Defining the molecular basis of inherited ciliopathies in the Amish

Abstract

Motile ciliopathies are a group of rare clinically and genetically heterogeneous disorders typified by chronic otosinopulmonary disease, laterality defects, male infertility and hydrocephalus. The diverse range of features exhibited in these disorders are associated with defects in the structure, maintenance or function of motile cilia, hair-like organelles that extend from the surface of cells to perform a diverse range of functions throughout the body. The work described in this thesis pertains to clinical, genomic and functional studies of motile ciliopathy disorders identified in genetically isolated Anabaptist communities from North America, with the aim of advancing clinical and scientific understanding of this group of conditions. The unique genetic make-up of the Anabaptist populations, where otherwise rare genetic variants may become enriched, greatly empowers genomic studies and enables the precise clinical relevance of these variants to be determined.

Chapter 3 describes the discovery of a novel motile ciliopathy disorder associated with biallelic variants in MNS1. This finding stemmed from clinical and genomic studies in a large Amish kindred with individuals affected by situs inversus and infertility in males, closely mirroring findings reported in an Mns1 knockout mouse. This chapter also documents preliminary collaborative studies identifying additional unrelated affected individuals expanding the phenotypic spectrum of this condition to include heterotaxy and possible otosinopulmonary disease.

Chapter 4 describes the identification of a Mennonite family with three female siblings affected by a motile ciliopathy disorder associated with a homozygous missense variant in DAW1. A further affected Palestinian child homozygous for a DAW1 nonsense variant, was subsequently identified via GeneMatcher. The clinical spectrum of disease in affected individuals included laterality defects and variable respiratory disease. Zebrafish studies showed that loss of daw1 resulted in reduced cilia motility and left-right patterning defects, consistent with the human phenotype. Additionally, analyses of cilia from affected individuals revealed that DAW1 plays an important role in assembling outer dynein arms to ciliary axonemes, corroborating a putative role in intraflagellar transport that was previously postulated in studies in algae. Together, these findings establish biallelic DAW1 variants as a cause of human motile ciliopathy.

Chapter 5 details extensive genomic studies defining a DIXDC1 nonsense founder variant as a novel cause of isolated laterality defects in an extended Amish kindred. Genetic investigations involved genome-wide single nucleotide polymorphism genotyping alongside whole genome and exome sequencing to identify the likely cause of disease. Preliminary data from zebrafish studies suggests that loss of dixdc1 in embryos causes laterality defects, concordant with the findings in the Amish pedigree. Investigation of cilia function in biopsies from affected individuals showed no abnormalities, suggesting that DIXDC1 acts via a cilia-independent mechanism. Ongoing work to delineate the role of DIXDC1 in determining laterality is also described.

The identification and functional characterisation of three new motile ciliopathy disease genes provides important new scientific insights into the pathomolecular causes of these disorders and improves clinical understanding of phenotypic spectrum of what is an expanding group of conditions. In turn, this facilitates improved diagnostic provision and clinical management strategies for affected individuals and their families ultimately improving healthcare outcomes.