Understanding Cystic Fibrosis

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Understanding Cystic Fibrosis as a genetic
disease
DNA Day 2009
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DNA Day 2009
2003 Human Genome Sequence Completed
1953 DNA Structure
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DNA

• Who?
• What?
• Where?
• When?
• Why?
• How?

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What is DNA?
"It's a history book - a narrative of the journey
of our species through time. It's a shop
manual, with an incredibly detailed blueprint for
building every human cell. And it's a
transformative textbook of medicine, with
insights that will give health care providers
immense new powers to treat, prevent and cure
disease."
-Francis Collins
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DNA
Cystic Fibrosis
?
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All living things contain DNA
DNA
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Where is DNA found?
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DNA is arranged in chromosomes that are found in
the cell nucleus.
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What is DNA made of?
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DNA is made of

• A sugar/phosphate backbone
• A nucleotide ladder which is comprised of
• A T C G

The nucleotide sequence is very important
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So, what is the difference between DNA and a gene?
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A gene is a unit of information that is encoded
in DNA based on the nucleotide sequence.
Gene
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The information in a gene is used to make a
proteins, which are the building blocks of
cellular machinery
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DNA contains all of the information necessary to
make a complete organism.
Central Dogma
DNA
Gene segment of DNA that tells the cell how to
make a certain protein.
transcription
RNA
Proteins work together to form the functional
machinery that makes up a cell.
translation
Protein
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DNA
How can knowing about DNA be used to treat a
disease, such as Cystic Fibrosis?
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Cystic Fibrosis is a genetic disorder which
affects the lungs and other organs
Genetic disorder Def A disease that is caused by
an abnormality in an individuals DNA.

• There are about 4000 known genetic disorders.
• The inheritance pattern of CF is autosomal
  recessive, which means
• A person must have 2 mutatated copies of the
  gene in order to have the disease, and
• One good copy of the gene will protect from the
  bad copy of the gene.

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Cystic Fibrosis A Brief History before genetics
In the Middle Ages Woe to that child which
kissed on the forehead tastes salty. He is
bewitched and will soon die.
1930s The disease was described by a physician,
Dr. Dorothy Andersen
1940s Pedigree analysis of families with CF
established that it was inherited.
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Symptoms of Cystic Fibrosis

• Increased salt in the sweat
• Thick mucus in airways and lungs
• Chronic Lung infections
• Inflammatory cells can cause permanent lung-cell
  damage.
• Digestive problems
• Lack of nutrient absorption by the pancreas

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In order to get a better understanding of CF,
lets focus on the lungs
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The lungs are lined with specialized epithelial
cells.
Cilia
Out to mouth
Dust
Dirt
Dust
Mucus gland
Mucus gland
Dust
Mucus
Mucociliary Escalator
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Mucociliary Escalator
Cilia
Mucous
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Cystic Fibrosis patients airways are dehydrated
and cannot clear mucus.
Healthy Airway
CF Airway
Normal mucus
Thick, dry mucus
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As a result, CF airways accumulate bacteria and
inflammatory cells.
Antibiotics are the most commonly used treatment
for CF.
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Is there a better way to treat Cystic Fibrosis?
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Remember what Francis Collins said about
DNA.. DNA will give health care providers
immense new powers to treat, prevent and cure
disease.
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1989 The Cystic Fibrosis Gene was Identified

• The gene mutated in CF is called the Cystic
  Fibrosis Transmembrane Conductance Regulator
  (CFTR)
• Scientists have identified 900 mutations in this
  gene!
• The most common mutation is the ?F508 mutation.

Chromosome 7
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How do mutations in the CFTR gene lead to thick
mucus and infections in the lung?
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Cystic Fibrosis Transmembrane Conductance
Regulator gene (CFTR)
Central Dogma
Chromosome 7
DNA
DNA
transcription
RNA
RNA
translation
CFTR protein
Protein
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The CFTR in Lung Epithelial Cells
Cilia
Cilia
the CFTR
cell nuclei
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CFTR functions in Lung Epithelial Cells
Lung Epithelial Cell
Chloride Ions
Cl-

Cl-
Cl-
Cl-

• Localized to the Plasma Membrane
• Functions as a Chloride Ion Channel

Cl-
Cl-
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In a healthy cell, the CFTR transports Chloride
Ions out of the cell
Remember, the cilia beat back and forth to move
dust and pathogens out of the airways..
Cl-
Dust
Cl-
Cl-
Cl-
Cl-
Dust
Healthy cell
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How is the CFTR protein related to hydrating the
airways?

• To answer this question, lets think about osmosis.

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Osmosis
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The same thing happens in your cells.
Cl-
Selectively permeable membrane
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Direction of water
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
High Cl- concentration within the cell
Low Cl- concentration within the cell
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So, why are the airways dehydrated in patients
with Cystic Fibrosis?
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Mutations in the CFTR gene blocks CFTR protein
function
Healthy cell
CFTR
Cystic fibrosis cell
Changes in the DNA sequence lead to a protein
that cannot reach the plasma membrane.
Mutant CFTR
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Review The CFTR protein is expressed on the
surface of lung epithelial cells.
Chloride Ions
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Healthy cell
CF cell
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Pseudomonas aeruginosa
Inflammatory Cell Infiltrates
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
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The Bronchoalveolar Lavage test allows
investigation of what is in the lung airways
The Bronchoscopy Procedure
Here is what a BALF looks like under the
microscope.
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Cystic Fibrosis Activity

• You be the investigator

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Investigate the BALF

• Do not open the tubes!!
• Analyze the contents of each tube for
• -Mucus consistency
• -Presence of inflammatory cells
• -Evidence of infection with Pseudomonas
  aeruginosa (bacterial cells)

A

• Record your findings on the worksheet provided.

inflammatory cell (macrophage)
bacterium (Pseudomonas)
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Who do think has Cystic Fibrosis?

• What would you do next to confirm the diagnosis?

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Sequence the DNA and look for mutations in the
cftr gene.

• There are 900 different mutations that have
  been found in the cftr gene.
• To save time, we have printed the DNA sequences
  from the sequencing reactions for you.

Remember the Central Dogma?
DNA RNA Protein
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How do mutations in the DNA sequence lead to
defective proteins?
DNA RNA Protein
DNA
AGG TCG GGT CAA CAA TGC
UCC AGC CCA GUU GUU ACG
RNA
Convert the DNA sequence into the RNA
intermediate.
DNA A T C G
RNA U A G C
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Now convert the mRNA message to a protein.
DNA RNA Protein
UCC AGC CCA GUU GUU ACG
mRNA
Amino acid sequence
Ser
Ser
Pro
Val
Val
Thr
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Which patient has the defective CFTR protein?
Now you know that mutations in the genes (or DNA)
can lead to changes in the protein.
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What would you do to treat Cystic Fibrosis?

• Use your imagination

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Gene Therapy for Cystic Fibrosis The Idea

• In theory, delivery of a wild type copy of the
  CFTR gene by using viral vectors would alleviate
  the disease..
• Major Obstacle the lung has evolved to be highly
  resistant to viral infection.

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Gene Therapy for Cystic Fibrosis Has Not Been
SuccessfulYET

• However, new developments in research are
  promising

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In September 2008 Scientists mutated the CFTR
gene in pigs!
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Hot off the Press!!!!! PNAS, March 10, 2009
Scientists discovered mutations in the
therapeutic virus which make it 10Xs more
infectious!
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A better gene therapy for CF?
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• How else could genetics help to treat Cystic
  Fibrosis?

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• Newborn Screening

• Prenatal carrier screening

• Used to determine whether potential parents are
  CF carriers

• Used for early detection of CF

• A recent study found that in the years following
  the introduction of optional prenatal screening,
  the number of newborns with CF declined.

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• Non gene based therapies have not been abandoned

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Surfing makes CF patients feel better Why?
This observation lead to the development of
saline mist therapies for CF.
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In the last 50 years, the life expectancy for a
person with Cystic Fibrosis has gone from 5 years
old to 36 years. Early diagnosis and improved
treatments are the reason people with CF can now
live 30 years longer.
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NC DNA DAY Activity Developers

• Ginnie Hench
• Jennifer James
• Anne White