Normal and Abnormal Hemoglobin Structure and Function

Question 1

Hemoglobin

1. Oxygen Affinity at [High] vs. [Low]
2. Binding Curve Shape
3. Factors favoring O2 dissociation
4. Factors favoring O2 binding

Answer 1

1. Hb has higher affinity for O2 when there is high oxygen present
2. Sigmoidal
3. Dissociation: favored by L__ow pH, High CO2, Low O2

and the presence of 2,3 BPG favors dissociation

4. Binding: High pH, Low CO2, High O2

Question 2

What subunits does Hb A₂ consist of?

Answer 2

two alpha and two delta subunits

(Delta are related to Beta, and are referred to as Beta-like Globins)

Question 3

What are the main hemoglobins found in the early embryo?

Answer 3

Hb Gower 1 (Z₂E₂), Hb Gower 2 (a₂E₂), and Hb Portland (Z₂y₂)

Question 4

What is the major hemoglobin type of the fetus?

Answer 4

HbF

Question 5

What is the main modification that allows HbF to have higher oxygen affinity than HbA?

Answer 5

In the gamma (y) chain, HbF contains a serine residue, which is less positive (than the histidine reside on the B chain in HbA) and therefore reduces the affinity of HbF for 2,3-BPG –> thus increasing the oxygen affinity of HbF

(The gamma (y) chain is also found in Hb Portland)

Question 6

What are the two globin chain familes, and which of the six globin chains falls into each? Which chromosome do each of the globin familes fall on?

Answer 6

alpha-like globins: alpha (a) and Z

***Chromosome 16***

Beta-like globins: Beta (B), gamma (y), Delta (D), and Epsilon (E)

***Chromosome 11***

Question 7

What are the two “obvious groups” of hemoglobinopathies?

Answer 7

1. Structural Variants
2. Thalassemias (i.e. imbalanced synthesis of alpha- and Beta-globin chains)

Question 8

What are the 3 categories of Structural Hemoglobin Variants?

Answer 8

1. Insoluble Complexes –> are oxidized to methemoglobin (Fe³⁺) and form hemichrome –> Heinz Bodies and hemolytic anemia
2. Increased/Decreased Oxygen Affinity
3. Increased Tendency to form Methemoglobin

Question 9

What rare Hb structural variant results in increased oxygen affinity?

Answer 9

Hb_Helsinki (Beta subunit mutation (Lys –> Met))

Question 10

What rare Hb structural variant results in decreased oxygen affinity?

Answer 10

Hb_{<strong>Kansas</strong>} (Beta subunit mutation (Asn –> Thr))

Question 11

What rare Hb structural variants more readily form methemoglobin?

Answer 11

Hb_{<strong>Boston</strong>} (alpha subunit mutation (distal His –> Tyr)

Hb_{Hyde Park} (Beta subunit mutation (proximal His –> Tyr)

Question 12

HbS

1. Disease
2. Mutation
3. Consequences
4. Hetero- vs. Homozygote
5. Treatment

Answer 12

Disease: Sickle Cell Disease

Mutation: Glutamate is replaced by valine at position 6 of the Beta-globin chain

Consequences: ***Deoxygenated HbS polymerizes*** –> distortion of shape of RBC, misshapen cells block microcirculation, cells lyse readily (Chronic Hemolytic Anemia)

Heterozygotes = Sickle Cell Trait

Homozygoets = Sickle Cell Disease

Treatment: Hydroxyurea (HU) (antineoplastic)—> increased HbF –> increased solubility of hemoglobin and decreased sickling of cells

***Reduced: sickling, painful crises, hospitalizations***

Question 13

HbC

1. Disease
2. Mutation
3. Consequences
4. Heterozygotes
5. Treatment

Answer 13

Disease: HbC Disease (restricted to West African Origin)

Mutation: Glutamate replaced by lysine at position 6 of the Beta-globin chain

Consequences: HbC does not polymerize and cells do not sickle

***HbC is less soluble than HbA and precipitates –> hemolytic anemia

Heterozygotes: Compound with both HbC and HbS is not uncommon

***HbSC is a MILDER disease than HbS***

Question 14

HbE

1. Disease
2. Mutation
3. Consequences
4. Hetero- vs. Homozygote
5. Treatment

Answer 14

Disease: HbE Disease (Common in Southeast Asia)

Mutation: Glutamate is replaced by lysine at position 26 of the Beta-globin chain

Consequences: Beta globin chain is not synthesized effectiely leading to imbalanced alpha and Beta-globin chain synthesis –> mild Thalassemia develops

Heterozygotes (HbE Trait) = Asymptomatic

Homozygotes (HbE Disease) = Microcytosis, Hypochromia, Mild Anemia

Question 15

What are the most common types of Thalassemias?

Answer 15

Question 16

alpha-Thalassemias

1. Genes
2. Types of Hb affected
3. Carrier States

Answer 16

Genes: 2 genes for a-globin on chromosome 16 (so 4 total copies)

Types of Hb affected: Embryonic (Hb Gower 2), Fetal, and Adult

***a-thalassemias manifest during development and adult life

Carrier States:

• 3 functional genes/1 defective –> No clinical signs
• 2 functional/2 defective –> Mild Thalassemic anemia
• 1 functional/3 defective –> Severe a-globin chain deficiency (Bart’s hemoglobin or HbH)
• 4 defective –> a-thalassemia –> only embryonic hemoglobins (Gower 1 and Portaland) can be produced –> Hemoglobin Bart’s Hydrops Fetalis _***Lethal Condition***_

Question 17

What is Bart’s hemoglobin?

Answer 17

When gamma (y)₄ tetramers form in fetus

-Poor oxygen carriers (due to High oxygen affinity)

Question 18

What is HbH?

Answer 18

When Beta (B)₄ tetramers form later in development

• Poor oxygen carriers (due to High oxygen affinity)
• Precipitates, shortening RBC life

Question 19

What is the most common molecular defect that gives rise to a-thalassemia?

Answer 19

Deletions

-Duplicated a-globin genes are found in a region of homology, and gene deletion appears to occur during homologous recombination events

Question 20

What happens when both a-globin genes are deleted?

Where is this most common?

Answer 20

Hemoglobin Bart’s Hydrops Fetalis Syndrome (a LETHAL condition)

Most common in Southeast Asia

Question 21

Hb_{Constant Spring} Mutation

1. Mutation Type
2. Results
3. Symptoms

Answer 21

Point Mutation

Results: T is replaced by C, so the normal TAA stop codon is bypassed, leading to a read through and increased a-globin chain length from 141 to 172

-Expected Hb_{constant spring} to comprise 2025% total Hb, however, only contributes 1-2%, as mRNA fro Hb_{constant spring} is unstalbe

Symptoms: like a⁺-thalassemia (moderate hemolytic anemia, splenomegaly) even though it has 2 of 4 functional globin genes and would be expected to only show mild thalassemic anemia

Question 22

Beta-thalassemias

1. Heterozygote vs Homozygote

Answer 22

***Only one gene for Beta-globin on chromosome 11***

Heterozygote defect = asymptomatic (lower MCV, lower MCH, increased HbA₂)

Homozygote defect = SEVERE phenotype

-Abnormal cell shape, Target cells, Hypochromia

Question 23

B-thalassemias: Generation by Deletion

1. Homologous Recombination Events

Answer 23

May delete B-globin gene or both B and Delta

May cause B⁺ thalassemia, B thalassemia, or DB-thalassemia

Question 24

B-thalassemias: Lepore hemoglobin

Answer 24

Recombinatnio events delete part of both B and D gloin

Generates Lepore fusion globin

• Functions poorly as a globin chain
• Hb_Lepore trait asymptomatic
• Hb_Lepore diseases rare and severe

Question 25

B-thalassemias: generation by Point Mutation

Answer 25

***Best characterized point mutations affect splicing***

T normally present is replaced with A, generating a decent match site to the 5’ splice donor site. Splicing occurs here, as well as the true splice site** –> **REDUCES the amount of correct message produced

Question 26

Screening Techniques: Electrophoresis

Answer 26

Typically used in neonatal screening for hemoglobinopathies

• Relatively cheap and easy
• Somewhat insensitive –> Difficulty testing premature infants

***Also issues with co-migration of some Hb variants***

Question 27

Screening Techniques: Isoelectric Focusing

1. When is it used?

Answer 27

Used during initial screen

Also can confirm with a second electrophoretic technique using agarose electrophoresis under acidic conditions

Question 28

What is the main screening technique that uses Molecular Biology?

When is it best used?

Answer 28

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)

Best suited for screening for common mutations (HbS) or for confirming the identity of a mutant hemoglobin