1. Sickle Cell Anemia: Tracking an Inherited Trait

2. 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.

3. 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)

4. under low oxygen levels under low oxygen levels
Low Oxygen Levels Cause Sickle Hemoglobin (S) to Polymerize Into Long Fibers
Hemoglobin A (normal)
under low oxygen levels
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.
Under low oxygen levels, normal (A) hemoglobin is very soluble and does not form polymers.

5. 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.
Sickle red blood cells are rigid and sticky. They live for only 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.
Normal
Sickle

6. 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.

7. 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.

8. Punnett Square For Offspring of Two Heterozygous ParentsAA SS AS
25% chance that offspring will be
AA: homozygous for normal gene (individual is unaffected)
50% chance that offspring will be
AS: heterozygous (trait); (individual is a sickle cell carrier)
SS: homozygous for defective gene (individual has sickle cell disease)
A: normal beta globin allele
S: sickle beta globin allele

9. Central Dogma of Molecular Biology
DNA
RNA
Protein
DNA is composed of nucleotides.
DNA is transcribed into RNA
RNA is composed of nucleotides.
RNA is translated into protein
Proteins are composed of amino acids.

10. 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 2 3 4 5 6 7
DNA
RNA
Protein

11. Because Sickle Cell Anemia is a genetic disease, we can study the disease at the level of the gene and the protein.

12. 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-

13. Digestion of beta globin DNA with Bsu36I
Normal beta globin gene
(531 base pairs)
Sickle beta globin gene
(531 base pairs)
CCTGAGG
CCTGTGG
Incubate with Bsu36I
Incubate with Bsu36I X +
(331 base pairs)
(200 base pairs)
(531 base pairs)

14. Where do you think restriction enzymes come from?

15. Now we can use an agarose gel to analyze cut DNA fragments. So
Now we can use an agarose gel to analyze cut DNA fragments. So.. What is gel electrophoresis?

16. 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)
SS: homozygous for sickle gene
500 bp +

17. Today’s Laboratory Experiment
Determine the genotype of 3 DNA samples (labelled X, Y, and Z). That is, are they AA, AS, or SS?