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Background Atrial fibrillation (AF) represents the most common clinical cardiac arrhythmia and substantially increases the risk of cerebral stroke, heart failure, and death. Although causative genes for AF have been identified, the genetic determinants for AF remain largely unclear. Objective This study aimed to investigate the molecular basis of AF in a Chinese kindred. Methods A 4-generation family with autosomal-dominant AF and other arrhythmias (atrioventricular block, sinus bradycardia, and premature ventricular contractions) was recruited. Genome-wide scan with microsatellite markers and linkage analysis as well as whole-exome sequencing analysis were performed. Electrophysiological characteristics and subcellular localization of the AF-linked mutant were analyzed using dual whole-cell patch clamps and confocal microscopy, respectively. Results A novel genetic locus for AF was mapped to chromosome 17q21.3, a 3.23-cM interval between markers D17S951 and D17S931, with a maximum 2-point logarithm of odds score of 4.2144 at marker D17S1868. Sequencing analysis revealed a heterozygous mutation in the mapping region, NM_005497.4:c.703A>T;p.(M235L), in the GJC1 gene encoding connexin45 (Cx45). The mutation cosegregated with AF in the family and was absent in 632 control individuals. The mutation decreased the coupling conductance in cell pairs (M235L/M235L, M235L/Cx45, M235L/Cx43, and M235L/Cx40), likely because of impaired subcellular localization. Conclusion This study defines a novel genetic locus for AF on chromosome 17q21.3 and reveals a loss-of-function mutation in GJC1 (Cx45) contributing to AF and other cardiac arrhythmias.

Background Catheter ablation (CA) is a promising option in most patients with refractory atrial fibrillation (AF). However, data on over 5 years' outcomes with larger numbers in hypertrophic cardiomyopathy (HCM) patients with AF have not been reported. We assessed the outcome of 120 HCM patients following CA compared with a non-CA group and general patients without AF matched by HCM type with a 61.9 ± 31.6-month follow-up. Methods and results A total of 120 patients (age 61 ± 9.8 years, female n = 43, 35.8%) with paroxysmal AF (n = 60, 50%) and persistent AF (n = 60, 50%) were enrolled. Of the 120 patients, 48 (40%) required redo procedures, and 82 (68.3%) were in sinus rhythm at the last evaluation. The composite clinical events rate following the initial CA was lower than that in the non-CA group (P = .023) and was also comparable to that in general patients without AF matched by HCM type (P = .729). Female (HR 2.358, 95% CI, 1.151-4.831; P = .019), NYHA functional class III-IV (HR 2.422, 95% CI, 1.032-5.685; P = .042) and left atrial diameter ≥50 mm (HR 3.319, 95% CI, 1.469-7.499; P = .004) were predictors of AF recurrence after multiple procedures. Conclusions CA was successful in restoring long-term sinus rhythm and improving symptomatic status in most HCM patients with refractory AF especially for those patients with small atrial size and mild symptoms. In addition, CA may contribute to the prevention of major clinical adverse events in the long-term clinical course.

Atrial fibrillation (AF), as the most common sustained cardiac arrhythmia, is associated with substantially increased morbidity and mortality. Aggregating evidence demonstrates that genetic defects play a crucial role in the pathogenesis of AF, especially in familial AF. Nevertheless, AF is of pronounced genetic heterogeneity, and in an overwhelming majority of cases the genetic determinants underlying AF remain elusive. In the current study, 162 unrelated patients with familial AF and 238 unrelated healthy individuals served as controls were recruited. The coding exons and splicing junction sites of the SHOX2 gene, which encodes a homeobox-containing transcription factor essential for proper development and function of the cardiac conduction system, were sequenced in all study participants. The functional effect of the mutant SHOX2 protein was characterized with a dual-luciferase reporter assay system. As a result, a novel heterozygous SHOX2 mutation, c.580C>T or p.R194X, was identified in an index patient, which was absent from the 476 control chromosomes. Genetic analysis of the proband's pedigree revealed that the nonsense mutation co-segregated with AF in the family with complete penetrance. Functional assays demonstrated that the mutant SHOX2 protein had no transcriptional activity compared with its wild-type counterpart. In conclusion, this is the first report on the association of SHOX2 loss-of-function mutation with enhanced susceptibility to familial AF, which provides novel insight into the molecular mechanism underpinning AF, suggesting potential implications for genetic counseling and individualized management of AF patients.

As the most prevalent form of human birth defect, congenital heart disease (CHD) contributes to substantial morbidity, mortality and socioeconomic burden worldwide. Aggregating evidence has convincingly demonstrated that genetic defects exert a pivotal role in the pathogenesis of CHD, and causative mutations in multiple genes have been causally linked to CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic components underpinning CHD in the overwhelming majority of patients remain obscure. In this research, a four-generation consanguineous family suffering from CHD transmitted in an autosomal dominant mode was recruited. By whole-exome sequencing and bioinformatics analyses as well as Sanger sequencing analyses of the family members, a new heterozygous SOX17 variation, NM_022454.4: c.553G > T; p.(Glu185*), was identified to co-segregate with CHD in the family, with complete penetrance. The nonsense variation was neither detected in 310 unrelated healthy volunteers used as controls nor retrieved in such population genetics databases as the Exome Aggregation Consortium database, Genome Aggregation Database, and the Single Nucleotide Polymorphism database. Functional assays by utilizing a dual-luciferase reporter assay system unveiled that the Glu185*-mutant SOX17 protein had no transcriptional activity on its two target genes NOTCH1 and GATA4, which have been reported to cause CHD. Furthermore, the mutation abrogated the synergistic transactivation between SOX17 and NKX2.5, another established CHD-causing transcription factor. These findings firstly indicate SOX17 loss-of-function mutation predisposes to familial CHD, which adds novel insight to the molecular mechanism of CHD, implying potential implications for genetic risk appraisal and individualized prophylaxis of the family members affected with CHD.

Congenital heart defect (CHD) is the most common type of birth defect in humans and a leading cause of infant morbidity and mortality. Previous studies have demonstrated that genetic defects play a pivotal role in the pathogenesis of CHD. However, the genetic basis of CHD remains poorly understood due to substantial genetic heterogeneity. In this study, the coding exons and splicing boundaries of the NR2F2 gene, which encodes a pleiotropic transcription factor required for normal cardiovascular development, were sequenced in 168 unrelated patients with CHD, and a novel mutation (c.247G > T, equivalent to p.G83X) was detected in a patient with double outlet right ventricle as well as ventricular septal defect. Genetic scanning of the mutation carrier's relatives available showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the index patient's pedigree displayed that the mutation co-segregated with CHD, which was transmitted as an autosomal dominant trait with complete penetrance. The nonsense mutation was absent in 230 unrelated, ethnically-matched healthy individuals used as controls. Functional deciphers by using a dual-luciferase reporter assay system revealed that the mutant NR2F2 protein had no transcriptional activity as compared with its wild-type counterpart. Furthermore, the mutation abrogated the synergistic transcriptional activation between NR2F2 and GATA4, another core cardiac transcription factor associated with CHD. This study firstly associates NR2F2 loss-of-function mutation with an increased susceptibility to double outlet right ventricle in humans, which provides further significant insight into the molecular mechanisms underpinning CHD, suggesting potential implications for genetic counseling of CHD families and personalized treatment of CHD patients.

Aggregating evidence suggests that genetic determinants play a pivotal role in the pathogenesis of the congenitally bicuspid aortic valve (BAV). BAV is of pronounced genetic heterogeneity, and the genetic components underlying BAV in an overwhelming majority of patients remain elusive. In the current study, the whole coding exons and adjacent introns, as well as 5′ and 3′ untranslated regions of the GATA4 gene, which codes for a zinc-finger transcription factor crucial for the normal development of the aortic valve, were screened by direct sequencing in 150 index patients with congenital BAV. The available family members of an identified mutation carrier and 300 unrelated, ethnically matched healthy individuals used as controls were also genotyped for GATA4 . The functional effect of the mutation was characterized using a dual-luciferase reporter assay system. As a result, a novel heterozygous GATA4 mutation, p.E147X, was identified in a family with BAV transmitted in an autosomal dominant pattern. The nonsense mutation was absent in 600 control chromosomes. Functional deciphers revealed that the mutant GATA4 protein lost transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation disrupted the synergistic transcriptional activation between GATA4 and NKX2.5, another transcription factor responsible for BAV. In conclusion, this study associates the GATA4 loss-of-function mutation with enhanced susceptibility to a BAV, thus providing novel insight into the molecular mechanism underpinning the BAV.

Dilated cardiomyopathy (DCM), the most common form of primary myocardial disease, is a leading cause of congestive heart failure and the most common indication for heart transplantation. Recently, NKX2-5 mutations have been involved in the pathogenesis of familial DCM. However, the prevalence and spectrum of NKX2-5 mutations associated with sporadic DCM remain to be evaluated. In this study, the coding regions and flanking introns of the NKX2-5 gene, which encodes a cardiac transcription factor pivotal for cardiac development and structural remodeling, were sequenced in 210 unrelated patients with sporadic adult-onset DCM. A total of 300 unrelated healthy individuals used as controls were also genotyped for NKX2-5. The functional effect of the mutant NKX2-5 was investigated using a dual-luciferase reporter assay system. As a result, two novel heterozygous NKX2-5 mutations, p.R139W and p.E167X, were identified in 2 unrelated patients with sporadic adult-onset DCM, with a mutational prevalence of approximately 0.95%. The mutations were absent in 600 referential chromosomes and the altered amino acids were completely conserved evolutionarily across species. Functional assays revealed that the NKX2-5 mutants were associated with significantly reduced transcriptional activity. Furthermore, the mutations abrogated the synergistic activation between NKX2-5 and GATA4 as well as TBX20, two other cardiac key transcription factors that have been causally linked to adult-onset DCM. This study is the first to associate NKX2-5 loss-of-function mutations with enhanced susceptibility to sporadic DCM, which provides novel insight into the molecular etiology underpinning DCM, and suggests the potential implications for the genetic counseling and personalized treatment of the DCM patients.

Congenital bicuspid aortic valve (BAV) represents the most common type of cardiac birth defect affecting 0.4‑2% of the general population, and accounts for a markedly increased incidence of life‑threatening complications, including valvulopathy and aortopathy. Accumulating evidence has demonstrated the genetic basis of BAV. However, the genetic basis for BAV in the majority of cases remains to be elucidated. In the present study, the coding regions and splicing donors/acceptors of the nuclear receptor subfamily 2 group F member 2 (NR2F2) gene, which encodes a transcription factor essential for proper cardiovascular development, were sequenced in 176 unrelated cases of congenital BAV. The available family members of the proband carrying an identified NR2F2 mutation and 280 unrelated, sex‑ and ethnicity‑matched healthy individuals as controls were additionally genotyped for NR2F2. The functional effect of the mutation was characterized using a dual‑luciferase reporter assay system. As a result, a novel heterozygous NR2F2 mutation, NM_021005.3: c.288C>A; p.(Cys96*), was identified in a family with BAV, which was transmitted in an autosomal dominant mode with complete penetrance. The nonsense mutation was absent from the 560 control chromosomes. Functional analysis identified that the mutant NR2F2 protein had no transcriptional activity. Furthermore, the mutation disrupted the synergistic transcriptional activation between NR2F2 and transcription factor GATA‑4, another transcription factor that is associated with BAV. These findings suggested NR2F2 as a novel susceptibility gene of human BAV, which reveals a novel molecular pathogenesis underpinning BAV.

Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia and substantially increases the risks of cerebral stroke, heart failure and death. Accumulating evidence has convincingly demonstrated the strong genetic basis of AF, and an increasing number of pathogenic variations in over 50 genes have been causally linked to AF. Nevertheless, AF is of pronounced genetic heterogeneity, and the genetic determinants underpinning AF in most patients remain obscure. In the current investigation, a Chinese pedigree with AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy was recruited. Whole exome sequencing and bioinformatic analysis of the available family members were conducted, and a novel heterozygous variation in the KLF15 gene (encoding Krüppel-like factor 15, a transcription factor critical for cardiac electrophysiology and structural remodeling), NM_014079.4: c.685A&gt, T, p.(Lys229*), was identified. The variation was verified by Sanger sequencing and segregated with autosomal dominant AF in the family with complete penetrance. The variation was absent from 300 unrelated healthy subjects used as controls. In functional assays using a dual-luciferase assay system, mutant KLF15 showed neither transcriptional activation of the KChIP2 promoter nor transcriptional inhibition of the CTGF promoter, alone or in the presence of TGFB1, a key player in the pathogenesis of arrhythmias and cardiomyopathies. The findings indicate KLF15 as a new causative gene responsible for AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy, and they provide novel insight into the molecular mechanisms underlying cardiac arrhythmias and hypertrophic cardiomyopathy.

Atrial fibrillation (AF) represents the most frequent form of sustained cardiac rhythm disturbance, affecting approximately 1% of the general population worldwide, and confers a substantially enhanced risk of cerebral stroke, heart failure, and death. Increasing epidemiological studies have clearly demonstrated a strong genetic basis for AF, and variants in a wide range of genes, including those coding for ion channels, gap junction channels, cardiac structural proteins and transcription factors, have been identified to underlie AF. Nevertheless, the genetic pathogenesis of AF is complex and still far from completely understood. Here, whole-exome sequencing and bioinformatics analyses of a three-generation family with AF were performed, and after filtering variants by multiple metrics, we identified a heterozygous variant in the ISL1 gene (encoding a transcription factor critical for embryonic cardiogenesis and postnatal cardiac remodeling), NM_002202.2: c.481G > T; p.(Glu161*), which was validated by Sanger sequencing and segregated with autosome-dominant AF in the family with complete penetrance. The nonsense variant was absent from 284 unrelated healthy individuals used as controls. Functional assays with a dual-luciferase reporter assay system revealed that the truncating ISL1 protein lost transcriptional activation on the verified target genes MEF2C and NKX2-5. Additionally, the variant nullified the synergistic transactivation between ISL1 and TBX5 as well as GATA4, two other transcription factors that have been implicated in AF. The findings suggest ISL1 as a novel gene contributing to AF, which adds new insight to the genetic mechanisms underpinning AF, implying potential implications for genetic testing and risk stratification of the AF family members.

Previous genome-wide association studies have demonstrated that single nucleotide polymorphisms in T‑box (TBX)5 are associated with increased susceptibility to atrial fibrillation (AF), and a recent study has causally linked a TBX5 mutation to atypical Holt-Oram syndrome and paroxysmal AF. However, the prevalence and spectrum of TBX5 mutations in patients with AF remain to be elucidated. In the present study, a cohort of 190 unrelated patients with idiopathic AF were prospectively recruited, with 400 unrelated healthy individuals recruited as controls. The coding exons and flanking introns of the TBX5 gene were sequenced in the participants. The functional characteristics of the mutant TBX5 were delineated in contrast with its wild‑type counterpart using a dual‑luciferase reporter assay system. As a result, a novel heterozygous TBX5 mutation, p.P132S, was identified in an index patient with AF, with a mutational prevalence of ~0.53%. Genetic analysis of the proband's family showed that the mutation co‑segregated with AF, and was transmitted in an autosomal dominant pattern. The missense mutation was absent in the 800 control chromosomes, and the altered amino acid was completely evolutionarily conserved across species. Functional analyses revealed that the mutant TBX5 had significantly reduced transcriptional activity. Furthermore, the mutation markedly decreased the synergistic activation between TBX5 and NK2 homeobox 5, another transcription factor which has been causatively linked to AF. The present study was the first, to the best of our knowledge, to report on the association between a TBX5 loss‑of‑function mutation and increased susceptibility to AF. These results provide novel insight into the molecular mechanism underpinning AF, and have potential implications in the development of novel prophylactic and therapeutic strategies for AF, the most common form of sustained cardiac arrhythmia.

Atrial fibrillation (AF), the most common type of cardiac rhythm disturbance encountered in clinical practice, is associated with substantially increased morbidity and mortality. Aggregating evidence demonstrates that abnormal cardiovascular development is involved in the pathogenesis of AF. A recent study has revealed that the TBX5 gene, which encodes a T-box transcription factor key to cardiovascular development, was associated with AF and atypical Holt-Oram syndrome. However, the prevalence and spectrum of TBX5 mutation in patients with lone AF remain unclear. In this study, the coding regions and splicing junction sites of TBX5 were sequenced in 192 unrelated patients with lone AF and 300 unrelated ethnically-matched healthy individuals used as controls. The causative potential of the identified TBX5 variation was evaluated by MutationTaster and PolyPhen-2. The functional effect of the mutant TBX5 was assayed by using a dual-luciferase reporter assay system. As a result, a novel heterozygous TBX5 mutation, p.H170D, was identified in a patient, with a mutational prevalence of approximately 0.52%. This mutation, which was absent in the 300 control individuals, altered the amino acid completely conserved evolutionarily across species, and was predicted to be disease-causing. Functional deciphers showed that the mutant TBX5 was associated with significantly reduced transcriptional activity when compared with its wild-type counterpart. Furthermore, the mutation significantly decreased the synergistic activation between TBX5 and NKX2-5 or GATA4. The findings expand the mutational spectrum of TBX5 linked to AF and provide new evidence that dysfunctional TBX5 may contribute to lone AF.

Background: The basic helix-loop-helix transcription factor HAND1 is essential for cardiac development and structural remodeling, and mutations in HAND1 have been causally linked to various congenital heart diseases. However, whether genetically compromised HAND1 predisposes to dilated cardiomyopathy (DCM) in humans remains unknown. Methods: The whole coding region and splicing junctions of the HAND1 gene were sequenced in 140 unrelated patients with idiopathic DCM. The available family members of the index patient carrying an identified mutation and 260 unrelated ethnically matched healthy individuals used as controls were genotyped for HAND1. The functional effect of the mutant HAND1 was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. Results: A novel heterozygous HAND1 mutation, p.R105X, was identified in a family with DCM transmitted as an autosomal dominant trait, which co-segregated with DCM in the family with complete penetrance. The nonsense mutation was absent in 520 control chromosomes. Functional analyses unveiled that the mutant HAND1 had no transcriptional activity. Furthermore, the mutation abolished the synergistic activation between HAND1 and GATA4, another crucial cardiac transcription factors that has been associated with various congenital cardiovascular malformations and DCM. Conclusions: This study firstly reports the association of HAND1 loss-of-function mutation with increased susceptibility to DCM in humans, which provides novel insight into the molecular mechanisms underpinning DCM.

As two members of the basic helix-loop-helix family of transcription factors, HAND1 and HAND2 are both required for the embryonic cardiogenesis and postnatal ventricular structural remodeling. Recently a HAND1 mutation has been reported to cause dilated cardiomyopathy (DCM). However, the association of a HAND2 mutation with DCM is still to be ascertained. In this research, the coding regions and splicing junction sites of the HAND2 gene were sequenced in 206 unrelated patients affected with idiopathic DCM, and a new heterozygous HAND2 mutation, NM_021973.2: c.199G > T; p.(Glu67*), was discovered in an index patient with DCM. The nonsense mutation was absent in 300 unrelated, ethnically-matched healthy persons. Genetic scan of the mutation carrier's family members revealed that the genetic mutation co-segregated with DCM, which was transmitted in an autosomal dominant fashion, with complete penetrance. Functional deciphers unveiled that the mutant HAND2 protein had no transcriptional activity. In addition, the mutation abrogated the synergistic transcriptional activation between HAND2 and GATA4 or between HAND2 and NKX2.5, two other cardiac transcription factors that have been implicated in DCM. These research findings firstly suggest HAND2 as a novel gene predisposing to DCM in humans, which adds novel insight to the molecular pathogenesis of DCM, implying potential implications in the design of personized preventive and therapeutic strategies against DCM.

Dilated cardiomyopathy (DCM) represents the most prevalent form of primary cardiomyopathy, and is the most common reason for heart transplantation and a major cause of congestive heart failure. Aggregating evidence demonstrates that genetic defects are associated with DCM, and a great number of mutations in >50 genes have been linked to DCM. However, DCM is a genetically heterogeneous disorder and the genetic components underpinning DCM in a significant proportion of patients remain unknown. In the present study, the coding exons and flanking exon‑intron boundaries of the T-Box 5 (TBX5) gene, which encodes a T‑box transcription factor required for normal cardiac development, were sequenced in 146 unrelated patients with sporadic DCM. The functional characteristics of the mutant TBX5 were assayed in contrast to its wild‑type counterpart by using a dual‑luciferase reporter assay system. As a result, a novel heterozygous TBX5 mutation, p.A143T, was identified in a patient with sporadic DCM. The missense mutation, which was absent in 400 control chromosomes, altered the amino acid that was completely conserved evolutionarily among species. Biological analyses revealed that the A143T mutation of TBX5 was associated with significantly decreased transcriptional activity on the promoter of the target gene atrial natriuretic factor (ANF), when compared to its wild‑type counterpart. Furthermore, the A143T mutation abolished the synergistic activation of the ANF promoter between TBX5 and GATA binding protein 4 (GATA4), another crucial transcriptional factor for heart development. To the best of our knowledge, this is the first report on the association of a TBX5 loss‑of‑function mutation with an enhanced susceptibility to sporadic DCM, providing novel insight into the molecular mechanisms of the pathogenesis of DCM and suggesting potential implications for the prenatal prophylaxis and personalized treatment of this commonest primary myocardial disease.

The cardiac T-box transcription factor TBX5 is crucial for proper cardiovascular development, and mutations in TBX5 have been associated with various congenital heart diseases and arrhythmias in humans. However, whether mutated TBX5 contributes to dilated cardiomyopathy (DCM) remains unclear. In this study, the coding exons and flanking introns of the TBX5 gene were sequenced in 190 unrelated patients with idiopathic DCM. The available family members of the index patient carrying an identified mutation and 200 unrelated ethnically matched healthy individuals used as controls were genotyped for TBX5. The functional characteristics of the mutant TBX5 were explored in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. As a result, a novel heterozygous TBX5 mutation, p.S154A, was identified in a family with DCM inherited in an autosomal dominant pattern, which co-segregated with DCM in the family with complete penetrance. The missense mutation was absent in 400 control chromosomes and the altered amino acid was completely conserved evolutionarily across various species. Functional assays revealed that the mutant TBX5 had significantly decreased transcriptional activity. Furthermore, the mutation markedly diminished the synergistic activation of TBX5 with NKX2-5 or GATA4, other two transcription factors causatively linked to DCM. This study firstly associates TBX5 loss-of-function mutation with enhanced susceptibility to DCM, providing novel insight into the molecular mechanisms of DCM, and suggesting the potential implications in the development of new treatment strategies for this common form of myocardial disorder.

Dilated cardiomyopathy (DCM), the most common form of primary myocardial disease, is an important cause of sudden cardiac death and heart failure and is the leading indication for heart transplantation in children and adults worldwide. Recent studies have revealed a strong genetic basis for idiopathic DCM, with many distinct genes causally implicated. Nevertheless, DCM is a genetically heterogeneous disorder and the genetic determinants underlying DCM in a substantial proportion of patients remain unclear. In this study, the whole coding exons and flanking introns of the GATA binding protein 5 (GATA5) gene, which codes for a zinc-finger transcription factor essential for cardiovascular development and structural remodeling, were sequenced in 130 unrelated patients with idiopathic DCM. The available relatives of the index patient carrying an identified mutation and 200 unrelated ethnically matched healthy individuals used as the controls were genotyped for GATA5. The functional characteristics of the mutant GATA5 were analyzed in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. As a result, a novel heterozygous GATA5 mutation, p.G240D, was identified in a family with DCM inherited in an autosomal dominant pattern, which co-segregated with DCM in the family with complete penetrance. The missense mutation was absent in 400 reference chromosomes and the altered amino acid was completely conserved evolutionarily across species. Functional analyses revealed that the GATA5 mutant was associated with significantly diminished transcriptional activity. This study firstly links GATA5 mutation to DCM, which provides novel insight into the molecular mechanisms of DCM, suggesting a potential molecular target for the prenatal prophylaxis and allele-specific treatment of DCM.

Dilated cardiomyopathy (DCM) is the most prevalent type of primary myocardial disease, which is the third most common cause of heart failure and the most frequent reason for heart transplantation. Aggregating evidence demonstrates that genetic risk factors are involved in the pathogenesis of idiopathic DCM. Nevertheless, DCM is of remarkable genetic heterogeneity and the genetic defects underpinning DCM in an overwhelming majority of patients remain unknown. In the present study, the whole coding exons and splice junction sites of the NKX2-5 gene, which encodes a homeodomain transcription factor crucial for cardiac development and structural remodeling, were sequenced in 130 unrelated patients with idiopathic DCM. The available relatives of the index patient harboring an identified mutation and 200 unrelated ethnically matched healthy individuals used as controls were genotyped for the NKX2-5 gene. The functional effect of the mutant NKX2-5 was characterized in contrast to its wild-type counterpart using a dual-luciferase reporter assay system. As a result, a novel heterozygous NKX2-5 mutation, p.S146W, was identified in a family with DCM inherited as an autosomal dominant trait, which co-segregated with DCM in the family with complete penetrance. Notably, the mutation carriers also had arrhythmias, such as paroxysmal atrial fibrillation and atrioventricular block. The missense mutation was absent in 400 reference chromosomes and the altered amino acid was completely conserved evolutionarily among species. Functional analysis revealed that the NKX2-5 mutant was associated with a significantly reduced transcriptional activity. The findings expand the mutational spectrum of NKX2-5 linked to DCM and provide novel insight into the molecular mechanisms underlying DCM, contributing to the antenatal prophylaxis and allele-specific management of DCM.

Bicuspid aortic valve (BAV) is the most common form of congenital cardiovascular defect in humans and is associated with substantial morbidity and mortality. Emerging evidence demonstrates that genetic risk factors play an important role in the pathogenesis of BAV. However, BAV is a genetically heterogenous disorder, and the genetic defects underpinning BAV in most patients remain to be identified. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeodomain-containing transcription factor essential for the normal development of the aortic valve, were sequenced in 142 unrelated patients with BAV. The available relatives of the mutation carrier and 200 unrelated healthy subjects used as controls were also genotyped for NKX2.5. The functional characteristics of the mutation were delineated by using a dual-luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.K192X, was identified in a family with BAV transmitted in an autosomal dominant pattern. The nonsense mutation was absent in 400 control chromosomes. Functional analyses revealed that the mutant NKX2.5 had no transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation abolished the synergistic transcriptional activation between NKX2.5 and GATA5, another transcription factor crucial for the aortic valvular morphogenesis. In conclusion, this study is the first to link an NKX2.5 loss-of-function mutation to enhanced susceptibility to human BAV, providing novel insight into the molecular mechanism of BAV and suggesting potential implications for genetic counseling and clinical care of families presenting with BAV.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia and is associated with substantially increased morbidity and mortality rates. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of AF and a number of AF-associated genes have been identified. Nevertheless, AF is a genetically heterogeneous disorder and the genetic components underpinning AF in an overwhelming majority of patients remain unclear. In this study, the entire coding exons and splice junction sites of the NK2 homeobox 6 (NKX2-6) gene, which encodes a homeodomain transcription factor important for cardiovascular development, were sequenced in 150 unrelated patients with lone AF, and a novel heterozygous NKX2-6 mutation, p.Q175H, was identified in an index patient. Genetic analysis of the available family members of the mutation carrier revealed that the mutation co-segregated with AF transmitted in an autosomal dominant pattern. The missense mutation was absent in the 200 unrelated ethnically matched healthy individuals used as controls and the altered amino acid was completely conserved evolutionarily among species. Due to unknown transcriptional targets of NKX2-6, the functional characteristics of the mutation as regards transcriptional activity were analyzed using NKX2-5 as a surrogate. Alignment between human NKX2-6 and NKX2-5 proteins displayed that the Q175H-mutant NKX2-6 was equivalent to the Q181H-mutant NKX2-5, and the introduction of Q181H into NKX2-5 significantly decreased its transcriptional activity at the atrial natriuretic factor promoter. The present study firstly associates genetically defective NKX2-6 with enhanced susceptibility to AF, providing novel insight into the molecular mechanisms underlying AF and suggesting potential strategies for the antenatal prophylaxis and personalized treatment of AF.

Dilated cardiomyopathy (DCM) is a common primary myocardial disease leading to congestive heart failure, arrhythmia and sudden cardiac death. Increasing studies demonstrate substantial genetic determinants for DCM. Nevertheless, DCM is of substantial genetic heterogeneity, and the genetic basis for DCM in most patients remains unclear. The present study was sought to investigate the association of a genetic variant in the ZBTB17 gene with DCM. A cohort of 158 unrelated patients with idiopathic DCM and a total of 230 unrelated, ethnically matched healthy individuals used as controls were recruited. The coding exons and splicing boundaries of ZBTB17 were sequenced in all study participants. The functional effect of the mutant ZBTB17 was characterized by a dual-luciferase reporter assay system. A novel heterozygous ZBTB17 mutation, p.E243X, was discovered in an index patient. Genetic scan of the mutation carrier's available relatives showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the proband's pedigree revealed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation was absent in the 460 control chromosomes. Functional assays demonstrated that the truncated ZBTB17 protein had no transcriptional activity as compared with its wild-type counterpart. This study firstly associates ZBTB17 loss-of-function mutation with enhanced susceptibility to DCM in humans, which provides novel insight into the molecular mechanism underpinning DCM, implying potential implications for genetic counseling and personalized management of DCM.

Background:The MADS-box transcription factor myocyte enhancer factor 2C (MEF2C) is required for the cardiac development and postnatal adaptation and in mice-targeted disruption of theMEF2Cgene results in dilated cardiomyopathy (DCM). However, in humans, the association ofMEF2Cvariation with DCM remains to be investigated.Methods:The coding regions and splicing boundaries of theMEF2Cgene were sequenced in 172 unrelated patients with idiopathic DCM. The available close relatives of the index patient harboring an identifiedMEF2Cmutation and 300 unrelated, ethnically matched healthy individuals used as controls were genotyped forMEF2C. The functional effect of the mutant MEF2C protein was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system.Results:A novel heterozygous MEF2C mutation, p.Y157X, was detected in an index patient with adult-onset DCM. Genetic screen of the mutation carrier’s family members revealed that the mutation co-segregated with DCM, which was transmitted as an autosomal dominant trait with complete penetrance. The non-sense mutation was absent in 300 control individuals. Functional analyses unveiled that the mutant MEF2C protein had no transcriptional activity. Furthermore, the mutation abolished the synergistic transactivation between MEF2C and GATA4 as well as HAND1, two other transcription factors that have been associated with DCM.Conclusions:This study indicatesMEF2Cas a new gene responsible for human DCM, which provides novel insight into the mechanism underpinning DCM, suggesting potential implications for development of innovative prophylactic and therapeutic strategies for DCM, the most prevalent form of primary myocardial disease.

BACKGROUND The zinc finger transcription factor CASZ1 plays a key role in cardiac development and postnatal adaptation, and in mice, deletion of the CASZ1 gene leads to dilated cardiomyopathy (DCM). However, in humans whether genetically defective CASZ1 contributes to DCM remains unclear. METHODS The coding exons and splicing junction sites of the CASZ1 gene were sequenced in 138 unrelated patients with idiopathic DCM. The available family members of the index patient harboring an identified CASZ1 mutation and 200 unrelated, ethnically matched healthy individuals used as controls were genotyped for CASZ1. The functional characteristics of the mutant CASZ1 were analyzed in contrast to its wild-type counterpart using a luciferase reporter assay system. RESULTS A novel heterozygous CASZ1 mutation, p.K351X, was identified in an index patient with DCM. Genetic analysis of the mutation carrier's family showed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation, which was absent in 400 referential chromosomes, altered the amino acid that was highly conserved evolutionarily. Biological investigations revealed that the mutant CASZ1 had no transcriptional activity. CONCLUSIONS The current study reveals CASZ1 as a new gene responsible for human DCM, which provides novel mechanistic insight and potential therapeutic target for CASZ1-associated DCM, implying potential implications in improved prophylactic and therapeutic strategies for DCM, the most common type of primary myocardial disease.

Dilated cardiomyopathy (DCM), the most prevalent form of primary heart muscle disease, is the third most common cause of heart failure and the most frequent reason for cardiac transplantation. Mounting evidence has demonstrated that genetic risk factors are crucial in the pathogenesis of DCM. However, DCM is genetically heterogeneous, and the genetic basis of DCM in a large majority of cases remains unclear. In the current study, the coding exons and flanking introns of the GATA6 gene, which encodes a zinc‑finger transcription factor essential for cardiogenesis, was sequenced in 140 unrelated patients with DCM, and two novel heterozygous mutations, p.C447Y and p.H475R, were identified in two index patients with DCM, respectively. Analysis of the pedigrees showed that in each family the mutation co-segregated with DCM transmitted in an autosomal-dominant pattern, with complete penetrance. The missense mutations were absent in 400 control chromosomes and predicted to be disease-causing by MutationTaster or probably damaging by PolyPhen-2. The alignment of multiple GATA6 proteins across species revealed that the altered amino acids were completely conserved evolutionarily. The functional assays showed that the mutated GATA6 proteins were associated with significantly reduced transcriptional activation in comparison with their wild-type counterpart. To the best of our knowledge, this is the first study on the association of GATA6 loss-of-function mutations with enhanced susceptibility to familial DCM, which provides novel insight into the molecular mechanism of DCM and suggests potential implications for the antenatal prophylaxis and allele-specific treatment of DCM.

OBJECTIVE: This study aimed to identify novel PITX2c mutations responsible for idiopathic atrial fibrillation. METHODS: A cohort of 210 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy individuals used as controls were recruited. The whole coding exons and splice junctions of the PITX2c gene, which encodes a paired-like homeobox transcription factor required for normal cardiovascular morphogenesis, were sequenced in 210 patients and 200 control subjects. The causative potentials of the identified mutations were automatically predicted by MutationTaster and PolyPhen-2. The functional characteristics of the PITX2c mutations were explored using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous PITX2c mutations (p.Q105L and p.R122C) were identified in 2 of the 210 unrelated patients with idiopathic atrial fibrillation. These missense mutations were absent in the 400 control chromosomes and were both predicted to be pathogenic. Multiple alignments of PITX2c protein sequences across various species showed that the altered amino acids were highly evolutionarily conserved. A functional analysis demonstrated that the mutant PITX2c proteins were both associated with significantly reduced transcriptional activity compared with their wild-type counterparts. CONCLUSION: The findings of this study associate PITX2c loss-of-function mutations with atrial fibrillation, supporting the hypothesis that dysfunctional PITX2c confers enhanced susceptibility to atrial fibrillation and suggesting potential implications for early prophylaxis and allele-specific therapy for this common arrhythmia.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia in humans and is responsible for substantial morbidity and mortality worldwide. Emerging evidence indicates that abnormal cardiovascular development is involved in the pathogenesis of AF. In this study, the coding exons and splice sites of the NKX2-5 gene, which encodes a homeodomain-containing transcription factor essential for cardiovascular genesis, were sequenced in 146 unrelated patients with lone AF as well as the available relatives of the mutation carriers. A total of 700 unrelated ethnically matched healthy individuals used as controls were genotyped. The disease-causing potential of the identified NKX2-5 variations was predicted by MutationTaster and PolyPhen-2. The functional characteristics of the mutant NKX2-5 proteins were analyzed using a dual-luciferase reporter assay system. As a result, two heterozygous NKX2-5 mutations, including a previously reported p.E21Q and a novel p.T180A mutation, were identified in two families with AF transmitted in an autosomal dominant pattern. The mutations co-segregated with AF in the families with complete penetrance. The detected substitutions, which altered the amino acids highly conserved evolutionarily across species, were absent in 700 control individuals and were both predicted to be causative. Functional analyses demonstrated that the NKX2-5 mutants were associated with significantly decreased transcriptional activity compared with their wild-type counterpart. The findings expand the spectrum of NKX2-5 mutations linked to AF and provide additional evidence that dysfunctional NKX2-5 may confer vulnerability to AF, suggesting the potential benefit for the early prophylaxis and personalized treatment of AF.

Congenital heart disease (CHD) is the most common form of birth defect and is the leading noninfectious cause of infant death. A growing body of evidence demonstrates that genetic risk factors are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic defects underlying CHD in an overwhelming majority of patients remain unclear. In this study, the whole coding region and splice junction sites of the PITX2c gene, which encodes variant 3 of paired-like homeodomain transcription factor 2 crucial for normal cardiovascular morphogenesis, were sequenced in 382 unrelated patients with CHD, and 2 novel heterozygous mutations, p.W147X and p.N153D, were identified in 2 unrelated patients with CHD, respectively, including a 1-year-old male patient with double outlet right ventricle in combination with ventricular septal defect and a 4-year-old female patient with ventricular septal defect. The mutations were absent in 400 control chromosomes and were both predicted to be disease-causing by MutationTaster. Multiple alignments of PITX2c proteins across species displayed that the altered amino acids were completely conserved evolutionarily. Functional analysis revealed that the mutated PITX2c proteins were associated with a significantly reduced transactivational activity compared with their wild-type counterpart. These findings provide a novel insight into the molecular mechanisms implicated in CHD, suggesting potential implications for the antenatal prophylaxis and allele-specific treatment of CHD.

Congenital atrial septal defect (ASD) and progressive atriventricular block (AVB) are the two most common phenotypes linked to NK2 homeobox 5 (NKX2.5) mutations in animals and humans. However, the prevalence and spectrum of NKX2.5 mutation in patients with ASD and AVB remain to be elucidated. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeobox‑containing transcription factor essential for development of the heart, were sequenced in a cohort of 62 unrelated patients with ASD and AVB, and subsequently in a mutation carrier's available family members. As controls, 300 unrelated, ethnically‑matched healthy individuals were recruited, who were also genotyped for NKX2.5. The functional consequence of the mutant NKX2.5 was evaluated in contrast to its wild‑type counterpart using a dual‑luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.Q181X, was identified in an index patient with ASD and AVB, with a prevalence of ~1.61%. Genetic analysis of the proband's pedigree revealed that the mutation co‑segregated with ASD and AVB with complete penetrance. The nonsense mutation, which eliminated partial homeobox and the carboxyl terminus, was absent in the 600 control chromosomes. Functional evaluation showed that the NKX2.5 mutant had no transcriptional activity. Furthermore, the mutation disrupted the synergistic activation between NKX2.5 and GATA binding protein 4, another cardiac core transcription factor associated with ASD. The results of the present study expand the spectrum of NKX2.5 mutations linked to ASD and AVB, and indicated that NKX2.5 loss‑of‑function mutations are an uncommon cause of ASD and AVB in humans.

Dilated cardiomyopathy (DCM) is the most prevalent form of primary cardiomyopathy in humans and is a leading cause of heart failure and sudden cardiac death. Genetic abnormalities have been demonstrated to be a major contributor to the development of DCM. However, DCM is a genetically heterogeneous disease, and the genetic basis underlying DCM in a significant proportion of patients remains unclear. In the current study, the coding exons and splicing junction sites of the T‑Box 20 (TBX20) gene, which encodes a T‑box transcription factor essential for cardiac morphogenesis and structural remodeling, were sequenced in 115 unrelated patients with idiopathic DCM, and a novel heterozygous mutation, p.E143X, was identified in one patient. Genetic analysis of the mutation carrier's pedigree indicated that the nonsense mutation was present in all the living family members with DCM, and also in a female patient with a congenital atrial septal defect. The mutation, which was predicted to generate a truncated protein with only the N‑terminus and a fraction of the T‑box domain remaining, was absent in 800 control chromosomes. Functional assays using a dual‑luciferase reporter assay system revealed that the truncated TBX20 protein had no transcriptional activity in contrast to its wild‑type counterpart. Furthermore, the mutation abolished the synergistic activation between TBX20 and NK2 homeobox 5, or between TBX20 and GATA binding protein 4. The observations of the current study expand the mutation spectrum of TBX20 associated with DCM and congenital heart disease (CHD), which provide novel insight into the molecular mechanisms underlying DCM and CHD, suggesting the potential implications for the effective and personalized treatment of these diseases.

BACKGROUND The MADS-box transcription factor myocyte enhancer factor 2C (MEF2C) is required for the cardiac development and postnatal adaptation and in mice-targeted disruption of the MEF2C gene results in dilated cardiomyopathy (DCM). However, in humans, the association of MEF2C variation with DCM remains to be investigated. METHODS The coding regions and splicing boundaries of the MEF2C gene were sequenced in 172 unrelated patients with idiopathic DCM. The available close relatives of the index patient harboring an identified MEF2C mutation and 300 unrelated, ethnically matched healthy individuals used as controls were genotyped for MEF2C. The functional effect of the mutant MEF2C protein was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. RESULTS A novel heterozygous MEF2C mutation, p.Y157X, was detected in an index patient with adult-onset DCM. Genetic screen of the mutation carrier's family members revealed that the mutation co-segregated with DCM, which was transmitted as an autosomal dominant trait with complete penetrance. The non-sense mutation was absent in 300 control individuals. Functional analyses unveiled that the mutant MEF2C protein had no transcriptional activity. Furthermore, the mutation abolished the synergistic transactivation between MEF2C and GATA4 as well as HAND1, two other transcription factors that have been associated with DCM. CONCLUSIONS This study indicates MEF2C as a new gene responsible for human DCM, which provides novel insight into the mechanism underpinning DCM, suggesting potential implications for development of innovative prophylactic and therapeutic strategies for DCM, the most prevalent form of primary myocardial disease.

Tetralogy of Fallot (TOF) represents the most common form of cyanotic congenital heart disease and accounts for significant morbidity and mortality in humans. Emerging evidence has implicated genetic defects in the pathogenesis of TOF. However, TOF is genetically heterogeneous and the genetic basis for TOF in most patients remains unclear. In this study, the GATA4 gene were sequenced in 52 probands with familial TOF, and three novel heterozygous mutations, including A9P and L51V both located in the putative first transactivational domain and N285S in the C-terminal zinc finger, were identified in three probands, respectively. Genetic analysis of the pedigrees demonstrated that in each family the mutation cosegregated with TOF with complete penetrance. The missense mutations were absent in 800 control chromosomes and the altered amino acids were highly conserved evolutionarily. Functional analysis showed that the GATA4 mutants were consistently associated with diminished DNA-binding affinity and decreased transcriptional activity. Furthermore, the N285S mutation completely disrupted the physical interaction between GATA4 and TBX5. To our knowledge, this report associates GATA4 loss-of-function mutations with familial TOF for the first time, providing novel insight into the molecular mechanism involved in TOF and suggesting potential implications for the early prophylaxis and allele-specific therapy of TOF.

The cardiac transcription factor GATA4 is essential for cardiac development, and mutations in this gene have been implicated in a wide variety of congenital heart diseases in both animal models and humans. However, whether mutated GATA4 predisposes to dilated cardiomyopathy (DCM) remains unknown. In this study, the whole coding region and splice junction sites of the GATA4 gene was sequenced in 110 unrelated patients with idiopathic DCM. The available relatives of the index patient harboring an identified mutation and 200 unrelated ethnically matched healthy individuals used as controls were genotyped. The functional effect of the mutant GATA4 was characterized in contrast to its wild-type counterpart using a luciferase reporter assay system. As a result, a novel heterozygous GATA4 mutation, p.C271S, was identified in a family with DCM inherited as an autosomal dominant trait, which co-segregated with DCM in the family with complete penetrance. The missense mutation was absent in 400 control chromosomes and the altered amino acid was completely conserved evolutionarily among species. Functional analysis demonstrated that the GATA4 mutant was associated with significantly decreased transcriptional activity and remarkably reduced synergistic activation between GATA4 and NKX2-5, another transcription factor crucial for cardiogenesis. The findings provide novel insight into the molecular mechanisms involved in the pathogenesis of DCM, suggesting the potential implications in the prenatal diagnosis and gene-specific treatment for this common form of myocardial disorder.