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Identification of novel genes and mechanisms for PCD and male infertility

Periodic Reporting for period 1 - PCDfert (Identification of novel genes and mechanisms for PCD and male infertility)

Reporting period: 2018-09-01 to 2020-08-31

Primary ciliary dyskinesia (PCD) is a heterogenous respiratory disease, which prevalence in Europe is estimated as 1 in 10,000. Improved availability of DNA testing would provide a definitive diagnostic tool for patients, since PCD is underdiagnosed due to lack of knowledge of genetic causes as well as sometimes inconclusive pathology-based tests based upon cilia beating and ultrastructure. This is partly due to its genetic heterogeneity since causal PCD mutations in >40 genes account for 70% of cases. Furthermore, although male infertility has been linked to PCD, it does not affect all patients. Therefore, it is important to identify novel PCD genes and establish the effect of specific PCD mutations on male infertility.
The first project objective was to provide training for the Researcher in implementing a patient cohort study for identification of causative mutations in human health issues. Causative mutations for Finnish PCD patient cohort were identified enabling development of DNA tests for diagnostics.
Second objective was the characterization of sperm phenotypes in PCD patients. During the project the Researcher organized the sperm sample collection and analysis. Sperm tail structural changes were characterized for patients with known gene mutations.
In the third objective the Researcher familiarized with super resolution microscopy techniques during studies of ciliary transition zone components in sperm. Role of intraflagellar transport (IFT) in transition zone formation was studied using Tctex1d2 mutant mouse model and Cep164 conditional knock-out demonstrated the importance of transition fibers in sperm tail formation.
The MSCA fellowship has enabled development of a collaboration network with world leading scientists and clinicians in the field of cilia research and expertise in clinical studies and human molecular biology. For the first time the link between ciliopathies and male infertility and related molecular mechanisms have been investigated consistently. This has produced unique and timely opportunities to prepare research funding applications for further experiments in the field. Results gained from the fellowship provide insights into protein transport mechanisms during spermiogenesis and genetic causes of PCD and related male infertility.
The PCDfert project aimed to increase knowledge and understanding of motile ciliopathies, male fertility and biological processes related to sperm tail formation. This was achieved through training and creation of collaboration opportunities for the research fellow. Results have been communicated to researchers, clinicians and the general public through seminar, conference and workshop presentations. For sperm sample collection and characterization of the male fertility in PCD patients, we updated the ethics for sperm sample collections, organized site approvals for patient recruitment (primarily from the Royal Brompton Hospital RBH, London and Seacroft Hospital, Leeds) and for subsequent sample collection and sperm quality analysis (Hammersmith Hospital, London). We were able to start sperm collection and recruited >25 adult male PCD patients for the study. Unfortunately, the Covid-19 pandemic stopped sperm collection, but the preliminary results show different sperm phenotypes depending on the mutated gene. This has revealed a stratification of motile ciliopathy patients into a range of more or less severely affected male germ cells according to genotype and factors still remaining to be elucidated. These results underline the importance of identification of the real prevalence of male infertility in PCD as well as the exact causes of sperm defects. We applied for additional funding (BBSRC) to continue sperm collection and analysis in 2021 – this significant funding was successfully awarded (01/21). The developed pipeline and knowledge can be utilized in a recently established national PCD adult clinical service for the UK, where centers will be able to inform patients of their fertility status. A review article about known effects of PCD mutations on male fertility has been published in collaboration with Dr Amelia Shoemark and Prof Michael Loebinger at RBH. Several additional papers are in preparation for publications.
Novel PCD gene variant identification was performed for a Finnish PCD patient cohort. Gene panel and whole exome sequencing was utilized for candidate variant detection. Loss-of-function variants in PCD candidate genes CFAP300 and HYDIN were identified as causative mutations explained 50% of studied Finnish cases. We have developed a Sanger sequencing based method for diagnosis of PCD caused by these variants within the Finnish population.
For functional studies of transition zone formation specific antibodies were tested in human sperm samples and during mouse spermiogenesis. For the first time we have tested and developed methods for localization of proteins within the spermatid transition zone and investigated the effect of inhibited IFT on transition zone formation. Super resolution microscopy imaging was used for protein localization in wild type and Tctex1d2 (component of retrograde IFT dynein complex) mutant mouse model to establish the transition zone structure during spermiogenesis. These results show novel data with differential localization of transition zone proteins in the sperm head tail connecting piece. The importance of functional IFT in transition zone formation was revealed, in addition to the requirement for specialized protein transport to the sperm tail. Furthermore, a new collaboration was established with Newcastle University, to investigate the specific role of transition fiber protein CEP164 in spermiogenesis.
During the fellowship the Researcher has been able to train in ciliopathy related subjects and methods, improved knowledge and skills in microscopy and high speed video imaging and gained knowledge to conduct research in collaboration with clinicians and hospital settings. The expertise of the Researcher has enabled development of systematic studies of male fertility in PCD patients and experiments to resolve the similarities and differences in cilia/sperm tail development. The Researcher has been able to develop an extensive international collaboration network in the field of cilia research, especially in wider Europe through engagement in the pan-European BEAT-PCD network (previous COST Action and current European Respiratory Society Clinical Research Collaboration).
The project has provided extensive training and collaboration opportunities for the Researcher. Several new collaborations have been established, which have led to a successful funding application for BBSRC. This includes identification of genetic causes of non-obstructive azoospermia, which was started during MSCA fellowship resulting in causative mutations in two genes to date. Socio-economic impact of the project is the development of diagnostic tools for PCD and male infertility.The project has provided insights into the role of protein transport during sperm tail development and genetic causes of motile ciliopathies. These results and developed sample collection and analysis pipelines enable understanding of causes of male infertility and development of diagnostics and management tools for PCD in Finland and male infertility worldwide. Seven peer-reviewed publications are in preparation for publication or have been published.
Markers for detection of different axonemal components in human sperm
Transition zone protein localization in human sperm and mouse spermatids
The effect of TCTEX1D2 depletion on axonemal and transition zone protein localization in sperm
Different dynein arm content in motile cilia and sperm tail