Hematology 425 Hemoglobinopathies

Hematology 425 Hemoglobinopathies Russ Morrison November 13, 2006

Hemoglobin – NL review • A review of normal adult Hgb production reminds us that • Heme is a protoporphyrin ring with Fe2+ (ferrous iron) • Globin is a polypeptide chain • Hgb molecule has 2 alpha and 2 non-alpha globin molecules • Globin chains bind heme after release from the ribosomes and then pair off

Hemoglobin – NL review • An alpha chain and a non-alpha chain pair to form a dimer • Two dimers combine to form tetramers completing formation of the Hgb molecule • NL Adult Hgbs: • HgbA (2 alpha and 2 beta chains) • HgbA2 (2 alpha and 2 delta chains) • HgbF (2 alpha and 2 gamma chains)

Hemoglobinopathies • Hemoglobinopathies are inherited disorders where genetic mutations alter either the structure or the rate of synthesis of a particular globin chain • Divided into 2 categories • Hemoglobinopathies, amino acid sequence is altered • Thalassemias, reduce or completely eliminate the production of one or more globin chains (Chapter 25)

Hemoglobinopathies • There are approximately 700 Hgb variants • Molecular abnormalities may cause mutant hemoglobins in any adult hemoglobin chain (alpha, beta, gamma, or delta) • Molecular abnormalities are characterized as follows: • Substitution of one amino acid for another (valine for glutamic acid in HgbS

Hemoglobinopathies • Substitution of more than one amino acid (glutamic acid for valine and aspartate for asparagine in HgbC-Harlem) • Deletion of one or more amino acids (glutamic acid deletion in Hgb Leiden) • Fusions of Hgb chains (δβ in Hgb Lepore) • Extension of an amino acid chain (Hgb Constant Spring with 31 additional amino acid residues)

Hemoglobinopathies • The impact of the amino acid changes is variable • Many of the changes are clinically insignificant because there is no detectable physiologic effect • Most are associated with beta-chain abnormalities and more patients with beta-chain abnormalities demonstrate clinical disease

Hemoglobinopathies • Table 24-2 in the text lists clinically significant abnormal hemoglobins • The most frequently occurring, as well as the most severe, of the abnormal Hgbs is hemoglobin S

Hemoglobinopathies • Abnormal Hgbs may demonstrate heterozygous or homozygous inheritance patterns • In homozygous beta hemoglobinopathies, the variant Hgb is the major component and normal Hgb (HgbA) is absent. Examples include sickle cell disease (HgbSS) and Hgb C disease (HgbCC)

Hemoglobinopathies • In heterozygous beta-hemoglobinopathies, the variant hemoglobin is usually present in lesser amounts than normal HgbA • Some cases demonstrate equal amounts of HgbA and the abnormal Hgb, examples are HgbC trait (HgbAC) and Hgb S trait (HgbAS) • In both cases, Hgbs F and A2 are usually normal structurally

Hemoglobinopathies • In heterozygous alpha- hemoglobinopathies, an abnormalityin the alpha chain will affect all three normal adult hemoglogin types (A, F, A2) and 6 different hemoglobin types are found, three of which are normal and three abnormal • Examples of heterozygous alpha types include HgbD-Baltimore and HgbM-Boston

Hemoglobinopathies • Hemoglobinopathies are genetic disorders in which a structural abnormality results from the alteration of DNA genetic code for one or more globin chain producing a qualitative difference • Thalassemias are similar except that the genetic alteration inhibits or completely eliminates production of a globin chain, hence a quantitative difference

Hemoglobinopathies • Results of Hgb changes depends on the amino acids involved and their position in the molecule • Hemoglobinopathies are divided into 4 groups: • Abnormal hemoglobins that result in hemolytic anemia, such as HgbS and the unstable Hgbs • Methemoglobinemia, such as HgbM (ferric iron, increased affinity for oxygen)

Hemoglobinopathies • Hemoglobins with either increased or decreased oxygen affinity • Abnormal Hgbs with no clinical or functional effect • Functional classification of Hgb variants is summarized in Table 24-3 in the text

Hemoglobinopathies • As hemoglobins were discovered and reported, they were designated by letters of the alphabet (HbA, HbF) • Soon, it was discovered that there would not be enough letters and a geographic notation was added (HbGPhiladelphia) • Designation may also include the variant chain, sequential number and nature of the substitution (examples in Table 24-3)

Hemoglobinopathies – Hgb S • Sickle Cell anemia is a relatively recent discovery having first been described in 1910 with a student suffering from severe anemia • Sickle cell diseases are the most comon hemoglobinopathies and include a group of hereditary disorders characterized by the presence of only HbSS or HbS in combination with another Hgb beta-chain mutation

Hemoglobin S • The most common variants of the disease are HbSC and HbS-β-thalassemia • Genes that carry information for globin chain synthesis are located at specific loci on C16 and C11 • Alpha genes are located on C16, while Beta chain coding is located on the short arm of C11 • Each chain has 2 loci resulting in Hgb variants that are inherited as autosomal codominants with one gen coming from each parent

Hemoglobin S • Patients with Sickle Cell disease (SS, SC or S/β-thal) have inherited a sickle (S) gene from one parent and either an S, C or β-thalassemia gene from the other. • Individuals who are homozygous have more severe disease than those who are hetorzygous for HgbS • Inheritance is typical as deomonstrated by Pnnnett square predictions

Hemoglobin S • Hgb S is defined by a β chain substitution of glutamic acid by valine at the 6th position • The carrier rate of Hgb S is between 20 and 40% in Africa • Approximately 12% of African Americans carry the gene • The homozygous state (HbSS) is the most severe with an incidence of 1 in 375 African-American births

Hemoglobin S • Sickle cell trait occurs at the rate of 8% in the African-American population • HbSC occurs at a rate of 0.12% and HbS/ β-thal occurs in 0.16% of African Americans • Sickle cell anemia may also be found in persons from the Middle East, India and the Mediterranean (distribution fig 24-3)

Hemoglobin S • Hgb S can also be found in persons from the Caribbean and from Central and South America • HbS is soluble in the RBC and as long as the cell is oxygenated it remains a biconcave disk • Upon deoxygenation, the Hgb becomes less soluble, liquid crystals of HbS form and cellular sickling occurs

Hemoglobin S • In homozygotes, the sickling begins when Oxygen saturation drops below 85% • In heterozygotes, oxygen saturation must be below 40% for sickling to occur • Upon sickling, the blood becomes more viscous, pH is reduced and there is an increase in 2,3-BPG • Sickling results in occlusion of capillaries and arterioles and infarction of surrounding tissues

Hemoglobin S • Sickle cells come in two types • Reversible, cells that undergo hgb polymerization, increased viscosity and change shape upon deoxygenation; the cells are normal in shape, viscosity and presence of polymers when oxygenated • Vaso-occlusive complications are blamed on these cells who travel into the microvasculature when oxygenated and then become distorted while becoming deoxygenated in the periphery

Hemoglobin S • The second type of sickle cell is termed irreversible. • Irreversible cells do not change their shape regardless of the change in environment and hemoglobin polymerization • Irreversible cells are seen on the PB smear as elongated sickle cells with a point at each end

Hemoglobin S- clinical features • Symptoms can vary from asymptomatic to potentially lethal • Symptoms characteristically develop during the second half of the first year of life as patients are protected by HgbF • As beta chains of HbS replace gamma chains of HbF, the progressive problems related to increasing HbS develop

Hemoglobin S- clinical features • Only 4 variants of HgbS inheritance are known to be clinically significant (HbSS, HbSC, HbS-β0thal and HbS-β+thal) • These 4 forms carry high morbidity and mortality • Average life expectancy has improved and is now 45 years for the HbSS patient and 65 years for the HbSC patient

Hemoglobin S- clinical features • Many victims of sickle cell disease undergo episodes of recurring pain termed sickle crises, the pathogenesis of which is not understood • The hallmark of sickle cell disease is vaso-occlusion, with acute, severe pain resulting in visits to the emergency room • Triggers of crises include acidosis, hypoxia, dehydration, infection and fever, exposure to cold

Hemoglobin S- clinical features • Pain most often occurs in the bones, lungs, liver, spleen, penis, eyes and central nervous system • Frequency of painful symptoms varies from none to as many as 6 per year and last for 4-5 days • Repeated infarcts of the spleen result in the spleen’s diminishment due to scarring

Hemoglobin S- clinical features • Chronic hemolytic anemia is characterized by • Shortened RBC survival • Decreased hgb and hct • Elevated reticulocyte count • Jaundice • Clinical features of sickle cell disease are summarized in table 24-4 of the text

Hemoglobin S- Incidence With Malaria and G6PD Deficiency • The frequency of the sickle gene occurs in parallel to the incidence of Plasmodium falciparum and appears to offer protection from the parasite • One explanation is that the infected cell is quickly sickled and destroyed, reducing the number of organisms and increasing available time for immunity to develop

Hemoglobin S- Incidence With Malaria and G6PD Deficiency • It has also been suggested that G6PD deficiency plays some type of protective role with malarial parasites • This correlation has not been confirmed by scientific study

Hemoglobin S – Lab Diagnosis • Anemia of SCD is that of a chronic hemolytic anemia with normocytic, normochromic RBCs • PB smear shows marked poikilocytosis and anisocytosis with normal, sickled, target and nucleated RBCs, a few spherocytes, basophilic stippling, Pappenheimer bodies and HJ bodies

Sickle Cell - EM

Hemoglobin S – Lab Diagnosis • The diagnosis is made by demonstration of the insolubility of S Hgb in the deoxygenated form with a sickle cell screen • If the screening test is positive, the presence of hemoglobin S is confirmed using electrophoresis

Hemoglobin S – Treatment • Supportive care (transfusion, analgesics) has been the hallmark of treatment • Current research aimed toward modifying the genetic pathogenesis of sickle cell disease looks promising • Neonatal screening, childhood prophylactic penicillin, bone marrow transplantation and other treatments may extend the life of the sickle disease patient

Hemoglobin S – Treatment • Transfusion should only be used for prevention of the complications of sickle cell disease (increasing the RBC mass) • Red cell exchange processes with HbAA blood may be used when compications arise during surgery • Bone marrow transplantation is generally performed in children with severe complications

Hemoglobin S – Prognosis • Median life expectancy for men and women with homozygous SCD is 42 and 48 years, respectively • Heterozygote SC disease imparts a life expectancy of 60 and 68 years for men and women, respectively • Individuals with HbSS are discouraged from jobs that require strenuous physical exertion, exposure to high altitudes or extreme environmental temperature changes

Sickle Cell Trait • Heterozygotes (HbAS) are described as having sickle cell trait • Individuals are generally asymptomatic, having no significant clinical or hematologic manifestations • However, under extreme hypoxic conditions, sickling and the associated complications may occur

Sickle Cell Trait • Conditions such as severe respiratory infections, unpressurized flights at high altitude and anesthesia may trigger these complications • PB smear is normal (maybe a few target cells) • Sickle cell screen (solubility test) will be positive • Electrophoretograms demonstrate approximately 40% HgbS • No treatment is required for this condition and life expectancy is not affected

Hemoglobin C & E • Hemoglobins C and E are the second most common hemoglobinopathies • They cause mild hemolysis in the homozygous state • In heterozygous inheritance patterns, they are asymptomatic • HgbCC patients will often demonstrate tetragonal crystals that may be seen inside and outside the RBC membrane on a PB smear

Hemoglobin C & E • HbEE results in a mild microcytic anemia • No treatment is usually required for patients homozygous for HbC or HbE as the patient’s health is usually not affected

Hemoglobin C Disease

HbS in Combination With Other Hgb Variants • In the double heterozygous state, where a different abnormal chain is inherited from each parent, varying severity of HA may occur • HbSC is a double heterozygous syndrome where both beta-globin chains are affected by amino acid subsititutions • Incidence of HbSC is 1 in 833 births in the US

HbSC • HbSC disease resembles mild SCD • Childhood growth and development are delayed compared to normal children • Significant symptoms usually do not occur until the teenage years • May exhibit all of the vaso-occlusive symptoms of sickle cell disease • Episodes are less frequent and damage is less disabling

HbSC • Life expectancy for men in the US is 60 years and for women, 68 years • Sickle screen is positive (solubility test) • Confirmation is by electrophoresis • Treatment is similar to those patients with sickle cell disease

HbS/β-Thalassemia • This is the most common form of double heterozygosity in patients of Mediterranean descent and is second most frequent after HbSC • Syndrome is similar to mild SCD • If there is no production of beta globin (S-β0-thal), clinical course is similar to HbSS anemia

HbS/β-Thalassemia • If there is production of beta globin (S-β+-thal) patients tend to have a milder disease than those with HbSC • These patients have more HbS than HbA, microcytosis, hemolytic anemia, abnormal peripheral blood morphology and splenomegaly

Unstable Hemoglobin Variants • Approximately 200 variants of unstable hemoglobin have been described • Most are beta-chain variants, while some are alpha-chain variants • Few gamma-and delta-chain variants exist • Most of the unstable Hgb variants have no clinical significance, though the majority have increased oxygen affinity • About 25% are responsible for a variable hemolytic anemia

Unstable Hemoglobin Variants • This syndrome, unstable hemoglobin disease, occurs at or just after birth • Inherited as an autosomal dominant • All patients are heterozygous as the homozygous condition results in death • Instability of the Hgb molecule has several causes: • Substitution in the interior of the molecule of the polar for a nonpolar pocket

Hematology 425 Hemoglobinopathies