Wei-Yi Fang

Novel connexin40 missense mutations in patients with familial atrial fibrillation.

AIMS This research was aimed at screening connexin40, a cardiac gap junction protein alpha 5, for genetic defects in patients with familial atrial fibrillation (AF). METHODS The subjects included 218 unrelated families with lone AF and 200 ethnically matched unrelated healthy individuals as controls. The entire coding region of the connexin40 gene was sequenced initially in 218 unrelated probands with familial AF. The relatives of mutation carriers and 200 controls were subsequently genotyped for the presence of mutations identified in probands. RESULTS Three novel connexin40 mutations, p.V85I, p.L221I, and p.L229M, were identified in 3 of 218 unrelated AF families, respectively. These heterozygous missense mutations co-segregated with AF in the families and were absent in the 200 unrelated control subjects. A cross-species alignment of connexin40 protein sequences revealed that the altered amino acids were completely conserved evolutionarily. CONCLUSION The findings expand the spectrum of mutations in connexin40 linked to AF and provide new insight into the molecular aetiology involved in the pathogenesis of AF.

Prevalence and spectrum of PITX2c mutations associated with familial atrial fibrillation

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice, accounting for approximately one-third of hospitalizations for heart rhythm disorders. The prevalence of AF is estimated to be 1% in the general population and increases markedly with advancing age, rising from about 0.5% of people in their fifties to nearly 10% of the octogenarians (1) .A F is associated with substantial morbidity and mortality. It confers a 5-fold increased risk of stroke and a double risk of death (1). AF generally occurs secondary to various cardiac and systemic disorders, including hypertension, cor-

GATA4 loss-of-function mutations in familial atrial fibrillation

BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major source of the substantially increased morbidity and mortality. Growing studies demonstrate that genetic defects play pivotal roles in a subgroup of AF. However, AF is a genetically heterogeneous disorder and the molecular basis of AF in a majority of cases remains unknown. METHODS The whole coding region of the GATA4 gene, which encodes a zinc-finger transcription factor essential for cardiogenesis, was analyzed in 130 unrelated probands with AF in contrast to 200 unrelated ethnically matched healthy individuals used as controls. The available family members of the probands harboring the identified mutations were genotyped. The functional effect of the mutant GATA4 was characterized using a luciferase reporter assay system. RESULTS Two novel heterozygous GATA4 mutations, p.S70T and p.S160T, were identified in 2 unrelated families with AF inherited as an autosomal dominant trait, respectively, which co-segregated with AF in each family with complete penetrance. Functional analysis showed that the mutations of GATA4 were associated with a significantly decreased transcriptional activity. CONCLUSION The findings provide new insight into the molecular mechanism involved in the pathogenesis of AF, suggesting the potential implications in the genetic diagnosis and gene-specific therapy of this common arrhythmia.

Mutational spectrum of the GATA5 gene associated with familial atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia seen in clinical practice, with an estimated prevalence of 1-2% in the general population. The incidence of AF increases dramatically with age, ranging from less than 1% in patients under 60 years of age to almost 10% in those aged 80 and over (1). AF is associated with substantial morbidity, mortality and health care burden. AF confers a five-fold increased risk of stroke, and about 15-20% of all strokes result from this tachycardia. AF also accounts for an approximately two-fold increase in risk of death, and a third of all hospitalizations for cardiac rhythm disturbances (1-3). AF frequently arises from diverse cardiac and systemic disorders, including hypertension, coronary artery disease, valvular heart disease, and hyperthyroidism (1). However, in 30% to 45% of AF cases, an underlying cause cannot be detected by routine methods, a condition usually defined as idiopathic or lone AF, of which at least 15% have a positive family history, hence termed familial AF (1). There is now growing evidence demonstrating that genetic defects play an important role in the pathogenesis of AF and multiple genes involved in AF have been identified (4). Nevertheless, AF is genetically heterogeneous and the genetic determinants of AF remain largely unclear. Recent studies highlight a key role for several cardiac transcrip- tion factors, including NKX2-5, GATA4, GATA5 and GATA6, in the cardiogenesis (5,6), and mutations in NKX2-5, GATA4 and GATA6 have been causally implicated in congenital heart diseases and AF (7-12). GATA5 is another member of the GATA family, and its expression and functions overlap with those of GATA4 and GATA6 during cardiovascular development, especially in regulation of target gene expression synergistically with NKX2-5, which points to the likely association of functionally impaired GATA5 with AF (5,6). To assess the prevalence and spectrum of GATA5 mutations in patients with familial AF, 130 unrelated index patients with familial AF identified among the Chinese Han population were included in this study. The control population comprised 200 unrelated ethnically matched healthy individuals. All subjects were appraised by medical history, physical examination, electrocardiography, and echocardiogra- phy. The study subjects were clinically classified using a consistently applied set of definitions (10). The baseline demographic and clinical characteristics of the study population are summarized in Table 1 .T he study protocol was reviewed and approved by the local institutional ethics committee and written informed consent was obtained from all participants prior to study. Peripheral venous blood specimens were taken from all subjects and genomic DNA was extracted as described previously (10). According to the genomic DNA sequence of GATA5 (GenBank accession no. NT_011362), the primer sequences were designed as shown in Table 2. The coding exons (exons 2-7) and their flanking splice junction sites of GATA5 were screened for genetic variations by means of polymerase chain reaction, followed by DNA sequencing with Big Dye chemistry under an ABI 3130 XL DNA Analyzer.

GATA4 loss-of-function mutations underlie familial tetralogy of fallot.

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.

A novel GATA5 loss-of-function mutation underlies lone atrial fibrillation

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with significantly increased morbidity and mortality. Cumulative evidence highlights the importance of genetic defects in the pathogenesis of AF. However, AF is of remarkable heterogeneity and the genetic determinants of AF in a vast majority of patients remain illusive. In this study, the coding exons and splice junctions of the GATA5 gene, which encodes a zinc-finger transcription factor essential for normal cardiogenesis, were sequenced in 118 unrelated patients with lone AF. The available relatives of the index patient carrying an identified mutation and 200 unrelated ethnically-matched healthy individuals used as controls were genotyped. The functional effect of the mutant GATA5 was characterized in contrast to its wild-type counterpart using a luciferase reporter assay system. As a result, a novel heterozygous GATA5 mutation, p.W200G, was identified in a family with AF inherited as an autosomal dominant trait. The mutation was absent in 200 control individuals and the altered amino acid was completely conserved evolutionarily across species. Functional analysis showed that the mutation of GATA5 was associated with a significantly decreased transcriptional activity. These findings provide novel insight into the molecular mechanism involved in AF, suggesting potential implications for the early prophylaxis and gene-specific therapy of AF.

Novel Germline GJA5/Connexin40 Mutations Associated with Lone Atrial Fibrillation Impair Gap Junctional Intercellular Communication

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia worldwide. Here, we investigate the molecular and cellular mechanisms of lone AF‐linked germline mutations in the connexin40 (Cx40) gene, GJA5. The entire coding region of GJA5 was sequenced in 68 unrelated patients with lone AF. A novel germline heterozygous missense mutation in Cx40 (p.I75F) was identified in one index patient. The mutation was also present in the probands father with lone AF but was not found in the unaffected family members who were examined and 200 unrelated healthy control individuals. Electrophysiological studies revealed no electrical coupling of the cell pairs expressing the mutant alone and a significant reduction in gap junction coupling conductance when the mutant was coexpressed with wild‐type (wt) Cx40 or Cx43. Interestingly, another lone AF‐linked Cx40 mutant p.L229M did not show any apparent coupling defect when expressed alone or together with wt Cx40 but specifically reduced the gap junction coupling when coexpressed with wt Cx43. This study is the first to demonstrate that the germline familial mutations in Cx40 impair the gap junctions through different mechanisms, which may predispose the mutant carriers to AF.

Novel GATA4 mutations in lone atrial fibrillation

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia and is a major cause of morbidity and mortality. Previous studies have established genetic defects as a risk factor for AF in a minority of patients. However, AF is of substantial genetic heterogeneity and the molecular determinants for AF in a majority of cases remain unclear. In this study, the entire coding sequence and splice junctions of GATA4, which encodes a zinc-finger transcription factor essential for cardiogenesis, were sequenced in 160 unrelated patients with lone AF. A total of 200 unrelated ethnically matched healthy individuals were used as controls. The available relatives of the patient carrying an identified mutation were genotyped. The functional characteristics of the mutant GATA4 were analyzed using a luciferase reporter assay system. As a result, two novel heterozygous GATA4 mutations of p.G16C and p.H28D, were identified in 2 unrelated families with AF, respectively, which co-segregated with AF in each family with complete penetrance. Functional analysis demonstrated that the mutations of GATA4 were associated with a significantly decreased transcriptional activity. The findings expand the mutation spectrum of GATA4 linked to AF and provide novel insight into the molecular mechanism involved in the pathogenesis of AF.

A Novel NKX2.5 Loss-of-Function Mutation Associated With Congenital Bicuspid Aortic Valve

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.

GATA6 loss-of-function mutation in atrial fibrillation

Atrial fibrillation (AF) is the most common type of sustained cardiac arrhythmia and is associated with substantial morbidity and mortality. Increasing evidence demonstrates that hereditary defects are involved in the pathogenesis of AF. However, AF is of remarkable genetic heterogeneity, and the heritable components responsible for AF in the majority of patients remain unclear. In this study, the entire coding region of the GATA6 gene, which encodes a zinc-finger transcription factor crucial for cardiogenesis, was sequenced in 138 unrelated patients with lone AF, and a novel heterozygous GATA6 mutation, c.704A > C equivalent to p.Y235S, was identified in a patient. The detected substitution, which altered the amino acid highly conserved evolutionarily across species, was absent in 200 unrelated ethnically matched healthy individuals, and was predicted to be disease-causing by MutationTaster. Genetic analysis of the available relatives of the mutation carrier showed that in the family the variation co-segregated with the disease transmitted as an autosomal dominant trait, with complete penetrance. The functional analysis performed using a luciferase reporter assay system revealed that the mutant GATA6 protein resulted in significantly decreased transcriptional activity compared with its wild-type counterpart. These findings provide novel insight into the molecular pathophysiology implicated in AF, suggesting the potential implications in the prophylactic strategy and effective therapy for this common arrhythmia.