Introduction

Congenital heart disease (CHD), a structural defect that arises from abnormal development of the heart or major cardiothoracic blood vessels, is the most prevalent of all congenital malformations in humans, with an estimated prevalence of 1 % in live neonates worldwide [17, 21]. It is the most common cause of infant death caused by birth defects, with approximately 27 % of infants who died of a birth defect having a heart defect. [21]. Clinically, congenital cardiovascular deformities are usually categorized into more than 20 different types with specific anatomic or hemodynamic lesions, including ventricular septal defect (VSD), atrial septal defect, patent ductus arteriosus, endocardial cushion defect, double outlet right ventricle (DORV), tetralogy of Fallot, persistent truncus arteriosus, aortic coarctation, coronary artery anomalies, valvular pulmonary stenosis, pulmonary atresia, abnormal pulmonary venous return, interrupted aortic arch, transposition of the great arteries, and hypoplastic left heart syndrome, of which VSD is the most common form of CHD in children [5, 21, 36, 39, 69, 86]. Distinct kinds of CHDs may occur separately or concomitantly with each other, leading to degraded quality of life, poor exercise tolerance, brain injury, pulmonary hypertension, reduced lung function, impaired muscle function, subclinical hypothyroidism, autonomic nervous dysfunction, aortic aneurysm or dissection, infective endocarditis, thromboembolism, heart failure, arrhythmias, and even cardiac death [1, 3, 6, 8, 14–16, 19, 20, 29, 32–34, 40, 45–48, 51, 55, 57, 59, 60, 62, 66, 67, 87]. Although striking progress in medical treatment of newborns with CHD has contributed to an increasing number of adult survivors, unfortunately, the late morbidity and mortality are still markedly increased in the survivors [49, 68, 70, 71]. Therefore, CHD has conferred a vast economic burden on patients’ families and healthcare systems, and the socioeconomic burden is anticipated to increase in the future as the CHD adults accrue [72]. Despite the high prevalence and important clinical significance, in an overwhelming majority of cases, the etiologies responsible for CHD remain unknown.

Cardiac morphogenesis from a straight tube to a four-chambered heart experiences a complex dynamic biological process that mandates a precise spatial and temporal cooperation of cardiac cell commitment, differentiation, proliferation, and migration, and both environmental and genetic pathogenic factors may disarrange this process of cardiogenesis, resulting in various CHDs [4, 13, 24, 37, 53, 54, 65, 74]. Recently, there is compelling evidence that demonstrates the genetic origin of CHD, and a growing number of mutations in more than 60 genes have been shown to cause CHD [2, 4, 9, 12, 18, 22, 27, 28, 31, 35, 41, 42, 56, 58, 61, 63, 75–78, 82–85]. Nevertheless, these causative genes can only explain the CHDs in a small proportion of patients and in most patients the genetic basis underpinning CHD is still to be revealed.

Chromosome 22q11.2 deletion syndrome (22q11DS), which is caused by a heterozygous multi-gene deletion, is a relatively common genetic disorder, affecting 1 in 4000 live births. CHDs are a prominent part of the 22q11DS phenotype, with an incidence of about 80 % in infants with 22q11DS [44]. Additionally, 22q11DS is found in a small percentage of patients with double outlet right ventricle. The TBX1 gene, a member of the T-box gene family of DNA-binding transcription factors, is mapped to the 22q11.2 and has been identified to be associated with the cardiac phenotype of 22q11.2 DS, including tetralogy of Fallot, truncus arteriosus, and interrupted aortic arch [30, 38, 43, 44]. Interestingly, mutations of the TBX1 gene have been found in some patients featuring 22q11DS who are otherwise devoid of the 22q11.2 deletion [81] and also found in non-syndromic CHD patients [26, 80]. These data strongly suggest TBX1 as an important candidate gene for human CHD.

Materials and Methods

Ethics

This study was performed in conformity to the ethical principles of the revised Declaration of Helsinki (Somerset West, Republic of South Africa, 1996). The study protocol was reviewed and approved by the local institutional ethics committee of Tongji Hospital, Tongji University (the ethical approval number for cases and controls: LL(H)-09-07; the date for the approval: July 27, 2009), and written informed consent was obtained from the parents of each patient and control prior to study.

Study Participants

A cohort of 230 unrelated children suffered from CHD was enrolled. The available family members of the proband carrying an identified TBX1 mutation were also included. All patients underwent a comprehensive clinical evaluation, including individual and familial histories, medical records, complete physical examination, 12-lead electrocardiogram, and two-dimensional transthoracic echocardiography with color flow Doppler. Cardiac catheterization, angiography, chest X-ray, and cardiac magnetic resonance imaging were performed only if there was a strong clinical indication. Medical records were also reviewed in the case of deceased or unavailable relatives. CHD was confirmed by imaging and/or direct view during cardiac surgery. A total of 200 non-CHD individuals from the same geographic area, who were matched to the CHD patients in ethnicity and gender, were recruited as the controls. After obtaining informed written parental consent, approximately 0.5–2 ml of peripheral venous blood sample was taken from each study participant, and the genomic DNA was extracted from peripheral venous blood leukocytes using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Sequencing of TBX1

The coding exons and flanking introns of the TBX1 gene (including isoforms A, B and C) were sequenced in 230 unrelated CHD patients. The available relatives of the index patient carrying an identified TBX1 mutation and 200 unrelated control individuals were subsequently genotyped for TBX1. The referential genomic DNA sequence of TBX1 was derived from nucleotide (Accession No. NC_000022.11), a gene sequence database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/nucleotide/). With the aid of the online Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), the primer pairs used to amplify the coding regions and splice junction sites of TBX1 by polymerase chain reaction (PCR) were designed as shown in Table 1. The PCR was conducted using HotStar Taq DNA polymerase (Qiagen GmbH, Hilden, Germany) on a Veriti Thermal Cycler (Applied Biosystems, Foster, CA, USA), with standard conditions and concentrations of reagents. Both strands of each PCR product were sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) under an ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems). The DNA sequences were analyzed with the DNA Sequencing Analysis Software v5.1 (Applied Biosystems). Additionally, an identified sequence variation was queried in the single-nucleotide polymorphism (SNP) database at NCBI (http://www.ncbi.nlm.nih.gov/), the human gene mutation (HGM) database (http://www.hgmd.org/), and the 1000 Genome Project (1000 GP) database (http://www.1000genomes.org/) to confirm its novelty.

Table 1 The primers to amplify the coding exons and flanking introns of TBX1
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Multiple Alignments of TBX1 Amino Acid Sequences Among Various Species

Conservation of the amino acid altered by the identified mutation was estimated by aligning human TBX1 to chimpanzee, monkey, dog, mouse, rat, zebrafish, and frog TBX1 using the HomoloGene and Show Multiple Alignment links on the NCBI’s website (http://www.ncbi.nlm.nih.gov/homologene).

Prediction of the Pathogenic Potential of a Novel TBX1 Sequence Variation

The causative potential of a novel TBX1 sequence variation was predicted by MutationTaster (an online program at http://www.mutationtaster.org), which automatically gave a probability for the variation to be either a disease-causing mutation or a benign polymorphism. Of note, here the p value is the probability of the correct prediction, i.e., a p value close to one indicates high accuracy of the prediction.

Plasmids and Site-Directed Mutagenesis

The recombinant expression plasmid TBX1-pcDNA3.1, which contains the full-length cDNA of TBX1 isoform C, was constructed as described previously [80]. For generation of the 4 × T-pGL4.25 luciferase reporter vector (4 × T-luc), four conserved T-half sites “ATTTCACACCT” were oriented head to tail, similar to those reported by Sinha et al. [64], synthesised and subcloned into the KpnI-HindIII sites in the pGL4.25 [luc2CP/minP] plasmid (Promega). The mutant TBX1 expression vector was generated by using a QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with a complementary pair of primers and with the wild-type TBX1-pcDNA3.1 used as the template. The mutant TBX1 was sequenced to confirm the desired mutation and to exclude any other sequence variations.

Cell Transfection and Luciferase Assays

The transient cell transfection was performed with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10 % fetal calf serum (Invitrogen) and seeded in 12-well plates (2 × 105) before transfection. Twenty-four hours after plating, the COS-7 cells at about 75 % confluence were co-transfected with 0.4 μg of wild-type or mutant TBX1-pcDNA3.1, 1.0 μg of 4 × T-luc, and 0.04 μg of pGL4.75 (hRluc/CMV, Promega), a Renilla luciferase reporter plasmid used as an internal control. For co-transfection experiments, 0.2 μg of wild-type TBX1-pcDNA3.1 together with 0.2 μg of mutant TBX1-pcDNA3.1 or 0.2 μg of empty pcDNA3.1 vector was used in the presence of 1.0 μg of 4 × T-luc and 0.04 μg of pGL4.75. The cells were harvested 48 h after transfection. The Firefly and Renilla luciferase activities were measured with the Dual-Glo luciferase assay system (Promega). The activity of the Firefly luciferase was normalized to that of the Renilla luciferase. At least three independent experiments were performed in triplicate for wild-type and mutant TBX1.

Statistical Analysis

Continuous variables are expressed as means ± standard deviations (SD). Student’s unpaired t test was used to compare the continuous variables between two groups. Comparison of the categorical variables between two groups was made by using Pearson’s χ 2 test or Fisher’s exact test when appropriate. A two-tailed p < 0.05 was considered to indicate statistical difference.

Results

Baseline Clinical Characteristics of the Study Population

A cohort of 230 unrelated patients with CHD was clinically investigated in contrast to a total of 200 unrelated non-CHD control individuals (102 males and 98 females, with no family history of CHD in the control individuals). All the patients had confirmed CHD, while the control individuals had no evidence of structural cardiac abnormalities. None of the study participants had established environmental risk factors for CHD, such as maternal illness and drug use in the first trimester of pregnancy, parental smoking, and long-term exposure to chemical toxicants and ionizing radiation. There is no difference in either gender or ethnicity between patient and control groups. The baseline clinical characteristics of the study population are summarized in Table 2.

Table 2 Baseline clinical characteristics of the studied patients with congenital heart disease
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Identification of a Novel TBX1 Mutation

By sequence analysis of the coding exons and exon–intron boundaries of TBX1, a heterozygous sequence variation was identified in 1 of 230 unrelated CHD patients, with a mutational prevalence of roughly 0.43 %. Specifically, a substitution of thymine for cytosine in the first nucleotide of codon 277 (c.829C > T), predicting the transition of glutamine into a premature stop codon at amino acid position 277 (p.Q277X), was identified in an index patient with DORV and a large subpulmonary VSD, who underwent surgical treatment 1 week after birth. The sequence electropherograms showing the identified heterozygous TBX1 variation in contrast to its corresponding control sequence are shown in Fig. 1a. The schematic diagrams of the wild-type TBX1C and mutant TBX1 proteins showing the structural domains and location of the mutation detected in this study are presented in Fig. 1b. The variation was neither observed in 400 control chromosomes nor found in the SNP, HGM, and 1000 GP databases, which were consulted again on November 22, 2014, indicating a novel mutation.

Fig. 1
figure 1

A novel TBX1 mutation associated with congenital heart disease. a Sequence electropherograms showing the TBX1 mutation in contrast to its corresponding control. The arrow indicates the heterozygous nucleotides of C/T in the proband from family 1 (mutant) or the homozygous nucleotides of C/C in the corresponding control individual (wild type). The rectangle means the nucleotides constituting a codon of TBX1. b Schematic diagrams of the wild-type human TBX1 isoform C and mutant human TBX1 protein structures. The mutation associated with congenital heart disease is predicted to produce a truncated protein with only 276 amino acids at the amino-terminus left. NH2, amino-terminus; NLS, nuclear location signal; TAD, transcriptional activation domain; and COOH, carboxyl-terminus. c Pedigree structure of the family with congenital heart disease. Family is designated as family 1. Family members are identified by generations and numbers. Square indicates male family member; circle, female member; closed symbol, affected member; open symbol, unaffected member; symbol with a slash, the deceased member; arrow, proband; ‘‘+’’, carrier of the heterozygous mutation; and ‘‘−’’, non-carrier

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Genetic analysis of the proband’s families displayed that the nonsense mutation was present in all affected family members alive, but absent in unaffected family members examined. Analysis of the pedigree demonstrated that the mutation co-segregated with subpulmonary VSD transmitted in an autosomal dominant pattern and with complete penetrance. There are no other anomalies consistent with 22q11 deletion syndrome, and there is no history of speech delay or learning disability in the affected family members. The pedigree structure of the family is shown in Fig. 1c. The phenotypic characteristics and status of TBX1 mutation of the affected family members are listed in Table 3.

Table 3 Phenotypic characteristics and status of TBX1 mutation of the affected pedigree members
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Multiple Alignments of TBX1 Protein Sequences Among Various Species

As shown in Fig. 2, a cross-species alignment of multiple TBX1 protein sequences showed that the altered glutamine at amino acid position 277 of human TBX1 was completely conserved evolutionarily, implying that the amino acid is functionally important.

Fig. 2
figure 2

Alignment of multiple TBX1 protein sequences across various species. The altered glutamine at amino acid position 227 of human TBX1 protein is completely conserved evolutionarily among species

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Causative Potential of the Identified TBX1 Sequence Variation

The TBX1 sequence variation of c.829C > T was predicted to be disease-causing, with a p value of 1.0000, supporting that Q277X-mutant TBX1 contributes to the occurrence of CHD in these mutation carriers.

Functional Assay of the Q277X-mutant TBX1

As shown in Fig. 3, the Q277X-mutant TBX1 had no transcriptional activity compared with its wild-type counterpart (t = 4.8028, p = 0.0086). When wild-type TBX1 was co-expressed with the same amount of Q277X-mutant TBX1, the induced transcriptional activation was significantly reduced compared with the wild-type TBX1 (t = 4.2947, p = 0.0127).

Fig. 3
figure 3

Transcriptional activation defect of TBX1 resulted from Q277X mutation. Transcriptional activation of the 4 × T-luciferase reporter in cultured COS-7 cells by wild-type TBX1 (WT) or mutant (Q277X), alone or together, showed significantly diminished transcriptional activity by the mutant protein. The activity of the Firefly luciferase was normalized for transfection efficiency to that of Renilla luciferase. The results are shown as the mean and standard deviations of three independent experiments performed in triplicate. ** and * indicate p < 0.01 and p < 0.05, respectively, when compared with wild-type TBX1 (0.4 μg)

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Discussion

In the current study, a novel heterozygous mutation in TBX1, p.Q277X, was identified in a family with CHD. The mutant allele was present in all affected family members available but absent in unaffected relatives examined and 400 referral chromosomes from an ethnically matched control population. A cross-species alignment of multiple TBX1 protein sequences showed that the altered amino acid was completely conserved evolutionarily. The p.Q277X variation was predicted to be a disease-causing mutation by MutationTaster, and the functional analysis revealed that the mutant TBX1 had no transcriptional activity. Hence, it is very likely that mutated TBX1 predisposes these mutation carriers to CHD. To the best of our knowledge, this is the first report on the association of TBX1 loss-of-function mutation with enhanced susceptibility to DORV and VSD in humans.

In the developing mammalian heart, 6 of the 17 family members, including TBX1, TBX18, and TBX20 of the TBX1 subfamily, and TBX2, TBX3, and TBX5 of the TBX2 subfamily, are expressed and required in a combinatorial fashion in different progenitor pools as well as in different compartments [25]. The human TBX1 gene is located on chromosome 22q11.21, the center of the 22q11DS chromosomal region, which acts in the pharyngeal mesoderm to maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and septated outflow tract, playing a key role in the elongation of the cardiac tube at the anterior pole [10, 25]. To date, three alternatively spliced transcripts, TBX1A (NM_080646.1), TBX1B (NM_005992.1), and TBX1C (NM_080647.1), have been found in humans that differed in their terminal exons. However, analysis of the gene expression levels in human tissues and comparison of the human and mouse genomic sequences showed that TBX1C is the major transcript with the nuclear location signal and the transcriptional activation domain and is highly homologous to mouse Tbx1 [23]. Besides, the apparently pathologic TBX1 mutations identified to date reside on exons 3–8 shared by isoforms A, B, and C or on exon 9C specific to isoform C, with no mutation on exons 9A and 9B specific to isoforms A and B. This is consistent with TBX1C having the primary biological function [52]. Three isoforms of TBX1 proteins share an evolutionarily conserved T-box homeodomain that recognizes and binds to a consensus DNA motif, ATTTCACACCT. The homeodomain is located at amino acid positions 109–302 and is predominantly involved in target DNA binding as well as interaction with other transcription factors [25]. The TBX1 mutation of p.Q277X identified in this study is predicted to generate a truncated protein with amino-terminus along with partial T-box homeodomain left (Fig. 1b) and thus may be anticipated to disable TBX1 by interfering with its binding to target DNA, nuclear distribution, and transcriptional activation.

In order to ascertain the functional consequence of the p.Q277X mutation in TBX1, the major transcript TBX1C was chosen as a representative of TBX1, and introduction of p.Q277X mutation into TBX1C abolished the transcriptional activation of TBX1C. These functional data suggest that haploinsufficiency or dominant-negative effect caused by TBX1 mutation is potentially an alternative pathological mechanism of CHD. Additionally, as the mutation is predicted to lead to a nonsense mutation, it is likely that the mutant mRNA undergoes nonsense-mediated decay, and in that case, it is likely that the mutation results in haploinsufficiency and does not have a dominant-negative effect [50]. However, at present, we cannot assay the mutant protein expression in Q277X-mutant patients due to the inaccessibility to cardiac tissue samples from these patients.

Previous investigations have verified that TBX1 can form complexes with such transcriptionally cooperative partners as NKX2.5 and SRF to synergistically mediate multiple important genes that are expressed in the heart during embryogenesis, including PITX2, FOXA2, FGF8, and FGF10 [25], and loss-of-function mutations in several genes, such as PITX2 and NKX2.5, have been causally linked to CHD including DORV and VSD [4, 11, 73, 76, 83, 84]. Therefore, genetically defective TBX1 may increase the vulnerability to CHD by reducing the expression of such genes essential for cardiovascular genesis.

Association of functionally compromised Tbx1 with increased susceptibility to CHD has been established in experimental animals. In mice, Tbx1 was highly expressed in the mesoderm and endoderm of the pharyngeal arches, and in the outflow tract. Genetic lineage analysis revealed that Tbx1-positive cells of the pharyngeal mesoderm contributed extensively to the outflow tract myocardium, endocardium, and mesenchymal cushions, indicating that Tbx1 plays a key role in an anterior subdomain of the second heart field [25]. Although mice heterozygous for deletion of Tbx1 presented mild anomalies, homozygous Tbx1-null mice died at birth with severe defects in the derivatives of the pharyngeal apparatus, of which cardiac defects included persistent truncus arteriosus, VSD, and mispatterning of the coronaries. Conditional knockout of Tbx1 from the pharyngeal endoderm and mesoderm, respectively, led to a spectrum of cardiovascular defects resembling those of the Tbx1-null mutants. Furthermore, mesodermal re-expression of Tbx1 in a null background corrected most of these defects, highlighting the pivotal role of Tbx1 for proper cardiovascular development [79]. In addition, Tbx1 function in the pharyngeal mesoderm was also required for survival, differentiation, and migration of the neural crest, suggesting a vital role in the development of pharyngeal arch artery [7]. Taken together, these findings support that TBX1 plays a crucial role in human cardiovascular development.

In conclusion, this study firstly associates TBX1 loss-of-function mutation with DORV and VSD, which expands the TBX1 mutation spectrum linked to CHD and provides novel insight into the molecular pathogenesis of CHD, implying the potential implications for genetic counseling and development of new preventive strategies for CHD.