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Congenital heart disease (CHD) is one of most prevalent birth defects in the world. However, the underlying molecular mechanism(s) have not been fully understood. Here we report that increased CHD susceptibility is associated with genetic polymorphisms for de novo nucleotide biosynthesis in northern Chinese population, which has been reported with lower plasma folate levels. Nine tagSNPs of four genes (GART, ATIC, MTHFD1 and SHMT1) in de novo nucleotide biosynthesis were sequenced in 802 sporadic CHDs patients and 1,093 controls from two Han Chinese populations, located in north China (Shandong) and South China (Shanghai). Six SNPs were found to be significantly associated with CHDs or septation defects only in the Shandong population dataset, but none displayed significant association with any CHDs in the Shanghai population dataset as well as in the combined dataset. We also showed that the minor A allele of rs7279549 in GART reduced transcriptional activity and displayed lower affinity for unknown transcription factor(s), demonstrating the allele is a functional risk factor for CHD in Shandong population. Our study indicates that dysregulation of de novo nucleotide biosynthesis pathway may conditionally contribute to CHD pathogenesis in northern Chinese.

The genetic heterogeneity of non-syndromic hearing loss (NSHL) has hampered the identification of its pathogenic mutations. Several recent studies applied targeted genome enrichment (TGE) and massively parallel sequencing (MPS) to simultaneously screen a large set of known hearing loss (HL) genes. However, most of these studies were focused on familial cases. To evaluate the effectiveness of TGE and MPS on screening sporadic NSHL patients, we recruited 63 unrelated sporadic NSHL probands, who had various levels of HL and were excluded for mutations in GJB2, MT-RNR1, and SLC26A4 genes. TGE and MPS were performed on 131 known HL genes using the Human Deafness Panel oto-DA3 (Otogenetics Corporation., Norcross, GA). We identified 14 pathogenic variants in STRC, CATSPER2, USH2A, TRIOBP, MYO15A, GPR98, and TMPRSS3 genes in eight patients (diagnostic rate = 12.7%). Among these variants, 10 were novel compound heterozygous mutations. The identification of pathogenic mutations could predict the progression of HL, and guide diagnosis and treatment of the disease.

Objectives The posterior lateral line (PLL) system in zebrafish has recently become a model for investigating tissue morphogenesis. PLL primordium periodically deposits neuromasts as it migrates along the horizontal myoseptum from head to tail of the embryonic fish, and this migration requires activity of various molecular mechanisms. Histone deacetylases (HDACs) have been implicated in numerous biological processes of development, by regulating gene transcription, but their roles in regulating PLL during embryonic development have up to now remained unexplored. Material and methods In this study, we used HDAC inhibitors to investigate the role of HDACs in early development of the zebrafish PLL sensory system. We further investigated development of the PLL by cell-specific immunostaining and in situ hybridization. Results Our analysis showed that HDACs were involved in zebrafish PLL development as pharmacological inhibition of HDACs resulted in its defective formation. We observed that migration of PLL primordium was altered and accompanied by disrupted development of PLL neuromasts in HDAC inhibitor-treated embryos. In these, positions of PLL neuromasts were affected. In particular, the first PLL neuromast was displaced posteriorly in a treatment dose-dependent manner. Primordium cell proliferation was reduced upon HDAC inhibition. Finally, we showed that inhibition of HDAC function reduced numbers of hair cells in PLL neuromasts of HDAC inhibitor-treated embryos. Conclusion Here, we have revealed a novel role for HDACs in orchestrating PLL morphogenesis. Our results suggest that HDAC activity is necessary for control of cell proliferation and migration of PLL primordium and hair cell differentiation during early stages of PLL development in zebrafish.

Congenital heart defects (CHDs) are one of the most common human birth defects worldwide. TBX20 is a crucial transcription factor for the development of embryonic cardiovascular system. Previous studies have demonstrated that mutations in the TBX20 coding region contribute to familial and sporadic CHD occurrence. However, it remains largely unknown whether variants in the TBX20 regulatory region are also related to CHDs. In this study, we sequenced the 2 kb region upstream of the TBX20 transcription start site in 228 CHD patients and 292 controls in a Han Chinese population. Among the 8 single nucleotide polymorphisms (SNPs) identified, six SNPs are in strong linkage disequilibrium and the minor alleles are associated with lower CHD risk (for rs10235849 chosen as tag SNP, p = 0.0069, OR (95% CI) = 0.68 (0.51–0.90)). Functional analysis showed that the minor alleles have lower transcriptional activity than major alleles in both human heart tissues and three cell lines. The electrophoretic mobility shift assay suggested that TBX20 minor alleles may exhibit higher binding affinity with certain transcription repressors. Our results indicate that a moderately lower TBX20 activity potentially reduces CHD risk in the Han Chinese population, providing new insight in the study of CHD etiology.

Homocysteine is an independent risk factor for various cardiovascular diseases. There are two ways to remove homocysteine from embryonic cardiac cells: remethylation to form methionine or transsulfuration to form cysteine. Cystathionine β-synthase (CBS) catalyzes the first step of homocysteine transsulfuration as a rate-limiting enzyme. In this study, we identified a functional variant -4673CG (rs2850144) in the CBS gene promoter region that significantly reduces the susceptibility to congenital heart disease (CHD) in a Han Chinese population consisting of 2 340 CHD patients and 2 270 controls. Individuals carrying the heterozygous CG and homozygous GG genotypes had a 15% (odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.75-0.96, P = 0.011) and 40% (OR = 0.60, 95% CI = 0.49-0.73, P = 1.78 × 10(-7)) reduced risk to develop CHD than the wild-type CC genotype carriers in the combined samples, respectively. Additional stratified analyses demonstrated that CBS -4673CG is significantly related to septation defects and conotruncal defects. In vivo detection of CBS mRNA levels in human cardiac tissues and in vitro luciferase assays consistently showed that the minor G allele significantly increased CBS transcription. A functional analysis revealed that both the attenuated transcription suppressor SP1 binding affinity and the CBS promoter hypomethylation specifically linked with the minor G allele contributed to the remarkably upregulated CBS expression. Consequently, the carriers with genetically increased CBS expression would benefit from the protection due to the low homocysteine levels maintained by CBS in certain cells during the critical heart development stages. These results shed light on unexpected role of CBS and highlight the importance of homocysteine removal in cardiac development.Cell Research advance online publication 18 September 2012; doi:10.1038/cr.2012.135.

BACKGROUND: Genome-wide association studies on components of the one-carbon metabolic pathway revealed that human vitamin B12 levels could be significantly influenced by variations in the fucosyltransferase 2 (FUT2), cubilin (CUBN), and transcobalamin-I (TCN1) genes. An altered vitamin B12 level is an important factor that disturbs the homeostasis of the folate metabolism pathway, which in turn can potentially lead to the development of congenital heart disease (CHD). Therefore, we investigated the association between the variants of vitamin B12-related genes and CHD in Han Chinese populations. METHODS AND RESULTS: Six variants of the vitamin B12-related genes were selected for analysis in two independent case-control studies, with a total of 868 CHD patients and 931 controls. The variant rs11254363 of the CUBN gene was associated with a decreased risk of developing CHD in both the separate and combined case-control studies. Combined samples from the two cohorts had a significant decrease in CHD risk for the G allele (OR = 0.48, P = 1.7×10⁻⁵) and AG+GG genotypes (OR = 0.49, P = 4×10⁻⁵), compared with the wild-type A allele and AA genotype, respectively. CONCLUSIONS: Considering the G allele of variant rs11254363 of the CUBN gene was associated with an increased level of circulating vitamin B12. This result suggested that the carriers of the G allele would benefit from the protection offered by the high vitamin B12 concentration during critical heart development stages. This finding shed light on the unexpected role of CUBN in CHD development and highlighted the interplay of diet, genetics, and human birth defects.

Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.

SMAD7 is a general antagonist of TGF-β signaling and has been found to be involved in cardiogenesis in mouse models, but its role in human congenital heart disease (CHD) has yet to be investigated. To examine if SMAD7 is associated with CHD, we conducted a case-control study in the Han Chinese population. Exon1 and exon4 of SMAD7, which encode the functional MH1 and MH2 domains, were directly sequenced in 1,201 sporadic CHD patients and 1,116 control individuals. A total of 18 sequence variations were identified. Two common variants rs3809922 and rs3809923 are located at exon4 of SMAD7, and were found in strong linkage disequilibrium with each other (r 2 = 0.93). We analyzed the association of these two loci with CHD in 3 independent subgroup case-control studies, and found that in some subgroups, rs3809922 and rs3809923 were significantly associated with CHD through genetic model analysis. In the combined data set, TT genotype in rs3809922 significantly increased the risk of CHD compared with CC and CT, while GG genotype in rs3809923 significantly increased the risk of CHD compared with CC and CG, particularly in the recessive model. In addition, haplotype analyses showed that haplotype TG significantly increased the risk of CHD (P = 6.9×10−6); this finding supports the results from the analyses based on single locus. According to data from the 1000 Genomes Project, the frequencies of the two risk alleles varied greatly between populations worldwide, which indicate the identified associations might have a population difference. To our knowledge, this is the first report that genetic variants in SMAD7 influence susceptibility to CHD risk.

Sensorineural hearing loss (SNHL) is one of the most common sensory defects in humans. Hair cells are vulnerable to various ototoxic insults. Effective prevention of hair cell loss remains an unmet medical need. Apoptotic hair cell death, which involves active regulation of transcription, accounts for the majority of aminoglycoside-induced hair cells loss. As one of the important epigenetic covalent modifications, histone methylation is involved in the regulation of gene expression, development and reaction to injury. In particular, H3K9 dimethylation (H3K9me2) is critical for euchromatin gene silencing. In the present study, we examined the roles of two highly homologous histone methyltransfereases responsible for this modification, G9a/G9a-like protein (GLP), in the reaction to aminoglycoside-induced hair cell damage. We observed a rapid increase of H3K9me2 upon hair cell damage in organotypic cochlear cultures. Treatment with the G9a/GLP-specific inhibitors, BIX01294 or UNC0638, reduced the level of H3K9me2 and prevented hair cells from death. Local delivery of BIX01294 also prevented neomycin-induced in vivo auditory hair cell loss in the organ of Corti in a mouse damage model. It is unlikely that BIX01294 functions through blocking aminoglycoside absorption as it does not interfere with aminoglycoside uptaking by hair cells in the organotypic cochlear cultures. Our data revealed a novel role of histone methylation in otoprotection, which is of potential therapeutic value for SNHL management.