HAEMOGLOBINOPATHIES Dr.Mousa Qasim 16-11-2015

HAEMOGLOBINOPATHIES These diseases are caused by mutations affecting the genes encoding the globin chains of the haemoglobin molecule.

Normal haemoglobin is comprised of two alpha and two non-alpha globin chains. Alpha globin chains are produced throughout life, including in the fetus, so severe mutations may cause intrauterine death.

Production of non-alpha chains varies with age; fetal haemoglobin (HbF-αα/γγ) has two gamma chains, while the predominant adult haemoglobin (HbA-αα/ββ) has two beta chains. Thus, disorders affecting the beta chainsdo not present until after 6 months of age. A constant small amount of haemoglobin A2 (HbA2-αα/δδ, usually less than 2%) is made from birth.

Normal hemoglobins in the red cell consist of Hb A, Hb F, and Hb A2. The protein sequences are DNA coded on Chromosome 11 for the beta, delta and gamma chains. The alpha chains are coded on Chromosome 16. The beta variants such as Hb S, Hb C, and Hb D all occur from a mutation on Chromosome 11.

Quantitative abnormalities – thalassaemias In quantitative abnormalities (the thalassaemias), there are mutations causing a reduced rate of production of one or other of the globin chains, altering the ratio of alpha to non-alpha chains. In alpha-thalassaemia excess beta chains are present, whilst in beta-thalassaemia excess alpha chains are present. The excess chains precipitate, causing red cell membrane damage and reduced red cell survival.

The thalassaemias Thalassaemia is an inherited impairment of haemoglobin production, in which there is partial or complete failure to synthesise a specific type of globin chain. In alpha-thalassaemia, disruption of one or both alleles on chromosome 16 may occur, with production of some or no alpha globin chains. In beta-thalassaemia, defective production usually results from disabling point mutations causing no (β0) or reduced (β–) beta chain production.

Beta-thalassaemia Failure to synthesise beta chains (beta-thalassaemia) is the most common type of thalassaemia, most prevalent in the Mediterranean area. Heterozygotes have thalassaemia minor, a condition in which there is usually mild anaemia and little or no clinical disability, which may be detected only when iron therapy for a mild microcytic anaemia fails.

Homozygotes (thalassaemia major) either are unable to synthesise haemoglobin A or at best produce very little; after the first 4-6 months of life they develop profound hypochromic anaemia.

Thalassemia inherited in an autosomal recessive pattern,

Pathogenesis 1-Defective globin-chain synthesis in β-thalassemia causes both decreased normal hemoglobin production and the production of a relative excess of α chains. 2-The decrease in normal hemoglobin synthesis results in a hypochromic anemia.

3-the excess α chains form insoluble α-chain complexes and cause hemolysis. In mild thalassemic syndromes, the excess α chains are insufficient to cause significant hemolysis, and the primary finding is a microcytic anemia. In severe forms of thalassemia, hemolysis occurs both in the periphery and in the marrow, with intense secondary expansion of the marrow production of red cells.

4-The expansion of the marrow space causes severe skeletal abnormalities. 5- the ineffective erythropoiesis also provides a powerful stimulus to absorb iron from the intestine

CLINICAL FEATURES: 1.Sever anaemia:- overlap after the first 4 months of life, many pt with B-Thalassaeinia major (Coolley s Anaemia) are transfusion dependent, the pt develop iron overlapped, in untreated pt this usually lead to death in 2nd decade. 2. Bone changes:- due to expansion of bone marrow space and growth retardation 3. Chipmunk facies:- due to increase erythropoiesis 4. Skin:- copper colour from paller,icterus and melanin deposition. 5. hepatosplenomegally:- may be massive

DIAGNOSTIC FEATURES:- *major: . Profound hypochromic anaemia • Evidence of RBC dysplasia • Erythroblastosis • Absence or gross reduction ofHbA • Raised level HbF • Evidence that both parents have Thalassaemia minor.

Hair on end skull in B-thalassemia major

*minor: . Mild anaemia . Microcystic hypochromic erythrocyte(non-iron deficient) . Some target cell -punctate basophilia . Raised resistant of RBC to osmotic lysis . Increase HbA2 fraction . Evidence that one parents have Thalassaemia minor

HPLC pattern of hemoglobin from a patient with heterozygous Beta Thalassem

Target cells and microcytosis

TREATMENT of B-Thalassaemia MAJOR: -Erythropoiesis failure Allogenic bone marrow transplantation From human leukocyte antigen (HLA)-c compatible sibling Transfusion to maintain Hb>10g/dl folic acid 5mg daily - Iron Overload **iron therapy forbidden **desfenioxamine therapy -Splenomegaly causing mechanical problems or excessive transfusion needs, the treatment will be Splenctomy.

** TREATMENT of B-Thalassaemia MINOR: Do not need to be treated but in certain regions in which the incidence increased, screening for B-Thalassaemia combined with counseling and it is an important role to decrease the incidence of the disease

**Prevention: It is possible to identify a fetus with homozygous B-Thalassaemia by obtaining chorionic villous material for DNA analysis sufficiently early in pregnancy to allow termination. This examination is only appropriate if both parents are known to be carrier (beta -Thalassaemia minor) and will accept a termination.

Alpha-thalassaemia: The reduction or absence of alpha-chain synthesis is common in Southeast Asia. There are two alpha gene loci on chromosome 16 and therefore four alpha genes. If one is deleted there is no clinical effect. If two are deleted there may be a mild hypochromic anaemia. If three are deleted the patient has haemoglobin H disease and if all four are deleted the baby is stillborn (hydrops fetalis). Haemoglobin H is a beta-chain tetramer formed from the excess of chains. It is functionally useless. Treatment of haemoglobin H disease is similar to that of beta-thalassaemia of intermediate severity

ALPHA-THALASSAEMIA Cause Age and sex incidence Genetics Presentation Failure of production of haemoglobin alpha chains due to gene deletion Age and sex incidence Both sexes from birth onward Genetics Two alpha-chain genes from each parent Presentation Hydrops fetalis if all genes deleted Haemoglobin H if three genes deleted Mild hypochromic microcytic anaemia if two genes deleted Treatment Hydrops fetalis: none available Haemoglobin H: no specific therapy required; avoid iron therapy; folic acid if necessary

Qualitative abnormalities – abnormal haemoglobins In qualitative abnormalities (called the abnormal haemoglobins),there is a functionally important alteration in the amino acid structure of the polypeptide chains of the globin chains. Several hundred such variants are known; they were originally designated by letters of the alphabet, e.g. S, C, D or E, but are now described by names usually taken from the town or district in which they were first described

The best-known example is haemoglobin S, found in sickle-cell anaemia The best-known example is haemoglobin S, found in sickle-cell anaemia. Mutations around the haem-binding pocket cause the haem ring to fall out of the structure and produce an unstable haemoglobin. These substitutions often change the charge of the globin chains, producing different electrophoretic mobility, and this forms the basis for the diagnostic use of haemoglobin electrophoresis to identify haemoglobinopathies.

SICKLE-CELL ANAEMIA

SICKLE-CELL ANAEMIA . Is type of Anemia results from single Glutamic acid to Valine substitution at position 6 of β globin p.p chain. . Inherited as Autosomal recessive trait. . Homozygotes only produce abnormal β chain that make HbS (termed SS) & it results in clinical syndrome of sickle cell dis. . Heterozygote produces mixture Of normal & abnormal β chain makes normal HbA & HbS(termedAS) clinically asymptomatic sickle cell trait.

Epidemiology The heterozygote frequency is over 20% in tropical Africa . In black American populations, sickle-cell trait has a frequency of 8%. Individuals with sickle-cell trait are relatively resistant to the lethal effects of falciparum malaria in early childhood. homozygous patients with sickle-cell anaemia do not have correspondingly greater resistance to falciparum malaria.

PATHOGENESIS: . When HbS is deoxygenated, the molecule of Hb polymerizes to form Pseudo crystalline structures Called TACTOIDS, these distort red cell membrane and produce Characteristic sickle-shaped cells.

This polymerization is reversible when reoxygenation occurs otherwise the distorted RBC membrane Is irreversibly sickled. The greater concentration of sickled cell Hb in individual cell, the more easily tactoids are formed, But this process may be enhanced by presence of other Hb. HbC participates in polymerization more readily than HbA. HbF strongly inhibits polymerization

CLINICAL FEATURES: Sickling is precipitated by: 1.Hypoxia 2.Acidosis 3.Dehydration 4.Infection

Irreversible sickled cells have short survival & plug vessels in microcirculation.This results in a no. of acute syndromes (Crises) & chronic organic damage. 1.Vaso-Occlusive Crises: (PAINFULL) Plugging of small vessels in bone produces acute sever bone pain. This affects areas of active marrow, the hands & feet in children so called (Dactylitis) or femora, humeri, ribs, pelvis and vertebrae in adult. Patients usually have a systemic response that includes tachycardia, sweating, fever and this type is the most common crisis.

2.Sickle chest syndrome: May follow on from a vaso-occlusive crises & it is the most common cause of death in adult sickled disease. Bone marrow infarction results in fat emboli to the lungs which cause sickling and infarction leading to Ventilatory failure if not treated.

3.Sequestration Crises: Thrombosis of venous outflow causes loss of function & acute painful enlargement. Spleen is most common site in children. massive splenic enlargement may result in sever anemia & circulatory collapse and death. Recurrent splenic sickling in childhood result in infarction and adults may have no functional spleen & the liver may undergo sequestration and sever pain will occur due to capsular stretching.

4.Aplastic Crises: Infection of adult with human erythrovirus 19 results in sever but self-limiting RBC aplasia. This produce a very low Hb that may cause Heart Failure. unlike all other sickle crises the reticulocytes count is low.

INVESTIGATIONS: 1. Compensated anemia: Hb% 6-8 g/dl 2. Blood film: sickled cells, target cells, features of hyposplenism 3. Reticulocytosis 4. Sickling test: exposing red cells to reducing agent (Na dithionite): -HbA clear solution -HbS turbid solution due to polymerization. We can't distinguish between sickle trait & disease.

5. Definitive diagnosis requires Hb electrophoresis that demonstrates: 1-no HbA. 2- 2-20 % Hbf . 3- predominance of HbS. 4-Both parents of affected individual will be sickle trait

Pattern of hemoglobin electrophoresis from several different individuals. Lanes 1 and 5 are hemoglobin standards. Lane 2 is a normal adult. Lane 3 is a normal neonate. Lane 4 is a homozygous HbS individual. Lanes 6 and 8 are heterozygous sickle individuals. Lane 7 is a SC disease individual.

TREATMENT: 1. All pt. with sickle dis. should receive prophylaxis with Daily Folic acid & Penicilln V to protect against pneumococcal infection. 2. Pt. should be vaccinated against pneumococcal & where available Hemophillus influenza & hepatitis B. 3. Vaso-occlusive Crises: aggressive rehydration, Oxygen therapy, adequate analgesia (opiates) and antibiotics.

4. Transfusion with fully genotyped blood: Simple top-up transfusion is used in sequestration or Aplastic Crises. Regular transfusion program to suppress HbS production & maintain HbS level below 30% may be indicated in recurrent sever complications such as CVA in children or chest syndromes in adults. Exchange transfusion to replace HbS to HbA usually used in life-threaten crises or in preparation patient to surgery.

5. A high level of Hbf inhibit polymerization & Sickling 5. A high level of Hbf inhibit polymerization & Sickling. So patient with sickle cell disease & Hbf will have mild clinical course with few crises. Some agents are able to induce the synthesis of Hbf to reduce the frequency of sever crises.

6. Allogenic stem- cell Transplants from HLA matched siblings have been performed but this procedure appears to be potentially curative.

7-The most significant advance in the therapy of sickle cell anemia has been the introduction of hydroxyurea as a mainstay of therapy for patients with severe symptoms. Hydroxyurea (10–30 mg/kg per day) increases fetal hemoglobin and may also exert beneficial affects on RBC hydration, vascular wall adherence, and suppression of the granulocyte and reticulocyte counts; dosage is titrated to maintain a white cell count between 5000 and 8000 per L..

Hydroxyurea should be considered in: 1- patients experiencing repeated episodes of acute chest syndrome . 2- more than three crises per year requiring hospitalization.

8-Gene therapy for sickle cell anemia is being intensively pursued, but no safe measures are currently available. 9- Agents blocking RBC dehydration or vascular adhesion, such as clotrimazole or magnesium, may have value as an adjunct to hydroxyurea therapy, pending the completion of ongoing trials. Combinations of clotrimazole and magnesium are being evaluated

Prognosis In Africa few children with sickle-cell anaemia survive to adult life without medical attention. Even with standard medical care, approximately 15% die by the age of 20 years and 50% by the age of 40 years.

Thank you