The Genetics of Heterotaxy Syndrome A dissertation submitted to the Division of Graduate Studies and Research, University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Developmental Biology by Jason R. Cowan Master of Science, University of British Columbia, Vancouver, 2007 Committee Chair: Stephanie M. Ware, M.D., Ph.D. Robert B. Hinton Jr., M.D. Linda M. Parysek, Ph.D. S. Steven Potter, Ph.D. Aaron M. Zorn, Ph.D. Molecular & Developmental Biology Graduate Program College of Medicine, University of Cincinnati Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio, USA, 2015 ABSTRACT Congenital heart defects (CHDs) are the greatest cause of infant morbidity and mortality worldwide, occurring in roughly 8 per 1000 live births (~1%). Heterotaxy, a multiple congenital anomaly syndrome resulting from failure to establish left-right (L-R) asymmetry, is characterized by diverse, complex CHDs. Heterogeneous in presentation and etiology, heterotaxy serves as a complex and growing focal point for cardiovascular genetic research. In the two decades since the zinc finger transcription factor, ZIC3, was first identified as a cause of X-linked heterotaxy, mutations in nearly twenty genes with L-R patterning functions have been detected among patients with heterotaxy. Nevertheless, despite considerable progress, genetic causes for heterotaxy remain largely uncharacterized. With an estimated 70-80% of heterotaxy cases still unexplained, there remains enormous potential for novel gene discovery. In this dissertation, we have balanced gene discovery efforts aimed at identifying and characterizing novel causes of heterotaxy with studies into the mechanisms governing ZIC3- related heterotaxy. In order to identify novel genetic causes of heterotaxy, array-based single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) screens for copy number variation (CNVs) were completed in a large and carefully phenotyped cohort of 225 patients with heterotaxy and CHDs. Identified CNVs with pathogenic potential ranged in size from large unbalanced translocations to smaller, kilobase-scale abnormalities. Over 35 rare CNVs were found to encompass 165 genes of possible interest as heterotaxy candidates. Top candidates were screened for L-R patterning functions by morpholino loss of function experiments in Xenopus laevis. We describe results from these analyses and identify the platelet isoform of phosphofructokinase-1 (PFKP) as a novel genetic cause of heterotaxy. Results from these studies collectively confirm a high yield for array-based testing of patients with heterotaxy, and support use of these technologies for identification of novel causative genes. ii Previous genetic analyses have suggested that nearly 75% of all X-linked familial and 1% of all sporadic heterotaxy cases can be attributed to mutations in ZIC3. To date, most reported mutations have been identified in five tandemly repeated zinc finger domains in the first two exons. Many of these mutations have demonstrated functional consequences in ZIC3 nuclear transactivation and subcellular localization. The pathogenic potentials of flanking N- and C- terminal mutations are, however, less certain. Therefore, in order to further define the functional significance of mutations occurring throughout the ZIC3 gene, the full ZIC3 coding region and associated splice junctions was sequenced in a cohort of 440 unrelated patients with assorted situs anomalies and CHDs. Of the 11 mutations identified, 8 were novel, including 5 occurring in non-zinc finger domains. For functional studies, we supplemented these 11 mutations with 4 previously reported variants of uncertain significance. Aberrant cytoplasmic shuttling and decreased luciferase reporter transactivation were observed for all mutations affecting zinc finger domains but not for mutations in terminal regions. Results from these analyses significantly expanded the ZIC3 mutation spectrum, supported a higher than expected mutation yield in patients with sporadic heterotaxy (3.8% vs. 1% overall; 5.2% in affected males), and suggested alternative pathogenic mechanisms for mutations affecting non-zinc finger domains. iii iv DEDICATION: To my friends and family for their love and support. To Michael Hester, Kristin Bell, and Thomas Acciani for their Thursdays and Wednesdays. And to the Prog Gods for all that they do. I may never find all the answers. I may never understand why. I may never prove what I know to be true, But I know that I still have to try. Dream Theater Metropolis Pt. 2: Scenes from a Memory v Table of Contents ABSTRACT .................................................................................................................................................. ii CHAPTER 1: Introduction ........................................................................................................................... 1 1.1. THE HETEROTAXY CLINICAL SPECTRUM .............................................................................. 1 1.2. HETEROTAXY AND CONGENITAL HEART DISEASE ............................................................. 3 1.3. HETEROTAXY IS GENETICALLY HETEROGENEOUS ............................................................ 4 1.4. GENETIC TESTING FOR HETEROTAXY AND CONGENITAL HEART DEFECTS ............... 7 1.5. ZIC3 AS A GENETIC CAUSE OF HETEROTAXY ..................................................................... 10 1.5.1. The ZIC3 mutation spectrum .................................................................................................... 10 1.5.2. ZIC3 expression and function ................................................................................................... 12 1.5.3. ZIC3 structure ........................................................................................................................... 14 1.6. THE MOLECULAR MECHANISMS OF LEFT-RIGHT PATTERNING .................................... 16 1.6.1. The node, cilia, and nodal flow ................................................................................................. 17 1.6.2. Nodal signaling ......................................................................................................................... 22 1.6.3. Flux or flow? ............................................................................................................................. 24 1.7. XENOPUS AS A MODEL SYSTEM FOR STUDYING HETEROTAXY ................................... 25 1.8. EXPERIMENTAL RATIONALE AND SUMMARY OF MAJOR FINDINGS ............................ 26 1.9. FIGURES ......................................................................................................................................... 29 Figure 1.9.1. ZIC3 structure ................................................................................................................ 29 1.10. REFERENCES .............................................................................................................................. 30 CHAPTER 2: Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects ............................................................................................................................... 48 2.1. ABSTRACT ..................................................................................................................................... 48 2.2. INTRODUCTION ........................................................................................................................... 49 2.3. MATERIALS AND METHODS ..................................................................................................... 53 2.3.1. Patient recruitment and phenotypic classification ..................................................................... 53 2.3.2. Chromosome microarray analysis (CMA) ................................................................................ 54 2.3.3. CNV prioritization .................................................................................................................... 55 2.3.4. In vitro fertilization and Xenopus laevis embryo staging .......................................................... 56 2.3.5. Morpholino design .................................................................................................................... 56 2.3.6. pfkp and pitrm1 knockdown ...................................................................................................... 57 2.3.7. Synergy and rescue ................................................................................................................... 58 2.3.8. Left-right marker analyses ........................................................................................................ 59 vi 2.4. RESULTS ........................................................................................................................................ 60 2.4.1. Genetic analyses of patients with heterotaxy identify rare copy number variants .................... 60 2.4.2. Knockdown of pfkp results in left-right patterning defects in Xenopus laevis .......................... 62 2.5. DISCUSSION .................................................................................................................................. 65 2.5.1. Copy number variant analyses identify novel heterotaxy gene candidates ............................... 65 2.5.2. PFKP as a novel cause of heterotaxy