Analysis of CFTR gene mutations and intragenic polymorphisms in chronic respiratory and gastrointestinal disorders with an yet unknown etiology and in severe disorders of human reproduction

• Elucidation of the role of known / novel, ethnically specific, CFTR gene mutations / polymorphisms in the pathogenesis of idiopathic acute / chronic pancreatitis and in chronic respiratory diseases of yet unknown etiology and in severe disorders of human reproduction.
• Detection of novel CFTR mutations and intragenic polymorphisms regulating the phenotypic expression of CFTR, including the study of mechanisms of CFTR alternative splicing, in these diseases.
• Study of identified modifier genes that affect CFTR expression and screening for their mutations in our population.
• Analysis of cDNA from pancreatic / lung / testicular biopsies for gene expression studies.
• Improvement of the pathogenetic process, of diagnosis, treatment and prenatal /postnatal prevention of cystic fibrosis in CFTR-mutation positive patients and their families.
• The implementation of expertise of an invited senior visitor, the training of junior visitor, postdoc and/or PhD students in our center, the organization of training of our two young researchers in other research center.
• The organization of one workshop on the methods of DGGE, OLA and QFPCR for the CFTR mutation detection and analysis of different types of intragenic polymorphism in the CFTR gene.
• The organization of a conference with international participation on CFTR gene mutations and intragenic polymorphisms in idiopathic acute / chronic pancreatitis, in chronic respiratory diseases of yet unknown etiology and in severe disorders of human reproduction.

Description of contents, the workplan, the steps, the approach or the methodology:

This type of study is already running for 3 years at our CF Center within the frame of a domestic research grants IGA MH CR # 2899-5, 3526-3 and INCO-BIOMED (OK 192).

1. Ascertained patients are being clinically characterized, followed by specialized genetic counseling and elimination of other non-CFTR related affections of the pancreas, lung and of the male reproductive system by all appropriate biochemical, ultrasound and CT examinations.
2. Moreover, hereditary pancreatitis will be excluded by clinical examinations and molecular genetic testing of common mutations in the cationic trypsinogen gene. This will provide an optimal selection of pediatric and adult patients with acute and chronic pancreatitis and with uncharacterized lung disorders for the analysis of pathogenetic impact of CFTR mutations / polymorphisms.
3. Selected patients will be tested for common CFTR mutations, that account at present for over 95% of all CFTR mutations in our population in classical CF.
4. Subsequently, DGGE scanning of the entire coding region of the CFTR gene in patients with yet unidentified mutations will be performed. Samples with altered DGGE migration patterns will be sequenced by F-PCR on the ABI 310 Genescanner.
5. In selected patients cDNA studies will be initiated from pancreatic, lung and testicular tissue biopsies and from biopsies of other tissues, including derived cell cultures in CFTR-mutation positive patients. The impact of intron 8 5/7/9T, TG/n/ alleles and exon 10 1540A/G polymorphism (M470V) will also be studied in these patients compared to their prevalence in normal, healthy, random controls.
6. All patients with detected CFTR mutations will undergo repeated sweat chloride testing in order to exclude atypical presentations of cystic fibrosis. In all patients with CFTR mutations / polymorphisms (known and novel) the comparison of their frequency with data from a random control population will be followed by thorough genotype-phenotype correlations.
7. All our investigations have been approved by the University Hospital Motol (Charles University Prague) Ethical Committee and follows all its guidelines (inform consent forms, counseling). Our research is in accordance with the ethical laws of our country and it also complies with respective EU Ethical Guidelines and Regulations.
8. The implementation of an expertise of invited senior visitor within months 0 - 12. The short training of a junior visitor within months 12-36 and training of postdoc and/or PhD student for 6 - 12 months in our center since month 12 of the project. The arrangement of training of two young researcher for 6 - 12 months in other cooperating EU center within months 12 - 30 of the project.
9. Coordination activities of this type of research for Central and Eastern European countries, similar to our current EU ECCACF (Belgium)- INCO-Biomed activities.
10. The organization of a Workshop on the methods of DGGE and OLA within month 12 - 24.
11. The organization of a EU conference with international participation on CFTR gene mutations and intragenic polymorphisms in idiopathic acute / chronic pancreatitis and in chronic respiratory diseases of yet unknown etiology.

Partners from the Czech Republic:

1. Integration of the National Cystic Fibrosis Center with the Department of Reproductive Genetics and Department of Reproductive Medicine of the Institute of Biology and Medical Genetics and University Hospital Motol and with 2nd Pediatric Clinic in improved treatment of CF if future gene therapy will be possible.
2. The National Cystic Fibrosis Center cooperates with all major pediatric and adult gastroenterology / surgery, otorhinolaryngology and pneumology departments in the country that assures a representative cohort of affected patients.

Partners from the EU countries:

• The collaboration with the Centers for Medical Genetics involved in the frame of EU INCO-BIOMED programme - Institute of Human Genetics, Hannover (Prof. Dr. J. Schmidtke, Dr. T. Doerk).
• Center of Medical Genetics, Catholic University of Leuven (Prof. J. J. Cassiman, H. Cuppens - ECCACF Consortium).
• Department of Human Genetics, University of Verona (Prof. P. F. Pignatti)
• ETSBO - CDTS - INSERM, Brest, France (Prof. C. Ferec)
• Cancer Research Institute Hospital Duran y Reynalds, Barcelona, Spain (Prof. X. Estivill)
• Department of Pediatric Genetics, Manchester University, UK (Dr. M. Schwartz)
• Department of Genetics, INSERM U32, Paris - Creteile, France (Prof. Dr. M. Goosens)
• Within the frame of international Science and Technology cooperation between Czech Republic and the Medical School of the University of Ljubljana, Slovenia, (Prof. Dr. Damian Glavac).
• With the Dept. of Gastroenterology, University of Liverpool, Prof. Dr. J. Neoptolemos- EUROPAC
• with the Institute of Human Genetics, Humboldt University, Berlin, Germany (Dr. R. Varon)

Partners from  the USA and Canada:

• Dept. of Molecular Genetics, Hospital for Sick Children, University of Toronto, Canada, Dr. J. Zielenski.
• Department of Human Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA (Prof. Dr. G. R. Cutting).