Holt-Oram Research Articles

An Algorithm for Management of Radial Longitudinal Deficiency.

Lack of specific guidelines in the literature renders management of radial longitudinal deficiency (RLD) exceedingly diverse. This diversity in treatment approaches can lead to variability in outcomes and pose a challenge in determining the most effective interventions for each individual case of RLD. Surgeons must carefully consider the unique characteristics and needs of each patient when developing a treatment plan for this complex condition. Around 40% of cases are linked to syndromes like Holt-Oram syndrome, thrombocytopenia absent radius (TAR) syndrome, Fanconi anaemia and VACTERL. Hand surgeons must be knowledgeable about these conditions, as they may be the first to identify these syndromes in children. Associated elbow stiffness can also be a crucial factor in treatment decision-making. Although this condition has been studied for more than 10 decades, the surgical treatment that is currently available is not completely successful in restoring 'normal' wrist function and appearance. Centralisation and radialisation procedures continue to be the cornerstones of surgical treatment. Many innovative approaches include altering the biomechanics of this condition with bony or soft tissue procedures and microvascular techniques. Understanding the proximal limb changes is crucial in the holistic management of this challenging malformation. This article presents a treatment algorithm based on literature evidence and our clinical experience. Level of Evidence: Level V (Therapeutic).

Single-nuclei transcriptomics reveals TBX5-dependent targets in a patient with Holt-Oram syndrome.

Single-nuclei transcriptomics reveals TBX5-dependent targets in a patient with Holt-Oram syndrome.

Abstract 4120919: Cell physiology and multi-omics analysis revealed disordered embryogenesis as early as mesoderm specification in models of Holt-Oram Syndrome

Introduction: Congenital heart diseases (CHDs) are the leading cause of childhood morbidity and mortality. The dysregulation of several cardiac transcription factors (TFs) leads to CHD. The coordination of several cardiac TFs is required and essential for cardiogenesis. However, the mechanisms to acquire its cardiac cell identity remain unclear. Hypothesis: Mutant Tbx5 dysregulates embryogenesis during mesoderm specification, prior to its expression in the tissues in Holt-Oram syndrome. Methods: Using cellular physiology and multiomics analysis in both human ES cells and a zebrafish model of TBX5 germline mutation. we evaluated embryonic structure and function, prior to the onset of gastrulation in the context of both heterozygous and homozygous TBX5 mutation. Results: Zebrafish time course transcriptome profiles over gastrulation revealed that loss of Tbx5 impacted transcriptional profiles at the blastula stage. Single cell RNA sequencing (scRNA-seq) on 16243 zebrafish blastula stage cells showed loss of Tbx5 impacted transcription noise at the blastula stage prior to the expression of zygotic Tbx5 with associated effects on chromatin accessibility using omni-assay for transposase-accessible chromatin (ATAC) and evidence of aberrant Wnt signaling even at the blastula stage. Embryo-wide cell structure and function were abnormal in both Tbx5 mutant zebrafish heterozygotes or homozygotes. To validate Undifferentiated human ES cell H3K4me3 Cleavage Under Targets&Release Using Nuclease (CUT&RUN) also showed abnormal Wnt signaling in TBX5 homozygous mutation and TBX5 CUT&RUN showed aberrant mesoderm pathway. Calcium imaging analysis demonstrated the lowest excitation frequency in TBX5 homozygous mutation prior to mesoderm specification. TBX5 homozygous human ES derived mesoderm cells exhibited low expression levels of mesoderm marker genes compared to TBX5 wild type mesoderm. Conclusion: Integrating single cell physiology and multi-omics technologies, we idneifified fundamental dysregulation of embryogenesis in mutant cardiac-restricted gene disorder prior to mesoderm specification or the zygotic expression of the mutant gene. These findings suggest that Tbx5 started to determine cell fate prior to mesoderm specification by altering chromatin accessibilities and histone modification. Tbx5 affected mesoderm differentiation by influencing Wnt signaling and cell physiology.

Elucidating the roles of SOD3 correlated genes and reactive oxygen species in rare human diseases using a bioinformatic-ontology approach.

Superoxide Dismutase 3 (SOD3) scavenges extracellular superoxide giving a hydrogen peroxide metabolite. Both Reactive Oxygen Species diffuse through aquaporins causing oxidative stress and biomolecular damage. SOD3 is differentially expressed in cancer and this research utilises Gene Expression Omnibus data series GSE2109 with 2,158 cancer samples. Genome-wide expression correlation analysis was conducted with SOD3 as the seed gene. Categorical SOD3 Pearson Correlation gene lists incrementing in correlation strength by 0.01 from ρ≥|0.34| to ρ≥|0.41| were extracted from the data. Positively and negatively SOD3 correlated genes were separated for each list and checked for significance against disease overlapping genes in the ClinVar and Orphanet databases via Enrichr. Disease causal genes were added to the relevant gene list and checked against Gene Ontology, Phenotype Ontology, and Elsevier Pathways via Enrichr before the significant ontologies containing causal and non-overlapping genes were reviewed with a literature search for possible disease and oxidative stress associations. 12 significant individually discriminated disorders were identified: Autosomal Dominant Cutis Laxa (p = 6.05x10-7), Renal Tubular Dysgenesis of Genetic Origin (p = 6.05x10-7), Lethal Arteriopathy Syndrome due to Fibulin-4 Deficiency (p = 6.54x10-9), EMILIN-1-related Connective Tissue Disease (p = 6.54x10-9), Holt-Oram Syndrome (p = 7.72x10-10), Multisystemic Smooth Muscle Dysfunction Syndrome (p = 9.95x10-15), Distal Hereditary Motor Neuropathy type 2 (p = 4.48x10-7), Congenital Glaucoma (p = 5.24x210-9), Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome (p = 3.77x10-16), Classical-like Ehlers-Danlos Syndrome type 1 (p = 3.77x10-16), Retinoblastoma (p = 1.9x10-8), and Lynch Syndrome (p = 5.04x10-9). 35 novel (21 unique) genes across 12 disorders were identified: ADNP, AOC3, CDC42EP2, CHTOP, CNN1, DES, FOXF1, FXR1, HLTF, KCNMB1, MTF2, MYH11, PLN, PNPLA2, REST, SGCA, SORBS1, SYNPO2, TAGLN, WAPL, and ZMYM4. These genes are proffered as potential biomarkers or therapeutic targets for the corresponding rare diseases discussed.

When the heart and hands tell a story: an intriguing case of Holt–Oram syndrome

BackgroundHolt–Oram syndrome is a rare genetic disorder caused by a mutation in the TBX5 gene, combining skeletal and cardiac malformations. Vital prognosis depends essentially on cardiac involvement, while skeletal malformations determine functional prognosis.Case presentationWe describe the case of a young patient aged 49, with no particular history, who presented to the emergency department with de novo congestive heart failure. Clinical examination revealed not only signs of heart failure, but also malformations such as triphalangia of the left thumb, prono-supination defects of both forearms and dorsolumbar scoliosis. The electrocardiogram showed that an atypical atrial flutter and transthoracic echocardiography revealed an atrial septal defect. We also performed a spinal scan to assess the severity of the scoliosis. Genetic studies confirmed a TBX5 gene mutation in the patient, and family screening revealed no similar cases in the family. Management consisted mainly of pharmacological treatment of heart failure, in addition to scoliosis management.ConclusionHolt–Oram syndrome is a rare genetic disorder which should be suspected in the presence of any upper limb anomaly associated with cardiac malformation and confirmed by genetic study. A family investigation is necessary after diagnosis, because of autosomal dominant inheritance.

Functional characterization vs in silico prediction for TBX5 missense and splice variants in Holt-Oram syndrome

PurposePredicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome is an autosomal condition characterized by the association of radial and heart defects, due to variants in TBX5. Most variants are predicted to be truncating and result in haploinsufficiency. The pathogenicity of missense or splice variants is harder to demonstrate. MethodsFourteen TBX5 variants of uncertain significance (5 missense, 9 splice) and 6 likely pathogenic missense variants were selected for functional testing, depending on the variant-type (immunolocalization, western blot, reporter assays, minigene splice assays, and RT-PCR). Results were compared with in silico predictions. ResultsFunctional tests allowed to reclassify 9/14 variants of uncertain significance in TBX5 as likely pathogenic, confirming their role in Holt-Oram syndrome. We demonstrated loss of function (n = 8) or gain of function (n = 1) for 9 of the 11 missense variants, whereas no functional impact was shown for the 2 variants: p.(Gly195Ala) and p.(Ser261Cys), as suggested by contradictory predictions of in silico approaches. Of 9 splice variants predicted to affect splicing by SpliceAI, we observed partial or complete exon skipping (n = 6), intron retention (n = 2) or exon shortening (n = 1), inducing frame shifting with premature stop codons. ConclusionBioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate American College of Medical Genetics and Genomics classification in human rare diseases.

Advances in genetic research on Non-syndromic congenital joint synostosis

Congenital joint synostosis (CJS) is a functional impairment resulting from failure in joint morphogenesis during embryonic development. Clinically, it may be classified as syndromic (sCJS) and non-syndromic (nsCJS) disorders. Common sCJS include chromosomal disorders such as Klinefelter syndrome and single-gene disorders like Apert/Pfeiffer/Crouzon syndromes, Holt-Oram syndrome, Ehlers-Danlos syndrome, and Radial-ulnar synostosis with thrombocytopenia, presenting with multiple system/organ anomalies. By contrast, nsCJS manifest with only joint abnormalities, affecting one or multiple joints. This review has focused on human nsCJS and its genetic etiology. To date, variants in seven genes (NOG, GDF5, FGF9, GDF6, FGF16, SMAD6, and MECOM) have been identified as causative factors for nsCJS. This review has focused on such genes and provided a comprehensive review for the clinical phenotypes, genetic patterns, common variants, and underlying mechanisms associated with nsCJS based on a literature review. In addition, it has also analyzed other candidate genes for nsCJS within the context of relevant signaling pathways involved in joint morphogenesis.

Holt-Oram Syndrome – A Case Report and a Short Review

Holt-Oram syndrome also known as the heart-hand syndrome type 1, is an autosomal dominant rare congenital disorder, manifested as a conglomeration of various skeletal and cardiac abnormalities. The list of differential diagnoses of such abnormalities is extensive but careful examination can point towards a specific diagnosis even without genetic testing in a resource-limited country. We present a sporadic case of Holt-Oram syndrome with OS-ASD highlighting the diagnostic problem without genetic testing and discussing differentiating salient features of most of the syndromic associations.

Anesthetic management of an infant with Holt-Oram syndrome.

Anesthetic management of an infant with Holt-Oram syndrome.

Acute Post Streptococcal Glomerulonephritis with Persistent Hypertension in a Child of Holt-oram Syndrome: A Case Report

Holt-Oram syndrome is a rare congenital autosomal dominant disorder caused by a mutation in the TBX5 gene. It is characterised by upper limb abnormalities and congenital heart lesions, such as Atrial Septal Defect (ASD), typically affecting children. In this case, a sixyear-old boy presented with facial puffiness and decreased urine output following an upper respiratory infection. Upon examination, he was found to have Holt-Oram syndrome, a rare genetic disorder characterised by abnormalities in the bones of the upper limbs and congenital heart defects. He developed acute Post Streptococcal Glomerulonephritis (PSGN) following a streptococcal infection, resulting in persistent hypertension. Each condition independently poses significant health challenges, and their simultaneous occurrence complicates the clinical picture considerably. Following detailed investigations and management, the child required continued treatment with two antihypertensive medications upon discharge. The need for dual therapy in a paediatric patient highlights the severity of his condition and the challenges in managing it effectively. Close follow-up is essential to monitor disease progression and ensure optimal outcomes. This case features both a genetic disorder (Holt-Oram syndrome due to a TBX5 gene mutation) and an immune-mediated renal disease (PSGN), representing a rare intersection of genetic and postinfectious pathologies. The co-existence of these two conditions is unusual and complicates the clinical picture, necessitating a tailored approach to management. This case underscores the need for a multidisciplinary approach involving cardiology, nephrology, and genetics. Coordinated care is crucial for monitoring disease progression, managing complications, and ensuring comprehensive treatment.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copynumber variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Human Genetics of Tetralogy of Fallot and Double-Outlet Right Ventricle.

Tetralogy of Fallot (TOF) and double-outlet right ventricle (DORV) are conotruncal defects resulting from disturbances of the second heart field and the neural crest, which can occur as isolated malformations or as part of multiorgan syndromes. Their etiology is multifactorial and characterized by overlapping genetic causes. In this chapter, we present the different genetic alterations underlying the two diseases, which range from chromosomal abnormalities like aneuploidies and structural mutations to rare singlenucleotide variations affecting distinct genes. For example, mutations in the cardiac transcription factors NKX2-5, GATA4, and HAND2 have been identified in isolated TOF cases, while mutations of TBX5 and 22q11 deletion, leading to haploinsufficiency of TBX1, cause Holt-Oram and DiGeorge syndrome, respectively. Moreover, genes involved in signaling pathways, laterality determination, and epigenetic mechanisms have also been found mutated in TOF and/or DORV patients. Finally, genome-wide association studies identified common singlenucleotide polymorphisms associated with the risk for TOF.

Abstract 18674: Deep Phenotyping Arrhythmogenic MVP With Imaging and Genotyping Reveals Important Shared Genetic Etiology With Arrhythmia and Cardiomyopathy Genes

Introduction: Mitral valve prolapse (MVP) has a high population prevalence 2-4% with an arrhythmogenic subtype presenting with premature ventricular complexes (PVC) ventricular tachycardia (VT) and sudden death. There is a continued search for markers of MVP to help risk stratify patients. Hypothesis: Arrhythmogenic MVP shares genetic etiology with Mendelian causes of arrhythmia and cardiomyopathy Methods: Consecutive adult patients with arrhythmogenic MVP patients prospectively evaluated Results: A total of 33 cases (age 55±15 years, female 51.5%) presented with history of hypertension (33%), diabetes (3%), coronary disease (12%), atrial fibrillation (45%), bileaflet MVP (58%) and syncope (18%). ECG findings include inverted T-waves in inferior leads in 6%, pleomorphic PVC in 6%, Ventricular couplets in 51.5%, non-sustained VT in 73% and sustained VT in 33%; High PVC burden was seen in 55% of cases with burden 15±14%. Echocardiography noted LV ejection fraction of 55±8%, mitral regurgitations in 70% (severe 18%, moderate 27%, mild 24%), mitral annular disjunction in 39%, and Pickelhaube sign in 9% of cases. Cardiac magnetic resonance showed LVESV 113±119mm, LVEDV 117±70mm, and late Gadolinium enhancement in 48% with highest prevalence in basal inferolateral segment. PET-CT performed in 17 cases and showed fluorodeoxyglucose uptake in 24% cases, mostly in basal inferolateral segment as well. Genetic testing was performed in 23 cases and reported a putative pathogenic variant in 21.7%: FLNC (Exemplar case, Figure 1 ) cause of dilated cardiomyopathy, TBX5 cause of Holt-Oram syndrome, DSP cause of arrhythmogenic cardiomyopathy , TTN causes of dilated cardiomyopathy, MTO1 cause of mitochondrial hypertrophic cardiomyopathy. Conclusion: Full evaluation of arrhythmogenic MVP with multimodality imaging and genetic testing reveals high burden of fibrosis, inflammation, and candidate pathogenic variants.

Correction of a deleterious TBX5 mutation in an induced pluripotent stem cell line (DHMi004-A-1) using a completely plasmid-free CRISPR/Cas 9 approach

TBX5 is a transcription factor which plays an essential role at different checkpoints during cardiac differentiation. However, regulatory pathways affected by TBX5 still remain ill-defined. We have applied the CRISPR/Cas9 technology using a completely plasmid-free approach to correct a heterozygous causative “loss-of function” TBX5 mutation in an iPSC line (DHMi004-A), that has been established from a patient suffering from Holt-Oram syndrome (HOS). This isogenic iPSC line, DHMi004-A-1, represents a powerful in vitro tool to dissect the regulatory pathways affected by TBX5 in HOS.

Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.

Loss of the Tbx5 gene in the ventricular conduction system induces spontaneous ventricular arrhythmias

The ventricular conduction system (VCS) and the working myocardium (WM) derive from the same myogenic progenitors during the embryonic development. The VCS development is regulated by numerous genes including the T-box transcription factor 5 (Tbx5). In human, Tbx5 mutations cause Holt-Oram syndrome, an autosomal dominant disease associated with heart abnormalities such as arrhythmias. In adult mice, the deletion of the Tbx5 gene provoked a slowing of ventricular conduction due to a loss of the gap junction Connexin 40 and dysfunction of cardiac channels. However, it is still unknown how Tbx5 deletion affects the development and the function of the VCS. The aim is to study the consequences of the Tbx5 deletion in embryonic progenitors of the ventricular conduction system on cardiac function in adult mice. Conditional deletion of Tbx5 alleles in mice was induced by expressing the Cre recombinase under the control of the Connexin 40 (Cx40) gene. We studied two groups of mutants: Tbx5 deletion at embryonic days E13-14 targeting both the ventricular conduction system and the working myocardium (“VCS + WM” group; n = 40) and mice deleted at E18-P2 targeting only the ventricular conduction system (“VCS” group; n = 17). Surface ECGs were achieved monthly until 3 months of age, when immunostaining was performed on the left VCS. Immunostaining showed a reduced expression of the two major connexines in the ventricle (Cx40 and Cx43) and a reduced number of conductive cells in both mutants, suggesting a defective maturation of the VCS (Fig. 1). Furthermore, the loss of Cx43 was correlated with that of Tbx5, reflecting a direct transcriptional effect. Surface ECG revealed a significant increase of the ventricular activation duration in both mutants (P < 0.01). Moreover, premature ventricular contractions (PVCs) and a left QRS axis deviation were observed in both mutants. Finally, these PVCs disappeared with age in both mutants under resting conditions, and are unmasked when the heart is challenged with a β-1 adrenergic agonist. Our results provide new insights into Tbx5 targets in the VCS. Tbx5 deletion impacts the maturation and the anatomy of the VCS as proved by the deviation of the ventricular electrical axis, which even occurs when the VCS is already formed. Tbx5 appears to have a direct impact on the Gja1 gene, encoding the gap junction Cx43, which is reduced at the junction between the VCS and the WM in both mutants and may be responsible for PVCs.

Congenital pseudoarthrosis of the clavicle co-existing with tetralogy of Fallot: Another variant of the Holt-Oram syndrome?

Congenital pseudoarthrosis of the clavicle is a rare condition characterized by a defect in the clavicle. Upper limb anomalies have been documented to occur in association with congenital heart malformations. However, there is no documented report of congenital heart malformation associated with congenital pseudoarthrosis of the clavicle.&#x0D; Methods: We present a 9yr old boy with left clavicular pseudoarthrosis coexisting with tetralogy of Fallot.

A novel de novo nonsense mutation in SALL4 causing duane radial ray syndrome: a case report and expanding the phenotypic spectrum

BackgroundSALL4, a member of the SALL genes family, encodes a zinc-finger transcriptional factor that either activates or represses gene transcription depending on cell type during embryonic development. SALL4 mutations cause extremely variable conditions including Duane-radial ray (DRR), Okihiro, Holt-oram, Acro-renal ocular and IVIC syndromes, all with autosomal dominant inheritance pattern. However, all these syndromes with different terminologies are actually the same entity termed SALL4 related disorders.Case presentationHerein, we examine an Iranian patient suspected to DRR syndrome which has not been previously described in the population. Whole-exome sequencing (WES) was performed to examine pathogenic genes in the proband. Subsequently, Sanger sequencing was used to confirm the mutation found. To elucidate the effects of the identified mutation, clinical data of patient was collected. Morever, the possible impact of the mutation found on the corresponding protein was evaluated using bioinformatics tools. WES identifed a novel de novo heterozygous nonsense mutation in exon 2 of SALL4 gene (c.712 C > T:p.Q238X). Subsequently, segregation and phenotype-genotype correlation analysis as well as in-silico approaches confirmed the autosomal dominance inheritance and disease-causing nature of the identified mutation. In addition, studied patient had features not described previously, including kyphoscoliosis, dimple presacral sinus, barrel chest and artric disc (C6–C7). These manifestations could be additional characteristics of the growing phenotypic spectrum of SALL4 related disorders.ConclusionOur findings could extend the pathogenic mutations and phenotypic spectrum of SALL4 related disorders. Such reports can also aid to conduct genetic counseling, prenatal diagnosis and clinical management for individuals at high risk of SALL4 related disorders.

363 - Holt-oram syndrome and its variable phenotypic expression: a multi-generational case series

363 - Holt-oram syndrome and its variable phenotypic expression: a multi-generational case series