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Endocrine Exam 3 > 39 - Normal and Abnormal Hemoglobin > Flashcards

39 - Normal and Abnormal Hemoglobin Flashcards

1
Q

What is the structure of normal hemoglobin?

A
  • Hb is a tetramer of two alpha-type globin and two beta-type globin chains
  • The most abundant form is Hb A (alpha2-beta2)
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2
Q

Describe the contact of hemoglobin subunits within the molecule

A
  • There is extensive contact between the subunits in the hemoglobin molecule
  • The subunit-subunit interactions are stabilized by (hydrogen bonds, salt bridges, hydrophobic interactions, van der waals attractions)

The extensive contacts between the different subunits of the Hb molecule has important consequences for Hb function

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3
Q

Describe the oxygen binding properties of hemoglobin

A
  • Hb has four subunits, each containing a heme prosthetic group
  • Heme groups do NOT bind to O2 with equal affinity
  • Hb does NOT bind to O2 efficienctly at low O2 concentration
  • As O2 levels increase, Hb becomes more efficient at binding O2
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4
Q

Describe the oxygen binding affinity curve

A

Remember, as O2 levels increase, Hb becomes more efficient at binding O2
- This is evident from the sigmoid shape of the binding curve

See slide 3 for image

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5
Q

Describe the difference in binding to myoglobin

A
  • In contrast, myoglobin binds O2 with high affinity at low O2 concentration and exhibits a hyperbolic O2 binding curve

See slide 3 for image

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6
Q

How abundant is Hb A?

A
  • Hb A comprises 97% of the total hemoglobin in adults
  • The remainder is mostly Hb A2 (alpha2-delta2)
  • Different Hb species are found at different stages during development
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7
Q

Describe the progression of Hb changes during development

A

Hb forms expressed in early embryonic development

  • Hb Gower 1 (ζ2ε2)
  • Hb Gower 2 (α2ε2)
  • Hb Portland (ζ2γ2)

All these forms are replaced by Hb F which becomes the major fetal Hb form

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8
Q

What changes allow the Hb F to eventually be replaced by Hb A?

A
  • As development progresses, there is a switch from gamma-chain synthesis to beta-chain synthesis
  • This leads to the replacement of Hb F with Hb A
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9
Q

Describe the concentration of Hb F by the end of the first year of life

A

Hb F comprises

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10
Q

What is the significance of the fetal forms of Hb?

A
  • The embryo lacks a functional circulatory system in early development
  • Hb Gower 1, Gower 2 and Portland must capture O2 from the mother
  • The way they accomplish this is by having a VERY HIGH O2 AFFINITY ***
  • Hb F has a higher oxygen affinity than Hb A as well
  • This allows oxygen to continue to flow from the mother to the fetus
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11
Q

What structural difference accounts for the difference in the oxygen binding affinity between Hb F and Hb A?

A
  • Here, the difference in oxygen affinity arises because of differences in the amino acid sequence between the β-subunit of Hb A and the γ-subunit of Hb F.
  • A histidine residue in the beta chain sequence is replaced with a serine residue in the gamma-chain sequence.
  • This histidine is involved in the binding of 2,3-BPG
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12
Q

How does this contribute to oxygen affinity?

A
  • This histidine is involved in the binding of 2,3-BPG.
  • The replacement of histidine with serine removes positive charge from the 2,3-BPG binding site, reducing the affinity of Hb F for 2,3-BPG.
  • This reduced 2,3-BPG affinity increases the oxygen affinity of HbF.
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13
Q

What is the only thing that is written out on the slides and probably the main point you need to focus on here?

A

A histidine residue in the β-chain of Hb A is required for 2,3-BPG binding is replaced with a serine in the γ-chain of Hb F
- When you bind 2,3-BPG, it is harder to bind oxygen

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14
Q

What are the two families of hemoglobin genes?

A
  • Alpha-like globulins

- Beta-like globulins

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15
Q

Describe alpha-like globulins

A

Alpha-like globulins

  • Includes alpha and zeta globulin genes
  • Alpha-like globin gnees are clustered on chromosome 16
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16
Q

Describe beta-like globulins

A

Beta-like globulins

  • Includes alpha, gamma, delta and epsilon globulin genes
  • The beta-like globulin genes are clustered on chromosome 11
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17
Q

What is a “pseudo gene”?

A

A “pseudo gene” contains multiple mutations and cannot produce a functional protein product

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18
Q

Describe the control of hemoglobin gene expression

A
  • Poorly understood
  • We don’t know the overall structure of the globin gene loci
  • We do know that regulatory elements upstream of the alpha and beta globulin loci give rise to high-level, tissue-specific expression
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19
Q

What are the two upstream regulatory segments?

A
  • HS-40 (found upstream of the ALPHA-like globin genes)

- LCR (found upsteram of the BETA-like globin genes)

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20
Q

How are the upstream regulatory segments turned on and off?

A
  • Regulatory elements upstream of individual genes bind to specific factors
  • The factors are still poorly understood
  • We do know about the EKLF or erythroid Kruppel-like factor
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21
Q

What is the EKLF (erythroid Kruppel-like factor)

A

EKLF

  • Enriched during specific phase of development
  • Activates the beta-globin gene expression
  • Involved in the gene switching from gamma (Hb F) to beta (Hb A)
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22
Q

What are the categories of hemoglobinopathies?

A

Hemoglobinopathies

  • More than 1000 are identified
  • They are broken down into fairly simple groups
  • The two groups are structural variants and thalassemias ***
23
Q

Describe the structural variant hemoglobinopathies

A

Structural variants

  • Mutations that produce UNSTABLE HEMOGLOBINS
  • This often forms a hemichrome and precipitates as a “heinz body” ***
  • Heinz bodies are hemoglobins with altered oxygen affinity
  • These hemoglobins will form methemoglobin more easily (Hb with Fe3+)
24
Q

Describe thalassemias

A
  • Imbalanced globin chain synthesis

- If you don’t synthesize enough alpha or beta, you have an imbalance

25
Q

What is the most common structural variant?

A

Hb S

- Found in sickle cell disease

26
Q

Describe sickle cell disease

A
  • glutamate replaced with valine*** at position six of the β-globin chain
  • Valine does not want to be exposed on the surface, it wants to be protected
  • Deoxygenated Hb S polymerizes to protect valine
  • The polymerization distorts the shape of red cells
  • Misshappen cells block the microcirulation
  • These misshapen cells will lyse readily, leading to chronic hemolytic anemia
  • Heterozygotes have the “sickle cell train”
  • Homozygotes have “sickle cell disease”
27
Q

What is hydroxyurea?

A

An antineoplastic agent which is the treatment of choice for sickle cell disease in adults ***

28
Q

What does hydroxyurea do?

A
  • It increases the expression of Hb F (we don’t know why) ***
  • This promotes hemoglobin solubility
  • It only increases Hb F from a fraction of a percent to a slight amount more
  • Not a huge increase, but it does increase the solubility of Hb
  • This reduces the painful “sickling” crises and hospitalizations
  • The mechanism of increasing Hb F is uncertain, but could be related to stimulating the sub-population of Hb F-producing cells, upregulating the promoter of specific Hb F production or generation of nitric oxide
29
Q

What is Hb C disease?

A
  • Another structural variant of hemoglobin
  • Restricted to those of West African origin
  • glutamate replaced with a lysine** at position six of the β-globin chain **
  • Hb C does not polymerize and cells do not sickle
  • Hb C less soluble than Hb A and precipitates
  • Less flexible red cells have reduced lifespan
  • Hemolytic anemia results
30
Q

What do we call a patient with a Hb C and a Hb S trait?

A
  • Compound heterozygotes with both Hb C and Hb S traits not uncommon
  • Called Hb SC disease
  • Similar to, but somewhat milder than Hb S disease (sickle cell disease)
31
Q

What Hb E disease?

A

Another structural variant

  • Common in Southeast Asia
  • Glutamate at position twenty six of the β-globin chain is replaced with lysine***
  • Mutant β-globin chain is not synthesized effectively ***
  • Imbalanced α- and β-globin chain synthesis
  • Makes more alpha than beta and a mild thalassemia develops
  • Heterozygotes (Hb E trait) are asymptomatic
  • Homozygotes (Hb E disease) which consists of microcytosis, hypochromia, and typically a mild anemia
32
Q

What are thalassemias?

A
  • Hemoglobin is a tetramer of two different types of globin chain
  • Reduced synthesis of either type of chain reduces amount of functional tetramer formed
  • This results in anemia
33
Q

What are the most common thalassemias?

A

α-thalassemias and β-thalassemias

  • Either NO alpha or NO beta is produced
  • NO functional globin chain produced from affected locus

α+-thalassemias and β+-thalassemias
- SOME alpha or beta is produced, but a reduced amount of the globin chain is produced from the affected locus

34
Q

Describe alpha-thalassemias

A
  • Two genes for alpha globin are present on chromosome 16
  • α-globin chains found in embryonic (Hb Gower 2; α2ε2), fetal (α2γ2), and adult hemoglobins (α2β2 and α2δ2)
  • α-thalassemias manifest during development and in adult life
35
Q

What are the characteristics of the carrier state in alpha-thalassemia?

A

3 functional α-globin genes/1 defective gene
- typically no clinical signs

2 functional α-globin genes/2 defective genes
- mild thalassemic anemia

36
Q

What are the actual states of alpha-thalassemia?

A
  • 1 functional α-globin gene/3 defective genes

- 4 defective α-globin genes

37
Q

Describe the characteristics of alpha-thalassemia with 1 functional α-globin gene/3 defective genes

A

1 functional α-globin gene/3 defective genes

  • severe α-globin chain deficiency
  • γ4-tetramers (Bart’s hemoglobin) form in fetus
  • β4-tetramers (Hb H) form later in development
    • -> Bart’s Hb and Hb H are poor oxygen carriers
    • -> Hb H precipitates, shortening red cell life
38
Q

Describe the characteristics of alpha-thalassemia with 4 defective alpha-globin genes

A
  • α-thalassemia
  • only embryonic hemoglobins Gower 1 (ζ2ε2) and Portland (ζ2γ2) can be produced
  • Lethal condition
  • Hemoglobin Bart’s hydrops fetalis syndrome
39
Q

How do alpha-thalassemias arise?

A
  • α-thalassemias most often arise by deletion***
  • α-globin genes found in region of homology
  • deletion occurs by homologous recombination ***
  • misalignment and reciprocal crossovers at meiosis
40
Q

How else can alpha-thalassemias arise?

A

Point mutation

  • Various different point mutations identified
  • In Hb(constant spring) a T is replaced by a C
  • This is the one we will focus on
41
Q

What happens in Hb(constant spring) where a T is replaced by a C?

A
  • stop codon converted to codon for g- lutamine
  • read-through into normally non-coding mRNA occurs
  • α-globin chain length increased from 141 to 172 amino acids
42
Q

What percent of Hb in the body will be affected by this point mutation?

A
  • We would expect Hb(constant spring) to comprise 20 – 25% total Hb
  • But it only contributes 1-2%
  • This is because mRNA for Hb(constant spring) is UNSTABLE ***
  • It should be making a longer chain, but it isn’t able to because it is so unstable

Overall, Hb(constant spring) BEHAVES like an alpha+-thalassemia (a reduction in alpha chain)

43
Q

Describe beta-thalassemias

A
  • There is only one gene for β-globin on chromosome 11

- There is only one carrier state and one disease state

44
Q

Describe the carrier state of beta-thalassemia

A
  • 1 functional β-globin gene/1 defective β-globin gene
  • Typically asymptomatic
  • Lowered MCV (mean corpuscular volume), lowered MCH (mean corpuscular hemoglobin) and increased level of Hb A2
45
Q

Describe the disease state of beta-thalassemia

A
  • Homozygotes for β-globin gene defects (or compound heterozygotes) generally have quite severe phenotype
  • Abnormally-shaped cells
  • Target cells
  • Hypochromia
46
Q

How do beta-thalassemias arise?

A

By deletion during homologous recombination events

  • may delete just β-globin gene or both β and δ
  • may find β+-thalassemia, β-thalassemia, δβ-thalassemia
  • variable phenotype dependent upon level of residual β-globin expression remaining
47
Q

What is Lepore hemoglobin?

A
  • A form of hemoglobin that forms in beta-thalassemias
  • Occurs when recombination events delete part of both β- and δ-globin genes
  • Generates a Lepore fusion globin
  • It functions poorly as a globin chain
  • HbLepore trait asymptomatic (heterozygote)
  • HbLepore disease rare and severe (homozogote)
48
Q

What are the two ways we have discussed beta-thalassemias occurring so far?

A
  • Deletion during homologous recombination events

- Recombination leading to the Lapore fusion globin chain

49
Q

What is the other type of mutation that can lead to a Beta-thalassemia?

A

Point mutation

  • This point mutation affects SPLICING ***
  • There is a point mutation which changes a T to an A
  • This generates a “match” to the 5’ splice site
  • Splicing now occurs at the point mutation and again at the 5’ splice site
  • This reduces the amount of correct message that is produced (it cuts out some of the AA sequence that should have been included)
50
Q

How can we screen for hemoglobinopathies?

A

Electrophoretic techniques typically used in neonatal screening for hemoglobinopathies

  • This is relatively cheap and easy, somewhat insensitive though
  • The insensitivity makes it hard to test premature infants (they might have it, but it doesn’t show up)
51
Q

What specific electrophoesis test do we do initially use to screen?

A
  • Often isoelectric focusing used in initial screen
  • Electrophoresis is performed in pH gradient gel
  • Proteins stop migrating at their isoelectric point
  • This can confirm with second electrophoretic technique e.g. agarose electrophoresis under acidic conditions
52
Q

How can we screen using molecular biology? Why would we do this?

A
  • These tests are best suited for screening for common mutations (such as that generating Hb S), or for confirming the identity of a mutant hemoglobin.
  • One commonly-used technique is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
  • For example, PCR-RFLP can be used to screen for sickle cell trait and sickle cell disease.
53
Q

How is the molecular biology testing done?

A
  • PCR primers are designed to amplify a region of the β-globin gene containing the point mutation (A → T; Glu → Val) that generates Hb S
  • This mutation also removes a recognition site for the restriction endonuclease MstII
  • . Therefore, the restriction fragments generated from MstII digestion of the amplification product differ when one compares normal individuals with individuals who have the sickle cell disease or sickle cell trait.

Look at slide 24-25 for a depiction of this

Endocrine Exam 3 (13 decks)

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