Ciliopathy disease gene identification by whole exome medical resequencing

Lead Research Organisation: University of Leeds
Department Name: School of Medicine

Abstract

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.

Technical Summary

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.

Planned Impact

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 chaenge 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.

Publications

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Cuvertino S (2020) A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome. in Genetics in medicine : official journal of the American College of Medical Genetics

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Mans DA (2014) Medical genetics of ciliopathies. in Journal of pediatric genetics

 
Description 100K Genome Project: outreach
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description diagnosis and treatment of neurodevelopmental conditions
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Our research has enabled a new recognition of the broad range of phenotype for neurodevelopmental conditions such as Joubert syndrome. The identification of new genes provided more accurate prognostic testing and genetic counselling, with national NHS service testing for both Meckel-Gruber and Joubert syndromes now based at Yorkshire Regional Genetics Service. New services based on clinical whole exome sequencing has enabled testing of about 40-60 patients per annum for both the UK and for international referrals.
 
Description recessive conditions
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact greater investment in clinical genetics and training of genetic counsellors, greater awareness of recessive conditions and rare diseases
 
Description Bilateral BBSRC-SFI (structure-function relationships in the ciliary transition zone)
Amount £576,400 (GBP)
Funding ID BB/P007791/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2021
 
Description MRC project grant (functional genomics)
Amount £856,000 (GBP)
Funding ID MR/M000532/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description PhD Training Fellowship for Clinicians "Developing variant interpretation pipelines for inherited retinal diseases and ciliopathies: using medical genomics to improve diagnostic yield"
Amount £250,000 (GBP)
Funding ID UNS81212 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description Pre-clinical testing of new therapeutic treatments for cystic kidney disease
Amount £200,000 (GBP)
Funding ID GN2628 
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 06/2021
 
Title MKS patient cohort 
Description cohort of ciliopathy patients, sampled for DNA with assessment of mutation status and clinical phenotype 
Type Of Material Biological samples 
Year Produced 2007 
Provided To Others? Yes  
Impact role of RPGRIP1L in determining a retinal phenotype in ciliopathies; mutation screening in other ciliopathy genes eg ARL13B, TMEM216, TMEM138. TMEM237 etc 
 
Title WES variant analysis and filtering 
Description developed standard workflow for analysis of variants from WES experiments based on existing publically-available databases (EVS, 1000 Genomes etc) and in-house ethnically-matched exomes bespoke Perl scripts developed to allow automated annotation of candidate genes, pathogenic potential of variants (from Condel, Polyphen etc), segregation analysis in small families with recessive or dominant inheritance patterns 
Type Of Material Improvements to research infrastructure 
Year Produced 2011 
Provided To Others? Yes  
Impact Parry DA,et al. (2013). SAMS, a syndrome of short stature, auditory canal atresia, mandibular hypoplasia and skeletal abnormalities is a unique neurocristopathy caused by mutations in Goosecoid. Am. J. Hum. Genet. in press Khan K, et al. (2012). Next generation sequencing identifies mutations in Atonal homolog 7 (ATOH7) in families with global eye developmental defects. Hum Mol Genet. 21: 776-83. Logan CV, et al. (2011). Mutations in MEGF10, a regulator of satellite cell myogenesis, cause early-onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD). Nat. Genet. 43: 1189-1192 
URL http://autozygosity.org
 
Title effectors of ciliogenesis 
Description list of validated candidate genes implicated in ciliogenesis, cilia maintenance and cilia length growth as a result of a whole genome cell-based reverse genetics visual screen primary list: ca. 600 genes secondary screen list: 174 genes tertiary validated genes: ca. 42 genes selected functional candidates: 14 genes other date includes RNA-Seq expression data from non-ciliated vs. ciliated cell-lines 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact methodlogy of assessing cell numbers in the screen described in this publication: Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172. Wheway G, et al. (2015). An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat. Cell. Biol. 17: 1074-87 
URL http://www.syscilia.org
 
Title effectors of ciliogenesis 
Description list of validated candidate genes implicated in ciliogenesis, cilia maintenance and cilia length growth as a result of a whole genome cell-based reverse genetics visual screen primary list: ca. 600 genes secondary screen list: 174 genes tertiary validated genes: ca. 42 genes selected functional candidates: 14 genes other date includes RNA-Seq expression data from non-ciliated vs. ciliated cell-lines 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Wheway G*, Schmidts M*, Mans DA*, Szymanska K*, Nguyen T-MT*, ...69 others... Doherty D+, Mitchison HM+, Roepman R+, Johnson CA+ (2015). An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat. Cell. Biol. 17: 1074-87 
 
Description medical resequencing of ciliopathy genes 
Organisation Birmingham Women's Hospital
Department Clinical Genetics
Country United Kingdom 
Sector Hospitals 
PI Contribution ascertained fmailies affected with ciliopathies, screened known genes, collated clinical data, verified mutations, exluded polymorphic variants
Collaborator Contribution access the high-throughput resequencing technology and techniques for assessing the pathogenic potential of missense variants; whole exome sequendcing of ciliopathy patients
Impact the A229T change in the RPGRIP1L gene is associated with retinal features in ciliopathies; published in Nat. Genet. 2009
Start Year 2006
 
Description medical resequencing of ciliopathy genes 
Organisation Duke University
Department Department of Cell Biology
Country United States 
Sector Academic/University 
PI Contribution ascertained fmailies affected with ciliopathies, screened known genes, collated clinical data, verified mutations, exluded polymorphic variants
Collaborator Contribution access the high-throughput resequencing technology and techniques for assessing the pathogenic potential of missense variants; whole exome sequendcing of ciliopathy patients
Impact the A229T change in the RPGRIP1L gene is associated with retinal features in ciliopathies; published in Nat. Genet. 2009
Start Year 2006
 
Description medical resequencing of ciliopathy genes 
Organisation Leeds Teaching Hospitals NHS Trust
Department Yorkshire Regional Genetics Service & Clinical Genetics
Country United Kingdom 
Sector Hospitals 
PI Contribution ascertained fmailies affected with ciliopathies, screened known genes, collated clinical data, verified mutations, exluded polymorphic variants
Collaborator Contribution access the high-throughput resequencing technology and techniques for assessing the pathogenic potential of missense variants; whole exome sequendcing of ciliopathy patients
Impact the A229T change in the RPGRIP1L gene is associated with retinal features in ciliopathies; published in Nat. Genet. 2009
Start Year 2006
 
Description medical resequencing of ciliopathy genes 
Organisation Newcastle University
Department Institute of Genetic Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution ascertained fmailies affected with ciliopathies, screened known genes, collated clinical data, verified mutations, exluded polymorphic variants
Collaborator Contribution access the high-throughput resequencing technology and techniques for assessing the pathogenic potential of missense variants; whole exome sequendcing of ciliopathy patients
Impact the A229T change in the RPGRIP1L gene is associated with retinal features in ciliopathies; published in Nat. Genet. 2009
Start Year 2006
 
Description medical resequencing of ciliopathy genes 
Organisation Sheffield Children's NHS Foundation Trust
Department Clinical Genetics
Country United Kingdom 
Sector Hospitals 
PI Contribution ascertained fmailies affected with ciliopathies, screened known genes, collated clinical data, verified mutations, exluded polymorphic variants
Collaborator Contribution access the high-throughput resequencing technology and techniques for assessing the pathogenic potential of missense variants; whole exome sequendcing of ciliopathy patients
Impact the A229T change in the RPGRIP1L gene is associated with retinal features in ciliopathies; published in Nat. Genet. 2009
Start Year 2006
 
Description zebrafish models of human disease 
Organisation Newcastle University
Department Institute of Genetic Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution candidates genes with putative pathogenic mutations for recessive developmental or cardiac conditions are modelled in wild-type zebrafish embryos using morpholino knockdown, or novel mutations are knocked-in using the CRISPR-Cas9 system
Collaborator Contribution access to zebrafish husbandry and microinjection facilities; assessment of embryo phenotypes
Impact two manuscripts in the drafting stage, reporting novel genes mutated in a retinal dystrophy and Meckel-Gruber syndrome (a severe ciliopathy), and a form of primary ciliary dyskinesia that manifests only with cardiac heterotaxy
Start Year 2013
 
Description zebrafish models of human disease 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution candidates genes with putative pathogenic mutations for recessive developmental or cardiac conditions are modelled in wild-type zebrafish embryos using morpholino knockdown, or novel mutations are knocked-in using the CRISPR-Cas9 system
Collaborator Contribution access to zebrafish husbandry and microinjection facilities; assessment of embryo phenotypes
Impact two manuscripts in the drafting stage, reporting novel genes mutated in a retinal dystrophy and Meckel-Gruber syndrome (a severe ciliopathy), and a form of primary ciliary dyskinesia that manifests only with cardiac heterotaxy
Start Year 2013
 
Description zebrafish models of human disease 
Organisation University of Sheffield
Department MRC Centre for Developmental and Biomedical Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution candidates genes with putative pathogenic mutations for recessive developmental or cardiac conditions are modelled in wild-type zebrafish embryos using morpholino knockdown, or novel mutations are knocked-in using the CRISPR-Cas9 system
Collaborator Contribution access to zebrafish husbandry and microinjection facilities; assessment of embryo phenotypes
Impact two manuscripts in the drafting stage, reporting novel genes mutated in a retinal dystrophy and Meckel-Gruber syndrome (a severe ciliopathy), and a form of primary ciliary dyskinesia that manifests only with cardiac heterotaxy
Start Year 2013
 
Title NHS service testing for ciliopathies 
Description First use of clinical whole exome sequencing by the Yorkshire Regional Genetics Service for the diagnosis of ciliopathies (primary ciliary dyskinesia, Meckel-Gruber syndrome, Joubert syndrome). New services based on clinical whole exome sequencing has enabled testing of about 40-60 patients per annum for both the UK and for international referrals. 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Wide-scale adoption
Year Development Stage Completed 2013
Development Status Under active development/distribution
Impact Improved diagnosis, prognosis and genetic counselling for ciliopathy patients and their families 
URL http://www.leedsth.nhs.uk/a-z-of-services/molecular-genetics/test-pages/meckel-and-joubert-syndromes...
 
Description BioJapan2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues
Year(s) Of Engagement Activity 2017
 
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact About 50 attendees to the Cafe Scientifique meeting in Feb 2020; I presented on recent advances in the diagnosis and treatment of inherited blindness, and was able to answer questions and speak to patients and supporters after the formal presentation.
Year(s) Of Engagement Activity 2020
 
Description Cilia in Development and Disease 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact contributed to organization and programme for these international conferences

new research collaborations were started as a result of these conferences
Year(s) Of Engagement Activity 2012,2014
 
Description Ciliopathy Alliance UK 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Supporters
Results and Impact promoted support group attendance at the international conference "Cilia 2012" in London 2012

continuing links with support groups (Joubert Syndrome UK and Alstrom Syndrome UK)
Year(s) Of Engagement Activity 2009,2010,2011,2012
 
Description FASEB "Polycystic Kidney Disease" meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact to facillitate and establish research collabirations, with critique of current research
Year(s) Of Engagement Activity 2017
 
Description ISN Forefronts Symposium on PKD 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact a general overview of my group's work on Meckel-Gruber syndrome and other ciliopathies, to an audience of researchers and clincians working on Polycystic Kidney Disease

the meeting was very useful as an informal venue to establish new collaborations with some existing collaborators
Year(s) Of Engagement Activity 2008
 
Description Johnson Lab Twitter feed 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Twitter feed for the lab group which reports on our professional activities and events
Year(s) Of Engagement Activity 2020
URL https://twitter.com/johnsoncilialab
 
Description Joubert syndrome scientific meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact imporant discussion on the improved methods for diagnosis of Joubert syndrome and other ciliopathies, as well as new insights into pathogenic mechanisms

promoting new collaborations with workers in the field of Joubert syndrome
Year(s) Of Engagement Activity 2013
 
Description Midbrain/Hindbrain Malformations and Hydrocephalus workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact important opportunity to compare methods of diagnosis for ciliopathies and provided new insights into development of the cerebullum and posterior fossa structures

initiated new collaborations as part of on-going discussions
Year(s) Of Engagement Activity 2014
 
Description PEGS Boston 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues
Year(s) Of Engagement Activity 2018
 
Description UK Cilia Club 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact informal research club for reseachers in the north of England and Scotland; first meeting was in Sheffield Sept 2014, with on-going meetings planned for every 6 months in Leeds, Newcastle adn Edinburgh

new collaboration with University of Newcastle
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
 
Description autozygosity.org website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact a website inititated and maintained by Prof. David Bonthron at the University of Leeds to summarize our work on autozygosity mapping of recessive inherited conditions

increased clinical collaboration and publicity
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012,2013,2014
URL http://www.autozygosity.org
 
Description genetics and cell biology of Meckel-Gruber syndrome 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact a overview of the molecular genetics and cell biology of ciliopathies, focussing on Meckel-Gruber syndrome, to an audience of medical and clinical geneticists

useful to establish clinical collaborations on other ciliopathies (notably family ascertainment for Ivemark syndrome)
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012,2013,2014
 
Description school out-reach activities 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact discussed careers in medical research with about 60 Year 12 and 13 students on a visit and presentation at their school; 10 had a follow-on visit to the lab to discuss research methods
Year(s) Of Engagement Activity 2015,2016,2017,2018