Beta-Thalassemia Major

DISCOVERY OF HEMOGLOBIN

1. Hemoglobin was discovered by Hünefeld in 1840.
2. Hemoglobin (Hb) is an iron-containing oxygen-transport metalloprotein in the red blood cells (rbc's) of all vertebrates (with the exception of the fish family Channichthyidae) as well as the tissues of some invertebrates.
3. Hemoglobin has the formula C2952H4664O832N812S8Fe4. 
4. Each hemoglobin molecule has 4 iron (Fe) atoms.
5. The role of hemoglobin in the blood was elucidated by French physiologist Claude Bernard.
6. Hemoglobin carries oxygen in the blood from the respiratory organs (lungs or gills) to the rest of the body (ie. the tissues). 
7. Hemoglobin is red because it contains heme, which is a bright cherry red molecule.
8. Hemoglobin is a bright cherry red molecule which can undergo oxygenation and deoxygenation reactions.
9. Hemoglobin's reversible oxygenation was described a few years later (1851-1859) by Felix Hoppe-Seyler.
10. Presence of oxygenated hemoglobin gives blood a bright red colour and the smell of fresh blood as present in hospital corridors.
11. Presence of deoxygenated hemoglobin gives blood a dark red-brown colour and this blood stinks of dead bodies (corpses) if left to stand at room temperature.
12. Cells and tissues require oxygen for aerobic glycolysis. Complete biological oxidation produces energy (ATP), carbon dioxide (CO2) and water (H2O).
13. Hemoglobin + CO2 = carbaminohemoglobin. Carbaminohemoglobin carries carbon dioxide
14. Hemoglobin is also found outside red blood cells and their progenitor lines. 
15. Other cells that contain hemoglobin include the A9 dopaminergic neurons in the substantia nigra, macrophages, alveolar cells, and mesangial cells in the kidney. In these tissues, hemoglobin has a non-oxygen-carrying function as an antioxidant and a regulator of iron metabolism.
16. In 1959, Max Perutz determined the molecular structure of hemoglobin by X-ray crystallography.

HEMOGLOBIN GENES AND HEME & HEMOGLOBIN SYNTHESES

Hemoglobin forms in the developing rbc (reticulocytes) in the bone marrow (BM).

Steps in heme & hemoglobin synthesis:

1. Glycine + succinyl-CoA = delta-aminolevulinic acid (d-ALA)
2. d-ALA exits into cytoplasm
3. d-ALA is reacted upon by enzymes
4. forms protoporphyrin
5. protoporphyrin re-enters mitochondria
6. Iron (Fe) is added to protoporphyrin
7. forms heme
8. Globin is made at ribosomes of rbc's and involves 2 chromosomes. Alpha globin chains are made by Chromosome 16. Beta globin chains are made by Chromosome 11.
9. Heme + Globin = Hemoglobin
10. Lead (Pb) is an inhibitor of heme and hemoglobin synthesis

HEMOGLOBIN STRUCTURE AND FUNCTION

1. Hemoglobin helps to carry oxygen to body tissues.
2. Hemoglobin helps to carry carbon dioxide for disposal by the lungs.
3. Rbc's contain hemoglobin.
4. The level of hemglobin in blood varies for males and females.
5. Males perform more physical activities and have higher hemoglobin levels in blood.
6. Females perform less physical activities and have lower hemoglobin levels in blood.
7. Hemoglobin has a quaternary structure characteristic of many multi-subunit globular proteins.
8. There are 4 types of globin chains: alpha (a), beta (b), delta (d) and gamma (g)
9. Hemoglobin consists of 4 subunits: 2 alpha globins + 2 beta/delta/gamma globins
10. Fetal hemoglobin is Hb F.
11. Adult hemoglobins are Hb A1 and Hb A2
12. Hb A1 = a2 b2 (alpha2 beta2)
13. Hb A2 = a2 d2 (alpha2 delta2)
14. Hb F = a2 g2 (alpha2 gamma2)
15. Hemoglobin can be saturated with oxygen molecules (oxyhemoglobin), or desaturated with oxygen molecules (deoxyhemoglobin).

POSTNATAL GENETICS:
(1) GLOBIN CHAINS IN HEMOGLOBINS
(2) HEMOGLOBIN VARIANTS

1. A globin chain is a polypeptide.
2. The amino acid sequence of a globin chain is determined by DNA sequences called genes.
3. There are 4 globin chains: alpha (a), beta (b), delta (d) and gamma (g)
4. Different globin chains are synthesized at different times during fetal development and till adult stage.
5. There is more than one hemoglobin gene.
6. The main form of hemoglobin present in adult man is hemoglobin A (HbA).
7. In humans, hemoglobin A is coded for by the genes, HBA1, HBA2, and HBB.
8. Hemoglobin contains 2 alpha globin chains and 2 beta, 2 delta or 2 gamma globin chains
9. Thus, many configurations of globin chains are possible:
10. Hb A1 = a2 b2 (alpha2 beta2) (heterotetramer, α2β2)
11. Hb A2 = a2 d2 (alpha2 delta2)
12. Hb F = a2 g2 (alpha2 gamma2) (HbF, α2γ2)
13. Fetal hemoglobin is Hb F.
14. Adult hemoglobins are Hb A1 and Hb A2
15. Normal hemoglobin types found in adults are; 

• Hemoglobin A (Hb A), which is 95-98% of hemoglobin found in adults, 
• Hemoglobin A2 (Hb A2), which is 2-3% of hemoglobin found in adults, and 
• Hemoglobin F (Hb F), which is found in adults up to 2.5%. It  is the primary hemoglobin that is produced by the fetus during pregnancy.

     16. In the embryo:

• Hb Gower 1 (ζ₂ε₂)
• Hb Gower 2 (α₂ε₂)
• Hemoglobin Portland I (ζ₂γ₂)
• Hemoglobin Portland II (ζ₂β₂).

     17. In the fetus: Hemoglobin F (α₂γ₂) 

      18. After birth:

• Hemoglobin A (α₂β₂) – The most common with a normal amount over 95%.
• Hemoglobin A2 (α₂δ₂) – δ chain synthesis begins late in the third trimester and, in adults, it has a normal range of 1.5–3.5%.
• Hemoglobin F (α₂γ₂) – In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. However, the level of Hb F can be elevated in persons with sickle-cell disease and beta-thalassemia.

    19. Variant forms that cause disease:

• Hemoglobin D-Punjab – (α₂β^D₂) – A variant form of hemoglobin.
• Hemoglobin H (β₄) – A variant form of hemoglobin, formed by a tetramer of β chains, which may be present in variants of α thalassemia.
• Hemoglobin Barts (γ₄) – A variant form of hemoglobin, formed by a tetramer of γ chains, which may be present in variants of α thalassemia.
• Hemoglobin S (α₂β^S₂) – A variant form of hemoglobin found in people with sickle cell disease. There is a variation in the β-chain gene, causing a change in the properties of hemoglobin, which results in sickling of red blood cells.
• Hemoglobin C (α₂β^C₂) – Another variant due to a variation in the β-chain gene. This variant causes a mild chronic hemolytic anemia.
• Hemoglobin E (α₂β^E₂) – Another variant due to a variation in the β-chain gene. This variant causes a mild chronic hemolytic anemia.
• Hemoglobin AS – A heterozygous form causing sickle cell trait (SCT) with one adult gene and one sickle cell disease gene
• Hemoglobin SC disease – A compound heterozygous form with one sickle gene and another encoding Hemoglobin C. Hemoglobin Hopkins-2 - A variant form of hemoglobin that is sometimes viewed in combination with Hemoglobin S to produce sickle cell disease.

      20. List of known hemoglobin variants

• Hb Kansas
• Hb S
• Hb C
• Hb E
• Hb D-Punjab
• Hb O-Arab
• Hb G-Philadelphia
• Hb Hasharon
• Hb Lepore
• Hb M
• Hb F
• Hb Hope
• Hb Pisa
• Hb J
• Hb N-Baltimore

HEMOGLOBIN F (Hb F)

1. Fetal hemoglobin (HbF, α2γ2) is found in the developing fetus, and binds oxygen with greater affinity than adult hemoglobin A.
2. Hemoglobin F (Hb F) is the primary hemoglobin that is produced by the fetus during pregnancy.
3. The levels can be normal to increased in beta thalassemia. 
4. Hemoglobin F frequently increases in individuals with sickle cell anemia and sickle cell-beta thalassemia. 
5. Individuals with sickle cell and increase of Hb F have a milder case of the disease. 
6. There are situations where the Hb F is increased. This rare condition is called Hereditary Persistence of Fetal Hemoglobin (HPFH).
7. HPFH is a group of disorders where the Hemoglobin F is increased without signs or clinical features of thalassemia. 
8. Some different ethnic groups have different mutations that cause HPFH. 
9. Hb F can also be increase by acquired conditions that involve the red blood cells. 
10. Elevated Hemoglobin F levels are also associated with Leukemia and myeloproliferative disorders.

HEMOGLOBIN H

1. Hemoglobin H (Hb H) increases the affinity for oxygen. 
2. Hb H holds onto the oxygen instead of releasing it into tissue and cells. 
3. Hb H usually occurs in some alpha thalassemia and is composed of four beta globin (protein) chains (beta tetramer, β4). 
4. This variant is usually produced in response to a severe shortage of alpha chains, and usually cause beta chains to function abnormally.

HEMOGLOBINOPATHY VS THALASSEMIA

1. Hemoglobinopathy is a hereditary condition involving an abnormality in the structure of hemoglobin.
2. Mutations in the genes for the hemoglobin protein in a species result in hemoglobin variants. Many of these mutant forms of hemoglobin cause no disease. Some of these mutant forms of hemoglobin, however, cause a group of hereditary diseases termed the hemoglobinopathies. The best known hemoglobinopathy is sickle-cell disease (SCD).
3. Hemoglobin variants are a part of the normal embryonic and fetal development. They may also be pathologic mutant forms of hemoglobin in a population, caused by variations in genetics. Some well-known hemoglobin variants, such as sickle-cell anemia (SCA), are responsible for diseases and are considered hemoglobinopathies. Other variants cause no detectable pathology, and are thus considered non-pathological variants.
4. Thalassemia is a reduced or no production of a or b globin chain. The a and b globin chains have normal structures.

HEMOGLOBIN CONTENT OF ERYTHROCYTES

1. Hemoglobin carries oxygen to tissues.
2. Hemoglobin carries carbon dioxide back to the lungs for expulsion (expiration).
3. Hemoglobin is not synthesized (made) in red blood cells (erythrocytes).
4. Hemoglobin synthesis occurs in nucleated reticulocytes.
5. Hemoglobin synthesis is regulated (controlled).
6. Erythrocytes have a half-life of 120 days (ie they survive approximately 120 days before they are cleared from the blood circulation).
7. When old erythrocytes are removed and broken down, hemoglobin in them too is broken down (degraded).
8. New hemoglobin will need to be synthesized to replace that lost.
9. If hemoglobin synthesis is slowed (or lags behind), there will be either insufficient hemoglobin, lack of hemoglobin or defective hemoglobin in the erythrocytes.

THALASSEMIAS

1. Thalassemias are a group of genetic blood disorders 
2. Thalassemias are inherited blood disorders
3. Thalassemia patients make normal globin chains, but at reduced rates
4. These blood disorders have defective production of hemoglobin
5. The thalassemias are autosomal recessive disorders which result in reduced production of one or more of the subunits of hemoglobin
6. There are 2 forms of thalassemia: alpha- and beta-thalassemia
7. There are two forms of beta-thalassemia: thalassemia minor and thalassemia major (also called Cooley's anemia)

ALPHA-THALASSEMIA

1. Reduced production of alpha globin chains
2. Results from gene deletion
3. Alpha-thalassemias have reduced production of alpha-globin chains to make new hemoglobin
4. The resulting hemoglobin molecule has either 1 or no alpha-globin chain; 
5. The resulting hemoglobin configuration can be b2 or ab2, which are defective hemoglobins.
6. Hemoglobin with b2 configuration occurs in alpha-thalassemia major.
7. Hemoglobin with ab2 configuration occurs in alpha-thalassemia minor.

ALPHA THALASSEMIA GENE DELETION

1. The alpha globin gene is present on Chromosome 16.
2. Alpha thalassemia results from gene deletion.
3. ONE alpha globin gene deletion is asymptomatic.
4. TWO alpha globin gene deletions, can be either from the same chromosome (cis deletion) or from different chromosomes (trans deletion)
5. Cis deletion is worse than trans deletion. 
6. Inheritance of cis deletion from both parents by the offspring is dangerous as it results in severe alpha thalassemia (no alpha globin chain).
7. THREE alpha gene deletions results in Hb H, with b2 dimer and b4 tetramer.
8. FOUR alpha gene deletions results in Hb Bart's, with g4 tetramer.

TREATMENT OF ALPHA THALASSEMIA

1. alpha carrier and Hb H don't need treatment as they are fine.

BETA THALASSEMIA

1. Reduced or no production of beta globin chains
2. It is genetically determined
3. The beta globin gene is located on a chromosome 11 (ie an autosome, not a sex chromosome, not X or Y)
4. Two copies are derived in the child, one from the father, and one from the mother
5. Each chromosome has an allele for beta globin
6. Each allele codes for beta globin chain, one from the father, one from the mother
7. Beta thalassemia also occurs as a result of gene mutation 

BETA THALASSEMIA GENE MUTATIONS

1. There are 2 types of beta globin gene mutations: B+ and B0
2. In B+ gene mutation, the gene is capable of producing beta globin chain even though at reduced rate; there is some production of beta globin chain
3. In B0 mutation, the gene is incapable of producing beta globin chain; there is no production of beta globin chain
4. Possible genetic combinations are thus: B+B+, B+B0, B0B+, and B0B0
5.         B+B+ = produce beta globin chain .... beta thalassemia major
6.         B+B0 or B0B+  = produce beta globin chain ... beta thalassemia minor
7.         B0B0 = do not produce beta globin chain .... beta thalassemia major

BETA-THALASSEMIA MAJOR & MINOR

1. Beta-thalassemias are of 2 types - thalassemia major and thalassemia minor
2. Thalassemia major is also called Cooley's anemia
3. Production of beta-globin chain of hemoglobin is reduced or none
4. The resulting hemoglobin molecule has either a2 or a2b, which are hemoglobins with reduced beta globin or no beta globin
5. Hemoglobin with a2 configuration occurs in beta-thalassemia major.
6. Hemoglobin with a2b configuration occurs in beta-thalassemia minor.
7. Beta thalassemia minor is a2BB+ with mild anemia and increased Rbc count
8. Beta thalassemia major is a2B+B+ or a2B0B0 with severe anemia and increased Rbc count
9. In B0B0, there is no beta globin production; there will be excess alpha globin chains
10. When there is no beta globin production, and there is excess alpha globins, 4 alpha globin chains combine to form an alpha globin tetramer, which in turn forms ineffective hemoglobin. This results in ineffective erythropoiesis.

THE BONE MARROW

1. When rbc count is low, the bone marrow compensates by producing more rbc's.
2. Rbc's with alpha globin tetramers (a4) are abnormal and are destroyed while still in the bone marrow.
3. Any rbc's with alpha globin tetramers (a4) that escaped from the bone marrow and are released into the circulation, are trapped by the endothelial system (eg  spleen) and are destroyed, thus removing them from the systemic circulation ... leading to anaemia.
4. Any rbc's containing alpha4 tetramer will be destroyed ... leading to anaemia.
5. When the body senses a rapid reduction in rbc count, it tries to make more rbc's in the bone marrow.
6. The bone marrow will become hyperactive in order to make rbc's.
7. The bone marrow is now hyperactive ... trying to make a lot of new rbc's even though beta globin synthesis is reduced.
8. When the bone marrow is active and increasing its mass (expanding), it compresses the cortex, thus thinning the cortex.
9. The thick cortex gives bone strength. Once the cortex is thinner, bone strength is reduced and the bones are weakened, and can possibly lead to pathological fracture. This can happen in thalassemia patients. The body will try to produce rbc's using all the bone marrow in the body.
10. Normally after birth, erythropoiesis is limited to a few bones - bone marrow of the central bones - of the skull, ribs, vertebra and a few long bones, ribs. However, in beta thalasemia with anemia, almost all bones with bone marrow will try to make rbc's.
11. As a result, when the bone marrow cavity expands rapidly to produce more rbc's in the skull and the cortex thins, the skull marrow will crack, giving a hair-on-end or crew cut appearance. Expansion of the bone marrow of the face will give a chipmunk appearance. These are findings in thalassemia patients.

VIRAL INFECTIONS IN THALASSEMIA PATIENTS

1. Parvovirus P19 infection halts rbc production for 1-2 weeks in normal healthy persons, as we have a large reserve of rbc's.
2. However, in beta thalassemia major patients, Parvovirus P19 infection is dangerous. They hardly have any rbc reserve and need all the rbc's in the bone marrow.

GEOGRAPHIC ORIGIN OF THE THALASSEMIAS

1. The Indians are classified as Caucasanoids. They are Aryans of Italian and Greek origins. Thus, they inherit beta-thalassemia from their Mediterranean ancestors.
2. The Malays of Indian heritage also inherit beta-thalassemia from their Indian Mediterranean ancestors. Beta-thalassemia is common among this Malay population in Malaysia.

THALASSEMIA COMMON CLINICAL FEATURES

1. Anaemia
2. Manifestation of increased hemopoiesis in face and skull
3. Extramedullary hemopoiesis in liver and spleen results in nucleated rbc's
4. Hepatosplenomegaly

BETA-THALASSEMIA MAJOR SYMPTOMS

1. fatigue, weakness, or shortness of breath
2. a pale appearance (pallor) or a yellow color to the skin (jaundice)
3. irritability
4. deformities of the facial bones
5. slow growth (retarded growth, short for stature)
6. a swollen abdomen
7. dark urine

INVESTIGATIONS OF THALASSEMIA

1. Microscopy
2. Skull radiography
3. Hemoglobin

MICROSCOPY OF ERYTHROCYTES IN THALASSEMIA

1. The microscope is the best way to examine erythrocyte appearances.
2. Erythrocytes with insufficient or deficient hemoglobin will not look normal when examined under the microscope.
3. Microscopic findings are diagnostic.
4. Pale erythrocytes point to anemia.
5. "Mexican hats" or target cells (or targets) point to thalassemia.
6. Small erythrocytes (microcytic) point to iron deficiency - microcytic anemia.
7. Big pale erythrocytes (macrocytic) point to vitamin B deficiency - macrocytic anemia.
8. Spiky erythrocytes (echinocytes) have spike-like projections on their surfaces.
9. Spindle-shaped erythrocytes
10. Sickle-shaped erythrocytes occur in sickle cell anemia (SCA)
11. Extramedullary hemopoiesis (occurs in liver and spleen) will lead to nucleated rbc's.
12. Liver and spleen are not equipped to make rbs'c. So these organs will make immature nucleated rbc's which are seen in the circulation.
13. Microcytic hypochromic anaemia
14. Normal rbc's are round biconcave discs, with 2/3 red due to hemoglobin and 1/3 pale due to less hemogloin
15. Target cells are seen thalassemia. Target cells lack tensile strength and rbc biconcavity has blebs (outgrowths) and are filled with hemoglobin. The rbc's now look like target cells, like bull's eye

TREATMENT OF BETA THALASSEMIA

1. Blood transfusion. Blood transfusion bags contain iron (Fe); 1 bag = 250 mg iron. When we transfuse patients at 4 weeks interval, we also supply them with extra iron (blood > rbc > Hb > rbc's are degraded in 120 days and free iron is released). Iron has no specific way for excretion. So iron is deposited in tissues, leading to hemosiderosis or hemochromatosis. So, iron overload results. A lot of problems thus result.
2. Iron chelation therapy

External links

Hemoglobin
https://en.wikipedia.org/wiki/Hemoglobin
https://youtu.be/M4cKGWP12w4

HBB
https://en.wikipedia.org/wiki/HBB

Hemoglobin variants
https://en.wikipedia.org/wiki/Hemoglobin_variants

Hemoglobinopathies
https://youtu.be/89feCoBXRGE

Thalassemia

https://www.medicinenet.com/beta_thalassemia/article.htm
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943116/pdf/JBB2010-706872.pdf
http://www.tandfonline.com/doi/full/10.1080/00313020600922538?scroll=top&needAccess=true

Theodora Lam and Brenda S.A. Yeoh. 2004. Negotiating ‘home’ and ‘national identity’: Chinese-Malaysian transmigrants in Singapore
http://onlinelibrary.wiley.com/doi/10.1111/j.1467-8373.2004.00235.x/full

Videos - Thalassemia

https://youtu.be/39tW_o3SqDM
https://youtu.be/uowfqUc4ems
https://youtu.be/M4cKGWP12w4