Transcript Snímek 1

Cystic fibrosis
•
the most common autosomal recessive (AR) disorder among
Caucasians, carriers of cystic fibrosis are not affected by the disease
•
carrier frequency - 1 in 25 individuals of Northern European descent
– ethnic variability
– affecting both males and females equally
•
CFTR - cystic fibrosis transmembrane conductance regulator
gene, localized on chromosome 7
•
mutation in the CFTR gene
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CFTR encodes a chloride channel; transport of chloride ions across
plasma membrane of epithelial cells that line the lung airways,
pancreas, sweat glands, and other tissues
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disorder of epithelial ion transport
Disease
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chronic and progressive disease
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pulmonary disease – damage to the lungs because of accumulation of thick
mucus, improper function of water secretion and its cleaning function,
breathing difficulties, frequent resp. infections, permanent lung damage
– Viscous mucus in the lungs – chronic airway infection (pneumonia,
Pseudomonas, Cephacia)
– Damage to lungs shortens life expectancy – 30 - 40 years
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digestive system
– pancreatic secretion – leading to digestion problems, malabsorption of
proteins and fats, retained enzymes causes fibrosis
– liver – plugging of small bile ducts, cirrhosis
– meconium ileus – intestinal obstruction (15-20% CF babies)
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reproductive system
– improper formation of the vas deferens leading to male sterility
•
sweat glands
– uptake of chloride from sweat ducts – absence of functional CFTR
causes increased sodium chloride content, excess salt loss
Molecular causation of CF
CFTR protein - cystic fibrosis transmembrane conductance regulator
• cAMP-regulated chloride channel
• transport of chloride ions across plasma membrane of epithelial cells (lungs,
pancreas, sweat glands, and other tissues)
• mutations in the CFTR gene
Cllumen
TM – transmembrane
TM1
TM2
NBD1
cytoplasm
NBD2
spanning domains
NBD-nucleotide binding domains (ATP)
R
R-regulation domain (cAMPdep.)
CFTR mutations
• There is one the most commonly inherited allele which is known
to cause up to 70% of all cystic fibrosis cases. This mutation is
called the delta – F508 mutation
• Δ F508 allele has 3 nucleotides deletion, which code for the
amino acid phenylalanine (F) in the 508 position of the amino
acid sequence of the chloride transporter
• There are twelve other common mutations with a combined
frequency of ~15% (in total 1967 mutations,
http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html)
• Reverse hybridization kit (34 mutation) – detects about 91% of
patients of Czech origin
Mutation in the CFTR gene
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•
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germinal mutations
somatic mutations have not been described so far
de novo mutation – rarely
distribution of mutation shown population specificity
Hemochromatosis
• autosomal recessive disease affecting iron metabolism, carrier
frequency - 1 in 15 individuals (Czech population), incomplete
penetrance
• excessive iron absorption, its deposition in organs (mainly
parenchymal) and subsequent damage of the organism
• hepatopathy (cirrhosis, hepatocellular carcinoma)
• diabetes mellitus, arthropathy, hypogonadism, kardiomyopathy,
amenorhea
• serum iron, transferrin saturation, ferritin, liver biopsy
• repeated phlebotomy
HFE gene mutations
RFLP
Restriction Fragment Length Polymorphism
restriction analysis of DNA
by its digestion with restriction
endonucleases (RE) in specific
restriction sites
in the case the sequence
difference (polymorphism)
creates or disturbs a specific
site for RE, after restriction,
fragments with different sizes
are formed
RFLP
Restriction Fragment Length Polymorphism
• Restriction endonucleases (RE)
– known about 2100 bacterial RE
– RE recognize variously short nucleotides sequences (4,6,8), in which
then they digest covalent phosphodiester bonds
• Principle of the analysis
– starting DNA (genomic DNA, PCR product)
– digestion with a restriction enzyme into fragments with different sizes
– fragments electrophoresis separation
Sequence of C282Y mutation
(hemochromatosis)
5´- TGGCAAGGGTAAACAGATCCctctcctcatccttcctctttcctgtcaagtgccctcctttggtgaaggtgacacatcatgtgacctcttca
gtgaccacactacggtgtcgggccttgaactactacccccagaacatcaccatgaagtggctgaaggataagcagccaatggatgccaaggagttcgaac
GCcaggtgg
ctaaagacgtattgcccaatggggatgggacctaccagggctggataaccttggctGTACcccctggggaagagcagagatatacGT
agcacccaggcctggatcagcccctcattgtgatctggggtatgtgactgatgagagccaggagctgagaaaatctattgggGGTTGAGAGGAGTGC
CTGAGgaggtaattatggcagtgagatgaggatctgctctttgttagggggtgggctgagggtggcaatcaaaggctttaacttgctttttctgttttagagccctca
ccgtctggcaccctagtcattggagtcatcagtgga – 3´
Primers
restriction endonucleasis RsaI restriction site (GTAC)
mutation C282Y (GTGC → GTAC)
RFLP – mutation C282Y - ELFO
RFLP – mutation C282Y+H63D
(hemochromatosis)
• MIX – per 1 sample
• 17 ul H2O
• 2 ul buffer
• 10 ul PCR product
• 1 ul restriction endonuclease (add on ice)
C282Y: restriction endonuclease RsaI
H63D: restriction endonuclease BclI
Evaluation of the results of the RFLP analysis
Mutation C282Y (v bp)
Healthy homozygote :
250 140
Homozygote with mutation : 250
Heterozygote:
250 140
111
111
Evaluation of the results of the RFLP analysis
Mutation H63D (v bp)
Healthy homozygote:
Homozygote with mutation:
Heterozygote:
208
208
138
70
138
70
Detection of ΔF508 mutation
in CFTR gene
This technique depends on the specificity of PCR primers
ASO-PCR:
3 primers are made:
General primer (G)
Specific primer to normal sequence (N)
Specific primer to mutated sequence (M)
TARGET SEQUENCE
G
X
N
M
PCR reaction is performed in 2 tubes:
PCR mix M0 contains primer G and primer N
PCR mix M1 contains primer G and primer M
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
PCR – deltaF508 (cystic fibrosis)
PCR mix 0:
• water
• buffer
• dNTP mix
• MgCl2
• primers M0
• DNA
8,5 µl
2,0 µl
4,0 µl
2,0 µl
1,2 µl
2,0 µl
• Taq polymerase0,4 µl
(on ice)
PCR mix 1:
• water
• buffer
• dNTP mix
• MgCl2
• primers M1
• DNA
8,5 µl
2,0 µl
4,0 µl
2,0 µl
1,2 µl
2,0 µl
• Taq polymerase0,4 µl
(on ice)
Evaluation of the results of the CFTR gene analysis
Mutation ΔF508 detection contingent upon the cystic fibrosis
Allele specific PCR + gel electrophoresis
M0 – PCR reaction detects non-mutated allele
M1 – PCR reaction detects mutated allele
M0 M1
M0 M1
M0 M1 M0 M1
M0 M1
PCR product for control gene
PCR product for CFTR gene (ΔF508)
Unutilized PCR chemicals
(may not always be visible)
healthy
homozygote
heterozygote
homozygote
with mutation