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A novel TBX5 mutation predisposes to familial cardiac septal defects and atrial fibrillation as well as bicuspid aortic valve

Abstract

TBX5 has been linked to Holt-Oram syndrome, with congenital heart defect (CHD) and atrial fibrillation (AF) being two major cardiac phenotypes. However, the prevalence of a TBX5 variation in patients with CHD and AF remains obscure. In this research, by sequencing analysis of TBX5 in 178 index patients with both CHD and AF, a novel heterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was identified in one index patient with CHD and AF as well as bicuspid aortic valve (BAV), with an allele frequency of approximately 0.28%. Genetic analysis of the proband’s pedigree showed that the variation co-segregated with the diseases. The pathogenic variation was not detected in 292 unrelated healthy subjects. Functional analysis by using a dual-luciferase reporter assay system showed that the Gly193*-mutant TBX5 protein failed to transcriptionally activate its target genes MYH6 and NPPA. Moreover, the mutation nullified the synergistic transactivation between TBX5 and GATA4 as well as NKX2-5. Additionally, whole-exome sequencing analysis showed no other genes contributing to the diseases. This investigation firstly links a pathogenic variant in the TBX5 gene to familial CHD and AF as well as BAV, suggesting that CHD and AF as well as BAV share a common developmental basis in a subset of patients.

Keywords:
Congenital heart disease; atrial fibrillation; bicuspid aortic valve; molecular genetics; TBX5

Introduction

As the most prevalent type of human birth defect, congenital heart defect (CHD) occurs in about 1% of all live neonates, accounting for nearly a third of all forms of developmental abnormalities (Benjamin et al., 2019Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR et al. (2019) Heart disease and stroke statistics-2019 update: A report from the American Heart Association. Circulation 139:e56-e528.; Oliveira-Brancati et al., 2020Oliveira-Brancati CIF, Ferrarese VCC, Costa AR and Fett-Conte AC (2020) Birth defects in Brazil: Outcomes of a population-based study. Genet Mol Biol 43:e20180186.). Although minor CHD may resolve spontaneously (Benjamin et al., 2019Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR et al. (2019) Heart disease and stroke statistics-2019 update: A report from the American Heart Association. Circulation 139:e56-e528.), serious CHD may lead to poor health-related quality of life (Amedro et al., 2018Amedro P, Bajolle F, Bertet H, Cheurfi R, Lasne D, Nogue E, Auquier P, Picot MC and Bonnet D (2018) Quality of life in children participating in a non-selective INR self-monitoring VKA-education programme. Arch Cardiovasc Dis 111:180-188., 2019Amedro P, Gavotto A, Legendre A, Lavastre K, Bredy C, De La Villeon G, Matecki S, Vandenberghe D, Ladeveze M, Bajolle F et al. (2019) Impact of a centre and home-based cardiac rehabilitation program on the quality of life of teenagers and young adults with congenital heart disease: The QUALI-REHAB study rationale, design and methods. Int J Cardiol 283:112-118.; Boukovala et al., 2019Boukovala M, Müller J, Ewert P and Hager A (2019) Effects of congenital heart disease treatment on quality of life. 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Heart Fail Clin 14:569-577.; Chan et al., 2019Chan J, Collins RT II, Hall M and John A (2019) Resource utilization among adult congenital heart failure admissions in pediatric hospitals. Am J Cardiol 123:839-846.), ventricular or supraventricular dysrhythmia (Labombarda et al., 2017Labombarda F, Hamilton R, Shohoudi A, Aboulhosn J, Broberg CS, Chaix MA, Cohen S, Cook S, Dore A, Fernandes SM et al. (2017) Increasing prevalence of atrial fibrillation and permanent atrial arrhythmias in congenital heart disease. J Am Coll Cardiol 70:857-865.; Barry et al., 2018Barry OM, Gauvreau K, Rhodes J, Reichman JR, Bourette L, Curran T, O'Neill J, Pymm JL and Alexander ME (2018) Incidence and predictors of clinically important and dangerous arrhythmias during exercise tests in pediatric and congenital heart disease patients. JACC Clin Electrophysiol 4:1319-1327.; Hernández-Madrid et al., 2018Hernández-Madrid A, Paul T, Abrams D, Aziz PF, Blom NA, Chen J, Chessa M, Combes N, Dagres N, Diller G et al. (2018) Arrhythmias in congenital heart disease: a position paper of the European Heart Rhythm Association (EHRA), Association for European Paediatric and Congenital Cardiology (AEPC), and the European Society of Cardiology (ESC) Working Group on Grown-up Congenital heart disease, endorsed by HRS, PACES, APHRS, and SOLAECE. Europace 20:1719-1753.; Fuchs et al., 2019Fuchs SR, Smith AH, Van Driest SL, Crum KF, Edwards TL and Kannankeril PJ (2019) Incidence and effect of early postoperative ventricular arrhythmias after congenital heart surgery. Heart Rhythm 16:710-716.), and death (Lynge et al., 2018Lynge TH, Jeppesen AG, Winkel BG, Glinge C, Schmidt MR, Søndergaard L, Risgaard B and Tfelt-Hansen J (2018) Nationwide study of sudden cardiac death in people with congenital heart defects aged 0 to 35 years. Circ Arrhythm Electrophysiol 11:e005757.; Moore et al., 2018Moore B, Yu C, Kotchetkova I, Cordina R and Celermajer DS (2018) Incidence and clinical characteristics of sudden cardiac death in adult congenital heart disease. Int J Cardiol 254:101-106.; Yu C et al., 2018Yu C, Moore BM, Kotchetkova I, Cordina RL and Celermajer DS (2018) Causes of death in a contemporary adult congenital heart disease cohort. Heart 104:1678-1682.). Although vast advance in cardiac surgery allows over 90% of CHD newborns to survive into adulthood, it results in an increasing adult CHD population, and mow CHD adults outnumber CHD children (Bouma and Mulder, 2017Bouma BJ and Mulder BJ (2017) Changing landscape of congenital heart disease. Circ Res 120:908-922.; Benjamin et al., 2019Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR et al. (2019) Heart disease and stroke statistics-2019 update: A report from the American Heart Association. Circulation 139:e56-e528.). Moreover, the late complications and mortality substantially increase in adult CHD patients (Bouma and Mulder, 2017Bouma BJ and Mulder BJ (2017) Changing landscape of congenital heart disease. Circ Res 120:908-922.;Spector et al., 2018Spector LG, Menk JS, Knight JH, McCracken C, Thomas AS, Vinocur JM, Oster ME, St Louis JD, Moller JH and Kochilas L (2018) Trends in long-term mortality after congenital heart surgery. J Am Coll Cardiol 71:2434-2446.; Trusty et al., 2018Trusty PM, Slesnick TC, Wei ZA, Rossignac J, Kanter KR, Fogel MA and Yoganathan AP (2018) Fontan surgical planning: previous accomplishments, current challenges, and future directions. J Cardiovasc Transl Res 11:133-144.). Despite clinical importance, the etiologies of CHD in the majority of cases are still elusive.

Cardiogenesis undergoes a highly complex biological process, and both environmental and genetic pathogenic factors can perturb this finely regulated process, leading to CHD (Patel and Burns, 2013Patel SS and Burns TL (2013) Nongenetic risk factors and congenital heart defects. Pediatr Cardiol 34:1535-1555.; Pierpont et al., 2018Pierpont ME, Brueckner M, Chung WK, Garg V, Lacro RV, McGuire AL, Mital S, Priest JR, Pu WT, Roberts A et al. (2018) Genetic basis for congenital heart disease: revisited: A scientific statement from the American Heart Association. Circulation 138:e653-e711.; Shabana et al., 2020Shabana NA, Shahid SU and Irfan U (2020) Genetic contribution to congenital heart disease (CHD). Pediatr Cardiol 41:12-23.). The well-established environmental factors underlying CHD include maternal conditions (such as innutrition, viral infection and endocrine disorder) and exposures to toxic chemicals, therapeutic drugs, or ionizing radiation during pregnancy (Patel and Burns, 2013Patel SS and Burns TL (2013) Nongenetic risk factors and congenital heart defects. Pediatr Cardiol 34:1535-1555.). However, increasing studies underscore the genetic defects underpinning CHD, and variations in over 70 genes, encompassing those encoding transcription factors, signaling molecules, and sarcomeric proteins, have been involved in CHD (Bashamboo et al., 2018Bashamboo A, Eozenou C, Jorgensen A, Bignon-Topalovic J, Siffroi JP, Hyon C, Tar A, Nagy P, Sólyom J, Halász Z et al. (2018) Loss of function of the nuclear receptor NR2F2, encoding COUP-TF2, causes testis development and cardiac defects in 46,XX children. 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Interestingly, TBX5 variations have recently been involved in atrial fibrillation (AF), the most common sustained cardiac arrhythmia (January et al., 2014January CT, Wann LS, Alpert JS, Calkins H, Cigarroa JE, Cleveland JC Jr, Conti JB, Ellinor PT, Ezekowitz MD, Field ME et al. (2014) 2014 AHA/ACC/HRS guideline for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society. J Am Coll Cardiol 64:e1-e76.). Postma et al., (2008Postma AV, van de Meerakker JB, Mathijssen IB, Barnett P, Christoffels VM, Ilgun A, Lam J, Wilde AA, Lekanne Deprez RH and Moorman AF (2008) A gain-of-function TBX5 mutation is associated with atypical Holt-Oram syndrome and paroxysmal atrial fibrillation. Circ Res 102:1433-1442.) reported that a TBX5 gain-of-function mutation caused an atypical Holt-Oram syndrome (HOS), with AF being the predominant clinical phenotype. Ma et al., (2016Ma JF, Yang F, Mahida SN, Zhao L, Chen X, Zhang ML, Sun Z, Yao Y, Zhang YX, Zheng GY et al. (2016) TBX5 mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians. Cardiovasc Res 109:442-450.) identified multiple loss-of-function mutations in TBX5 in multiple patients affected with AF. Wang et al., (2016Wang ZC, Ji WH, Ruan CW, Liu XY, Qiu XB, Yuan F, Li RG, Xu YJ, Liu X, Huang RT et al. (2016) Prevalence and spectrum of TBX5 mutation in patients with lone atrial fibrillation. Int J Med Sci 13:60-67.) found a novel loss-of-function mutation in TBX5 in a case with AF. Guo et al., (2016Guo DF, Li RG, Yuan F, Shi HY, Hou XM, Qu XK, Xu YJ, Zhang M, Liu X, Jiang JQ et al. (2016) TBX5 loss-of-function mutation contributes to atrial fibrillation and atypical Holt-Oram syndrome. Mol Med Rep 13:4349-4356.) uncovered a new TBX5 loss-of-function mutation in an index patient with idiopathic AF. These observational results highlight the pronounced genetic heterogeneity of CHD and AF, which makes it justifiable to investigate the prevalence of TBX5 variations in patients with both CHD and AF, and unveil the molecular mechanism of CHD and AF resulted from novel TBX5 variations.

Material and Methods

Study participants

This study subjects comprised 178 unrelated adult patients suffering from both CHD and AF, who were consecutively recruited between February 2015 and March 2019Ma L, Wang J, Li L, Qiao Q, Di RM, Li XM, Xu YJ, Zhang M, Li RG, Qiu XB et al. (2019) ISL1 loss-of-function mutation contributes to congenital heart defects. Heart Vessels 34:658-668. from the Chinese Han population. Diagnosis of CHD and various kinds of AF was made as described previously (Wang et al., 2016Wang ZC, Ji WH, Ruan CW, Liu XY, Qiu XB, Yuan F, Li RG, Xu YJ, Liu X, Huang RT et al. (2016) Prevalence and spectrum of TBX5 mutation in patients with lone atrial fibrillation. Int J Med Sci 13:60-67.; Li et al., 2018bLi N, Wang ZS, Wang XH, Xu YJ, Qiao Q, Li XM, Di RM, Guo XJ, Li RG, Zhang M et al. (2018b) A SHOX2 loss-of-function mutation underlying familial atrial fibrillation. Int J Med Sci 15:1564-1572.; Ma et al., 2019Ma L, Wang J, Li L, Qiao Q, Di RM, Li XM, Xu YJ, Zhang M, Li RG, Qiu XB et al. (2019) ISL1 loss-of-function mutation contributes to congenital heart defects. Heart Vessels 34:658-668.). The patients with rheumatic heart disease, ischemic heart disease, essential hypertension, or other recognized risk factors for AF were excluded. The patients with AF occurred after cardiac surgery were also ruled out from the present investigation. If available, the relatives of the probands were also enrolled. The control individuals were 292 unrelated adult healthy persons, who were enlisted from the same geographic area during the same time period. The healthy controls were matched with the affected individuals for ethnicity, sex and age. All study participants were subject to comprehensive medical evaluation, including familial histories, medical histories, physical examination, trans-thoracic echocardiogram, standard 12-lead electrocardiogram, and routine biological tests. This investigation was conducted in accordance with the ethical principles stated in the Declaration of Helsinki. The protocol used in this study was reviewed and approved by the Human Ethics Committee of the Shanghai Chest Hospital, Shanghai, China. Informed consent was obtained from the study participants prior to sample collection.

Genetic analyses

Blood samples were collected from each study subject. Genomic DNA of each test subject was purified from blood cells with the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The coding exons and splicing donors/acceptors of TBX5 were amplified from each study participant’s genomic DNA by polymerase chain reaction (PCR) for a variation scan by PCR-sequencing. The PCR primers were designed as described elsewhere (Zhang et al., 2015Zhang XL, Qiu XB, Yuan F, Wang J, Zhao CM, Li RG, Xu L, Xu YJ, Shi HY, Hou XM et al. (2015) TBX5 loss-of-function mutation contributes to familial dilated cardiomyopathy. Biochem Biophys Res Commun 459:166-171.). Each PCR mixture was prepared in a thin-walled PCR tube with a total volume of 25 μL containing 50 ng of genomic DNA, 0.2 mM dNTPs (Qiagen, Hilden, Germany), 1 × Buffer (Qiagen), 1 × Q solution (Qiagen), 0.5 μM of each primer, and 0.02 U/μL of HotStar Taq DNA Polymerase (Qiagen). PCR was carried out on a Veriti® 96-Well Thermocycler (Applied Biosystems, Foster, CA, USA). The PCR program was set as follows: initial pre-denaturation at 95 °C for 15 min followed by 35 thermal cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s and extension at 72 °C for 1 min, with final extension at 72 °C for 8 min. The amplified products were fractionated by electrophoresis on a 1.2% agarose gel, and isolated utilizing the QIAquick Gel Extraction Kit (Qiagen). The purified amplicons were subjected to PCR-sequencing under an ABI 3730 XL DNA Analyzer (Applied Biosystems), with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s instructions. The detected sequence variant was validated by bi-directional re-sequencing of an independent PCR-generated amplicon from the same subject. For an identified TBX5 variation, the 1000 Genomes Project database The 1000 Genomes Project database, The 1000 Genomes Project database, http://www.1000genomes.org (May 6, 2020).
http://www.1000genomes.org...
(http://www.1000genomes.org), the Genome Aggregation DatabaseThe Genome Aggregation Database,The Genome Aggregation Database, https://gnomad.broadinstitute.org (May 6, 2020).
https://gnomad.broadinstitute.org...
(https://gnomad.broadinstitute.org), and the Single Nucleotide Polymorphism databaseThe Single Nucleotide Polymorphism database, The Single Nucleotide Polymorphism database, http://www.ncbi.nlm.nih.gov/snp (May 6, 2020).
http://www.ncbi.nlm.nih.gov/snp...
(http://www.ncbi.nlm.nih.gov/snp) were queried to check its novelty.

In addition, in order to rule out the potential causative effects of other genes on the diseases, whole-exome sequencing (WES) analysis of the mutation carrier’s family members was performed as described previously (Xu et al., 2019Xu YJ, Wang ZS, Yang CX, Di RM, Qiao Q, Li XM, Gu JN, Guo XJ and Yang YQ (2019) Identification and functional characterization of an ISL1 mutation predisposing to dilated cardiomyopathy. J Cardiovasc Transl Res 12:257-267.). In brief, 2 μg of DNA from each family member was utilized to construct an exome library with the SureSelectXT Human All Exon V6 Kit (Agilent Technologies, Santa Clara, CA, USA), and which sequenced on the Solexa Genome Analyzer (GA) IIx platform (Illumina, San Diego, CA, USA), according to the manufacturer’s protocols. Raw image files were processed by the Illumina pipeline to call bases and generate the reads set. By using SOAPaligner, reads were aligned with the human reference genome. Variations of single nucleotide polymorphisms, insertions and deletions were identified by Genome Analysis Toolkit. The identified variants in known genes were classified according to the recommended guidelines (Xu et al., 2019Xu YJ, Wang ZS, Yang CX, Di RM, Qiao Q, Li XM, Gu JN, Guo XJ and Yang YQ (2019) Identification and functional characterization of an ISL1 mutation predisposing to dilated cardiomyopathy. J Cardiovasc Transl Res 12:257-267.). The candidate disease-causing variations found by WES were checked by Sanger sequencing.

Expression plasmid constructs and site-targeted mutagenesis

The wild-type TBX5 expression plasmid TBX5-pcDNA3.1 was constructed as described elsewhere (Zhang et al., 2015Zhang XL, Qiu XB, Yuan F, Wang J, Zhao CM, Li RG, Xu L, Xu YJ, Shi HY, Hou XM et al. (2015) TBX5 loss-of-function mutation contributes to familial dilated cardiomyopathy. Biochem Biophys Res Commun 459:166-171.). The mutant-type TBX5-pcDNA3.1 was produced via PCR-based site-targeted mutagenesis with a complimentary pair of primers and the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. The mutant was selected by DpnI (NEB, Hitchin, UK) and was fully sequenced to confirm the desired mutation and to exclude any other unwanted sequence variations. The eukaryotic expression vectors GATA4-pSSRa and NKX2-5-pEFSA, and the natriuretic peptide precursor A-luciferase (NPPA-luc) reporter vector, which expresses Firefly luciferase, were kind gift from Dr. Ichiro Shiojima, at the Department of Cardiovascular Science and Medicine, Chiba University, Japan. The α-myosin heavy chain 6-luciferase (MYH6-luc) reporter plasmid, which expresses Firefly luciferase, was created as described previously (Chen et al., 2017Chen HX, Zhang X, Hou HT, Wang J, Yang Q, Wang XL and He GW (2017) Identification of a novel and functional mutation in the TBX5 gene in a patient by screening from 354 patients with isolated ventricular septal defect. Eur J Med Genet 60:385-390.).

Cell culture, plasmid transfection and luciferase analysis

COS-7 cells (derived from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were grown in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), as well as penicillin (100 U/mL) and streptomycin (100 μg/mL), in an atmosphere with 5% CO2 at 37 °C. COS-7 cells were seeded in 24-well plates, at a density of 2 × 105 per cell before transfection. Plasmids were transfected into cells 24 h after plating with the Lipofectamine 3000 reagent (Invitrogen) according to the product description. To balance transfection efficiency, the internal control plasmid pGL4.75 (Promega), which expresses the Renilla luciferase, was co-transfected. Specifically, COS-7 cells were transiently transfected with empty pcDNA3.1 (1.0 μg), or wild-type TBX5-pcDNA3.1 (1.0 μg), or mutant TBX5-pcDNA3.1 (1.0 μg), or wild-type TBX5-pcDNA3.1 (0.5 μg) plus empty pcDNA3.1 (0.5 μg), or wild-type TBX5-pcDNA3.1 (0.5 μg) plus mutant TBX5-pcDNA3.1 (0.5 μg), together with MYH6-luc (1.5 μg) and pGL4.75 (0.04 μg). To analyze the synergistic transactivation, the same amount (0.6 μg) of each expression vector (empty pcDNA3.1, wild-type TBX5-pcDNA3.1, mutant TBX5-pcDNA3.1, NKX2-5-pEFSA, GATA4-pSSRa) was used singly or in combination, in the presence of NPPA-luc (1.0 μg) and pGL4.75 (0.04 μg). The transfected cells were cultured for 48 h, and then were harvested and lysed. The Firefly luciferase and Renilla luciferase activities were measured under the GloMax-96 Microplate Luminometer (Promega) by utilizing the Dual-Glo Luciferase Assay System (Promega), following the manufacturer’s manual. The activity of the promoter was presented as fold activation (ratio) of Firefly luciferase relative to Renilla luciferase. Each transfection experiment was conducted in triplicate for three times, and the results for promoter activity were given as mean ± standard deviation (SD) of three experiments in triplicate.

Statistics

Differences in promoter activities between two groups were compared using the Student’s t-test, or one-way ANOVA with Tukey’s post hoc test, when indicated, with a p<0.05 indicating significant difference.

Results

Baseline characteristics of the study patients

In this investigation, a total of 178 unrelated cases suffering from CHD and AF (105 males, with a mean age of 33 years at initial diagnosis of AF) were clinically analyzed in contrast to a total of 292 unrelated control people (173 males, with a mean age of 33 years). The included cases had both echocardiograph-documented CHD and electrocardiogram-documented AF, while the controls had normal echocardiographs and electrocardiograms, with no evidence of cardiac diseases. All the 178 patients had positive family histories of CHD and AF; whereas none of the 292 control individuals had a positive family history of CHD or AF. No study participants had known traditional pathogenic factors for CHD or AF. There was no significant difference between case and control groups in gender, age or ethnicity. The baseline features of the 178 cases affected with CHD and AF are summarized in Table 1.

Table 1 -
Demographic and baseline clinical characteristics of the 178 patients with familial congenital heart disease and atrial fibrillation.

Detection of a causative TBX5 mutation

By sequencing the whole coding regions and flanking introns of the TBX5 gene, a heterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was detected in one out of the 178 patients affected with CHD and AF, with an allele frequency of ~0.28% in the patient population. The variation carrier had positive family histories of CHD and AF as well as bicuspid aortic valve (BAV). Genetic studies of the variation carrier’s available family members revealed that the variation co-segregated with ASD and AF as well as BAV, which were transmitted as autosomal dominant traits. In addition, two family members (II-1 and III-1) had also congenital VSD. The sequence chromatograms illustrating the heterozygous TBX5 variation of c.577G>T and its wild-type control sequence are given in Figure 1A. The schematic diagrams showing the structural domains of wild-type and mutant TBX5 proteins are illustrated in Figure 1B. The pedigree structure of the family with CHD and AF as well as BAV is shown in Figure 1C. The phenotypic characteristics as well as mutational status for TBX5 of the affected family members are presented in Table 2. The nonsense mutation was absent from 296 control people, and was not found in the 1000 Genomes Project databaseThe 1000 Genomes Project database, The 1000 Genomes Project database, http://www.1000genomes.org (May 6, 2020).
http://www.1000genomes.org...
, the Genome Aggregation DatabaseThe Genome Aggregation Database,The Genome Aggregation Database, https://gnomad.broadinstitute.org (May 6, 2020).
https://gnomad.broadinstitute.org...
, or the Single Nucleotide Polymorphism database (accessed on May 6, 2020The Single Nucleotide Polymorphism database, The Single Nucleotide Polymorphism database, http://www.ncbi.nlm.nih.gov/snp (May 6, 2020).
http://www.ncbi.nlm.nih.gov/snp...
), indicating its novelty. Besides, similar with previous studies (Al-Qattan and Abou Al-Shaar, 2015Al-Qattan MM and Abou Al-Shaar H (2015) Molecular basis of the clinical features of Holt-Oram syndrome resulting from missense and extended protein mutations of the TBX5 gene as well as TBX5 intragenic duplications. Gene 560:129-136.; Chen et al., 2017Chen HX, Zhang X, Hou HT, Wang J, Yang Q, Wang XL and He GW (2017) Identification of a novel and functional mutation in the TBX5 gene in a patient by screening from 354 patients with isolated ventricular septal defect. Eur J Med Genet 60:385-390.), no more c.577G>T variation was detected in either cases or controls. Thus, the allele frequency of TBX5 variation identified in this study was 1/356 (0.28%) in patients and 0/584 (0%) in controls.

Figure 1 -
A new TBX5 mutation responsible for familial heart defect and atrial fibrillation. (A) Sequence chromatograms illustrating the TBX5 heterozygous mutation from the proband (mutant) and its homozygous wild-type control from a healthy individual (wild type). An arrow points to the heterozygous nucleotides of G/T or the homozygous nucleotides of G/G. (B) Schematic drawings showing the structural domains of the TBX5 proteins. NH2, amino-terminus; NLS1, nuclear location signal 1; TBX, T-box; TAD, transcriptional activation domain; NLS2, nuclear location signal 2; COOH, carboxyl-terminus. (C) Pedigree structure of the family suffering from congenital heart defect and atrial fibrillation. Family members are recognized by generations as well as numbers. Circles mean female members; squares, male family member; closed symbols, affected members; open symbols, unaffected members; the symbol with a slash, the deceased member; the arrow beside the closed square, the index patient; “+”, carriers of the TBX5 mutation; “-”, non-carriers.

Table 2 -
Phenotypic features and TBX5 mutation status of the family members with congenital heart defect and atrial fibrillation as well as bicuspid aortic valve.

Additionally, WES analysis of the genomic DNAs from two affected family members (II-4 and III-4) and one unaffected family member (II-3) of the proband who harbored an identified TBX5 mutation was carried out, and an average of 12,973 exonic variants ranging from 11,652 to 14,395 was detected for each family member. A total of 742 exonic variants were shared by both affected subjects, of which 262 were autosomal, heterozygous non-synonymous, nonsense, and splice site variants. After filtered, only the variation c.577G>T in TBX5 was verified by Sanger sequencing and demonstrated to co-segregate with CHD and AF as well as BAV in the family.

No transactivational function of the mutant TBX5 protein

As shown in Figure 2, the same amount (1.0 μg) of wild-type and Gly193*-mutant TBX5 plasmids transcriptionally activated the MYH6 promoter by ~12 fold and ~1 fold, respectively (comparison between wild type and mutant: t = 8.07389, p = 0.00128). When half the amount of wild-type and Gly193*-mutant TBX5 plasmids (each 0.5 μg) was used, the resultant transcriptional activity was ~6-fold (comparison between wild type plus empty plasmid and wild type plus mutant: t = 3.91627, p = 0.01730).

Figure 2 -
Functional failure of TBX5 caused by the mutation. Activation of α-myosin heavy chain 6 promoter-driven luciferase in cultured COS-7 cells by wild-type or Gly193*-mutant TBX5, singly or together, revealed that the Gly193*-mutant TBX5 protein had no transcriptional activity. Transfection experiments for each plasmid were carried out in triplicates and the results are expressed as means with standard deviations. Here ## and # indicate p<0.01 and p<0.02, respectively, in comparison with wild-type TBX5.

No synergistic effect between mutant TBX5 and NKX2-5 as well as GATA4

As shown in Figure 3, wild-type and Gly193*-mutant TBX5 activated the NPPA promoter by ~7 fold and ~1 fold, respectively (comparison between wild type and mutant: t = 9.24975, p = 0.00076). In combination with wild-type NKX2-5, wild-type and Gly193*-mutant TBX5 activated the NPPA promoter by ~30 fold and ~5 fold, respectively (comparison between wild type and mutant: t = 9.36360, p = 0.00072); while together with wild-type GATA4, wild-type and Gly193*-mutant TBX5 transcriptionally activated the NPPA promoter by ~22 fold and ~4 fold, respectively (comparison between wild type and mutant: t = 9.51139, p = 0.00068).

Figure 3 -
Disrupted synergistic transactivation between mutant TBX5 and NKX2-5 as well as GATA4. The synergistic transactivation of the promoter of natriuretic peptide precursor A in cultured cells by TBX5 and NKX2-5 as well as GATA4 was ablated by the Gly193* mutation. Transfection experiments for each plasmid were done in triplicates, with means and standard deviations shown. Here the symbols a, b and c all indicate p<0.001, in comparison with their wild-type counterparts.

Discussion

In the current investigation, a novel heterozygous TBX5 variation, NM_000192.3: c.577G>T; p.(Gly193*), was discovered in a family with CHD and AF as well as BAV. The variation was absent in the 584 reference chromosomes as well as in such population databases as the 1000 Genomes Project databaseThe 1000 Genomes Project database, The 1000 Genomes Project database, http://www.1000genomes.org (May 6, 2020).
http://www.1000genomes.org...
, the Genome Aggregation DatabaseThe Genome Aggregation Database,The Genome Aggregation Database, https://gnomad.broadinstitute.org (May 6, 2020).
https://gnomad.broadinstitute.org...
, and the Single Nucleotide Polymorphism databaseThe Single Nucleotide Polymorphism database, The Single Nucleotide Polymorphism database, http://www.ncbi.nlm.nih.gov/snp (May 6, 2020).
http://www.ncbi.nlm.nih.gov/snp...
. Functional explorations showed that Gly193*-mutant TBX5 lost transcriptional activity on the MYH6 and NPPA promoters. Moreover, the mutation disrupted the synergistic transcriptional effect between TBX5 and GATA4 as well as NKX2-5. Additionally, WES analysis showed no other genes contributing to the diseases of the family. These observational results indicate that the pathogenic variation in the TBX5 gene predisposes to CHD and AF as well as BAV.

In humans, TBX5 is located on chromosome 12q24.1, which encodes a 518-amino acid protein. The TBX5 protein harbors four functionally important domains, including a T-box domain (TBX; amino acids 56-236), which functions to bind target DNAs and interact with other proteins; a transcriptional activation domain (TAD; amino acids 339-379), which is responsible for transactivation of target genes; and two nuclear localization signals (NLS) including NLS1 (amino acids 78-90) and NLS2 (amino acids 325-340), which were essential for nuclear localization (Steimle and Moskowitz1, 2017Steimle JD and Moskowitz1 IP (2017) TBX5: A key regulator of heart development. Curr Top Dev Biol 122:195-221.). Previous studies have corroborated that TBX5 is highly expressed in the hearts of humans and vertebrates, encompassing the endocardium, myocardium, and epicardium of embryonic and adult hearts, and its expression is much higher in atria than in ventricles during embryogenesis, where it plays a key role in cardiovascular morphogenesis and postnatal heart remodeling (Steimle and Moskowitz1, 2017Steimle JD and Moskowitz1 IP (2017) TBX5: A key regulator of heart development. Curr Top Dev Biol 122:195-221.). Recent research has validated that TBX5 transcriptionally regulates expression of many target genes, including NPPA, GJA5, MYH6 and SCN5A, separately or together with GATA4, GATA6, NKX2-5, MEF2C and TBX20 (Steimle and Moskowitz1, 2017Steimle JD and Moskowitz1 IP (2017) TBX5: A key regulator of heart development. Curr Top Dev Biol 122:195-221.), and variations in TBX5 and its target genes as well as cooperative partners have been reported to result in CHD and/or AF in humans (Postma et al., 2008Postma AV, van de Meerakker JB, Mathijssen IB, Barnett P, Christoffels VM, Ilgun A, Lam J, Wilde AA, Lekanne Deprez RH and Moorman AF (2008) A gain-of-function TBX5 mutation is associated with atypical Holt-Oram syndrome and paroxysmal atrial fibrillation. Circ Res 102:1433-1442.; Mahida, 2014Mahida S (2014) Transcription factors and atrial fibrillation. Cardiovasc Res 101:194-202.; Guo et al., 2016Guo DF, Li RG, Yuan F, Shi HY, Hou XM, Qu XK, Xu YJ, Zhang M, Liu X, Jiang JQ et al. (2016) TBX5 loss-of-function mutation contributes to atrial fibrillation and atypical Holt-Oram syndrome. Mol Med Rep 13:4349-4356.; Ma et al., 2016Ma JF, Yang F, Mahida SN, Zhao L, Chen X, Zhang ML, Sun Z, Yao Y, Zhang YX, Zheng GY et al. (2016) TBX5 mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians. Cardiovasc Res 109:442-450.; Wang et al., 2016Wang ZC, Ji WH, Ruan CW, Liu XY, Qiu XB, Yuan F, Li RG, Xu YJ, Liu X, Huang RT et al. (2016) Prevalence and spectrum of TBX5 mutation in patients with lone atrial fibrillation. Int J Med Sci 13:60-67.; Li and Yang, 2017Yang B, Zhou W, Jiao J, Nielsen JB, Mathis MR, Heydarpour M, Lettre G, Folkersen L, Prakash S, Schurmann C et al. (2017) Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve. Nat Commun 8:15481.; Campbell and Wehrens, 2018Campbell HM and Wehrens XHT (2018) Genetics of atrial fibrillation: an update. Curr Opin Cardiol 33:304-310.). In the current investigation, the pathogenic variation detected in patients with familial CHD and AF as well as BAV was predicted to produce a truncating TBX5 protein with most functional domains lost, and functional explorations revealed that the mutant TBX5 protein failed to transcriptionally activate target genes. Moreover, the pathogenic variation ablated the synergistic transactivation between TBX5 and NKX2-5 as well as GATA4. These data indicate that TBX5 haploinsufficiency is a molecular mechanism of CHD and AF as well as BAV in a subset of patients.

It might be ascribed to the aberrant cardiovascular genesis that TBX5 deficiency contributes to CHD and AF. In mice, TBX5 is abundantly expressed in entire cardiac crescent, heart tube, left ventricle, vena cavae, common atrium, and cardiac central conduction system, encompassing atrioventricular bundle and bundle branches (Bruneau et al., 1999Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG and Seidman CE (1999) Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev Biol 211:100-108.; Moskowitz et al., 2004Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE and Seidman JG (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116.). Homozygous deletion of Tbx5 led to murine embryonic death because of failure to undergo cardiac looping as well as left ventricular and sinoatrial hypoplasia; while heterozygous Tbx5-bull mice showed ASD, VSD, left ventricular hypoplasia, endocardial cushion defect, and conduction system anomalies, encompassing atrioventricular conduction blocks and bundle branch blocks (Bruneau et al., 1999Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG and Seidman CE (1999) Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev Biol 211:100-108.; Bruneau et al., 2001Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE et al. (2001) A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106:709-721.; Moskowitz et al., 2004Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE and Seidman JG (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116.). In addition, in murine hearts Tbx5 haploinsufficiency also markedly reduced the transcription of multiple target genes, including Nppa and Cx40 (Bruneau et al., 2001Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE et al. (2001) A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106:709-721.). Moreover, adult-restricted Tbx5-mutant mice demonstrated spontaneous AF, and in Tbx5-deficient atrial cardiomyocytes, action potential abnormalities occurred due to a decreased SERCA2-mediated sarcoplasmic reticulum calcium uptake (Dai et al., 2019Dai W, Laforest B, Tyan L, Shen KM, Nadadur RD, Alvarado FJ, Mazurek SR, Lazarevic S, Gadek M, Wang Y et al. (2019) A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice. Elife 8:e41814.). In human beings, TBX5 is highly expressed in embryonic and postnatal hearts (Hatcher et al., 2000Hatcher CJ, Goldstein MM, Mah CS, Delia CS and Basson CT (2000) Identification and localization of TBX5 transcription factor during human cardiac morphogenesis. Dev Dyn 219:90-95.), and a number of TBX5 loss- or gain-of-function mutations have been causally linked to HOS, including CHD and AF as well as cardiac block (Al-Qattan and Abou Al-Shaar, 2015Al-Qattan MM and Abou Al-Shaar H (2015) Molecular basis of the clinical features of Holt-Oram syndrome resulting from missense and extended protein mutations of the TBX5 gene as well as TBX5 intragenic duplications. Gene 560:129-136.). Taken collectively, these findings suggest that genetically defective TBX5 enhances the susceptibility to CHD and AF in humans, and underscore that TBX5 dosage must be precisely regulated to avoid heart disorders.

Notably, previous studies have causally linked TBX5 variations to various cardiovascular malformations, including ASD, VSD, atrioventricular septal defect, pulmonary stenosis, hypoplastic left ventricle, mitral valve anomaly (Gharibeh et al., 2018Gharibeh L, Komati H, Bossé Y, Boodhwani M, Heydarpour M, Fortier M, Hassanzadeh R, Ngu J, Mathieu P, Body S et al. (2018) GATA6 regulates aortic valve remodeling, and its haploinsufficiency leads to right-left type bicuspid aortic valve. Circulation 138:1025-1038.). In the current investigation, the affected family members had also BAV, in addition to ASD, VSD and AF, thus expanding the phenotypic spectrum linked to mutant TBX5. Given that loss-of-function mutations in multiple transcriptional partners of TBX5 (Balistreri et al., 2019Balistreri CR, Forte M, Greco E, Paneni F, Cavarretta E, Frati G and Sciarretta S (2019) An overview of the molecular mechanisms underlying development and progression of bicuspid aortic valve disease. J Mol Cell Cardiol 132:146-153.), encompassing GATA6 (Gharibeh et al., 2018Gharibeh L, Komati H, Bossé Y, Boodhwani M, Heydarpour M, Fortier M, Hassanzadeh R, Ngu J, Mathieu P, Body S et al. (2018) GATA6 regulates aortic valve remodeling, and its haploinsufficiency leads to right-left type bicuspid aortic valve. Circulation 138:1025-1038.; Xu et al., 2018Xu YJ, Di RM, Qiao Q, Li XM, Huang RT, Xue S, Liu XY, Wang J and Yang YQ (2018) GATA6 loss-of-function mutation contributes to congenital bicuspid aortic valve. Gene 663:115-120.), GATA4 (Yang et al., 2017Yang B, Zhou W, Jiao J, Nielsen JB, Mathis MR, Heydarpour M, Lettre G, Folkersen L, Prakash S, Schurmann C et al. (2017) Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve. Nat Commun 8:15481.; Li et al., 2018Li RG, Xu YJ, Wang J, Liu XY, Yuan F, Huang RT, Xue S, Li L, Liu H, Li YJ et al. (2018c) GATA4 loss-of-function mutation and the congenitally bicuspid aortic valve. Am J Cardiol 121:469-474.c), GATA5 (Padang et al., 2012Padang R, Bagnall RD, Richmond DR, Bannon PG and Semsarian C (2012) Rare non-synonymous variations in the transcriptional activation domains of GATA5 in bicuspid aortic valve disease. J Mol Cell Cardiol 53:277-281.; Bonachea et al., 2014Bonachea EM, Chang SW, Zender G, LaHaye S, Fitzgerald-Butt S, McBride KL and Garg V (2014) Rare GATA5 sequence variants identified in individuals with bicuspid aortic valve. Pediatr Res 76:211-216.; Shi et al., 2014Shi LM, Tao JW, Qiu XB, Wang J, Yuan F, Xu L, Liu H, Li RG, Xu YJ, Wang Q et al. (2014) GATA5 loss-of-function mutations associated with congenital bicuspid aortic valve. Int J Mol Med 33:1219-1226.), NKX2-5 (Qu et al., 2014Qu XK, Qiu XB, Yuan F, Wang J, Zhao CM, Liu XY, Zhang XL, Li RG, Xu YJ, Hou XM et al. (2014) A novel NKX2.5 loss-of-function mutation associated with congenital bicuspid aortic valve. Am J Cardiol 114:1891-1895.), and TBX20 (Luyckx et al., 2019Luyckx I, Kumar AA, Reyniers E, Dekeyser E, Vanderstraeten K, Vandeweyer G, Wünnemann F, Preuss C, Mazzella JM, Goudot G et al. (2019) Copy number variation analysis in bicuspid aortic valve-related aortopathy identifies TBX20 as a contributing gene. Eur J Hum Genet 27:1033-1043.), have been related to BAV, it is very likely that mutated TBX5 contributes to BAV by reducing expression of the target genes related to BAV morphogenesis in synergy with these partners.

Conclusions

This investigation causally links TBX5 loss-of-function mutation to CHD, AF and BAV for the first time, which highlights the key role of abnormal cardiovascular development in the pathogenesis of CHD, AF and BAV, implying potential implications for individualized prophylaxis and management of patients with CHD and AF as well as BAV.

Acknowledgments

The authors thank the study participants for their dedication to this investigation. This work was financially supported by grants from the National Natural Science Foundation of China (81670305, 81600228, 81470372), the Science and Technology Support Project of Medical Guidance, Shanghai, China (19411971900), the Natural Science Foundation of Shanghai, China (18ZR1423400), the Program of Health and Family Planning Commission of Shanghai, China (201740064 and 20154Y0026), and the Key Project of Shanghai Fifth People’s Hospital, Fudan University, China (2018WYZD05).

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Internet Resources

Data availability

Data citations

The 1000 Genomes Project database, The 1000 Genomes Project database, http://www.1000genomes.org (May 6, 2020).

The Genome Aggregation Database,The Genome Aggregation Database, https://gnomad.broadinstitute.org (May 6, 2020).

The Single Nucleotide Polymorphism database, The Single Nucleotide Polymorphism database, http://www.ncbi.nlm.nih.gov/snp (May 6, 2020).

Publication Dates

  • Publication in this collection
    13 Nov 2020
  • Date of issue
    2020

History

  • Received
    07 May 2020
  • Accepted
    19 Sept 2020
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