Clinical information
The study was approved by the institutional review board of Jiangxi maternal and child health hospital, Nanchang, China. All the enrolled subjects provided written informed consent. A family from the Jiangxi province, China, comprising 25 family members (11 males, 12 females, and 2 fetuses) across three generations (Fig. 1a) were included in the study. Our proband (II:5) was a 31-year-old female who was 15 weeks pregnant at her first visit to our hospital. She was slightly tachypneic and reported occasional fatigue since the age of 25 years old. She performed a transthoracic color echocardiogram examination at 30 years old and revealed mild aortic valve stenosis accompanied with mild mitral, tricuspid, and aortic regurgitation. She denied any surgical or pharmaceutical interventions. Her husband was 32 years old (II:6) and declared no cardiac symptoms or family history of heart diseases. Their first child (III:7) was a CHD girl with left and right ventricles dilatation. The girl then experienced sudden cardiac death (SCD) at 2 years of age. The second child (III:8) was detected with no heartbeat at 9 weeks of pregnancy using a Doppler fetal monitor, and subsequently, the pregnancy was terminated. The third child (III:9) was a 2-year-old girl with no symptoms of cardiac disorders. She was not diagnosed with any heart abnormalities using echocardiography.
The proband’s other family members were interviewed, and cardiac symptoms, such as tachypnea, shortness of breath, and fatigue, were reported. Four females and one male were identified with clinical symptoms (Fig. 1a). The proband’s mother (I:2) died in her late fifties due to sudden cardiac arrhythmia. Evident atrial septal aneurysms were detected in the proband’s sisters (II:1, II:7, and II:9) using echocardiography. Left coronary artery dilation was screened in the patient II:1 by echocardiography and then identified using coronary arteriography. Other cardiac anomalies, such as mild mitral valves prolapse with mitral and pulmonic regurgitation, and left atrial and ventricular dilatation, were found in II:7 and II:9, respectively. III:11 was a 3-year-old boy who was diagnosed with mild left ventricular and right atrial dilation as well as mild mitral valves prolapse with mitral regurgitation using echocardiography. Transabdominal fetal echocardiography was performed on the proband’s fetus (III:10) at 15 weeks of pregnancy, and no cardiac abnormality was detected. The heart defects in the individuals enrolled in this study are summarized in Table 1.
Mutation detection
An autosomal dominant inheritance pattern was suggested on the basis of vertical transmission of CHD in the family. Peripheral blood was obtained from 13 family members (II:1, II:3, II:5, II:6, II:7, II:8, II:9, III:3, III:4, III:5, III:9, III:11, and III:13). Genomic DNA was extracted from the peripheral blood lymphocytes using the QIAamp DNA blood mini kit (Qiagen).
To determine the causative mutation in the family, WES of the proband (II:5) was performed as previously described with minor modifications [6]. Two micrograms of the genomic DNA from the proband II:5 was used for human whole-exome analysis with paired-end-sequencing at 100× resolution. Libraries were constructed using the SureSelect Human All ExonV7 kit (Agilent Technologies, USA) and sequenced on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA), as per the manufacturer’s instructions. The reads were aligned to the human reference genome (University of California Santa Cruz, UCSC hg19) using SOAPaligner. Single-nucleotide polymorphism (SNP) and indel (insertion or deletion) identification was performed using SAMtools and/or the Genome Analysis Toolkit (GATK), and SNPs with a read depth > 4 and quality > 20 were used for subsequent analyses. SNPs and indels were annotated using SeattleSeq annotation. Known polymorphisms in the dbSNP (https://www.ncbi.nlm.nih.gov/snp/) (minor allele frequency, > 0.01) and 1000 genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) (genotype frequency, > 0.005) databases as well as synonymous single-nucleotide variants and variants not located in exonic or splicing regions were excluded. Pathogenicity of the obtained variants was predicted using Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml) [7], SIFT (https://sift.bii.a-star.edu.sg/) [8], and MutationTaster (http://www.mutationtaster.org/) [9].
Approximately 99.71% of the sequencing reads were mapped to the human genome hg19, with mean 186.76× sequencing depth. A heterozygous variant in TAB2 (c.C446G, NM_015093.5), which results in a premature stop codon with early termination of protein translation at residue 149 (p.S149X), passed the filtering criteria. Using Sanger sequencing, this variant was detected in all the affected family members (II:1, II:5, II:7, II:9, and III:11); it was absent in the unaffected individuals (II:3, II:6, II:8, III:3, III:4, III:5, III:9, and III:13) (Fig. 1b). The variant, with no record in 1000 genomes, the Exome Variant Server (EVS; http://evs.gs.washington.edu/EVS/), the Genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/), and the HGMD databases, was not detected in our 531 control cohorts using high-resolution melting analysis (SsoFast EvaGreen, Bio-Rad). TAB2 protein sequences (from Danio rerio to Homo sapiens) were obtained from the protein database of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). Alignments of TAB2 protein family members using cluster 2.0 software [10] revealed that the affected amino acid is evolutionarily conserved (Fig. 2). According to the latest recommendation by ACMG and AMP on sequence variants interpretation, the c.C446G variant in TAB2 would be classified as “pathogenic”, having met the requirements of fulfilling one very strong (PVS1), one moderate (PM2) and three supporting (PP1, PP3 and PP4) criteria (Fig. 3) [5].
A prenatal molecular genetic diagnosis for the fetus (III:10) was recommended for evaluating the risk for CHD. With complete informed consent of the proband (II:5) and her husband (II:6), a prenatal diagnosis of the fetus (III:10) was performed at 19 weeks of pregnancy. The amniocytes was obtained by amniocentesis, and fetal genomic DNA (III:10) was extracted using the QIAamp DNA mini kit (Qiagen). A heterozygous TAB2 c. C446G variant was detected, indicating that the fetus had a high risk for CHD.