GATA6 loss-of-function mutation in atrial fibrillation

European Journal of Medical Genetics 55 (2012) 520e526

Contents lists available at SciVerse ScienceDirect

European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg

Original article

GATA6 loss-of-function mutation in atrial fibrillation Yi-Qing Yang a, *,1, Li Li b,1, Juan Wang c,1, Xian-Ling Zhang d, Ruo-Gu Li d, Ying-Jia Xu d, Hong-Wei Tan d, Xin-Hua Wang d, Jin-Qi Jiang e, Wei-Yi Fang d, Xu Liu d a

Department of Cardiovascular Research, Shanghai Chest Hospital, Medical College of Shanghai Jiaotong University, 241 West Huaihai Road, Shanghai 200030, China Key Laboratory of Arrhythmias, Ministry of Education, Tongji University School of Medicine, Shanghai 200092, China c Department of Cardiology, East Hospital, Tongji University School of Medicine, Shanghai 200120, China d Department of Cardiology, Shanghai Chest Hospital, Medical College of Shanghai Jiaotong University, 241 West Huaihai Road, Shanghai 200030, China e Department of Emergency, Shanghai Chest Hospital, Medical College of Shanghai Jiaotong University, 241 West Huaihai Road, Shanghai 200030, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 December 2011 Accepted 9 June 2012 Available online 26 June 2012

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. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Atrial fibrillation Genetics Transcription factor

1. Introduction Atrial fibrillation (AF) is the most common cardiac arrhythmia seen in clinical practice, affecting 1%e2% of the general population. The prevalence of AF increases progressively with advancing age, ranging from less than 1% in individuals under 60 years of age to nearly 10% in octogenarians [1]. According to the Framingham Heart Study, the lifetime risk for development of AF is approximately 25% in those who have reached the age of 40 [2]. The chaotic electrical activity of the atria, often resulting in an irregular ventricular rhythm, is associated with substantial morbidity and mortality independent of other known risk factors. AF accounts for a 2-fold increased risk of death, and confers a 5-fold increase in the risk of thromboembolic stroke, and one in five of all strokes is attributed to this tachycardia [3,4]. AF is also responsible for

* Corresponding author. Tel.: þ86 21 62821990; fax: þ86 21 62821105. E-mail address: [email protected] (Y.-Q. Yang). 1 These authors contributed equally to the work. 1769-7212/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmg.2012.06.007

tachycardia-induced cardiomyopathy, left ventricular dysfunction or even congestive heart failure, degraded quality of life, and reduced exercise capacity [1]. AF has traditionally been regarded as a complication arising from various cardiac and systemic conditions, including hypertensive heart disease, valvular heart disease, coronary artery disease, cardiomyopathy, hyperthyroidism, and even electrolyte imbalance [1]. However, in 30%e45% of the cases, especially in the young, AF occurs in the absence of overt underlying cardiovascular pathologies and systemic disorders and is hence called lone AF [1], of which at least 15% of patients present with a positive family history, so diagnosed as familial AF [5]. Rapidly aggregating genetic epidemiological studies from around the world have consistently substantiated the familial aggregation of AF and a significantly increased risk of developing AF in the close relatives of patients with AF, suggesting a very strong genetic basis for AF [6e12]. Genome-wide genetic linkage analysis with polymorphic markers mapped susceptibility loci for AF on human chromosomes 10q22, 6q14e16, 11p15.5, 5p15, 10p11eq21, and 5p13, of which AF-causing mutations in two genes, including KCNQ1 on chromosome 11p15.5 and NUP155 on chromosome 5p13,

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

521

were identified and characterized [13]. Analyses of candidate genes and genome-wide association studies revealed a growing number of AF-related genes, including KCNE2, KCNE3, KCNE5, KCNA5, KCNJ2, KCNH2, KCNN3, GJA1, GJA5, NPPA, PITX2, ZFHX3, SCN5A, SCN1B, SCN2B, and SCN3B [13,14]. However, these established AFassociated genes seem to be relatively rare causes of AF, and in the vast majority of AF patients the genetic defects predisposing to AF remain to be identified. It is well known that abnormal embryological development of the atrial myocardium, in particular the myocardial sleeves clothing the systemic venous tributaries and the pulmonary veins at their junctions with the atrial chambers, is a major anatomic substrates for AF [15]. Recent studies highlight the essential role for several transcription factors, including NKX2-5 and GATA4, in the cardiogenesis [15,16], and mutations in NKX2-5 and GATA4 have been causally implicated in congenital heart diseases and AF [17e21]. GATA6 is another member of the GATA family, and its expression and functions overlap with those of GATA4 during cardiovascular development, especially in regulation of target gene expression synergistically with NKX2-5, which suggests the potential association of mutated GATA6 with AF [22].

family members and 200 unrelated healthy control individuals were also screened for the presence of mutation. The PCR was carried out using HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany) on a PE 9700 Thermal Cycler (Applied Biosystems, Foster, CA, USA) with standard conditions and concentrations of reagents. Amplified products were purified with the QIAquick Gel Extraction Kit (Qiagen). Both strands of each PCR product were sequenced with a BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) under an ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems). The sequencing primers were those designed previously for specific region amplifications. DNA sequences were viewed and analyzed with the DNA Sequencing Analysis Software v5.1 (Applied Biosystems). The variant was validated by resequencing of an independent PCR-generated amplicon from the subject and met our quality control threshold with a call rate exceeding 99%. Additionally, an identified variant was searched in NCBI’s single nucleotide polymorphism (SNP) database (http:// www.ncbi.nlm.nih.gov/SNP/) and Exome Variant Server (http:// evs.gs.washington.edu/EVS/) to confirm its novelty.

2. Materials and methods

Multiple GATA6 protein sequences across various species were aligned using the online program ClustalW2 (http://www.ebi.ac. uk/Tools/msa/clustalw2/).

2.1. Study participants A cohort of 138 unrelated patients with lone AF was identified among the Chinese Han population and included in current study. In addition, if a GATA6 mutation was identified in a patient, the mutation carrier’s available relatives were also included. The control population comprised a total of 200 ethnically matched unrelated healthy individuals. Peripheral venous blood samples were prepared and clinical data including medical records, electrocardiogram and echocardiography reports were collected and reviewed. The study subjects were clinically classified using a consistently applied set of definitions [5,20]. The study protocol was approved by the local institutional ethics committee and written informed consent was obtained from all participants before any research procedures are performed. 2.2. Mutation screening Genomic DNA from all participants was extracted from blood lymphocytes with Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The referential genomic DNA sequence of GATA6 derived from GenBank: Genetic sequence database at the National Center for Biotechnical Information (NCBI) (GenBank ID: EF444980). With the help of on-line Primer 3 software (http:// frodo.wi.mit.edu/), the primer pairs used to amplify the coding exons (exons 2e7) and introneexon junctions of GATA6 by polymerase chain reaction (PCR) were designed as shown in Table 1. The GATA6 gene was screened for mutation by PCR-sequencing in 138 unrelated patients with lone AF. Samples from a carrier’s available Table 1 The intronic primers to amplify the coding exons and exoneintron boundaries of GATA6. Exon

Forward primer (50 e30 )

Reverse primer (50 e30 )

Size (bp)

2-a 2-b 2-c 3 4 5, 6 7

ttg,tta,acc,cgt,cga,tct,cc tgc,tgt,tca,ctg,acc,tcg,ac ccg,aca,gcc,ctc,cat,acg ggc,caa,gga,gaa,aag,ctc,ag tct,tgg,ccc,aga,aaa,gtc,ag ctg,gga,tta,gag,gcg,tga,gc att,tct,cct,gcc,ctg,ggt,ct

gcg,agg,gtc,tgg,tac,atc,tc ctg,gga,gag,tag,ggg,aag,c gaa,aac,agg,gcc,cga,gtg gtt,gga,aca,gcc,ggg,aca,g tca,ttt,gct,gat,tct,ttg,taa,ctg ttt,act,aga,gag,cag,ccc,agt ctg,cac,aaa,agc,aga,cac,ga

543 466 539 485 387 473 382

2.3. Alignment of multiple GATA6 protein sequences

2.4. Prediction of the disease-causing potential of a GATA6 sequence variation The disease-causing potential of a GATA6 sequence variation was predicted by MutationTaster (an online program at http://www. mutationtaster.org), automatically giving a probability for the alteration to be either a pathogenic mutation or a benign polymorphism. Notably, the P value used here is the probability of the prediction rather than the probability of error as used in t-test statistics, i.e. a value close to 1 indicates a high ’security’ of the prediction. 2.5. Plasmids and site-directed mutagenesis The recombinant expression plasmid pcDNA3-hGATA6 was kindly provided by Dr. Angela Edwards-Ghatnekar, from the Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA. The atrial natriuretic factor (ANF)-luciferase reporter plasmid, which contains the 2600-bp 50 -flanking region of the ANF gene, namely ANF(-2600)Luc, was kindly provided by Dr. Ichiro Shiojima, from the Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan. The human connexin40 (CX40)-luciferase reporter vector, which contains the proximal promoter region of the CX40 gene from position 177 to þ98 relative to the transcription start site, namely CX40(-177/ þ98)-Luc, was constructed as described previously [23]. Briefly, genomic DNA from an individual with the genotype 44GG/þ71AA was amplified by PCR for the reporter plasmid. After digestion with restriction endonucleases of BamH I and Hind III, the PCR product was inserted into the region between BamH I and Hind III restriction sites of the pGL3 plasmid (Promega), which contained the gene of firefly luciferase. The inserted region of the construct was sequenced to confirm no PCR errors. The identified mutation was introduced into the wild-type GATA6 using a QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with a complementary pair of primers. The mutant was sequenced to confirm the desired mutation and to exclude any other sequence variations.

522

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

2.6. Luciferase assays HEK-293 cells were cultured in DMEM supplemented with 10% fetal calf serum. The ANF(2600)-Luc and CX40(177/þ98)-Luc reporter constructs and an internal control reporter plasmid pGL4.75 (hRluc/CMV, Promega) were used in transient transfection assays to examine the transcriptional activation function of the GATA6 mutant. HEK-293 cells were transfected with 0.4 mg of wildtype or mutant pcDNA3-hGATA6 expression vector, 0.4 mg of ANF(2600)-Luc or CX40(177/þ98)-Luc reporter construct, and 0.04 mg of pGL4.75 control reporter vector using PolyFect Transfection Reagent (Qiagen). For co-transfection experiments, 0.2 mg of wild-type pcDNA3-hGATA6, 0.2 mg of mutant pcDNA3-hGATA6 or empty vector pcDNA3, 0.4 mg of ANF(2600)-Luc or CX40(177/ þ98)-Luc, and 0.04 mg of pGL4.75 were used. Firefly luciferase and Renilla luciferase activities were measured with the Dual-Glo luciferase assay system (Promega) 48 h after transfection. The activity of the ANP or CX40 promoter was presented as fold activation of Firefly luciferase relative to Renilla luciferase. Three independent experiments were performed at minimum for wildtype and mutant GATA6. 2.7. Statistics Data are given as means  SD. Continuous variables were tested for normality of distribution and student’s unpaired t test was used for comparison of numeric variables between two groups. Comparison of the categorical variables between two groups was performed using Pearson’s c2 test or Fisher’s exact test when appropriate. A 2tailed P value < 0.05 was defined as being statistically significant. 3. Results 3.1. Characteristics of the study subjects A cohort of 138 unrelated patients with lone AF was recruited and clinically evaluated in contrast to a total of 200 ethnically matched unrelated healthy individuals. None of them had apparent traditional risk factors for AF. There were no significant differences between patient and control groups in baseline characteristics including age, gender, body mass index, blood pressure, fasting blood glucose, serum lipid, left atrial dimension, left ventricular ejection fraction, heart rate at rest, as well as life style (data not shown). At the present study visit, fourteen patients were also diagnosed with hypertension in accordance to the criterion that the average systolic or diastolic blood pressure (2 readings made after 5 min of rest in the sitting position) was 140 mm Hg or 90 mm Hg, respectively, but at the time of initial diagnosis of AF, their blood pressures were normal. The baseline clinical characteristics of the 138 patients with lone AF are summarized in Table 2. 3.2. GATA6 mutation A heterozygous mutation in GATA6 was identified in 1 of 138 patients. The total population prevalence of GATA6 mutation based on AF cohort was roughly 0.72%. Specifically, A substitution of cytosine (C) for adenine (A) in the second nucleotide of codon 235 (c.704A > C), predicting the transition of tyrosine (Y) into serine (S) at amino acid 265 (p.Y235S), was detected in a patient with lone AF. The sequence chromatograms showing the observed heterozygous GATA6 mutation of c.704A > C compared with control sequence are shown in Fig. 1A. A schematic diagram of GATA6 depicting the structural domains and the location of the identified mutation is presented in Fig. 1B. The mutation that was absent in the control population was neither reported in the NCBI’s SNP database nor

Table 2 Baseline demographics and clinical characteristics of study population. Parameters

Statistics

Baseline demographics Age at first diagnosis of atrial fibrillation (years) Male (n, %) Body mass index (kg/m2) Left ventricular ejection fraction (%) Left atrial diameter (mm)

48 84 23 63 38

Personal history of atrial fibrillation (n, %) Type of atrial fibrillation at presentation Paroxysmal Persistent Permanent Positive family history of atrial fibrillation History of cardioversion

109 (79) 21 (15) 8 (6) 34 (25) 76 (55)

Medical history (n, %) History of syncope History of pacemaker Arterial hypertension Diabetes Hyperlipidemia Thromboembolic complication

12 (9) 5 (4) 14 (10) 6 (4) 13 (9) 9 (7)

Medications (n, %) Aspirin Warfarin Amiodarone Beta-blocker Calcium channel blocker Digitalis

30 75 66 32 17 36

 14 (61) 3 5 4

(22) (54) (48) (23) (12) (26)

found in the Exome Variant Server. Genetic scan of the mutation carrier’s family showed that the mutation was present in all affected living family members, but absent in unaffected family members examined. Analysis of the pedigree demonstrated that the mutation co-segregated with AF transmitted as an autosomal dominant trait in the family with a complete penetrance. The pedigree structure of the family is illustrated in Fig. 1C. The phenotypic characteristics and results of genetic screening of the affected family members are listed in Table 3. Importantly, the identified sequence variation altered the amino acid conserved to some extent evolutionarily across species (Fig. 2) and was automatically predicted to be a disease-causing mutation by MutationTaster, with a p value of 0.91, implying that mutated GATA6 is potentially the cause of AF in this family. No other SNPs in the altered region were found in the MutationTaster database. Interestingly, congenital cardiac defects were confirmed by medical records of previous catheter-based closure and surgical repair in 2 AF patients, including an atrial septal defect in patient III-3 and a ventricular septal defect in patient IV-2. Additionally, one previously reported sequence variation (c.43G > C) predicting a transversion of glycine into arginine at amino acid 15 (p.G15R) was identified [24]. This nucleotide change occurred in 3 of 138 AF patients (2.17%) and in 5 of 200 control individuals (2.50%). Four other sequence polymorphisms in the introns and the 30 UTR of GATA6 were found in both AF patients and control subjects, of which three sequence variants have been previously reported in the NCBI’s SNP database. No significant differences were observed between the AF patients and control subjects in any of their allele frequencies. All the sequence variants and their allele frequencies are listed in Table 4. 3.3. Transcriptional activity of the GATA6 mutant The transcriptional activation characterization of the mutated GATA6 in HEK-293 cells was explored using one of its direct cardiac downstream target genes, ANF, as a luciferase reporter, and the

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

523

Fig. 1. GATA6 Y235S mutation associated with atrial fibrillation. A, Sequence electropherogram showing the GATA6 variation in comparison with its control. The arrow indicates the heterozygous nucleotides of C/A in the proband (mutant) or the homozygous nucleotides of A/A in the corresponding control individual (wild-type). The square denotes the nucleotides comprising a codon of GATA6. B, schematic representation of GATA6 protein structure with the atrial fibrillation related mutation indicated. The mutation found in patients with atrial fibrillation is shown above the structural domains. NH2 means amino-terminus; TAD, transcriptional activation domain; ZF, zinc finger; NLS, nuclear localization signal; COOH, carboxyl-terminus. C, Pedigree structure of the family with atrial fibrillation. Family members are identified by generations and numbers. Squares indicate male family members; circles, female members; symbols with a slash, the deceased members; closed symbols, affected members; open symbols, unaffected members; stippled symbols, members with phenotype undetermined; arrow, proband; “þ”, carrier of the heterozygous missense mutation; and “”, non-carrier.

Table 3 Phenotypic characteristics and status of GATA6 mutation of the affected pedigree members. Subject information

Phenotype

Electrocardiogram

Echocardiogram

Genotype GATA6 mutation

Identity

Gender

Age at time of study (years)

Age at diagnosis of AF (years)

AF (Classification)

Heart rate (beats/min)

QRS interval (ms)

QTc

LAD (mm)

LVEF (%)

Family 1 I-2 II-4 II-5 III-3 III-5 IV-2

F F M M M F

62a 68 63 46 40 20

35 42 48 40 32 20

Permanent Persistent Paroxysmal Paroxysmal Paroxysmal Paroxysmal

74 82 105 90 72 78

108 100 96 122 94 96

460 450 447 458 424 437

40 38 36 37 35 37

58 65 67 60 64 65

Y235S NA þ/ þ/ þ/ þ/ þ/

AF ¼ atrial fibrillation; F ¼ female; M ¼ male; QTc ¼ corrected QT interval; N/A ¼ not available or not applicable; LAD ¼ left atrial dimension; LVEF ¼ left ventricular ejection fraction; a ¼ Age at death; þ ¼ presence of mutation; e ¼ absence of mutation.

524

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

Fig. 2. Alignment of multiple GATA6 protein sequences across species. The altered amino acid of p.Y235 is conserved to some extent evolutionarily across species.

activity of the ANF promoter was presented as fold activation of Firefly luciferase relative to Renilla luciferase. The same amounts of wild-type (0.4 mg) and Y235S-mutant GATA6 (0.4 mg) activated the ANF promoter by w10-fold and w5-fold, respectively. When the same amount of wild-type GATA6 (0.2 mg) was cotransfected with Y235S-mutant GATA6 (0.2 mg), the induced activation of the ANF promoter was w7-fold, in contrast to w8-fold activation of the ANF promoter by wild-type GATA6 (0.2 mg) alone. When the CX40(177/ þ98)-Luc reporter was used instead of the ANP(2600)-Luc reporter, a similar result was obtained. These results suggest that the GATA6 mutation has a significantly reduced activation activity compared with wild-type counterpart (as shown in Fig. 3). 4. Discussion In the present study, a novel heterozygous GATA6 mutation, p.Y235S, was identified in a family with lone AF. The mutation cosegregated with AF in the family and was absent in the 400 normal chromosomes. A cross-species alignment of multiple GATA6 protein sequences displayed that the altered amino acid was relatively conserved evolutionarily. Functional analysis revealed that the p.Y235S mutation of GATA6 was associated with a significantly decreased transcriptional activation. Therefore, it is very likely that compromised GATA6 is responsible for AF in this family. To our knowledge, this is the first description of the relationship between a loss-of-function mutation in GATA6 and susceptibility to AF. GATA factors constitute a family of transcriptional regulatory factors characteristic of their ability to bind to the consensus DNA sequence “GATA”. To date six GATA family members have been discovered in vertebrates, of which GATA4, GATA5 and GATA6 are expressed in a similar pattern in the developing heart [25]. In humans the GATA6 gene maps to chromosome 18q11.1eq11.2, encoding a protein composed of 595 amino acids [26]. Northern analysis and in situ hybridization show that in human GATA6 is expressed at exceptionally high level in the embryonic heart and continues to express at high levels in the fetal and adult hearts [27,28]. Topologically GATA6 contains transcriptional activation domain, zinc finger domain, and nuclear localization signal [25].

Table 4 GATA6 sequence variations identified in this study. Location

Nucleotide

Amino acid

Allele frequencies Patients

Controls

Exon 2 Exon 2 Intron 2 Intron 2 30 UTR 30 UTR

c.43G > C c.704A > C c.133685C > T c. 133660C > T c.þ72G > A c.þ77G > A

p.G15R p.Y235S

(0.022) (0.007) (0.022) (0.007) (0.333) (0.087)

(0.025) (0.000) (0.015) (0.010) (0.250) (0.130)

3/138 1/138 3/138 1/138 46/138 12/138

5/200 0/200 3/200 2/200 50/200 26/200

The GATA6 mutation of p.Y235S identified in this study is located in transcriptional activation domain, which is essential for the normal function of GATA6, thus may be expected to exert direct influence on transcriptional activation by GATA6. Previous studies have demonstrated that heterozygous missense mutants maybe act in a dominant inhibitory fashion on wild-type homeoproteins, such as MIX1, XVENT2, and NKX2-5 [29e31]. The dominant inhibitory effect of mutant proteins is partially ascribed to their ability to homo- or heterodimerize with other homeoproteins, and these mutant-wild-type homeoprotein complexes may change the transcriptional activity [29e31]. Therefore, to elucidate whether the induction of AF by the GATA6 mutation is mediated by haploinsufficiency or by dominant negative effect, we co-transfected the same amounts of wild-type GATA6, alone or together with the mutant, in the presence of ANF or CX40 promoter. The expression of the mutant inhibited wildtype-induced activation of the ANF or CX40 promoter, suggesting that the mutant functions in a dominant-negative manner. However, it remains to be determined how the mutant suppresses the transcriptional activity of wild-type GATA6. It has been validated that GATA6 is an upstream transcriptional regulator of several genes expressed during cardiac development, including ANF and CX40 [28], and mutations in these downstream

Fig. 3. Transcriptional activity of the mutant GATA6 on ANF or Cx40 promoter. HEK-293 cells were transfected with 0.4 mg of wild-type or mutant pcDNA3-hGATA6 expression vector, 0.4 mg of ANF(2600)-Luc or CX40(177/þ98)-Luc reporter construct, and 0.04 mg of pGL4.75 control reporter vector. For co-transfection experiments, 0.2 mg of wild-type pcDNA3-hGATA6, 0.2 mg of mutant pcDNA3hGATA6 or 0.2 mg of empty vector pcDNA3, 0.4 mg of ANF(2600)-Luc or CX40(177/þ98)-Luc, and 0.04 mg of pGL4.75 were used. The activity of the ANF or CX40 promoter was presented as fold activation of firefly luciferase relative to Renilla luciferase. Values are the mean  SD of data from three independent experiments performed in triplicate. ** and * represent P < 0.005 and P < 0.01, respectively, when compared with the wild-type GATA6 (0.4 mg).

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

genes have been implicated with AF [32e35]. In this study, the functional effect of the AF-associated GATA6 mutation of p.Y235S was investigated by transcriptional activity assays and the results showed a significantly decreased transcriptional activity on both ANF and CX40, consistent with the loss-of function effect of AFassociated mutations in ANF and CX40 as well as another GATA6 mutation underlying congenital cardiovascular anomalies [32e36]. These findings indicate that loss-of-function effect resulting from GATA6 mutation is potentially an alternative pathophysiological mechanism involved in AF. The discovery that dysfunctional GATA6 predisposes to AF may be likely due to the abnormal development of the pulmonary vein myocardium [37]. The pulmonary venous vessels are ensheathed by a myocardial layer known as the pulmonary myocardial sleeve, which has been shown to be involved in the initiation and perpetuation of AF by several possible pathological mechanisms including intrinsic pacemaker activity and properties that facilitate reentrance [38,39]. Genetic-labeling lineage tracing studies have shown that NKX2-5 is expressed in the atria and pulmonary myocardium and is essential for embryonic development of the localized formation of the sinoatrial node. NKX2-5 functions as a repressor of the sinoatrial node lineage gene program, thus limiting pacemaker activity to the sinus node and the atrioventricular node [40]. Therefore, as a transcriptionally cooperative partner of NKX2-5 [22], GATA6, when mutated, may contribute to formation of the atrial electrophysiological substrate prone to AF. Interestingly, congenital cardiac septal defects were observed in two AF patients carrying the p.Y235S mutation of GATA6. Since some congenital cardiac structural defects may close spontaneously, we cannot rule out the possibility that some of the mutation carriers had smaller cardiac septal defects that closed shortly after birth on their own. Additionally, some AF associated mutations in NKX2-5 and GATA4 were also implicated in congenital cardiovascular anomalies [17e21]. These results imply that AF may share, at least partially, a common molecular pathway with congenital heart disease. In conclusion, the current study links the cardiac transcription factor GATA6 to AF, which provides novel insight into the molecular mechanisms involved in the pathogenesis of AF. Acknowledgements We are greatly indebted to the participants for their participation in the study. This work was supported in part by grants from the National Natural Science Fund of China (81070153, 81000082 and 30570768), the National Basic Research Program of China (2012CB966803 and 2010CB912604), the Personnel Development Foundation of Shanghai, China (2010019), the Fundamental Research Funds for the Central Universities for Lili, and the Key Program of Basic Research of Shanghai, China (10JC1414000, 10JC1414001 and 10JC1414002). References [1] V. Fuster, L.E. Rydén, D.S. Cannom, H.J. Crijns, A.B. Curtis, K.A. Ellenbogen, J.L. Halperin, G.N. Kay, J.Y. Le Huezey, J.E. Lowe, S.B. Olsson, E.N. Prystowsky, J.L. Tamargo, L.S. Wann, S.C. Smith Jr., S.G. Priori, N.A. Estes 3rd, M.D. Ezekowitz, W.M. Jackman, C.T. January, J.E. Lowe, R.L. Page, D.J. Slotwiner, W.G. Stevenson, C.M. Tracy, A.K. Jacobs, J.L. Anderson, N. Albert, C.E. Buller, M.A. Creager, S.M. Ettinger, R.A. Guyton, J.L. Halperin, J.S. Hochman, F.G. Kushner, E.M. Ohman, W.G. Stevenson, L.G. Tarkington, C.W. Yancy, American College of Cardiology Foundation/American Heart Association Task Force, 2011 ACCF/ AHA/HRS focused updates incorporated into the ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, Circulation 123 (2011) e269ee367. [2] D.M. Lloyd-Jones, T.J. Wang, E.P. Leip, M.G. Larson, D. Levy, R.S. Vasan, R.B. D’Agostino, J.M. Massaro, A. Beiser, P.A. Wolf, E.J. Benjamin, Lifetime risk

[3]

[4] [5]

[6] [7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15] [16]

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25] [26]

[27]

[28]

525

for development of atrial fibrillation: the Framingham Heart Study, Circulation 110 (2004) 1042e1046. E.J. Benjamin, P.A. Wolf, R.B. D’Agostino, H. Silbershatz, W.B. Kannel, D. Levy, Impact of atrial fibrillation on the risk of death: the Framingham Heart Study, Circulation 98 (1998) 946e952. P.A. Wolf, R.D. Abbott, W.B. Kannel, Atrial fibrillation as an independent risk factor for stroke: the Framingham Study, Stroke 22 (1991) 983e988. D. Darbar, K.J. Herron, J.D. Ballew, A. Jahangir, B.J. Gersh, W.K. Shen, S.C. Hammill, D.L. Packer, T.M. Olson, Familial atrial fibrillation is a genetically heterogeneous disorder, J. Am. Coll. Cardiol. 41 (2003) 2185e2192. P.T. Ellinor, D.M. Yoerger, J.N. Ruskin, C.A. MacRae, Familial aggregation in lone atrial fibrillation, Hum. Genet. 118 (2005) 179e184. D.O. Arnar, S. Thorvaldsson, T.A. Manolio, G. Thorgeirsson, K. Kristjansson, H. Hakonarson, K. Stefansson, Familial aggregation of atrial fibrillation in Iceland, Eur. Heart J. 27 (2006) 708e712. M.J. Junttila, M.J. Raatikainen, J.S. Perkiömäki, K. Hong, R. Brugada, H.V. Huikuri, Familial clustering of lone atrial fibrillation in patients with saddleback-type ST-segment elevation in right precordial leads, Eur. Heart J. 28 (2007) 463e468. I.E. Christophersen, L.S. Ravn, E. Budtz-Joergensen, A. Skytthe, S. Haunsoe, J.H. Svendsen, K. Christensen, Familial aggregation of atrial fibrillation: a study in Danish twins, Circ. Arrhythm. Electrophysiol. 2 (2009) 378e383. Y.Q. Yang, X.L. Zhang, X.H. Wang, H.W. Tan, H.F. Shi, W.Y. Fang, X. Liu, Familial aggregation of lone atrial fibrillation in the Chinese population, Intern. Med. 49 (2010) 2385e2391. C.S. Fox, H. Parise, R.B. D’Agostino Sr, D.M. Lloyd-Jones, R.S. Vasan, T.J. Wang, D. Levy, P.A. Wolf, E.J. Benjamin, Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring, JAMA 291 (2004) 2851e2855. S.A. Lubitz, X. Yin, J.D. Fontes, J.W. Magnani, M. Rienstra, M. Pai, M.L. Villalon, R.S. Vasan, M.J. Pencina, D. Levy, M.G. Larson, P.T. Ellinor, E.J. Benjamin, Association between familial atrial fibrillation and risk of new-onset atrial fibrillation, JAMA 304 (2010) 2263e2269. S. Mahida, S.A. Lubitz, M. Rienstra, D.J. Milan, P.T. Ellinor, Monogenic atrial fibrillation as pathophysiological paradigms, Cardiovasc. Res. 89 (2011) 692e700. M.F. Sinner, P.T. Ellinor, T. Meitinger, E.J. Benjamin, S. Kääb, Genome-wide association studies of atrial fibrillation: past, present, and future, Cardiovasc. Res. 89 (2011) 701e709. M.T. Mommersteeg, V.M. Christoffels, R.H. Anderson, A.F. Moorman, Atrial fibrillation: a developmental point of view, Heart Rhythm. 6 (2009) 1818e1824. M. Salazar, F. Consoli, V. Villegas, V. Caicedo, V. Maddaloni, P. Daniele, G. Caianiello, S. Pachón, F. Nuñez, G. Limongelli, G. Pacileo, B. Marino, J.E. Bernal, A. De Luca, B. Dallapiccola, Search of somatic GATA4 and NKX2.5 gene mutations in sporadic septal heart defects, Eur. J. Med. Genet. 54 (2011) 306e309. I. Gutierrez-Roelens, L. De Roy, C. Ovaert, T. Sluysmans, K. Devriendt, H.G. Brunner, M. Vikkula, A novel CSX/NKX2-5 mutation causes autosomal-dominant AV block: are atrial fibrillation and syncopes part of the phenotype? Eur. J. Hum. Genet. 14 (2006) 1313e1316. L.H. Boldt, M.G. Posch, A. Perrot, M. Polotzki, S. Rolf, A.S. Parwani, M. Huemer, A. Wutzler, C. Ozcelik, W. Haverkamp, Mutational analysis of the PITX2 and NKX2-5 genes in patients with idiopathic atrial fibrillation, Int. J. Cardiol. 145 (2010) 316e317. M.G. Posch, L.H. Boldt, M. Polotzki, S. Richter, S. Rolf, A. Perrot, R. Dietz, C. Ozcelik, W. Haverkamp, Mutations in the cardiac transcription factor GATA4 in patients with lone atrial fibrillation, Eur. J. Med. Genet. 53 (2010) 201e203. Y.Q. Yang, M.Y. Wang, X.L. Zhang, H.W. Tan, H.F. Shi, W.F. Jiang, X.H. Wang, W.Y. Fang, X. Liu, GATA4 loss-of-function mutations in familial atrial fibrillation, Clin. Chim. Acta 412 (2011) 1825e1830. J.Q. Jiang, F.F. Shen, W.Y. Fang, X. Liu, Y.Q. Yang, Novel GATA4 mutations in lone atrial fibrillation, Int. J. Mol. Med. 28 (2011) 1025e1032. Y. Zhang, N. Rath, S. Hannenhalli, Z. Wang, T. Cappola, S. Kimura, E. Atochina-Vasserman, M.M. Lu, M.F. Beers, E.E. Morrisey, GATA and Nkx factors synergistically regulate tissue-specific gene expression and development in vivo, Development 134 (2007) 189e198. J.M. Juang, Y.R. Chern, C.T. Tsai, F.T. Chiang, J.L. Lin, J.J. Hwang, K.L. Hsu, C.D. Tseng, Y.Z. Tseng, L.P. Lai, The association of human connexin 40 genetic polymorphisms with atrial fibrillation, Int. J. Cardiol. 116 (2007) 107e112. K. Kodo, T. Nishizawa, M. Furutani, S. Arai, E. Yamamura, K. Joo, T. Takahashi, R. Matsuoka, H. Yamagishi, GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 13933e13938. A. Brewer, J. Pizzey, GATA factors in vertebrate heart development and disease, Expert Rev. Mol. Med. 8 (2006) 1e20. E. Suzuki, T. Evans, J. Lowry, L. Truong, D.W. Bell, J.R. Testa, K. Walsh, The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals, Genomics 38 (1996) 283e290. I.C. Huggon, A. Davies, C. Gove, G. Moscoso, C. Moniz, Y. Foss, F. Farzaneh, P. Towner, Molecular cloning of human GATA-6 DNA binding protein: high levels of expression in heart and gut, Biochim. Biophys. Acta 1353 (1997) 98e102. M.C. Rémond, G. Iaffaldano, M.P. O’Quinn, N.V. Mezentseva, V. Garcia, B.S. Harris, R.G. Gourdie, C.A. Eisenberg, L.M. Eisenberg, GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling, Am. J. Physiol. Heart Circ. Physiol. 301 (2011) H1952eH1964.

526

Y.-Q. Yang et al. / European Journal of Medical Genetics 55 (2012) 520e526

[29] P.E. Mead, I.H. Brivanlou, C.M. Kelley, L.I. Zon, BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1, Nature 382 (1996) 357e360. [30] D. Onichtchouk, A. Glinka, C. Niehrs, Requirement for Xvent-1 and Xvent-2 gene function in dorsoventral patterning of Xenopus mesoderm, Development 125 (1998) 1447e1456. [31] H. Kasahara, B. Lee, J.J. Schott, D.W. Benson, J.G. Seidman, C.E. Seidman, S. Izumo, Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease, J. Clin. Invest. 106 (2000) 299e308. [32] D.M. Hodgson-Zingman, M.L. Karst, L.V. Zingman, D.M. Heublein, D. Darbar, K.J. Herron, J.D. Ballew, M. de Andrade, J.C. Burnett Jr., T.M. Olson, Atrial natriuretic peptide frameshift mutation in familial atrial fibrillation, N. Engl. J. Med. 359 (2008) 158e165. [33] M.H. Gollob, D.L. Jones, A.D. Krahn, L. Danis, X.Q. Gong, Q. Shao, X. Liu, J.P. Veinot, A.S. Tang, A.F. Stewart, F. Tesson, G.J. Klein, R. Yee, A.C. Skanes, G.M. Guiraudon, L. Ebihara, D. Bai, Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation, N. Engl. J. Med. 354 (2006) 2677e2688. [34] Y.Q. Yang, X.L. Zhang, X.H. Wang, H.W. Tan, H.F. Shi, W.F. Jiang, W.Y. Fang, X. Liu, Connexin40 nonsense mutation in familial atrial fibrillation, Int. J. Mol. Med. 26 (2010) 605e610.

[35] Y.Q. Yang, X. Liu, X.L. Zhang, X.H. Wang, H.W. Tan, H.F. Shi, W.F. Jiang, W.Y. Fang, Novel connexin40 missense mutations in patients with familial atrial fibrillation, Europace 12 (2010) 1421e1427. [36] X. Lin, Z. Huo, X. Liu, Y. Zhang, L. Li, H. Zhao, B. Yan, Y. Liu, Y. Yang, Y.H. Chen, A novel GATA6 mutation in patients with tetralogy of Fallot or atrial septal defect, J. Hum. Genet. 55 (2010) 662e667. [37] M.T. Mommersteeg, N.A. Brown, O.W. Prall, C. de Gier-de Vries, R.P. Harvey, A.F. Moorman, V.M. Christoffels, Pitx2c and Nkx2-5 are required for the formation and identity of the pulmonary myocardium, Circ. Res. 101 (2007) 902e909. [38] M. Haïssaguerre, P. Jaïs, D.C. Shah, A. Takahashi, M. Hocini, G. Quiniou, S. Garrigue, A. Le Mouroux, P. Le Métayer, J. Clémenty, Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins, N. Engl. J. Med. 339 (1998) 659e666. [39] S. Nattel, Basic electrophysiology of the pulmonary veins and their role in atrial fibrillation: precipitators, perpetuators, and perplexers, J. Cardiovasc. Electrophysiol. 14 (2003) 1372e1375. [40] M.T. Mommersteeg, W.M. Hoogaars, O.W. Prall, C. de Gier-de Vries, C. Wiese, D.E. Clout, V.E. Papaioannou, N.A. Brown, R.P. Harvey, A.F. Moorman, V.M. Christoffels, Molecular pathway for the localized formation of the sinoatrial node, Circ. Res. 100 (2007) 354e362.