congenital heart disease is the most prevalent form of developmental abnormality in newborns , with an estimated prevalence of 1% , and is the leading non - infectious cause of infant mortality , with more than 29% of neonates who die of a birth defect having a cardiovascular deformity . congenital cardiovascular anomaly is clinically classified into at least 18 different types , with many additional anatomic variations , of which ventricular septal defect ( vsd ) is the most common type . vsd occurs in 3060% of all children , with various kinds of congenital cardiovascular deformations , and accounts for 1416% of birth defects that require an invasive procedure within the first year of life [ 13 ] . congenital vsd can occur by itself or in combination with other cardiac malformations such as atrial septal defect , tetralogy of fallot , and patent ductus arteriosis . regardless of other potential conditions that accompany vsd , isolated moderate - to - large vsd with persistent left - to - right shunting of blood may give rise to cardiac enlargement , ventricular dysfunction , pulmonary hypertension , eisenmenger s syndrome , delayed fetal brain development , arrhythmias , and even sudden cardiac death in the absence of surgical or catheter - based repair [ 613 ] . despite the high incidence and the significant association of vsd with substantial morbidity and mortality in human , the etiology of vsd remains largely unclear . abnormally developed interventricular septum is implicated in a heterogeneous , complex pathogenic process , for which both environmental risk factors and genetic defects may be responsible [ 2,1416 ] . recently , growing evidence points to a crucial role of the zinc finger transcription factor gata4 in the cardiogenesis . the human gata4 gene maps to chromosome 8p23.1-p22 and comprises 7 exons encoding a protein of 442 amino acids . therefore , gata4 has been one of prime candidate genes in identifying the molecular components underlying structural congenital heart defects . presently , over 22 germline mutations in the coding exons of the gata4 gene have been identified in patients with a wide variety of congenital heart anomalies , including vsd , atrial septal defect , atrioventricular septal defect , tetralogy of fallot , and endocardial cushion defect [ 2033 ] . nevertheless , vsd is genetically heterogeneous and the heritable determinants leading to vsd in the majority of cases are still to be identified . a cohort of 230 unrelated patients with vsd , including 205 cases with apparently sporadic vsd and 25 index patients with familial vsd , was identified among a chinese han population . subjects were evaluated by individual and familial history , review of the medical records , complete physical examination , 12-lead electrocardiogram ( ecg ) and two - dimensional transthoracic echocardiography with color flow doppler . all patients had a classic form of vsd , with a defect diameter of > 3 mm and nearly all patients underwent cardiac catheterization and , if required , cardiac surgery . the available family members of the probands harboring identified gata4 mutations were enrolled and evaluated by medical history , medical records , physical examination , ecg and echocardiography with color flow doppler . a total of 200 ethnically matched unrelated healthy individuals , recruited from the general population , were used as controls to screen for likely mutations in gata4 . peripheral venous blood specimens from vsd patients , available relatives of the mutation carriers , and control individuals were prepared . the study protocol was reviewed and approved by the local institutional ethics committee and written informed consent was obtained from all participants or their guardians prior to investigation . genomic dna from all participants was extracted from blood lymphocytes with wizard genomic dna purification kit ( promega ) . genotyping gata4 in the available relatives of an index patient carrying an identified mutation and the 200 ethnically matched unrelated healthy control individuals was conducted subsequently . nc_000008 ) . by the aid of on - line primer 3 software ( http://frodo.wi.mit.edu ) , the primer pairs used to amplify the coding exons and exon / intron boundaries of gata4 by polymerase chain reaction ( pcr ) were designed , as shown in table 1 . the pcr was carried out using hotstar taq dna polymerase ( qiagen ) on a pe 9700 thermal cycler ( applied biosystems ) , with standard conditions and concentrations of reagents . amplified products were analyzed on 1% agarose gels stained with ethidium bromide and purified with qiaquick gel extraction kit ( qiagen ) . both strands of each pcr product were sequenced with a bigdye terminator v3.1 cycle sequencing kit ( applied biosystems ) under an abi prism 3130 xl dna analyzer ( applied biosystems ) . the dna sequences were viewed and analyzed with the dna sequencing analysis software v5.1 ( applied biosystems ) . the variant was validated by re - sequencing an independent pcr - generated amplicon from the subject and met our quality control thresholds with a call rate > 99% . the multiple gata4 protein sequences across species were aligned using the online program of clustalw ( http://www.bioinformatics.nl/tools/clustalw.html/ ) . a cohort of 230 unrelated patients with vsd , including 205 cases with apparently sporadic vsd and 25 index patients with familial vsd , was identified among a chinese han population . subjects were evaluated by individual and familial history , review of the medical records , complete physical examination , 12-lead electrocardiogram ( ecg ) and two - dimensional transthoracic echocardiography with color flow doppler . all patients had a classic form of vsd , with a defect diameter of > 3 mm and nearly all patients underwent cardiac catheterization and , if required , cardiac surgery . the available family members of the probands harboring identified gata4 mutations were enrolled and evaluated by medical history , medical records , physical examination , ecg and echocardiography with color flow doppler . a total of 200 ethnically matched unrelated healthy individuals , recruited from the general population , were used as controls to screen for likely mutations in gata4 . peripheral venous blood specimens from vsd patients , available relatives of the mutation carriers , and control individuals were prepared . the study protocol was reviewed and approved by the local institutional ethics committee and written informed consent was obtained from all participants or their guardians prior to investigation . genomic dna from all participants was extracted from blood lymphocytes with wizard genomic dna purification kit ( promega ) . genotyping gata4 in the available relatives of an index patient carrying an identified mutation and the 200 ethnically matched unrelated healthy control individuals was conducted subsequently . nc_000008 ) . by the aid of on - line primer 3 software ( http://frodo.wi.mit.edu ) , the primer pairs used to amplify the coding exons and exon / intron boundaries of gata4 by polymerase chain reaction ( pcr ) were designed , as shown in table 1 . the pcr was carried out using hotstar taq dna polymerase ( qiagen ) on a pe 9700 thermal cycler ( applied biosystems ) , with standard conditions and concentrations of reagents . amplified products were analyzed on 1% agarose gels stained with ethidium bromide and purified with qiaquick gel extraction kit ( qiagen ) . both strands of each pcr product were sequenced with a bigdye terminator v3.1 cycle sequencing kit ( applied biosystems ) under an abi prism 3130 xl dna analyzer ( applied biosystems ) . the dna sequences were viewed and analyzed with the dna sequencing analysis software v5.1 ( applied biosystems ) . the variant was validated by re - sequencing an independent pcr - generated amplicon from the subject and met our quality control thresholds with a call rate > 99% . the multiple gata4 protein sequences across species were aligned using the online program of clustalw ( http://www.bioinformatics.nl/tools/clustalw.html/ ) . a cohort of 230 unrelated patients with vsd was enrolled and clinically evaluated in contrast to a cohort of 200 ethnically matched unrelated healthy individuals as controls . the baseline clinical characteristics of the 230 unrelated patients with vsd are summarized in table 2 . direct sequencing of the coding exons of the gata4 gene was conducted after pcr amplification of genomic dna from the 230 unrelated vsd patients . the total population prevalence of gata4 mutations based on the cohort patients was approximately 1.74% . specifically , displacement of adenine by guanine in the second nucleotide of codon 55 of the gata4 gene ( c.164a > g ) , equivalent to the replacement of glutamine by arginine at amino acid 55 ( p.q55r ) , was identified in the proband from family 1 . substitution of adenine for guanine in the first nucleotide of codon 96 of the gata4 gene ( c.286g > a ) , predicting the transition of glycine to arginine at amino acid position 96 ( p.g96r ) , was identified in the index patient from family 2 . a change of adenine into guanine in the second nucleotide of codon 197 of the gata4 gene ( c.590a > g ) , corresponding to the transversion of asparagine to serine at amino acid residue 190 ( p.n197s ) , was identified in the proband from family 3 . a gata4 sequence variation of c.1211a > g , resulting in the conversion of lysine into arginine at amino acid 404 ( p.k404r ) , was identified in the proband from family 4 . the sequence chromatograms showing the detected heterozygous gata4 mutations in comparison with control sequences are shown in figure 1 . the variants were not detected in 200 control individuals and they are not described in the human snp database ( http://www.ncbi.nlm.nih.gov/snp/ ) . genetic scan of the available family members of the mutation carriers demonstrated that in each family the variant was present in all affected family members , but absent in unaffected family members who were tested . analysis of the pedigrees showed that each mutation co - segregated with vsd in the family with complete penetrance . the phenotypic characteristics and results of genetic screening of the affected pedigree members are listed in table 3 . a cross - species alignment of multiple gata4 protein sequences showed that the affected amino acids of q55 , g96 , n197 , and k404 were completely conserved among mammals , as shown in figure 3 , suggesting that these amino acids are functionally important . a cohort of 230 unrelated patients with vsd was enrolled and clinically evaluated in contrast to a cohort of 200 ethnically matched unrelated healthy individuals as controls . the baseline clinical characteristics of the 230 unrelated patients with vsd are summarized in table 2 . direct sequencing of the coding exons of the gata4 gene was conducted after pcr amplification of genomic dna from the 230 unrelated vsd patients . the total population prevalence of gata4 mutations based on the cohort patients was approximately 1.74% . specifically , displacement of adenine by guanine in the second nucleotide of codon 55 of the gata4 gene ( c.164a > g ) , equivalent to the replacement of glutamine by arginine at amino acid 55 ( p.q55r ) , was identified in the proband from family 1 . substitution of adenine for guanine in the first nucleotide of codon 96 of the gata4 gene ( c.286g > a ) , predicting the transition of glycine to arginine at amino acid position 96 ( p.g96r ) , was identified in the index patient from family 2 . a change of adenine into guanine in the second nucleotide of codon 197 of the gata4 gene ( c.590a > g ) , corresponding to the transversion of asparagine to serine at amino acid residue 190 ( p.n197s ) , was identified in the proband from family 3 . a gata4 sequence variation of c.1211a > g , resulting in the conversion of lysine into arginine at amino acid 404 ( p.k404r ) , was identified in the proband from family 4 . the sequence chromatograms showing the detected heterozygous gata4 mutations in comparison with control sequences are shown in figure 1 . the variants were not detected in 200 control individuals and they are not described in the human snp database ( http://www.ncbi.nlm.nih.gov/snp/ ) . genetic scan of the available family members of the mutation carriers demonstrated that in each family the variant was present in all affected family members , but absent in unaffected family members who were tested . analysis of the pedigrees showed that each mutation co - segregated with vsd in the family with complete penetrance . the phenotypic characteristics and results of genetic screening of the affected pedigree members are listed in table 3 . a cross - species alignment of multiple gata4 protein sequences showed that the affected amino acids of q55 , g96 , n197 , and k404 were completely conserved among mammals , as shown in figure 3 , suggesting that these amino acids are functionally important . in the present study , 4 novel heterozygous missense mutations of gata4 were identified in 4 families with congenital vsd . in each family , the mutation was present in all the affected family members alive but absent in unaffected relatives tested and 400 normal chromosomes from a matched control population . a cross - species alignment of gata4 protein sequences demonstrates that the altered amino acids were highly conserved evolutionarily . therefore , it is very likely that mutated gata4 is involved in the pathogenesis of vsd in these families . gata transcription factors are a family of transcription factors characterized by the ability to bind to the consensus dna sequence gata. to date , 6 members of the gata family have been identified in vertebrates , of which gata4 , gata5 and gata6 are expressed mainly in the developing heart and in several endodermal lineages . structurally , gata4 comprises 2 transcriptional activation domains ( tad1 , amino acids 174 ; tad2 , amino acids 130177 ) , 2 zinc finger domains ( zf1 , amino acids 215240 ; zf2 , amino acids 270294 ) , 1 nuclear localization signal ( nls , amino acids 295324 ) , and 1 c - terminus ( c - ter , amino acids 325442 ) . the c - terminal zf1 is required for dna sequence recognition and binding to the consensus motif , while the n - terminal zf2 is responsible for sequence specificity and stability of protein - dna binding . the gata4 mutation p.q55r identified in this study is located in tad1 , and thus may be expected to exert direct influence on the transactivating activity of gata4 . the other 3 gata4 mutations , including p.g96r and p.n197s located in the neighbor of tads and p.k404r located in the c - terminus , may indirectly influence the transcriptional activity of gata4 by altering the molecular conformation . our results are supported by the reports of other gata4 mutations associated with congenital cardiac septal defects . presently , at least 13 out of 22 germline mutations identified in the coding region of the gata4 gene ( p.h28y , p.a66 t , p.p163s , p.e216d , p.g296s , p.g296r , p.q316e , p.s377 g , p.v380 m , p.p407q , p.a411v , p.d425n , and p.h436y ) have been associated with isolated or syndromic vsd , showing that although gata4 mutations underlie a long list of cardiac developmental aberrations , one of the most common phenotypes ascribed to mutations of the gata4 gene is vsd [ 2033 ] . however , the prevalence of gata4 mutations varies significantly in different cohorts of individuals with congenital heart diseases . according to the 12 reports on the prevalence of gata4 mutations in different cohorts of patients with vsd , the detection frequencies of gata4 mutations are 12.50% ( 2/16 ) , 10.00% ( 5/50 ) , 6.90% ( 2/29 ) , 3.74% ( 4/107 ) , 3.23% ( 1/31 ) , 2.47% ( 12/486 ) , 1.67% ( 2/120 ) , 1.48% ( 2/135 ) , 0.80% ( 5/628 ) , 0.49% ( 1/205 ) , 0.48% ( 1/210 ) , and 0% ( 0/99 ) , and the prevalence in the compound population is 1.75% ( 37/2116 ) . similar to these findings , the mutational prevalence in our vsd cohort was 1.74% ( 4/230 ) , suggesting that gata4 mutations are an uncommon cause of congenital cardiovascular defects . additionally , based on known mutation sites , no mutation hot spots exist within the gata4 gene and the penetrance of different gata4 mutations differs considerably , which may be ascribed to genetic backgrounds , environmental modifiers , epigenetic regulations , or even phenotypic ascertainment bias . furthermore , the remarkable genetic heterogeneity of vsd was supported by a failure to detect mutations in nearly 98% of our cohort patients . mutations in other transcription factors associated with cardiogenesis , such as nkx2 - 5 , tbx5 , tbx20 , and gata6 , have also been identified in patients with vsd . in addition , mutations in cardiac structural proteins as troponin i type 3 ( tnni3 ) and alpha myosin heavy chain ( myh6 ) were identified in vsd patients . therefore , genetic analysis of these candidate genes in our cohort patients with vsd is warranted . however , these vsd - associated genes have also been reported to result in other cardiac or even extracardiac defects that underlie the clinical heterogeneity and the suggestive roles of the established genotype - phenotype relations of these genes . interestingly , nkx2 - 5 mutations were mainly reported to cause atrial septal defect and atrioventricular block , implying the essential role of nkx2 - 5 in the morphogenesis of the heart and in the construction of the cardiac conduction system [ 4749 ] . moreover , tbx5 mutations were predominantly found to cause holt - oram syndrome , which is clinically characterized by upper limb and heart defects . despite the variable clinical manifestations , upper limb abnormalities were always present , highlighting the pivotal role of tbx5 in the development of both heart and upper limbs . in the present study , a compound phenotype of vsd and atrioventricular block was observed in 3 out of 230 patients . genetic screening of nkx2 - 5 in the 3 patients was performed , but no non - synonymous variants were found , largely excluding the possibility of nkx2 - 5 as a cause for vsd combined with atrioventricular block in these patients . association of compromised gata4 with increased susceptibility to vsd has been demonstrated in animal experiments . in the embryonic hearts of knock - down chicks generated by using small interfering rnas targeted to gata4 , the bilateral myocardial rudiments failed to travel to the midline , resulting in the formation of 2 separate hearts in lateral positions , an anomaly of cardia bifida . in mice , gata4 is one of the earliest transcription factors expressed in developing cardiac cells and continues to be expressed abundantly in cardiomyocytes throughout the life of mice . homozygous gata4-deficient mice died between day 7.0 to 9.5 and analysis of the gata4-null embryo substantiated the lethal failure to form a linear heart tube . transgenic mice expressing gata4 mutants demonstrated a wide variety of cardiac malformations , including septal defects , right ventricular hypoplasia , endocardial cushion defect , tetralogy of fallot , double outlets of the right ventricle , and cardiomyopathy , similar to the anomalies seen in humans . taken together , these experimental results from animals suggest that gata4 mutations underlie a wide variety of congenital cardiac abnormalities , including vsd in humans . the findings link novel gata4 mutations to vsd and provide additional insight into the molecular etiology associated with vsd , suggesting potential implications for the prophylaxis and therapy of vsd .