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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.

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.

As the most common form of birth defect in humans, congenital heart disease (CHD) is associated with substantial morbidity and mortality in both children and adults. Increasing evidence demonstrates that genetic defects play a pivotal role in the pathogenesis of CHD. However, CHD is of great heterogeneity, and in an overwhelming majority of cases, the genetic determinants underpinning CHD remain elusive. In the present investigation, the coding exons and flanking introns of the CASZ1 gene, which codes for a zinc finger transcription factor essential for the cardiovascular morphogenesis, were sequenced in 172 unrelated patients with CHD. As a result, a novel heterozygous CASZ1 mutation, p.L38P, was identified in an index patient with congenital ventricular septal defect (VSD). Genetic scanning of the mutation carrier's available family members revealed that the mutation was present in all affected patients but absent in unaffected individuals. Analysis of the proband's pedigree showed that the mutation co-segregated with VSD, which was transmitted as an autosomal dominant trait with complete penetrance. The missense mutation, which altered the amino acid that was highly conserved evolutionarily, was absent in 200 unrelated, ethnically-matched healthy subjects used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant CASZ1 had significantly reduced transcriptional activity as compared with its wild-type counterpart. To the best of our knowledge, the current study firstly identifies CASZ1 as a new gene predisposing to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD and a potential therapeutic target for CASZ1-associated CHD, suggesting potential implications for personalized prophylaxis and therapy of CHD.

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.

Congenital heart disease (CHD) is the most prevalent developmental abnormality in humans and is the most common non-infectious cause of infant morbidity and mortality. Increasing evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous, and the genetic determinants underpinning CHD in most patients remain unknown. In this study, the whole coding region of the PITX2 gene (isoform c) was sequenced in 185 unrelated patients with CHD. The available relatives of a mutation carrier and 300 unrelated healthy individuals used as controls were also genotyped for PITX2. The functional characteristics of the mutation were delineated by using a dual-luciferase reporter assay system. As a result, a novel heterozygous PITX2 mutation, p.Q102L, was identified in a patient with tetralogy of Fallot (TOF). Genetic analysis of the index patient's pedigree showed that the mutation co-segregated with TOF. The mutation was absent in 600 reference chromosomes. Biochemical analysis revealed that the Q102L-mutant PITX2 is associated with significantly reduced transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation markedly decreased the synergistic activation between PITX2 and NKX2-5. This study firstly associates PITX2 loss-of-function mutation with increased susceptibility to TOF, providing novel insight into the molecular mechanism of CHD.

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.

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.

Congenital heart disease (CHD) is the most common form of birth defect and is the most prevalent non-infectious cause of infant death. A growing body of evidence documents that genetic defects are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic basis underpinning CHD in an overwhelming majority of patients remain unclear. In this study, the coding exons and flanking introns of the Nkx2.6 gene, which codes for a homeodomain-containing transcription factor important for normal cardiovascular development, were sequenced in 320 unrelated patients with CHD, and two novel heterozygous Nkx2.6 mutations, p.V176M and p.K177X, were identified in two unrelated patients with CHD, respectively, including a patient with tetralogy of Fallot and a patient with double outlet of right ventricle and ventricular septal defect. The mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across species. Due to unknown transcriptional targets of Nkx2.6, the functional consequences of the identified mutations at transcriptional activity were evaluated by using Nkx2.5 as a surrogate. Alignment between human Nkx2.6 and Nkx2.5 proteins showed that V176M-mutant Nkx2.6 was equivalent to V182M-mutant Nkx2.5 and K177X-mutant Nkx2.6 was equal to K183X-mutant Nkx2.5, and introduction of V182M or K183X into Nkx2.5 significantly diminished its transcriptional activating function when compared with its wild-type counterpart. To our knowledge, this is the first report on the association of Nkx2.6 loss-of-function mutation with increased susceptibility to tetralogy of Fallot or double outlet of right ventricle and ventricular septal defect, providing novel insight into the molecular mechanism of CHD.

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.

The occurrence, correlation and clinical outcome of intra-procedural stent thrombosis (IPST) in patients undergoing primary percutaneous coronary intervention (PCI) in the setting of acute coronary syndromes (ACS) have not been previously described. This retrospective study comprised a review of