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[Sousse]Mutation screening of DNAH5 gene in a Tunisian family with Primary Ciliary Dyskinesia (PCD) in order to confirm the diagnosis of the pathology
Tunisia (ASSOCIA-MED) - University of Sousse/ Faculty of Medecine of Sousse, Sousse
Laboratory of Biochemistry. Faculty of Medicine of Sousse. Street Mohamed Karoui, 4000 – Sousse -TUNISIA
Professor Zohra SOUA
Professor Zohra SOUA
Type of Research Project
- Basic science
What is the background of the project?
Primary Ciliary Dyskinesia (PCD) is a rare hereditary ciliopathy. It is responsible of muccociliary clearance defect which leads to recurrent infections of the upper and lower respiratory airways, bronchiectasis, sinusitis and otitis. The diagnosis of this pathology is mainly based on the study of ciliary ultrastructural defects through Transmission Electron Microscopy. However, this technique is only available in highly specialized laboratories and has weaknesses regarding the detection of subtle ciliary defects. Moreover, PCD has a very heterogeneous genetic background. To date, 39 different genes are associated to PCD. According to literature, DNAH5; a gene encoding a ciliary structural protein, is the most frequently mutated in Primary Ciliary Dyskinesia patients. Particularly, 5 exons of DNAH5 have been reported as the most frequently mutated regions of the gene. The study of these hotspots could be a cost-effective method to identify mutations in PCD patients and thus confirm their diagnosis.
What is the aim of the project?
The aim of this study is to confirm Primary Ciliary Dyskinesia (PCD) diagnosis through mutational screening of DNAH5 hotspot exons.
What techniques and methods are used?
Stages of this project are as follows: 1. Genomic DNA extraction and purification by Salting-out method 2. Assessment of the extracted DNA quality and quantity using Nanodrop 3. Genomic DNA quality evaluation through agarose gel electrophoresis 4. DNA amplification by Polymerase chain reaction (PCR) 5. Evaluation of PCR results by agarose gel and polyacrylamide gel electrophoresis 6. Screening of DNAH5 selected exons through PCR-SSCP technique (Single Strand Confirmation Polymorphisim) 7. Sequencing of exons harboring variants detected by SSCP 8. Interpretation of results and conclusions.
What is the role of the student?
What are the tasks expected to be accomplished by the student?
The student will learn how to: 1. Plan scientific experiments 2. Prepare and sterilize solutions required for the experiments 3. Use and manipulate the laboratory tools 4. Prepare genomic DNA by Salting-out method 5. Perform agarose gel and polyacrylamide gel electrophoresis of DNA 6. Perform DNA amplification by PCR technique 7. Screen mutations using SSCP technique 8. Use bio-informatics tools to analyze sequencing results 9. Analyze and discuss the experiments results 10. The results will be afterwards transmitted to the clinicians
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Preliminary readings are highly required. Thus, scientific articles on PCD will be provided for the student. The student will also benefit from explanations provided by their tutors and the laboratory personnel.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic knowledge about genetic diseases, structure of eukaryotic genes and DNA amplification by Polymerase chain reaction (PCR) are required. Theoretical knowledge of: - Structure of eukaryotic genes - The central dogma of molecular biology (transcription and translation of the genetic information) - Types of genetic mutations - Amplification of DNA by Polymerase chain reaction (PCR)
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students
- Djakow; J.; Svobodová; T.; Hrach; K.; Uhlík; J.; Cinek; O.; & Pohunek; P. (2012). Effectiveness of sequencing selected exons of DNAH5 and DNAI1 in diagnosis of primary ciliary dyskinesia. Pediatric Pulmonology; 47(9); 864–875. http://doi.org/10.1002/ppul.22520
- Praveen; K.; Davis; E. E.; & Katsanis; N. (2015). Unique among ciliopathies: primary ciliary dyskinesia; a motile cilia disorder. F1000Prime Reports; 7(March).
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