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

< Link to animations about the disease.

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).