TBX5 variants and cardiac phenotype: A systematic review of the literature and a novel variant.

T-Box Transcription Factor 5 (TBX5) variants are associated with Holt-Oram syndrome. Holt-Oram syndrome display phenotypic variability, regarding upper limb defects, congenital heart defects, and arrhythmias. To investigate the genotype-phenotype relationship between TBX5 variants and cardiac disease, we performed a systematic review of the literature. Through the systematic review we identified 108 variants in TBX5 associated with a cardiac phenotype in 277 patients. Arrhythmias were more frequent in patients with a missense variant (48% vs 30%, p = 0.009) and upper limb abnormalities were more frequent in patients with protein-truncating variants (85% vs 64%, p = 0.0008). We found clustering of missense variants in the T-box domain. Furthermore, we present a family with atrial septal defects. By whole exome sequencing, we identified a novel missense variant p.Phe232Leu in TBX5. The cardiac phenotype included atrial septal defect, arrhythmias, heart failure, and dilated cardiomyopathy. Clinical examination revealed subtle upper limb abnormalities. Thus, the family corresponds to the diagnostic criteria of Holt-Oram syndrome. We provide an overview of cardiac phenotypes associated with TBX5 variants and show an increased risk of arrhythmias associated to missense variants compared to protein-truncating variants. We report a novel missense variant in TBX5 in a family with an atypical Holt-Oram syndrome phenotype.

Novel de novo missense mutation in the interferon regulatory factor 6 gene in an Italian infant with IRF6-related disorder.

BACKGROUND: Congenital maxillomandibular syngnathia is a rare craniofacial anomaly leading to difficulties in feeding, breathing and ability to thrive. The fusion may consist of soft tissue union (synechiae) to hard tissue union. Isolated cases of maxillomandibular fusion are extremely rare, it is most often syndromic in etiology. CASE PRESENTATION: Clinical management of a female newborn with oromaxillofacial abnormities (synechiae, cleft palate, craniofacial dysmorphisms, dental anomaly) and extraoral malformations (skinfold overlying the nails of both halluces, syndactyly, abnormal external genitalia) is presented. The associated malformations addressed to molecular genetic investigations revealing an interferon regulatory factor 6 (IRF6)-related disorder (van der Woude syndrome/popliteal pterygium syndrome). A novel de novo heterozygous mutation in exon 4 of IRF6 gene on chromosome 1q32.2, precisely c.262A > G (p.Asn88Asp), was found. Similarities are discussed with known asparagine missense mutations in the same codon, which may alter IRF6 gene function by reduced DNA-binding ability. A concomitant maternal Xp11.22 duplication involving two microRNA genes could contribute to possible epigenetic effects. CONCLUSIONS: Our reported case carrying a novel mutation can contribute to expand understandings of molecular mechanisms underlying synechiae and orofacial clefting and to correct diagnosing of incomplete or overlapping features in IRF6-related disorders. Additional multidisciplinary evaluations to establish the phenotypical extent of the IRF6-related disorder and to address family counseling should not only be focused on the surgical corrections of syngnathia and cleft palate, but also involve comprehensive otolaryngologic, audiologic, logopedic, dental, orthopedic, urological and psychological evaluations.

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

BACKGROUND: 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. METHODS: 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]). RESULTS: 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. CONCLUSIONS: 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.

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.

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.

Position effects at the FGF8 locus are associated with femoral hypoplasia.

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.

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.

TBX3 and TBX5 duplication: A family with an atypical overlapping Holt-Oram/ulnar-mammary syndrome phenotype.

Holt-Oram syndrome (HOS) is a rare, autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. A wide spectrum of TBX5 mutations have been reported previously, most resulting in a null allele leading to haploinsufficiency. TBX5 gene duplications have been previously reported in association with typical and atypical HOS phenotypes. Ulnar-Mammary syndrome (UMS) is a distinct rare, autosomal dominant condition caused by mutations in the TBX3 gene. TBX5 and TBX3 are physically linked in cis on human chromosome 12 and contiguous chromosome 12q24 deletions comprising both TBX5 and TBX3 genes have been previously reported but to our knowledge, duplications have never been described. We report on a large German family with at least 17 affected individuals over 6 generations bearing a duplication at 12q24.21 identified on array-CGH comprising both TBX5 and TBX3 genes. Affected patients are presenting with HOS and UMS symptoms, consisting of variable limb anomalies involving the radial and the ulnar rays and cardiac findings such as congenital heart defects, persistent arterial duct or aortic stenosis, and non-classical symptoms, such as supernumerary nipples and cardiomyopathy. Fluorescence in situ hybridisation confirmed a tandem duplication at the 12q24.21 locus. This is the first report of a contiguous TBX3/TBX5 duplication associated with HOS/UMS phenotype.

A novel homozygous RIPK4 variant in a family with severe Bartsocas-Papas syndrome.

Bartsocas-Papas syndrome (BPS) is a rare autosomal recessive disorder characterized by popliteal pterygia, syndactyly, ankyloblepharon, filiform bands between the jaws, cleft lip and palate, and genital malformations. Most of the BPS cases reported to date are fatal either in the prenatal or neonatal period. Causative genetic defects of BPS were mapped on the RIPK4 gene encoding receptor-interacting serine/threonine kinase 4, which is critical for epidermal differentiation and development. RIPK4 variants are associated with a wide range of clinical features ranging from milder ectodermal dysplasia to severe BPS. Here, we evaluated a consanguineous Turkish family, who had two pregnancies with severe multiple malformations compatible with BPS phenotype. In order to identify the underlying genetic defect, direct sequencing of the coding region and exon-intron boundaries of RIPK4 was carried out. A homozygous transversion (c.481G>C) that leads to the substitution of a conserved aspartic acid to histidine (p.Asp161His) in the kinase domain of the protein was detected. Pathogenicity predictions, molecular modeling, and cell-based functional assays showed that Asp161 residue is required for the kinase activity of the protein, which indicates that the identified variant is responsible for the severe BPS phenotype in the family.

The clinical-phenotype continuum in DYNC1H1-related disorders-genomic profiling and proposal for a novel classification.

Mutations in the cytoplasmic dynein 1 heavy chain gene (DYNC1H1) have been identified in rare neuromuscular (NMD) and neurodevelopmental (NDD) disorders such as spinal muscular atrophy with lower extremity dominance (SMALED) and autosomal dominant mental retardation syndrome 13 (MRD13). Phenotypes and genotypes of ten pediatric patients with pathogenic DYNC1H1 variants were analyzed in a multi-center study. Data mining of large-scale genomic variant databases was used to investigate domain-specific vulnerability and conservation of DYNC1H1. We identified ten patients with nine novel mutations in the DYNC1H1 gene. These patients exhibit a broad spectrum of clinical findings, suggesting an overlapping disease manifestation with intermixed phenotypes ranging from neuropathy (peripheral nervous system, PNS) to severe intellectual disability (central nervous system, CNS). Genomic profiling of healthy and patient variant datasets underlines the domain-specific effects of genetic variation in DYNC1H1, specifically on toleration towards missense variants in the linker domain. A retrospective analysis of all published mutations revealed domain-specific genotype-phenotype correlations, i.e., mutations in the dimerization domain with reductions in lower limb strength in DYNC1H1-NMD and motor domain with cerebral malformations in DYNC1H1-NDD. We highlight that the current classification into distinct disease entities does not sufficiently reflect the clinical disease manifestation that clinicians face in the diagnostic work-up of DYNC1H1-related disorders. We propose a novel clinical classification for DYNC1H1-related disorders encompassing a spectrum from DYNC1H1-NMD with an exclusive PNS phenotype to DYNC1H1-NDD with concomitant CNS involvement.

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.

High-throughput transcriptome analysis reveals potentially important relationships between lncRNAs and genes in broilers affected by Valgus-varus Deformity (Gallus gallus).

Valgus-varus Deformity (VVD) is an outward or inward deviation of the tibiotarsus or tarsometatarsus, which results in physical distress of chickens and economic loss in poultry industry. While the etiology and pathogenesis of VVD at the molecular level are still not fully understood so far. Here, based on a case/control design with VVD birds and normal birds, we identified genes and lncRNAs which associated with VVD using RNA sequencing. Transcriptome analysis revealed 231 differentially expressed mRNAs and 23 differentially expressed lncRNAs between case and control of leg cartilage. We identified the cis- and trans-regulatory targets of the differentially expressed lncRNAs, and we constructed a functional lncRNA-mRNA co-expression network. Analysis of the network showed that the differentially expressed mRNAs and the target genes of the differentially expressed lncRNAs were enriched in the signaling pathways associated with bone development, including p53, MAPK, Toll-like receptor, Jak-STAT, Hedgehog, and PPAR. The expression levels of DENND4A, FGF10, FGF12 and BMP3 were also determined in cartilage and other six tissues. Overall, our study predicted the mRNAs and lncRNAs related with leg diseases by transcriptome analyses, which might contribute to understand the etiology and pathogenesis of VVD. It established the foundation for the further research on the function of -mRNAs and lncRNAs in skeleton development.

A newly recognized multiple malformation syndrome with caudal regression associated with a biallelic c.402G>A variant in TBX4.

The T-box4 (TBX4) gene (OMIM *601719) belongs to the T-box family of transcription regulators that share a conserved homology domain and are expressed at specific sites during various stages of embryonic development. Tbx4 has been found to be a crucial transcriptional regulator in embryonic hindlimb development in animal models. Monoallelic variants in the TBX4 gene are reported to be associated with skeletal defects of the pelvis and lower limbs. We report here a fetus with a novel multiple malformation syndrome associated with sacrococcygeal agenesis, bilateral lower limb aplasia, hypoplastic left heart, bilateral lung hypoplasia, hydroureteronephrosis, and nonimmune fetal hydrops, found to have a homozygous nonsense variant in the TBX4 gene. We propose that biallelic variants in the TBX4 gene are associated with a severe syndromic phenotype of sacrococcygeal agenesis and lower limb reduction defects.

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.

HDAC4 and 5 repression of TBX5 is relieved by protein kinase D1.

TBX5 is a T-box family transcription factor that regulates heart and forelimb development in vertebrates and functional deficiencies in this protein result in Holt-Oram syndrome. Recently, we have shown that acetylation of TBX5 potentiates its activity and is important for heart and limb development. Here we report that class II histone deacetylases HDAC4 and HDAC5 associate with TBX5 and repress its role in cardiac gene transcription. Both HDAC4 and HDAC5 deacetylate TBX5, which promotes its relocation to the cytoplasm and HDAC4 antagonizes the physical association and functional cooperation between TBX5 and MEF2C. We also show that protein kinase D1 (PRKD1) relieves the HDAC4/5-mediated repression of TBX5. Thus, this study reveals a novel interaction of HDAC4/5 and PRKD1 in the regulation of TBX5 transcriptional activity.

The RIPK4-IRF6 signalling axis safeguards epidermal differentiation and barrier function.

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.

The role of ESCO2, SALL4 and TBX5 genes in the susceptibility to thalidomide teratogenesis.

Thalidomide is widely used for several diseases; however, it causes malformations in embryos exposed during pregnancy. The complete understanding of the mechanisms by which thalidomide affects the embryo development has not yet been obtained. The phenotypic similarity makes TE a phenocopy of syndromes caused by mutations in ESCO2, SALL4 and TBX5 genes. Recently, SALL4 and TBX5 were demonstrated to be thalidomide targets. To understand if these genes act in the TE development, we sequenced them in 27 individuals with TE; we verified how thalidomide affect them in human pluripotent stem cells (hPSCs) through a differential gene expression (DGE) analysis from GSE63935; and we evaluated how these genes are functionally related through an interaction network analysis. We identified 8 variants in ESCO2, 15 in SALL4 and 15 in TBX5. We compared allelic frequencies with data from ExAC, 1000 Genomes and ABraOM databases; eight variants were significantly different (p < 0.05). Eleven variants in SALL4 and TBX5 were previously associated with cardiac diseases or malformations; however, in TE sample there was no association. Variant effect prediction tools showed 97% of the variants with potential to influence in these genes regulation. DGE analysis showed a significant reduction of ESCO2 in hPSCs after thalidomide exposure.