1. Hemoglobinopathies, Thalassemias Prof. Dr. S. Sami Kartı

2. Types of inherited hemolytic anemias Structural Hemoglobinopathies: Synthesis of a structurally abnormal hemoglobin protein (globin chain) Thalassemias: Quantitative abnormality (decreased synthesis) of a globin chain Enzyme Defects: Absent or decreased function of a metabolic enzyme Membrane Defects: Abnormalities in the proteins that make up the cytoskeleton of the cell membrane

3. The structural hemoglobinopathies Definition Structural hemoglobinopathies (often simply called hemoglobinopathies) are caused by mutations in the gene for a globin chain, resulting in the synthesis of a structurally abnormal hemoglobin. In most cases, this is due to substitution of a single amino acid. The majority of hemoglobinopathies that are clinically detected are β-chain mutations. The hemoglobinopathies are inherited in an autosomal fashion homozygotes for the mutation have clinical disease, but heterozygotes are asymptomatic or have mild disease.

5. Terminology and Nomenclature The normal hemoglobins are hemoglobin A (α2 β2), hemoglobin A2 (α2 δ2), and hemoglobin F (fetal hemoglobin; α2 γ2). In the adult, hemoglobin A represents ≥95% of the hemoglobin, A2 represents ≤3 to 4%, and F represents ≤1 to 2%. Sickle hemoglobin is designated S because erythrocytes with hemoglobin S can transform into a sickle shape.

6. Hemoglobin genotype and phenotype (I) Genotype: The genotypic designation is based upon the specific globin chains that are present. Heterozygous sickle cell would be designated α2 ββS (two normal chains, one normal chain, one chain with the sickle mutation). Homozygous sickle cell anemia would be designated α2 β2 S (two normal chains, two chains with the sickle mutation). Compound heterozygosity for hemoglobin S and hemoglobin C would be designated α2 βS βC.

7. Hemoglobin genotype and phenotype (II) Phenotype: The phenotypic designation is based on the hemoglobin types that are present. The letter designation for each of the hemoglobins is given, and the hemoglobin in highest concentration should be given first. Therefore, heterozygous sickle cell would be designated hemoglobin AS (since hemoglobin A is usually present in greater concentration than hemoglobin S). Homozygous sickle cell would be designated hemoglobin SS. Double heterozygosity for hemoglobins S and C would be designated hemoglobin SC. A person who is heterozygous for both hemoglobin S and thalassemia, where the hemoglobin S concentration is greater than that of hemoglobin A, would be designated hemoglobin SA.

8. Diagnosis of Hemoglobinopathies Hemoglobin electrophoresis, which separates hemoglobins based on differences in size and electrical charge (the patient should not have been transfused for at least 90 days before the test) Other tests –sickle solubility tests –isopropanol test for unstable hemoglobins –supravital (Heinz body) stains for denatured hemoglobins Reference hemoglobinopathy laboratories can perform more complex tests, –isoelectric focusing –electrophoresis of separated globin chains –Globin chain synthesis analysis;

9. Sickle Cell Anemia (Hemoglobin S)

10. Types of disease Heterozygosity for hemoglobin S (hemoglobin AS) is designated sickle cell trait. Sickle cell anemia is the preferred term for people who are homozygous for hemoglobin S (hemoglobin SS). The term sickle cell disease indicates patients with clinical evidence of sickling and includes sickle cell anemia (hemoglobin SS), sickle/hemoglobin C (hemoglobin SC; α2 βS βC ), and sickle/-thalassemia (α2 βS βThal ). Since people with sickle trait are asymptomatic, sickle cell trait is not considered a sickle cell disease.

11. Epidemiology In parts of Africa, approximately 10 to 40% of the population is heterozygous for hemoglobin S Approximately 8% of African Americans are heterozygous for hemoglobin S (sickle trait), and approximately 1 in 400 to 600 are homozygous (sickle cell anemia) There are separate pockets of hemoglobin S in Turkey, along the Mediterranean coast (Sicily, southern Italy, and northern Greece), in Saudi Arabia, and in India These are areas where falciparum malaria is endemic, suggesting that hemoglobin S arose as a protective mechanism against malaria

12. Pathophysiology (I) The abnormality in Hb S is substitution of valine for glutamic acid at the sixth amino acid position (6 Glu Val ) Deoxygenated hemoglobin S tends to polymerize into long rigid structures, which distort the cell into the characteristic sickle shape Anything that causes deoxygenation of hemoglobin predisposes to sickling, including hypoxia, acidosis, and increased temperature The polymerization of hemoglobin S is reversible, and cells that have sickled may return to normal shape with reoxygenation

14. Pathophysiology (II) The repeated cycles of sickling and unsickling damage the cell, and, eventually, the erythrocytes becomes irreversibly sickled The rigid elongated sickle cells obstruct small blood vessels, resulting in tissue infarction Sickled erythrocytes are also “sticky” and adhere to endothelial cells, predisposing to thrombosis Common sites of infarction include the spleen, bone and bone marrow, the medulla of the kidney, mesenteric vessels, and pulmonary vessels

15. Homozygous Hemoglobin S (Sickle Cell Anemia) The severity of illness in sickle cell anemia (hemoglobin SS) is highly variable and can vary even within families Many children become symptomatic in infancy after 3 to 4 months of age (before that time they are protected by the high levels of hemoglobin F) Other people have very mild disease and may not be diagnosed until adulthood. The reasons for this variability are not clear; the level of hemoglobin F is a factor (an increase in hemoglobin F decreases the severity of sickle cell disease), but other factors also appear to be important

16. Sickle Cell Diseases Other Than Sickle Cell Anemia People who are heterozygous for both hemoglobin S and some other hemoglobinopathy (hemoglobin C, hemoglobin D Los Angeles [also called D Punjab], or hemoglobin O Arab), or who are heterozygous for hemoglobin S and thalassemia, are often symptomatic and may have many of the same complications as homozygous sickle cell anemia People with sickle-β+ thalassemia, in which some normal β-globin chain is produced by the thalassemia gene, tend to have relatively mild disease People with sickle-βo thalassemia, in which there is a complete absence of –β globin synthesis by the thalassemia gene, have severe disease that closely resembles homozygous sickle cell anemia

17. Sickle Cell Crises (I) Three major categories of sickle cell complications have been designated sickle crises: Acute vaso-occlusive (painful) crises: Painful crises are the most common type of crisis and are believed to be caused by occlusion of small blood vessels, with consequent infarction of tissues. Pain can occur in the abdomen, bones, joints, or muscles. Young children often present with pain involving the hands or feet (the hand-foot syndrome or dactylitis); long bones and the abdomen are more common sites of pain in adults. Sequestration crises: Sequestration crisis can occur during childhood, usually during the first 3 to 4 years, before the spleen has become infarcted. The spleen suddenly becomes enlarged and engorged with blood; this can sequester a major portion of the total blood volume and can be fatal.

18. Sickle Cell Crises (II) Acute aplastic crises: This occurs as a complication of infections, usually but not always due to parvovirus B19. Acute parvovirus B19 infection causes a transient halt in production of erythrocytes, which usually lasts about 5 to 7 days. –In normal individuals, in which erythrocytes are being replaced at the rate of about 1% per day, the transient drop in hemoglobin is not significant. Since red cell survival is greatly decreased in patients with sickle cell anemia (10–20 days, compared with 120 days in normal individuals), the hemoglobin drops much more quickly (up to 1 g/dL per day), and without transfusions, the marked exacerbation of anemia can be fatal. Recovery of hematopoiesis usually occurs after about 7 days.

19. Other Complications of Sickle Cell Disease Infections Cerebrovascular accidents Acute chest syndrome (acute lung syndrome) Altered splenic function and splenic infarcts Renal disease Priapism Gallstones Leg ulcers Aseptic necrosis of the femoral heads Retinopathy Complications of pregnancy

21. Diagnosis of Sickle Hemoglobin (I) CBC and blood smear is normal in patients with sickle trait In sickle cell anemia hemoglobin is typically 5 to 8 g/dL. MCV is normal. The blood smear shows target cells and the characteristic sickled erythrocytes. Howell-Jolly bodies may be present after splenic infarction, and nucleated red blood cells (RBCs) may be present. Patients with S/β-thalassemia show prominent target cells and microcytosis, and the MCV is decreased.

22. Diagnosis of Sickle Hemoglobin (II) Common tests for sickle hemoglobin include sickle solubility tests and hemoglobin electrophoresis. Sickle solubility tests depend on the decreased solubility of deoxygenated hemoglobin S in high- molarity phosphate buffers. The solubility test is usually positive if hemoglobin S is more than 10 to 20% of the hemoglobin. It is positive in patients with both sickle trait and sickle cell anemia and therefore cannot be used to distinguish homozygous from heterozygous hemoglobin S.

23. Diagnosis of Sickle Hemoglobin (III) Fetal hemoglobin interferes with polymerization of hemoglobin S, and the sickle solubility test can give a false-negative result if hemoglobin F makes up more than ~10 to 20% of hemoglobin in the sample. The other test commonly used to detect hemoglobin S is hemoglobin electrophoresis, usually performed at an alkaline pH on cellulose acetate. Electrophoresis can be used to distinguish between sickle trait and sickle cell anemia and can diagnose other sickle cell diseases such as hemoglobin SC. Typically, a person with homozygous sickle cell anemia has >80% hemoglobin S, with the remainder being hemoglobin A2 and hemoglobin F. Hemoglobin A (α2 β2) is completely absent

24. Treatment of Sickle Cell Disease General supportive measures, Folic acid supplementation Prophylactic penicillin (should be started as soon as possible after birth and continued at least through age 5 years). Psychological counseling (decreases anxiety related to having a chronic and potentially lethal disease, can help patients deal with chronic pain and increase their level of function, and improves compliance with therapy). Vaccination against S. pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis.

25. Treatment of acute painful vaso-occlusive crises The key measures are adequate hydration and pain control. Pain in patients with sickle cell anemia should be managed aggressively, last 4 to 5 days on average, Be careful to exclude other illnesses that may cause or mimic vaso-occlusive crises (osteomyelitis, other infections, stroke, acute appendicitis or other intra- abdominal processes). Morphine is generally the preferred parenteral agent for severe pain. Meperidine (Demerol) should be avoided because seizures may occur due to accumulation of meperidine metabolites. Bolus corticosteroids (methylprednisolone) have been suggested to be beneficial; Long-acting nonsteroidal anti-inflammatory agents may also be helpful. Patient-controlled anesthesia is a useful approach.

26. Red cell transfusions Should be considered for acute cerebrovascular accidents, intractable or recurrent episodes of the acute chest syndrome, or priapism that does not respond to conservative measure Exchange transfusion allows the hemoglobin S concentration to be decreased rapidly Patients who have had a cerebrovascular accident should be considered for chronic transfusions to maintain the hemoglobin S level below 30% to prevent recurrence. These transfusions should be continued for at least 3 years Transfusion therapy is associated with significant risks, including iron overload, alloimmunization (antibodies to red cell antigens), hemolytic transfusion reactions, and infections. Infections should be treated aggressively

27. Other therapies Hydroxyurea increases the percent of fetal hemoglobin in the erythrocytes of many patients, which interferes with polymerization of hemoglobin S and therefore decreases sickling. The Hydroxyurea has been shown to decrease the number of painful crises, hospitalizations, red cell transfusions, and episodes of the acute chest syndrome in patients with sickle cell anemia. Acute chest syndrome is treated with supplemental oxygen, empiric antibiotics pending culture results, adequate analgesia, and transfusions as needed Stem cell therapy (bone marrow transplant). Because of the possible mortality associated with BMT, transplants should be reserved for patients at increased risk of serious complications of their disease.

28. Hemoglobin C Hemoglobin C has a substitution of lysine for glutamic acid at the sixth amino acid of the β globin chain. Like hemoglobin S, hemoglobin C is capable of polymerizing into crystals when deoxygenated. Heterozygous Hb C (hemoglobin C trait) is clinically silent. No treatment is required for hemoglobin C trait or homozygous hemoglobin C.

29. THE THALASSEMIAS quantitative abnormality of globin chain synthesis Classic thalassemia is characterized by a complete lack of globin chain synthesis or a decreased amount of a structurally normal globin chain The thalassemias are extremely heterogeneous, both in clinical manifestations and molecular (genetic) basis. Hundreds of different mutations have been identified in patients with thalassemia The majority of thalassemias involve either the α or β globin chains. Cases involving both the δ and β globin chains (δβ thalassemia) or all three β-like chains (γδβ thalassemia) do occur but are rare There is a high prevalence of thalassemia in areas with endemic malaria, including parts of Africa, the Mediterranean basin, the Middle East, India, Southeast Asia, and southern China

30. Pathophysiology The decrease in globin chain synthesis has two consequences: Decreased hemoglobin synthesis, resulting in anemia and microcytosis Aggregation of the excess free globin chains produced by the non-thalassemic gene (α chains in the case of β-thalassemia, β chains in the case of α-thalassemia). The aggregates of unpaired globin chains attach to and damage the erythrocyte cell membrane, resulting in hemolysisIn severe cases, the majority of erythroid precursors are destroyed in the marrow (ineffective erythropoiesis).

31. Complications of Thalassemias (I) Chronic anemia: Chronic anemia causes growth retardation, delayed sexual maturation, cardiac dilatation and congestive heart failure, decreased work capacity, and all of the other complications associated with chronic anemia. Marked expansion of the bone marrow: The bone marrow becomes greatly expanded due to marked erythroid hyperplasia. Hypertrophy of the frontal bones results in frontal bossing. Hypertrophy of the maxillae results in prominent cheeks and dental malocclusions, giving a characteristic “chip-munk” facies. Thinning of the cortex of the vertebrae and long bones results in fractures. Extramedullary hematopoiesis causes enlargement of the spleen and liver. Foci of extramedullary hematopoiesis may occur in soft tissues (myeloid tumors), and paravertebral masses may cause spinal cord compression.

32. Complications of Thalassemias (II) Iron overload: There is chronic hyperabsorption of iron by the gastrointestinal tract, driven by the chronic erythropoiesis, and this is exacerbated by RBC transfusions. Iron deposition in the heart causes cardiomyopathy and cardiac arrhythmias. Deposition in the liver causes portal fibrosis and may result in hepatic cirrhosis. Patients with hepatic cirrhosis are at risk of developing hepatocellular carcinoma (hepatoma) Chronic hemolysis: Chronic hemolysis causes splenomegaly, hepatomegaly, and bilirubin gallstones. Hypersplenism may develop, increasing transfusion requirements.

33. β-Thalassemia Pathophysiology There is a single gene for the β globin chain on chromosome 11 The mutation may result in a complete lack of β chain synthesis (βo thalassemia) or a decrease in β chain synthesis (β+ thalassemia). The severity of illness depends on how much β chain is synthesized and whether the person is heterozygous or homozygous for the mutation.

34. β-thalassemias main clinical syndromes β-Thalassemia minor: Heterozygosity for β-thalassemia results in a mild clinical syndrome designated β-thalassemia minor. The hemoglobin and the MCV are mildly or moderately decreased (hemoglobin ~9–12 g/dL and MCV ~65–75 fL), and the patient has few symptoms or complications. β-Thalassemia major (Cooley’s anemia): Homozygosity for β- thalassemia results in a severe clinical syndrome characterized by severe anemia and microcytosis (hemoglobin ~3–5 g/dL and MCV <65 fL), total or near total absence of Hb A, marked ineffective erythropoiesis, marked expansion of the bone marrow with skeletal complications, splenomegaly, and iron overload due to hyperabsorption of iron β-Thalassemia intermedia: β-Thalassemia intermedia is homozygous β-thalassemia that is not transfusion dependent. It is genetically and clinically heterogeneous. The hemoglobin is intermediate between β-thalassemia major and minor (~6–9 g/dL), as is the incidence of clinical complications.

35. Diagnosis of Thalassemia A microcytic anemia that is not due to iron deficiency is most likely thalassemia Check the serum ferritin or serum iron/transferrin/transferrin saturation If the results do not indicate iron deficiency, start a workup for thalassemia Ethnic background and family history, Examination of a well-stained blood smear (microcytosis, hypochromia, and target cells, may be basophilic stippling) Hemoglobin electrophoresis (the presence of increased hemoglobin A2)(The mean hemoglobin A2 level in β-thalassemia is about 5% (normal is less than about 3%)

36. Treatment of Thalassemia red cell transfusion Patients with thalassemia minor usually do not require transfusion or other specific therapy Patients with thalassemia intermedia may or may not require transfusions to prevent complications of anemia and erythroid hyperplasia Patients with severe thalassemia are frequently maintained on hypertransfusion regimens - periodic red cell transfusions to maintain the blood hemoglobin >9 to 10 g/dL (some centers try to keep the hemoglobin ≥12 g/dL). This prevents the skeletal complications of thalassemia by shutting off the erythropoietin-driven erythroid hyperplasia, allows normal growth and sexual development, and delays the onset of splenomegaly Complications of transfusion include alloimmunization, iron overload, and transfusion-transmitted infections, particularly viral hepatitis.

37. Treatment of Thalassemia iron chelation Iron overload is a serious complication of transfusion therapy (each red cell unit contains 200–250 mg of iron), and chelation therapy is required to prevent the development of secondar hemochromatosis. Patients usually start continuous subcutaneous infusions of deferoxamine in the evening and continue overnight. This mobilizes the iron so that it is excreted in the urine. Deferoxamine can cause painful reactions at the site of injection. Other possible side effects include cataracts and hearing loss Oral iron chelators (deferiprox and desferrioxamine ??) is used succesfully. The side effects are granulocytopenia and renal impairment

38. Treatment of Thalassemia other therapies Splenomegaly eventually develops in patients with severe thalassemia even with transfusion therapy, and splenectomy will be required. The usual indication for splenectomy is an increase in transfusion requirement. Patients should be immunized against S. pneumoniae, H. influenzae, and N. meningitidis prior to splenectomy. Bone marrow transplant is potentially curative for thalassemia. Patients with related HLA-matched donors and severe thalassemia should be considered for BMT early, before serious complications occur (particularly before hepatic fibrosis or hepatomegaly develop).