Based on “Hemoglobin, the Oxygen Carrier” Fundamentals of Biochemistry and “Sickle Cell Anemia: A Fictional Reconstruction” by Debra Stamper (National Center for Case Study Teaching in Science)

The Patient:

A 10-year old black male child named Michael Jones was admitted to the hospital because he was experiencing severe chest pain. He had been hospitalized on several previous occasions for vaso-occlusive episodes that caused him to experience severe pain that could not be managed with non-prescription drugs such as ibuprofen. He was slightly jaundiced, short of breath and easily tired, and feverish. A chest x-ray was taken and was abnormal. An arterial blood sample showed a PO2 value of 6 kPa (normal is 10 – 13 kPa).

As Michael’s blood sample was waiting for processing a lab technician noticed that the red blood cells at the bottom of the tube had a strange, elongated shape. When the technician mixed the blood the red blood cells returned to their normal shape.

A portion of Michael’s blood was smeared onto a microscope slide and the following picture was obtained:

Furthermore, the cells were lysed, run on a non-denaturing (ie. simple) electrophoresis gel to separate the proteins, and stained for hemoglobin. Lanes 1 – 3 are standards, lane 4 is from Michael’s blood, and lane 5 is a reference sample of “normal” blood. The following gel was obtained:

Question 1:

What is the most likely diagnosis for Michael?

A. Carbon monoxide poisoning

B. Dietary anemia (low intake of iron)

C. Pernicious anemia (due to a vitamin B12 deficiency)

D. Sickle cell anemia

Question 2:

Why does the hemoglobin from Michael’s blood not run as far on the electrophoresis gel as the normal sample?

A. Sickle hemoglobin (HbS) is a longer protein than normal hemoglobin (HbA) so does not travel as far in the gel.

B. HbS aggregate together and since this was a non-denaturing gel there are multiple copies of HbS trying to run together through the gel.

C. HbS has been cleaved as compared to HbA so runs differently.

D. HbS has a different overall net charge than HbA, which causes it to run differently through the gel.

Question 3:

What is the best explanation for the observation of abnormally shaped red blood cells at the bottom of Michael’s blood sample that appear to correct themselves when the sample was mixed?

A. The sickle shape is more pronounced when the hemoglobin is in the T state, which would be more likely at the bottom of the tube due to lower oxygen availability.

B. The sickle shape is more pronounced when the hemoglobin is in the R state, which would be more likely at the bottom of the tube due to lower oxygen availability.

C. The sickle cells are more dense than normal shaped cells so they settle to the bottom of the tube.

D. The lab technician was seeing things, there were no differences in the cells at the bottom of the tube.

Question 4:

In the gel, which lane would look the most similar to what would be obtained if Michael’s parents had their blood sampled?

A. Lane 1

B. Lane 2

C. Lane 3

The doctors treated Michael in part by administering 100% oxygen (normal air is ~20% oxygen), which alleviated some of his symptoms.

Question 5:

Why would 100% oxygen be an effective treatment for Michael?

A. HbS can only bind 1 oxygen per Hb molecule so more oxygen is required to allow for effective transport to the rest of the body.

B. HbS has a lower affinity for oxygen so more oxygen is required to get HbS fully saturated with oxygen.

C. High oxygen will activate the release of BPG to result in more oxygen being delivered to the tissues.

D. High oxygen will cause other oxygen carriers to be activated.

A medical student shadowing the case speaks up about a new drug treatment for sickle cell anemia that they had read about. The drug is hydroxyurea, and is thought to function by stimulating the affected person’s synthesis of fetal hemoglobin (HbF). HbF is composed of two α chains and two g chains and is expressed in a developing fetus beginning at the third month of gestation and continues up through birth when the expression of the g chain, and therefore HbF, declines. In a clinical study, patients who took hydroxyurea showed a 50% reduction in frequency of hospital admissions for severe pain and abnormal chest x-rays.

Question 6:

Why would increasing the synthesis of HbF alleviate the symptoms of sickle cell anemia?

A. HbF has a higher affinity for oxygen than HbS so is a more effective oxygen transporter.

B. HbF is composed of α and g chains and the mutation that causes sickle cell anemia is on the β

chain so HbF would not be affected.

C. Additional Hb, of any type, in the blood would increase the ability to transport oxygen.

D. All of the above contribute to the alleviation of symptoms.

The mutation that causes sickle cell anemia is a single nucleotide polymorphism (SNP) where only one base differs in the DNA sequence between HbS and HbA. That one nucleotide difference however causes a change in the amino acid sequence where a Valine is incorporated in HbS while a Glutamate is incorporated in HbA.

Question 7:

What class of amino acid is valine?

A. Non polar

B. Aromatic

C. Polar, uncharged

D. Negatively charged

E. Positively charged

Question 8:

What class of amino acid is glutamate?

A. Non polar

B. Aromatic

C. Polar, uncharged

D. Negatively charged

E. Positively charged

Question 9:

Why would a Glu à Val mutation cause so many problems in the functioning of the protein?

A. Changes the covalent peptide bonds of the protein, thus changing the folding pattern.

B. Changes the non-covalent interactions between amino acid side chains, thus changing the

folding pattern.

C. Changes the amount of the protein that is synthesized.

D. Only changes protein function if the mutation is at a critical location, such as the oxygen binding

site of Hb.

The doctors involved in Michael’s case get involved in a conversation with a colleague at a conference shortly after Michael was discharged from the hospital. The colleague tells them that she had “cured” a patient of sickle cell anemia by performing a bone marrow transplant.

Question 10:

Would a bone marrow transplant “cure” sickle cell anemia?

A. Yes, the transplanted bone marrow cells would produce red blood cells with normal HbA so the

patient would not express HbS or sickle cell anemia symptoms.

B. No, the patient would still express the mutated gene even if they had no symptoms.

C. No, the patient would still pass the HbS gene on to any offspring so they are not “cured”.

D. All of these statements are correct, it depends on your definition of “cured”.