Non-syndromic Congenital Heart Disease Research Articles

Expanding the phenotypic spectrum of NOTCH1 variants: clinical manifestations in families with congenital heart disease.

Pathogenic variants in NOTCH1 are associated with non-syndromic congenital heart disease (CHD) and Adams-Oliver syndrome (AOS). The clinical presentation of individuals with damaging NOTCH1 variants is characterized by variable expressivity and incomplete penetrance; however, data on systematic phenotypic characterization are limited. We report the genotype and phenotype of a cohort of 33 individuals (20 females, 13 males; median age 23.4 years, range 2.5-68.3 years) from 11 families with causative NOTCH1 variants (9 inherited, 2 de novo; 9 novel), ascertained from a proband with CHD. We describe the cardiac and extracardiac anomalies identified in these 33 individuals, only four of whom met criteria for AOS. The most common CHD identified was tetralogy of Fallot, though various left- and right-sided lesions and septal defects were also present. Extracardiac anomalies identified include cutis aplasia (5/33), cutaneous vascular anomalies (7/33), vascular anomalies of the central nervous system (2/10), Poland anomaly (1/33), pulmonary hypertension (2/33), and structural brain anomalies (3/14). Identification of these findings in a cardiac proband cohort supports NOTCH1-associated CHD and NOTCH1-associated AOS lying on a phenotypic continuum. Our findings also support (1) Broad indications for NOTCH1 molecular testing (any familial CHD, simplex tetralogy of Fallot or hypoplastic left heart); (2) Cascade testing in all at-risk relatives; and (3) A thorough physical exam, in addition to cardiac, brain (structural and vascular), abdominal, and ophthalmologic imaging, in all gene-positive individuals. This information is important for guiding the medical management of these individuals, particularly given the high prevalence of NOTCH1 variants in the CHD population.

Association of MTR gene polymorphisms with the occurrence of non-syndromic congenital heart disease: a case–control study

To exhaustively explore the association of infant genetic polymorphisms of methionine synthase (MTR) gene with the risk of non-syndromic congenital heart disease (CHD). A hospital-based case–control study involving 620 CHD cases and 620 health controls was conducted from November 2017 to March 2020. Eighteen SNPs were detected and analyzed. Our date suggested that the genetic polymorphisms of MTR gene at rs1805087 (GG vs. AA: aOR = 6.85, 95% CI 2.94–15.96; the dominant model: aOR = 1.77, 95% CI 1.35–2.32; the recessive model: aOR = 6.26, 95% CI 2.69–14.54; the addictive model: aOR = 1.81, 95% CI 1.44–2.29) and rs2275565 (GT vs. GG: aOR = 1.52, 95% CI 1.15–1.20; TT vs. GG: aOR = 4.93, 95% CI 1.93–12.58; the dominant model: aOR = 1.66, 95% CI 1.27–2.17; the recessive model: aOR = 4.41, 95% CI 1.73–11.22; the addictive model: aOR = 1.68, 95% CI 1.32–2.13) were significantly associated with the higher risk of CHD. And three haplotypes of G-A-T (involving rs4659724, rs95516 and rs4077829; OR = 5.48, 95% CI 2.58–11.66), G-C-A-T-T-G (involving rs2275565, rs1266164, rs2229276, rs4659743, rs3820571 and rs1050993; OR = 0.78, 95% CI 0.63–0.97) and T-C-A-T-T-G (involving rs2275565, rs1266164, rs2229276, rs4659743, rs3820571 and rs1050993; OR = 1.60, 95% CI 1.26–2.04) were observed to be significantly associated with risk of CHD. Our study found that genetic polymorphisms of MTR gene at rs1805087 and rs2275565 were significantly associated with higher risk of CHD. Additionally, our study revealed a significant association of three haplotypes with risk of CHD. However, the limitations in this study should be carefully taken into account. In the future, more specific studies in different ethnic populations are required to refine and confirm our findings.Trial registration: Registration number: ChiCTR1800016635; Date of first registration: 14/06/2018.

Copy number variation-associated lncRNAs may contribute to the etiologies of congenital heart disease

Copy number variations (CNVs) have long been recognized as pathogenic factors for congenital heart disease (CHD). Few CHD associated CNVs could be interpreted as dosage effect due to disruption of coding sequences. Emerging evidences have highlighted the regulatory roles of long noncoding RNAs (lncRNAs) in cardiac development. Whereas it remains unexplored whether lncRNAs within CNVs (CNV-lncRNAs) could contribute to the etiology of CHD associated CNVs. Here we constructed coexpression networks involving CNV-lncRNAs within CHD associated CNVs and protein coding genes using the human organ developmental transcriptomic data, and showed that CNV-lncRNAs within 10 of the non-syndromic CHD associated CNVs clustered in the most significant heart correlated module, and had highly correlated coexpression with multiple key CHD genes. HSALNG0104472 within 15q11.2 region was identified as a hub CNV-lncRNA with heart-biased expression and validated experimentally. Our results indicated that HSALNG0104472 should be a main effector responsible for cardiac defects of 15q11.2 deletion through regulating cardiomyocytes differentiation. Our findings suggested that CNV-lncRNAs could potentially contribute to the pathologies of a maximum proportion of 68.4% (13/19) of non-syndromic CHD associated CNVs. These results indicated that explaining the pathogenesis of CHD associated CNVs should take account of the noncoding regions.

Association between placental DNA methylation and fetal congenital heart disease.

Congenital heart disease (CHD) is a worldwide problem with high morbidity and mortality. Early diagnosis of congenital heart disease is still a challenge in clinical work. In recent years, few studies indicated that placental methylation may be predictors of CHD. More studies are needed to confirm the association between placental methylation and CHD. The aim of this study was to investigate the association between prenatal placental DNA methylation and CHD. Placental tissues were obtained from four fetuses during the second trimester with isolated, non-syndromic congenital heart disease, including three cases with double outlet right ventricle (DORV) and one case with tetralogy of Fallot (TOF), and four unaffected fetuses as controls. The Illumina Infinium Human Methylation 850K BeadChip assay was employed to identify differential methylation sites (DMSs) and differential methylation regions (DMRs). Differential methylation was evaluated by comparing the β-values for individual CpG loci in cases vs. controls. In addition, the function of genes was assessed through KEGG enrichment analysis, Gene Ontology (GO) analysis and KEGG pathway analysis. Compared with the control group, we identified 9625 differential methylation genes on 26,202 DMSs (p < 0.05), of which 6997 were hyper-methylation and 2628 were hypo-methylation. The top 30 terms of GO biological process and KEGG enrichment analysis of DMSs were connected with multiple important pathways of heart development and disease. Ten differentially methylated regions and the genes related to DMRs, such as TLL1, CRABP1, FDFT1, and PCK2, were identified. The deformity caused by the loss of function of these genes is remarkably consistent with the clinical phenotype of our cases. The DNA methylation level of placental tissue is closely associated with fetal congenital heart disease.

Neuropsychological features in children and adults with congenital heart disease: an exploratory data analysis

ABSTRACT We aimed to recognize clinically meaningful patterns among patients with congenital heart disease to support clinical decision-making and better classification in practice. This research was a secondary analysis of data from the Congenital Heart Disease Genetic Network Study conducted from December 2010 to November 2014 in the United States. The analytic dataset included 6002 patients ≥1 year of age with non-syndromic congenital heart disease. For each patient, features included demographic, clinical, maternal and paternal characteristics. We clustered patients to identify subgroups that shared similarities in their clinical features. The performance of the clustering algorithm was evaluated with a random forest. Next, we used the apriori algorithm to generate clinical rules from patients’ characteristics. The clustering algorithm identified two discernible groups of patients. The two classes of patients were different in maternal diabetes and in neuropsychological indicators [Accuracy (95% CI) = 97.1% (96.2, 97.8), area under the ROC curve = 96.8%]. Our rule extraction suggested the presence of clinical pictures with high lift values among patients with maternal diabetes or with seizure, depression, attention-deficit hyperactivity disorder, anxiety, developmental delay, learning disability and speech problem. Beyond the age of 1 year, maternal diabetes and neuropsychological characteristics identify two clusters of patients with congenital heart disease. These characteristics have the potential of being incorporated into the current systems for the classification of congenital heart disease.

Investigation of Copy Number Variation in South African Patients With Congenital Heart Defects.

Congenital heart disease (CHD) is a leading non-infectious cause of pediatric morbidity and mortality worldwide. Although the etiology of CHD is poorly understood, genetic factors including copy number variants (CNVs) contribute to the risk of CHD in individuals of European ancestry. The presence of rare CNVs in African CHD populations is unknown. This study aimed to identify pathogenic and likely pathogenic CNVs in South African patients with CHD. Genotyping was performed on 90 patients with nonsyndromic CHD using the Affymetrix CytoScan HD platform. These data were used to identify large, rare CNVs in known CHD-associated genes and candidate genes. We identified eight CNVs overlapping known CHD-associated genes (GATA4, CRKL, TBX1, FLT4, B3GAT3, NSD1) in six patients. The analysis also revealed CNVs encompassing five candidate genes likely to play a role in the development of CHD (DGCR8, KDM2A, JARID2, FSTL1, CYFIP1) in five patients. One patient was found to have 47, XXY karyotype. We report a total discovery yield of 6.7%, with 5.6% of the cohort carrying pathogenic or likely pathogenic CNVs expected to cause the observed phenotypes. In this study, we show that chromosomal microarray is an effective technique for identifying CNVs in African patients diagnosed with CHD and have demonstrated results similar to previous CHD genetic studies in Europeans. Novel potential CHD genes were also identified, indicating the value of genetic studies of CHD in ancestrally diverse populations.

SLC2A3 variants in familial and sporadic congenital heart diseases in a Chinese Yunnan population.

Solute carrier family 2member 3 (SLC2A3), is a member of a superfamily of transport protein genes. SLC2A3 played an important role in embryonic development. Previous research reported SLC2A3 duplication was reportedly associated with congenital syndromic heart defects. However, it is not clear whether the gene is associated with non-syndromic congenital heart disease. Our study aimed to elucidate the relationship between its variation and congenital heart disease. Genomic DNA extracted from the peripheral blood leukocytes of two families with CHD were sequenced with whole-exome sequencing to identify variations, used Sanger sequencing to investigate SLC2A3 variants in 494 Chinese patients with CHD and 576healthy unrelated individuals. In members from the two families, three with CHD had the SLC2A3 (rs3931701) C>T variant. Of the 494 patients with CHD, 394had gene variants (86had the TT type and 308had the CT type). Of the 576healthy controls, 272 participants had gene variants (36had the TT type and 236had the CT type). The TT type [p<0.0001, odds ratio (OR) =7.262, 95% confidence interval (CI) =4.631-11.388] and CT type (p<0.0001, OR =3.967, 95% CI =2.991-5.263) of SLC2A3 (rs3931701) significantly increased the risk of sporadic ASD in a Chinese Yunnan population. Single nucleotide variations of SLC2A3, particularly, the SLC2A3 (rs3931701) C>T variant increased the risk of CHD among the studied population.

Functional analysis of novel genetic variants of NKX2-5 associated with nonsyndromic congenital heart disease.

NKX2-5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N-terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine-rich domain (p.P247A). Variant-p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N-terminal variants-p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT-PCR. Nonetheless, variant p.R95L affected the synergy of NKX2-5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes-Irx4, Mef2c, Bmp10, Myh6, Myh7, and Myocd is also observed in response to NKX2-5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N-terminal variants supports the emerging role of this region during cardiac-developmental pathways.

The Predisposing Risk Factors for Non-syndromic Congenital Heart Disease: A Case-Control Study

Objectives: Despite the importance of congenital heart disease (CHD) in the population, the risk factors predisposing infants to be born with non-syndromic CHD are not well understood yet. Materials and Methods: In a case-control study, we recruited consecutive 109 infants with non-syndromic CHD who were referred to our pediatric cardiac care clinic and then compared them with 117 infants without CHD (2015-2016). Paternal, maternal, and neonatal demographic information, maternal past medical history, and antenatal history were recorded for each child. To investigate the potential risk factors for developing CHD, infants with and without CHD were compared in terms of study variables. In a second analysis, preterm infants were excluded and term infants were compared regarding the study variables. Results: The findings revealed that higher maternal and paternal education were both associated with a lower risk of CHD (odds ratio [OR]: 0.47, 95% confidence interval [CI]: 0.24-0.93, P=0.031, and OR: 0.45, 95% CI: 0.23-0.89, P=0.023, respectively). The family history of CHD in the first- or second-degree relatives of infants was significantly associated with CHD (OR: 3.56, 95% CI: 1.35-9.40, P=0.007). Several parameters were more prevalent in the CHD group, including having lower birth weight, having preterm birth, being the fourth birth order or higher, and not receiving maternity care under the supervision of a gynecologist. However, higher birth order and lower birth weight were not associated with CHD in exclusively term infants. Finally, a higher maternal educational level was related to lower CHD in term infants even after adjusting for a family history of CHD and preconception diabetes mellitus (OR: 5.45, 95% CI: 1.71-17.37, P=0.004). Conclusions: Our study findings demonstrate the need for a more enhanced primary care program, especially in patients with poor financial status and a family history of CHD.

A Non-coding HES1 Variant Predisposes Children to Congenital Heart Disease in Chinese Population.

Background: As a key component in the NOTCH signaling pathway, HES1 plays an important role in vertebrate heart development. Variants in the HES1 coding sequence are known to be associated with congenital heart disease (CHD). However, little is known about HES1 non-coding sequence variants and their association with the risk of developing CHD.Method and Results: We initially analyzed the non-coding sequence of the HES1 gene in 12 unrelated CHD families by direct sequencing and identified a previously unreported promoter region variant (NM_005524.4: c.−1279−1278 insAC, rs148941464) in the HES1 gene in four CHD families. The homozygous variant in patients was inherited from carrier parents with normal phenotypes, indicating a likely recessive genetic model. Given that the HES1 gene is predicted to be likely to exhibit haploinsufficiency (%HI: 11.44), we hypothesized that the HES1 homozygous variant is a genetic risk factor underlying CHD. We then carried out sequencing of this HES1 variant in 629 sporadic non-syndromic CHD cases and 696 healthy controls and performed association analysis. Interestingly, we observed a significant association of the homozygous HES1 promoter variant with CHD (18.92% of cases vs. 9.91% of controls; OR: 2.291, 95% CI: 1.637-3.207, p = 9.72 × 10−7). No significant association with CHD was observed for the HES1 promoter heterozygous variant (p > 0.05). However, association analysis tests of the HES1 homozygous variant with each subtype of CHD revealed that this homozygous variant was strongly associated with transposition of the great arteries (TGA) (OR: 3.726, 95% CI: 1.745-7.956, p = 0.0003). Moreover, the prevalence of HES1 homozygous variants in CHD patients with TGA (27.66%) was significantly higher than that in patients with other CHD subtypes or controls. Similar results were observed in a replication group of TGA (n = 64). Functional studies demonstrated that the homozygous variant in the HES1 promoter can disrupt its ability to bind RXRA, an inhibitory transcription factor, which results in abnormally high expression of the HES1 gene, indicating that this variant harbors gain-of-function effects.Conclusions: Our findings reveal that the non-coding homozygous variant in the HES1 promoter has a gain-of-function effect and is associated with an increased risk of CHD development, especially the severe TGA subtype.

Genetic Etiology of Left-Sided Obstructive Heart Lesions: A Story in Development.

Congenital heart disease is the most common congenital defect observed in newborns. Within the spectrum of congenital heart disease are left‐sided obstructive lesions (LSOLs), which include hypoplastic left heart syndrome, aortic stenosis, bicuspid aortic valve, coarctation of the aorta, and interrupted aortic arch. These defects can arise in isolation or as a component of a defined syndrome; however, nonsyndromic defects are often observed in multiple family members and associated with high sibling recurrence risk. This clear evidence for a heritable basis has driven a lengthy search for disease‐causing variants that has uncovered both rare and common variants in genes that, when perturbed in cardiac development, can result in LSOLs. Despite advancements in genetic sequencing platforms and broadening use of exome sequencing, the currently accepted LSOL‐associated genes explain only 10% to 20% of patients. Further, the combinatorial effects of common and rare variants as a cause of LSOLs are emerging. In this review, we highlight the genes and variants associated with the different LSOLs and discuss the strengths and weaknesses of the present genetic associations. Furthermore, we discuss the research avenues needed to bridge the gaps in our current understanding of the genetic basis of nonsyndromic congenital heart disease.

An update on the CHDGKB for the systematic understanding of risk factors associated with non-syndromic congenital heart disease

The Congenital Heart Disease Genetic Knowledge Base (CHDGKB) was established in 2020 to provide comprehensive knowledge about the genetics and pathogenesis of non-syndromic CHD (NS-CHD). In addition to the genetic causes of NS-CHD, environmental factors such as maternal drug use and gene-environment interactions can also lead to CHD. There is a need to integrate this information into a platform for clinicians and researchers to better understand the overall risk factors associated with NS-CHD. The updated CHDGKB contains the genetic and non-genetic risk factors from over 4200 records from PubMed that was manually curated to include the information associated with NS-CHD. The current version of CHDGKB, named CHD-RF-KB (KnowledgeBase for non-syndromic Congenital Heart Disease-associated Risk Factors), is an important tool that allows users to evaluate the recurrence risk and prognosis of NS-CHD, to guide treatment and highlight the precautions of NS-CHD. In this update, we performed extensive functional analyses of the genetic and non-genetic risk information in CHD-RF-KB. These data can be used to systematically understand the heterogeneous relationship between risk factors and NS-CHD phenotypes.

MLPA � first-tier Screening Assay in Newborns with Nonsyndromic Congenital Heart Disease

Congenital heart disease(CHD) is the most frequent malformative pathology seen in newborns, with an incidence of 10/1000 births, and is considered a major cause of neonatal morbidity and mortality. About one third of congenital heart disease cases are of genetic origin, particular copy number variations being described as possible nonsyndromic and syndromic congenital heart disease causes. Here, we set out to find whether the MLPA technique could be used as a first-tier screening assay in newborns with apparently nonsyndromic CHDs, and thus to genetically confirm the CHD diagnosis. The study cohort included 60 newborns diagnosed with apparently nonsyndromic congenital heart disease, recruited for a period of 18 months. MLPA analysis was performed using the SALSA MLPA P311 and P250 kits. 10 newborns (16.67%) showed known genetically relevant copy number variations, namely three patients with 22q11.21 deletion, that were diagnosed with DiGeorge syndrome, and seven patients with a probable single exon 8p23.1 duplication that will be subjected to further molecular testing, in order to correctly assess their diagnosis. We can conclude that the screening of patients with apparently nonsyndromic congenital heart disease may lead to their early and correct diagnosis, and thus them benefitting from the detection of clinically relevant copy number variations using the MLPA technique.

Down-regulation of the insulin signaling pathway by SHC may correlate with congenital heart disease in Chinese populations.

Congenital heart disease (CHD) is one of the most common and severe congenital defects. The incidence of fetal cardiac malformation is increased in the context of maternal gestational diabetes mellitus (GDM). Therefore, we wanted to determine whether abnormalities in the insulin signaling pathway are associated with the occurrence of nonsyndromic CHD (ns-CHD). We used digital gene expression profiling (DGE) of right atrial myocardial tissue samples from eight ns-CHD patients and four controls. The genes potentially associated with CHD were validated by real-time fluorescence quantitative PCR analysis of right atrial myocardial tissues from 37 patients and 10 controls and the H9C2 cell line. The results showed that the insulin signaling pathway, which is mediated by the SHC gene family, was inhibited in the ns-CHD patients. The expression levels of five genes (PTPRF, SHC4, MAP2K2, MKNK2, and ELK1) in the pathway were significantly down-regulated in the patients' atrial tissues (P<0.05 for all). In vitro, the H9C2 cells cultured in high glucose (33 mmol/l) expressed less SHC4, MAP2K2, and Elk-1 than those cultured in low glucose (25 mmol/l). Furthermore, the high glucose concentration down-regulated the 25 genes associated with blood vessel development based on Gene Ontology (GO) term enrichment analyses of RNA-seq data. We considered that changes in the insulin signaling pathway mediated by SHC might be involved in the heart development process. This mechanism might account for the increase in the incidence of fetal cardiac malformations in the context of GDM.

CHDGKB: a knowledgebase for systematic understanding of genetic variations associated with non-syndromic congenital heart disease.

Congenital heart disease (CHD) is one of the most common birth defects, with complex genetic and environmental etiologies. The reports of genetic variation associated with CHD have increased dramatically in recent years due to the revolutionary development of molecular technology. However, CHD is a heterogeneous disease, and its genetic origins remain inconclusive in most patients. Here we present a database of genetic variations for non-syndromic CHD (NS-CHD). By manually literature extraction and analyses, 5345 NS-CHD-associated genetic variations were collected, curated and stored in the public online database. The objective of our database is to provide the most comprehensive updates on NS-CHD genetic research and to aid systematic analyses of pathogenesis of NS-CHD in molecular level and the correlation between NS-CHD genotypes and phenotypes. Database URL: http://www.sysbio.org.cn/CHDGKB/

Integration of Large-Scale Genomic Data Sources With Evolutionary History Reveals Novel Genetic Loci for Congenital Heart Disease.

Most cases of congenital heart disease (CHD) are sporadic and nonsyndromic, with poorly understood etiology. Rare genetic variants have been found to affect the risk of sporadic, nonsyndromic CHD, but individual studies to date are of only moderate sizes, and none to date has incorporated the ohnolog status of candidate genes in the analysis. Ohnologs are genes retained from ancestral whole-genome duplications during evolution; multiple lines of evidence suggest ohnologs are overrepresented among dosage-sensitive genes. We integrated large-scale data on rare variants with evolutionary information on ohnolog status to identify novel genetic loci predisposing to CHD. We compared copy number variants present in 4634 nonsyndromic CHD cases derived from publicly available data resources and the literature, and >27 000 healthy individuals. We analyzed deletions and duplications independently and identified copy number variant regions exclusive to cases. These data were integrated with whole-exome sequencing data from 829 sporadic, nonsyndromic patients with Tetralogy of Fallot. We placed our findings in an evolutionary context by comparing the proportion of vertebrate ohnologs in CHD cases and controls. Novel genetic loci in CHD cases were significantly enriched for ohnologs compared with the genome (χ2 test, P<0.0001, OR =1.253 [95% CI, 1.199-1.309]). We identified 54 novel candidate protein-coding genes supported by both: (1) copy number variant and whole-exome sequencing data; and (2) ohnolog status. We have identified new CHD candidate loci, and show for the first time that ohnologs are overrepresented among CHD genes. Incorporation of evolutionary metrics may be useful in refining candidate genes emerging from large-scale genetic evaluations of CHD.

EPAS 1, congenital heart disease, and high altitude: disclosures by genetics, bioinformatics, and experimental embryology.

The high-altitude environment is a challenge for human settlement. Low oxygen concentrations, extreme cold, and a harsh arid climate are doubtlessly challenges for the colonization of the Tibetan plateau. I am delighted to comment on the article of Pan et al. (2018) on mutations in endothelial PAS domain-containing protein 1 (EPAS1) in congenital heart disease in Tibetans. In humans, the EPAS1 gene is responsible for coding EPAS1 protein, an alias of which is HIF2α, an acronym for hypoxia-inducible factor 2 alpha. EPAS1 is a type of hypoxia-inducible factors, which are collected as a group of transcription factors involved in body response to oxygen level. EPAS1 gene is active under hypoxic conditions and plays an essential role in the development of the heart and in the management of the catecholamine balance, mutations of which have been identified in neuroendocrine tumors. In this article, Pan et al. investigated Tibetan patients with and without non-syndromic congenital heart disease. They identified two novel EPAS1 gene mutations, of which N203H mutation significantly affected the transcription activity of the vascular endothelial growth factor (VEGF) promoter, particularly in situations of hypoxia. VEGF is a downstream target of HIF-2 (other than HIF-1), and the expression levels of either HIF-1α or HIF-2α correlate positively to VEGF expression. Pan et al.’s data may be of incitement to further evaluate protein–protein interaction and using experimental animal models. Moreover, it may also be a stimulus for setting up genetic epidemiologic studies for other populations living at high altitudes.

Genetic polymorphisms in MTR are associated with non-syndromic congenital heart disease from a family-based case-control study in the Chinese population

Genetic polymorphisms of folate pathway genes have been reported to be associated with congenital heart diseases (CHDs); however, the results remain conflicting. We conducted a family-based case-control study, which included160 CHD case-parent triads and 208 control-parent triads to explore the association of 18 genetic variants of seven folate metabolism-related genes with the risk of CHDs. The MTR C allele of rs1770449 (OR = 1.961, 95%CI: 1.379–2.788) and the MTR A allele of rs1050993 (OR = 1.994, 95%CI: 1.401–2.839) in infants were associated with an increased risk of CHDs. Over-transmission of SNPs rs1770449 and rs1050993 and haplotype CAA (rs1770449-rs1805087-rs1050993) in MTR were detected in total CHDs. The above mentioned associations of MTR with CHDs were also observed in septal defects and conotruncal heart defects subgroups. Without maternal periconceptional folate intake, the risk of CHDs among women carrying the rs1770449 “CT or CC” genotype or the rs1050993 “AG or AA” genotype in MTR was 3.262(95%CI: 1.656–6.429) or 3.263(95%CI: 1.656–6.429) times greater than the aOR in women carrying wild genotype, respectively. Our study suggests that MTR polymorphisms (rs1770449 and rs1050993) may be associated with the risk of CHDs and modify the relation between maternal folate intake and CHDs.

Mutations in EPAS1 in congenital heart disease in Tibetans.

EPAS1 encodes HIF2 and is closely related to high altitude chronic hypoxia. Mutations in the EPAS1 coding sequence are associated with several kinds of human diseases, including syndromic congenital heart disease (CHD). However, whether there are rare EPAS1 coding variants related to Tibetan non-syndromic CHD have not been fully investigated. A group of 286 Tibetan patients with non-syndromic CHD and 250 unrelated Tibetan healthy controls were recruited from Qinghai, China. Sanger sequencing was performed to identify variations in the EPAS1 coding sequence. The novelty of identified variants was confirmed by the examination of 1000G and ExAC databases. Control samples were screened to establish that the rare candidate variants were specific to the Tibetan patients with non-syndromic CHD. Bioinformatics software was used to assess the conservation of the mutations and to predict their effects. The effect of EPAS1 mutations on the transcription of its target gene, VEGF, was assessed by dual-luciferase reporter assay. The mammalian two-hybrid assay was used to study the protein interactions between HIF2 and PHD2 or pVHL. We identified two novel EPAS1 mutations (NM_001430: c.607A>C, p.N203H; c.2170G>T, p.G724W) in two patients. The N203H mutation significantly affected the transcription activity of the VEGF promoter, especially in conditions of hypoxia. The N203H mutation also showed enhanced protein–protein interactions between HIF2 and PHD2, and HIF2 and pVHL, especially in conditions of hypoxia. However, the G724W mutation did not demonstrate the same effects. Our results indicate that EPAS1 mutations might have a potential causative effect on the development of Tibetan non-syndromic CHD.

Implication of GATA4 synonymous variants in congenital heart disease: A comprehensive in-silico approach

Synonymous variations, previously considered as neutral, are recently shown to have a significant impact on mRNA structure and stability thereby affecting protein expression and function. Their role in disease pathogenesis is also emerging. GATA4 is an important transcription factor involved in cardiac development and a well-known candidate gene associated with congenital heart disease (CHD). In the present study, we sought to conduct molecular screening of GATA4 gene in 285 sporadic and non-syndromic CHD cases. We identified four synonymous (c.27C>A, c.822C>T, c.1233G>A and c.1263C>T) and two intronic variants (g.83217T>G & g.85012T>A) in GATA4. Extensive computational analysis using widely acceptable tools i.e., Mfold, Human Splicing Finder (HSF) and Codon Usage bias was performed with a view to understand their putative downstream effects on GATA4 function. Mfold, a mRNA structure prediction tool showed the alterations of the mRNA structure and stability due to synonymous variants. Similarly, HSF also confidently predicted effect on the cis-acting regulatory elements of splicing due to four synonymous and one donor site intronic variants. Additionally, a significant change in ‘Relative Synonymous Codon usage (RSCU) frequencies’ and ‘log ratio of codon usage frequencies’ of variant codon was also noted that might affect the rate of translation. This study establishes that the synonymous variants are possibly associated with disease phenotype in CHD patients. Comprehensive computational analysis, using well-established web based tools, is suggestive of their potential downstream molecular effects on the structure, stability and expression of GATA4 protein.