Heterotaxy, a birth defect involving randomized left-right patterning of visceral organs, is frequently associated with complex congenital heart disease (CHD), a reflection of the importance of left-right patterning in formation of asymmetries in the four-chamber heart. Heterotaxy (HTX) patients have unexplained higher morbidity and mortality, often with increased postsurgical respiratory complications. This may reflect the common requirement for motile cilia, both in embryonic left-right patterning and also mucus clearance in the airway. We recently showed 42% of HTX patients with CHD (HTX/CHD) have airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD), a recessive disorder associated with laterality defects and sinopulmonary disease due to mucus clearance defects caused by immotile/dyskinetic cilia in the airway. Significantly, exome sequencing showed HTX patients with CD (HTX/CD) are enriched for novel/rare coding variants (RCV) in genes known to cause PCD and other cilia related genes. In this application, we will functionally assay 53 cilia candidate genes identified in 39 HTX/CD patients by exome sequencing analysis. We will assess the effects of gene knockdown on airway cilia motility using a novel assay with reciliating human airway epithelial cells. To assay gene function required for left-right patternin, antisense MO knockdown in zebrafish embryos will be carried out to examine heart and gut looping. Genes shown to disrupt airway cilia motility and cause HTX after knockdown will be further tested to determine whether the RCVs are pathogenic. Specifically we will examine whether expression of the RCV can rescue the HTX phenotype elicited by MO gene knockdown in the zebrafish embryo. Given all of the RCVs identified in HTX/CD patients were heterozygous, we hypothesize a multigenic model of disease, which will be tested by examining the phenotypes of double heterozygous mouse and zebrafish mutants with two-gene combinations observed in the HTX-CD patients. We will examine for evidence of digenic interactions by assaying motile cilia function and visceral organ situs in the double heterozygous mutants. These experiments will interrogate 8 digenic combinations that make use of 9 novel mouse mutants recovered from our ongoing mouse mutagenesis screen, and 7 other digenic combinations in zebrafish using existing mutant lines and de novo production of 4 zebrafish knockout lines by TALENs gene disruption. Finally, to establish genotype-phenotype correlation in ciliary motion defects, we will develop software for quantitative classification of ciliary motin defects using a computational approach with computer vision and machine learning algorithms for visual pattern recognition. Using this software, we will determine whether different RCVs are associated with different ciliary motion defects. This will provide insights into structure-functio relationships in the regulation of cilia motility. This software, to be made available as an online tool, will have translational potential for clinical evaluation of patient airway ciliary motion daa. Together, these studies will establish functional assays and software that can elucidate the genetic etiology of CHD/HTX.
Patients with heterodoxy, a birth defect involving abnormal left-right patterning, have some of the most complex congenital heart defects. Such patients exhibit unexplained higher morbidity and mortality, often with increased postsurgical respiratory complications. As motile cilia function is required both for airway mucus clearance and embryonic left-right patterning, we will develop functional assays and software to assess cilia candidate genes containing rare coding variants identified in heterodoxy patients for their role in airway cilia motility and left-right patterning of organ asymmetries.
|Li, You; Yagi, Hisato; Onuoha, Ezenwa Obi et al. (2016) DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia. PLoS Genet 12:e1005821|
|San Agustin, Jovenal T; Klena, Nikolai; Granath, Kristi et al. (2016) Genetic link between renal birth defects and congenital heart disease. Nat Commun 7:11103|
|Guimier, Anne; Gabriel, George C; Bajolle, Fanny et al. (2015) MMP21 is mutated in human heterotaxy and is required for normal left-right asymmetry in vertebrates. Nat Genet 47:1260-3|
|Quinn, Shannon P; Zahid, Maliha J; Durkin, John R et al. (2015) Automated identification of abnormal respiratory ciliary motion in nasal biopsies. Sci Transl Med 7:299ra124|