Haemoglobin Molecule and Thalassaemia

Question 1

What’s the normal concentration of haemoglobin?

Answer 1

120-165g/L

around 90mg/kg produced or destroyed per day

Question 2

When does haemoglobin synthesis occur?

Answer 2

during development of RBCs

begins in pro-erythroblast stage

• 65% in erythroblast stage
• 35% in reticulocyte stage

Question 3

Where are the components of haemoglobin made?

Answer 3

haem - mitochondria

globin - ribosomes

1. Haem:
   - Transferrin transports the ferrous to the RBC or the ferrous is liberated from the ferritin molecules.
   - Glycine, B6 and Succinyl CoA create delta-ALA which then undergoes a few moderations outside the mitochondria and then passes back in as proto-porphyrin.
   - Proto-porphyrin -> haem which binds with the globins.
2. Globin:
   - Amino acids are used in ribosomes to create the globin chains.
3. Haemoglobin:
   - Globins and haem associate

Question 4

What is haem?

Answer 4

Haem is also contained in other proteins – e.g. myoglobin and cytochromes.

Haem is the SAME in all molecules (only globins change).

Haem is a combination of a proto-porphyrin ring and central ferrous – ferro-proto-porphyrin.

It is synthesised mainly in the mitochondria with the main enzyme being ALAS

Question 5

What makes up the globin molecule?

Answer 5

There are 8 functional globin chains arranged in 2 clusters:
beta-cluster - beta, gamma, delta, epsilon - encoded on Chr11, p-arm (short arm)

alpha -cluster - alpha, zeta - encoded on Chr16, p-arm (short arm)

Question 6

Describe the globin gene expression and switching

Answer 6

alpha- is made relatively early and stays high throughout.

beta - is equal and opposite to gamma and becomes dominant after birth.

gamma - is equal and opposite to beta and is dominant pre-natal.

delta- production begins mid-natal and remains low forever.

epsilon and zeta - is equal and opposite to alpha and levels drop ~0 after 8 weeks.

Question 7

Describe the globin abundance in adults

Answer 7

HbA (a2b2) is the most common – 96-98%.

HbA2 (a2d2) is the second most common – 1.5-3.2%.

HbF (a2g2) is the least common – 0.5-0.8%.

Question 8

Describe the structure of globin

Answer 8

Primary - a (141aa), non-a- globins (146aa).

Secondary – 75% of a and b chains show a helical arrangement.

Tertiary – approx. sphere with a hydrophilic surface and a hydrophobic core, contain a haem pocket.

Question 9

What makes 2.3-DPG ?

Answer 9

muscle cells to increase dissociation of oxygen

Question 10

What affects the position of the ODC?

What would cause a right shift?

Answer 10

2, 3-DPG conc., pH, CO2 conc., structure of Hb

Right shift – High H+, High 2, 3-DPG, High CO2, HbS.

Question 11

What is haemoglobinopathis?

Answer 11

dude to structural variants of haemoglobin/ defects in globin chain synthesis (thalassaemia)

Most common inherited single gene disorder worldwide.

Question 12

How can you classify thalassaemia?

Answer 12

Globin type affected

Clinical severity
Minor or “trait”
Intermedia
Major

• now classified as transfusion dependent or non tranfusion dependent

There are 4 alpha clusters in total (alpha1 and alpha2 on each chromosome 16) but only 2 beta clusters

Question 13

What happens on beta - thalassamia?

Answer 13

autosomal recessive inheritance.

Deletion or mutation in b-globin chains – reduced or absent production of beta-globins.

Inheritance:

• When 2 beta trait have a child, there is a 25% chance of a beta-major offspring.
• Beta Thal Intermedia can also come about when one partner has a beta+ mutation which is less severe.

b0 = deletion of one beta globin-encoding gene.

b+ = mutation of one beta-globin encoding gene

Question 14

How is b-thalassamia diagnosed in the laboratory?

Answer 14

FBC – microcytic Hypochromia and increased RBCs relative to Hb.

Film – Target cells, Poikilocytosis (shape change) but NO anisocytosis (unequal size).

Hb EPS (electrophysiology studies)/HPLC (high performance liquid chromatography):

• a-thal – normal HbA2 and HbF, ±HbH.
• b-thal – raised HbA2 and HbF.

Globin chain synthesis/DNA studies:
- Genetic analysis for b-thal mutations and Xmn1 polymorphisms (b-thal) and a-thal genotypes (in all cases)

Question 15

What is the beta-thalassaemia trait?

Answer 15

is a carrier trait and often asymptomatic.

Diagnosis usually made by blood film showing hypochromic microcytic blood cells and raised HbA2 and HbF.

Question 16

What is (beta-) thalassaemia major?

What would you see on a blood film?

Answer 16

Carry 2 abnormal copies of the beta-globin gene.

Results in severe anaemia and requires regular blood transfusions.

Clinical representation after 4-6 months.

Blood film:
- Anaemia, irregularly contracted cells, hypochromic cells, a-chain precipitates, nucleated RBCs, iron inclusions (Pappenheimer bodies).

Question 17

What are the clinical features and complications of b- thalassaemia?

Answer 17

Clinical representation:

• Severe anaemia presenting after 4 months, hepatosplenomegaly, film (Hypochromia, Poikilocytosis, NRBCs (nucleated RBC), bone marrow (erythroid hyperplasia), extra-medullary haematopoiesis.
• Can show frontal bossing and maxilla bossing in the face due to extra-medullary haematopoiesis.

Clinical features:
Chronic fatigue
failure to thrive 
jaundice
late puberty
skeletal deformity
splenomegaly
iron overload

Complications:
Cholelithiasis
biliary sepsis
cardiac/liver failure
Endocrinopathies

• Most of the clinical complications of beta-thal come from the iron overload which is achieved in two ways:
• Non-transfusion dependant iron overload – ineffective erythropoiesis so iron excess is not utilised.
• Transfusion iron overload – many transfusions lead to iron overload.
• Largest cause of death in patients with beta-thal-major is cardiac failure.

Question 18

What is the treatment for thalassaemia major?

Answer 18

Regular blood transfusions
- Require 2-4 weekly transfusions but if a high requirement is needed, consider splenectomy.

Iron chelation therapy (removal of iron)
- Started after 10-12 transfusions or when serum ferritin >1000mcg/L.
- Audiology and ophthalmology screening is needed before starting.
- 3 forms of iron chelating drugs:
> DFO, Deferiprone, Deferasirox.
> Deferasirox has limited clinical experience

Other – splenectomy, supportive medical care, hormone therapy, hydroxyurea (boost HbF), bone marrow transplant.

Prophylaxis is required in splenectomy patients – immunisation and AB.

• Infection:
• Patients that have a high iron content are more prone to Yersinia infection and other gram- infections (these bacteria have an iron-loving nature)

Question 19

How can you monitor iron overload?

Answer 19

Serum levels of ferritin - >2500 associated with increased complications, and check 3-monthly if transfused otherwise annually.

Liver biopsy – rarely performed.

MRI T2 cardiac and hepatic - <20ms – increased risk of impaired LF function, check annually or 3-6-monthly if cardiac dysfunction.

Ferriscan (R2MRI) – non-invasive, <3mg/g is normal, >15mg/g associated with cardiac disease

Question 20

What is alpha - thalassamia?

Answer 20

Due to a deletion or mutation in alpha globin genes – reduced or absent alpha globins

Affects both foetus and adult (alpha is in ALL globin variants)

Severity depends upon number of chains affected

Excess beta and gamma chains will form tetramers of HbH (beta excess) and HbBarts (gamma excess)

Question 21

What are the problems with treatment?

Answer 21

*in other countries

Lack of awareness and experience of the problems
availability of blood
cost and compliance with iron chelation
availability of and cost of bone marrow transplants

Question 22

How can you screen for and prevent thalassaemia?

Answer 22

counselling and health education for thalassamia, family members and general public

extended family screening

pre-marital screening?

discourage marriage between relatives?

antenatal testing

pre-natal diagnosis (CVS)