The haemoglobin molecule and thalassaemia

Question 1

• function of RBC
• average number of RBCs
• how much Hb do they contain?
• properties of RBCs

Answer 1

Carry oxygen from lungs to tissues 
Transfer CO2 from tissues to lungs
3.5-5 x 1012 /L
Contain haemoglobin (Hb)
Each red cell contains approximately 640 million molecules of Hb
Do not have nucleus or mitochondria

Question 2

• normal conc of Hb in adults
• how much is produced and destroyed every day?
• how much iron does each gram of Hb contain?

Answer 2

Found exclusively in RBCs
MW 64-64.5 kDa
Normal concentration in adults:120-165g/L
Approximately 90 mg/kg produced and destroyed in the body every day
Each gram of Hb contains 3.4mg Fe

Question 3

when does haemoglobin synthesis occur?

Answer 3

Synthesis occurs during development of RBC and begins in pro-erythroblast:

65% erythroblast stage
35% reticulocyte stage

Question 4

difference between reticulocyte and erythroblast

Answer 4

reticulocyte- immature RBC not containing a nucleus

erythroblast- immature RBC containing a nucleus

Question 5

what is the basic structure of haemoglobin

Answer 5

It is a composite molecule consisting of FOUR haem groups and FOUR globin
chains

Each haem group is bound to an Fe2+

Each haemoglobin molecule can bind
FOUR oxygen molecules

Question 6

where are Haem and Globin synthesised?

Answer 6

• Haem (synthesised in mitochondria which contain the enzyme ALAS)
• Globin (synthesised in ribosomes)

Question 7

where can haem also be found?

Answer 7

also contained in other proteins eg myoglobin, cytochromes, peroxidases, catalases, tryptophan

Question 8

how is globin synthesises?

Answer 8

Various types (alpha, beta, delta, gamma) which combine with haem to form different haemoglobin molecules

Eight functional globin chains, arranged in two clusters:

Question 9

what chromosomes are used for globin genes?

Answer 9

b- cluster (b, g, d and e globin genes) on the short arm of chromosome 11
a- cluster (a and z globin genes) on the short arm of chromosome 16

Question 10

why is the arrangement of globin genes unusual?

Answer 10

this is an exception to the one gene=one protein hypothesis. There are TWO alpha globin genes from each parent, so in total there are FOUR alpha globin genes

Question 11

describe globin gene expression and switching

Answer 11

– Globin Gene Expression and Switching:
§ a - is made relatively early and stays high throughout.
§ b - is equal and opposite to g and becomes dominant after birth.
§ g - is equal and opposite to b and is dominant pre-natal.
§ d - production begins mid-natal and remains low forever.
§ e and z - is equal and opposite to a and levels drop ~0 after 8 weeks.

Question 12

how is haemoglobin synthesised?

Answer 12

1. Haem:
   a. Transferrin transports the ferrous to the RBC or the ferrous is liberated from the ferritin molecules.
   b. 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.
   c. Proto-porphyrin à haem which binds with the globins.
2. Globin:
   a. Amino acids are used in ribosomes to create the globin chains.
3. Haemoglobin:
   a. Globins and haem associate.

Question 13

what is the most abundant haemoglobin?

Answer 13

§ 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 14

what is the globin structure (primary, secondary and tertiary)

Answer 14

§ 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 15

what is the difference between oxygenated and deoxygenated haemoglobin?

Answer 15

§ Haemoglobin has the highest affinity to oxygen when the binding is loose (cooperativity) – more O2 means greater binding of O2.

Question 16

what does 2,3 DPG do to the affinity for oxygen

Answer 16

2, 3-DPG is made by muscle cells to increase dissociation of oxygen.

Question 17

describe the oxygen dissociation curve

Answer 17

§ Sigmoid shape – binding of one molecule facilitates binding of another.
o Greater unloading across a range of low pressures.

Question 18

what does the position of the ODC depend on?

Answer 18

Position of the ODC depends on – 2, 3-DPG conc., pH, CO2 conc., structure of Hb.

Question 19

define P50

Answer 19

P50 = 26.6mmHg (partial pressure of O2 at which Hb is half saturated with O2).

Question 20

define Haemoglobinopathies

Answer 20

Haemoglobinopathies – a genetic disorder characterised by a defect of globin chain synthesis.or structural variants of haemoglobin

Question 21

how can thalassaemia be classified?

Answer 21

o Globin type affected.

Clinical severity:
§ Minor or “trait”.
§ Intermedia.
§ Major

Question 22

how many alpha and beta clusters are there?

Answer 22

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

Question 23

what is beta thalassaemia?

Answer 23

autosomal recessive inheritance.

§ Deletion or mutation in b-globin chains – reduced or absent production of beta-globins.
§ Inheritance:
o When 2 beta trait have a child, there is a 25% chance of a beta-major offspring.
o 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 24

what laboratory diagnosis can be used to diagnose beta thalassaemia?

Answer 24

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): 
o a-thal – normal HbA2 and HbF, ±HbH. 
o b-thal – raised HbA2 and HbF. 

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

Question 25

beta thalassaemia trait- what is it?

Answer 25

it is a carrier trait and often asymptomatic

-diagnosis usually made by blood film showing hypochromic microcytic blood cells and raised HbA2 and HbF

Question 26

what is beta thalassaemia major?

Answer 26

Carry 2 abnormal copies of the beta-globin gene.

Results in severe anaemia and requires regular blood transfusions.

Clinical representation after 4-6 month

Question 27

what would you see in the blood film of someone with beta thalassaemia major?

Answer 27

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

Question 28

what is the clinical representation of someone with beta thalassaemia major?

Answer 28

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.

Question 29

what are the clinical features of beta thalassaemia major?

Answer 29

Chronic fatigue, failure to thrive, jaundice, late puberty, skeletal deformity, splenomegaly, iron overload.

Question 30

what are complications of beta thalassaemia major?

Answer 30

Cholelithiasis, biliary sepsis, cardiac/liver failure, Endocrinopathies

Question 31

where do most of the clinical complications of beta-thal major come from?

Answer 31

on dependant iron overload – ineffective erythropoiesis so iron excess is not utilised.
o Transfusion iron overload – many transfusions lead to iron overload.
o Largest cause of death in patients with beta-thal-major is cardiac failure.

Question 32

what is the treatment on offer for someone with beta-thal?

Answer 32

Treatment:
o Regular blood transfusions.
o Iron chelation therapy (removal of iron).
o Other – splenectomy, supportive medical care, hormone therapy, hydroxyurea (boost HbF), bone marrow transplant.
§ Prophylaxis is required in splenectomy patients – immunisation and AB

Question 33

what is the treatment on offer for someone with beta-thal?

Answer 33

Treatment:
o Regular blood transfusions (phenotyped red cells, regular transfusion 2-4weekly)
o Iron chelation therapy (removal of iron).
o Other – splenectomy, supportive medical care, hormone therapy, hydroxyurea (boost HbF), bone marrow transplant.
§ Prophylaxis is required in splenectomy patients – immunisation and AB

Question 34

what is a potential risk for someone with beta-thal major?

Answer 34

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 35

describe iron chelation therapy:

Answer 35

Iron chelation therapy:
§ 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.

Question 36

how can you monitor iron overload?

Answer 36

Monitoring iron overload:
o Serum levels of ferritin - >2500 associated with increased complications, and check 3-monthly if transfused otherwise annually.
o Liver biopsy – rarely performed.
o MRI T2 cardiac and hepatic - <20ms – increased risk of impaired LF function, check annually or 3-6-monthly if cardiac dysfunction.
o Ferriscan (R2MRI) – non-invasive, <3mg/g is normal, >15mg/g associated with cardiac disease.

Question 37

what can thalassaemia be co-inherited with?

Answer 37

§ Thalassaemia mutations can be co-inherited with other complications – such as SCD and beta-thal.
§ Co-inherited Beta-Thal:
o Sickle Beta Thalassaemia.
o HbE Beta Thalassaemia – very common in SE Asia and can be as severe as beta-thal major.

Question 38

what is alpha thalassaemia?

Answer 38

o Due to a deletion or mutation in alpha globin genes – reduced or absent alpha globins.
o Affects both foetus and adult (alpha is in ALL globin variants).
o Severity depends upon number of chains affected.
o Excess beta and gamma chains will form tetramers of HbH (beta excess) and HbBarts (gamma excess).

Question 39

what is the dominant Hb in sickle b thalassaemia?

Answer 39

As little or no HbA is being produced in these patients HbS will be the dominant haemoglobin and will precipitate as it does in homozygote sickle cell patients

Question 40

comparison of currently available iron chelators

Answer 40

check slides