Holt-Oram Research Articles

MiR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

P1801Multiple members with an isolated atrial septal defect phenomenon presented in a family with Holt-Oram syndrome

Abstract Background Holt-Oram syndrome (HOS), characterized by upper limb malformations, congenital heart diseases (CHD) and/or cardiac conduction abnormalities, is an autosomal dominant disease. Almost all the HOS patients suffer from cardiac and limb abnormalities at the same time. Among them, atrial septal defect (ASD) is the most common cardiac anomaly. The unusual phenotype in a pedigree including multiple members with isolated ASD and a few with isolated limb deformity attracted attention. Aim To detect mutant genes of ASD in this pedigree and make a definitive diagnosis. To investigate the mutant type of the gene and illustrate the possible mechanism of heterogeneous phenotype. Methods Echocardiography, electrocardiography and physical examination were given to a four-generation Chinese HOS family. Of all 11 patients in this pedigree, eight patients had isolated ASD and one patient had isolated finger deformity. In addition, two patients suffered from both diseases (Figure. 1). Whole Exome Sequencing was performed on the proband and his relatives including three with ASD only, one with finger deformity only and one normal person. Sanger sequencing was performed on biological relatives in this pedigree to valid rare variants. Single nucleotide polymorphisms and insertions/deletions were identified using the GATK program. Pathogenicity was predicted in software like SIFT, Ployphen-2, MutationTaster and CADD_phred. Results An exon site mutation (c.100dupG:p.Ala34fs) of TBX5 was detected in all the patients of this pedigree. This mutation site is located in front of T-box and most probably leads to haploinsufficiency of TBX5 protein. In addition, only 42 variants (1 splicing single nucleotide variants (SNV), 15 insertions/deletions, 24 nonsynonymous SNVs, 1 stopgain SNVs and 1 unknown) were found in 29 genes, which are related to cardiac disease. Figure 1 Conclusions Our study revealed a mutation (c.100dupG:p.Ala34fs) of TBX5 and further demonstrated the possibility of HOS-related TBX5 mutation in patients with congenital ASD only. This study clarified the diagnosis of this pedigree, provided genetic counseling and promoted the prevention and treatment of HOS. Further research is needed on mechanisms that may lead to phenotypic variation between and within families, such as random monoallelic expression. Acknowledgement/Funding None

Abstract 602: Mir-182-5p is a Conserved Downstream Effector of Tbx5 Involved in Heart Development and Arrhythmia in Zebrafish

Background: TBX5 mutations cause Holt-Oram syndrome (HOS) characterized by upper limb and cardiac malformations, but can also contribute to early-onset of atrial fibrillation. Focusing on miRNAs involved in TBX5 regulatory circuits with a cardiac relevant role, we identified miR-182-5p, belonging to miR-183 cluster, found upregulated in Tbx5-depleted hearts of mouse and zebrafish embryos. Methods: To functionally analyse the miR-182-5p role in developing heart, miR-182-5p was dysregulated in zebrafish zygotes of Tg(Myl7:EGFP) and Tg(myl7:gCaMP) transgenic lines. To stably deregulate miR-182-5p in zebrafish heart we exploited the Gal4/UAS system to restrict the miR-182 expression into cardiac context. For physiological analyses we performed the mechanogram of cardiac contraction and electrocardiogram recording. To understand miR-182-5p downstream regulation, in silico analyses, followed by ddPCR/real-time quantifications on dissected zebrafish hearts and rescue experiments both in transient and stable miR-182-5p overexpressing zebrafish embryos were performed. Results: Depletion of Tbx5 from cardiomyocytes increased the expression of miR-182 cluster family that is controlled by Kruppel-like factor 4 (KLF4), a transcription factor repressed by Tbx5. Both transient and stable upregulation of miR-182 in zebrafish affect heart morphology, calcium handling and the onset of arrhythmia while its cardiac-specific downregulation decreases cardiac defects in zebrafish HOS hearts. Expression analyses on selected miR-182-5p putative targets revealed that several calcium channel proteins resulted downregulated in miR-182-5p overexpressing hearts. Transgenic zebrafish line stably overexpressing miR-182-5p in the heart manifested arrhythmia overtime with or without cardiac structural defects. Conclusion: We identified miR-182-5p as a potential suitable target to interfere in the circuit between upstream genetic abnormalities and downstream effectors leading to arrhythmia occurrence.

Tbx5 inhibits hedgehog signaling in determination of digit identity.

Dominant TBX5 mutation causes Holt-Oram syndrome (HOS), which is characterized by limb defects in humans, but the underlying mechanistic basis is unclear. We used a mouse model with Tbx5 conditional knockdown in Hh-receiving cells (marked by Gli1+) during E8 to E10.5, a previously established model to study atrial septum defects, which displayed polydactyly or hypodactyly. The results suggested that Tbx5 is required for digit identity in a subset of limb mesenchymal cells. Specifically, Tbx5 deletion in this cell population decreased cell apoptosis and increased the proliferation of handplate mesenchymal cells. Furthermore, Tbx5 was found to negatively regulate the Hh-signaling activity through transcriptional regulation of Ptch1, a known Hh-signaling repressor. Repression of Hh-signaling through Smo co-mutation in Tbx5 heterozygotes rescued the limb defects, thus placing Tbx5 upstream of Hh-signaling in limb defects. This work reveals an important missing component necessary for understanding not only limb development but also the molecular and genetic mechanisms underlying HOS.

Holt-Oram Sendromlu Hastada Primum Tip Atriyal Septal Defekt ve Patent Foramen Ovale Birlikteliği

Holt-Oram Sendromlu Hastada Primum Tip Atriyal Septal Defekt ve Patent Foramen Ovale Birlikteliği

DNA‐binding specificity of TBX5 and its coding variants associated with Holt‐Oram Syndrome

Transcription factors (TFs) are important sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. Evaluation of the DNA‐binding properties of transcription factors is fundamental for understanding their possible roles in controlling gene regulatory networks. TBX5 is a transcription factor that has been identified as an important regulator in organ development. TBX5 mutations have been associated to Holt‐Oram Syndrome, a genetic disorder that affects upper limb formation and often the heart. We aim to determine the specificity of DNA‐binding of TBX5 and its T‐Box DNA‐binding domain and how it changes when the protein mutates. We have cloned TBX5 and its T‐Box domain that contains a His‐ and Strep‐Tag affinity tags that we plan to use for purifying after we overexpress it in E. coli cells. We will then analyze the DNA‐binding specificity of TBX5 and the changes it conveys when it mutates using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential enrichment (HT‐SELEX), as this will allow us to better understand the impact of TBX5 mutations on its function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Combined Use of Ultrasound and Fetal Magnetic Resonance Imaging for a Comprehensive Fetal Neurological Assessment in Fetal Congenital Cardiac Defects: Scientific Impact Paper No. 60.

Heart problems are common in newborn babies, affecting approximately 5-10 in 1000 babies. Some are more serious than others, but most babies born with heart problems do not have other health issues. Of those babies who have a serious heart problem, almost 1 in 4 will have heart surgery in their first year. In the UK, pregnant women are offered a scan at around 20weeks to try and spot any heart problems. In most cases there is not a clear reason for the problem, but sometimes other issues, such as genetic conditions, are discovered. In recent years the care given to these babies after they are born has improved their chances of surviving. However, it is recognised that babies born with heart problems have a risk of delays in their learning and development. This may be due to their medical condition, or as a result of surgery and complications after birth. In babies with heart problems, there is a need for more research on ultrasound and magnetic resonance imaging (MRI) to understand how the brain develops and why these babies are more likely to have delays in learning and development. This paper discusses the way ultrasound and MRI are used in assessing the baby's brain. Ultrasound is often used to spot any problems, looking at how the baby's brain develops in pregnancy. Advances in ultrasound technologies have made this easier. MRI is well-established and safe in pregnancy, and if problems in the brain have been seen on ultrasound, MRI may be used to look at these problems in more detail. While it is not always clear what unusual MRI findings can mean for the baby in the long term, increased understanding may mean parents can be given more information about possible outcomes for the baby and may help to improve the counselling they are offered before their baby's birth.

Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Holt–Oram syndrome – Case series of two reports

Holt–Oram syndrome is a rare genetic autosomal dominant disorder which affects the upper limbs and heart. It is also known as “heart–hand” syndrome or “atriodigital dysplasia.” The present article describes the clinical and radiological images of the features of Holt–Oram syndrome in two patients.

Unilateral and isolated absence of opponens pollicis and adductor pollicis: Could it be Cavanagh's syndrome?

Functions of the human hand such as grasping and releasing require synchronous action of thenar muscles and thereby require considerable mechanical complexity. Isolated absence of thenar muscles is often reported in association with syndromes such as Cavanagh's syndrome or Holt–Oram syndrome (in addition to cardiac defects). During routine anatomical dissection of an approximately 55-year-old male formalin-embalmed cadaver, we observed a unique variation in the left palm where opponens pollicis and adductor pollicis muscle were completely devoid of muscle fibers and solely constituted by fibrous tissue. We could not make out any other nervous/vascular/musculoskeletal abnormalities or signs of surgical procedures on the left superior extremity. We followed a step-wise approach rule out possible clinical syndromes and etiologies. The presumable hypothesis would be focal deficiency in the proliferation of premyogenic cells in the limb bud. This rare variation would be of paramount importance to plastic surgeons who can offer surgical correction (tendon transfers) if presented at earlier ages.

Síndrome de Holt-Oram y Trastorno del Espectro Autista. A propósito de un caso

El Síndrome de Holt-Oram o displasia atriodigital es un trastorno de herencia autosómica dominante con un 100% de penetrancia que afecta a uno de cada 100000 nacimientos, causado por la mutación del gen TBX5 (12q24.1), responsable de la cardiogénesis y de la formación de los miembros superiores, por lo que se caracteriza por la presencia de anomalías y malformaciones esqueléticas y cardiológicas. Se describe el caso de un varón de 8 años con diagnóstico de Síndrome de Holt-Oram y de Trastorno del Espectro Autista.

Holt-Oram syndrome: clinical and molecular description of 78 patients with TBX5 variants.

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterised by the association of congenital heart defect (CHD), with or without rhythm disturbances and radial defects, due to TBX5 variants. The diagnosis is challenged by the variability of expression and the large phenotypic overlap with other conditions, like Okihiro syndrome, TAR syndrome or Fanconi disease. We retrospectively reviewed 212 patients referred for suspicion of HOS between 2002 and 2014, who underwent TBX5 screening. A TBX5 variant has been identified in 78 patients, representing the largest molecular series ever described. In the cohort, 61 met the previously described diagnostic criteria and 17 have been considered with an uncertain HOS diagnosis. A CHD was present in 91% of the patients with a TBX5 variant, atrial septal defects being the most common (61.5%). The genotype-phenotype study highlights the importance of some critical features in HOS: the septal characteristic of the CHD, the bilateral and asymmetric characteristics of the radial defect and the presence of shoulder or elbow mobility defect. Besides, 21 patients presented with an overlapping condition. Among them, 13 had a typical HOS presentation. We discuss the strategies that could be adopted to improve the molecular delineation of the remaining typical patients.

A transcriptomics analysis of the Tbx5 paralogues in zebrafish.

TBX5 is essential for limb and heart development. Mutations in TBX5 are associated with Holt-Oram syndrome in humans. Due to the teleost specific genome duplication, zebrafish have two copies of TBX5: tbx5a and tbx5b. Both of these genes are expressed in regions of the lateral plate mesoderm and retina. In this study, we perform comparative RNA sequencing analysis on zebrafish embryos during the stages of lateral plate mesoderm migration. This work shows that knockdown of the Tbx5 paralogues results in altered gene expression in many tissues outside of the lateral plate mesoderm, especially in the somitic mesoderm and the intermediate mesoderm. Specifically, knockdown of tbx5b results in changes in somite size, in the differentiation of vasculature progenitors and in later patterning of trunk blood vessels.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

Electrical disorders in atrial septal defect: genetics and heritability.

Atrial septal defect (ASD) is one of the most common types of congenital heart diseases (CHDs). Most ASDs occur sporadically, but some are inherited and associated with cardiac conduction defects such as atrioventricular block (AVB) or bundle branch block. Mutations in genes encoding transcription factor gene TBX5 and NKX2-5, were found in Holt-Oram syndrome (HOS) and ASD with atrioventricular (AV) conduction defects, respectively. HOS is characterized by upper limb anomaly in addition to ASD and AVB (heart-hand syndrome). ASD associated with NKX2-5 is rare but is reported to cause sudden cardiac death (SCD) or cardiomyopathy. We provide a review of these two diseases.

Holt-Oram Syndrome: A Variety of Heart-Hand Syndrome

Holt-Oram syndrome (HOS) is a heart-upper limb malformation complex , is a rare autosomal dominant disorder, caused by a mutation in the TBX5 gene located on chromosome 12 and near complete penetrance but variable expression. Holt and Oram first described this syndrome in 1960. Approximately 40% of cases represent new mutations. The syndrome is associated with defective development of cardiac structures that results in atrial septal defect(ASD), most commonly the secundum type; heart block of varying degree or both.The syndrome is characterized by cardiac malformations and aplasia or hypoplasia of the thumb. The incidence of HOS is estimated at 1:100,000 live births. In the literature, it is also known as atriodigital syndrome, heart-hand syndrome, upper limb-cardiovascular syndrome, cardiac-limb syndrome, or cardiomelic syndrome. Herein, we report a 45-year-old female case of HOS presenting a large ostium secundum type ASD along with congenitally fusion of carpal bones in both hands and absence of right first metacarpal bone with triphalagial thumb where as hypoplastic proximal phalange. In left hand, hypoplastic first metacarpal bone and hypoplastic thumb with syndactyly of both thumb with index finger.University Heart Journal Vol. 13, No. 2, July 2017; 67-71

T-box genes and retinoic acid signaling regulate the segregation of arterial and venous pole progenitor cells in the murine second heart field.

The arterial and venous poles of the mammalian heart are hotspots of congenital heart defects (CHD) such as those observed in 22q11.2 deletion (or DiGeorge) and Holt-Oram syndromes. These regions of the heart are derived from late differentiating cardiac progenitor cells of the Second Heart Field (SHF) located in pharyngeal mesoderm contiguous with the elongating heart tube. The T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion syndrome gene, regulates SHF addition to both cardiac poles from a common progenitor population. Despite the significance of this cellular addition the mechanisms regulating the deployment of common progenitor cells to alternate cardiac poles remain poorly understood. Here we demonstrate that Tbx5, mutated in Holt-Oram syndrome and essential for venous pole development, is activated in Tbx1 expressing cells in the posterior region of the SHF at early stages of heart tube elongation. A subset of the SHF transcriptional program, including Tbx1 expression, is subsequently downregulated in Tbx5 expressing cells, generating a transcriptional boundary between Tbx1-positive arterial pole and Tbx5-positive venous pole progenitor cell populations. We show that normal downregulation of the definitive arterial pole progenitor cell program in the posterior SHF is dependent on both Tbx1 and Tbx5. Furthermore, retinoic acid (RA) signaling is required for Tbx5 activation in Tbx1-positive cells and blocking RA signaling at the time of Tbx5 activation results in atrioventricular septal defects at fetal stages. Our results reveal sequential steps of cardiac progenitor cell patterning and provide mechanistic insights into the origin of common forms of CHD.

Investigation of T-box gene function in patterning second heart field progenitor cells

The heart is derived from two sources of progenitor cells: the first heart field giving rise to the left ventricle and atria, and the Second Heart Field (SHF) that progressively contributes to the right ventricle, outflow tract and parts of the atria. Perturbation of SHF cell addition leads to a spectrum of congenital heart defects, exemplified by conotruncal and atrioventricular septal defects observed in mouse embryos lacking the T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion (DiGeorge) syndrome candidate gene. Tbx1 is expressed in the SHF where it regulates proliferation, differentiation and epithelial properties. Tbx1 is also required for the segregation of distinct anterior and posterior subpopulations of the SHF giving rise to arterial and venous pole myocardium, respectively. Here we show using genetic lineage tracing and immunofluorescence labeling that a second T-box gene Tbx5 , implicated in Holt-Oram syndrome, is activated in Tbx1-positive pharyngeal mesoderm in response to retinoic acid signaling at the onset of progenitor cell segregation. Loss of retinoic acid signaling results in failure to establish the posterior SHF domain and atrioventricular septal defects. SHF cells coexpressing Tbx1 and Tbx5 subsequently downregulate Tbx1 expression, in a Tbx1-dependent manner, leading to the emergence of a transcriptional boundary between anterior and posterior SHF domains. Analysis of Tbx5 lineage and conditional loss of function implicate de novo activation of Tbx5 in boundary formation. Our results provide new insights into progenitor cell patterning during SHF deployment and the origins of common forms of congenital heart defects.

Abductor digiti minimi opponensplasty for a patient with Cavanagh’s syndrome: A case report

Thumb hypoplasia only in the intrinsic thenar muscles is a rare condition; this defect might be accompanied with cardiac diseases (Holt-Oram syndrome), ocular anomalies, and vascular anomalies of the hand and wrist (Okihiro syndrome). In addition, this condition may be detected in hypereosinophilic syndrome (HES), which gives rise to other hand anomalies, as well. Unilateral or bilateral absence of isolated thenar muscles is another rare abnormality.In this study, we presented a case of Cavanagh’s syndrome in an eight-year-old boy with right-sided thenar hypoplasia who had difficulty in thumb opposition. For the treatment, the patient underwent abductor digiti minimi (ADM) opponensplasty. After three years of follow-up, he regained thumb opposition and was symptom-free. Care must be taken to avoid misdiagnosis of thenar atrophy/hypoplasia with carpal tunnel syndrome in case of children.

Holt-Oram Syndrome With Multiple Cardiac Abnormalities

Holt-Oram syndrome (HOS) is a rare monogenic disorder characterized by upper limb abnormalities, congenital heart defects and/or conduction abnormalities. It is determined by mutations of TBX5 gene and is inherited in an autosomal dominant manner. Penetrance is complete, but variable expressivity is present, which gives sometimes diagnostic difficulties. Our case is a young adult with a personal history of preaxial polydactyly operated in infancy, multiple cardiac malformations (atrial septal defect, bicuspid aortic valve, left ventricular non-compaction) and radiologic findings consistent with HOS. Family history is negative for HOS. In conclusion, we present a case of HOS diagnosed in the adult period to highlight the diagnostic problems for the proband and the family and the importance of an early diagnostic.