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Chemokines mediate leucocyte migration and homeostasis, and are key targets in inflammatory diseases including atherosclerosis, cytokine storm and chronic auto-immune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines overcoming redundancy, and are effective in several pre-clinical disease models. Their clinical development has not progressed due to concerns regarding potential immunogenicity, parenteral delivery and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple-chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange mass spectrometry to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand 8 (CCL8) and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native mass spectrometry approaches showed that BK1.1 binds CCL8, CCL7 and CCL18, and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2 and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2 and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

Ticks are hematophagous arachnids that parasitize mammals and other hosts, feeding on their blood. Ticks secrete numerous salivary factors that enhance host blood flow or suppress the host inflammatory response. The recruitment of leukocytes, a hallmark of inflammation, is regulated by chemokines, which activate chemokine receptors on the leukocytes. Ticks target this process by secreting glycoproteins called Evasins, which bind to chemokines and prevent leukocyte recruitment. This review describes the recent discovery of numerous Evasins produced by ticks, their classification into two structural and functional classes, and the efficacy of Evasins in animal models of inflammatory diseases. The review also proposes a standard nomenclature system for Evasins and discusses the potential of repurposing or engineering Evasins as therapeutic anti-inflammatory agents., Highlights Chemokines are mammalian proteins that are secreted at the sites of tissue insult and stimulate trafficking of leukocytes to the affected tissues, a key component of the inflammatory response. Evasins are tick salivary glycoproteins that bind to chemokines, thereby suppressing the host inflammatory response, an apparent mechanism to prolong the residence times of ticks on their hosts. Bioinformatics searches of salivary transcriptomic and genomic sequence databases and yeast surface display screening methods have enabled discovery of Evasins produced by numerous tick species spread across at least three genera. Two families of Evasins (Classes A and B) have been identified. Evasins from the two families have different 3D structures, different conserved sequence features (including patterns of disulfide bonds), and selectivity for different families of chemokines (CC and CXC, respectively). Evasins each bind to several (or many) chemokines but different Evasins have distinct selectivities for target chemokines. Structures, mutational experiments, and sequence comparisons are beginning to reveal the critical elements of Evasins for chemokine recognition. Evasins show efficacy in animal models of inflammatory diseases, suggesting that either natural Evasins or engineered variants have potential as therapeutic anti-inflammatory agents.

Myocardial fibrosis is a severe global health problem due to its prevalence in all forms of cardiac diseases and direct role in causing heart failure. The discovery of efficient antifibrotic compounds has been hampered due to the lack of a physiologically relevant disease model. Herein, we present a disease model of human myocardial fibrosis and use it to establish a compound screening system. In the Biowire II platform, cardiac tissues are suspended between a pair of poly(octamethylene maleate (anhydride) citrate) (POMaC) wires. Noninvasive functional readouts are realized on the basis of the deflection of the intrinsically fluorescent polymer. The disease model is constructed to recapitulate contractile, biomechanical, and electrophysiological complexities of fibrotic myocardium. Additionally, we constructed a heteropolar integrated model with fibrotic and healthy cardiac tissues coupled together. The integrated model captures the regional heterogeneity of scar lesion, border zone, and adjacent healthy myocardium. Finally, we demonstrate the utility of the system for the evaluation of antifibrotic compounds. The high-fidelity in vitro model system combined with convenient functional readouts could potentially facilitate the development of precision medicine strategies for cardiac fibrosis modeling and establish a pipeline for preclinical compound screening., The cardiomyocytes/fibroblasts ratio is modulated to develop cardiac fibrosis-on-a-chip. An integrated model is constructed to mimic the interaction between scar lesion and adjacent healthy tissue.

Chemokines function via G-protein coupled receptors in a robust network to recruit immune cells to sites of inflammation. Due to the complexity of this network, targeting single chemokines or receptors has not been successful in inflammatory disease. Dog tick saliva contains polyvalent CC-chemokine binding peptides termed evasins 1 and 4, that efficiently disrupt the chemokine network in models of inflammatory disease. Here we develop yeast surface display as a tool for functionally identifying evasins, and use it to identify 10 novel polyvalent CC-chemokine binding evasin-like peptides from salivary transcriptomes of eight tick species in Rhipicephalus and Amblyomma genera. These evasins have unique binding profiles compared to evasins 1 and 4, targeting CCL2 and CCL13 in addition to other CC-chemokines. Evasin binding leads to neutralisation of chemokine function including that of complex chemokine mixtures, suggesting therapeutic efficacy in inflammatory disease. We propose that yeast surface display is a powerful approach to mine potential therapeutics from inter-species protein interactions that have arisen during evolution of parasitism in ticks.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Congenital cardiovascular malformation is a common birth defect incorporating abnormalities of the outflow tract and aortic arch arteries, and mice deficient in the transcription factor AP-2&alpha, (Tcfap2a) present with complex defects affecting these structures. AP-2&alpha, is expressed in the pharyngeal surface ectoderm and neural crest at mid-embryogenesis in the mouse, but the precise tissue compartment in which AP-2&alpha, is required for cardiovascular development has not been identified. In this study we describe the fully penetrant AP-2&alpha, deficient cardiovascular phenotype on a C57Bl/6J genetic background and show that this is associated with increased apoptosis in the pharyngeal ectoderm. Neural crest cell migration into the pharyngeal arches was not affected. Cre-expressing transgenic mice were used in conjunction with an AP-2&alpha, conditional allele to examine the effect of deleting AP-2&alpha, from the pharyngeal surface ectoderm and the neural crest, either individually or in combination, as well as the second heart field. This, surprisingly, was unable to fully recapitulate the global AP-2&alpha, deficient cardiovascular phenotype. The outflow tract and arch artery phenotype was, however, recapitulated through early embryonic Cre-mediated recombination. These findings indicate that AP-2&alpha, has a complex influence on cardiovascular development either being required very early in embryogenesis and/or having a redundant function in many tissue layers.

Inflammation, is driven by a network comprising cytokines, chemokines, their target receptors and leukocytes, and is a major pathologic mechanism that adversely affects organ function in diverse human diseases. Despite being supported by substantial target validation, no successful anti-chemokine therapeutic to treat inflammatory disease has yet been developed. This is in part because of the robustness of the chemokine network, which emerges from a large total chemokine load in disease, promiscuous expression of receptors on leukocytes, promiscuous and synergistic interactions between chemokines and receptors, and feedforward loops created by secretion of chemokines by leukocytes themselves. Many parasites, including viruses, helminths and ticks, evade the chemokine network by producing proteins that bind promiscuously to chemokines or their receptors. Evasins - three small glycoproteins identified in the saliva of the brown dog tick - bind multiple chemokines, and are active in several animal models of inflammatory disease. Over 50 evasin homologs have recently been identified from diverse tick species. Characterization of the chemokine binding patterns of evasins show that several have anti-chemokine activities that extend substantially beyond those previously described. These studies indicate that evasins function at the site of the tick bite by reducing total chemokine load. This not only reduces chemokine signaling to receptors, but also interrupts feedforward loops, thus disabling the chemokine network. Taking the lead from nature, a goal for the development of new anti-chemokine therapeutics would be to reduce the total chemokine load in disease. This could be achieved by administering appropriate evasin combinations or by smaller peptides that mimic evasin action.

Inflammation, is driven by a network comprising cytokines, chemokines, their target receptors and leukocytes, and is a major pathologic mechanism that adversely affects organ function in diverse human diseases. Despite being supported by substantial target validation, no successful anti-chemokine therapeutic to treat inflammatory disease has yet been developed. This is in part because of the robustness of the chemokine network, which emerges from a large total chemokine load in disease, promiscuous expression of receptors on leukocytes, promiscuous and synergistic interactions between chemokines and receptors, and feedforward loops created by secretion of chemokines by leukocytes themselves. Many parasites, including viruses, helminths and ticks, evade the chemokine network by producing proteins that bind promiscuously to chemokines or their receptors. Evasins - three small glycoproteins identified in the saliva of the brown dog tick - bind multiple chemokines, and are active in several animal models of inflammatory disease. Over 50 evasin homologs have recently been identified from diverse tick species. Characterization of the chemokine binding patterns of evasins show that several have anti-chemokine activities that extend substantially beyond those previously described. These studies indicate that evasins function at the site of the tick bite by reducing total chemokine load. This not only reduces chemokine signaling to receptors, but also interrupts feedforward loops, thus disabling the chemokine network. Taking the lead from nature, a goal for the development of new anti-chemokine therapeutics would be to reduce the total chemokine load in disease. This could be achieved by administering appropriate evasin combinations or by smaller peptides that mimic evasin action.

Introduction There is strong evidence from familial recurrence studies for a genetic predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF). TOF is the most common, cyanotic congenital heart disease (CHD) phenotype yet the cause for the majority of cases remains elusive. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Methods and Results 829 TOF patients underwent whole exome sequencing (WES), the largest cohort of non-syndromic TOF patients reported to date. The prevalence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database (gnomAD) and a scaled combined annotation-dependent depletion (CADD) score of ≥20. Clustering analysis of variants revealed that two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P Conclusion In summary, the NOTCH1 locus is the most frequent site of genetic variants predisposing to non-syndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients. Conflict of Interest None

International audience; Generation of widely differing and specialized cell types from a single totipotent zygote involves large-scale transcriptional changes and chromatin reorganization. Pioneer transcription factors play key roles in programming the epigenome and facilitating recruitment of additional regulatory factors during successive cell lineage specification and differentiation steps. Here we show that Isl1 acts as a pioneer factor driving cardiomyocyte lineage commitment by shaping the chromatin landscape of cardiac progenitor cells. Using an Isl1 hypomorphic mouse line which shows congenital heart defects, genome-wide profiling of Isl1 binding together with RNA- and ATAC-sequencing of cardiac progenitor cells and their derivatives, we uncover a regulatory network downstream of Isl1 that orchestrates cardiogenesis. Mechanistically, we show that Isl1 binds to compacted chromatin and works in concert with the Brg1-Baf60c-based SWI/SNF complex to promote permissive cardiac lineage-specific alterations in the chromatin landscape not only of genes with critical functions in cardiac progenitor cells, but also of cardiomyocyte structural genes that are highly expressed when Isl1 itself is no longer present. Thus, the Isl1/Brg1-Baf60c complex plays a crucial role in orchestrating proper cardiogenesis and in establishing epigenetic memory of cardiomyocyte fate commitment.

Rationale: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. Objective: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. Methods and Results: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4 , surpassed thresholds for genome-wide significance (assigned as P −8 ) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%–6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%–3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1 , FLT4 and the well-established TOF gene, TBX1 , we identified potential association with variants in several other candidates, including RYR1 , ZFPM1 , CAMTA2 , DLX6 , and PCM1 . Conclusions: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4 . Together, variants in these genes are found in almost 7% of TOF patients.

© 2019 American Society of Human Genetics The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.

AimsFamilial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Here, we use whole exome sequencing to assess the prevalence of rare, potentially deleterious variants in candidate genes previously associated with both syndromic and non-syndromic TOF, in the largest cohort of non-syndromic TOF patients reported to date.Methods & Results829 non-syndromic TOF patients underwent whole exome sequencing. A systematic review of the literature was conducted which revealed 77 genes in which mutations had been reported in patients with TOF. The presence of rare, deleterious variants in the 77 candidate genes was determined, defined by a minor allele frequency of ≤ 0.001 and scaled combined annotation-dependent depletion (CADD) score of ≥ 20. We found a clustering of heterozygous rare, deleterious variants inNOTCH1(P=1.89E-15),DOCK6(P=2.93E-07),MYOM2(P= 7.35E-05),TTC37(P=0.016),MESP1(P=0.024) andTBX1(P=0.039), after correcting for multiple testing.NOTCH1was most frequently found to harbour deleterious variants. Changes were observed in 49 patients (6%; 95% confidence interval [CI]: 4.5% - 7.8%) and included six truncating/frameshift variants and forty missense variants. Sanger sequencing of the unaffected parents of thirteen cases identified fivede novovariants. Variants were not confined to a single functional domain of the NOTCH1 protein but significant clustering of variants was evident in the EGF-like repeats (P=0.018). ThreeNOTCH1missense variants (p.G200R, p.C607Y andde novop.N1875S) were subjected to functional evaluation and showed a reduction in Jagged1 ligand-induced NOTCH signalling. p.C607Y, which exhibited the most significant reduction in signalling, also perturbed S1 cleavage of the NOTCH1 receptor in the Golgi.ConclusionTheNOTCH1locus is a frequent site of genetic variants predisposing to non-syndromic TOF with 6% of patients exhibiting rare, deleterious variants. Our data supports the polygenic origin of TOF and suggests larger studies may identify additional loci.

International audience; To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ß11% of the cases (113 variants in 107/986 individuals: ß8% of the individuals had a likely pathogenic loss-of-function [LoF] variant, whereas ß3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%–15% yield from array CGH alone.

Recurrent rearrangements of chromosome 1q21.1 that occur via non-allelic homologous recombination have been associated with variable phenotypes exhibiting incomplete penetrance, including congenital heart disease (CHD). However, the gene or genes within the ~1 Mb critical region responsible for each of the associated phenotypes remains unknown. We examined the 1q21.1 locus in 948 patients with tetralogy of Fallot (TOF), 1488 patients with other forms of CHD and 6760 ethnically matched controls using single nucleotide polymorphism genotyping arrays (Illumina 660W and Affymetrix 6.0) and multiplex ligation-dependent probe amplification. We found that duplication of 1q21.1 was more common in cases of TOF than in controls [odds ratio (OR) 30.9, 95% confidence interval (CI) 8.9-107.6); P = 2.2 × 10(-7)], but deletion was not. In contrast, deletion of 1q21.1 was more common in cases of non-TOF CHD than in controls [OR 5.5 (95% CI 1.4-22.0); P = 0.04] while duplication was not. We also detected rare (n = 3) 100-200 kb duplications within the critical region of 1q21.1 in cases of TOF. These small duplications encompassed a single gene in common, GJA5, and were enriched in cases of TOF in comparison to controls [OR = 10.7 (95% CI 1.8-64.3), P = 0.01]. These findings show that duplication and deletion at chromosome 1q21.1 exhibit a degree of phenotypic specificity in CHD, and implicate GJA5 as the gene responsible for the CHD phenotypes observed with copy number imbalances at this locus.

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.

AIMS: Heterozygous mutations in the transcription factor Nkx2.5 indicate a genetic cause for congenital heart diseases (CHDs) in human beings. The present study aimed to assess the prevalence of NKX2.5 mutations in Italian patients with sporadic non-syndromic and syndromic CHD, as well as to appraise any genotype-phenotype correlations. METHODS: One hundred Italian patients affected with CHD (90 had sporadic non-syndromic CHD and 10 had syndromic CHD) were screened for NKX2.5 mutations. The coding region and flanking regions involved in gene splicing of the CSX/NKX2.5 gene were amplified from genomic DNA by PCR, and mutational analysis was performed using denaturing high performance liquid chromatography and DNA sequencing. RESULTS: One previously reported NKX2.5 mutation (c.73C>T, p.R25C) was identified in two of the 100 CHD patients (2%). We have detected the p.R25C alteration in a woman showing aneurysm of the membranous septum, aortic coarctation and bicuspid aortic valve, that was a different phenotype from those previously reported, and for the first time in a patient with syndromic CHD with Down's syndrome (posterior ventricular septal defect, atrial septal defect, left superior cava vein ' sinus, and patent ductus arteriosus). CONCLUSION: Our results confirm that NKX2.5 mutations are not a common cause of CHD; furthermore, the p.R25C variation may increase susceptibility to development of CHD in patients with and without chromosomal abnormalities.

The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, minor allele frequency ~0.25% (UK)) associated with plasma triglyceride (TG) levels (−1.43 s.d. (s.e.=0.27 per minor allele (P-value=8.0 × 10−8)) discovered in 3,202 individuals with low read-depth, whole-genome sequence. We replicate this in 12,831 participants from five additional samples of Northern and Southern European origin (−1.0 s.d. (s.e.=0.173), P-value=7.32 × 10−9). This is consistent with an effect between 0.5 and 1.5 mmol l−1 dependent on population. We show that a single predicted splice donor variant is responsible for association signals and is independent of known common variants. Analyses suggest an independent relationship between rs138326449 and high-density lipoprotein (HDL) levels. This represents one of the first examples of a rare, large effect variant identified from whole-genome sequencing at a population scale., Population-based genome sequencing provides an increasingly rich resource for the identification of low-frequency, large effect variants associated with clinically important phenotypes. Timpson et al. use UK10K data to identify a variant of the APOC3 gene strongly associated with plasma triglyceride levels.

Congenital heart defects (CHDs) have a neonatal incidence of 0.8–1% (refs. 1,2). Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (~2.7%)3, suggesting a considerable role for de novo mutations (DNMs) and/or incomplete penetrance4, 5. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of 'syndromic' patients with extra-cardiac manifestations6, 7. We exome sequenced 1,891 probands, including both syndromic CHD (S-CHD, n = 610) and nonsyndromic CHD (NS-CHD, n = 1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs but not inherited PTVs in known CHD-associated genes, consistent with recent findings8. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study finds evidence for distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

Therapeutic angiogenesis is a major goal ofregenerative medicine, but no clinically approved small molecule exists that enhancesnew blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitroangiogenesis, promoting branching morphogenesis, and tubule remodeling. The pro-angiogenic phenotype is mediated by Retinoic Acid Receptor (RAR) but not Retinoic X Receptor(RXR) activation, and is characterized by secretion of the pro-angiogenic factors Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGFA), Plasminogen Activator, Urokinase (PLAU) and Placental Growth Factor (PGF), and reduced secretion of the antiangiogenic factor Pentraxin-3 (PTX3) from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing.

CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.

Embryonic development in normal and genetically modified mice is commonly analysed by histological sectioning. This procedure is time-consuming, prone to artefact, and results in the loss of three-dimensional (3D) information. Magnetic resonance imaging (MRI) of embryos has the potential of non-invasively acquiring a complete 3D data set. Published methods have used spin-echo techniques with inherently high signal-to-noise ratio (SNR); however, they required either perfusion of the embryo with a contrast agent, or prolonged acquisition times to improve contrast and resolution. Here, we show that a standard preparation (i.e. paraformaldehyde fixation) of 15.5 days post-coitum embryos followed by MRI using a fast gradient-echo sequence with T 1-weighting achieves high resolution and high throughput for investigating mouse embryonic anatomy. 3D data sets were acquired in overnight experiments (

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells. © 2011 Chen et al.

Summary International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The 'Deciphering the Mechanisms of Developmental Disorders' (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

Rationale : 22q11 deletion syndrome arises from recombination between low-copy repeats on chromosome 22. Typical deletions result in hemizygosity for TBX1 associated with congenital cardiovascular disease. Deletions distal to the typically deleted region result in a similar cardiac phenotype but lack in extracardiac features of the syndrome, suggesting that a second haploinsufficient gene maps to this interval. Objective : The transcription factor HIC2 is lost in most distal deletions, as well as in a minority of typical deletions. We used mouse models to test the hypothesis that HIC2 hemizygosity causes congenital heart disease. Methods and Results : We created a genetrap mouse allele of Hic2. The genetrap reporter was expressed in the heart throughout the key stages of cardiac morphogenesis. Homozygosity for the genetrap allele was embryonic lethal before embryonic day E10.5, whereas the heterozygous condition exhibited a partially penetrant late lethality. One third of heterozygous embryos had a cardiac phenotype. MRI demonstrated a ventricular septal defect with over-riding aorta. Conditional targeting indicated a requirement for Hic2 within the Nkx2.5+ and Mesp1 + cardiovascular progenitor lineages. Microarray analysis revealed increased expression of Bmp10 . Conclusions : Our results demonstrate a novel role for Hic2 in cardiac development. Hic2 is the first gene within the distal 22q11 interval to have a demonstrated haploinsufficient cardiac phenotype in mice. Together our data suggest that HIC2 haploinsufficiency likely contributes to the cardiac defects seen in distal 22q11 deletion syndrome.

Background Patent ductus arteriosus (PDA) is one of the most common congenital heart defects. Transcription factor AP-2 beta (TFAP2B) mutations are associated with the Char syndrome, a disorder associated with PDA, and with facial and fingers abnormalities. Recently, we identified two TFAP2B mutations in two families without Char syndrome phenotype, c.601+5G>A and c.435-438delCCGG, and these TFAP2B mutations were associated with familial isolated PDA. The aim of this study was to identify the effects of these mutations on TFAP2B function. Methods Plasmids containing the wild-type or mutated TFAP2B were constructed and transfected in cells. Plasmids containing the TFAP2B coactivator, Cpb/p300-interacting transactivator 2 (CITED2), was also transfected. TFAP2B expression was detected by luciferase expression and by Western blot analysis. Results These mutations resulted in loss of transactivation function, which could not be improved by Cpb/p300-interacting transactivator 2. The c.601+5G>A mutated gene did not express any protein, whereas the c.435-438delCCGG mutation did not impact the transactivation function activated by the wild-type TFAP2B. Conclusions These results suggest that a haploinsufficiency effect of TFAP2B could be involved in familial isolated PDA. © 2014 Elsevier Inc. All rights reserved.

Cardiac failure affects 1.5% of the adult population and is predominantly caused by myocardial dysfunction secondary to coronary vascular insufficiency. Current therapeutic strategies improve prognosis only modestly, as the primary cause – loss of normally functioning cardiac myocytes – is not being corrected. Adult cardiac myocytes are unable to divide and regenerate to any significant extent following injury. New cardiac myocytes are, however, created during embryogenesis from progenitor cells and then by cell division from existing cardiac myocytes. This process is intimately linked to the development of coronary vasculature from progenitors originating in the endothelium, the proepicardial organ and neural crest. In this review, we systematically evaluate approx. 90 mouse mutations that impair heart muscle growth during development. These studies provide genetic evidence for interactions between myocytes, endothelium and cells derived from the proepicardial organ and the neural crest that co-ordinate myocardial and coronary vascular development. Conditional knockout and transgenic rescue experiments indicate that Vegfa, Bmpr1a (ALK3), Fgfr1/2, Mapk14 (p38), Hand1, Hand2, Gata4, Zfpm2 (FOG2), Srf and Txnrd2 in cardiac myocytes, Rxra and Wt1 in the proepicardial organ, EfnB2, Tek, Mapk7, Pten, Nf1 and Casp8 in the endothelium, and Bmpr1a and Pax3 in neural crest cells are key molecules controlling myocardial development. Coupling of myocardial and coronary development is mediated by BMP (bone morphogenetic protein), FGF (fibroblast growth factor) and VEGFA (vascular endothelial growth factor A) signalling, and also probably involves hypoxia. Pharmacological targeting of these molecules and pathways could, in principle, be used to recreate the embryonic state and achieve coupled myocardial and coronary vascular regeneration in failing hearts.

Background: 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome in humans, characterized by cardiovascular defects such as interrupted aortic arch, outflow tract defects, thymus and parathyroid hypo- or aplasia, and cleft palate. Heterozygosity of Tbx1, the mouse homolog of the candidate TBX1 gene, results in mild defects dependent on genetic background, whereas complete inactivation results in severe malformations in multiple tissues. Results: The loss of function of two Sprouty genes, which encode feedback antagonists of receptor tyrosine kinase (RTK) signaling, phenocopy many defects associated with 22q11DS in the mouse. The stepwise reduction of Sprouty gene dosage resulted in different phenotypes emerging at specific steps, suggesting that the threshold up to which a given developmental process can tolerate increased RTK signaling is different. Tbx1 heterozygosity significantly exacerbated the severity of all these defects, which correlated with a substantial increase in RTK signaling. Conclusions: Our findings suggest that TBX1 functions as an essential component of a mechanism that protects the embryo against perturbations in RTK signaling that may lead to developmental defects characteristic of 22q11DS. We propose that genetic factors that enhance RTK signaling ought to be considered as potential genetic modifiers of this syndrome. Developmental Dynamics 241:1310–1324, 2012. © 2012 Wiley Periodicals, Inc.

This chapter discusses imaging cardiac developmental malfunctions in the mouse embryo. Studies of human genetic architecture cannot provide a mechanistic understanding of gene function in cardiac development, and analysis of gene-gene and gene-environment interactions in humans remains a challenge. These considerations necessitate the use of animal models, the most appropriate and widely used being the mouse. Like the human, the mouse has a four-chambered heart with a septated outflow tract, left-sided great arteries, and parallel pulmonary and systemic circulations. Common cardiac malformations such as septal, outflow tract, and aortic arch and pulmonary trunk defects can thus be identified in mouse embryos, and mouse mutations typically recapitulate the cardiac malformations observed in patients with mutations in the same genes (reviewed in Schneider and Bhattacharya, 2004). Imaging such a complex and dynamic structure as the developing heart plays a crucial role in such studies, underlying accurate assessment of phenotype. New anatomical imaging techniques such as optical projection tomography, high resolution episcopic microscopy and magnetic resonance imaging provide ways to supplement conventional histology with comprehensive image data, yielding three-dimensional models that allow systematic, quantitative comparisons of structure. These imaging methods have complementary strengths, and by combining them it is possible to envisage a powerful cardiac phenotyping pipeline. © 2010 Elsevier Inc. All rights reserved.

AIMS: The sinus venous myocardium, comprising the sinoatrial node (SAN) and sinus horns (SH), is a region subject to congenital malformations and cardiac arrhythmias. It differentiates from symmetric bilateral mesenchymal precursors, but morphological, molecular, and functional left/right differences are progressively established through development. The role of the laterality gene Pitx2 in this process is unknown. We aimed to elucidate the molecular events driving left/right patterning in the sinus venosus (SV) myocardium by using a myocardial Pitx2 knockout mouse. METHODS AND RESULTS: We generated a myocardial specific Pitx2 knockout model (cTP mice). cTP embryos present several features of Pitx2 null, including right atrial isomerism with bilateral SANs and symmetric atrial entrance of the systemic veins. By in situ hybridization and optical mapping analysis, we compared throughout development the molecular and functional properties of the SV myocardium in wt and mutant embryos. We observed that Pitx2 prevents the expansion of the left-SAN primordium at the onset of its differentiation into myocardium; Pitx2 promotes expansion of the left SH through development; Pitx2 dose-dependently represses the autorhythmic properties of the left SV myocardium at mid-gestation (E14.5); Pitx2 modulates late foetal gene expression at the left SH-derived superior caval vein. CONCLUSION: Pitx2 drives left/right patterning of the SV myocardium through multiple developmental steps. Overall, Pitx2 plays a crucial functional role by negatively modulating a nodal-type programme in the left SV myocardium.

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10(-5)). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ∼5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ∼4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias. ispartof: American Journal of Human Genetics vol:91 issue:3 pages:489-501 ispartof: location:United States status: published