Abstract The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction ...of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.
Objective
Epilepsy‐associated developmental lesions, including malformations of cortical development and low‐grade developmental tumors, represent a major cause of drug‐resistant seizures requiring ...surgical intervention in children. Brain‐restricted somatic mosaicism has been implicated in the genetic etiology of these lesions; however, many contributory genes remain unidentified.
Methods
We enrolled 50 children who were undergoing epilepsy surgery into a translational research study. Resected tissue was divided for clinical neuropathologic evaluation and genomic analysis. We performed exome and RNA sequencing to identify somatic variation and we confirmed our findings using high‐depth targeted DNA sequencing.
Results
We uncovered candidate disease‐causing somatic variation affecting 28 patients (56%), as well as candidate germline variants affecting 4 patients (8%). In agreement with previous studies, we identified somatic variation affecting solute carrier family 35 member A2 (SLC35A2) and mechanistic target of rapamycin kinase (MTOR) pathway genes in patients with focal cortical dysplasia. Somatic gains of chromosome 1q were detected in 30% (3 of 10) of patients with Type I focal cortical dysplasia (FCD)s. Somatic variation in mitogen‐activated protein kinase (MAPK) pathway genes (i.e., fibroblast growth factor receptor 1 FGFR1, FGFR2, B‐raf proto‐oncogene, serine/threonine kinase BRAF, and KRAS proto‐oncogene, GTPase KRAS) was associated with low‐grade epilepsy‐associated developmental tumors. RNA sequencing enabled the detection of somatic structural variation that would have otherwise been missed, and which accounted for more than one‐half of epilepsy‐associated tumor diagnoses. Sampling across multiple anatomic regions revealed that somatic variant allele fractions vary widely within epileptogenic tissue. Finally, we identified putative disease‐causing variants in genes not yet associated with focal cortical dysplasia.
Significance
These results further elucidate the genetic basis of structural brain abnormalities leading to focal epilepsy in children and point to new candidate disease genes.
Congenital heart disease (CHD) is the most common type of birth defect with family- and population-based studies supporting a strong genetic cause for CHD. The goal of this study was to determine ...whether a whole exome sequencing (WES) approach could identify pathogenic-segregating variants in multiplex CHD families.
WES was performed on 9 kindreds with familial CHD, 4 with atrial septal defects, 2 with patent ductus arteriosus, 2 with tetralogy of Fallot, and 1 with pulmonary valve dysplasia. Rare variants (<1% minor allele frequency) that segregated with disease were identified by WES, and variants in 69 CHD candidate genes were further analyzed. These selected variants were subjected to in silico analysis to predict pathogenicity and resulted in the discovery of likely pathogenic mutations in 3 of 9 (33%) families. A GATA4 mutation in the transactivation domain, p.G115W, was identified in familial atrial septal defects and demonstrated decreased transactivation ability in vitro. A p.I263V mutation in TLL1 was identified in an atrial septal defects kindred and is predicted to affect the enzymatic functionality of TLL1. A disease-segregating splice donor site mutation in MYH11 (c.4599+1delG) was identified in familial patent ductus arteriosus and found to disrupt normal splicing of MYH11 mRNA in the affected individual.
Our findings demonstrate the clinical utility of WES to identify causative mutations in familial CHD and demonstrate the successful use of a CHD candidate gene list to allow for a more streamlined approach enabling rapid prioritization and identification of likely pathogenic variants from large WES data sets.
URL: https://clinicaltrials.gov; Unique Identifier: NCT0112048.
Congenital heart defects affect ∼2% of live births and often involve malformations of the semilunar (aortic and pulmonic) valves. We previously reported a highly penetrant
p.Gly296Ser mutation in ...familial, congenital atrial septal defects and pulmonic valve stenosis and showed that mice harboring the orthologous G295S disease-causing mutation display not only atrial septal defects, but also semilunar valve stenosis. Here, we aimed to characterize the role of Gata4 in semilunar valve development and stenosis using the
mouse model. GATA4 is highly expressed in developing valve endothelial and interstitial cells. Echocardiographic examination of
mice at 2 months and 1 year of age identified functional semilunar valve stenosis predominantly affecting the aortic valve with distal leaflet thickening and severe extracellular matrix (ECM) disorganization. Examination of the aortic valve at earlier postnatal timepoints demonstrated similar ECM abnormalities consistent with congenital disease. Analysis at embryonic timepoints showed a reduction in aortic valve cushion volume at embryonic day (E)13.5, predominantly affecting the non-coronary cusp (NCC). Although total cusp volume recovered by E15.5, the NCC cusp remained statistically smaller. As endothelial to mesenchymal transition (EMT)-derived cells contribute significantly to the NCC, we performed proximal outflow tract cushion explant assays and found EMT deficits in
embryos along with deficits in cell proliferation. RNA-seq analysis of E15.5 outflow tracts of mutant embryos suggested a disease state and identified changes in genes involved in ECM and cell migration as well as dysregulation of Wnt signaling. By utilizing a mouse model harboring a human disease-causing mutation, we demonstrate a novel role for GATA4 in congenital semilunar valve stenosis.This article has an associated First Person interview with the joint first authors of the paper.
Recent characterizations of pioneer transcription factors provide insights into their structures and patterns of chromatin recognition associated with their roles in cell fate commitment and ...transformation. Intersecting with these basic science concepts, identification of pioneer factors (PFs) fused together as driver translocations in childhood cancers raises questions of whether these fusions retain the fundamental ability to invade repressed chromatin, consistent with their monomeric PF constituents. This study defines the cellular and chromatin localization of PAX3-FOXO1, an oncogenic driver of childhood rhabdomyosarcoma (RMS), derived from a fusion of PFs. To quantitatively define its chromatin-targeting functions and capacity to drive epigenetic reprogramming, we developed a ChIP-seq workflow with per-cell normalization (pc-ChIP-seq). Our quantitative localization studies address structural variation in RMS genomes and reveal insights into inactive chromatin localization of PAX3-FOXO1. Taken together, our studies are consistent with pioneer function for a driver oncoprotein in RMS, with repressed chromatin binding and nucleosome-motif targeting.
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•The fusion oncoprotein PAX3-FOXO1 binds to both active and repressed chromatin•PAX3-FOXO1-binding sites are adjacent to H3K9me3 domains•PAX3-FOXO1 engages partial DNA motifs at early timepoints•PAX3-FOXO1 can bind stably to inaccessible chromatin without inducing accessibility
Molecular Genetics; Systems biology; Transcriptomics
Heart valves are organized connective tissues of high mechanical demand. They open and close over 100,000 times a day to preserve unidirectional blood flow by maintaining structure-function ...relationships throughout life. In affected individuals, structural failure compromises function and often leads to regurgitant blood flow and progressive heart failure. This is most common in degenerative valve disease due to age-related wear and tear, or congenital malformations. At present, the only effective treatment of valve disease is surgical repair or replacement and this is often impermanent and requires anti-coagulation therapy throughout life. Therefore, there is a critical need to discover new alternatives. A promising therapeutic area is tissue regeneration and in non-valvular tissues this requires a tightly regulated genetic "growth program" involving cell proliferation. To explore this in heart valves, we performed RNA-seq analysis to compare transcriptional profiles of aortic valve tissue isolated from mice during stages of growth (postnatal day (PND) 2) and adult maintenance (4 months). Data analysis reveals distinct mRNA profiles at each time point and pathway ontology identifies associated changes in biological functions. The PND2 aortic valve is characterized by extensive cell proliferation and expression of mRNAs related to the extracellular matrix (ECM). At 4 months, proliferation is not significant and a differential set of ECM-related genes are expressed. Interestingly there is enrichment of the defense response biological process at this later time point. Together, these data highlight the unique transcriptome of the postnatal valve during stages of growth and maturation, as well as biological functions associated with adult homeostatic valves. These studies create a platform for future work exploring the molecular programs altered in the onset of heart valve disease after birth and provide insights for the development of mechanistic-based therapies.
Rhabdoid tumors (RTs) of the brain (atypical teratoid/rhabdoid tumor; AT/RT) and extracranial sites (most often the kidney; RTK) are malignant tumors predominantly occurring in children, frequently ...those with
SMARCB1
germline alterations. Here we present data from seven RTs from three pediatric patients who all had multi-organ involvement. The tumors were analyzed using a multimodal molecular approach, which included exome sequencing of tumor and germline comparator and RNA sequencing and DNA array-based methylation profiling of tumors.
SMARCB1
germline alterations were identified in all patients and in all tumors. We observed a second hit in
SMARCB1 via
chr22 loss of heterozygosity. By methylation profiling, all tumors were classified as rhabdoid tumors with a corresponding subclassification within the MYC, TYR, or SHH AT/RT subgroups. Using RNA-seq gene expression clustering, we recapitulated the classification of known AT/RT subgroups. Synchronous brain and kidney tumors from the same patient showed different patterns of either copy number variants, single-nucleotide variants, and/or genome-wide DNA methylation, suggestive of non-clonal origin. Furthermore, we demonstrated that a lung and abdominal metastasis from two patients shared overlapping molecular features with the patient’s primary kidney tumor, indicating the likely origin of the metastasis. In addition to the
SMARCB1
events, we identified other whole-chromosome events and single-nucleotide variants in tumors, but none were found to be prognostic, diagnostic, or offer therapeutic potential for rhabdoid tumors. While our findings are of biological interest, there may also be clinical value in comprehensive molecular profiling in patients with multiple rhabdoid tumors, particularly given the potential prognostic and therapeutic implications for different rhabdoid tumor subgroups demonstrated in recent clinical trials and other large cohort studies.
Primary spinal cord tumors contribute to ≤ 10% of central nervous system tumors in individuals of pediatric or adolescent age. Among intramedullary tumors, spinal ependymomas make up ~ 30% of this ...rare tumor population. A twelve-year-old male presented with an intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. Gross total resection and histopathology revealed a World Health Organization (WHO) grade 2 ependymoma. He recurred eleven months later with extension from C2 through T1-T2. Subtotal resection was achieved followed by focal proton beam irradiation and chemotherapy. Histopathology was consistent with WHO grade 3 ependymoma. Molecular profiling of the primary and recurrent tumors revealed a novel amplification of the MYC (8q24) gene, which was confirmed by fluorescence in situ hybridization studies. Although MYC amplification in spinal ependymoma is exceedingly rare, a newly described classification of spinal ependymoma harboring MYCN (2p24) amplification (SP-MYCN) has been defined by DNA methylation-array based profiling. These individuals typically present with a malignant progression and dismal outcomes, contrary to the universally excellent survival outcomes seen in other spinal ependymomas. DNA methylation array-based classification confidently classified this tumor as SP-MYCN ependymoma. Notably, among the cohort of 52 tumors comprising the SP-MYCN methylation class, none harbor MYC amplification, highlighting the rarity of this genomic amplification in spinal ependymoma. A literature review comparing our individual to reported SP-MYCN tumors (n = 26) revealed similarities in clinical, histopathologic, and molecular features. Thus, we provide evidence from a single case to support the inclusion of MYC amplified spinal ependymoma within the molecular subgroup of SP-MYCN.
An ascending aortic aneurysm (AscAA) is a life-threatening disease whose molecular basis is poorly understood. Mutations in NOTCH1 have been linked to bicuspid aortic valve (BAV), which is associated ...with AscAA. Here, we describe a potentially novel role for Notch1 in AscAA. We found that Notch1 haploinsufficiency exacerbated the aneurysmal aortic root dilation seen in the Marfan syndrome mouse model and that heterozygous deletion of Notch1 in the second heart field (SHF) lineage recapitulated this exacerbated phenotype. Additionally, Notch1+/- mice in a predominantly 129S6 background develop aortic root dilation, indicating that loss of Notch1 is sufficient to cause AscAA. RNA sequencing analysis of the Notch1.129S6+/- aortic root demonstrated gene expression changes consistent with AscAA. These findings are the first to our knowledge to demonstrate an SHF lineage-specific role for Notch1 in AscAA and suggest that genes linked to the development of BAV may also contribute to the associated aortopathy.
Osteosarcoma remains one of the deadliest cancers in pediatrics and young adults. We administered two types of immunotherapies, oncolytic virotherapy and immune checkpoint inhibition, to two murine ...osteosarcoma models and observed divergent results. Mice bearing F420 showed no response, whereas those with K7M2 showed prolonged survival in response to combination therapy. K7M2 had higher expression of immune-related genes and higher baseline immune cell infiltrates, but there were no significant differences in tumor mutational burden or predicted MHC class I binding of nonsynonymous mutations. Instead, we found several mouse endogenous retrovirus sequences highly expressed in K7M2 compared with F420. T cell tetramer staining for one of them, gp70, was detected in mice with K7M2 but not F420, suggesting that endogenous retrovirus proteins are targets for the anti-tumor immune reaction. Given prior observations of endogenous retrovirus expression in human osteosarcomas, our findings may be translatable to human disease.
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•K7M2 but not F420 responds to viroimmunotherapy•K7M2 and F420 exhibit similar tumor mutational burdens•K7M2 shows much higher expression of endogenous retrovirus genes including gp70•Tetramer staining suggest gp70 is the immune target in K7M2
Cancer; Immunology
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