cystic fibrosis cf. cysticfibrosis cystic fibrosis the most common autosomal recessive (ar) disorder...
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Cystic fibrosisCystic fibrosisCFCF
CysticCystic fibrosisfibrosis• the most common autosomal recessive (AR) disorder among Caucasians• chronic and progressive disease• median at death is ~ 35 years
OrgansOrgans AffectedAffected by CFby CF
Lung: Lung: thick accumulations of mucus, breathing difficulties,thick accumulations of mucus, breathing difficulties, frequent resp. infectious, permanent lung damage frequent resp. infectious, permanent lung damage
Pancreas: Pancreas: exocrine pancreatic insufficiencyexocrine pancreatic insufficiency malabsorption of proteins and fatsmalabsorption of proteins and fats
Liver:Liver: plugging of small bile ducts, cirrhosis plugging of small bile ducts, cirrhosis
GIT:GIT: intestinal obstruction-Meconium ileus intestinal obstruction-Meconium ileus (15-20% CF babies)(15-20% CF babies)
Reproduction: Reproduction: improper formation of Vas deferens improper formation of Vas deferens sterility sterility (95% CF male)(95% CF male)
Skin:Skin: CF patients have salt crystal formation on their skin CF patients have salt crystal formation on their skin((sweat excessivelysweat excessively))
MolecularMolecular causationcausation ofof CFCF
NBD-nucleotide binding domains (ATP)
R
NBD1 NBD2
TM1 TM2
Cl-
• 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
R-regulation domain (cAMPdep.)
TM- transmembrane spanning domainslumen
cytoplasm
Mutation in the CFTRMutation in the CFTR genegene • germinal mutations • somatic mutations have not been described so far• de novo mutation – rarely• distribution of mutation shown population
specificity
F508del = delta F508 = F508del = delta F508 = ΔF508ΔF508 • 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
RNBD1 NBD2
TM1 TM2
Cl-
…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…
508
Detection of ΔF508 mutation in CFTR geneDetection 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)
M
C
TARGET SEQUENCE
NX
Detection of ΔF508 mutation in CFTR geneDetection of ΔF508 mutation in CFTR gene
C/N C/NC/NC/M C/MC/M
1 2 1 2 1 2
HomozygousNo Mutation
HeterozygousCarrier
Homozygousfor Mutation
DNA sample in placed in 2 PCR tubes:
Tube 1 contains primer C and primer NTube 2 contains primer C and primer M
Detection of ΔF508 mutation in CFTR geneDetection of ΔF508 mutation in CFTR gene
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
PC R Mix
1
PC R Mix
0
NCDNAmarker
Patient 1 2 3 + + +
+ - +
Detection of ΔF508 mutation in CFTR geneDetection of ΔF508 mutation in CFTR gene
PCR Mix 0 / Mix 1:
Water 8.5µlTaq buffer 2.0µldNTP mix 4.0µlMg2+ (MgCl2) 2.0µlDNA 2.0µlPrimer mix M0 / M1 1.2µl
Taq polymerase (on ice) 0.4 µl
Total 20.0µl