The study is carried out within the OPUS 16 project, led by prof. Ewa Zietkiewicz
Project title: „Unsolved issues in molecular basis of primary ciliary dyskinesia (PCD), a motile ciliopathy: identification of large genomic deletions and transcript isoforms”
Ciliopathies are a growing group of inherited diseases caused by dysfunction of primary or motile cilia, evolutionary conserved organelles present on the surface of many eukaryotic cells. During the recent years, intensive research efforts have been directed to understand genetics and molecular basis of ciliopathies, but so far, many areas remain unknown or unexplored. Primary ciliary dyskinesia (PCD), whose symptoms include recurrent airway infections, male infertility and situs inversus, is a flagship ciliopathy caused by a hereditary dysfunction of motile cilia. Genetically heterogeneous, PCD is caused by mutations in genes encoding proteins, which either form structural and functional elements of motile cilia, or are essential for cilia formation. Among hundreds of cilia-related genes (ciliome genes), over 40 have been found, mutations in which cause PCD (further referred to as PCD genes). In spite of a substantial progress achieved in elucidating molecular basis of PCD pathogenesis, mutations underlying the disease remain unknown in ~30% of patients. It is still not known, whether this is due to the presence of unknown pathogenic variants, lying outside the usually tested positions in known PCD genes, or to the presence of mutations in yet-unknown PCD genes. Application of modern sequencing techniques, such as whole exome sequencing (WES) technology, to the unsolved PCD cases is not always conclusive – only in some of these patients, biallelic mutations in relevant genes are found. In others, WES screening does not identify any pathogenic variants consistent with PCD. This can be due to the limitations of WES technology, which is not able to detect large genomic insertions/deletions (indels) or aberrant splicing events caused by mutations activating cryptic splice sites in introns. In such cases, other techniques have to be employed to explain the unknown genetic cause of the disease. Large genomic indels can be detected using copy-number sensitive techniques. Some pathogenic variants related to aberrant splicing cannot be identified by the genomic sequence analysis alone, and analysis of the ciliated cells transcriptome (e.g. by RNAseq) needs to be employed. Proper interpretation of RNAseq findings requires comprehension of the role of ciliary gene transcripts isoforms at different stages of ciliated cells differentiation in healthy individuals. The aim of the current project is to extend the current knowledge on molecular causes of PCD. Based on the data from WES-based analysis performed in PCD patients, we will study in detail the genome and transcriptome of those PCD patients, in whom WES did not show conclusive results. Our goals include: identification of pathogenic variants resulting in structural changes of the genomic sequence, e.g. large-scale genomic deletions; identification of aberrant splicing events in PCD genes; elucidation of the relevance of different isoforms of PCD genes transcripts at different stages of motile cilia biogenesis.
Research project methodology. Whole genome sequencing will be performed in DNA from selected PCD WES-negative patients. For RNAseq, RNA isolated from the respiratory epithelial (RE) cells from WES-negative PCD patients and from healthy individuals will be used; the cells will be derived from the primary samples and/or differentiated in air-liquid interface (ALI) cultures. Downstream analysis of new mutations, candidate genes and transcripts indicated by the bioinformatic analysis of WGS or RNAseq data will include targeted genome screening, quantitative RT-PCR, cilia imaging in the relevant biological material, and functional analysis of the candidate genes in an animal model (RNAi in planaria).
Predicted tasks in the project:
1. Active participation in the realization of project goals.
2. Supervising master students.
3. Participation in writing scientific papers, presenting results during seminars and conferences.
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