The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Microtia is a congenital malformation characterized by a small, abnormally shaped auricle (pinna) ranging in severity. Congenital heart defect (CHD) is one of the comorbid anomalies with microtia. However, the genetic basis of the co-existence of microtia and CHD remains unclear. Copy number variations (CNVs) of 22q11.2 contribute significantly to microtia and CHD, respectively, thus suggesting a possible shared genetic cause embedded in this genomic region. In this study, 19 sporadic patients with microtia and CHD, as well as a nuclear family, were enrolled for genetic screening of single nucleotide variations (SNVs) and CNVs in 22q11.2 by target capture sequencing. We detected a total of 105 potential deleterious variations, which were enriched in ear- or heart-development-related genes, including TBX1 and DGCR8. The gene burden analysis also suggested that these genes carry more deleterious mutations in the patients, as well as several other genes associated with cardiac development, such as CLTCL1. Additionally, a microduplication harboring SUSD2 was validated in an independent cohort. This study provides new insights into the underlying mechanisms for the comorbidity of microtia and CHD focusing on chromosome 22q11.2, and suggests that a combination of genetic variations, including SNVs and CNVs, may play a crucial role instead of single gene mutation.

TBX1 is systematically conserved in the T-box transcription factor family and regulates craniofacial muscle development during various stages of myogenesis, including commitment, proliferation, terminal differentiation, and survival. However, the role and mechanism by which TBX1 regulates the myogenic development of myoblasts remains unclear. In our study, we overexpressed TBX1 in mouse C2C12 myoblasts using a lentivirus method. We found that TBX1 inhibited cell proliferation and muscle differentiation, which had no effect on apoptosis. During myogenic differentiation, we also found that TBX1 overexpressing cells regulate myogenic differentiation by upregulating the expression levels of Smad2 and Smad3 and downregulating the expression level of MEF2C. After treatment with a specific inhibitor of Smad3 (SIS3), the myogenic differentiation of wild-type and TBX1 overexpressing cells increased. Thus, TBX1 may regulate myoblast muscle differentiation by enhancing the expression of Smad2 and Smad3. TBX1 may be a therapeutic target for muscular dystrophy.

UNASSIGNED: Autism spectrum disorder (ASD) is a developmental disorder that can cause substantial social, communication, and behavioral challenges. Genetic factors play a significant role in ASD, where the risk of ASD has been increased for unclear reasons. Twin studies have shown important evidence of both genetic and environmental contributions in ASD, where the level of contribution of these factors has not been proven yet. It has been suggested that copy number variation (CNV) duplication and the deletion of many genes in chromosome 22 (Ch22) may have a strong association with ASD. This study screened the CNVs in Ch22 in autistic Saudi children and assessed the candidate gene in the CNVs region of Ch22 that is most associated with ASD.
UNASSIGNED: This study included 15 autistic Saudi children as well as 4 healthy children as controls; DNA was extracted from samples and analyzed using array comparative genomic hybridization (aCGH) and DNA sequencing.
UNASSIGNED: The aCGH detected (in only 6 autistic samples) deletion and duplication in many regions of Ch22, including some critical genes. Moreover, DNA sequencing determined a genetic mutation in the TBX1 gene sequence in autistic samples. This study, carried out using aCGH, found that six autistic patients had CNVs in Ch22, and DNA sequencing revealed mutations in the TBX1 gene in autistic samples but none in the control.
UNASSIGNED: CNV deletion and the duplication of the TBX1 gene could be related to ASD; therefore, this gene needs more analysis in terms of expression levels.

The major vessels in mammals that take blood away from the heart and deliver it to the arms and the head take their origin from the aortic arch and are derived from the arteries formed within the embryonic pharyngeal arches. These pharyngeal arch arteries, initially symmetrical, form in a cranial to caudal sequence within the pharyngeal mesenchyme. They then undergo a complex process of remodeling to produce the asymmetrical brachiocephalic arteries as seen in the adult. A complex interaction between the tissues of the pharyngeal arches and the genes they express is required to ensure that arterial formation and remodeling is able to proceed normally. If this process is disrupted, life-threatening congenital cardiovascular malformations can occur, such as interruption of the aortic arch, isolation of individual arteries, or so-called vascular rings. Here, using state-of-the-art imaging techniques, we describe the morphogenesis of the arteries in humans and mice and the cardiovascular defects in the Tbx1 mutant mouse model. We provide details of the process of remodeling, clarifying also the morphogenesis of the external carotid artery and the so-called \"migration\" of the left subclavian artery.

UNASSIGNED: Tbx1 mutant mice are a widely used model of 22q11.2 deletion syndrome (22q11.2DS) because they manifest a broad spectrum of physical and behavioral abnormalities that is similar to that found in 22q11.2DS patients. In Tbx1 mutants, brain abnormalities include changes in cortical cytoarchitecture, hypothesized to be caused by the precocious differentiation of cortical progenitors. The objectives of this research are to identify drugs that have efficacy against the brain phenotype, and through a phenotypic rescue approach, gain insights into the pathogenetic mechanisms underlying Tbx1 haploinsufficiency.
UNASSIGNED: Disease model: Tbx1 heterozygous and homozygous embryos. We tested the ability of two FDA-approved drugs, the LSD1 inhibitor Tranylcypromine and Vitamin B12, to rescue the Tbx1 mutant cortical phenotype. Both drugs have proven efficacy against the cardiovascular phenotype, albeit at a much reduced level compared to the rescue achieved in the brain.
UNASSIGNED: In situ hybridization and immunostaining of histological brain sections using a subset of molecular markers that label specific cortical regions or cell types. Appropriate quantification and statistical analysis of gene and protein expression were applied to identify cortical abnormalities and to determine the level of phenotypic rescue achieved.
UNASSIGNED: Cortical abnormalities observed in Tbx1 mutant embryos were fully rescued by both drugs. Intriguingly, rescue was obtained with both drugs in Tbx1 homozygous mutants, indicating that they function through mechanisms that do not depend upon Tbx1 function. This was particularly surprising for Vitamin B12, which was identified through its ability to increase Tbx1 gene expression.
UNASSIGNED: To our knowledge, this is only the second example of drugs to be identified that ameliorate phenotypes caused by the mutation of a single gene from the 22q11.2 homologous region of the mouse genome. This one drug-one gene approach might be important because there is evidence that the brain phenotype in 22q11.2DS patients is multigenic in origin, unlike the physical phenotypes, which are overwhelmingly attributable to Tbx1 haploinsufficiency. Therefore, effective treatments will likely involve the use of multiple drugs that are targeted to the function of specific genes within the deleted region.

TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1.
Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1.
We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6.
Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.

BACKGROUND: DNA methylation is the best epigenetic mechanism for explaining the interactions between nutrients and genes involved in intrauterine growth and development programming. A possible contributor of methylation abnormalities to congenital heart disease is the folate methylation regulatory pathway; however, the mechanisms and methylation patterns of VSD-associated genes are not fully understood.
OBJECTIVE: To determine if maternal dietary intake of folic acid (FA) is related to the methylation status (MS) of VSD-associated genes (AXIN1, MTHFR, TBX1, and TBX20).
METHODS: Prospective case-control study; 48 mothers and their children were evaluated. The mothers\' dietary variables were collected through a food frequency questionnaire focusing on FA and the consumption of supplements with FA. The MS of promoters of genes was determined in the children.
RESULTS: The intake of FA supplements was significantly higher in the control mothers. In terms of maternal folic acid consumption, significant differences were found in the first trimester of pregnancy. Significant differences were observed in the MS of MTHFR and AXIN1 genes in VSD and control children. A correlation between maternal FA supplementation and MS of AXIN1 and TBX20 genes was found in control and VSD children, respectively.
CONCLUSIONS: A lower MS of AXIN1 genes and a higher MS of TBX20 genes is associated with FA maternal supplementation.

Congenital heart disease (CHD) is the most common life-threatening congenital anomaly. CHD occurs due to defects in cardiovascular development, and the majority of CHDs are caused by a multifactorial inheritance mechanism, which refers to the interaction between genetic and environmental factors. During embryogenesis, the cardiovascular system is derived from at least four distinct cell lineages: the first heart field, second heart field, cardiac neural crest, and proepicardial organ. Understanding the genes involved in each lineage is essential to uncover the genomic architecture of CHD. Therefore, we provide an overview of recent research progress using animal models and mutation analyses to better understand the molecular mechanisms and pathways linking cardiovascular development and CHD. For example, we highlight our recent work on genes encoding three isoforms of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) that regulate various vital and developmental processes, which have genetic redundancy during cardiovascular development. Specifically, IP3R1 and 2 have redundant roles in the atrioventricular cushion derived from the first heart field lineage, whereas IP3R1 and 3 exhibit redundancy in the right ventricle and the outflow tract derived from the second heart field lineage, respectively. Moreover, 22q11.2 deletion syndrome (22q11DS) is highly associated with CHD involving the outflow tract, characterized by defects of the cardiac neural crest lineage. However, our studies have shown that TBX1, a major genetic determinant of 22q11DS, was not expressed in the cardiac neural crest but rather in the second heart field, suggesting the importance of the cellular interaction between the cardiac neural crest and the second heart field. Comprehensive genetic analysis using the Japanese genome bank of CHD and mouse models revealed that a molecular regulatory network involving GATA6, FOXC1/2, TBX1, SEMA3C, and FGF8 was essential for reciprocal signaling between the cardiac neural crest and the second heart field during cardiovascular development. Elucidation of the genomic architecture of CHD using induced pluripotent stem cells and next-generation sequencing technology, in addition to genetically modified animal models and human mutation analyses, would facilitate the development of regenerative medicine and/or preventive medicine for CHD in the near future.