Ciliopathy disease gene identification by whole exome medical resequencing

Cilia are finger-like projections from cells that act as a cellular "antenna" to detect and respond to chemical or mechanical cues. One important cue is fluid flow during the process of establishing asymmetry during early embryogenesis, the formation of the kidney nephron, and the formation of other tubular structures such as the neural tube. A group of comparatively common inherited conditions called "ciliopathies" can arise when the structure or function of cilia are defective. For example, cystic kidney disease is a common feature of many ciliopathies, often leads to kidney failure, and is therefore a significant cause of childhood disease and death. The most severe ciliopathy ("Meckel-Gruber syndrome") also involves problems in the development of the brain and spinal cord (called "neural tube defects") that arise during growth of babies in the womb. Reduced or absent mucus clearance from the lungs is the main consequence in other ciliopathies ("primary ciliary dyskinesia"), which causes recurrent respiratory infections and progressive damage to the respiratory system. In many ciliopathies, there are disturbances in the patterns of left-right organization of the lungs and heart, which are also seen in congenital heart defects (CHD), the most common birth defect.

This research proposes to identify many of the remaining disease genes that are defective ("mutated") in this group of ciliopathies. To achieve this aim, we will take advantage of recent exciting advances in genetic technology that allow us to analyze the entire coding parts of a patient's genome ("whole exome sequencing"). We are uniquely placed to do this work and have a proven track record of success in this field: we have formed excellent research partnerships with families and their clinicians in the local community to participate in gene identification studies, and we have the appropriate state-of-the-art technology, computer analysis tools and experience.

The identification of a new ciliopathy gene 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 the disease mechanism of the ciliopathy and into the normal function of the disease gene, which can lead to new treatments. We also expect new insights into the causes of other, more common conditions such as polycystic kidney disease, spina bifida and congenital heart defects.

We have ascertained and sampled DNA from about 250 individuals with a broad range of known or suspected ciliopathy phenotypes, but with a common theme of left-right laterality defects. These patients are confirmed to be mutation negative for known genes, or have been excluded from linkage to known loci in multiplex or consanguineous families. About 50% of disease genes for Meckel-Gruber syndrome and primary ciliary dyskinesia still remain to be identified, and many families with inherited forms of left-right laterality defects have an unknown genetic aetiology. We estimate that this research will identify a minimum of 10-12 new genes, comprising the majority of the remaining genes in this group of conditions. To discover new ciliopathy genes, we will perform whole exome sequencing for 100 individuals, including trios with an orphan ciliopathy. We will use our own successful bioinformatics workflow to distinguish putative pathogenic mutations from benign polymorphisms, even in the context of genetic heterogeneity. We will prioritize potential pathogenic variants in functional candidate genes on the basis of putative function, and our own data from a reverse genetics screen for ciliogenesis. Collaboration with other groups will allow us identify further mutations in a new gene for cohorts of patients with a ciliopathy and/or a retinal degeneration. This will also delineate an allelic series for a new gene, and may define genotype-phenotype correlations. We will collaborate with external groups to assess zebrafish embryo morphant phenotypes for 2 or 3 newly-identified ciliopathy disease genes. In some instances (for example if we need to prove the pathogenicity of a missense mutation), we will perform straight-forward functional characterization of the protein. This will include sub-cellular localization using confocal immunofluorescence microscopy and the effect of RNAi or a missense mutation on ciliogenesis or protein localization.

Whole exome sequencing is very likely to improve the rate of molecular diagnosis in children with a known or suspected ciliopathy, and reduce 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. For example, patients with congenital heart disease (CHD) and heterotaxy have a high postsurgical morbidity and mortality, often developing respiratory complications, so prescreening for ciliary dysfunction or mutations in a ciliopathy gene may be of great potential benefit for CHD patients. CHD is the most common birth defect, affecting 1 in 150 live births or 3600 patients a year in the UK.
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. In our experience, once a disease gene has been identified, genetic testing on a research basis can be applied immediately, and translation into service provision through an accredited NHS diagnostic lab can be made available in months. As a direct outcome of our own previous research, we have established UK-wide testing for Alström syndrome, Aicardi-Goutières syndrome and Meckel-Gruber syndrome (through the UK Genetic Testing Network and based at Yorkshire Regional Clinical Genetics Service). From our experiences of establishing NHS service testing using next-generation clonal sequencing at Yorkshire Regional Clinical Genetics Service, we fully expect that whole exome sequencing ("clinical exomes") will out-perform conventional diagnostic testing in terms of speed, sensitivity and cost. There are therefore considerable potential cost savings for NHS Trusts, but the fundamental challenge in analyzing exome sequence data is then distinguishing pathogenic mutations from benign polymorphisms, particularly in the context of genetically heterogeneous conditions such as the ciliopathies. Our ability to overcome this challenge will inform the debate on the eventual clinical utility and clinical validity of "clinical exomes", and the probable widespread adoption of whole exome sequencing as a routine diagnostic strategy. In particular, we are keen to retain key researchers with key bioinformatic skills so that their knowledge can stimulate the adoption of clinical bioinformatics expertise, and the use of standardized databases of normal and pathogenic genomic variation. The development of a robust interface between research and diagnostic development work is therefore an indirect but important added benefit of this research, which should encourage the implementation of NGS for the diagnosis of genetic diseases in other clinical centres.
The identification of further novel ciliopathy disease genes may also enable the future rational design of therapeutics to modify or treat cystic kidney disease or ciliopathy disease progression, or the long-term outlook of patients with these conditions. Since most of these conditions are autosomal recessive and are the result of absence of normal protein, rather than the presence of an abnormal protein, they can in principle be corrected by gene-replacement, a therapeutic approach now undergoing Phase II clinical trials in major centres in the US, UK and elsewhere. Patients in whom mutations are found can therefore be given a clearer prognosis and prioritised for these new treatments, making recessive disease a top priority for further characterization.