Functional genomics identification and characterization of novel disease genes, mechanisms and pathways of ciliogenesis

Cilia are small structures which protrude from the surface of most animal cells like antennae. Like antennae, they receive signals from other cells and their surroundings, and help the cell behave appropriately. This is especially important in development, and defects in cilia lead to a range of human developmental diseases called "ciliopathies". These conditions range from severe, lethal conditions that involve complex defects in multiple organs including the brain, to retinitis pigmentosa (RP) which is a form of hereditary, progressive sight loss. Scientists still do not fully understand how cilia help to control brain and retina development. Although these conditions are individually rare, collectively they are a common cause of morbidity and mortality in babies and young children but remain difficult to diagnose and treat.

This research proposes to identify genes that are either defective ("mutated") in ciliopathies, or otherwise contribute to the formation of a cilium either individually or as part of a pathway. To achieve this aim, we will take advantage of recent exciting advances in genetic technology that allow us to evaluate the contribution of every human gene to cilia formation ("reverse genetics screen"). We are uniquely placed to do this work and the team of investigators have a proven track record of success in this field: we have formed excellent research partnerships with families, their clinicians and other workers in the field to participate in gene identification studies; we have the appropriate state-of-the-art technology, image analysis tools and experience; and we have already produced and validated the very large data-set from the screen that we now wish to exploit more extensively in the present research proposal. We will study key genes ("screen hits") and their contributions to cilia formation, and, in particular we will use special cell systems that more closely model brain and retinal tissue. To do this, we will use both mouse and human stem cells differentiated into neuronal precursors, neurons and retinal cells. Importantly, this approach allows us to study cilia in retinal tissue derived from patients with RP.

The identification of new genes in cilia formation provides two major benefits. Firstly, accurate genetic testing then becomes possible for patients and families, with improvements in diagnosis and genetic counselling. Secondly, important and often unexpected scientific insights are made into disease mechanisms and into the normal function of the disease gene that can lead to new treatments. In particular, we have preliminary evidence from the screen that certain forms of RP, for which the disease mechanism has been unclear previously, may be caused by defective cilia formation in the retina. A better understanding of these processes may provide opportunities for developing drugs or new treatments to prevent disease progression in common forms of retinal degeneration. We also expect new insights into the causes of other common conditions such as polycystic kidney disease, spina bifida and congenital heart defects.

This proposal builds upon our recent results from the first whole-genome reverse genetics screen to identify genes involved in ciliogenesis. This has used high-throughput siRNA-based visual screening, with secondary and validation screens identifying 68 candidate genes. Our proposal integrates existing and new functional genomics data-sets with variant data from existing gene discovery programmes, secondary screens to validate hits, and protein-protein interaction data to confirm that potential ciliary proteins bind to retinitis pigmentosum (RP)-associated spliceosome components. We will then use tertiary screens that include functional annotation with cell biology in physiologically-relevant cell models or zebrafish mutagenesis models. Specific aims are to understand the novel roles of G-protein-coupled receptors (GPCRs) and pre-mRNA processing factors (PRPFs) in ciliogenesis, and how loss of these can lead to neurodevelopmental defects or retinal degeneration, by using stem cell-derived model systems. We will study the role of hit GPCRs in ciliogenesis during neural progenitor differentiation of mouse embryonic stem (ESCs) into neuroepithelial cells and/or radial glial cells. To study the potential ciliary roles of hit PRPFs in a physiologically-relevant tissue, we will use differentiated retinal pigmentary epithelium and photoreceptor-induced cultures derived from induced pluripotent stem cells (iPSCs). Importantly, this approach allows us to study cilia in retinal tissue derived from patients with RP. We aim to demonstrate the clinical utility and validity of an integrated "systems medicine" approach and its ability to make relevant predications about disease causality. The research will complete the functional annotation of genes contributing to ciliogenesis, developing our understanding of the basic biology of primary cilia. It may also identify novel therapeutic strategies to slow disease progression for ciliopathies or RP.

Recent advances have improved the molecular diagnosis of children with a known or suspected ciliopathy, and reduced the burden and disruption of a "diagnostic odyssey" for families. For clinicians, the identification of a new gene can also reduce misdiagnosis or late diagnosis, inform the establishment of proper care pathways for ciliopathy patients, and prioritize those patients that can most benefit from future targeted therapies. Although ciliopathies are a heterogenous group of rare inherited conditions, collectively they are a common cause of perinatal morbidity and mortality (1 in 500 births) and remain difficult to diagnose and treat. For example, in the UK, about 4000 patients require renal replacement therapy (dialysis and transplantation) as a result of cystic kidney disease that leads to kidney failure. There are currently no preventative treatments or new therapeutic interventions for cystic kidney disease that may modify disease progression or the long-term outlook of patients.

The identification of new genes and disease pathways provides insights into normal human physiology and development, and the underlying mechanism of common conditions such as obesity, hypertension, cystic kidney disease and congenital heart disease thus benefiting the wider population. In families with a ciliopathy, the identification of a novel disease gene immediately permits the offer of genetic testing to at-risk relatives. Carrier tests and prenatal diagnosis can also encourage informed reproductive choices for families. Recently, we have established NHS service testing for ciliopathies (including Meckel-Gruber syndrome, Joubert syndrome and primary ciliary dyskinesia) using next-generation clonal sequencing at Yorkshire Regional Clinical Genetics Service. However, this work is often limited by the extreme genetic heterogeneity of the ciliopathies, and the inability to interpret "private" mutations in single families as pathogenic or variants of unknown significance.

To solve this problem we have used a functional genomics strategy to evaluate the contribution of every human gene to ciliogenesis. Our data-set has already identified new disease genes, mechanisms and pathways including unexpected roles for pre-mRNA processing factors (PRPFs). PRPF mutations are the second most common cause of autosomal dominant retinitis pigmentosum (RP) affecting up to 1 in 3500 people. We are very confident that our functional genomics screen has high specificity for ciliary processes and that our multidisciplinary approach allows the functional annotation of many new genes. We will integrate functional genomics with WES variant data from existing gene discovery programmes, followed by cell biology in physiologically-relevant cell or zebrafish mutant models. We aim to demonstrate the clinical utility and validity of integrated "systems medicine" annotation and its ability to make relevant predications about disease causality. We are also keen to retain key researchers with key skills in functional genomics so that their expertise can stimulate the adoption of the broader "systems medicine" approach. The development of a strong interface between gene discovery, functional annotation and diagnostic development work is therefore an indirect but important added benefit of this research.

The identification of new disease genes and pathways may enable the future rational design of therapeutics to modify or treat cystic kidney disease, retinal degeneration or ciliopathy disease progression, or improve the long-term outlook of patients with these conditions. Since these conditions result from absence of normal protein, they can in principle be corrected by gene-replacement, therapeutic approaches now undergoing Phase II clinical trials. Patients in whom mutations are found can therefore be given a clearer prognosis and prioritised for these new treatments, making inherited disease a top priority for further characterization.