Mutations In TBX5 Research Articles

Interaction between Tbx1 and HoxD10 and connection with TGFβ-BMP signal pathway during kidney development

Renal malformations are commonly found among patients carrying a 22q11 deletion which renders loss of Tbx1 gene, an important transcriptional factor implicated in a number of developmental processes. Smad1 is known to interact with Tbx1, but the exact mechanism remains unknown. In this study, we have measured the expression of Tbx1 in both murine and human tissues using RT-PCR, and analyzed its protein product and protein-protein interactions with Western blotting and immunoprecipitation assays. Precipitated proteins were verified with mass spectrometry. As discovered, Tbx1 binds with Hoxd10. Tbx1 and Hoxd10 genes also have similar expression profiles during murine kidney development. Based on homology between mouse and human, we hypothesized that such interaction also exists in human. Through a RNA interference experiment using a human embryonic kidney HEK293 cell line, we demonstrated that TBX1 can alter TGF-β/BMP, an important signaling pathway, through interacting with HOXD10. Above findings may shed light on the mechanism of TBX1 mutations leading to renal malformations found in patients carrying a 22q11 deletion.

Modifying Mendel

Congenital cardiovascular malformations (CVMs) are the most common birth defect, affecting approximately 8 per 1000 live births. Roughly 25% of CVMs occur in the context of multiple congenital anomalies or as part of a genetic syndrome, while the other 75% of individuals present as an isolated, nonsyndromic CVM.1 Genomic disorders comprise the majority of syndomic CVMs, exemplified by aneuploidies such as trisomy 21 (Down syndrome) or monosomy X (Turner syndrome) and copy number variations (CNVs) such as deletion 22q11.2 (Velocardiofacial syndrome) and deletion 7q11.23 (Williams syndrome). There are also a few phenotypically well-characterized syndromes with CVMs that occur due to pathogenic variants in a single gene. These include the group of Noonan, Costello, and Cardiofaciocutaneous syndromes resulting from mutations in genes coding for proteins of the Ras pathway, and Holt-Oram syndrome which is caused by mutations in the gene TBX5 .2–4 Article see p 301 These single-gene syndromes are good examples of how human genetic studies have been very successful at identifying genes causing typical, highly penetrant Mendelian diseases. A few individuals with isolated, nonsyndromic CVMs possess this mode of inheritance, and such families have proven useful at identifying CVM causing genes such as NOTCH1 and ZIC3. 5,6 However, these types of families are few and far between. The vast majority of families have a strong genetic component for their CVM, measured by high recurrence rates and high heritability,7 but a complex pattern of inheritance that may include reduced penetrance, polygenic inheritance (genetic burden with susceptibility thresholds), and combinations of gene and environment interactions and modifiers. Even aneuploidies known to cause CVMs, such as trisomy 21 (T21), have far from universal occurrence of CVMs. For example, although CVMs in general, and atrioventricular septal defects in particular, are known to be associated with T21, …

The ulnar-mammary syndrome gene,Tbx3, is a direct target of the retinoic acid signaling pathway, which regulates its expression during mouse limb development

TBX3, a member of the T-box transcription factor gene family, is a transcriptional repressor that is required for the development of the heart, limbs, and mammary glands. Mutations in TBX3 that result in reduced functional protein lead to ulnar-mammary syndrome, a developmental disorder characterized by limb, mammary gland, tooth, and genital abnormalities. Increased levels of TBX3 have been shown to contribute to the oncogenic process, and TBX3 is overexpressed in several cancers, including breast cancer, liver cancer, and melanoma. Despite its important role in development and postnatal life, little is known about the signaling pathways that modulate TBX3 expression. Here we show, using in vitro and in vivo assays, that retinoic acid (RA) activates endogenous TBX3 expression, which is mediated by an RA-receptor complex directly binding and activating the TBX3 promoter, and we provide evidence that this regulation may be functionally relevant in mouse embryonic limb development. Our data identify TBX3 as a direct target of the RA signaling pathway and extend our understanding of the role and regulation of TBX3 in limb development.

Endothelial Neuropilin Disruption in Mice Causes DiGeorge Syndrome-Like Malformations via Mechanisms Distinct to Those Caused by Loss of Tbx1

The spectrum of human congenital malformations known as DiGeorge syndrome (DGS) is replicated in mice by mutation of Tbx1. Vegfa has been proposed as a modifier of DGS, based in part on the occurrence of comparable phenotypes in Tbx1 and Vegfa mutant mice. Many additional genes have been shown to cause DGS-like phenotypes in mice when mutated; these generally intersect in some manner with Tbx1, and therefore impact the same developmental processes in which Tbx1 itself is involved. In this study, using Tie2Cre, we show that endothelial-specific mutation of the gene encoding the VEGFA coreceptor neuropilin-1 (Nrp1) also replicates the most prominent terminal phenotypes that typify DGS. However, the developmental etiologies of these defects are fundamentally different from those caused by absence of TBX1. In Tie2Cre/Nrp1 mutants, initial pharyngeal organization is normal but subsequent pharyngeal organ growth is impaired, second heart field differentiation is normal but cardiac outflow tract cushion organization is distorted, neural crest cell migration is normal, and palatal mesenchyme proliferation is impaired with no change in apoptosis. Our results demonstrate that impairment of VEGF-dependent endothelial pathways leads to a spectrum of DiGeorge syndrome-type malformations, through processes that are distinguishable from those controlled by Tbx1.

TBX5 intragenic duplication: a family with an atypical Holt–Oram syndrome phenotype

Holt-Oram syndrome (HOS) is a rare autosomal dominant heart-hand syndrome due to mutations in the TBX5 transcription factor. Affected individuals can have structural cardiac defects and/or conduction abnormalities, and exclusively upper limb defects (typically bilateral, asymmetrical radial ray defects). TBX5 mutations reported include nonsense, missense, splicing mutations and exon deletions. Most result in a null allele and haploinsufficiency, but some impair nuclear localisation of TBX5 protein or disrupt its interaction with co-factors and downstream targets. We present a five generation family of nine affected individuals with an atypical HOS phenotype, consisting of ulnar ray defects (ulnar hypoplasia, short fifth fingers with clinodactyly) and very mild radial ray defects (short thumbs, bowing of the radius and dislocation of the radial head). The cardiac defects seen are those more rarely reported in HOS (atrioventricular septal defect, hypoplastic left heart syndrome, mitral valve disease and pulmonary stenosis). Conduction abnormalities include atrial fibrillation, atrial flutter and sick sinus syndrome. TBX5 mutation screening (exons 3-10) identified no mutations. Array comparative genomic hybridisation (CGH) revealed a 48 kb duplication at 12q24.21, encompassing exons 2-9 of the TBX5 gene, with breakpoints within introns 1-2 and 9-10. The duplication segregates with the phenotype in the family, and is likely to be pathogenic. This is the first known report of an intragenic duplication of TBX5 and its clinical effects; an atypical HOS phenotype. Further functional studies are needed to establish the effects of the duplication and pathogenic mechanism. All typical/atypical HOS cases should be screened for TBX5 exon duplications.

Lethal arrhythmias in Tbx3 -deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis

TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.

Holt–Oram syndrome: novel TBX5 mutation and associated anomalous right coronary artery

The Holt-Oram syndrome was confirmed in an asymptomatic 36-year-old man by a novel TBX5-gene mutation (exon 8 acceptor splicing site, c.663-1G greater than A). Computed tomography showed an atrial septal defect and an anomalous right coronary artery crossing between the aorta and pulmonary arteries. Surgery corrected the septal defect and the initial segment of the anomalous vessel was unroofed and enlarged. Anomalous coronary arteries were not previously described in the Holt-Oram syndrome patients and should be added to the list of possible associated cardiac defects.

The face of Ulnar Mammary syndrome?

Ulnar Mammary syndrome (UMS) is an autosomal disorder caused by haploinsufficiency of the TBX3 gene. There is marked intrafamilial variation in expression of the syndrome. We present one three generation family in which the proband has absence of the right ulna and third, fourth and fifth rays in her right hand. Her mother and maternal grandmother have more subtle anomalies while all have a similar facial appearance with a broad nasal tip, a broad jaw, a prominent chin and a tongue frenulum. They have a single base pair insertion (c. 992dup) in TBX3. We compare faces from the handful of published UMS patients which include photographs, this family and four other cases with TBX3 mutations. All have similarities in appearance which we suggest could alert clinicians to the possibility of a TBX3 mutation if individuals present with more subtle features of UMS such as postaxial polydactyly, isolated 5th finger anomalies, delayed puberty in males, breast hypoplasia or short stature with or without growth hormone deficiency.

Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

BackgroundThe tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.MethodsWild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.ResultsApoptosis-related genes (bad, bax, and bcl2), and cell cycle-related genes (cdk2, pcna, p27, and p57) showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.ConclusionOur study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos.

Síndrome de Holt-Oram: descripción de 7 casos

FUNDAMENTAL AND OBJECTIVE: Holt-Oram syndrome (HOS) is a heart-hand disease with an autosomal dominant inheritance pattern. About 85% of the affected patients present de novo mutations in the TBX5 gene. The aim of this study is to propose a molecular strategy to diagnose patients with clinical suspicion of HOS. A sequence analysis of 7 patients from exon 2 to exon 8 of the TBX5 gene was performed. MLPAp179 and MLPAp180 were performed in those cases in which no mutation was found. p.Arg270X and p.Ala34Glyfsx27 mutations were identified in 2 cases. These cases fulfilled the strict clinical criteria, had a family history of HOS and had similar clinical features. In other three cases, MLPA results showed deletions of the GLI3 coding region. In order to increase the TBX5 mutation detection rate, an exhaustive physical examination focused on the strict clinical criteria may be necessary to rule out clinical overlapping syndromes. We propose that molecular analysis of GLI3 may be performed in patients with clinical suspicion of HOS without mutations in TBX5.

Tbx1 regulatesVegfr3and is required for lymphatic vessel development

Lymphatic dysfunction causes several human diseases, and tumor lymphangiogenesis is implicated in cancer spreading. TBX1 is the major gene for DiGeorge syndrome, which is associated with multiple congenital anomalies. Mutation of Tbx1 in mice recapitulates the human disease phenotype. In this study, we use molecular, cellular, and genetic approaches to show, unexpectedly, that Tbx1 plays a critical role in lymphatic vessel development and regulates the expression of Vegfr3, a gene that is essential for lymphangiogenesis. Tbx1 activates Vegfr3 transcription in endothelial cells (ECs) by binding to an enhancer element in the Vegfr3 gene. Conditional deletion of Tbx1 in ECs causes widespread lymphangiogenesis defects in mouse embryos and perinatal death. Using the mesentery as a model tissue, we show that Tbx1 is not required for lymphatic EC differentiation; rather, it is required for the growth and maintenance of lymphatic vessels. Our findings reveal a novel pathway for the development of the lymphatic vessel network.

Comparing Pumps

Comparing Pumps

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.

Tbx4 and Tbx5 Acting in Connective Tissue Are Required for Limb Muscle and Tendon Patterning

SummaryProper functioning of the musculo-skeletal system requires the precise integration of bones, muscles and tendons. Complex morphogenetic events ensure that these elements are linked together in the appropriate 3D configuration. It has been difficult, however, to tease apart the mechanisms that regulate tissue morphogenesis. We find that deletion of Tbx5 in forelimb (or Tbx4 in hindlimbs) specifically affects muscle and tendon patterning without disrupting skeletal development thus suggesting that distinct cues regulate these processes. We identify muscle connective tissue as the site of action of these transcription factors and show that N-Cadherin and β-Catenin are key downstream effectors acting in muscle connective tissue regulating soft-tissue morphogenesis. In humans, TBX5 mutations lead to Holt-Oram syndrome, which is characterised by forelimb musculo-skeletal defects. Our results suggest that a focus on connective tissue is required to understand the aetiology of diseases affecting soft tissue formation.

Identification and characterisation of the developmental expression pattern of tbx5b, a novel tbx5 gene in zebrafish

Tbx5 is a T-box transcription factor that has been characterised in most vertebrate lineages and is widely expressed during the development of various embryonic structures, including the heart, the eyes and the anterior set of paired appendages (tetrapod forelimbs and fish pectoral fins). Mutations in TBX5 cause Holt–Oram syndrome, an autosomal dominant human “heart-hand” condition characterised by upper limb and heart malformations. In zebrafish, embryos with compromised tbx5 function show a complete absence of pectoral fins, whereas heart and eye development are not so highly disturbed. Here, we identify a new tbx5 gene in zebrafish that we have called tbx5b. This duplicate gene is present in all teleost genomes whose sequence is available, suggesting it resulted from the teleost-specific genome duplication event that took place during fish evolution. We show that tbx5b has lost the characteristic forelimb/pectoral fin expression of Tbx5 genes but has retained the eye and heart expression, partially overlapping with that of its paralogue, now referred to as tbx5a. Functional redundancy of tbx5a and tbx5b in the eye and heart would therefore explain the mild phenotypes observed during development of these organs in fish embryos with compromised tbx5a function.

Great vessel development requires biallelic expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice

Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/-;Chd7+/- double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

BackgroundOstium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause...

T-box 3 is expressed in the adult mouse hypothalamus and medulla

Using microarray analysis, in situ hybridization and immunocytochemistry, we found that the transcription factor TBX3 is produced in three discrete neuronal populations of the adult mouse brain, the arcuate nucleus (including in NPY but not dopaminergic neurons), the histaminergic tuberomammillary nucleus and in cholinergic neurons of the solitary tract nucleus. The immunoreactive protein had a nuclear location in these neurons, consistent with its function as a transcription factor. Although the function of tbx3 in these neurons is unknown, a review of the literature strongly suggests that these neuronal populations may be abnormal in Ulnar-Mammary syndrome patients with tbx3 mutations, explaining previously overlooked phenotypes in this syndrome, such as obesity, sexual dysfunction and possibly sleep abnormalities.

Functional Role of Transcriptional Factor TBX5 in Pre-mRNA Splicing and Holt-Oram Syndrome via Association with SC35

TBX5 is a T-box transcriptional factor required for cardiogenesis and limb development. TBX5 mutations cause Holt-Oram syndrome characterized by congenital heart defects and upper limb deformations. Here we establish a novel function for TBX5 in pre-mRNA splicing, and we show that this function is relevant to the pathogenesis of Holt-Oram syndrome, providing a novel pathogenic mechanism for the disease. Proteomics in combination with affinity purification identifies splicing factor SC35 as a candidate TBX5-associating protein. Co-immunoprecipitation and glutathione S-transferase pulldown assays confirm the complex formation between TBX5 and SC35. TBX5 can bind to RNA homopolymers (polyribonucleotides) and to the 5'-splice site, which overrides the binding of SC35 to the same RNA. Overexpression of TBX5 increases the efficiency of pre-mRNA splicing and regulates alternative splice site selection. However, co-expression of TBX5 and SC35 antagonizes each other's positive effect on splicing. The most severe TBX5 mutation, G80R, with complete penetrance of the cardiac phenotype, strongly affects pre-mRNA splicing, whereas other mutations with incomplete penetrance of the cardiac phenotype, including R237Q, do not alter the splicing activity of TBX5. This study establishes TBX5 as the first cardiac gene and the first human disease gene with dual roles in both transcriptional activation and pre-mRNA splicing.

S06-01 Cardiac transcription factors in development and disease

The homeodomain factor Nkx2-5 is expressed in cardiac progenitor cells of the first and second heart fields, and in differentiating cardiomyocytes and conduction cells. NKX2-5 mutations cause congenital heart disease (CHD) and conduction disease (CD) in humans. Nkx2-5 acts as both a repressor and an activator, and one of its early roles is to repress, through a negative feedback loop, the expression of the Bmp2 gene, which encodes one of the upstream inducers of cardiogenesis and is itself a repressor of cardiac progenitor cell proliferation. In this way Nkx2-5 orchestrates a transition between a period of cardiac induction and one of heart progenitor cell expansion and deployment. We have also studied the Nkx2-5 transcriptional cofactor, Tbx20, a member of the T-box transcription factor family, and shown that Tbx20 mutations also cause CHD as well as adult-onset dilated cardiomyopathy (DCM). The T-box DNA binding domain of Tbx20 has unique biophysical properties, with its tertiary hydrophobic interactions existing in a state of dynamic equilibrium akin to a molten globule, a state that may facilitate formation and/or stability of transcriptional complexes. Gain-of-function mutations can exacerbate this state. These studies address CHD at the molecular, cellular and organ level and contribute to an understanding of CHD origins and patient management.