Assessment of Heart Laterality Defects in Zebrafish to Study Variants of Uncertain Significance in Primary Ciliary Dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is an autosomal recessive orphan disease (OMIM#244400) characterized by motile ciliary dysfunction. These hairlike organelles are responsible for the mucociliary clearance of the lungs, and varying degrees of infections in the upper respiratory tract—including the inner ear, nasal passage, and lungs—are common in affected children present with PCD. Moreover, ciliary function is critical for embryonic development, and defects in cilia can lead to situs anomalies, which are sometimes associated with congenital heart disease. To date, more than 50 genes have been implicated in the etiology of PCD, each affecting different parts of the motile ciliary apparatus. Testing via multi- or single-gene panels is recommended for confirmation of diagnosis, which enables timely treatment initiation and familial risk counselling. Unfortunately, for a significant proportion of children with clinical features consistent with PCD, a molecular diagnosis cannot be established. For many of these children, genetic testing returns a variant of uncertain significance (VUS) in a known PCD gene. Hence, there is a pressing need to develop strategies to validate unresolved PCD variants for their pathogenicity. The overall aim of this project is to resolve VUSs in children with suspected but genetically unconfirmed PCD by use of zebrafish (Danio rerio). As a key PCD gene in humans, DNAAF1 has a dnaaf1 homolog in zebrafish, making this model highly relevant for studying human PCD phenotypes. By employing gene knock-down technology (antisense morpholino oligonucleotides; MO), we established and characterized PCD-specific developmental readouts for the zebrafish gene dnaaf1, which affect different aspects of motile ciliary ultrastructure. We observed an increase in ventral body curvature and hydrocephalus in embryos with dnaaf1-MO, with 38% of dnaaf1-MO knockdown embryos showing heart-laterality defects. These phenotypic outcomes not only provide a concrete framework for assessing PCD-related developmental defects in zebrafish but also offer a platform for validating VUSs in human PCD genes. By performing co-injection experiments with patient-derived VUSs and examining the resulting phenotypic alterations, we can directly link specific genetic variants to observable PCD-like traits, offering a robust methodology for determining the pathogenicity of previously uncharacterized variants. This approach aims to enhance the accuracy of genetic diagnoses in PCD and provide new insights into its molecular mechanisms.