THALASSAEMIA

DEFINITION, CLASSIFICATION, PATHOGENESIS AND COMPLICATIONS OF THIS DISORDER.

PRESENTATION BY NDEGE MERCY MAKENA,H31/21842/2007,

Q. 219

SUPERVISOR: DR. OKEMWA

What do you understand by the term “ Thalassaemia ”? Classify the disorder and discuss the pathogenesis and complications of this disorder.

DISCUSSION POINTS:

Definition and understanding of thalassaemia. Classification of thalassaemia. Pathogenesis. Complications.

HAEMOGLOBIN

Definition, structure and function

Haemoglobin is a large molecule found in red blood cells. It is a conjugated protein that is comprised of four haem

groups. It is a tetramer. Chromosome coding is on number 16 for alpha chains(α)

and number 11 for non-alpha chains(β,δ,γ). Functions of haemoglobin include:

a) Oxygen and carbon dioxide transport.

b) Acid base balance.

Reference Values

Female : 11.5 – 16.5 g/dl

Male : 13.5 – 18.0 g/dl

Infants ( full term, cord blood) : 13.5 – 19.5 g/dl

Children, 3 months : 9.5 – 13.5 g/dl

Children, 1 year : 10.5 – 13.5 g/dl

Children, 3-6 years : 12.0 – 14.0 g/dl

Children, 10-12 years : 11.5 – 14.5 g/dl

Haemoglobin structure has: Primary structure – as alpha chain. Secondary structure – folding peptide chain. Tertiary structure – folding protein – niche H. Quantenerary structure – coming together of four

chains.

Haemoglobin STRUCTURAL FORMULAAdultHb A1

Hb A2

- α2β2

- α2δ2

FetalHb FHb Bart’s

- α2γ2

- γ4

EmbryonicHb Gower 1Hb Gower 2Hb Portland

- ζ2ε2

- α2ε2

- ζ2γ2

The human haemoglobin molecule is made up of two pairs of globin chains, each with a haem group attached. Above are the seven different globin chains. HbA1 is found in adults and children, HbF is the predominant haemoglobin

in fetal life and just after bith, Hb Portland is transient and HbA2 and HbF are found in small quantities in adult life.

Haemoglobin Disorders

They can be divided into:a) Qualitative abnormalities e.g. HbS,

HbC, HbE. b) Quantitative abnormalities e.g.

Thalassaemia.

1)Qualitative abnormalities, also known as hemoglobinopathies are due to abnormal structural haemoglobin variants. In sickle cell anaemia, HbS, the red cells take a sickle shape. This is caused by substitution of valine for glutamic acid in position 6 in the β chain.

Others:

Increased haemoglobin affinity for oxygen e.g. Hb Capetown. This causes pure red cell polycythemia.

Decreased haemoglobin affinity for oxygen e.g. Hb Kansas.

2)Quantitative abnormalities are due to the deficient synthesis of one or more of the polypeptide chains of haemoglobin. Also known as thalassaemias. They are

generally inherited as alleles of one or more of the globin genes located on the chromosomes.

Haemoglobin disorders generally result from either the synthesis of an abnormal haemoglobin or due to reduced rate of synthesis of normal α- or β-globin chains. (the α – and β –thalassaemias.)

THALASSAEMIA

HISTORY

Thalassaemia or thalassemia is a combination of two Greek words, thalassa(sea) and haema(blood). In 1925, Colley and Lee described this syndrome in Mediterranean region. In 1938, Caminopetros observed that parents of a thalassemic child had abnormal osmotic fragility test. In 1940, Wintrobe observed basophilic stippling in healthy individuals and described thalassemia minor.

DEFINITION

Thalassemia is an inherited autosomal recessive blood disease. Genetic defect could be either due to mutation or deletion, that results in reduced rate of synthesis or lack of synthesis of one of the globin chains that make haemoglobin.

In a healthy individual, the amount of haemoglobin produced is generally normal but in individuals with thalassemia, the genetic defect results in production of an abnormally low quantities of a given haemoglobin chain or chains.

Haemoglobin is deficient due to:

Defective synthesis of a polypeptide chain e.g. in β-thalassemia, where there is β chain deficiency.

Complimentary chain formed is in excess and its precipitation results to its removal by the spleen.

CLASSIFICATION

Thalassemia are classified depending on the chain that is affected.(α and β )

There are many forms of thalassemia. Each type has many different subtypes. Both the alpha (α-) and beta (β-) thalassemia have these two forms.

Thalassemia major – which is a severe and transfusion dependent disorder.

Thalassemia minor – which is a symptomless carrier state.

GEOGRAPHICAL DISTRIBUTION

Alpha thalassemia : Sub-saharan Africa, Mediterranean, Middle East, Sri-Lanka, South China Thailand, Indonesia, South-east Asia.

Beta thalassemia : Broad belt from Mediterranean and parts of North and West Africa through the Middle East and Indian sub-continents to South-east Asia. Highest in

Yugoslavia, Romania, south of former USSR and south of China. Also in Thailand and Indonesia.

ALPHA ( α ) THALASSEMIA.

There are four α thalassemia syndromes. These are: Silent carrier : (-α/αα) α - thalassemia trait : less affected(-α/-α) ,

more affected(--/αα) Hb H disease : (--/-α) Hydrops fetalis : (-/-)

They reflect the inheritance of molecular defects affecting the function of:

1) One of the two globin genes on a single chromosome in silent carrier.

2) Two of globin genes in a single chromosome in α -thalassemia trait.

3) Three globin genes in Hb H disease.4) Four of the α-globin genes in Hb Bart’s

hydrops fetalis. The structural gene for α-globin is duplicated on

chromosome 16, and each diploid cell contains four copies of the α- globin gene. The α chain synthesis is directed by two genes , α1 and α2, on chromosome 16, and β and δ

chain synthesis by single β and δ genes on chromosome 11.

Gamma(γ) chain synthesis is directed by two genes Gγ and Aγ also on chromosome 11.Four chains are associated in the form of a tetramer. The α1β1 (and equivalent α2β2) contact is the strongest and involves many amino acids with interlocking side chains. The α1β2 (and equivalent α2β1) contact is less extensive and the contact between similar chains is relatively weak.

α-thalassemias result in decreased alpha-globin production, hence fewer alpha-globin chains produced, resulting in an excess of β chains in adults and excess γ (gamma) chains in newborns.

α-thalassemia minor is found in individuals who are heterozygotes for the α-thalassemia gene. It is symptomless and difficult to detect.

There is another gene which alone is silent but which when combined with one α-thalssemia,leads to mild anaemia. This is Hb H disease.

Some mutations abolish expression of an α-globin gene(αo) wheras others reduce expression of the gene to a variable degree(α+).

These syndromes are usually a result of deletions of one or more genes although approximately 20% are non- deletional.

Hb Constant Spring and Hb Gun Hill

Hb Constant Spring is a non- deletional form of α- thalassemia caused by mutations affecting the termination of the translation process. This gives rise to an elongated unstable chain (rare).

Deletion of a portion of the amino acid sequence causes Hb Gun Hill.

Point mutations causing dysfunction of genes also occurs.

α-Thalassemia forms associated with mental retardation

Involves mutation in a gene on chromosome 16 (ATR-16) or on chromosome X. The latter affects boys causing greater mental retardation and some physical abnormalities. ATR-16 causes mild mental retardation.

Myelodysplasia in elderly patients.

BETA ( β ) THALASSEMIA

Many different mutations cause β -thalassemia and related disorders. Most thalassemias of type β are a result of point mutations affecting the β-globin gene on chromosome 11,

but some large deletions are also known. The effect of different mutations varies greatly.

β- thalassemia syndromes: β- thalassemia silent carrier. β- thalassemia trait.(β+) Thalassemia intermedia. Thalassemia major.(βo , homozygous β

thalassemia, Cooley’s anaemia) Some of the rare mutations (e.g. exon 3 and β globin

gene) are so severe that they can produce the clinical syndrome of thalassemia intermedia in the heterozygous state(β –thalassemia trait). Others are mild alleles that produce thalassemia intermedia in the homozygous or compound heterozygous state(βo -thalassemia) , and some are so mild that they are completely hematologically silent(silent carrier), with normal MCV and HbA2 in heterozygous states(β –thalassemia trait). In between are the great majority of β -thalassemia traits(β+)and β -thalassemia major(βo)alleles, which cause β thalassemia major in homozygous or compound heterozygous state.

PATHOGENESIS

In thalassemia Heinz body formation is the principle mechanism of haemolysis.

In thalassemia, there is a relative excess of either β- chains in α- chain deficiency (alpha- thalassemia) or α- chains in β- chain deficiency (beta- thalassemia). The chains do not form tetramers but rather they bind to the red cell membranes producing membrane damage and at high concentrations they form toxic aggrgates.

Chains in excess accumulate and precipitate in erythroblasts and mature red cell precursors in the bone marrow, resulting in severe ineffective erythropoiesis. The few red cells that manage to leave the bone marrow are laden with precipitated chains and these are rapidly

removed by the reticuloendothelial system. This leads to haemolysis. Intermedullary red cell destruction shortens the red cell life span and causes peripheral hemodilution.

Capacity of individual patients to synthesize γ- globin modulates the clinical severity. This is because it improves red cell production. Severity of thalassemias is determined by the degree of globin chain imbalance rather than by the absolute level of either α- or β-globin synthesis.

Coinicident inheritance of an α thalassemia mutation also reduces chain imbalance in patients with homozygous or heterozygous β-thalassemia.

Clinical syndromes associated with thalassemia arise from the combined consequences of:

1) Inadequate haemoglobin production. This causes anaemia with hypochromia and microcytosis.2) Unbalanced accumulation of one type of globin chain. This leads to ineffective erythropoiesis followed by haemolysis.

Severity of disease depends on the nature of the mutation.Mutations are characterized as:

a) β thalassemia major (βo) : if the mutation prevents any formation of β chains (which is the most severe form of β thalassemia. )

- It occurs in one of four offspring if both parents are carriers of the β-thalassemia trait.- Mutations may be within the gene complex itself or in the promoter or enhancer region.- Thalassemia major is often as a result of inheritance of two different mutations each affecting β-globin synthesis (compound heterozygotes).e.g. sickle cell/β- thalassemia.- It is transfusion dependent. - Severe β-thalassemia is also called Cooley’s anaemia.

b) β thalassemia intermedia : if the mutation allows some β chain formation to occur. Mild form of β thalassemia major.They do not need regular transfusions. May be due to homozygous β-thalassemia with production of more Hb F than usual, mild defects in β chain synthesis, or by β-thalassemia trait of unusual severity etc. No regular transfusion requirement.

c) β-thalassemia trait (β thalassemia minor or β+ thalassemia) : this is a heterozygous state for the β-thalassemia gene. It s characterized by a moderate reduction in β chain synthesis. Carriers of this syndrome are asymptomatic except in cases of stress e.g. pregnancy, where they become more anaemic.

DELTA (δ) THALASSEMIAS

3% of adult haemoglobin is made of alpha and delta chains. Mutations can occur which affect the ability of this gene to produce delta chains.

THALASSEMIA IN COMBINATION WITH OTHER HAEMOGLOBINOPATHIES.

Thalassemia can co-exist with other haemoglobinopathies. The most common of these are:a) Haemoglobin S/thalassemia- clinically similar to

sickle cell anaemia with the additional feature of splenomegally. Sickle/ βO- thalassemia trait is associated with severe sickle cell disease. If it is

sickle/β+ thalassemic allele it gives rise to mild sickling disorder.

b) Haemoglobin C/thalassemia- in causes a moderately severe haemolytic anaemia with splenomegally if its βo and a milder disease if its β+.

c) Haemoglobin E/thalassemia- clinically similar to thalassemia major or thalassemia intermedia.

Structural haemoglobin variants related to thalassemia include:

Ustable haemoglobin e.g. Hb Sydney. (This haemoglobin is detected by the presence of abnormal haemoglobin precipitates called Heinz bodies.)

Hereditary persistence of fetal hemoglobin.Major transition from fetal to adult haemoglobin occurs in perinatal period. Most abundant haemoglobin is HbA1

but there are small amounts of HbA2 and HbF. HbF is restricted to F cells but the amount of HbF per cell can be increased in certain conditions particularly if there is rapid bone marrow regeneration.

Congenital methaemoglobinaemia due to haemoglobin variants.

Thalassemic structural variants. These are characterized by both a biosynthetic defect and an abnormal structure. E.g. Lepore syndrome which is a hybrid haemoglobin molecule of δβ segments.Unequal crossing over produces δβ fusion genes.Can also be of γ and δ segments in anti-Lepore Hb.

CLINICAL PRESENTATIONS

1) β –thalassemia trait( β-thalassemia minor): mild to moderate erythroid hyperplasia,slight ineffective erythropoiesis, pregnancy, iron deficiency,folic acid deficiency, intercurrent illness worsens anaemia and tendency to develop gallstones.

2) α -thalassemia minor (silent carrier) :is symptomless and difficult to detect.

3) α –thalassemia trait : not usually associated with anaemia. However, there is the possibility of acquiring mild anaemia with microcytosis and hypochromia.

4) Silent carrier (δ or β):presence of small amounts of Hb Bart’s in cord blood.

5) β –thalassemia major: increase in Hb F, severe haemolytic anaemia, massive hepatosplenomegally,

abdominal swelling and symptoms due to pressure on surrounding organs.-Changes in skeletal system, characteristic mongoloid facies due to expansion of the marrow in the malar bones, x-ray changes in the skull, long bones, hands and feet,bossing of the skull with a hair on end appearance, cortical thinning causing pathological fractures and bone pain occurs, deformities of long bones due to osteoporosis and impairement of growth may result in small stature.-The menarche is often delayed, secondary sex characteristics is undeveloped, pericarditis related to iron deposition, extramedullary haemopoiesis, organ dysfunction due to iron overload due to regular tranfusion, pancreatic haemosiderosis causing diabetes and cirrhosis due to iron deposition in the liver, haemosiderosis of cardiac muscle causing arrhythmias,heart block and chronic congestive heart failure.- Occassional clinical features eg. jaundice, epistaxis, skin pigmentation, leg ulcers and gall stones. In children it causes anorexia, diarrhoea, loss of body fat, recurrent fever and severe anaemia which results in cardiac dilatation. If the spleen is removed severe infection can occur leading to septicaemia and eventually mortality.

-Oxygen depletion in the body becomes apparent within the first 6 months of life. Death occurs within a few years if left untreated.- It is blood transmission dependent.

6) α- thalassemia major : complete inability to produce α-chains. Major component is Hb Bart’s. Incompatible with life.

a)Hydrops fetalis:very little functional haemoglobin, still birth, death in utero or birth of affected fetus with

severe anaemia and oedema. It causes congestive heart failure. -Mothers of hydropic infants have a high incidence of toxaemia of pregnancy and postpartum hemorrhage due to massive placenta.

7) Hb H disease: red cells are sensitive to oxidative stress,

moderately severe haemolytic anaemia, icterus, splenomegally, folic acid deficiency, pigment gall stones, leg ulcers, increased susceptibility to infection, jaundice, moderate bone marrow erythroid hyperplasia, red cell fragmentation, hypochromia and microcytosis, severe deficit in functional haemoglobin and oxygen carrying capacity, premature destruction of red cells.

8) Thalassemia intermedia: no regular transfusion required.

9) α –thalassemia traits. αo –thalassemia trait:microcytic hypochromic indices. Haemoglobin is normal or slightly reduced. α+ –thalassemia trait can be completely silent or with borderline microcytosis with a slightly reduced or normal MCHC.

10) Mental retardation.

11) Myelodysplasia in elderly patients.

LAB FINDINGS

1) β –thalassemia trait: presence of target cells, basophilic stippling, elliptocytosis, normal red cell distribution width, normal MCHC, normal reticulocyte count, decreased Hb A2, increased Hb F or HbA1 , mild anaemia with microcyosis

and hypochromia or absent ,reduced red blood cells, inclusions in bone marrow, rare peripheral red cells, reduced MCV and poikilocytosis.

PHENOTYPES: a) High HbA2 β-thalassemiab) δβ-thalassemia has increased HbF and low HbA2

c) High HbA2, high HbF β-thalassemiad) Normal HbA2 β-thalassemia with normal or low HbA2

levels and hypochromic microcytic red cells.

2) α –thalassemia trait: erythrocytosis, HbA2 and Hb F are normal or low, : presence of target cells, basophilic stippling, elliptocytosis, normal red cell distribution width, mild anaemia with microcyosis and hypochromia ,normal MCHC, presence of Hb H inclusion bodies.

3) β –thalassemia major: presence of α- chain inlusions which appear as irregularly shaped bodies close to the nucleus of normoblasts, increased red cell count, low MCV, low MCH, increase in plasma volume,absence of HbA1 increased Hb A2 or Hb F, leptocytosis and prescence of nucleated red cells.

4) α- thalassemia major: increased red cell count, low MCV, low MCH, presence of Hb H inclusion bodies, hydrops

fetalis (hypochromic macrocytes, numerous nucleated red cells.)

5) Hb H disease: increased reticulocyte count, characterized by marked poikilocytosis(tear-drop poikilocytes) and anisochromasia, red cell fragments, red cells contain Hb H together with some Hb Bart’s.

6) β -thalssemia minor (heterozygous β thalassemia): normal haemoglobin or poikilocytosis and mild microcytic hypochromic anaemia with elevated HbF and Hb A2 .

Complications: untreated thalassemia major causes:

a) Heart failure

b) Liver problemsc) Increased susceptibility to infectionsd) Blood transfusion can help control

some symptoms but may result in too much iron which can damage the heart, liver and endocrine system.

Autopsy findings: a) Hydrops fetalis:

- extensive extramedullary hematopoiesis.

- Placental hypertrophy.b)Severe β- thalassemia:- extraordinary expansion of the bone marrow at the expense of bony structures.- extamedullary hematopoiesis.- iron deposition in almost all organs in children.

Methods for investigation of Thalassemia.

Full blood count and in some cases reticulocyte count. Hb A2 measurement by cellulose acetate

electrophoresis with elution or microcolumn chromatography.

Automated high performance liquid chromatography.(HPLC)

Quantitation and assessment of the distribution of Hb F.

Assessment of iron status. Demonstration of red cell inclusion bodies. DNA analysis.

TREATMENT

Thalassemia major often involves regular blood transfusion.

Chelation therapy eg. desferrioxamine, desferasirox. Folate supplements Allogenic bone marrow transplantation. Prenatal diagnosis. Splenectomy.

Vitamin supplementation( ascorbic acid, vitamin E, folic acid, trace metals.)

Gene therapy. Bone marrow transplantation may be considered if

affected infant is delivered safely. Intra-uterine transfusion of fetuses if treatment after

delivery is contemplated. At risk pregnancies should be considered for

termination. Pharmacologic manipulation of Hb F synthesis.

BENEFITS

-Thalassemia trait are prevalent in populations that evolved in humid climates where malaria was endemic. Thalassemias protected these people from malaria due to the blood cells’ easy degradation.(heterozygous advantage)

- Heterozygous β- thalassemia offers some protection against coronary heart disease.

-Protection against severe anaemia if there presence of the thalassemia trait.

REFERENCE POINTS:

Nathan and Oski’s Hematology of Infancy and Childhood (7TH Edition).

Postgraduate Haematology; Hoffbrand Catovsky Tuddenham.

Essential Haematology (5TH Edition). Dacie & Lewis Practical Haematology (10TH Edition) Wikipedia. NCBI- PubMed Health U.S National Library of

Medicine and National Institute of Health. University of Nairobi Level 2 MBChB lecture notes. General Pathology(5TH Edition) ; J.B Walter, M.S.

Israel. Basic Pathology; Robbins.