In this family, the father is of Han Chinese descent, while the mother belongs to the Miao ethnic minority group in China. The parents denied a consanguineous marriage and have a healthy daughter. No family history of genetic disorders was reported. The proband was a male fetus, the second child of the 26-year-old mother (gravida 5, para 2). He was diagnosed with CHD in utero at 25 weeks of gestation. Fetal echocardiography revealed several cardiac abnormalities, with a normal cardiac rate (144 bpm); these abnormalities included aortic stenosis with coarctation of the aortic arch (from aortic root diameter measuring 2.3 mm to an isthmus of 1.7 mm), mitral valve regurgitation with reduced mitral valve flow, a patent foramen ovale (left-to-right shunt), an enlarged left atrium (left 13 mm × 13 mm vs. right 11 mm × 11 mm), and an enlarged right ventricle (right 14 mm × 11 mm vs. left 15 mm × 8 mm) (Figures 1A,B). The mother did not terminate the pregnancy despite the obstetrician informing her of the risks. At 39 weeks of gestation, the obstetrician reported no other abnormalities except the CHD.

The proband was delivered with normal growth parameters (birth weight = 3,130 g, length = 50 cm) by cesarean section at 40 weeks of gestation due to severe preeclampsia and then transferred to neonatology for respiratory distress. Imaging examinations did n't reveal any significant developmental anomalies of the sternum, ribs, or spine, but they did reveal more severe CHD symptoms, including an atrial septal defect (3.9 mm), a patent ductus arteriosus aneurysm (DAA, 14 mm × 13 mm aneurysm), aortic stenosis, aortic arch dysplasia (3.8 mm), moderate mitral regurgitation, an enlarged right ventricle (right 30 mm × 15 mm vs. left 27 mm × 16 mm), and an enlarged left atrium (left 26 mm × 23 mm vs. right 23 mm × 19 mm) with reduced left ventricular systolic function (Figures 1C–E). On the ninth day of life, the proband was transferred to the neonatal intensive care unit (NICU) due to heart failure (N-terminal BNP >5,000 pg/ml, troponin = 2.450 μg/L), respiratory failure, gastrointestinal bleeding, and cardiogenic shock accompanied by metabolic disorders (hypoglycemia, hyperlactatemia, and hyperkalemia). Life support measures were initiated, including ventilator-assisted ventilation, milrinone injection, alprostadil injection, and nutritional support therapy. Although medical treatment improved his metabolic acidosis and internal hemorrhage, the proband unfortunately died prematurely within a month due to heart failure.

Karyotyping and chromosomal microarray analysis (CMA) did n't reveal any abnormalities during pregnancy. However, prenatal trio-WES of amniotic fluid identified compound heterozygosity in EVC2 gene, consisting of a paternal non-sense mutation (NM_147127.5: c. 2484G>A, p.W828Ter) and two maternal missense mutations (c.260A>G, p.E87G; c.650C>G, p.S217C) (Table 1, Figure 2A). These three EVC2 mutations were verified by Sanger sequencing (Figures 2B–D). The EVC2 p.W828Ter mutation has been indexed as pathogenic or likely pathogenic for EVCS (ClinVar: #553315), while the other two EVC2 mutations have n't been previously reported or indexed in the database. The EVC2 protein contains a glycosylation site at amino acid position 220 and an EVC2-like domain spanning amino acids 237–660. None of the three detected mutations are located within the known EVC2 protein functional domains. The non-sense mutation, EVC2 p.W828Ter, is located in exon 14 and is predicted to lead to non-sense-mediated mRNA decay (PVS1 criterion met). According to GnomAD, the allele frequency (AF) for p.W828Ter is 0.0003013 in the East Asian population (PM2_supporting criterion met), while the other two missense mutations have not been indexed (PM2_supporting). The non-sense mutation has been reported in trans with pathogenic variants (EVC2 p.R399X and c.871-2_894del) in two patients with EVCS (PM3 criterion met) (11, 12). Integrated analysis—including rare exome variant ensemble learner (REVEL), combined annotation dependent depletion (CADD), ClinPred, and Computational_evidence_ratio—suggests that p.E87G is more likely to be tolerable, whereas most pathogenicity predictions for p.S217C are detrimental (Table 1) (meeting the PP3 criterion). Thus, EVC2 p.W828Ter is classified as pathogenic (PVS1 + PM2_supporting + PM3), while EVC2 p.S217C (PM2_supporting + PM3 + PP3) and p.E87G (PM2_supporting + PM3) are classified as variants of uncertain significance (VUSs). Unfortunately, samples from the last three miscarriages were not preserved for further verification. Thus, it is supposed that the novel identified EVC2 genotype may have affected the cardiac development of the proband.

Trio-WES was performed using peripheral blood samples from the parents and amniotic fluid from the fetus. Genomic DNA was extracted from parental blood samples using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), while fetal DNA was extracted from the amniotic fluid. The sequencing libraries were prepared using the Agilent SureSelect Human All Exon v.6. After PCR amplification, the sequencing libraries were sequenced on the HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA). For all three individuals, the mean sequencing depth exceeded 175×, with more than 99.4% of variants covered at a depth greater than 20×.

Adapter sequences and low-quality reads were filtered out. The clean reads were then aligned to the human reference genome (GRCh37/hg19) using the Burrows–Wheeler Aligner (BWA). Variant calling was performed using the Genome Analysis Toolkit (GATK), followed by variant annotation using ANNOVAR. Candidate mutations were filtered based on AF (<1%), clinical phenotype, inheritance patterns, and pathogenicity classification.

Sanger sequencing was conducted to validate the candidate variants. The primer sequences used for amplification are the following: for EVC2 c. 2484G>A: forward 5′-TGCAGCAGGAGTGTTAGGTG-3′ and reverse 5′- CCTGCAGAACTCAGCCATGA-3′; for EVC2: c.260A>G, forward 5′-ACTGTGCACTAACGCTTCGG-3′ and reverse 5′-TTCACACTTCTGGATGAAAGTGC-3′; for EVC2: c.650C>G, forward 5′-ACATGCCTGACCCAGAACAC-3′ and reverse 5′-CGTGCTACCCTCCTTCTTCC-3′. Sequencing was performed using an ABI 3130 Genetic Analyzer (Applied Biosystems, CA, USA).