Week 4- Haemoglobinopathies

Question 1

How much Hb is in the average adult?

Answer 1

7 x 10^20 Hb molecules.

each RBC has 280 million and we have 5000 million RBCs/ml of blood

Question 2

Describe the HB molecule?

Answer 2

Two alpha and two beta polypetide chains. Each chain contains a ferrous iron.
Molecular weight= 64500 Da

Question 3

Steps of haemoblobin development? Each type at each stage?

Answer 3

Embryo-
Gower (3 weeks)- Zeta2E2
Portland (5 weeks)- Zeta2Gamma2
Gower (5 weeks)- Alpha2E2

Fetus- F (10 weeks)- Alpha2Gamma2

Adult
F (1%)  A2 (2-3%)=alpha2delta2   A (97%)=alpha2beta2

Question 4

With regards to Hb development, why do thalassemias have big consequences?

Answer 4

Thalassemias affect alpha and beta chains. Alpha and beta make up 97% of adult Hb so big consequences!
Especially alpha, as its found 5 weeks onwards.
However alpha has 4 genes, 2 on each chromosome. Beta only has 1. In alpha, 4 things have to go wrong for disease.

Question 5

Describe the chromosomes with Hb genes on?

Answer 5

Beta globin gene cluster- chromosome 11.
epsilon, 2 gammas (G+A), delta and beta.
The 2 gamma genes are active in fetal growth and produce Hb F. The adult gene, beta, takes over after birth.

Alpha globin gene cluster- chromosome 16
zeta 2, zeta 1, alpha 2, alpha 1. Each of the 4 alpha genes contribute to alpha globin synthesis.

(all functional globin genes have 3 exons and 2 introns)

Question 6

What is always at the first part of Hb?

Answer 6

Alpha and zeta are on the same chromosome so first part of HB always either zeta or alpha
e.g Gower1- zeta2E2
F- Alpha 2 gamma2

Question 7

How many types of gamma are there?

Answer 7

Two. differ by a glycine residue.

Question 8

What’s the difference between Hbopathies and thalassemia?

Answer 8

Haemoglobinopathies- synthesis of mutant Hb chains. e.g. sickle cell anaemia.
Thalassemia- decreased synthesis of WT Hb but has normal structure.

(can overlap-some abnormal globins are made at a reduced rate. These disorders of Hb lead to anaemia)

Question 9

What Hb do sickle patients have?

Answer 9

Sickle Hb (HbS) is different from normal Hb (HbA).
Single point mutation in beta chains.

Question 10

How do you get sickle cell anaemia?

Answer 10

Both beta genes have to have the mutation. (one on each chromosome). If only one is mutated you have sickle cell trait.
Substitution of valine for glutamic acid on position 6 in the beta chain of Hb.

Question 11

How much sickle Hb do sickle cell trait and sickle cell anaemia sufferers have?

Answer 11

Trait-> 20-45% HbS.

Disease-> 80% HbS

Question 12

What happens with sickle cells?

Answer 12

When sickle Hb gives up its oxygen to tissues, it sticks together to form long rods inside RBCs, making RBCs rigid and sickle shaped.
Sickle cells form crystal structure and get stuck, aren’t flexible. Usually 7 Hb molecules stick together to make a crystal.
HbS is insoluble and forms crystals when exposed t low oxygen tension.

Question 13

How can sickle cell anaemia lead to different fingers adn toes?
(extra reading)

Answer 13

Painful crises caused by occlusion of small blood vessels. Can occur in bones, lungs and spleen. Most serious events occur in the brain. In children the hand-foot syndrome is common when occlusions occur in small bones-> can lead to digits of varying lengths.

Question 14

What problems does sickly cell anaemia cause?

Answer 14

Patients suffer from sever anaemia and severe pain. Suffer crises:
Vaso-occlusive-block vessel
Visceral- when it affects your organs, can get pooling in organs.
Aplastic- no cell formation. (can be caused by parvovirus. Treated with transfusion.)
Haemolytic- fall in Hb and reticulocytosis.

Macrophages in spleen try phagocytose weird RBCs, causes enlargement and stress on spleen. Body compensates for lost RBCs, lots of precursors.

Question 15

How does the sickle cell gene protect from malaria?

extra reading

Answer 15

Have protection from plasmodium-malaria. 2011
Soares and team from Portugal have found that low levels of circulating haem in blood of sickle-cell gene carrying mice down, releasing small amounts of carbon monoxide. This limits the accumulation of haem after infection with the malaria parasite. This appears to prevent the disease.

Question 16

What are the types of sickle cell crises?

Answer 16

Vaso-occlusive:
Most common, can damage bones, lungs, CNS
Sickle cells adhere to neutrophils and endothelial

Visceral:
Sicklign within organs and pooling of blood can make anaemia worse

Aplastic:
caused by infection with parvovirus. Reduction in haemoglobin. Take folic acid to help cell production.

Haemolytic:
Increased rate of haemolysis. Fall in Hb.

Question 17

What were the findings of the sickle Hb? difference from normal Hb

Answer 17

1949 pauling showed that HbS had a higher isolectric point than Hba. (The ph at which it has no net charge.)

1957 INgram discovered that the HbS had a valine instead of a glutamic acid. Valine has no charge, glutamic acid has negative. makes valine stick out of Hb molecule. makes it sticky too.

Because valine has no charge, HbS 50 times less soluble. So crystallises

Question 18

What are sickle cells like in high and low tensions?

Answer 18

When oxygen is bound to th Hb it looks noral, when it loses oxygen at peripheral tissues it polymerises into long fibres of & Hbs long.
At low oxygen tension (pressure), HbS is insoluble and forms crystals.

Question 19

Sickle cell trait

Answer 19

carrier of sickle Hb gene.
One HbS and one HbA gene
Enough Hba-? RBCs are flexible and no anaemia.
RBCs look normal.

Question 20

How do you diagnose sickle cell?

Answer 20

Low Hb: 6-9 g/dL
Sickle cells and target cells in blood.
Blood is deoxygenated
Electrophoresis- no HbA is detected HbF levels 5-15%

Question 21

What are sickle cell tests and treatments?

Answer 21

1. In pregnancy samples obtained by chionic villus biopsy
   amniotic fluid cells can be used
   PCR amplifies DNA.
2. Treatment= prophylactic- folic acid, 5mg/week to increase cell production. vaccination- reduce infection rates.
   Treat crises= rest, hydrate, antibiotics
   Transfusions= suppressed HbS production. Stem cells.
   Hydroxyurea (15-20 mg/kg) can increase Hb levels and improve outcome.

Question 22

Give 5 Hb variants that are clinically significant? (some extra reading)

Answer 22

HbS- sickle cell. mutation in beta chain

HbC- west africa. lysine instead of valine. Rhomboidal crystal (instead of 7 long)

HbD- 2 groups D^punjab + D^non-punjab. If D punjab, inherited with sickle-> anaemia. Non punjab not severe.

HbE- mutation of glutamic acid-> lysine at position 26. Subjects get mild beta-thalassemia (homozygous)

HbH- form of alpha thalassemia, reduced formation of alpha chains. HbH disease.

Question 23

What is thalassemia?

Answer 23

Reduction in alpha or beta synthesis.

Question 24

How is thalassemia classified?

Answer 24

Which globin effected: alpha or beta.
rate of synthesis: alpha+ reduced synthesis. alpha0 no synthesis.
Genotype: e.g. heterozygous alpha+

Question 25

How many copies of the genes are there and how do they affect protein production- in thalassemia

Answer 25

Alpha globin genes- 4 copies- chromosome 16

Beta- 2 copies- chromosome 11

All copies are used to the same extent. i.e. if one copy of beta gene deleted then 50% protein made.

Question 26

What causes thalassemias?

Answer 26

Gene deletions. Also single base substitutions leading to mis-splicing of premRNA.

Question 27

Where are most alpha and beta thals most prominent?

Answer 27

Alpha= far east
Beta= Mediterranean

Question 28

What’s needed to make alpha globulin chain?

Answer 28

4 genes needed to make alpha globin chains ( 2 from each parent)

Question 29

What are the silent carriers and alpha thalassemia trait caused by?

Answer 29

Silent carrier- heterozygous alpha+ thalassemia. If one alpha gene deleted, then no signs of disease.

Alpha thalassemia trait-alpha+ homozygous or alpha0 heterozygous. 2 alpha genes deleted. Not enough alpha globulin produced. Suffer hypochromic anaemia. Microcytic cells.

Question 30

Dedcribe HbH disease

Answer 30

3 alpha genes deleted. Anaemia (moderate to severe) and hepatosplenomegaly.
Microcytosis, polychromasia, poikilocytosis, hypochromasia.

Question 31

Hb Barts Hydrops Fetalis

Answer 31

4 genes deleted. After 10 weeks, no functional Hbs. Haemoglobin Barts (gamma4) and Hbh (beta4) are synthesised. Incompatible with life.

Question 32

What happens when alpha globulin not made?

Answer 32

Accumulation of other globin chains. Insoluble aggregate. Damages erythroblast.

Question 33

Beta thalassemia- when are they a carrier and when anaemic?

Why is beta not as bad as alpha?

Answer 33

When only one affected- carrier.
If two affected you have anaemia.
Not as bad as alpha because HbB only needed for HbA. So HbF will rise to compensate.

Question 34

What are most beta thalassemias caused by?

Answer 34

Point mutations in beta globin gene or close to b globin gene.

Question 35

Symptoms and clinical findings of B thal. major?

Answer 35

Enlarged maxilla. Prominent frontal and parietal bones. Hyperplasia- bones trying to make too many cells.
Siderosis- deposits of iron in liver
Anaemia (severe)
Hepatosplenomegaly
Secondary iron overload *treated with iron chelation)
Infections

Question 36

What are genetics of beta thal. major?

Answer 36

Genes tend to be mutated rather than deleted. Mainly point mutations. Mutations spread through the gene and in promoter and enhancer regions.
Whereas alpha- mainly deletions, beta can be splicing, frameshift etc.

Question 37

B thalassemia intermedia

Answer 37

Thallassemia= moderately severe.
Patients don’t need regular transfusions.

Caused by: Homozygous B thal. where HbF is made more than usual. Mild defects in B chain synthesis.
Dominant B thal. B trait which is unusally severe. Survival rate is good nowadays.

Question 38

What are the clinical features of B-thalassemia intermedia?

Answer 38

Bone abnormalities
enlarged spleen or liver
Extramedullary erytrhopoiesis
Iron overload

Question 39

B thalassemia minor (trait)

Answer 39

Abnormal trait inherited from one parent. Mild anaemia-> asymptomatic.
High RBC.
Usually don’t know you have it.

Question 40

What are thalassemia treatments?

Answer 40

Blood transfusions.
-Rbc transfusion.
2-3 units every 4-6 weeks.

Iron chelation therapy
Comon drug= deferoxamin,
deferipone- 3 x daily, iron excreted in urine.
deferasirox- new iron chelator, once daily

Folic Acid- if diet is poor
Splenectomy
Immunisation
Bone marrow transplant- 80% success but very intense, not common

Question 41

Beta thalassemia diagnosis

Answer 41

Hypochromic, microcytic anaemia
Raised reticulocyte %
Electrophoresis-? increased levels of HbA and HbF
Dna analysis for mutations
Iron overload tests-> serum ferritin liver biopsy.

Question 42

Extra reading on thalassemia

Answer 42

4.83% of global population have globin variants.
2009 study thalassemia Lin Ye et al.
Induced pluripotent stem cells can be made from the patients somatic cells and the mutation in b-globin gene corrected by gene targeting. Then let the cells differentiate into haematopoietic cells that could make Haemoglobin and return to patient.