Holt-Oram Research Articles

Radial Ray Anomaly with Associated Ventricular Septal Defect - Case Report with Review of Literature.

Ultrasound examination is used for the assessment of abnormal findings on prenatal screening. Radial ray defect can be screened by using ultrasonography. Abnormal findings can be detected quickly by having the understanding of the etiology, pathophysiology and embryology. It is a rare congenital defect that may be isolated or associated with other anomalies including Fanconi's syndrome and Holt-Oram syndrome. We report the case of a 28-year-old woman (G2P1L1) who presented for routine antenatal ultrasound at 25 weeks 0 days according to the last menstrual period. The patient did not have any level-II antenatal anomaly scan done. An ultrasound was performed, and the gestational age according to the ultrasound scan was 24 weeks and 3 days. In this paper, we present a brief review of embryology and critical practical points, and report a rare case of radial ray syndrome with associated ventricular septal defect.

Holt-Oram syndrome: a rare clinical image.

Holt-Oram syndrome: a rare clinical image.

A Case Report on Holt Oram Syndrome

Holt Oram Syndrome (HOS) falls in rare prevalence category with probability of 0.7 in 100,000 live births. It is a rare autosomal dominant multiple malformation syndrome characterized by abnormalities affecting hands, wrists, arms,congenital heart defects and/or conduction problems. Genetic mutations observed in TBX5 gene is attributed as the main cause of HOS. Our patient in his late 40s was diagnosed with Holt Oram Syndrome. He presented with typical conditions of congenital heart abnormalities (ASD) and upper limb malformations.

Epidemiology of Congenital Hand Differences at a Tertiary Hospital in Southern India - Establishment of a New Registry and Assessment Using Both the Swanson/IFSSH and the Oberg, Manske and Tonkin Classifications.

Background: In resource challenged nations, the true magnitude of the congenital hand differences (CHD) remain unknown due to a lack of nation-wide surveillance system. We created a hospital-based registry system with the aim to determine the prevalence and distribution of CHD and compare the Swanson/IFSSH (SI) and Oberg, Manske and Tonkin (OMT) classifications. Methods: Data of children aged 0 to 18 years with CHD was entered into the online registry and classified based on the SI and OMT classifications from January 2018 to December 2021. The prevalence and distribution of CHD and risk factors like family history were summarised using descriptive analysis. Syndromic and heredity associations were further analysed using Fischer exact test and odds ratio. Clustered columns were used to compare the two classification systems. Results: A total of 307 patients with CHD presented during the study with a prevalence of 2.4/1,000 patients. Among them, 164 were unilateral, 21 were born of consanguineous marriages (7.6%) and 10 (3.6%) had a family history of CHD. Although not statistically significant, bilateral involvement was twice as likely to be hereditary compared to unilateral involvement. A total of 10 different syndromes were identified among 27 patients of which Holt-Oram syndrome was most common. The most common CHD was Radial longitudinal defificiency (111). A total of 82 (27%) children had other associated anomalies that could not be grouped as a syndrome. Two patients could not be classified using either SI or OMT classifications. Conclusions: The online registry established an efficient way to store and analyse data related to CHD. It provides new information on its prevalence in South India, which is similar to the existing literature. Most of the CHD can be grouped in both the SI and OMT classifications. However, there still remains some conditions that are unclassifiable. There is a need for a national registry of CDH for effective management, funding and research. Level of Evidence: Level IV (Epidemiological).

Holt-Oram Syndrome; A Case report

Holt-Oram syndrome is a rare genetic autosomal dominant disorder which affects the preaxial radial ray of the upper limbs and septation of the heart and/or cardiac conduction. The present article describes the clinical and radiological features of Holt–Oram syndrome in a Tanzanian patient.
 This case emphasises the importance of proper prenatal screening for congenital anomalies and counselling of the parents.
 

Prenatal Diagnosis and Management of Thrombocytopenia-Absent Radius Syndrome.

Prenatal Diagnosis and Management of Thrombocytopenia-Absent Radius Syndrome.

Pathophysiological study of the loss of function of the Tbx5 gene in the ventricular conduction system

Patients with Holt-Oram syndrome suffer from cardiac conduction disorders and are often associated with mutations in the gene encoding the cardiac transcription factor Tbx5. In mice, it has been shown that Tbx5 is essential for maintaining the structure and function of the atrioventricular node and His bundle. Conditional deletion of Tbx5 in the adult mouse heart is associated with conduction defects, slowing of the ventricular conduction system (VCS), QRS prolongation, bundle branch blocks and atrial fibrillation. However, there were no data on the morphological and functional consequences of a cardiac deletion of Tbx5 during embryonic development. The aim is to analyze the morphological and functional consequences in the adult heart of the deletion of the Tbx5 gene in the progenitor cells of the cardiac VCS at the fetal stage. Tbx5 gene was inactivated in VCS progenitors using Cx40-CreERT2:Tbx5 floxed/floxed transgenic mice. Cre-mediated recombination was induced by oral gavage of pregnant females at E13-14 during trabecular compaction. Surface ECGs were achieved monthly until 3 months of age, when immunofluorescence staining was performed on mouse hearts. In 3-week-old Tbx5Fl/Fl mice ( n = 16), ECG analysis showed a decrease in P- ( P < 0.01) and T-wave amplitudes ( P < 0.01) and an increased QRS interval duration ( P < 0.05) compared to controls ( n = 12). At 2 months, the mice did not show any worsening of the phenotype. In addition, severe functional defects in Tbx5Fl/Fl mice, including atrial susceptibility, extrasystoles, and arrhythmias were observed by 3 weeks of age. Moreover, left bundle branches block and slowing of ventricular depolarization were also detected in Tbx5Fl/Fl mice. A β-adrenergic stimulation revealed a large tendency for atrial fibrillation and extrasystoles in 88% of Tbx5Fl/Fl mice against 50% of Tbx5Fl/+ mice and 43% of control mice. In 2-month-old Tbx5Fl/Fl mice, Purkinje Fiber cells composing the fast-VCS are still present, however, they lose the expression of Contactin-2 and Connexin-40. Furthermore, we observed a drastic loss of Connexin-43 in Tbx5-deleted contractile cardiomyocytes that are close to the VCS, which may have resulted in decreased signal propagation. In this study, we demonstrated that Tbx5 deletion in VCS progenitors prevents their maturation and hence the expression of rapid conduction markers causing disturbances in the propagation of ventricular electrical activity.

TBX5 variant with the novel phenotype of mixed‑type total anomalous pulmonary venous return in Holt‑Oram Syndrome and variable intrafamilial heart defects.

Variants in T-box transcription factor 5 (TBX5) can result in a wide phenotypic spectrum, specifically in the heart and the limbs. TBX5 has been implicated in causing non-syndromic cardiac defects and Holt-Oram syndrome (HOS). The present study investigated the underlying molecular etiology of a family with heterogeneous heart defects. The proband had mixed-type total anomalous pulmonary venous return (mixed-type TAPVR), whereas her mother had an atrial septal defect. Genetic testing through trio-based whole-exome sequencing was used to reveal the molecular etiology. A nonsense variant was identified in TBX5 (c.577G>T; p.Gly193*) initially showing co-segregation with a presumably non-syndromic presentation of congenital heart disease. Subsequent genetic investigations and more complete phenotyping led to the correct diagnosis of HOS, documenting the novel association of mixed-type TAPVR with HOS. Finally, protein modeling of the mutant TBX5 protein that harbored this pathogenic nonsense variant (p.Gly193*) revealed a substantial drop in the quantity of non-covalent bonds. The decrease in the number of non-covalent bonds suggested that the resultant mutant dimer was less stable compared with the wild-type protein, consequently affecting the protein's ability to bind DNA. The present findings extended the phenotypic cardiac defects associated with HOS; to the best of our knowledge, this is the first association of mixed-type TAPVR with TBX5. Prior to the current analysis, the molecular association of TAPVR with HOS had never been documented; hence, this is the first genetic investigation to report the association between TAPVR and HOS. Furthermore, it was demonstrated that the null-variants reported in the T-box domain of TBX5 were associated with a wide range of cardiac and/or skeletal anomalies on both the inter-and intrafamilial levels. In conclusion, genetic testing was highlighted as a potentially powerful approach in the prognostication of the proper diagnosis.

EP News: Basic and Translational

van Ouwerkerk et al (Circulation February 3, 2022; https://doi.org/10.1161/CIRCULATIONAHA.121.054347, PMID 35113653) investigated the pathogenic missense variant p.G125R in the TBX5 transcription factor that causes Holt-Oram syndrome (HOS; hand-heart syndrome) and early onset of atrial fibrillation (AF). The TBX5-p.G125R variant was introduced in the mouse genome (Tbx5G125R). The authors observed a high incidence of atrial extrasystoles and atrioventricular conduction disturbances in patients with HOS.

Congenital Disorders of the Pediatric Thumb.

Surgical timing for pediatric trigger thumb treatment is controversial for numerous reasons including the potential for spontaneous resolution, the possibility of bilateral involvement, and anesthesia concerns regarding the developing brain. Hence, a reasonable approach is to delay the surgical procedure until the patient is ≥3 years of age. Preaxial polydactyly is usually unilateral and sporadic, with the most common reconstruction method consisting of excision of the diminutive thumb with preservation and soft-tissue reconstruction of the dominant thumb. The surgical procedure is typically performed around the patient age of 1 year to decrease the risks of anesthesia but allow reconstruction prior to the development of a tip-to-tip pinch. Triphalangeal thumb and thumb hypoplasia are often found in the setting of systemic anomalies such as Holt-Oram syndrome, thrombocytopenia absent radius syndrome, Fanconi anemia, VACTERL (vertebral anomalies, anal atresia, cardiac anomalies, tracheoesophageal fistula, renal defects, and limb anomalies), and/or Blackfan-Diamond anemia. As such, patients should receive adequate workup for these entities. A surgical procedure should be performed only once patients have been medically cleared. The status of the carpometacarpal joint in thumb hypoplasia determines whether reconstruction with first web space deepening, collateral ligament stabilization, and opponensplasty compared with index pollicization is performed.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

Prenatal diagnosis of Holt-Oram syndrome

Abstract Objectives To detect common congenital disorders in Holt-Oram syndrome. Case presentation We present a case of a 32 years old primigravida pregnant woman affected by Holt-Oram syndrome referred to our institution for second trimester routine anatomy scan. The ultrasound reported a bilateral aplasia radii, slightly curved ulna and bilateral twisted hand with four digital rays. A significant enlargement of the right atrium without tricuspid regurgitation was also detected. The patient refused the amniocentesis and the postnatal evaluation confirmed the diagnosis of Holt-Oram syndrome. Conclusions Holt-Oram syndrome is an autosomal dominant genetic condition. It is characterized by abnormalities in the bones of the upper limb and congenital heart malformation. The mutation can be inherited, but most cases result from a new mutation in patients without family history of the disorder.

Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene

A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.

A genotype-first analysis in a cohort of Mullerian anomaly.

Müllerian anomaly (M.A.) is a group of congenital anatomic abnormalities caused by aberrations of the development process of the Müllerian duct. M.A. can either be isolated or be involved in Mendelian syndromes, such as Dandy-Walker syndrome, Holt-Oram syndrome and Bardet-Biedl syndrome, which are often associated with both uterus and kidney malformations. In this study, we applied a genotype-first approach to analyze the whole-exome sequencing data of 492 patients with M.A. Six potential pathogenic variants were found in five genes previously related to female urogenital deformities (PKD1, SON, SALL1, BMPR1B, ITGA8), which are partially overlapping with our patients' phenotypes. We further identified eight incidental findings in seven genes related to Mendelian syndromes without known association with reproductive anomalies (TEK, COL11A1, ANKRD11, LEMD3, DLG5, SPTB, BMP2), which represent potential phenotype expansions of these genes.

Establishment of a patient-specific induced pluripotent stem cell line DHMi004-A from a male Holt-Oram syndrome patient with verified TBX5 mutation.

The Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder, mostly based on mutations in the TBX5 gene. Patients show malformation of at least one upper limb along with congenital heart defects. The established induced pluripotent stem cell (iPSC) line was generated from a patient displaying pronounced and typical features of HOS and carrying a single-nucleotide change c.920_C>A leading to an amino acid change from proline to threonine at amino acid position 85, which appeared de novo. Adipose fibroblasts from the patient were reprogrammed using Sendai virus. Pluripotency of the iPSCs was fully demonstrated.

Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. Coli

• Protein purification of a recombinant TBX5 fusion protein was performed. • Recombinant TBX5 protein maintains its secondary structure after purification. • Purified TBX5 protein has cell penetration and nuclear translocation potential. • Purified TBX5 protein is bioactive. The protein T-box transcription factor 5 (TBX5) regulates heart and forelimb development in vertebrates, and its deficiency results in Holt-Oram syndrome. Notably, TBX5 functions as a tumor suppressor protein and is a key transcription factor in the reprogramming cocktail used to induce the formation of cardiomyocytes from fibroblasts. Here, we report the expression and purification of a transducible version of recombinant human TBX5 protein from the prokaryotic host Escherichia coli ( E. coli ) under native conditions. First, the protein-coding, codon-optimized TBX5 nucleotide sequence was fused in-frame with three tags (octahistidine, transactivator of transcription, and nuclear localization sequence) at either end of the sequence for affinity purification, cellular uptake, and nuclear delivery. Subsequently, this codon-optimized gene was cloned in a protein expression vector and expressed in E. coli . Various expression parameters were screened to obtain the highest expression of this protein with maximum solubility, to enable purification from the supernatant fraction. Furthermore, the TBX5 fusion protein was purified as a pure (>90%) recombinant protein under native conditions. The purity and identity were confirmed by Coomassie staining and immunoblotting, respectively. Subsequently, the retention of the secondary structure after purification was analyzed and established through far UV circular dichroism spectroscopy. The cellular uptake and nuclear translocation ability of purified TBX5 fusion protein and its biological activity in human cells were demonstrated. The cell-permeant recombinant protein tool established in this study should help elucidate the biological role of human TBX5 in various cellular processes and pave the way for more detailed understanding of cardiac reprogramming and disease-specific studies.

Identification of deleterious single nucleotide polymorphism (SNP)s in the human TBX5 gene & prediction of their structural & functional consequences: An in silico approach

T-box transcription factor 5 gene (TBX5) encodes the transcription factor TBX5, which plays a crucial role in the development of heart and upper limbs. Damaging single nucleotide variants in this gene alter the protein structure, disturb the functions of TBX5, and ultimately cause Holt-Oram Syndrome (HOS). By analyzing the available single nucleotide polymorphism information in the dbSNP database, this study was designed to identify the most deleterious TBX5 SNPs through insilico approaches and predict their structural and functional consequences.Fifty-eight missense substitutions were found damaging by sequence homology-based tools: SIFT and PROVEAN, and structure homology-based tool PolyPhen-2. Various disease association meta-predictors further scrutinized these SNPs. Additionally, conservation profile of the amino acid residues, their surface accessibility, stability, and structural integrity of the native protein upon mutations were assessed. From these analyses, finally 5 SNPs were detected as the most damaging ones: [rs1565941579 (P85S), rs1269970792 (W121R), rs772248871 (V153D), rs769113870 (E208D), and rs1318021626 (I222N)]. Analyses of stop-lost, nonsense, UTR, and splice site SNPs were also conducted.Through integrative bioinformatics analyses, this study has identified the SNPs that are deleterious to the TBX5 protein structure and have the potential to cause HOS. Further wet-lab experiments can validate these findings.

Abstract 13259: IRX5 Transcription Factor Cooperate With TBX5/GATA4/NKX2-5 Complex to Regulate Several Human Cardiomyocyte Functions

Introduction: The transcription factor (TF) Iroquois homeobox 5 (Irx5) is a key patterning and differentiation regulator. In humans, IRX5 regulates the expression of the main cardiac sodium ion channel gene, SCN5A . However, its global mechanistic role in human cardiomyocytes is still to be fully understood. Hamamy syndrome patients, carrying IRX5 mutations, have defects in various organs including the heart and the limbs. Interestingly, heart and limbs defects are also found in Holt-Oram syndrome patients ( TBX5 mutations), suggesting a functional link between TBX5 and IRX5. Moreover, a cooperation of TBX5 with GATA4 and NKX2-5 in cardiac development and functions has already been shown. Hypothesis: We hypothesized that IRX5 may cooperate with the TF complex TBX5/GATA4/NKX2-5 to regulate key cardiac functions in humans. Methods &amp; Results: First, immunoprecipitations in HEK293 cells showed that IRX5 can bind to TBX5, GATA4 and NKX2-5, individually and together as a complex. Using five truncated forms of IRX5 protein, its homeodomain was identified as being the essential protein region for these interactions. Second, we showed by luciferase assays, that each combination of these TFs impacted differently SCN5A expression: e.g. by itself, NKX2-5 has a strong activator effect (increased activity by 10 fold vs . no TF) that is potentialized by IRX5 (12 fold vs . no TF) but inhibited by TBX5 (8 fold vs . no TF). Third, genes bound by IRX5 (n=2253) were identified by ChIP-Seq on cardiomyocytes derived from induced pluripotent stem cells generated from 2 healthy individuals. Analyzing published ChIP-Seq datasets for TBX5, GATA4 or NKX2-5 in the same cellular model, we identified 2990 genes that were bound by these 3 TFs, and associated with cardiac biological pathways, such as muscle contraction. Among these genes, 848 were also bound by IRX5 and were associated with the function of electrical activity and with the fibrosis signaling, suggesting a specific role for IRX5 in these cardiac processes. Conclusions: Overall, our data show new physical and functional interactions between IRX5 and 3 key cardiac TFs (TBX5, GATA4 and NKX2-5), and suggest an unexpected regulatory role for IRX5 in specific human heart functions.

HOLT-ORAM SYNDROME: RARE CASE SERIES

The upper limb malformations in association with congenital heart defects occurring as autosomal dominant disorder are seen in Holt-Oram syndrome. It is a very rare disorder which can be detected with early prenatal ultrasound checkups. Here we are reporting two cases of holt-oram syndrome

Identification of a novel TBX5 c.755 + 1G > A variant and related pathogenesis in a family with Holt-Oram syndrome.

The proband with congenital heart disease and abnormal thumb was clinically diagnosed as Holt–Oram syndrome (HOS). A novel variant, T‐box transcription factor 5 (TBX5) c.755 + 1 G > A, was identified in the proband via whole exome sequencing and validated using Sanger sequencing. Pedigree analysis and clinical examinations revealed three/seven individuals over three generations within the family, with features suggestive of HOS. Deep amplicon sequencing confirmed that the allele frequencies of the novel variant in the proband (III‐1), her brother (III‐2), and her mother (II‐2) were 50%, 48.3%, and 38.1%, respectively, indicating that III‐1 and III‐2 harbored heterozygous variants, while II‐2 harbored mosaic heterozygous variants. The minigene splicing assay showed that the novel variant affected the normal splicing of exon 7, resulting in the production of abnormal TBX5 transcripts. Reverse transcription‐quantitative polymerase chain reaction and western blot analyses revealed that the novel variant upregulated TBX5 expression at the transcriptional and translational levels. Nuclear localization assay demonstrated impaired nuclear localization of the mutant TBX5. Cell viability assay revealed the inhibition of cell activity by the mutant TBX5. Our findings indicate that the novel variant was potentially induced HOS, probably by causing aberrant splicing, reducing the enrichment of nuclear TBX5 protein, and inhibiting cellular proliferation.