Martina Brueckner, M.D. - US grants

The development of left-right asymmetry of the heart and viscera is a fundamental process in vertebrate embryogenesis. The left-right axis is established at a later stage of development than either the antero- posterior or dorsoventral axes and is first apparent in the formation of the asymmetric cardiac loop from the previously symmetric midline cardiac tube. The mechanism of determination of left-right asymmetry and normal cardiac and visceral situs remains unknown. This project takes a genetic approach to the problem by analyzing the recessively inherited mouse mutation, iv, that leads to loss of control over the development of normal cardiac and visceral situs: 50% of mice that are homozygous for the iv mutation have dextrocardia and situs inversus, 50% have levocardia nad situs solitus. To study the normal function of Iv, we are taking a "reverse genetic" approach. We have mapped Iv to a location on distal mouse chromosome 12 by its segregation in a backcross with respect on distal mouse chromosome 12 by its segregation in a backcross with respect to DNA markers defined by RFLPs (Restriction Fragment Length Polymorphisms). We are now planning to isolate and identify DNA sequences containing Iv. A physical map of the region is being generated by pulsed field gel analysis. Further clones that map into the region will be isolated by strategies that include production of a radiation fusion cell line, chromosomal microdissection followed by PCR amplification, and the cloning of fragments isolated from pulsed field gels. DNA clones encompassing the region will then be isolated from phage, cosmid or YAC libraries. THe clone containing Iv will be identified by functional analysis in transgenic mice, enabling the gene and its product to be characterized.

DESCRIPTION: An important question in developmental biology is how vertebrate embryos acquire left-right asymmetry. Recent work has shown that in early embryos two TGF family members, nodal and lefty, are expressed in the left but not right lateral plate mesoderm, proceeding the asymmetry in the developing heart tube. Mutations in mice resulting in abnormal left-right asymmetry include iv (inversus viscerum) and lgl (legless), both which randomize left-right asymmetry. As iv functions upstream of nodal and lefty, it is likel to function very early in development. The PI has used positional cloning to clone the iv gene, and has identified it as an axonemal dynein heavy chain, named left/right dynein (lrd). This dynein heavy chain displays a mis-sense mutation in iv, and it is deleted in lgl. The specific aims are designed to test the role of lrd in left/right asymmetry.

[unreadable] DESCRIPTION (provided by applicant): The development of non-random asymmetry along the left-right axis is a unique feature of vertebrate development. Defects in this process in mouse and man commonly affect the development of the heart and result in severe congenital cardiac anomalies. The goal of this proposal is to understand the mechanism by which embryonic cilia create and signal left-right positional information. Motility of embryonic cilia is driven by the axonemal dynein, left right dynein (lrd). Lrd is essential and specific to the development of left-right asymmetry. When it is defective, the normally motile monocilia found on the node (organizer) of the embryo at the time of gastrulation are paralyzed. Motile node cilia generate directional flow of the extraembryonic fluid surrounding the node (nodal flow), and there is no nodal flow in mice with defective lrd. These observations suggest a direct role for motile 9+0 node cilia in the initiation of LR asymmetry. The development of LR asymmetry is also abnormal in mice with defects in the polycystin gene Pkd2, which functions in kidney monocilia as a mechanotransducer by mediating an intracellular calcium signal in response to fluid flow in the renal tubule. We have shown that Polycystin-2 protein localizes to a subset of node monocilia that are non-motile. Finally, we observe an asymmetric perinodal calcium signal in e7.75 mouse embryos. These data suggest that embryonic cilia may be required to both create and sense nodal flow. In this proposal, we will first test whether there are two classes of monocilia at the node. First, we will study the structure of node monocilia by immunofluorescence and transmission electron microscopy. Using Cre-lox technology, we will generate mice that have deleted all node monocilia, and compare their LR development to that observed in mice lacking only motile node monocilia. We will determine the role of asymmetric calcium signaling in the development of LR asymmetry by observing the effect of mutations affecting nodal flow, and of artificial nodal flow, on the asymmetric perinodal calcium signal. The position of asymmetric perinodal calcium signaling in the pathway of LR development will be tested by examining the effect of artificially introduced calcium signals on LR phenotype in cultured embryos. Finally, we will evaluate the role of the calcium-permeable cation channel polycystin-2 in LR development. Pkd2-/- mice will be examined for morphology of node cilia, and for asymmetric perinodal calcium signals in order to determine whether nodal flow produces asymmetric perinodal calcium signals via the polycystin channel. [unreadable] [unreadable] [unreadable]

Congenital heart disease is the most common serious birth defect, affecting .8% of liveborn infants. Normal cardiac development depends on complex interplay between genetic and epigenetic factors. In particular, blood flow and cardiac function are essential for cardiac morphogenesis: however, the mechanism by which these mechanical signals are sensed and interpreted remains unclear. Cilia, which are also essential in the development of cardiac LR asymmetry via their function at the embryonic organizer (node), have recently been found to function as mechanosensors in other tubular, fluid-filled organs such as the kidney. We have identified a set of cilia, called cardiac cilia, in the mouse heart at e8.5 - e12.5, corresponding to the time in development extending from the onset of blood flow through valve formation and septation. The goal of this proposal is to define the mechanism by which cardiac cilia function directly in cardiac morphogenesis independent of their role in the generation of LR asymmetry. Mice with immotile, but structurally normal cilia have abnormal positioning of organs along the LR axis. Although intracardiac defects are observed in 7-50% of affected mice, a significant number survive to adulthood with structurally and functionally normal hearts. In contrast, mice with complete absence of cilia or ciliary sensing have severe cardiac defects with 100% penetrance that result in mid-gestational embryonic lethality independent of LR axis development. These observations suggest that cilia are required in cardiac development independently from their function in LR development. We hypothesize that cardiac cilia function as sensors for extracellular signlas such as flow, cardiac function or secreted ligands to affect morphogenesis. In Spec. Aim 1 of this proposal, we will define what cardiac cilia do: are they mechanosensors, hedgehog receptors or motile structures? To this end, the distribution and composition of cardiac cilia will be examined. The role of constitutive ciliary mutations on cardiac development will be evaluated by analysis of the cardiac phenotype of mouse embryos with mutations resulting in defective ciliary motility, ciliary biogenesis or ciliary mechanosensation. The cardiac defects and distribution of cilia will be evaluated in mouse embryos with a mutation resulting in an absent heart beat. The role of cilia in LR development will be distinguished from their intracradiac function. In Spec. Aim 2, we will identify where cardiac cilia exert their effect by using Cre-lox technology to delete cilia specifically from the epicardium, endocardium, pericardium and anterior heart field. Finally, in Spec. Aim 3 we will seek to define how cardiac cilia direct morphogenesis. Here, the downstream signaling pathway(s) connecting ciliary sensing with cardiac morphogenesis will be investigated by analyzing epithelial-mesenchymal transformation, proliferation and hedgehog signaling in mouse embryo hearts with mutations in ciliary function and biogenesis.

DESCRIPTION (provided by applicant): Congenital heart disease is the most common serious birth defect, affecting .8% of live born infants, and ample evidence in both humans and animal model systems supports a genetic basis for CHD. However, low recurrence rates, small sample size and limitation in genomic technology have provided a significant hurdle in defining the genetics of CHD. The goal of this proposal is to combine the state-of-the art genomic technology in the Lifton laboratory with the understanding of the developmental mechanism underlying CHD developed in the Brueckner laboratory to determine the genetic determinants of two types of CHD, Heterotaxy (Htx) and Aortic Arch Abnormalities (AAAs). We have used copy number variation (CNV) analysis in a pilot study of 288 patients with Htx and identified rare genie CNVs in -20%. Interestingly, the CNVs direct attention to novel candidate genes that cluster in 3 pathways previously identified to have a role in the development of LR asymmetry and vasculature: ciliary structure and function, TGF-P signaling and glycosylation. These observations suggest that by combining powerful genomic techniques and large patient cohorts with our understanding of the developmental pathways implicated in cardiac morphogenesis we will identify a genetic cause in a significant number of CHD patients. In Specific Aim 1, we will recruit and carefully phenotype >2,000 pts with all CHD from Yale, University of Rochester and University College London to share with the PCGC consortium. In Specific Aim 2, patients from the consortium with Htx and AAAs will first be analyzed for CNVs. Subsequently, sporadic Htx and AAA patients with no detectable copy-number changes can undergo whole exome sequencing to discover causative mutations. In Specific Aim 3, we will determine whether discrete genotype variants with shared, clinically defined Htx and AAA phenotypes have significantly different short and mid-term clinical outcomes. Here we will focus on three aspects of clinical outcome that have possible links to the causative developmental pathways: myocardial function and renal function, both of which have been associated with ciliary function in model animal systems, and aortic root size, which is prominently affected by TGF-P signaling in mice and humans. RELEVANCE (See instructions): Although a genetic etiology for congenital heart disease (CHD) has long been suspected, limitations in patient number, genomic technology and understanding of the biology governing heart development have hampered identification of generic causes of CHD. This proposal seeks to use state-of-the-art genomic technology to identify the cause of two types of major CHD, heterotaxy and aortic arch abnormalities.

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) occurs in approximately 8 out of 1000 live births and effects 1.3 million newborns per year worldwide. While there is evidence to indicate that CHD does have a genetic basis, most of CHD burden remains unexplained genetically. New genomics technologies can efficiently identify variations in the genomes of CHD patients, but only a small percentage have second unrelated alleles to validate them as disease causing. Therefore there is a pressing need to develop functional assays to evaluate these candidate genes for CHD. There are two main goals for these functional assays: 1) provide evidence supporting candidate genes as disease causing and 2) identify the mechanism for the candidate gene on normal development and the disease state. Here we develop Xenopus as a rapid model system for testing CHD genes and apply advanced optical imaging methods to detect cardiac phenotypes. Xenopus is as an important animal model of congenital heart disease: large numbers of embryos can be readily manipulated, protein expression can be knocked-down using antisense morpholino oligos, and the heart is easily visualized. To expand the CHD spectrum that can be modeling in Xenopus, we need better microscale cardiac imaging methods. During the R21 phase, we will test two technologies, optic coherence tomography (OCT) and our novel hemoglobin contrast subtraction angiography (HCSA) to demonstrate that microscale imaging of Xenopus can be used to screen CHD genomic hits. Optic coherence tomography is an optical imaging system that can capture microscopic structures at high acquisition speeds allowing high-resolution phenotyping of dynamic heart structures. Hemoglobin contrast subtraction angiography (HCSA) is a noninvasive, nondestructive, quantitative microangiographic method that exploits the hemoglobin as an endogenous flow contrast agent during color imaging enabling us to delineate abnormal structures as well as quantify biomechanical phenotypes. In the R33 phase, our overall goal is to apply these methods to facilitate detailed high-resolution structural phenotypin of tadpole hearts that can be used to quickly test CHD candidate genes for cardiac phenotypes. This will allow us to identify cardiac phenotypes in CHD candidate genes that have no previous role in cardiac development and serve as a springboard for future mechanistic studies.

? DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common survivable birth defect, affecting 0.7% of all liveborn infants worldwide. Management of CHD has made great strides, so that there are now more living adults with CHD than children, but many patients with complex CHD have long-term morbidity frequently manifesting as neurodevelopmental abnormalities (NDA). The causative mechanisms of CHD have been poorly understood although genetic factors are strongly believed to play a role. Dramatic advances in genomic technologies now allow systematic identification of specific genes and pathways that have very large effects on disease pathogenesis. As part of the PCGC, we have performed whole-exome sequencing on 1,300 parent-offspring trios selected from over 9,000 patients recruited into the PCGC study. The salient findings are that de novo mutations occurring in genes that are highly expressed in the heart account for at least 8% of the CHD cases studied, and disproportionately occur in patients with CHD classified as left ventricular outflow obstruction, and in patients with CHD and NDA. Among the genes with de-novo mutations in CHD there is a marked enrichment of mutations, particularly damaging mutations, in genes involved in chromatin modification, and these mutations are strongly biased toward CHD cases with NDA. Moreover, and very unexpectedly, we find highly significant overlap of genes with damaging mutations in CHD and in autism. We propose that genes involved in chromatin regulation are dosage sensitive for both heart and neurodevelopment, and that mutations in these genes can result in both CHD and NDA. By sequencing all genes in the chromatin modification set in 10,000 PCGC cases using rapid, inexpensive targeted sequencing via MIPS (molecular inversion probe sequencing) we will identify genes in this pathway that contribute to CHD and determine their specific relationship to NDA. Since the last funding cycle has enabled us to show that CHD has high locus heterogeneity, we will also continue to use cost-effective exome sequencing to identify additional genes in which de-novo mutations confer large effects on disease risk. In order to determine the spectrum of NDA resulting from chromatin modifier mutations, we will identify patients from the existing PCGC cohort with and without mutation in this gene set and perform neurodevelopmental testing. Finally, we will prospectively recruit patients under one year of age, test for chromatin modifier mutations, and follow their neurodevelopmental outcome. In summary, we expect these studies to identify an easily testable set of genes that can be evaluated rapidly and inexpensively to identify CHD patients at increased risk for ND abnormalities.

? DESCRIPTION (provided by applicant): Cilia are central to the development of vertebrate left-right (LR) asymmetry, and failure to establish normal LR asymmetry results in the human heterotaxy syndrome (Htx) and severe congenital heart disease (CHD). Htx is amongst the most lethal forms of CHD in humans, and mutations affecting at least 12 genes affecting cilia structure and function have been identified in patients with Htx and other CHD. This proposal investigates the cilium as a distinct calcium signaling compartment, and investigates the role intraciliary calcium plays in heart development. We will define the mechanism by which intraciliary calcium establishes cardiac LR asymmetry. In vertebrate LR development, cilia at the left-right organizer (LRO) beat to generate directional flow of extraembryonic fluid, which is sensed by cilia and directs asymmetrical gene expression. Several lines of evidence link calcium, cilia and LR development: the ciliary calcium channel polycystin-2 (Pkd2) at the LRO is essential for LR development, and in mouse and zebrafish, a left-biased increase of cytoplasmic calcium correlates with LR development and is dependent on both flow and Pkd2. Despite the established importance of cilia, flow, polycystins and cytoplasmic calcium signaling in LR development, the mechanisms linking these components, and further to the downstream signaling cascade driving asymmetric cardiac morphogenesis, are not understood. We have developed a novel method to visualize and manipulate intraciliary calcium in cultured cells and zebrafish embryos. In this proposal, we will use this to address the physiology and function of intraciliary calcium in LR and heart development. We hypothesize that the cilium is a distinct cellular compartment with respect to calcium signaling, and that intraciliary calcium is the link between ciliary motility at the LRO and asymmetric cardiac morphogenesis. In Aim 1, we will characterize intraciliary calcium in-vivo in cultured cells and zebrafish embryos and examine the response of intraciliary calcium to ciliary motility and whether intraciliary calcium requires the polycystin channel in LR development. In Aim 2, we will dissect how intraciliary calcium regulates LR development by analyzing how intraciliary calcium regulates asymmetric gene expression. In Aim 3, we will test whether the ankyrin-repeat protein Inversin transduces intraciliary calcium in cultured renal epithelial cells, zebrafish and mice with mutations affectin inversin. In summary, these experiments provide a new paradigm for ciliary signaling in LR and heart development, and develop techniques that will be broadly applicable to the study of cilia biology.

? DESCRIPTION (provided by applicant): Congenital heart disease is one of the major causes of infant mortality and morbidity in the US. However, we know little about the genetic causes of this disease. In order to better understand congenital heart disease, we analyzed the human genetics of cardiac malformations. In particular, we studied heterotaxy patients with congenital heart disease for copy number variations. Heterotaxy is a disorder of left-right patterning and alters cardiac development due to failure of cardiac looping morphogenesis. In a heterotaxy patient, we identified duplication in the NUP188 gene, which encodes a component of the nuclear pore complex known as a nucleoporin. We then modeled this cardiovascular disease in Xenopus by knocking down nup188, which recapitulated the human heterotaxy phenotype. The main goal of this proposal is to analyze the role of nucleoporins in left-right patterning and congenital heart disease. Our preliminary data suggest that nucleoporins are important for cilia. Cilia are critical regulators of left-right patterning and so loss of cilia could explain the left-ight phenotype. In this proposal, we have three main aims: 1) Analyze multiple nucleoporins to see if they also alter left-right patterning and cilia 2) use super-resolution imaging to define the structure of nucleoporins at the base of the cilium and 3) determine the mechanism by which nucleoporins contribute to the function of cilia.

Congenital Heart Disease (CHD) is the most common birth defect affecting 1% of all live born infants. While ~90% of patients with CHD survive into adulthood, there are many comorbidities that make CHD an increasingly significant public health problem. Genomic analyses of large cohorts of CHD patients have identified a significant genetic contribution to CHD, but the link between etiology and clinical outcome remains an important question. When I began my search for the cause of CHD, I identified the cilium as being central to left-right (LR) axis and cardiac development, and most recently, as part of the Pediatric Cardiac Genomics Consortium (PCGC), identified significant contributions from mutations affecting cilia and chromatin remodeling genes to human CHD. However, the question of how cilia dysfunction precisely influences CHD remains unanswered. I have assembled a group of co-investigators with expertise in mouse and zebrafish development, live-cell imaging, optogenetics and genomics to take a multi-pronged approach to understanding the central role of the cilium in heart development with the long-term goal of leveraging this data with ongoing genomic and clinical studies to improve clinical outcomes of CHD. First, we will resolve the long-standing question of how cilia instruct cardiac LR asymmetry. We will use single-cell RNAseq to define the cellular composition of the left-right organizer (LRO), a transient ciliated organ that is essential for instructing cardiac asymmetry. We will then establish the molecular mechanism linking cilia signaling at the LRO to cardiac LR development in mouse and zebrafish embryos. Together these experiments will uncover the mechanism by which an embryo determines LR asymmetry, and provide gene sets that will inform the search for human CHD candidate genes. Second, we will investigate the role of cilia in cardiac valve formation. We have found dynamic, flow-sensitive cilia in the presumptive atrio-ventricular valve region of the zebrafish heart, and will test the hypothesis that valve specification is driven by the mechanical forces occurring at interfaces between differentially contracting chambers, and that valve cilia are the mechanotransducers leading to changes in transcription of klf2/klf4 and downstream valve morphogenesis. Third, we will unravel the mechanism by which epigenetic factors influence cardiac development. The important role of chromatin remodeling genes in human CHD has raised the question whether any of these transcriptionally regulate cilia in heart development. We have already found that histone H2B monoubiquitination (H2BUb1) transcriptionally regulates cilia function at the LRO. We are now testing how H2BUb1 affects cardiac development in mouse embryos and human iPSC- derived cardiomyocytes through cilia-dependent and/or cilia-independent mechanism(s). Our long-term goal of translating the basic biologic and genomic data to clinical impact will be addressed by returning mechanistic and genetic data from our cilia work to the PCGC CHD genomics project so that the resulting discoveries lead to personalized medicine for patients with CHD. !

Congenital heart disease (CHD) is the most common birth defect and affects 1% of all live born infants, and genetics likely contribute to 90%. While ~90% of patients with CHD survive into adulthood, there are many comorbidities that make CHD an increasingly significant public health problem. During the first two project periods the PCGC has made significant progress in understanding the genetic architecture of CHD. The highlights of the genomics work are the identification of de-novo variants contributing to ~10% of CHD, the identification of chromatin remodeling genes contributing to 2.3% of all CHD and to a striking 28% of CHD that is associated with extracardiac and neurodevelopmental abnormalities, the identification of novel CHD genes underlying inherited CHD, and the contribution of genes involved in cilia structure and function to CHD. The other most significant finding from the PCGC genomic studies is the tremendous heterogeneity of CHD: over 440 genes contribute to CHD by a dominant inheritance mechanism alone. Combining these findings with previous data on the contribution of copy-number variants and aneuploidy identifies a likely genetic cause for ~40% of CHD. Our hypotheses are that many yet unidentified mutations affecting the genic region contribute to a significant portion of CHD, and that the specific mutations contributing to CHD impact outcomes. The two major questions addressed in this proposal are what is the genetic contribution to the ?missing 55%?, and how do specific mutations impact the cardiac and non-cardiac outcomes and clinical care of patients with CHD? In the setting of large genetic heterogeneity and variable phenotypic expressivity that characterizes CHD, answers to these questions will require very large patient cohorts with genotype and phenotype data. Aim 1 will define the genetic architecture of CHD through analysis of 30,000 enrolled patients with genomic data in the combined PCGC cohort. Patients are recruited by outreach to the entire Pediatric Cardiology community along with internet-based direct patient recruiting. This will be coupled with a cost-effective tiered sequencing strategy that starts with MIPs-based targeted sequencing on all probands and progresses to whole-exome and whole- genome sequencing in MIPs-negative patients. Since the eventual goal of the PCGC program is to use genomic data to improve clinical care of CHD patients, we will need to link genomic and outcome data efficiently. Aim 2 will establish a central PCGC data mining center to directly link phenotypic data from the EMR and STS database with genomic data. We will initially focus on two outcomes that are readily available in the EMR and are associated with significant morbidity in CHD patients: the potential role of cilia mutations in progressive valve dysfunction in single-ventricle patients and the potential role of chromatin modifier gene mutations to cancer risk in adult CHD survivors. The informatics paradigms developed for this project can then be applied to investigate potential genetic contribution to a wide range of other CHD outcomes.