The Haemoglobin Molecule and Thalassemia

Question 1

What are the different types of globin chain proteins?

Answer 1

Alpha, Beta, Gamma, Delta (and embryonic- epsilon and zeta)

Question 2

State the three different haemoglobins that are present in the human body.

Answer 2

HbA – alpha + beta = 95%
HbA2 – alpha + delta = 1-3.5%
HbF – alpha + gamma = trace .7%

Question 3

Describe how affinity of haemoglobin changes with oxygen binding and how this helps its role of oxygen transport.

Answer 3

The more oxygen binds, the greater the affinity of the haemoglobin for oxygen.
This is good because if deoxyhaemoglobin has a low affinity for oxygen(as no oxygen is already bound), it will only pick up oxygen if the oxygen saturation is very high (i.e. in the lungs) so it will not take up oxygen in the metabolically active tissues where the oxygen saturation is low and where the tissues need oxygen.
Similarly, oxyhaemoglobin has a high affinity for oxygen so it will only give up oxygen in environments where the oxygen saturation is very low (i.e. respiring tissues that need oxygen)

Question 4

State 3 factors that can shift the oxygen dissociation curve to the right.

Answer 4

Increase in 2,3-DPG
Increase in [H+]
CO2 (hypercapnia)

Question 5

What effect do HbS and HbF have on the oxygen dissociation curve?

Answer 5

HbS has a lower affinity for oxygen than HbA so it shifts the ODC to the right
HbF has a higher affinity for oxygen than HbA so it shifts the ODC to the left

Question 6

What is special about alpha globin genes?

Answer 6

There are TWO alpha globin genes from each parent so there are FOUR alpha globin genes in total.

Question 7

Which globin chains are present in early embryonic life but are switched off after about 3 months gestation?

Answer 7

Zeta and epsilon

Question 8

Which globins are present in foetal haemoglobin?

Answer 8

Alpha

Gamma

Question 9

When are the genes coding for the globin in foetal haemoglobin switched off?

Answer 9

It is decreased towards birth and in the first year after birth.
After 1 year of life, the normal adult pattern of haemoglobin synthesis would have been established.

Question 10

Which globin chains are present in HbA2?

Answer 10

Alpha

Delta

Question 11

On which chromosomes are the two globin gene clusters and which genes are present in each cluster?

Answer 11

Chromosome 16 – ALPHA cluster 
 TWO alpha genes  
 Zeta gene  
Chromosome 11 – BETA cluster 
 Beta gene  
 Gamma gene 
 Delta gene  
 Epsilon gene

Question 12

What is thalassemia?

Answer 12

Disorders in which there is a reduced production of one of the two types of globin chains in haemoglobin leading to imbalanced globin chain synthesis

Question 13

How is thalassemia classified in terms of clinical severity?

Answer 13

Minor: Thalassemia trait = asymptomatic carrier common variant with little clinical significance

Intermediate: Thalassemia intermedia
somewhere in between
can require transfusions or not

Major: Transfusion dependent – Thalassemia Major = fatal without transfusion

Question 14

What is the outcome of alpha thalassemia major?

Answer 14

Fatal in utero because alpha globin is needed to make HbF (alpha + gamma)

Question 15

Clinical presentation of thalassemia major?

Clinical features

Answer 15

a. Severe anaemia presenting at 4 months leading to Chronic fatigue
b. Failure to thrive
c. Jaundice
d. Hyperactive bone marrow
e. Delay in growth and puberty
f. Extramedullary haematopoiesis

g. Skeletal deformity (bone marrow expansion due to extramed haematopoiesis causing the distinctive facial features)
h. Hepatosplenomegaly
i. Iron overload (patients with thalassaemia absorb more iron in GI tract)

Question 16

Which 2 types of abnormal haemoglobin can result due to Alpha thalassaemia

Answer 16

Haemoglobin H and Hb barts

Lack of Alpha chain forms tetramers of Beta chains instead of 2 alpha and beta= HbH

Same but tetramer of gamma chain= Hb barts

Question 17

What is beta thalassemia major? Describe how the disease progresses.

Answer 17

Severe defect in both beta globin chains
The foetus will have no problem in utero because they have normal functioning HbF (which doesn’t need beta globin)
At around 2-3 month after birth, you get a transition from HbF to HbA
At this time the baby will become profoundly anaemic.
They will need life-long transfusions from this point onwards.

Question 18

State some features of thalassemia trait.

Answer 18

This the carrier state of thalassemia.
They may be mildly anaemic but they can also be normal.
Usually have a LOW MCH and LOW MCV

Question 19

Summarise the laboratory diagnosis of Thalassemia

Answer 19

Start simple:
-	FBC 
-	Film 
Complex tests:
-	Hb electrophoresis/ HPLC 
-	DNA studies eg PCR to look at genes for globin chains

Question 20

What types of mutation cause alpha thalassemia and beta thalassemia?

Answer 20

both can be caused by deletion or point mutation in the respective alpha/beta globin gene

Question 21

Which ethnic groups have a high prevalence of thalassemia?

Answer 21

Chinese 
South-east Asian 
Greek 
Turkish 
Cypriot

Question 22

Laboratory diagnosis: What features would you see when looking at FBC of thalassemia?

Answer 22

• Poikilocytosis (no anisocytosis)
• Target cells
• Hypochromasia
• Nucleated RBC

Question 23

What are the consequences of regular blood transfusions used to treat beta thalassemia major?

Answer 23

Iron overload: Chronic transfusion has been associated with iron accumulation. There’s not enough transferrin to bind to all the iron, the unbound iron can catalyse production of reactive oxygen species which can damage the liver (cirrhosis), heart (cardiac failure), endocrine organs (hypopituitarism, hypothyroidism, diabetes) etc.
Also increases chance of infection.

Question 24

Name three iron chelator drugs that can be used to treat iron overload.

Answer 24

Desferrioxamine (DFO/desferal)
Deferiprone
Deferasirox

Question 25

What are the key indicators when monitoring the iron overload in the management of thalassemia patients

Answer 25

1. Serum ferritin (but not that accurate as it is also an ACP that rises in inflammation etc) 2. Cardiac and hepatic MRI to measure iron levels in heart and liver Liver biopsy used to be done but not anymore

Question 26

Laboratory diagnosis: What features would you see when looking at Blood film of thalassemia?

Answer 26

target cells poikilocytosis (different shape) NO anisocytosis(different size) Microcytic and hypochromic (relate this to the FBC card earlier)

Question 27

Laboratory diagnosis: What is the most definitive test for thalassemia

Answer 27

DNA studies - PCR, sanger sequencing etc | Most alpha thalassemias are diagnosed this way because they are quite hard to diagnose (HPLC gives normal HbA2 and HbF)

Question 28

Complications of thalassemia

Answer 28

``` Cholelithiasis (gallstones) Biliary sepsis Cardiac failure endocrinopathies liver failure ```

Question 29

Treatments for thalassemia major (minor usually asymtomatic)

Answer 29

Main treatments: - Regular blood transfusions - Iron chelation therapy (remove XS iron) Other treatments: - Splenectomy (patients may have splenomegaly due to extramedullary hematopoiesis) - Supportive medical care for cardiac, liver problems - Hormone therapy, iron overload can cause hypogonadism - Hydroxyurea to boost HbF production - Bone marrow transplant

Question 30

Why are thalassemia patients prone to infection? What can be done to reduce the chances?

Answer 30

They tend to have iron overload and there are pathogens that thrive on iron especially gram - bacteria like YERSINIA Also, thalassemia patients could have splenectomy due to splenomegaly via extramedullar haematopoiesis which is an important immune organ. to prevent this, immunisation in splenectomised patients is good. (prophylaxis)

Question 31

Main concern for management of thalassemia patients

Answer 31

infection and monitor iron overload

Question 32

What are: Sickle b thalassemia HbE b thalassemia

Answer 32

Sickle b thal: inherits one sickle allele from one parent and b thal allele from the other parent NB sickle allele is also a mutation on the beta globin gene. So this disease is basically inheriting two different faulty beta globin alleles from the 2 parents HbE b thal: - Single point Mutation in beta chain, forming HbE which is an abnormal haemoglobin - Reduced synthesis of functional beta chain leads to beta thalassemia

Question 33

Mature rbc do not have

Answer 33

nuclues or mitochondria

Question 34

Synthesis of haemoglobin : how much in eryhthroblast stage and reticulocyte stage

Answer 34

65% erythroblast stage | 35% reticulocyte stage

Question 35

Strucgure of haemolgobin a

Answer 35

4 chains-- 2 a, 2 b-- type of chains may alter with different haemoglobin Haem group in centre with central iron ion-- ferrous form fe2+

Question 36

Structure of haemolgobin a

Answer 36

4 chains-- 2 a, 2 b-- type of chains may alter with different haemoglobin Haem group in centre with central iron ion-- ferrous form fe2+

Question 37

Where is haem synthesised

Answer 37

Mitochondria

Question 38

Where is globin synthesised

Answer 38

Ribosomes

Question 39

How is iron transported into the cell

Answer 39

Bound to transferrin, transferrin iron complex is endocytosed by vesciles and transported to mitochondria.

Question 40

Enzyme that regulates synthesis of haem and where is it located

Answer 40

Delta ALA -- Negative feedback step Located in mitochondria

Question 41

List 5 proteins in which haem can also be in apart from haemoglobin

Answer 41

Myoglobin, cytochromes, peroxidases, catalases, tryptophan

Question 42

What makes up haem component

Answer 42

Protoporphyrin ring with central iron atom-- ferroportoporphyrin

Question 43

Synthesis of globin-8 FUNCTIONAL GLOBIN CHAINS ARRANGED IN A AND B CLUSTER- list B cluster-- which chromosome and which arm and a cluster-- which chromosome and which arm

Answer 43

B cluster- B, Gamma, Delta, Epislon globin genes located on short arm of chromosome 11 A cluster- A1 , a2 and zeta globin genes on the short arm of chromosome 16

Question 44

which gene will

Answer 44

Foetus and adult

Question 45

Which stage of life will the chromosome 16 genes be responsible for

Answer 45

Embryo

Question 46

Which globin from the alpha cluster is the first to be produced

Answer 46

Zeta globin chains-- produced after conception, but also production stopped 7 weeks of gestation-- embroygenic

Question 47

a globin chain is produced when and until what happens if this isnt functioning properly

Answer 47

Pregestation and even through adult life embryonic death

Question 48

Which globin from the beta cluster is the first to be produced form what type of haemoglobin

Answer 48

gamma globin chain foetal haemoglobin

Question 49

When does gamma globin chain begin to tail off and which globin chaintakes over its place

Answer 49

Gamma globin starts to tail off few weeks postpartum and B globin chain starts to increase

Question 50

When does gamma globin chain begin to tail off and which globin chaintakes over its place

Answer 50

Gamma globin starts to tail off few weeks postpartum and B globin chain starts to increase

Question 51

Primary globin structure

Answer 51

a 141 AA | Non a 146 AA

Question 52

Secondary globin structure

Answer 52

75% a and B chains- helical arrangement

Question 53

Tertiary globin structure

Answer 53

Approximate sphere Hydrophilic surface (charged polar side chains), hydrophobic core Haem pocket

Question 54

How many globin chains in haemoglobin and ho many oxygens can haemoglobin bind to

Answer 54

4 globin chains 4 oxygens

Question 55

Phenomenon in which oxygen binding becomes easier after first

Answer 55

Postive cooperativity

Question 56

p50 2,3 DPG role which cells can high levels be found

Answer 56

when Hb is half saturated with O2 Promote deoxygenated state-- high levels found in metabolic cells

Question 57

What 4 factors does normal position of curve depend on

Answer 57

Concentration of 2,3-DPG H+ ion concentration (pH) CO2 in red blood cells Structure of Hb

Question 58

Left shift means? Vv

Answer 58

Greater affinity, less readily donating REDUCED CONCENTRATIONOF 2,3 DPG INCREASED pH Right shift-- less affinity, more readily donating oxygen INCREASED 2,3 DPB DECREASED pH

Question 59

Haemoglobinopathies-- 2 types are?

Answer 59

Structural variants of haemoglobin Defects in globin chain synthesis -- thalassaemia

Question 60

2 different ways of Classifying thalassaemia

Answer 60

Globin type affected Clinical severity- Minor trait-- asymptomatic Intermediate-- can require or not require transfusions Major-- require transfusions

Question 61

B Thalassaemia caused by

Answer 61

Deletion or mutation in B globin gene | Results in reduced or asbsent porduction of B globin chain

Question 62

Prevalence of B thalassaemia

Answer 62

Mainly in Mediterranean countries, Arabina peninusla, Iran, Indian subcontinent, Africa, Southern China, South East Asia

Question 63

Inheriting pattern of B thalassaemia

Answer 63

Autosomal Recessive mendelian ``` BB^0= Trait BB=Normal B^0B^0= Major B+ (Able to produce some betaglobin) BB+-- tRIAT B+B^0--BThal Intermedia ```

Question 64

Thalassaemia major cause

Answer 64

Carry 2 abnormal copies of the beta globin gene

Question 65

Thalassaemia Requires

Answer 65

``` regular blood transfusions Iron chelation therapy Splenectomy Supportive medical care Hormone therapy Hydroxyurea to boost HbF Bone marrow transplant ```

Question 66

When do you present

Answer 66

Clinical presentation usually after 4-6 months of life

Question 67

Other complications of B thalassaemia

Answer 67

Cholelithiasis and biliary sepsis Cardiac failure Endocrinopathies Liver failure

Question 68

How are transfusions carried out

Answer 68

Phenotyped red cells Regular transfusion 2-4 weekly If high requirement consider splenectomy

Question 69

Mangement of infection

Answer 69

Yersinia Other Gram negative sepsis Prophylaxis in splenectomised patients – immunisation and antibiotics

Question 70

Iron chelation therapy ALL 3 CAN BE USED IN COMBINATION

Answer 70

Start after 10-2 transfusions or when serum ferritin >1000 mcg/l Audiology and ophthalmology screening prior to starting Deferasirox (Exjade) Oral SE: rash, GI symptoms, hepatitis, renal impairment ``` DFO Long-established SE: vertebral dysplasia, pseudo-rickets, genu valgum, retinopathy, high tone sensorineural loss, increased risk of Klebsiella and Yersinia infection Compliance-- parenteral adminsitration Vitamin C ``` Deferiprone (Ferriprox) Oral Effective in reducing myocardial iron SE: GI disturbance, hepatic impairment, neutropenia, agranulocytosis, arthropathy

Question 71

Monitoring of iron overload

Answer 71

Serum ferritin -Acute phase protein Liver Biopsy- Rare T2 cardiac and hepatic MRI T2* cardiac and hepatic MRI <20ms – increased risk of impaired LF function Check annually or 3-6monthly if cardiac dysfunction -Ferriscan – R2 MRI Non-invasive quantitation of LIC Not affected by inflammation or cirrhosis

Question 72

HbE B Thalassaemia common where

Answer 72

Common combination in South East Asia

Question 73

Clinical Variation>

Answer 73

Clinically variable in expression can be as severe as B thalassaemia major

Question 74

Alpha Thalassaemia

Answer 74

Deletion or mutation in a globin gene(s) Reduced or absent production of a globin chains Affects both foetus and adult Excess Band Gamma chains form tetramers of HbH and Hb Barts respectively Severity depends on number of a globin genes affected

Question 75

Thalassaemia carrier

Answer 75

Thalassaemia minor / trait Carry a single abnormal copy of the beta globin gene Usually asymptomatic Mild anaemia

Question 76

Problems Associated with Treatment in Developing Countries

Answer 76

Lack of awareness of the problems Lack of experience of health care providers Availability of blood Cost and compliance with iron chelation therapy Availability of and very high cost of bone marrow transplant

Question 77

Screening and Prevention

Answer 77

``` Counselling and health education for thalassaemics, family members and general public Extended family screening Pre-marital screening? Discourage marriage between relatives? Antenatal testing Pre-natal diagnosis (CVS) ```

Question 78

Haemoglobinopathies offer protection against

Answer 78

Malaria-- make it hard for the parasite to enter rbc

Question 79

Lab diagnosis of B thalassaemia

Answer 79

FBC– Full blood count-- Microcytic Hypochromic indices Increased RBCs relative to Hb Film- Target cells, poikilocytosis but no anisocytosis Hb EPS / HPLC A-thal- Normal HbA2 & HbF, +/- HbH (LOSS OF 3 A GLOBIN GENES YOU MAY SEE ABRNOMAL HAEMOGLOBIN VARIANCE. IF YOU LOSE ALL 4- INCOMPATIBLE FOR LYF) B-thal- Raised HbA2 & raised HbF Globin Chain synthesis/ DNA studies Genetic analysis for β-thalassaemia mutations and XmnI polymorphism (in β-thalassaemias) and α-thalassaemia genotype (in all cases)