Identification and investigation of novel candidate genes for primary ciliary dyskinesia

Almost every cell of your body has a thin, hair-like outgrowth called a cilium. Cilia can be thought of as the "cells' antennae", through which the cell gains sensory information about its environment. As such, the cilium performs sensory functions essential to the development and physiology of many organs, including kidney, nervous system, sense organs, bone and pancreas. In addition to their sensory functions, some types of cilia are capable of bending or beating and are involved in fluid movement. Such 'motile cilia' are found for example on cells lining the airways for mucus movement and the fallopian tubes for wafting a new egg towards the uterus. Moreover, sperm cells swim by means of a beating flagellum, which is essentially a long motile cilium. All these cilia move by means of banks of 'motor proteins' within them. Primary ciliary dyskinesia is an inherited disease in which cilia are immotile or only partially motile due to failure of these motor proteins. It is quite rare overall, but in some communities it can occur at a frequency of up to 1:2200 individuals. Most noticeable symptoms relate to difficulties in clearing mucus, leading for instance to frequent and damaging chest infections. Severe cases also have situs inversus - in which organ positioning is disrupted (e.g. the heart is no longer on the left side of the chest). If diagnosed early, then treatment can be effective (such as physiotherapy to clear lungs), but diagnosis is difficult and requires specialist techniques. Discovering the genetic causes of PCD will ultimately aid understanding of the disease, aid diagnosis, and potentially provide a route to therapy.

Mutations in many different genes cause PCD. Some mutations are in genes for the motor proteins themselves. However, in some 50-60% of PCD families the underlying gene defect has not been discovered. The question we are addressing is how to accelerate the discovery of PCD-causing gene mutations. Our strategy, unexpectedly, is to look at the fruit fly, Drosophila melanogaster. The fruit fly is easy to rear and to study. Sophisticated genetic and cellular approaches can be used to discover genes that are required for motile cilia in Drosophila. We shall examine the effect of disrupting the function of these genes. This is quite straightforward to achieve because in Drosophila, motile cilia are required for senses and sperm, and so flies with defective motile cilia are easy to spot through obvious sensory deficits and male infertility. For studies into cilium biology it is cost-effective and ethically more acceptable to use Drosophila than more complex organisms where possible. Our research therefore helps to satisfy the goal of reducing reliance on animal research.

Ease of gene discovery and analysis is not sufficient. Just as important is the fact that the molecular machinery of the cilium is completely conserved between insects and 'higher' animals. Therefore, genes discovered to be important for ciliary motility in Drosophila are likely to be important in humans too. As a corollary, the genes we discover in Drosophila are prime suspects to be mutated in cases of PCD. So, even though the fruit fly doesn't have lungs, it provides a perhaps surprising route to discovering the genetic causes of PCD.

On the basis of the evidence we obtain in Drosophila, our collaborators will screen for mutations of newly identified genes in a panel of PCD families. If gene variants are found in such families, they would be hypothesised to cause the PCD in those families. But this conclusion would require further experimental verification before it becomes proof. Some of this verification will come from further analysis of the mutant defects in Drosophila using a range of molecular and cellular techniques, in order to define what exactly is going wrong with the motile cilia.

There is a need to identify the gene defects underlying Primary Ciliary Dyskinesia, a disease in which cilia have impaired motility. One approach is to sequence candidate motile cilia genes in PCD patients. Our recent discovery of the Drosophila transcription factor, Fd3F, and appreciation of its role in regulating the motile cilia gene expression programme provides an avenue for motile cilia gene discovery and validation using the power of Drosophila genetics. In Drosophila motile cilia are confined to sensory neurons (particularly auditory neurons) and sperm, so mutations causing loss of ciliary motility yield flies that are uncoordinated, deaf and male infertile. Their sensory neurons and sperm are readily accessible to structural and functional analysis.

We found that two novel Fd3F target genes (Zmynd10 and Heatr2) are required for motile cilia in Drosophila. Or collaborators then found PCD-associated mutations in their human orthologues. Our first aim is to analyse these two genes in Drosophila to provide functional validation to support the studies of the PCD-associated mutations by our collaborators. We shall examine cilium ultrastructure and look at the localisation of known motor proteins within the mutant sensory cilia and sperm. We shall investigate protein localisation through tagging and immunofluorescence.

Our second aim is to screen for further ciliary motility genes in Drosophila by gene expression profiling for Fd3F target genes and in vivo RNAi screening of these for motile ciliary defects. This will identify new candidate genes for subsequent analysis in PCD families by our collaborators. If homologous genes are indeed mutated in PCD families, then we shall again use Drosophila to provide supporting functional validation.

Medical impact

The potential to identify new genes involved in genetic disease is highly important for people suffering from rare genetic diseases, since for a patient there is nothing worse that not knowing why he/she is sick. Even if this does not lead directly to healing, understanding the cause of a genetic disease represents significant changes in the lives of affected people. We envisage the impact to be (1) the discovery of new genetic mutations in PCD (2) the discovery of novel aspects of motile cilia biology hitherto unappreciated as potential causes of PCD (3) the potential to improve timely diagnosis of PCD, improving clinical outcome (4) in the long term, providing possible routes to therapy.

The main impact will therefore be for (1) PCD sufferers and their families (2) The PCD Family Support Group charity (3) Clinicians and other medical staff associated with PCD detection and management (in the UK, the national diagnostic centres for PCD), (4) Clinical researchers investigating the aetiology of PCD and its possible amelioration.

The research has potential impact for other diseases, such as ciliopathies in general and deafness. For example, genes we identify could be molecular targets for possible therapeutic intervention to prevent age-related cilium/hair cell degeneration. The ciliary motility defects we investigate also have clear implications for male infertility research, which is frequently due to poor sperm quality, including defective motility.

Drug companies may be interested in the targets identified in this research and in the screening techniques we develop during the project. For instance, our systems biology screening strategy (functional genomics, RNAi and PPI modelling) can be more generally applied to identify genes required for mechanosensory function, and therefore may be new therapeutic targets for deafness.

Other commercial impact

Insects are major pests of agriculture and are vectors of disease. Our research has potential benefits for agrochemical and insect control research. Insect mechanosensory cilium function is a known insecticide target and is a research interest of Syngenta. Discussions with Syngenta have suggested that the ciliary motility genes we discover are candidate molecular targets of these insecticides. Apart from cilia biology, our computational tools have attracted general interest, e.g. on request, we adapted our PPI tool for use in a fungal crop pathogen analysis being carried out at Rothampsted.

Low fertility due to poor sperm quality is a major problem in animal husbandry (e.g. the artificial insemination industry). In many cases sperm immotility is a key problem. Our research will illuminate the molecular mechanisms of sperm motility and provide candidate genes/proteins that could be targeted to improve sperm motility.

Skills/training

The named PDRA on this programme is highly experienced but will benefit from training in new techniques. In the course of this project, she will undertake training in electron microscopy (for which we request funds). This will maintain her employability at the bench, which is her wish. Moreover, she will expand supervisory skills by being directly responsible for the junior technician on this proposal, as well as PhD/undergraduate project students working on related projects. The technician post will likely be filled by a newly qualified graduate, who will gain valuable experience that may well be a stepping stone to a PhD position.

Public engagement

The wider public is likely to be interested in this research from several points of view. The use of an insect as a biomedical research tool with direct relevance to disease is a fascinating and unexpected concept to many, and it is essential to communicate this as an important aspect of MRC's support for NC3Rs (which works with scientists to refine, reduce and wherever possible replace the use of animals in research).