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HAEMOGLOBINOPATHIES Disorders of globin synthesis rather than haem synthesis. Qualitative Disorders Abnormal haemoglobins are formed when the sequence of globin chain amino acids is altered. There is usually only a single amino acid substitution in one of the globin (polypeptide) chains. Quantitative Disorders Thalassaemias result from a lack of production of particular globin chains to maintain adequate Hb levels. Hereditary persistence of fetal haemoglobin (HPFH) results from a failure to switch from fetal to adult Hb.


GLOBIN CHAIN SYNTHESIS

Occurs on the ribosomes of developing red cells. Various inherited genes direct the process. Each gene results in a specific polypeptide chain. A nucleated red cell contains four alpha (α), two zeta (ζ), two beta (β), two delta (δ), two epsilon (ε) and four gamma (γ) genes.


• Alpha (α1, α2) and zeta genes are on chromosome 16. • Beta, delta, epsilon and gamma genes (γG, γA) on chromosome 11. • Products are alpha, zeta, beta, delta, epsilon and gamma chains. • Epsilon and zeta chains only appear during embryonic development. • These two chains, plus the alpha and gamma chains, form the embryonic haemoglobins: Hb Gower 1 (ζ2ε2), Hb Gower 2 (α2ε2) and Hb Portland (ζ2γ2). • Epsilon and zeta chains are produced till 3 months after conception.


• Gamma chain production is most active from the third fetal month until birth. The major haemoglobin in the fetus is Hb F (α2γ2). By two years, Hb F falls to less than 2%. • Gamma chains occur as a mixture of two types: G-gamma (Gγ) and A-gamma (Aγ). • Beta chain production rises gradually prenatally and reaches adult percentages by 6 months postnatally. • Normal adult haemoglobins are tetramers consisting of 2 alpha plus 2 non-alpha globin chains. • Adult red cells contain the following: 95-97% Hb A (α 2β2), 23% Hb A2 (α2δ2) and <2% Hb F (α2γ2).


• Each globin chain links with one haem molecule to form haemoglobin; 2α, 2β and 4 haem groups. • Alpha chains contain 141 linear amino acid residues. Beta chains contain 146. The order of amino acids is critical for Hb structure and function. Each amino acid is numbered according to its position in its chain. • The α1β1 contact is the strongest and involves many amino acids with interlocking side chains; the α1β2 contact is less extensive. Contacts between like chains are relatively weak. • Binding of haem into the haem pocket in each chain stabilizes the whole molecule. If the haem attachment is weakened, the globin chains dissociate into dimers or monomers.


ABNORMAL HAEMOGLOBINS (Structural variants) Most Hb variants are single amino acid substitutions in one type of globin chain. Variants are inherited as codominant traits. The substituted amino acid may lead to instability of the individual globin chain or the tetrameric structure of the Hb molecule. If the substitution affects the haem pocket, the iron atom may oxidise to give methaemoglobinaemia. Oxygen affinity is affected if the tetrameric structure is stabilised. Increased affinity results in polycythaemia. Polymerisation of Hb leads to red cell rigidity and affects the flow of cells through the microcirculation, e.g. Sickle cell disease.


SICKLE CELL DISEASE (Hb SS) • Homozygous for the abnormal gene that codes for Hb S. • Hb S results from a substitution of valine for glutamic acid at the sixth position from the NH2 terminal end of the β chain. • The structural formula for sickle disease (Hb SS) is

α2β26Glu-Val • Hb S polymerises when deoxygenated and forms tactoid polymers. • The polymers cause the red cell to deform into the sickle shape and reduce the cells ability to circulate. This leads to hypoxia, pain and infarction of organs. • Presence of Hb F (HPFH) modifies the severity of sickling.


Qualitative Haemoglobinopathies COOH lys glu 6

146

α2 β2 6Glu→Val = Hb S

val

glu pro thr leu his val

1

NH2

lys β chain

α2 β2 6Glu→Lys = Hb C


The term sickle cell disease applies not only to the homozygous (SS) but to mixed syndromes (compound heterozygotes) in which sickling occurs, e.g. SC, SD, SE, SO. The disease is prevalent in West and Central Africa, where the gene frequency may reach one in three. Significant incidence in Greece, Turkey, Southern Italy,


Hb SS is usually diagnosed early in life, when the level of Hb F declines. Patients have severe chronic haemolytic anaemia with many complications. Major manifestations are referred to as sickle crisis: Aplastic crisis - (low retics); marrow is overworked. Haemolytic crises - Hb falls, reticulocytosis, jaundice, splenomegaly / autosplenectomy. Vaso-occlusive crises - pain, tissue damage, necrosis.


CLINICAL FEATURES OF SICKLE DISEASE • Abnormal growth (bone and joint abnormalities, e.g. dactylitis and "hand-foot" syndrome). • Renal (pyelonephritis and renal necrosis). • Spleen and liver (autosplenectomy, hepatomegaly and jaundice). • Cardiopulmonary (enlarged heart, heart murmurs and pulmonary infarction). • Eye (retinal haemorrhages). • Leg ulcers. • Infections (major cause of mortality).


Dactylitis


Leg ulcers


LABORATORY DIAGNOSIS The anaemia of Hb SS is severe (60-80g /L). Red cell indices are normochromic and normocytic. PBF can be striking with numerous target cells, fragmented red cells, polychromasia, NRBCs and usually sickle cells. Siderotic granules and Howell Jolly bodies may be seen as a result of rapid red cell turnover and "stressed" erythropoiesis. The retic count ranges between 5 and 20% (except during an aplastic crisis). There may be neutrophilia (due to infection) with a shift to the left, and thrombocytosis (due to autosplenectomy).


The definitive test for Hb S is haemoglobin electrophoresis at alkaline pH, followed if necessary by electrophoresis at acid pH. Hbs D and G migrate to the same position as Hb S at alkaline pH but not at acid pH. The patient with Hb SS has no Hb A on electrophoresis (unless recently transfused). Hb S = >80%, Hb F = 1 to 20%, Hb A2 = approx. 3%.


Hb A Hb F Hb S

Hb A2, C


Solubility tests are available which cause the abnormal Hb S to precipitate. Sodium metabisulphite and sodium dithionite are reducing substances which will induce sickling in vitro. The test solution becomes opaque in a positive solubility test and lines behind the test tube are hidden. Lines can still be seen behind a negative test solution.

neg

pos


SICKLE CELL TRAIT Heterozygous form of the disease;

α2β1β16Glu-Val.

The carrier state (Hb AS) is practically asymptomatic. 45% Hb S in the red cell allows sickling to occur if the hypoxia is severe enough, e.g. anaesthetic accidents or very high altitude. The PBF in sickle cell trait is usually normal. Solubility screening tests are positive. Electrophoresis at alkaline pH shows 60% Hb A, 40% Hb S and usually elevated Hb A2 (mean 3.6%). At acid pH one band is present in the A position (Hb A + Hb A 2), while the other band moves to the S position.


HAEMOGLOBIN C Found almost exclusively in Negro populations. Hb C differs from normal Hb A by the single amino acid substitution of lysine for glutamic acid in the sixth position from the NH 2 terminal end of the β chain

α2β26Glu-Lys This is the same substitution point as in Hb S.


Homozygous Hb C occurs in West Africa (Northern Ghana and Nigeria) and is most common in Mali. Clinical manifestations are mild chronic haemolytic anaemia with associated splenomegaly and abdominal discomfort. Hb is usually >100 g/L. Red cell morphology is typically normocytic, normochromic, with numerous target cells (50 to 90%) and occasionally microspherocytes and fragmented cells. Haemoglobin C crystals (bar of gold crystals) occur particularly in splenectomized patients. The retic count is slightly increased.


Hb C red cell morphology: Target cells and â&#x20AC;&#x153;bar of goldâ&#x20AC;? crystals.


Alkaline Hb electrophoresis shows approximately 95% Hb C plus Hb A2, <7% Hb F and no Hb A. Haemoglobins E, O, C and A2 all migrate to the same position at alkaline pH. Hb C is isolated at acid pH. Haemoglobin C trait (α2β1β16Glu-Lys) is clinically asymptomatic. The only characteristic feature in the PBF is target cells. At alkaline pH, there is approximately 60% Hb A and 40% Hb C plus Hb A2.


HAEMOGLOBIN D Prevalent in parts of India, particularly the Punjab. Glutamine substituted for glutamic acid at position 121. Both heterozygous (α2β1β1121Glu-Gln) and homozygous (α2β2121GluGln ) states are asymtomatic. Compound heterozygous (Hb SD) is severe. The PBF is unremarkable, except for a few target cells. Hb D migrates electrophoretically with Hb S and Hb G at alkaline pH but migrates with Hb A at acid pH. Hb D is a nonsickling soluble haemoglobin.


Haemoglobin E • Prevalent in Burma, Thailand, Cambodia, Laos, Malaysia and Indonesia. • Lysine is substituted for glutamic acid at position 26. • The homozygous state (α2β226Glu-Lys) presents with mild anaemia, target cells, microcytosis and hypochromasia. • On alkaline electrophoresis there is 95 to 97% Hb E plus Hb A2 and the remainder is Hb F. Hb E migrates with Hb A2, Hb C and Hb O at alkaline pH. • It runs with Hb A at acid pH.


Hb E trait (α2β1β126Glu-Lys) is asymptomatic.

There is microcytosis, hypochromasia and target cells without anaemia.

Alkaline electrophoresis shows approximately 70% Hb A and 30% Hb E plus Hb A2.

Hb E is slightly unstable and has associated thalassaemic component. This is responsible for the microcytosis and the lower than expected value of Hb E in Hb AE.

Hb E may protect against malaria. P. falciparum multiplies more slowly in Hb E red cells.


S

SICKLE

VERY

VALINE

6

C

CELL

LIKELY

LYSINE

6

D

DIS

GIVES

GLUTAMINE

121

E

EASE

LYSIS

LYSINE

26


METHAEMOGLOBIN (Hi)   



Methaemoglobinaemia exists when Hi levels exceed 1% of the total Hb. Hi contains the oxidised ferric form of iron (Fe+++) rather than the ferrous form (Fe ++). The molecule is unable to bind oxygen and results in cyanosis and possibly mild haemolytic anaemia. The blood is chocolate-brown in colour.


There are three causes of methaemoglobinaemia: 1.

Hb M variants (dominant inheritance). Amino acid substitution stabilises iron in the ferric form.

2. NADH-diaphorase deficiency (recessive inheritance). 3.

Toxic substance (acquired).

Hb M variants can be detected electrophoretically and most can be distinguished from Hi A by their absorption spectra.


Hb VARIANTS WITH ALTERED OXYGEN AFFINITY

High-affinity Haemoglobins. There is a shift to the left in the oxygen dissociation curve. Oxygen is bound more readily and released less easily to the tissues. The result is hypoxia, which stimulates increased erythropoietin production resulting in a congenital polycythaemia.


Decreased Oxygen Affinity Haemoglobins. The oxygen dissociation curve shifts to the right. Oxygen is readily released to the tissues. As more oxygen is released per unit of haemoglobin, erythropoietin levels fall. This results in decreased Hb concentration and mild anaemia develops.


UNSTABLE HAEMOGLOBINS These are variants in which amino acid substitutions or deletions have weakened the binding forces that maintain the structure of the molecule. The instability may cause the Hb to denature and precipitate in the red cells to form Heinz bodies.


Most unstable haemoglobins are inherited as autosomal dominant disorders, however, new mutations can occur. Many have high oxygen affinity and therefore may not cause anaemia. Haemolysis varies considerably. Most patients have a mild compensated condition until "stressed" by infection or exposure to oxidative drugs.


Reticulocytosis is variable. Hypochromasia may be seen and the MCHC can be low because unstable Hb may be denatured and "pitted" out of the cell by macrophages in the spleen. Hb electrophoresis is usually not helpful. Isopropanol precipitation and heat denaturation tests are the procedures of choice.


HEREDITARY PERSISTENCE OF FETAL Hb (HPFH), i.e. persistence of Hb F synthesis into adult life. Different types of HPFH exist, depending on the genetic defect responsible: Swiss type (heterocellular). Individuals have up to 3% Hb F. There appears to be no abnormalities of the delta and beta genes. The condition is asymptomatic. Negro type (pancellular). Deletion of beta and delta globin genes (delta-beta thalassaemia). Hb F constitutes 100% of the total Hb in the homozygous state and 15 to 30% in the heterozygous. Clinically, homozygotes demonstrate features of thalassaemia minor, heterozygotes are asymptomatic. Greek type (pancellular). About 15% Hb F is present in the heterozygous state. The homozygous state has yet to be described.


Quantitation of Hb F is done by alkali denaturation, RID or ELISA. The Kleihauer acid elution test is used to determine the intracellular distribution of Hb F. Some cells may be positive for Hb F while others are negative (heterocellular). When all red cells contain Hb F the distribution is pancellular. Heterocellular distribution of Hb F


? ?

A F S C


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