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.

Langerhans cell histiocytosis and multiple reticulohistiocytomas in a patient with TAR syndrome: An association not previously described.

We describe a patient with thrombocytopenia-absent radius (TAR) syndrome, multisystemic Langerhans cell histiocytosis and multiple reticulohistiocytomas. A mutational study by massive sequencing identified the Val600Glu (V600E) BRAF mutation in the Langerhans cell histiocytosis lesions, but no molecular alterations were found in the reticulohistiocytoma lesions. The concomitant presence in the same patient of more than one type of histiocytosis from two different groups recognized in the most recent Histiocyte Society classification is an extremely rare event. Our case is the first reported case of multisystemic Langerhans cell histiocytosis and multiple reticulohistiocytomas in a patient with TAR syndrome.

The stability of Magoh and Y14 depends on their heterodimer formation and nuclear localization.

Reduced expression of the Y14 gene is a cause of Thrombocytopenia-absent radius (TAR) syndrome. This gene contains a conserved RNA recognition motif (RRM) in the central region and nuclear localization/export sequences (NLS/NES) in the N-terminal. Y14 and Magoh proteins form tight heterodimers and are the core of exon junction complexes (EJCs), which mediate various processes of mRNA metabolism after transcription. In this report, we found that protein expression levels of exogenously expressed Magoh L136R and Y14 L118R (leucine-to-arginine substitution at amino acid residue 136 and 118 respectively, that results in the formation of the complex being lost) are lower than their wild-types. This reduction is likely caused by protein levels, as no difference in mRNA levels was detected. Meanwhile, a cycloheximide chase assay determined that the degradation rates of Magoh L136R and Y14 L118R were faster than their wild-types. Both Y14 L118R and Magoh L136R lost the ability to form heterodimers with corresponding wild-type proteins. However, Y14 L118R is able to still localize in the nucleus which causes the stability of Y14 L118R to be higher than Magoh L136R. These results reveal that the stability of Magoh and Y14 is not only dependent on the heterodimer structure, but also dependent on nuclear localization.

Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome.

In historical attempts to treat morning sickness, use of the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment; however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide-induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome.

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

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

Acute myeloid leukemia in a patient with thrombocytopenia with absent radii: A case report and review of the literature.

Thrombocytopenia with absent radii (TAR) syndrome is a rare congenital disorder characterized by low platelet counts of various severity, bilateral absent radii but thumbs are usually present. TAR syndrome is not generally associated with bone marrow failure or malignancy. Janus kinase-2, myeloproliferative leukemia protein, and calreticulin are not mutated in TAR patients. Only four cases of leukemia were reported in TAR patients in the literature: three acute myeloid leukemia (AML) and one acute lymphoblastic leukemia. Of the three cases of AML found in TAR patient, only one was reported in an adult. We report a case of myelodysplastic syndrome progressing to AML with calreticulin driver mutation in an adult male with TAR syndrome who was successfully treated with hematopoietic allogeneic stem cell transplantation.

KLF13 is a genetic modifier of the Holt-Oram syndrome gene TBX5.

TBX5, a member of the T-box family of transcription factors, is a dosage sensitive regulator of heart development. Mutations in TBX5 are responsible for Holt-Oram Syndrome, an autosomal dominant disease with variable and partially penetrant cardiac defects suggestive of the existence of genetic and environmental modifiers. KLF13, a member of the Krüppel-like family of zinc finger proteins is co-expressed with TBX5 in several cardiac cells including atrial cardiomyocytes and cells of the interatrial septum. We report that KLF13 interacts physically and functionally with TBX5 to synergistically activate transcription of cardiac genes. We show that TBX5 contacts KLF13 via its T-domain and find that several disease-causing mutations therein have decreased KLF13 interaction. Whereas Klf13 heterozygote mice have no detectable cardiac defects, loss of a Klf13 allele in Tbx5 heterozygote mice significantly increases the penetrance of TBX5-dependent cardiac abnormalities including atrial, atrial-ventricular and ventricular septal defects. The results reveal for the first time combinatorial interaction between a T-box protein and a KLF family member and its importance for heart and possibly other organ development. The data also suggest that, in human, KLF13 may be a genetic modifier of the Holt-Oram Syndrome gene TBX5.

TBX5 loss-of-function mutation contributes to atrial fibrillation and atypical Holt-Oram syndrome.

Previous genome-wide association studies have demonstrated that single nucleotide polymorphisms in T­box (TBX)5 are associated with increased susceptibility to atrial fibrillation (AF), and a recent study has causally linked a TBX5 mutation to atypical Holt-Oram syndrome and paroxysmal AF. However, the prevalence and spectrum of TBX5 mutations in patients with AF remain to be elucidated. In the present study, a cohort of 190 unrelated patients with idiopathic AF were prospectively recruited, with 400 unrelated healthy individuals recruited as controls. The coding exons and flanking introns of the TBX5 gene were sequenced in the participants. The functional characteristics of the mutant TBX5 were delineated in contrast with its wild­type counterpart using a dual­luciferase reporter assay system. As a result, a novel heterozygous TBX5 mutation, p.P132S, was identified in an index patient with AF, with a mutational prevalence of ~0.53%. Genetic analysis of the proband's family showed that the mutation co­segregated with AF, and was transmitted in an autosomal dominant pattern. The missense mutation was absent in the 800 control chromosomes, and the altered amino acid was completely evolutionarily conserved across species. Functional analyses revealed that the mutant TBX5 had significantly reduced transcriptional activity. Furthermore, the mutation markedly decreased the synergistic activation between TBX5 and NK2 homeobox 5, another transcription factor which has been causatively linked to AF. The present study was the first, to the best of our knowledge, to report on the association between a TBX5 loss­of­function mutation and increased susceptibility to AF. These results provide novel insight into the molecular mechanism underpinning AF, and have potential implications in the development of novel prophylactic and therapeutic strategies for AF, the most common form of sustained cardiac arrhythmia.

The pathogenesis of congenital radial head dislocation/subluxation.

The pathogenesis of congenital radial head dislocation/subluxation is unknown and has not been previously investigated. In this review, we explore the pathogenesis and define five different primary insults: collagen abnormalities, abnormal endochondral ossification of the developing growth plate, abnormalities of forearm ossification outside the growth plate, disproportionate growth of the radius and ulna, and altered HOX D expression/activity. Finally, the clinical relevance of our review is discussed.

Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease.

Recent studies have identified the genetic underpinnings of a growing number of diseases through targeted exome sequencing. However, this strategy ignores the large component of the genome that does not code for proteins, but is nonetheless biologically functional. To address the possible involvement of regulatory variation in congenital heart diseases (CHDs), we searched for regulatory mutations impacting the activity of TBX5, a dosage-dependent transcription factor with well-defined roles in the heart and limb development that has been associated with the Holt-Oram syndrome (heart-hand syndrome), a condition that affects 1/100 000 newborns. Using a combination of genomics, bioinformatics and mouse genetic engineering, we scanned ∼700 kb of the TBX5 locus in search of cis-regulatory elements. We uncovered three enhancers that collectively recapitulate the endogenous expression pattern of TBX5 in the developing heart. We re-sequenced these enhancer elements in a cohort of non-syndromic patients with isolated atrial and/or ventricular septal defects, the predominant cardiac defects of the Holt-Oram syndrome, and identified a patient with a homozygous mutation in an enhancer ∼90 kb downstream of TBX5. Notably, we demonstrate that this single-base-pair mutation abrogates the ability of the enhancer to drive expression within the heart in vivo using both mouse and zebrafish transgenic models. Given the population-wide frequency of this variant, we estimate that 1/100 000 individuals would be homozygous for this variant, highlighting that a significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome.

Two patterns of thrombopoietin signaling suggest no coupling between platelet production and thrombopoietin reactivity in thrombocytopenia-absent radii syndrome.

BACKGROUND: Thrombocytopenia with absent radii syndrome is defined by bilateral radius aplasia and thrombocytopenia. Due to impaired thrombopoietin signaling there are only few bone marrow megakaryocytes and these are immature; the resulting platelet production defect improves somewhat over time. A microdeletion on chromosome 1q21 is present in all patients but is not sufficient to form thrombocytopenia with absent radii syndrome. We aimed to refine the signaling defect in this syndrome. DESIGN AND METHODS: We report an extended study of 23 pediatric and adult patients suffering from thrombocytopenia with absent radii syndrome in order to scrutinize thrombopoietin signal transduction by immunoblotting and gel electrophoretic shift assays. In addition, platelet immunotyping and reactivity were analyzed by flow cytometry. Results were correlated with clinical data including age and platelet counts. RESULTS: Two distinct signaling patterns were identified. Juvenile patients showed abrogated thrombopoietin signaling (pattern #1), which is restored in adults (pattern #2). Phosphorylated Jak2 was indicative of activation of STAT1, 3 and 5, Tyk2, ERK, and Akt, showing its pivotal role in distinct thrombopoietin-dependent pathways. Jak2 cDNA was not mutated and the thrombopoietin receptor was present on platelets. All platelets of patients expressed normal levels of CD41/61, CD49b, and CD49f receptors, while CD42a/b and CD29 were slightly reduced and the fibronectin receptor CD49e markedly reduced. Lysosomal granule release in response to thrombin receptor activating peptide was diminished. CONCLUSIONS: We show a combined defect of platelet production and function in thrombocytopenia with absent radii syndrome. The rise in platelets that most patients have during the first years of life preceded the restored thrombopoietin signaling detected at a much later age, implying that these events are uncoupled and that an unknown factor mediates the improvement of platelet production.

Functional analysis of novel TBX5 T-box mutations associated with Holt-Oram syndrome.

AIMS: Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations. METHODS AND RESULTS: Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners. CONCLUSION: Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.

Cadmium-induced postaxial forelimb ectrodactyly: association with altered sonic hedgehog signaling.

Administration of CdSO(4) to C57BL/6 mice at day 9.5 of gestation induces a high incidence of postaxial forelimb ectrodactyly in the offspring. We propose that Cd(2+) exposure impairs the process of anterior/posterior formation in the limb bud, a process that is directed by Sonic hedgehog (Shh) signaling. We show that exposure of the mouse embryo to Cd(2+) disrupts Shh signaling as measured by polarizing activity of mouse limb bud ZPA grafted to a host chick wing, and activity of a Gli:luciferase reporter exposed to limb bud lysates. Yet the expression of Shh and its translation are not affected by Cd(2+) exposure. We propose that teratogen exposure affects the processing of Shh in the cells in which it is made.