3 - Normal/abnormal Hb structure and fx Flashcards Preview

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Flashcards in 3 - Normal/abnormal Hb structure and fx Deck (44):

normal hemoglobin structure

- Hb is a tetramer of two α-type globin and two β- type globin chains
- Four subunits, each with a heme prosthetic group
- Extensive contacts exist between subunits
--- have important consequences for Hb function


most abundant form in adults

Hb A (α2β2)
--- comprises ~ 97% of total hemoglobin in adults

- remainder mostly Hb A2 (α2δ2)
--- delta globins are similar to B
- Different Hb species are found at different stages during development


subunit/subunit interactions are stabilized by

hydrogen bonds
salt bridges
hydrophobic interactions
Van der Waals attractions


Oxygen binding properties of hemoglobin

- The 4 heme groups do not bind O2 with equal affinity
- Hb does not bind O2 efficiently at low O2 concentration
- As O2 levels increase, Hb becomes more efficient at binding O2
- This effect is evident from the sigmoid shape of the binding curve
--- positive cooperativity


myoglobin O2 binding properties

myoglobin binds O2 with high affinity at low O2 concentration and exhibits a hyperbolic O2 binding curve


Types of Hb

- HbA1 (α2β2) (most common)
- Hb A2 (α2δ2)
- Hb Gower 1 (ζ2ε2), Hb Gower 2 (α2ε2), and Hb Portland (ζ2γ2)
- Hb F (α2γ2)


Hb types expressed early-on in development

Hb Gower 1 (ζ2ε2), Hb Gower 2 (α2ε2), and Hb Portland (ζ2γ2)


major fetal hemoglobin

Hb F (α2γ2)
--- replaces the early-on Hbs


As development progresses, reciprocal switch from ___ to ____

- switch from γ-chain synthesis to β-chain synthesis occurs
--- leads to replacement of Hb F with Hb A
--- Hb F comprises <2% of total Hb by the end of the first year of life


Why are the other forms of Hb in embryo significant?

embryo lacks functional circulatory system in early development
--- Hb Gower 1, Gower 2, and Portland must capture O2 from mother and have a very high oxygen affinity


Why is it important that Hb F has higher oxygen affinity than Hb A?

- allows oxygen flow from mother to fetus
--- a histidine residue in the β-chain required for 2,3-BPG binding is replaced with a serine in the γ-chain (binds less tightly to fetal Hb = higher O2 affinity)


The six globin genes fall into two families

α-like globins
--- α and ζ
β-like globins
--- β, γ, δ, and ε


α-like globin genes are clustered on chromosome ___

β-like globin genes are clustered on chromosome ___

The α-like globin genes are clustered on chromosome 16
The β-like globin genes are clustered on chromosome 11


ψ indicates ___

- pseudo gene
--- Contain multiple mutations and cannot produce a functional protein product


Control of hemoglobin gene expression

Poorly understood
- regulatory elements upstream of α- and β-globin loci confer high-level, tissue-specific expression
- regulatory elements upstream of individual genes bind specific factors


regulatory elements for a- and B-globin loci

HS-40 region found upstream of α-like globin genes
LCR (locus control region) found upstream of β-like globin genes



- Factor involved in regulation of expression
- (erythroid Kruppel-like factor)
--- enriched during specific phase of development
--- activates β-globin gene expression
--- involved in γ- to β-globin gene switching


types of Hemoglobinopathies

- over 1000 hemoglobinopathies
- famillies
--- structural variants
--- thalassemias


hemoglobinopathies: Structural variants

- mutations that produce unstable hemoglobins
--- often form hemichrome and precipitate as Heinz bodies
- hemoglobins with altered oxygen affinity
- hemoglobins that form methemoglobin (Fe3+) more easily


hemoglobinopathies: thalassemias

imbalanced globin chain synthesis


Some (rare) structural variants with altered oxygen affinity

- HbHelsinki β-subunit mutation (Lys→Met) at 2,3-BPG binding site
--- increased oxygen affinity
- HbKansas β-subunit mutation (Asn→Thr) at α1β2 contact site
--- decreased oxygen affinity


Some (rare) structural variants that readily form methemoglobin (Hb M)

- Hb MBoston α-subunit mutation (distal His→Tyr)
- Hb MHyde Park β-subunit mutation (proximal His→Tyr)

- Histidines present right where heme binds oxygen iron


Structural variants – Hb S

- sickle cell disease
- glutamate replaced with valine at position six of the β-globin chain
- deoxygenated Hb S polymerizes
--- leads to chronic hemolytic anemia
- heterozygotes have ‘sickle cell trait’
- homozygotes have ‘sickle cell disease’


Hydroxyurea (HU)

- antineoplastic agent
--- also treatment of choice for sickle cell disease
- increases expression of Hb F
--- promotes hemoglobin solubility
- reduces sickling, painful crises, hospitalizations
- mechanism uncertain


Structural variants – Hb C

- 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 (forms crystals)
--- less flexible red cells have reduced lifespan
-----> hemolytic anemia

- compound heterozygotes with both Hb C and Hb S traits not uncommon
--- Hb SC disease
-----> milder than Hb S disease


Structural variants – Hb E

- 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 (a mild thalassemia develops)

- Heterozygotes (Hb E trait) are asymptomatic
- Homozygotes (Hb E disease): microcytosis, hypochromia, typically mild anemia
--- typically an incidental finding



- Hemoglobin is a tetramer of two different types of globin chain
- Reduced synthesis of either type of chain reduces amount of functional tetramer formed
--- results in anemia


Most common thalassemias

α-thalassemias and β-thalassemias
--- includes º and +


α-thalassemias and β-thalassemias

NO functional globin chain produced from affected locus (sometimes referred to as αº- and βº-thalassemias)


α+-thalassemias and β+-thalassemias

REDUCED amounts of globin chain produced from affected locus



- Two genes for α-globin 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
-----> expressed all the way through development (in embryo, fetus, and adult)


α-thalassemias: carrier states

- 3 functional α-globin genes/1 defective gene
--- typically no clinical signs
- 2 functional α-globin genes/2 defective genes
--- mild thalassemic anemia


α-thalassemias: 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


α-thalassemias: 4 defective α-globin genes

- α-thalassemia
- only embryonic hemoglobins Gower 1 (ζ2ε2) and Portland (ζ2γ2) can be produced
- lethal condition
--- hemoglobin Bart’s hydrops fetalis syndrome


thalassemias: generation

- gene deletion
- point mutation


α-thalassemias: generation by deletion

- most often by gene deletion
--- deletion occurs by homologous recombination
-----> misalignment and reciprocal crossovers at meiosis


α-thalassemias: generation by point mutation

- Various different point mutations identified
- In HbConstant Spring a T is replaced by a C
--- stop codon converted to codon for glutamine
--- read-through into normally non-coding mRNA occurs
-----> α-globin chain length increased from 141 to 172 amino acids

- Expect HbConstant Spring to comprise 20 – 25% total Hb
--- but realistically only contributes 1 to 2%
-----> mRNA for HbConstant Spring is unstable
*--- HbConstant Spring behaves like an α+-



- Only one gene for β-globin on chromosome 11
--- 1 functional β-globin gene/1 defective β-globin gene
-----> typically asymptomatic
-----> lowered MCV, lowered MCH, increased level of Hb A2
--- homozygotes for β-globin gene defects (or compound heterozygotes) generally have quite severe phenotype
-----> selecting against the incompetent cells


Characteristics of Blood film from a patient with a β+-thalassemia

Abnormally-shaped cells
Target cells


β-thalassemias: generation by deletion

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


β-thalassemias: Lepore hemoglobin

- Recombination events delete part of both β- and δ-globin genes
- Generates Lepore fusion globin
--- functions poorly as a globin chain
--- HbLepore trait asymptomatic
--- HbLepore disease rare and severe


β-thalassemias: generation by point mutation

Best characterized point mutations affect splicing
--- Change of T to A generates consensus splice site motif within exon
- get a mix of products with correct and incorrect splice sites (reduces amount of correct message)


Screening techniques for hemoglobinopathies

- Electrophoretic techniques typically used in neonatal screening for hemoglobinopathies
--- cheap, easy, but somewhat insensitive
--- screens for lots of problems
- Often isoelectric focusing used in initial screen
--- elecrophoresis in pH gradient gel, protein stops at their isoelectric point
- Can confirm with second electrophoretic technique e.g. agarose electrophoresis under acidic conditions


Screening using molecular biology

- Polymerase chain reaction-restriction fragment length polymorphism
--- MstII cts at consensus sequence. Mutation in Hb S alters sequence so can't cut there making a different size
- only detects a specific problem