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Introduction

TAB2 encodes the TGF-β activated kinase 1 (MAP3K7) binding protein 2 (TAB2), which can self-phosphorylate and regulate chemokines and inflammatory signaling molecules. Therefore, TAB2 gene defects are primarily associated with inflammatory responses (1,2). Thienpont et al (3) demonstrated the presence of a 6q24-q25 deletion following array-based comparative genomic hybridization detection in seven patients with congenital heart disease (CHD), and further revealed that insufficient haploid dosage of TAB2 in this region can lead to abnormal human heart development. Mutations in TAB2 lead to congenital heart defects, non-syndromic, 2 (CHTD2; OMIM#614980). TAB2 results in varying types of cardiac abnormalities, such as mitral, tricuspid valve prolapse and aortic stenosis, while some patients may face sudden cardiac death (3,4). In addition to cardiac structural abnormalities, patients with CHTD2 may present dysmorphic facial, joint and skin phenotypes (5,6). Therefore, identifying the abnormal features caused by mutations of TAB2 aids in clinical diagnosis, especially in prenatal diagnosis and genetic counseling.

The present study performed trio-whole exome sequencing (trio-WES) on a Chinese family with short stature and CHD as the main clinical features. The patient's cardiac color Doppler ultrasound showed insufficient mitral and mild regurgitation. In addition, the patient exhibited growth restriction and dysmorphic facial features. The results of genetic testing indicated that a novel pathogenic mutation occurred in the TAB2 gene; this may lead to protein non-expression, as indicated by in vitro functional experiments. The present study further expanded the pathogenic mutation spectrum of TAB2.

Case report

A 3-year-old male child was the subject of the present study (his mother is G1P1). Prenatal ultrasound diagnosis at 24 weeks of pregnancy showed that the fetus had short legs. Vaginal delivery occurred at 41+6 weeks of pregnancy, there was no history of birth asphyxia, the Apgar score was unknown and birth weight was 3,200 g. After birth, the motor milestone development of the child was normal. At the age of 10 months, his parents noticed that his growth development lagged behind those of his peers. No special treatment was given, and the annual growth rate was unknown. During the study period of the present study, the child was hospitalized in The Department of Pediatric Endocrinology and Metabolic Disease (Children's Hospital of Fudan University, Anhui Hospital, Hefei, China) for the growth related treatment. The physical examination conducted on admission showed that the child was in good mental state. He had dysmorphic facial features with a wide forehead and low ear position, with a height of 89.5 cm [-3 standard deviation (SD)] and a weight of 12.7 kg (-1.5 SD). His bilateral scrotum was empty, and the testicles were not touched. His limbs moved freely, with normal muscle tension of the limbs and no bone deformities were observed. No obvious abnormalities were detected during the auxiliary examination. Color Doppler echocardiography showed mitral insufficiency with mild regurgitation; a closing gap of ~0.23 cm was observed, as shown in Fig. 1A and B. The parents of the child were healthy, and stated that they were non-consanguineously married, and had no family history of short stature and CHD.

Figure 1 Clinical and mutation information of the patient. (A) Color doppler echocardiography showing a misaligned closing point mitral valve and a closing gap of 0.23 cm (as shown by the arrow). (B) Color doppler echocardiography showing the thickening of the mitral valve leaflets (arrow). (C) Results of genetic detection suggesting that exon 4 of TAB2 has frameshift mutation c.1056delC/p.Ser353fsTer8, and that the parents are wild-type, indicating a de novo mutation. TAB2, TGF-β-activated kinase 1 (MAP3K7) binding protein 2.

Genetic testing

The family was tested using trio-WES. The work described in the present case report was approved by the Ethics Committee of the Children's Hospital of Fudan University Anhui Hospital (Hefei, China), and informed consent was obtained from the parents of the patient. Approximately 3 ml of peripheral blood was collected from the proband as well as his parents, DNA was extracted using the genomic DNA extraction kit (Tiangen Biochemical Technology Inc.). A DNA library was constructed using the xGen Exome Research Panel V2 Kit (Integrated DNA Technologies Inc.). The paired-end sequencing (150 bp) was run on the Hiseq 4000 platform (Illumina Inc.). The off-line data were filtered for quality control and screened using the Burrows-Wheeler Aligner sequence alignment method (refer to hg19 version) (7). The target sequence mutation sites were identified using the GATK v4.1.9 software (8) and annotated according to the public variation databases (dbSNP build 155, www.ncbi.nlm.nih.gov/snp/; ExAC v0.3.1, https://gnomad.broadinstitute.org/; and 1000 Genomes, www.internationalgenome.org/). Multiple biohazard prediction software were used to analyze the suspected variation to the TAB2 gene (SIFT build 37, https://sift.bii.a-star.edu.sg/; Polyphen-2 v2.3, http://genetics.bwh.harvard.edu/pph2/; and MutationTaster 2021, www.mutationtaster.org/).

The results of the genetic testing for the child, but not his parents, revealed a heterozygous base deletion mutation TC>T in TAB2 exon 4, resulting in a frameshift mutation after encoded amino acid 352, NM_001369506: c.1056delC, (p.Ser353fsTer8), suggesting that the frameshift mutation observed may be a de novo mutation of TAB2. The mutation was not included in the public database. According to the guidelines of the American College of Medical Genetics and Genomics, the mutation is rated as pathogenic, and the evidence rating is PVS1+PM2+PM6(9). Sanger sequencing was performed using genomic DNA isolated from the blood of the patient and his parents. Direct sequencing was performed on the ABI 3730XL Automatic Sequencer (Thermo Fisher Scientific, Inc.). Amplification was performed using the following conditions: 1 cycle of 95˚C for 2 min, followed by 39 cycles of 95˚C for 30 sec, 60˚C for 30 sec, 72˚C for 40 sec and a final extension at 72˚C for 5 min. Sanger sequencing confirmed the existence of this mutation (Fig. 1C).

Plasmid construction and protein expression

The pcDNA3.1 vector (Tsingke Biotech) was used to construct a TAB2-wild-type (WT) plasmid using Phanta® Max Super-Fidelity DNA Polymerase (cat. no. P505; Vazyme Biotech Co., Ltd.), according to the manufacturer's instructions (amplification primers: Forward, 5'-CTTGGTACCGAGCTCGGATCCATGGCCCAAGGAAGCCAC-3'; and Reverse, 5'-TGCTGGATATCTGCAGAATTCTCAGAAATGCCTTGGCATCTC-3'). The TAB2-mutant (MUT) plasmid was constructed using a Mut Express MultiS Fast Mutagenesis Kit V2 (cat. no. C215; Vazyme Biotech Co., Ltd.) (amplification primer: Forward, 5'-CCAGCACTTCTCTTCAGTCAATAGCCAGACCTTAA-3'; and Reverse, 5'-ACTGAAGAGAAGTGCTGGAGGTTCGAGGTCCAG-3'). The amplification conditions were as follows: Pre-denaturation at 95˚C for 3 min, denaturation at 95˚C for 15 sec, annealing at 60˚C for 15 sec, extension at 72˚C for 1 min (35 cycles in total), and a final extension at 72˚C for 10 min. The plasmids were then transfected into human 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). After the cells were fully lysed in cell lysis buffer (Beyotime Institute of Biotechnology), 80 µg of protein was extracted and analyzed using western blotting (WB). Ultramicro spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration, and 10% SDS-PAGE gel (Epizyme, Inc.) was used to concentrate and isolate 80 µg of protein. After using polyvinylidene difluoride to transfer the film, a rapid sealing solution (cat. no. P0220; Beyotime Institute of Biotechology) was used at 20˚C for 15 min. The primary antibodies TAB2 (cat. no. A9867; ABclonal Biotech Co., Ltd.) and β-actin (cat. no. 3700; Cell Signaling Technology, Inc.) were used at a dilution ratio of 1:1,000. The secondary antibodies used were HRP goat anti-rabbit IgG (H+L) (cat. no. AS014; ABclonal Biotech Co., Ltd.), anti-mouse IgG and HRP-linked antibody (cat. no. 7076; Cell Signaling Technology, Inc.) at a dilution ratio of 1:5,000. The WB bands were analyzed using BeyoECL (cat. no. P0018FS; Beyotime Institute of Biotechnology) and ImageJ software (v1.80; National Institutes of Health), and GraphPad Prism 8 (GraphPad Software, Inc.) was used to conduct the statistical analyses and create the figures. Each experiment was repeated thrice. P<0.05 was considered to indicate a statistically significant difference.

WB experiment results showed that the protein expression in the TAB2-WT group was 2.48 times higher compared with that in the blank vector group (NC group) (P=0.0016), whereas no significant difference in the expression was observed in the TAB2-MUT group (P=0.57), indicating that the mutation led to the non-expression of TAB2 (Fig. 2).

Figure 2 Western blotting results showing that protein expression in the TAB2-WT group was significantly higher compared with that in the blank plasmid group. No significant expression was observed in the TAB2-MUT group (P=0.57). This indicates that the mutant TAB2 protein was not expressed, and the mutant Ser353fsTer8 may have led to the degradation of the TAB2 protein in vivo, resulting in an insufficient haploid dose and disease expression. **P<0.01. TAB2, TGF-β-activated kinase 1 (MAP3K7) binding protein 2; NC, negative control; WT, wild-type; MUT, mutation; ns, not significant.

Discussion

The present case study investigated a Chinese child with unexplained growth restriction and CHD. The height and weight of the patient in infancy were lower compared with those of his peers; however, his developmental motor milestones did not appear to be abnormal relative to his peers. Until 3-years old, there was obvious growth restriction. In addition, the patient had mild mitral valve prolapse and mild to moderate mitral regurgitation at the age of two. At the age of three, the patient still showed mitral insufficiency and semi moderate regurgitation as determined by color Doppler echocardiography. A novel frameshift mutation of TAB2, p.Ser353fsTer8, was discovered through genetic testing. The parents were WT at this locus; thus, the mutation identified may be a de novo mutation. In vitro function experiments showed that this mutation led to the non-expression of the TAB2 protein, suggesting that the heart structural defects and short stature may be caused by the insufficient haploid dosage of TAB2.

So far, to the best of our knowledge, a total of 62 cases related to TAB2 mutation in 31 families have been reported worldwide (3-6,10-16). The main clinical manifestations of these cases are valve abnormalities (such as valve stenosis and mitral or aortic insufficiency), dilated cardiomyopathy (DCM), connective tissue diseases (such as growth restriction and hyperactivity of joints in children) and abnormal facial features (increased forehead width, wider eye distances, ptosis and lower ear positions). It is worth noting that when CHTD2 is associated with TAB2 deficiency, it is a non-syndromic CHD by definition (3). However, statistical studies have demonstrated that <30% of affected patients show isolated CHD characteristics (10,11). The clinical phenotype spectrum of CHTD2 patients with the syndrome expressed is not scattered. Excluding cardiac structural abnormalities, the clinical phenotypes are mainly concentrated in connective/skeletal tissue and facial features, with a small sample of patients exhibiting hypotonia and hearing loss (11). The reported TAB2 mutation is primarily located in exon 2 (NM_015093), and the majority of the mutant types are loss of function mutations, such as nonsense or frameshift mutations (3-6,10-16). The phenotypical differences between individuals appear not to be associated with the mutation sites or types, and carriers within the same family may exhibit different phenotypes. For example, Westphal et al (10) reported that both the patient and his father carried the TAB2 mutation c.878del, but his father did not show physical characteristics. The patient in the present study was initially treated for growth restriction, and no other abnormal manifestations were revealed during the clinical examination, except for cardiac structural abnormalities and very slight facial abnormalities. The results of genetic testing suggested that CHDT2 is caused by the novel frameshift mutation of TAB2. Therefore, multidisciplinary consultation, in conjunction with cardiovascular medicine department is necessary; furthermore, improved methodologies for the growth abnormalities (such as short stature) should also be formulated.

TAB2 is located on autosomal 6q25.1, and the encoded TAB2 protein has 693 amino acids. TAB2 plays an important role in interleukin-1 signaling pathway and embryonic development of heart tissue (3). Although it is not clear how TAB2 defects lead to cardiac structural or functional abnormalities mainly involving valves, studies have demonstrated that TAB2 mutations or copy number deletions lead to insufficient haploid dose, reduce the binding ability with TAK1 (MAP3K7 coding) or TRAF6 protein, block TAK1 protein activation, lead to NF-κB signal error regulation and slow the necrosis of cardiomyocytes (1,17). In addition to regulating the NF-κB signaling pathway, Yin et al (18) proposed a new regulatory mechanism, suggesting that TAB2 protein plays an indispensable role in mediating TAK1-RIPK1 interaction. When the TAB2 protein is deleted, it can promote RIPK1 kinase activation and induce RIPK1 kinase dependent apoptosis by destroying TAK1-RIPK1 interaction. Therefore, different individuals may have early- or late-onset cardiac structure issues or dysfunctions, which may be associated with the activation of TAK1-RIPK1 or the NF-κB signaling pathway regulation mediated by TAB2 defects.

At present, there is no effective treatment for CHTD2 resulting in individuals with CHD, DCM or cardiac dysfunction. This necessitates targeted treatment for the cause of these diseases. Understanding the pathogenesis of the disease is important for developing treatment strategies. The mechanism of the aforementioned TAB2-related cardiac phenotype expression may be caused by promoting the activation of RIPK1 kinase (17,18). Researchers inactivated Ripk1 in a mouse model with the TAB2 gene knocked out; this largely reversed the pathological changes, such as severe cardiac contractile dysfunction, ventricular dilatation, and cardiac hypertrophy in mice (18). Inhibiting the RIPK1 protein or regulating the activity of the TAK1-RIPK1 pathway are potential strategies for the treatment of TAB2-related cardiac phenotypes (19). However, the effectiveness of growth hormone therapy on TAB2-related growth restriction requires further clinical study. The patient mentioned in this case report was treated using growth hormones, but longer-term follow-ups are required to ascertain its therapeutic value.

In summary, the present study reported a Chinese child with atypical facial features, short stature and CHD. The genetic testing results showed that the novel frameshift mutation of TAB2 caused CHTD2. Functional experiments in vitro showed that the mutant p.Ser353fsTer8 may lead to protein non-expression. The current study adds additional information on the TAB2 mutation spectrum and emphasizes that the occurrence of growth restriction and cardiac disorder may be associated with TAB2 defects. For patients with confirmed CHTD2, the present study recommends multidisciplinary consultation and regular follow-ups for abnormalities in the growth, development, hearing, vision and connective tissue of the patient. However, the feasibility of using human growth hormone to treat TAB2-related growth restriction still needs to be evaluated.

Acknowledgements

Not applicable.

Funding

Funding: The funding for this research was provided by the Scientific Research Fund of Anhui Medical University (grant no. 2019xkj080).

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to data privacy considerations but are available from the corresponding author on reasonable request. TAB2 mutation information from this study has been deposited in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/submitters/508986).

Authors' contributions

DQ, XW and YC designed the experiments. DQ, XW, XX, YW and YZ collected and analyzed the clinical data. XW, JG and YC were responsible for the protein experiments. XX, YW and YZ analyzed the genetic data. JG and YC confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the World Medical Association (Declaration of Helsinki) and approved by the Ethics Committee of the Children's Hospital of Fudan University Anhui Hospital (approval no. EYLL-2018-020).

Patient consent for publication

The parents of patients looked at in this case signed and provided informed consent for participation in the study.

Competing interests

The authors declare that they have no competing interests.

References