Sickle Cell Disease

Question 1

What is a thalassemia?

Answer 1

Inherited autosomal recessive blood disorder characterized by abnormal formation of hemoglobin

Question 2

What does it mean to have sickle cell trait?

Answer 2

Means you are an heterozygote and carry the mutant ß globin gene

Question 3

What is the genetic explanation of SCD? What does this cause?

Answer 3

Glu6Val missense mutation in the β chain → disrupts secondary structure → hydrophobic interaction with AAs on the interface with other subunit to hide Val from solvent when it T-state → new shape reduces deoxyhemoglobin solubility → T-state aggregation → Hb forms long inflexible chains: sickled RBCs

Question 4

What are the 2 ways of detecting SCD?

Answer 4

1. Electrophoretic analysis of HbS

2. Screening for the hbS mutation

Question 5

Describe how electrophoretic analysis of HbS works.

Answer 5

The substitution of the negatively charged Glu with the neutral Val alters the electrical
property of the hemoglobin molecule such that it migrates more slowly toward the anode when placed in an electric field.

Question 6

Does the electrophoretic analysis of HbS correspond to SDS page?

Answer 6

No! The protein is in its globular form

Question 7

Describe how genetic screening of HbS works.

Answer 7

Used to be done with Southern blot, but now we use PCR w/ digestion of the DNA by restriction enzymes followed by gel electrophoresis

Question 8

What are restriction enzymes? What are the ones used in SCD detection?

Answer 8

They are nucleases that recognize specific sequences.

The mutation in the ß globin gene changes the recognition site for particular restriction enzymes

Question 9

How can you interpret the result of PCR for SCD detection?

Answer 9

• 3 smaller fragments: HbA

- 2 larger fragment: HbS

Question 10

What is the lifespan of a normal RBC vs sickled?

Answer 10

90-120 days vs 10-20 days

Question 11

What brings T-state tetramers together? What does this produce?

Answer 11

Hydrophobic interactions that produce HbS strands: 10/4 fiber (10 strands around a core of 4 strands)

Question 12

Explain the 2 step process of HbS aggregation and fiber formation

Answer 12

1. The T-state HbS reversibly associate w/ each other
2. A critical nucleation mass is obtained, which nucleates the formation of other fibers, which in turn nucleates the formation of other fibers: this is irreversible.

Question 13

What is the molecular threshold for sickle cell crisis?

Answer 13

The second step of HbS aggregation and fiber formation

Question 14

What does the first step of HbS aggregation and fiber formation depend on? What does this mean?

Answer 14

Concentration dependent: decrease in T-state HbS = longer time for the step 1 to occur (could reach the lungs in time to avoid a crisis)

Question 15

Explain the biochemical rationale for hydroxyurea use

Answer 15

The chemotherapeutic agent 5-azacytidine increases HbF by altering DNA methylation –> lowers HbS levels and prevents aggregations by preventing the mucleation mass from being reached

Question 16

What do butyrates do to SCD patients?

Answer 16

They increase HbF levels and work synergistically with hydroxyurea

Question 17

What is an additional benefit of taking hydroxyurea?

Answer 17

It produces nitric oxide, which acts as a potent vasodilator to:

1. Prevent vaso-occulsion
2. Prevent Hydroxyurea’s rxn with T-HbS to form metHbS
3. Reacts with hydroxyurea to form HbSNO, which delays the onset of aggregation

Question 18

Explain the biochemical rationale for cyanate use

Answer 18

It reacts with the N-termini of the alpha units, which are then unable to bind H+/CO2 (salt bridges and carbamate formation would stabilize the T-state)

Question 19

Is cyanate still used to treat SCD?

Answer 19

No

Question 20

Explain the biochemical rationale for hydration therapy use

Answer 20

Increase RBC volume to decrease HbS concentration

Question 21

What can SCD patients do to avoid an SCD crisis?

Answer 21

Avoidance of situations that stabilize the T-state:
- Elevated temperatures
- Low O2 levels (eg: high altitude)
- Rapid temperature changes
Others:
-Dehydration: increases RBC concentration
- Very low temperatures (causes vasoconstriction)
- Stress

Question 22

What happens to the 2,3-BPG concentration in response to anemia associated with SCD? Why? What is the result?

Answer 22

Concentration increases considerable to promote better delivery of O2 to the tissues
This complicates the situation because the higher levels stabilize the T-state

Question 23

Would trying to decrease the concentration of 2,3-BPG in SCD patients be a good way to help them in theory?

Answer 23

Yes, but has not been tested

Question 24

What are the 7 different genotypes of SCD?

Answer 24

1. HbSS (the most common form): homozygote for the S globin with usually a severe or moderately severe phenotype and with the shortest survival. It is typically associated with vascular obstruction and ischemia.
2. HbSA: heterozygote, is also known as sickle cell trait. Individuals with sickle cell trait are usually healthy, however if the individual’s parent also has another abnormal hemoglobin gene (thalassemia, hemoglobin C, hemoglobin D, hemoglobin E) they are likely to have SCD.
3. HbSc - intermediate
4. HbSB0 - Severe
5. HbSB+ - milder
6. HbS+F -asymptomatic
7. HbSE - Mild

Question 25

What is ischemia?

Answer 25

Insufficient blood supply to the organs

Question 26

What are some major complications of SCD?

Answer 26

• Damage to organs because of O2 deprivation
• Stroke
• Lack of blood in lungs: acute chest syndrome, COPD, pulmonary embolism
• Jaundice: sickle cells do not live as long as normal red blood cells and, therefore, they are dying faster than the liver can filter them out. Bilirubin (which causes the yellow color) from these broken down cells builds up in the system causing jaundice.
• Blindness
• Priapism: painful erection that can lead to impotence
• By the age of 5: asplenic: no spleen
• Susceptibility to develop bacterial infections because the spleen is not protecting them

Question 27

Why are SCD patients given prophylactic antibiotics (penicillin)?

Answer 27

To prevent bacterial infections. They are more susceptible to these because of the lack of spleen (or decreased performance of the spleen)

Question 28

What is the difference between acute and chronic transfusion therapies?

Answer 28

Acute: for episodic events or a needed medical intervention
Chronic: may be needed if complications due to SCD have occurred

Question 29

What is the difference between simple and exchange transfusions?

Answer 29

• Simple: transfusing healthy RBCs
• Exchange: removing and replacing the patient’s BV by pheresis to reduce the concentration of HbS without increasing the hematocrit

Question 30

What is a negative side effect of transfusions?

Answer 30

Iron overload in vital organs which can be very dangerous

Question 31

What is hypoxia?

Answer 31

Deficiency in the amount of oxygen reaching the tissues

Question 32

Why do heterozygous patients have a lower rate of fiber formation and precipitation?

Answer 32

Because they have a lower concentration of HbS in their blood

Question 33

Is decreasing erythrocyte volume to ease passage through capillaries a potential treatment for SCD patients?

Answer 33

NOPE

Question 34

Does the missense mutation in SCD patients affect Hb’s affinity for H+?

Answer 34

NOPE

Question 35

Does HbS polymerization dependent on how long the HbS molecules are in the T-state?

Answer 35

Yes!

Question 36

Does HbS polymerization dependent on how long the HbS molecules are in the R-state?

Answer 36

NOPE

Question 37

What happens when the conditions are right for sickling to occur?

Answer 37

There is a lag between the conditions and the onset

Question 38

Does HbS in the R-state have a lower affinity for O2 than HbA in the R-state?

Answer 38

NOPE