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