F. Sargent Cheever Chair
Department of Developmental Biology
Department of Pediatrics
Clinical Translational Science Institute
530 45th St.
8120 Rangos Research Center
Pittsburgh, PA 15201
Our research objectives are focused on elucidating the genetic causes and developmental mechanisms of human congenital heart disease (CHD). CHD is one of the most common birth defects and yet the causes of CHD is still not well understood. We have undertaken a research strategy comprising using mouse models to elucidate the genetic etiology of CHD andf the integration and validation of findings in mice with human clinical studies. Our goal is to take advantage of the genetic homogeneity of inbred mouse strains and their near identity in cardiovascular anatomy to pursue the genetic basis for CHD. Parallel studies clinically to confirm the relevance of these findings in mice to human disease. Our focus remains on gaining insights into the genetic basis and developmental etiology of CHD. We expect our studies may lead to more effective diagnostic and therapeutic strategies for improving the standard of care for patients with CHD. This may help improve outcome for infants and children with life threatening structural heart disease.
CHD is often ascribed to complex genetic interactions, but given the genetic heterogeneity in the human population, clinical studies with human subjects have been problematic. In contrast, studies in inbred laboratory mouse strains provide an ideal experimental system to investigate the genetic basis for CHD. The mouse genome is well characterized and can be easily manipulated. Moreover, mice like humans, have four chamber hearts with separate systemic vs. pulmonary circulation that together comprise the major substrates of human CHD. We have shown the efficacy of using noninvasive fetal ultrasound for high throughput screening of fetal heart structure and function and have recovered a plethora of mutations causing CHD in a large scale mouse ethylnitrosourea (ENU) mutagenesis screen conducted using fetal ultrasound scanning. We scanned nearly 14,000 mouse fetuses encompassing nearly 50% genome equivalent and have observed the majority of CHDs found clinically. Fifteen mutations were mapped, with 11 of the 15 genes identified. Seven of the 11 genes are not previously known to cause CHD, with two being entirely novel genes with no known function. Surprisingly, seven of the mutations recovered encode proteins required for formation and function of the cilium. As this phenotype based screen has no a priori gene bias, these findings have led to our current working hypothesis that genes encoding proteins required for structure and function of the cilium, i.e. the ciliome, comprise a central disease pathway in human CHD. Using these newly recovered mutant mice, studies are underway to elucidate the role of the cilium in cardiovascular development and disease.
Building on the infrastructure developed for mouse fetal cardiovascular phenotyping from our pilot ENU mutagenesis screen, we are now engaged in a five-year large scale saturation mouse ENU mutagenesis screen. The goal is to recover the majority of mutations causing CHD. This research program is being conducted as part of the NHLBI Cardiovascular Development Consortium (CvDC) and will entail the interrogation of 100,000 fetuses encompassing 4-5 genome equivalents. Mutation identification will utilize whole mouse genome sequencing using the next generation AB SOLiD sequencing system. The goal of these studies is to identify the majority of genes that may contribute to CHD, and to provide insights into the developmental pathways that contribute to human CHD. Of particular interest is further elucidation of the role of the "ciliome" in CHD. The outcome of this screen may provide the basis for generating a clinical diagnostic chip that may be used to genotype patients with CHD. The genetic stratification of patients with CHD may allow more effective clinical management of the disease, and ultimately may help improve outcome for patients with life threatening CHD.
To investigate the genetic basis for human CHD, we are undertaking human studies motivated by the findings from our mouse studies indicating a central role for the cilium in the pathogenesis of CHD. Thus, our mouse mutagenesis screen showed a high incidence of mutations involving genes of the ciliome. One mouse mutant we recovered exhibited a high incidence of complex CHD and heterotaxy due to a mutation in Dnah5, a motor dynein gene most frequently mutated in patients with primary ciliary dyskinesia (PCD) - a disorder associated with mucociliary clearance defects due to immotility or dyskinetic motion of cilia in the airway epithelia. These findings suggest ciliary dysfunction in the respiratory epithelia similar to those observed in PCD patients may also be found in patients with CHD associated with heterotaxy. This could contribute to the respiratory complications and worse post-surgical outcomes often seen in patients with CHD associated with heterotaxy. To test this hypothesis, we conducted a retrospective chart review to validate the notion that CHD with heterotaxy have increased postsurgical complications. Indeed our analysis showed prolonged hospitalization, increased intubation period and more failed extubations, as well as increased morbidity. This was associated with an increased risk for respiratory complications. Altogether these findings are consistent with the possible association of ciliary dysfunction with CHD and heterotaxy.
In light of these findings from the retrospective analysis, we are now conducting a prospective analysis with the functional assessments of the respiratory epithelia in CHD patients with heterotaxy. Our analysis showed a greater than 40% incidence of ciliary dysfunction in CHD patients with heterotaxy. Ciliary dysfunction was demonstrated by the finding of reduced levels of nasal nitric oxide (nNO) and ciliary motion defects associated with the nasal epithelia. These same changes are also observed in patients with primary ciliary dyskinesia (PCD). As these CHD patients are not otherwise clinically diagnosed as having PCD, we refer to these clinical manifestations as a variant form of ciliary dyskinesia, or vCD. Genetic studies are underway using sequence capture and next generation sequencing to interrogate over 1,000 genes in the ciliome in these CHD patients. Through these studies we hope to identify the disease causing genes in the ciliome that may play a role in CHD and contribute to the respiratory complications and worse outcomes observed in this clinical population.
Future outcome studies will provide further insights into the contribution of ciliary dysfunction to respiratory complications and increased mortality and morbidity in CHD patients with ciliary dysfunction. We are also further investigating whether ciliary dysfunction also may be found in patients with complex CHD without heterotaxy, as some of our mouse mutants with mutations affecting the ciliome show only very low incidence of heterotaxy. In addition, we have found some mutants with mutations causing heterotaxy can exhibit complex CHD without heterotaxy. Through these clinical studies, we hope to determine whether the cilia may comprise a central disease pathway underlying many forms of human CHD. Such findings will pave the way for clinical translational studies that may change the standard of care and perhaps yield diagnostic and therapeutic tools to help improve outcome for patients with CHD.￼
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