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

PURPOSE: Predicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome (HOS) 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. METHODS: Fourteen TBX5 variants of uncertain significance (VUS) (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. RESULTS: Functional tests allowed to reclassify 9/14 VUS in TBX5 as likely pathogenic, confirming their role in HOS. 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. CONCLUSION: Bioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate ACMG classification in human rare diseases.

Human Genetics of Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital cardiovascular malformation characterized by hypoplasia of the left ventricle, aorta, and other structures on the left side of the heart. The pathologic definition includes atresia or stenosis of both the aortic and mitral valves. Despite considerable progress in clinical and surgical management of HLHS, mortality and morbidity remain concerns. One barrier to progress in HLHS management is poor understanding of its cause. Several lines of evidence point to genetic origins of HLHS. First, some HLHS cases have been associated with cytogenetic abnormalities (e.g., Turner syndrome). Second, studies of family clustering of HLHS and related cardiovascular malformations have determined HLHS is heritable. Third, genomic regions that encode genes influencing the inheritance of HLHS have been identified. Taken together, these diverse studies provide strong evidence for genetic origins of HLHS and related cardiac phenotypes. However, using simple Mendelian inheritance models, identification of single genetic variants that "cause" HLHS has remained elusive, and in most cases, the genetic cause remains unknown. These results suggest that HLHS inheritance is complex rather than simple. The implication of this conclusion is that researchers must move beyond the expectation that a single disease-causing variant can be found. Utilization of complex models to analyze high-throughput genetic data requires careful consideration of study design.

Human Genetics of Atrial Septal Defect.

Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.

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 single nucleotide 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 single nucleotide polymorphisms associated with the risk for TOF.

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 copy number 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.

Identification of a novel TBX5 c.755 + 1 G > 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.

Combined clinic and home-based therapeutic approach for the treatment of bilateral radial deficiency for a young child with Holt-Oram syndrome: A case report.

BACKGROUND: Holt-Oram syndrome (HOS) is a rare, genetic condition characterized by the combination of congenital heart defect and hypoplasia in one or both upper extremities. Children with HOS commonly present with varied joint and limb involvement including radial longitudinal deficiency impacting hand function. Evidence-based guidelines regarding orthotic wear and therapeutic techniques are lacking. PURPOSE: The aim of this case report was to present the results of a long-term occupational therapy program for a patient with HOS pre and postpollicization. STUDY DESIGN: Case report. METHODS: A 4-month-old patient with bilateral radial longitudinal deficiencies began outpatient occupational therapy for custom orthosis fabrication and treatment which included long term clinic and home-based intervention. Techniques included passive range of motion, orthosis wear, therapeutic taping, and modified constraint induced movement therapy. Longitudinal assessment of musculoskeletal alignment and functional hand use was performed using goniometry for passive and active range of motion, the Assisting Hand Assessment (AHA), and The Thumb Grasp and Pinch Assessment (T-GAP). RESULTS: Improvement in passive and active range of motion was achieved as well as improved activity level function as measured by the AHA and T-GAP postpollicization and intervention. CONCLUSIONS: A combined clinic and home-based therapeutic approach can be effective for children with HOS to improve alignment and function pre and postpollicization to further enhance hand function. Comprehensive, long-term assessment is necessary to fully evaluate and communicate improvement.

Functional analysis of two novel TBX5 variants present in individuals with Holt-Oram syndrome with different clinical manifestations.

Holt-Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5-a gene encoding a transcription factor important for heart and skeletal development-are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.

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.

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.

Familial dilated cardiomyopathy associated with pathogenic TBX5 variants: Expanding the cardiac phenotype associated with Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is a rare, autosomal dominant disorder caused by heterozygous pathogenic variants in cardiac T-box transcription factor, TBX5. Classically, it is associated with upper limb malformations and variable cardiac abnormalities. Limb manifestations are considered to be invariably present, ranging in severity from limitation in movement, to triphalangeal thumbs, absent thumbs, shortened forearms, or phocomelia. Cardiac involvement is characterized by congenital heart defects, most commonly septal structural malformations, and conduction system disease. Recently, novel TBX5 variants have also been reported in association with dilated cardiomyopathy (DCM). In this context, we report eight individuals from four unrelated families, in whom pathogenic variants in TBX5 segregated with an atypical HOS phenotype. Affected individuals exhibit relatively mild skeletal features of HOS, with a predominant cardiac phenotype, which includes several individuals affected by non-ischaemic DCM. To our knowledge, these represent the first reported cases of DCM in families with skeletal features of HOS, some of whom also harbored variants previously linked to a classical HOS phenotype (p. Arg279* and p.Arg237Gln). This finding supports diverse roles of TBX5 in cardiovascular development and function, and confirms the importance of long-term cardiac surveillance for individuals affected by HOS. Furthermore, these families highlight the wide phenotypic variability of HOS, which may include comparatively mild upper limb findings in respect to cardiac manifestations.

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in tbx5-deficient zebrafish embryos.

BACKGROUND: Tbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5. RESULTS: Among the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group. CONCLUSIONS: These results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

Asymmetric cell convergence-driven zebrafish fin bud initiation and pre-pattern requires Tbx5a control of a mesenchymal Fgf signal.

Tbx5 plays a pivotal role in vertebrate forelimb initiation, and loss-of-function experiments result in deformed or absent forelimbs in all taxa studied to date. Combining single-cell fate mapping and three-dimensional cell tracking in the zebrafish, we describe a Tbx5a-dependent cell convergence pattern that is both asymmetric and topological within the fin-field lateral plate mesoderm during early fin bud initiation. We further demonstrate that a mesodermal Fgf24 convergence cue controlled by Tbx5a underlies this asymmetric convergent motility. Partial reduction in Tbx5a or Fgf24 levels disrupts the normal fin-field cell motility gradient and results in anteriorly biased perturbations of fin-field cell convergence and truncations in the pectoral fin skeleton, resembling aspects of the forelimb skeletal defects that define individuals with Holt-Oram syndrome. This study provides a quantitative reference model for fin-field cell motility during vertebrate fin bud initiation and suggests that a pre-pattern of anteroposterior fate specification is already present in the fin-field before or during migration because perturbations to these early cell movements result in the alteration of specific fates.

Holt-Oram Syndrome: A Rare Variant.

Holt-Oram syndrome is an autosomal dominant disorder, characterised by skeletal abnormalities of the upper limb associated with congenital heart defect, mainly atrial and ventricular septal defects. Skeletal defects exclusively affect the upper limbs in the preaxial radial ray distribution and are bilateral and asymmetrical. They range from clinodactyly, absent or digitalised thumb, hypoplastic or absent radii, and first metacarpal to hypoplastic ulna and carpal bone anomalies. Cardiac involvement ranges from asymptomatic conduction disturbances to multiple structural defects. Structural defects are seen in 75% of the cases and include both atrial and ventricular septal defect. More complex cardiac lesions such as Tetrology of Fallot, endocardial cushion defects, double outlet right ventricle, and total anomalous pulmonary venous return are observed uncommonly. An aneurysm of the interatrium septum is an infrequent finding in infants. It has been speculated that atrial septal aneurysm (ASA) is a direct source of thrombus formation. Paradoxical embolism of venous thrombi across a right to left shunt is possibly responsible for the cryptogenic stroke in a patient with ASA. However, coagulopathy associated with cyanotic congenital heart defect may also be contributory. Our patient had a rare association of complex cardiac lesion (tricuspid atresia, pulmonary stenosis, atrial septal aneurysm) with cardiac conductive defects and left parietal infarct along with the usual skeletal abnormalities.

Left Ventricular Non-compaction in Holt-Oram Syndrome.

Holt-Oram Syndrome is an autosomal dominant condition with complete penetrance and which involves upper limb skeletal and cardiac abnormalities. The latter can be structural defects or involve the conduction system. This report details the occurrence of left ventricular non-compaction in multiple family members with Holt-Oram Syndrome. It is recommended that patients with the Holt-Oram Syndrome be considered for comprehensive cardiac evaluation to exclude non-compaction cardiomyopathy as this may have significant prognostic implications.

Clinical expression of Holt-Oram syndrome on the basis of own clinical experience considering prenatal diagnosis.

OBJECTIVES: Holt-Oram syndrome manifests with defects of upper limbs, pectoral girdle and cardiovascular system. The aim of this paper was to present complex clinical picture of the syndrome and its variable expression on the example of the family diagnosed genetically on the neonatal ward, after proband's prenatal examination. MARETIAL AND METHODS: Nine family members were tested for TBX5 gene mutation. RESULTS: Four of family members were diagnosed with Holt-Oram syndrome and five had correct genetic test results. The diagnosis allowed to identify a genetic risk family and enabled to provide them with genetic counselling. CONCLUSIONS: Diagnosis of Holt-Oram syndrome is possible as early as in prenatal period and it can be verified by genetic tests.

Coexisting urogenital anomaly and duodenal atresia in two atypical Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder, characterized by upper limb dysplasia and congenital cardiac defect. We report two cases with HOS, first associated with renal agenesis, coronal hypospadias, urethral duplication and second associated with duodenal atresia and horseshoe kidney that have not been reported in English literature.

A unique TBX5 microdeletion with microinsertion detected in patient with Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and congenital heart defects and caused by numerous germline mutations of TBX5 producing preterminal stop codons. Here, we report on a novel and unusual heterozygous TBX5 microdeletion with microinsertion (microindel) mutation (c.627delinsGTGACTCAGGAAACGCTTTCCTGA), which is predicted to synthesize a truncated TBX5 protein, detected in a sporadic patient with clinical features of HOS prenatally diagnosed by ultrasonography. This uncommon and relatively large inserted sequence contains sequences derived from nearby but not adjacent templates on both sense and antisense strands, suggesting two possible models, which require no repeat sequences, causing this complex microindel through the bypass of large DNA adducts via an error-prone DNA polymerase-mediated translesion synthesis.

Clinical and molecular characterisation of Holt-Oram syndrome focusing on cardiac manifestations.

BACKGROUND: Holt-Oram syndrome is characterised by CHD and limb anomalies. Mutations in TBX5 gene, encoding the T-box transcription factor, are responsible for the development of Holt-Oram syndrome, but such mutations are variably detected in 30-75% of patients. METHODS: Clinically diagnosed eight Holt-Oram syndrome patients from six families were evaluated the clinical characteristics, focusing on the cardiac manifestations, in particular, and molecular aetiologies. In addition to the investigation of the mutation of TBX5, SALL4, NKX2.5, and GATA4 genes, which are known to regulate cardiac development by physically and functionally interacting with TBX5, were also analyzed. Multiple ligation-dependent probe amplification analysis was performed to detect exonic deletion and duplication mutations in these genes. RESULTS: All included patients showed cardiac septal defects and upper-limb anomalies. Of the eight patients, seven underwent cardiac surgery, and four suffered from conduction abnormalities such as severe sinus bradycardia and complete atrioventricular block. Although our patients showed typical clinical findings of Holt-Oram syndrome, only three distinct TBX5 mutations were detected in three families: one nonsense, one splicing, and one missense mutation. No new mutations were identified by testing SALL4, NKX2.5, and GATA4 genes. CONCLUSIONS: All Holt-Oram syndrome patients in this study showed cardiac septal anomalies. Half of them showed TBX5 gene mutations. To understand the genetic causes for inherited CHD such as Holt-Oram syndrome is helpful to take care of the patients and their families. Further efforts with large-scale genomic research are required to identify genes responsible for cardiac manifestations or genotype-phenotype relation in Holt-Oram syndrome.