Congenital heart defects (CHD), as the most common congenital anomaly, have been reported to be frequently associated with pathogenic copy number variants (CNVs). Currently, patients with CHD are routinely offered chromosomal microarray (CMA) testing, but the diagnostic yield of CMA on CHD patients has not been extensively evaluated based on a large patient cohort. In this study, we retrospectively assessed the detected CNVs in a total of 514 CHD cases (a 422-case clinical cohort from Boston Children's Hospital (BCH) and a 92-case research cohort from Shanghai Children’s Medical Center (SCMC)) and conducted a genotype-phenotype analysis. Furthermore, genes encompassed in pathogenic/likely pathogenic CNVs were prioritized by integrating several tools and public data sources for novel CHD candidate gene identification. Based on the BCH cohort, the overall diagnostic yield of CMA testing for CHD patients was 12.8(pathogenic CNVs)-18.5% (pathogenic and likely pathogenic CNVs). The diagnostic yield of CMA for syndromic CHD was 14.1-20.6% (excluding aneuploidy cases), whereas the diagnostic yield for isolated CHD was 4.3-9.3%. Four recurrent genomic loci (4q terminal region, 15q11.2, 16p12.2 and Yp11.2) were more significantly enriched in cases than in controls. These regions are considered as novel CHD loci. We further identified 20 genes as the most likely novel CHD candidate genes through gene prioritization analysis. The high clinical diagnostic yield of CMA in this study provides supportive evidence for CMA as the first-line genetic diagnostic tool for CHD patients. The CNVs detected in our study suggest a number of CHD candidate genes that warrant further investigation.

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute. We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children’s Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P < 0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes. CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions without known CHD genes suggests unknown CHD genes within these intervals. Here, we have shown that knockdown of SORBS2, a 4q interval gene, disrupted sarcomeric integrity of cardiomyocytes and caused reduced cardiomyocyte number in human embryonic stem cell differentiation model. Molecular analyses revealed decreased expression of second heart field (SHF) marker genes and impaired NOTCH and SHH signaling in SORBS2-knockdown cells. Exogenous SHH rescued SORBS2 knockdown-induced cardiomyocyte differentiation defects. Sorbs2^-/- mouse mutants had atrial septal hypoplasia/aplasia or double atrial septum (DAS) derived from impaired posterior SHF with a similar expression alteration. Rare SORBS2 variants were significantly enriched in a cohort of 300 CHD patients. Our findings indicate that SORBS2 is a regulator of SHF development and its variants contribute to CHD pathogenesis. The presence of DAS in Sorbs2^-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.

Congenital heart disease (CHD) is the most prevalent type of birth defect in human, with high morbidity in infant. Several genes essential for heart development have been identified. GATA4 is a pivotal transcription factor that can regulate the cardiac development. Many GATA4 mutations have been identified in patients with different types of CHD. In this study, the NKX2-5, HAND1 and GATA4 coding regions were sequenced in a family spanning three generations in which seven patients had CHD. Disease-causing potential variation in this family was evaluated by bioinformatics programs and the transcriptional activity of mutant protein was analyzed by the dual luciferase reporter assay. A novel GATA4 mutation, c.C931T (p.R311W), was identified and co-segregated with the affected patients in this family. The bioinformatics programs predicted this heterozygous mutation to be deleterious and the cross-species alignment of GATA4 sequences showed that the mutation occurred within a highly conserved amino acid. Even though it resided in the nuclear localization signal domain, the mutant protein didn’t alter its intracellular distribution. Nevertheless, further luciferase reporter assay demonstrated that the p.R311W mutation reduced the ability of GATA4 to activate its downstream target gene. Our study identified a novel mutation in GATA4 that likely contributed to the CHD in this family. This finding expanded the spectrum of GATA4 mutations and underscored the pathogenic correlation between GATA4 mutations and CHD.

An aqueous two-phase system (ATPS) consisting of poly(ethylene glycol-ran-propylene glycol) monobutyl ether (UCON)/phosphate was developed for the separation of the antioxidant peptides from pepsin hydrolysate of Whey Protein Isolate (WPI). The efficiency of the separation was evaluated based on the DPPH radical scavenging activity, ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP) of the separated peptides. The effects of some parameters on the partition of antioxidant peptides were investigated. An efficient separation of antioxidant peptides was achieved using ATPS with pH of 4.0, 4 mL of UCON solution (40%, w/w), 4 mL of KH₂PO₄ solution (15.5%, w/w), 2 mL of WPI hydrolysate and 0.40 g/10 mL of NaCl. Reversed-phase high-performance liquid chromatography (RP-HPLC), amino acid analyzer and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the purified peptides separated by the ATPS. The peptides in top phase were less polar than those in bottom phase. More antioxidative and hydrophobic amino acids were extracted to the top phase of ATPS, and the peptides with the amino acid sequences with antioxidant activities moved to the top phase as well. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by UCON/phosphate ATPS.

Polydactyly and syndactyly are congenital limb malformations that may occur either as non-syndromic or syndromic forms. In the present study, massively parallel sequencing was performed on a proband in a four-generation family with polydactyly and syndactyly to identify disease-causing variant(s). A pathogenic variant c.739C > T (p.Gln247^∗) in the glioma-associated oncogene family zinc finger 3 (GLI3) gene was identified and co-segregated with the affected members of the family. Firstly, we examined GLI3 mRNA and GLI3 protein levels in peripheral blood mononuclear cells (PBMCs) of patients carrying this variant. The results showed that the truncated GLI3 p.Gln247^∗ (c.739C > T) protein was detectable in patients and the GLI3 transcript and protein levels were not significantly altered in the PBMCs of patients compared with healthy controls. Furthermore, functional analysis showed that the truncated GLI3 p.Gln247^∗ (c.739C > T) protein variant could lead to cytoplasmic accumulation of mutant protein and loss of ability to bind to the Suppressor of Fused protein. Alterations in protein expression levels of core components of the Sonic hedgehog signaling pathway were also observed. Our study shows that this novel GLI3 variant contributes to the malformations in this family and provides evidence for the mechanism by which GLI3 c.739C > T (p.Gln247^∗) was implicated in the pathogenesis of polydactyly and syndactyly.