and more scn5a mutations were discovered to be related with dcm , such as a1180v , d1275n , w156x , r225w , and r222q .
we first reported finding a1180v , a single nucleotide mutation , c.3539c > t , that occurred in exon 20 of the scn5a gene in a three - generation chinese family with age - related dcm and progressive avb in 2008 . in that study , a1180v was detected in all studied patients and in six younger unaffected members of the pedigree .
experimental data revealed that a1180v channels exhibited a negative transform of voltage - dependent inactivation of the cardiac sodium channels , which lead to a rate - dependent sodium current reduction , and a moderate increase in late sodium current . correspondingly , our clinical study revealed qrs widening at high heart rates and qtc prolongation at rest in unaffected carriers .
these findings suggest that a1180v is a functional mutation that can contribute to the development of dcm .
however , data deposited in the dbsnp database   show that a1180v has been found in two small sample groups of the asian population . in the first sample group , a1180v
( dbsnp refsnp number , rs41310765 ) was present in a cohort of 47 han people ( r31_hanla ) who were part of the los angeles panel at a carrier frequency of 6.4% . in another sample group ( her_asian - panel ) of 16 individual asians
selected from the coriell cell repository , genetic sequencing yielded a carrier frequency of 6.2% ( http://www.ncbi.nlm.nih.gov/projects/snp/snp_ref.cgi?rs=41310765 ) . to date , the data obtained from the dbsnp database show that a1180v is virtually absent in other ethnic groups .
thus , these unpublished data suggest that a1180v might be a common variant in asians , which is inconsistent with our previous observations .   in order to further evaluate the pathogenic role of a1180v in familial dcm and make sure the frequency of a1180v among healthy han chinese
, we followed up the three - generation chinese family and expanded the control size to screen for a1180v . besides , we analyzed its frequency in other database as well .
we followed up all the a1180v carriers and noncarriers of the three - generation chinese family for 6 years .
all individuals were interviewed with the symptoms of cardiovascular disease , such as typical chest pain , palpitation , cyanosis , and syncope .
12-lead electrocardiogram ( ecg ) and echocardiography were performed on the family members to make a diagnosis .
460 unrelated healthy han chinese living in shanghai , ranging from 20 to 70 years old , were enrolled to the control group from a national high technology research and development program ( the national 863 program ) of china .
individuals with known cardiovascular risk factors other than age , gender , and smoking history were excluded .
the enrolled participants did not show any abnormalities under comprehensive clinical evaluation , including review of medical history , physical examination , 12-lead ecg , submaximal exercise ecg , and blood biochemical examinations .
each of the participants and individuals whose data are included here signed an informed consent form after a complete and clear explanation of the nature and purpose of the study .
the study was approved by the local ethics committee of zhongshan hospital , fudan university .  
dna was extracted from 2  ml venous blood according to kit procedure ( sigma , usa ) and stored at 80c .
a 20  ul mixture was prepared for each reaction and included 1x hotstartaq buffer , 2.0  mm mg , 0.2  mm dntp , 0.2  m of each primer , 1  u hotstartaq polymerase ( qiagen inc . ) and 1  l template dna .
the dna was amplified by cycling at 95c for 15  min ; 11 cycles of 94c for 15  s , 62c0.5c per cycle for 40  s , 72c for 1  mins ; 24 cycles of 94c for 15  s , 57c for 30  s , 72c for 1  mins ; 72c for 2  min .
the oligonucleotide sequences of the primers were as follows : 5-acccccatcatcgtagctctt-3 and 5-tcctgctctggcctccatac-3. the pcr products were purified by 1  u sap and 6  u exo i , and they were added into 8  l pcr products .
the mixture was incubated at 37c for 60  mins , followed by incubation at 70c for 10  mins .
reaction mixture included 2  l bigdye3.1 mix , 2  l sequencing primer ( 0.4  m , 5-acccccatcatcgtagctctt3 ) and 1 - 2  l purified pcr product . the cycling program was 96c for 1  min ; 28 cycles of 96c for 10  s , 50c for 5  s , 60c for 4  mins .
besides , we have excluded some familial data to make sure that the frequency of a1180v from 1000 genomes project was calculated from unrelated individuals .
comparison of the a1180v carrier frequency between our studies and the results of dbsnp database was tested by using the fisher 's exact test .
combined with our former study and the 6 years of clinical followup of the affected pedigree , we discovered that a1180v related dcm was preceded by progressive avb which mostly occurred in the third decade of life . during the follow - up period ,
the condition of an a1180v carrier ( ii-8 in figure 1 ) who had been diagnosed with dcm before aggravated gradually and she now was diagnosed with severe congestive heart failure . besides , iii-1 with a1180v , developed to dcm and complete atrioventricular block ( avb ) and another 3 carriers ( iii-2 , iii-3 , iii-13 ) progressed to different degree of avb .  
figure 2 displays changes of echocardiograms of iii-1 and ecg recordings of iii-2 in the follow - up period .
none of the individuals in the pedigree showed any sign of long - qt or brugada syndrome and other diseases .
after screening a1180v in 460 individuals , we found that a1180v was absent in this group ( 0/460 , 0% ) .  
figure 3(a ) gives an example of the sequencing results , and figure 3(b ) is a sequencing result of a a1180v carrier in 2007 .
the combined carrier frequency , 0% ( i.e. , 0 out of 660 cases ) , of a1180v from our studies remarkably suggests that a1180v is not a common variant among healthy chinese , and the data significantly differs from that in the dbsnp database ( p = 5.3  10 ) .
data from the 1000 genomes project revealed a carrier frequency of 0.51% ( i.e. , 1 out of 197 cases ) for a1180v among chinese participants .
the data was obtained by analyzing the sequencing results of 97 northern and 100 southern chinese adults .
since the southern chinese group was familial related , the data from known offspring were excluded from the calculation of the carrier frequency to make sure the value was calculated from unrelated individuals ( http://browser.1000genomes.org/homo_sapiens/variation/population?db=core;r=3:38616415-38617415;v=rs41310765;vdb=variation;vf=12532571 ) .
the data also dramatically differs from those deposited in the dbsnp database ( p = 0.013 ) .  
we have presented the followup and sequencing data identifying that a1180v of scn5a was a potential risk factor for dcm , and it is absent among healthy han chinese . compared with the former results in 2008 , 5 a1180v carriers exhibited age - related progressive avb and dcm , and the a1180v carrier frequency among han chinese in our study is 0% out of 660 control cases .
evidence is now being accumulated that scn5a mutations are associated with development of cardiac dilation [ 13 , 1618 ] .
mcnair et al . have reported that mutations in scn5a were detected in 1.7% of dcm families with a shared mechanism of disruption of the voltage - sensing mechanism of this channel leading to dcm . moreover ,
some scn5a mutations which have been found to be a primary factor of leading dcm were characterized by an age - related phenotype of cardiac dilation with different types of arrhythmia [ 11 , 20 ] . and the pathogenic characteristic of a1180v conformed to the pattern as well .
since the cardiac sodium channel is principally in charge of the fast depolarization of the myocardium , some suggested that the cardiac dilation may result from long - term arrhythmia [ 22 , 23 ] .
however , our experimental evidence indicated that a1180v could alter calcium homeostasis as a consequence of alterations in intracellular sodium concentrations , and the former condition might damage myocardium directly and subsequently lead to cardiac dilation .
our data have revealed that a1180v specifically appeared in the affected pedigree and was absent among healthy controls , however , why do such significant discrepancies exist for a1180v among different studies of the same ethnic population ?
( 1 ) different asian ethnic subgroups may have different genetic backgrounds that cause differing prevalence of a genetic variant .
for example , r1193q ( another scn5a variant ) is more prevalent in asians living in taiwan ( frequency~12% ) compared to asians living in japan ( ~2% ) .
most chinese - americans are descendants of people from southern china , so they may have a genetic background that differs slightly from the majority of the subjects enrolled in our studies .
the population of the shanghai area consists mostly of people who have immigrated from other areas of china .
( 2 ) relatively small sample sizes may also account for high carrier frequency of those data deposited in the dbsnp database .
( 3 ) the sequencing depth of different sequencing technologies varies greatly within and between experiments , and it may introduce bias as well . at present ,
lots of gene mutations were found playing pathogenic roles in both familial and sporadic diseases [ 26 , 27 ] , then another question is that whether a1180v would be a risk factor among sporadic dcm patients as well . in our recent studies of the distributions of candidate mutations in dcm - related genes in sporadic dcm patients conducted using high - throughput sequencing technology , we identified a1180v in 1 out of 68 patients with dcm .
the patient , a 41-year - old male , was diagnosed as having dcm with nyha class i , lvedd 78  mm , and lvef 24% , but no other diseases .
however , the patient carrying a1180v also carries other mutations in several dcm - related genes , including g1117s , p1119r , y498h of lama4 , w768s of rbm20 , p398s of vcl , and r659w of scn5a .
although we found that a1180v might also be a risk factor in sporadic dcm patients , we can not exclude the possibility that a1180v could work together with other gene mutations .
in addition to genetic factors , our previous study of the dcm pedigree carrying a1180v demonstrated that the a1180v channel lost current only at high heart rates , which suggested that physical activity and lifestyle that increased average daily heart rate might enhance the phenotype for a1180v carriers .
therefore , environmental factors may also play a role in expression of the pathogenic mechanism of a1180v . further studies of genetic and environmental factors and followup of a1180v carriers will provide more information about the relationship between the mutation and the risk of dcm .
studies of transgenic mice may also provide a step to test the hypothesis and to explore the underlying mechanism .
in conclusion , herein we provide evidence that the a1180v is a potential cause for dcm rather than a common variant in han chinese , although additional genetic or environmental factors may potentiate the risk for dcm in a1180v carriers .
 our previous study of a chinese family with dilated cardiomyopathy ( dcm ) suggested that a1180v of the cardiac sodium channel gene ( scn5a ) was associated with the disease within this family . according to data deposited in dbsnp , however 
 , a1180v has been found in some small samples of the asian population . in this study 
 , we followed up the affected pedigree and expanded the investigation of the prevalence of a1180v in 460 unrelated healthy han chinese . besides , we searched and analyzed it in other database as well . during the follow - up period , 1 a1180v carrier 's condition deteriorated alot , and another 4 carriers progressed to dcm or atrioventricular block ( avb ) . 
 we also found that the a1180v was absent among the 460 individuals ( 0% , 0/460 ) , and the carrier frequency of a1180v among chinese was about 0.51% obtained from the 1000 genome project . in conclusion , our finding suggests that a1180v is a potential risk factor for dcm , and it is extremely rare among healthy han chinese . 