Cystic fibrosis is the most common autosomal recessive disease among people of Northern European descent, with an average life expectancy of 30 years. It is caused by mutations in the CFTR gene, which codes for a chloride channel protein. The most common mutation, ΔF508, causes a processing defect that results in reduced chloride transport. Over 1000 mutations in CFTR have been identified. While treatments aim to address symptoms, gene therapy may someday deliver a corrected version of the gene.

1. 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

2. 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 CF
tests for every newborn
(in order to treat it earlier).
…before Jan.1, 2006 Kentucky
only tested for 5 inherited
diseases….now 29 !

3. 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

4. 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

5. 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).

6. Life expectancy has risen from a few years
to 30 years because of RESEARCH
Anyone know why
the rose is associated
With this disease?

7. 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?

8. In the old days,
doctors used to lick
newborn babies
(to see whether they
had salty skin)
Now they apply Pilocarpene
to 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)

9. 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

10. 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?

11. 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

12. How was this gene cloned? RFLP Analysis
….they looked at 330 different RFLPs to find these that
correlated with Cystic Fibrosis

14. Perhaps this larger band
is correlated with being a
“carrier”

15. http://www.mun.ca/biology/scarr/Somatic_Cell_Hybridization.htm
Somatic
Cell
Hybridization
pp. 131-136

16. 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

17. Somatic Cell Hybrids (human #3, #5, and #17)

18. With correct staining you
can distinguish Rodent
chromosomes from Human
ones, and even observe
translocation events.

19. 6 T 1 6 1T
Translocation
Exchange of
chromosome parts
Translocation Normal Normal Translocation

20. From page 136 here…
Page 136 (your book)
Which DNA region consistently
hybridizes?

21. From page 136 here…
Page 136 (your book)
Which DNA region consistently
hybridizes?
Region C

22. | | 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

23. Lanes A, B, C, D, E has DNA from the people
used to make the hybrid lines

24. Which human chromosome appears to consistently hybridize
with the TCRB and DOCRI-917 probes?

26. Which human chromosome appears to consistently hybridize
with the TCRB and DOCRI-917 probes?

27. Then used this probe (and others like it)
to screen Genomic Libraries and
cDNA Libraries to isolate the gene.

29. Normal

30. Normal Mutation Normal Mutation

31. Normal

32. < Link to animations about
the disease.

38. Truncation
Processing NBD & R
mutation
Hydrophobic
Regions
mRNA
stability
R.B. Moss, New approaches to cystic fibrosis, 2001

39. Truncation
Processing NBD & R
mutation
Hydrophobic
Regions
mRNA
stability
R.B. Moss, New approaches to cystic fibrosis, 2001
What
functional
part of
the protein
is F508?

41. The environment also affects
the severity & symptomology
of this disease:
• Smoking
• Second-hand Smoke
• Diet
• Pseudomonas exposure
• Income (more severe in low-income patients)

42. 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!

43. 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).