Download Sickle Cell Anemia: Tracking an Inherited Trait

Sickle Cell Anemia:
Tracking an
Inherited Trait
What is Sickle Cell Disease?
• Sickle cell disease refers to a group of inherited red blood cell
disorders.
• It is the most common genetic disease in the U.S.
– Approximately 70,000 to 80,0000 Americans have sickle cell disease
• It affects people of many nationalities including Italians,
Latin Americans, Greeks, Arabs, Asiatic Indians.
• Symptoms include: pain, fatigue, shortness of breath,
dizziness and many others.
Sickle Cell Anemia affects Hemoglobin, which
transports oxygen from the lungs to tissues
throughout the body
-Hemoglobin is a
soluble protein
contained in red
blood cells
-It consists of two
alpha subunit and
two beta subunits
-Each subunit binds
a molecule of oxygen
(O2)
Low Oxygen Levels Cause Sickle Hemoglobin (S)
to Polymerize Into Long Fibers
Hemoglobin A (normal)
under low oxygen levels
•Under low oxygen levels, normal (A)
hemoglobin is very soluble and does
not form polymers.
Hemoglobin S (sickle)
under low oxygen levels
•Form insoluble polymers making the red
blood cells more fragile than normal ones.
•Thus, they hemolyze which leads to severe
anemia and leads to sickle cell crises.
When Hemoglobin Polymerizes, the Red Blood
Cells Deform and Take on the Characteristic
Sickle Shape
• Normal red blood cells are round like doughnuts and move
through blood vessels easily to deliver oxygen to the tissues.
They live for about 120 days in circulation.
Normal
Sickle
Sickle red blood cells are
rigid and sticky. They live for only
10-30 days in circulation.
-When they try to go through small
blood vessels, they get stuck and
break apart. This causes pain and a
low red blood cell count, or anemia.
Clinical Features Of Sickle Cell Anemia
Blood vessels
Sickle cells
Normal cells
Blood flow is disrupted by the sickle cells and this leads to hemorrhages, infarctions –
tissue death caused by lack of oxygen.
Pain episodes result from these events.
There are periods of crisis during which the symptoms are worsened. These episodes
can be brought on by infection, dehydration, high altitude, or overexertion.
Sickle Cell Anemia is an Autosomal
Recessive Disease
AUTOSOMAL RECESSIVE means
that the gene carrying the mutation is
located on one of the autosomes
(chromosome pairs 1 through 22)
Beta Hemoglobin gene is on
chromosome 11
Therefore, males and females are
equally affected.
“Recessive” means that both copies
of the gene must have the mutation in
order for the person to have the
disease.
Punnett Square For Offspring of Two
Heterozygous Parents
A
A
S
AA
AS
S
AS
25% chance that offspring will be
AA: homozygous for normal gene (individual is
unaffected)
SS
50% chance that offspring will be
AS: heterozygous (trait); (individual is a sickle
cell carrier)
A: normal beta globin allele
S: sickle beta globin allele
25% chance that offspring will be
SS: homozygous for defective gene (individual
has sickle cell disease)
Central Dogma of Molecular Biology
DNA
DNA is composed
of nucleotides.
DNA is transcribed into RNA
RNA
RNA is composed
of nucleotides.
RNA is translated into protein
Protein
Proteins are composed
of amino acids.
Sickle cell disease is caused by a single base
mutation in the gene that codes for the beta
globin subunit of hemoglobin
Amino
Acid
Position
1
DNA
2
3
4
RNA
5
6
7
Protein
Because Sickle Cell Anemia is a
genetic disease, we can study the
disease at the level of the gene
and the protein.
At the DNA Level, We Can Analyze DNA Sequences by
Digesting DNA with Restriction Enzymes
Restriction enzymes are proteins that cut DNA at specific nucleotide
sequences.
The restriction enzyme Bsu36I cuts DNA with the sequence
CC^TGAGG.
CCTGAGG
Incubate with Bsu36I at 37C
-CC
TGAGG-
Digestion of beta globin DNA with Bsu36I
Normal beta globin gene
(531 base pairs)
CCTGAGG
Incubate
with Bsu36I
Sickle beta globin gene
(531 base pairs)
CCTGTGG
Incubate
with Bsu36I
+
(331 base pairs)
(200 base pairs)
(531 base pairs)
X
Where do you think restriction
enzymes come from?
Now we can use an agarose gel to
analyze cut DNA fragments.
So..
What is gel electrophoresis?
Analysis of Hemoglobin DNA by Gel Electrophoresis
after Bsu36I digestion
_
DNA
ladder
AA
uncut
AA
cut
SS
uncut
SS
cut
AS
uncut
AS
cut
1000 bp
AA: homozygous for normal gene
AS: heterozygous (trait)
500 bp
SS: homozygous for sickle gene
+
Today’s Laboratory Experiment
• Determine the genotype of 3 DNA
samples (labelled X, Y, and Z). That is,
are they AA, AS, or SS?