Heart And Neural Crest Derivatives Expressed 2 Research Articles

Molecular Genetic Study on HAND2 Gene Promoter in Ventricular Septal Defect.

Ventricular septal defect (VSD), the most common type of congenital heart disease (CHD), is primarily caused by cardiac dysplasia. Heart and neural crest derivatives expressed 2 (HAND2) participates in developing the right heart. The loss of HAND2 expression in humans is closely connected with ventricular septal defects. We used a case-control study to analyze the genetic variations in the HAND2 promoter region in VSD patients and controls. Some statistical analysis methods were used to analyze the association of single nucleotide polymorphisms (SNPs) with VSD. The dual-luciferase reporter assay and electrophoretic mobility shift assay (EMSA) were used to conduct functional analysis and molecular mechanism study of genetic variations. Through sequencing, we identified nine genetic variants in patients with VSD. The SNP rs2276940 G>T and rs2276941 G>A were associated with an increased risk of VSD. The dual-luciferase reporter assay showed that SNP rs2276940 G>T and rs138531627 C>G decreased the transcriptional activity of the HAND2 promoter. Transcription factors (TFs) predicting suggested that all three SNPs may change the binding of TFs. The result of EMSA showed that rs138531627 C>G may create a new binding site for TFs while rs2276940 G>T enhanced the binding affinity for TFs. These results indicated that genetic variants of the HAND2 promoter may increase the risk of VSD, and the molecular mechanism may be the change of the binding affinity of TFs.

A Genome-Wide Association Study of the Chest Circumference Trait in Xinjiang Donkeys Based on Whole-Genome Sequencing Technology.

Animal genotyping by means of genome-wide association studies is important for connecting phenotypes of interest with their underlying genetics in livestock. However, the use of whole genome sequencing to investigate chest circumference (CC) in donkeys has rarely been reported. We aimed to use the genome-wide association study approach to detect significant single nucleotide polymorphisms (SNPs) and key genes associated with chest circumference traits in Xinjiang donkeys. We assessed 112 Xinjiang donkeys in this study. The chest circumference of each was measured 2 h before milking. We re-sequenced blood samples from the Xinjiang donkeys, and genome-wide association study analyses were performed using a mixed model with the PLINK, GEMMA, and REGENIE programs. We tested 38 donkeys for candidate SNPs for genome-wide association study using three software programs. Additionally, 18 SNP markers reached genome-wide significance (p < 1.61 × 10-9). On the basis of these, 41 genes were identified. Previously proposed candidate genes for CC traits were supported by this study, including NFATC2 (Nuclear Factor of Activated T Cells 2), PROP1 (PROP Paired-Like Homeobox 1), UBB (Ubiquitin B), and HAND2 (Heart and Neural Crest Derivatives Expressed 2). These promising candidates provide a valuable resource for validating potential meat production genes and will facilitate the development of high-yielding Xinjiang donkey breeds through marker-assisted selection or gene editing.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells.

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Transcriptional regulation of LGALS9 by HAND2 and FOXO1 in human endometrial stromal cells in women with regular cycles.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was upregulated by heart and neural crest derivatives expressed 2 (HAND2) and downregulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on Day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after a 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling

Aims/hypothesisAdipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis.MethodsHuman white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests.ResultsWe found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR–HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression.Conclusions/interpretationIn summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice.Data availabilityArray data have been submitted to the GEO database at NCBI (GSE148699).Graphical abstract

Artesunate Suppresses the Proliferation and Development of Estrogen Receptor-α-Positive Endometrial Cancer in HAND2-Dependent Pathway.

Endometrial cancer (EC) is a common leading cause of cancer-related death in women, which is associated with the increased level of estrogen in the body. Artesunate (ART), an active compound derived from Artemisia annua L., exerted antitumor properties in several cancer types. However, the role of artesunate and the molecular basis on EC remains unclear. Here, we aimed to explore the effects and mechanisms of artesunate. Our results identified that estrogen receptor-α (ER-α) was a key factor for the type I EC (ER-α-positive), which might suppress the downstream LKB1/AMPK/mTOR pathway. Besides, we found ART significantly inhibited tumor proliferation in a dose-dependent manner. Mechanistic studies identified that ART led to tumor cell apoptosis and cell cycle arrest by downregulating the ER-α expression and activating the LKB1/AMPK/mTOR pathway. In addition, we found ART could increase the expression of heart and neural crest derivatives expressed 2 (HAND2) in the ER-α-positive EC cells, which could interact with ER-α. Through the gain-and loss-function experiments, we showed that over expression of HAND2 repressed the proliferation and migration of ER-α-positive EC cells via inhibition of ER-α expression. HAND2 knockdown increased ER-α expression and alleviated the antitumor effect of ART in vitro and in vivo. Overall, our study first showed that ART could be an effective antitumor agent through modulating ER-α-mediated LKB1/AMPK/mTOR pathway in the HAND2 dependent manner. Our findings provide an effective therapeutic agent for ER-α-positive EC treatment.

Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2.

Congenital heart disease (CHD) is the most common birth defect worldwide. Long non-coding RNAs (lncRNAs) have been implicated in many diseases. However, their involvement in CHD is not well understood. This study aimed to investigate the role of dysregulated lncRNAs in CHD. We used Gene Expression Omnibus data mining, bioinformatics analysis, and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD. Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2 (HAND2). Moreover, lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation. Overall, these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.

Functional analysis of the congenital heart disease‑associated GATA4 H436Y mutation invitro.

Congenital heart disease (CHD) is the most common type of developmental defect, with high rates of morbidity in infants. The transcription factor GATA-binding factor 4 (GATA4) has been reported to serve a critical role in embryogenesis and cardiac development. Our previous study reported a heterozygous GATA4 c.1306C>T (p.H436Y) mutation in four Chinese infants with congenital heart defects. In the present study, functional analysis of the GATA4 H436Y mutation was performed in vitro. The functional effect of GATA4 mutation was compared with GATA4 wild-type using a dual-luciferase reporter assay system and immunofluorescence. Electrophoretic mobility-shift assays were performed to explore the binding affinity of the mutated GATA4 to the heart and neural crest derivatives expressed 2 (HAND2) gene. The results revealed that the mutation had no effect on normal nuclear localization, but resulted in diminished GATA-binding affinity to HAND2 and significantly decreased gene transcriptional activation. These results indicated that this GATA4 mutation may not influence cellular localization in transfected cells, but may affect the affinity of the GATA-binding site on HAND2 and decrease transcriptional activity, thus suggesting that the GATA4 mutation may be associated with the pathogenesis of CHD.

Upregulation of Fibroblast Growth Factors Caused by Heart and Neural Crest Derivatives Expressed 2 Suppression in Endometriotic Cells: A Possible Therapeutic Target in Endometriosis.

Several features exist that distinguish endometriotic cells from eutopic endometrial cells. Progesterone resistance is one of the main distinguishing features, although how progesterone resistance affects the phenotype of endometriotic cells is not fully elucidated. Heart and neural crest derivatives expressed 2 (HAND2) is a transcriptional factor that plays an important role in maintaining endometrial function in a progesterone-dependent manner. Therefore, we explored whether progesterone-dependent HAND2 is implicated in the progression of endometriosis. HAND2 was less expressed by endometriotic tissues compared to endometrial tissues. Suppression of HAND2 expression induced fibroblast growth factor 1 (FGF1), FGF2, and FGF9 in endometriotic stromal cells and consequently enhanced migration and invasion capacity. AZD4547, a FGF receptor inhibitor, diminished the migration and invasion of endometriotic cells in vitro. In the murine model of endometriosis, AZD4547 showed suppressive effects on the development of endometriotic lesions at a relatively low concentration. In conclusion, we demonstrated that FGF1, FGF2, and FGF9 are downstream effectors of HAND2 in endometriotic cells. Since HAND2-dependent FGFs play roles in enhancing invasive capacity of endometriotic cells, our results suggest that FGF receptor inhibitors, such as AZD4547, can be promising therapeutic targets for endometriosis.

Chronic Exposure of Mice to Bisphenol-A Alters Uterine Fibroblast Growth Factor Signaling and Leads to Aberrant Epithelial Proliferation.

Uterine epithelial proliferation is regulated in a paracrine manner by a complex interplay between estrogen (E) and progesterone (P) signaling, in which E stimulates proliferation and P inhibits it. Perturbation of steroid hormone signaling within the uterine milieu could contribute to the development of endometrial hyperplasia and cancer. It is well established that bisphenol-A (BPA) is an endocrine-disrupting chemical with weak estrogenic effects, although little is known about how it affects steroid hormone signaling in the adult uterus. Because BPA acts as a weak E, we hypothesized that chronic exposure to BPA would create an imbalance between E and P signaling and cause changes in the uterus, such as aberrant epithelial proliferation. Indeed, exposure to an environmentally relevant dose of BPA had a uterotrophic affect. BPA-treated mice showed increased proliferation, notably in the glandular epithelium, which are sites of origin for endometrial hyperplasia and cancer. Increased proliferation appeared to be mediated through a similar mechanism as E-induced proliferation, via activation of the fibroblast growth factor receptor pathway and phosphorylation of the ERK1/2 mitogen-activated protein kinases in the epithelium. Interestingly, BPA reduced expression of heart and neural crest derivatives expressed 2 (HAND2), a known mediator of the antiproliferative effects of P. BPA also increased methylation of a CpG island in the Hand2 gene promoter, suggesting that BPA may promote epithelial proliferation through epigenetic silencing of antiproliferative factors like HAND2. Collectively, these findings establish that chronic exposure to BPA impairs steroid hormone signaling in the mouse uterus, and may contribute to the pathogenesis of uterine hyperplasia and cancer.

Endometrial Stromal and Epithelial Cells Exhibit Unique Aberrant Molecular Defects in Patients With Endometriosis

Endometriosis is a chronic inflammatory disease that causes pain and infertility in women of reproductive age. To investigate the pathologic pathways in endometrial stromal and epithelial cells that contribute to the manifestation of endometriosis. In vitro cellular and molecular analyses of isolated eutopic endometrial stromal and epithelial cells. Eutopic stromal and epithelial cells from endometriotic and normal patients were isolated by fluorescence-activated cell sorting for paired sibling RNA sequencing and microRNA microarray. Aberrant pathways were identified using ingenuity pathway analysis networks and confirmed with in vitro modulation of the affected pathways in stromal and epithelial cell cultures. Both stromal versus epithelial cell types and paired endometriotic versus normal samples exhibited distinct hierarchical clustering. Compared to normal samples, there were 151 and 215 differentially expressed genes in the endometriotic stromal and epithelial populations, respectively, and concomitantly 9 and 16 differentially expressed microRNAs. Overall, endometriotic stromal and epithelial cells revealed distinct defects. In endometriotic stromal cells, key decidualization genes Zinc finger E-box Binding protein 1 (ZEB1), Heart And Neural crest Derivatives expressed 2 (HAND2), WNT4, and Interleukin 15 (IL-15) were found to be downregulated and Periostin (POSTN) and Matrix Metallopeptidase 7 (MMP7) were upregulated. Specifically, ZEB1 was downregulated in stromal cells by aberrant elevation in miR-200b. In contrast, ZEB1 was found to be upregulated in endometriotic epithelial cells through associated upregulation of transforming growth factor β1 (TGFβ1), inducer of the TGFβ1-Bone Morphogenetic Protein 2 (BMP2)-MMP2-Prostaglandin-endoperoxide Synthase 2 (COX2)-ZEB1 pathway, which activates epithelial-mesenchymal transition. Manifestation of endometriosis involves dysregulation of unique molecular pathways within the diseased endometrial stromal and epithelial cells in the endometrium. Targeting the cell type-specific defects may offer a novel approach to treating endometriosis.

Long non-coding RNA HAND2-AS1 inhibits invasion and metastasis in endometrioid endometrial carcinoma through inactivating neuromedin U

Endometrioid endometrial carcinoma (EEC) is one of the common causes of cancer-related mortality in women. Mounting evidences suggest that long noncoding RNAs (lncRNAs) function in multiple cancers. In this study, we discovered that HAND2-AS1, a lncRNA transcribed antisense adjacent to Heart and Neural Crest Derivatives Expressed 2 (HAND2) in chromosome 4q33-34, is significantly down-regulated in EEC. HAND2-AS1 and HAND2 were frequently down-regulated in EEC tissues, especially in poor differentiated tumor tissues. Down-regulation of HAND2-AS1 and HAND2 was correlated with tumor grade, lymph node metastasis and recurrence of EEC patients. HAND2-AS1 and HAND2 were co-downregulated by promoter DNA hypermethylation in EEC. Overexpression of HAND2-AS1 in EEC cells demonstrated that HAND2-AS1 suppressed migration and invasion of EEC cells. Similarly, overexpression of HAND2 also inhibited migration and invasion EEC cells indicating that HAND2-AS1 and HAND2 had a concordant role in the progression of EEC. However, HAND2 was not regulated by HAND2-AS1 in EEC. Furthermore, the anti-tumorigenic effect of HAND2-AS1 was mediated by down-regulating NMU, which has an oncogenic role in EEC. Our findings therefore provide the first evidence that HAND2-AS1 is a critical tumor suppressor in EEC.

Abstract 3448: LncRNA HAND2-AS1 inactivates neuromedin U (NMU) and inhibits tumor invasion and metastasis in endometrioid endometrial carcinoma

Abstract Background Endometrioid endometrial carcinoma (EEC) is one of the common cause of cancer-related mortality in women. Despite progress in diagnostics and treatment of EEC, its prognosis remains poor. Mounting evidence suggest that long noncoding RNAs (lncRNAs) function in mutiple human cancers. Aberrant lncRNA expression may predict tumor outcome of patients and have served as diagnostic or prognostic markers. In this study, we investigated the expression levels and functions of lncRNAs in EEC. Methods Differentially expressed lncRNAs involved in EEC were identified by using publically available RNA-Seq data. The expression of 18 dysregulated lncRNA candidates was verified in 5 NE tissues, 5 EEC tissues and 5 EEC cell lines (HEC1-A, HEC1-B, AN3CA, KLE and RL95-2) by real-time polymerase chain reaction (PCR). Further, we selected the most misexpressed lncRNA and confirmed the expression level of the lncRNA in 59 EEC tissues and 24 NE tissues by real-time PCR and correlated the lncRNA expression levels with the clinical pathological characteristics. The promoter methylation assay was used to analyze the methylation level of the lncRNA in EEC. The lncRNA methylation status was confirmed by bisulfite genomic sequencing. Cell proliferation assays, wound healing assays, and invasion and migration assays were performed to determine the biological functions of the lncRNA in EEC cells. To discover the direct targets of the lncRNA in EEC, we performed RNA-sequence analysis in EEC cells overexpressed with the lncRNA and target genes were further studied by functional studies in vitro (knockdown assay and overexpression rescue assay). Results We discovered that HAND2-AS1, a lncRNA transcribed antisense adjacent to Heart and Neural Crest Derivatives Expressed 2 (HAND2), was significantly downregulated lncRNA in EEC. HAND2-AS1 and HAND2 was frequently downregulated in EEC tissues, especially in poor differentiated tumor tissues. Downregulation of HAND2-AS1 and HAND2 was correlated with tumor grade, lymph node metastasis and recurrence of EEC patients. HAND2-AS1 and HAND2 was co-downregulated by promoter hypermethylation in EEC. HAND2-AS1 suppressed EEC cell migration and invasion but not cell growth. Similarly, HAND2 also inhibited EEC cell migration and invasion indicating that HAND2-AS1 and HAND2 have a concordant role in the progression of EEC. Moreover, the anti-tumorigenic effect of HAND2-AS1 was mediated by downregulating NMU, which had an oncogenic role in EEC. Conclusions Our findings provide the first evidence that HAND2-AS1 is a critical tumor suppressor in EEC and may constitute a prognostic biomarker in EEC. Note: This abstract was not presented at the meeting. Citation Format: Xueying Yang, Yu Zhao, Kun Sun, Yuying Li, Jiajian Zhou, Jianzhang Wang, Hao Sun, Chi Chiu Wang, Joseph Kwong, Huating Wang, Tony Kwok Hung Chung. LncRNA HAND2-AS1 inactivates neuromedin U (NMU) and inhibits tumor invasion and metastasis in endometrioid endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2017-3448

Chronic Exposure to Bisphenol A Affects Uterine Function During Early Pregnancy in Mice.

Environmental and occupational exposure to bisphenol A (BPA), a chemical widely used in polycarbonate plastics and epoxy resins, has received much attention in female reproductive health due to its widespread toxic effects. Although BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In this study, we addressed the effect of prolonged exposure to an environmental relevant dose of BPA on embryo implantation and establishment of pregnancy. Our studies revealed that treatment of mice with BPA led to improper endometrial epithelial and stromal functions thus affecting embryo implantation and establishment of pregnancy. Upon further analyses, we found that the expression of progesterone receptor (PGR) and its downstream target gene, HAND2 (heart and neural crest derivatives expressed 2), was markedly suppressed in BPA-exposed uterine tissues. Previous studies have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor and the MAPK signaling pathways and inhibiting epithelial proliferation. Interestingly, we observed that down-regulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with enhanced activation of fibroblast growth factor and MAPK signaling in the epithelium, thus contributing to aberrant proliferation and lack of uterine receptivity. Further, the differentiation of endometrial stromal cells to decidual cells, an event critical for the establishment and maintenance of pregnancy, was severely compromised in response to BPA. In summary, our studies revealed that chronic exposure to BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.

A novel HAND2 loss-of-function mutation responsible for tetralogy of Fallot

Congenital heart disease (CHD), the most common type of developmental abnormality, is associated with substantial morbidity and mortality in humans worldwide. The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed 2 (HAND2), has been demonstrated to be crucial for normal cardiovascular development in animal models. However, whether a genetically defective HAND2 contributes to congenital heart disease (CHD) in humans remains to be explored. In this study, the entire coding region and splicing boundaries of the HAND2 gene were sequenced in a cohort of 145 unrelated patients with CHD. A total of 200 unrelated, ethnically-matched healthy individuals used as controls were also genotyped for HAND2. The functional effect of the mutant HAND2 was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. As a result, a novel heterozygous HAND2 mutation, p.L47P, was identified in a patient with tetralogy of Fallot (TOF). The misense mutation, which altered the amino acid conserved evolutionarily among species, was absent in 400 control chromosomes. Functional analyses unveiled that the mutant HAND2 had a significantly decreased transcriptional activity. Furthermore, the mutation markedly reduced the synergistic activation between HAND2 and GATA4 or NKX2.5, other two cardiac key transcription factors involved in the pathogenesis of CHD. To the best of our knowledge, this study is the first to report the association of a HAND2 loss-of-function mutation with an increased vulnerability to TOF in humans, which provides novel insight into the molecular mechanism underpinning CHD, suggesting potential implications for the genetic counseling of families with CHD.