Cystic Fibrosis pp. 303-305 CF is the most common autosomal-recessive disease
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Cystic Fibrosis pp. 303-305 • CF is the most common autosomal-recessive disease among people of Northern European descent • Average life expectancy ≈ 30 years • 1 of every 3200 births in the U.S. has the disease • 1 of every 31 Americans is a carrier • When 2 carriers have children = 25% chance of the disease 1 / 31 x 1 / 31 = 1 / 961 x ¼ = 1 / 3844
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Cystic Fibrosis pp. 303-305 CF is the most common autosomal-recessive disease among people of Northern European descent Average life expectancy ≈ 30 years 1 of every 3200 births in the U.S. has the disease 1 of every 31 Americans is a carrier - PowerPoint PPT Presentation
Transcript of Cystic Fibrosis pp. 303-305 CF is the most common autosomal-recessive disease
Cystic Fibrosis pp. 303-305
• CF is the most common autosomal-recessive disease among people of Northern European descent
• Average life expectancy ≈ 30 years
• 1 of every 3200 births in the U.S. has the disease
• 1 of every 31 Americans is a carrier
• When 2 carriers have children = 25% chance of the disease
1/31 x 1/31 = 1/961 x ¼ = 1/3844
Cystic Fibrosis pp. 303-305
First shows up in babies as:
• Persistent diarrhea• Frequent pneumonia• Chronic coughing• Salty skin• Poor growth
http://www.cff.org/publications/videos_by_request/#Faces_of_CF
More states are now requiring CFtests for every newborn
(in order to treat it earlier).
…before Jan.1, 2006 Kentucky only tested for 5 inherited
diseases….now 29 !
Cystic Fibrosis
Average Life Expectancy ≈ 30 years
1. Chronic Pulmonary Disease (lungs) ….lots of lung infections…breathing difficulty
2. Pancreatic Insufficiency (lack of digestive enzymes secreted by pancreas into intestine ….intestinal blockage)
3. Lots of chloride in sweat (not dangerous per se)
4. Male Sterility
Cystic Fibrosis
Third gene to be cloned by “Positional Cloning”(other two: Chronic Granulomatous & Muscular Dystrophy)
1989 - Toronto, Canada
At a cost of $200 million (US)
Gene is huge = 190,000 bp (24 exons)
Codes for a Transmembrane Chloride Transporter(CFTR = CF Transmembrane Conductance Regulator)
About 1000 known mutations, but 70% of the cases involve a single type, called F508
Cystic Fibrosis
Codes for a Transmembrane Chloride Transporter
• Water transport follows chloride transport so this mutation interferes with mucous secretions (water dilutes mucous).
• In Homozygous Recessives = total Loss of Function. But there are lots of interactions with other genes & the
environment.
• Up until the 1940s, death typically occurred in first few years.
• In Heterozygotes, it might provide a selective advantage where high rates of infantile diarrhea is common (Europe).
Life expectancy has risen from a few years to 30 years because of RESEARCHRESEARCH
Anyone know whythe rose is associated
With this disease?
The quickest and cheapest way to test for CF is the
“Sweat Test”
…collect perspiration & test for high chloride levels
How was this gene cloned?
In the old days, doctors used to lick
newborn babies(to see whether they
had salty skin)
Now they apply Pilocarpeneto the skin – wait 30 minutes- and measure chloride levels.
Jaborandi (Pilocarpus pennatifolius)
Native to Brazil….made the native Amazonians “jabber”
Also, commonly used to treat glaucoma (high intraocular pressure)
How was this gene cloned?
Pedigree Analysis…its more difficult with recessive alleles !…and when it causes sterility !…and when it is lethal !
Needs to be done hand-in-hand with molecular techniques
1) Which RFLP Marker is most closely linked to the mutation causing this disease?
2) Can you see where a ‘cross-over’ event has occurred?
E+ E- E- E-
B- B+ B- B- H+ H- X H- H+
S+ S+ S- S-
CF CF Carrier Carrier
E+ E- E- E- E- E- B- B- B+ B- B+ B- H+ H+ H- H- H+ H- S+ S- S+ S- S+ S-
Cystic Healthy CF Fibrosis Carrier
How was this gene cloned?
1) Which RFLP Marker is most closely linked to the mutation causing this disease?
2) Can you see where a ‘cross-over’ event has occurred?
E+ E- E- E-
B- B+ B- B- H+ H- X H- H+
S+ S+ S- S-
CF CF Carrier Carrier
E+ E- E- E- E- E- B- B- B+ B- B+ B- H+ H+ H- H- H+ H- S+ S- S+ S- S+ S-
Cystic Healthy CF Fibrosis Carrier
How was this gene cloned?
The HindIII site
How was this gene cloned? RFLP Analysis
….they looked at 330 different RFLPs to find these that correlated with Cystic Fibrosis
Perhaps this larger bandis correlated with being a
“carrier”
http://www.mun.ca/biology/scarr/Somatic_Cell_Hybridization.htm
SomaticCell
Hybridization
pp. 131-136
Somatic Cell Hybridization
“Somatic cell hybrids are culture lines that contain the entire complement of the mouse genome and a few human chromosomes. These culture lines are developed by mixing human and rodent cells in the presence of the Sendai virus. The virus facilitates the fusing of the two cell types to form a hybrid cell. For a reason that is not entirely known, most, but not all, human chromosomes are lost from the hybrid cell lines. Usually a few human chromosomes are retained. Because the human and rodent chromosomes can be distinguished by chromosome staining techniques, it can be determined which human cells are retained with a specific cell line.”Phillip McClean. 1997. North Dakota State University
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/genomic/genomic3.htm
Somatic Cell Hybrids (human #3, #5, and #17)
With correct staining you can distinguish Rodent
chromosomes from Human ones, and even observe
translocation events.
6 T 1 6 1T
Translocation
Exchange ofchromosome parts
Translocation Normal Normal Translocation
From page 136 here…
Page 136 (your book) Which DNA region consistentlyhybridizes?
From page 136 here…
Page 136 (your book) Which DNA region consistentlyhybridizes?
Region C
| | 3.6 | 5.0 | 5.8 | 8.9 | | | E E E E E H* E E E
Probe for Southern Blotting for CF
Another group of researchers used this probe…
This HindIII site correlates with CF
Lanes A, B, C, D, E has DNA from the people used to make the hybrid lines
Which human chromosome appears to consistently hybridizewith the TCRB and DOCRI-917 probes?
Which human chromosome appears to consistently hybridizewith the TCRB and DOCRI-917 probes?
Then used this probe (and others like it) to screen Genomic Libraries and
cDNA Libraries to isolate the gene.
Normal
Normal Mutation Normal Mutation
Normal
Truncation
Processing NBD & Rmutation Hydrophobic
Regions
mRNAstability
R.B. Moss, New approaches to cystic fibrosis, 2001
Truncation
Processing NBD & Rmutation Hydrophobic
Regions
mRNAstability
R.B. Moss, New approaches to cystic fibrosis, 2001
What functional part of the protein is F508?
The environment also affects the severity & symptomology
of this disease:
• Smoking• Second-hand Smoke
• Diet• Pseudomonas exposure
• Income (more severe in low-income patients)
Conventional Therapy:
• Antibiotics • Inhalers to reduce mucus blockage in lungs• Medicines to reduce inflammation in lungs (e.g. corticosteroids, ibuprofen)• Pancreatic enzymes• Vitamins• Extra calories
Average 20 medications
per day!
Gene Therapy ?
Deliver a correct version of the gene to the affected tissue using a vector.
• Retroviruses = RNA viruses that integrate their code into the genome of host.
• Adenoviruses = DNA viruses that remain in the nucleus of the host as a separate entity (transcription of their genes occurs).
