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Cystic fibrosis CF

Cystic fibrosis CF. Cystic fibrosis. the most common autosomal recessive (AR) disorder among Caucasians chronic and progressive disease median at death is ~ 35 years. Organs Affected by CF Lung: thick accumulations of mucus, breathing difficulties,

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Cystic fibrosis CF

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  1. Cystic fibrosisCF

  2. Cysticfibrosis • the most common autosomal recessive (AR) • disorder among Caucasians • chronic and progressive disease • median at death is ~ 35 years OrgansAffectedby CF Lung: thick accumulations of mucus, breathing difficulties, frequent resp. infectious, permanent lung damage Pancreas: exocrine pancreatic insufficiency malabsorption of proteins and fats Liver: plugging of small bile ducts, cirrhosis GIT: intestinal obstruction-Meconium ileus (15-20% CF babies) Reproduction: improper formation of Vas deferens sterility (95% CF male) Skin: CF patients have salt crystal formation on their skin (sweat excessively)

  3. Cl- TM1 TM2 NBD1 NBD2 R MolecularcausationofCF • mutations in the CFTR gene • CFTR gene coding for chloride channel protein: cystic fibrosis transmembrane conductance regulator – located on the plasma membrane of epithelial cells of the lungs, pancreas, sweat glands, and other tissues • cAMP regulated chloride channel TM- transmembrane spanning domains lumen cytoplasm NBD-nucleotide binding domains (ATP) R-regulation domain (cAMPdep.)

  4. Mutation in the CFTRgene • germinal mutations • somatic mutations have not been described so far • de novo mutation – rarely • distribution of mutation shown population specificity

  5. 508 …E   N   I I F  G   V    S  Y   D…  …..GAA AAT ATC ATC TTT GGT GTT TCC TAT GAT…. …..GAA AAT ATC ATT GGT GTT TCC TAT GAT…. …E   N   I I G   V    S  Y   D…  Cl- TM1 TM2 NBD1 NBD2 R F508del = delta F508 = ΔF508 • the most common mutation among Caucasians(70%) • deletion of three basepairs in exon 10 of DNA • resulting in deletion of a Phe F508 from CFTR protein

  6. TARGET SEQUENCE X C N M Detection of ΔF508 mutation in CFTR gene • This technique depends on the specificity of PCR primers • 3 primers are made: General primer (C) Normal specific primer (N) Mutation specific primer (M)

  7. C/N C/M C/N C/M C/N C/M 1 2 1 2 1 2 Homozygous No Mutation Heterozygous Carrier Homozygous for Mutation Detection of ΔF508 mutation in CFTR gene DNA sample in placed in 2 PCR tubes: Tube 1 contains primer C and primer N Tube 2 contains primer C and primer M

  8. Detection of ΔF508 mutation in CFTR gene Patient 1 2 3 DNA marker NC + + + P C R M i x 0 STS gen (P3,4) – control of PCR process CFTR gen (P0,2) – general primer – primer specific to sequence without mutation + - + STS gen (P3,4) –control of PCR process CFTR gen (P1,2) –general primer – primer specific to mutated sequence P C R M i x 1

  9. Detection of ΔF508 mutation in CFTR gene PCR Mix 0 / Mix 1: Water 8.5µl Taq buffer 2.0µl dNTP mix 4.0µl Mg2+ (MgCl2) 2.0µl DNA 2.0µl Primer mix M0 / M1 1.2µl Taq polymerase (on ice) 0.4 µl Total 20.0µl

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