3.4 Hemoglobin Structure, Hemoglobinopathies (Sickle Cell, Thalassemia), and Variants

Hemoglobin Structure, Hemoglobinopathies, and Thalassemia

Normal Hemoglobin Structure and Synthesis

Hemoglobin is a complex, tetrameric protein that is responsible for the transport of oxygen from the lungs to the tissues and the return of carbon dioxide from the tissues to the lungs. A normal hemoglobin molecule consists of four globular protein subunits (globins), each containing a heme group. Each heme group is a porphyrin ring with a central ferrous (Fe2+) iron atom capable of reversibly binding one oxygen molecule. Thus, a single hemoglobin tetramer can bind up to four molecules of oxygen.

The synthesis of globin chains is tightly regulated and involves two gene clusters. The alpha-globin gene cluster is located on chromosome 16 and contains the genes for alpha and zeta chains. Since humans inherit two alleles from each parent, there are a total of four alpha genes ($\alpha\alpha/\alpha\alpha$). The beta-globin gene cluster is located on chromosome 11 and contains the genes for beta, gamma, delta, and epsilon chains. There are only two beta genes (one from each parent).

Throughout development, the type of hemoglobin produced changes to adapt to the oxygen affinity requirements of the embryo, fetus, and adult.

• Embryonic Hemoglobins: Gower 1, Gower 2, and Portland are present during early embryonic development.
• Fetal Hemoglobin (HbF): Composed of two alpha and two gamma chains ($\alpha_2\gamma_2$). HbF is the predominant hemoglobin during fetal life because its decreased affinity for 2,3-bisphosphoglycerate (2,3-BPG) allows it to extract oxygen from the maternal circulation. At birth, HbF makes up about 60-90% of total hemoglobin, but it rapidly declines to less than 1-2% by 6 months of age.
• Adult Hemoglobins:
  • HbA: Composed of two alpha and two beta chains ($\alpha_2\beta_2$). It is the major adult hemoglobin, comprising >95% of total hemoglobin.
  • HbA2: Composed of two alpha and two delta chains ($\alpha_2\delta_2$). It comprises approximately 1.5-3.5% of total hemoglobin in normal adults.

Qualitative Hemoglobinopathies

Hemoglobinopathies are genetic disorders characterized by the production of structurally abnormal globin chains. These are qualitative defects, typically resulting from a single amino acid substitution due to a point mutation.

Hemoglobin S (Sickle Cell Disease and Trait)

HbS is the most common and clinically significant hemoglobin variant. It results from a single point mutation in the beta-globin gene where the polar amino acid glutamic acid is replaced by the nonpolar amino acid valine at the 6th position of the beta chain ($\beta^6\text{ Glu}\rightarrow\text{Val}$).

This single amino acid substitution causes a profound change in the physical properties of the hemoglobin molecule. When deoxygenated, HbS molecules polymerize into rigid, elongated tactoids. These tactoids distort the red blood cell into the characteristic crescent or "sickle" shape. Sickled cells are inflexible and tend to occlude the microvasculature, leading to vaso-occlusive crises, tissue ischemia, and infarction (e.g., autosplenectomy). They are also prematurely destroyed by the reticuloendothelial system, causing a severe chronic hemolytic anemia.

• Sickle Cell Trait (HbAS): The heterozygous state. Patients are generally asymptomatic because the presence of normal HbA prevents significant polymerization of HbS under normal physiological conditions. In vitro sickling tests (e.g., sodium metabisulfite) or solubility tests (using sodium dithionite) will be positive.
• Sickle Cell Anemia (HbSS): The homozygous state. The peripheral smear shows sickle cells (drepanocytes), target cells, Howell-Jolly bodies (due to functional asplenia), and nucleated red blood cells.

Hemoglobin C

HbC is another common variant, particularly in populations of West African descent. It results from a substitution of glutamic acid by lysine at the 6th position of the beta chain ($\beta^6\text{ Glu}\rightarrow\text{Lys}$). Unlike HbS, HbC does not polymerize upon deoxygenation. Instead, it tends to crystallize, especially in oxygenated conditions or when the cell is dehydrated.

• HbC Trait (HbAC): Asymptomatic, mild targeting on the peripheral smear.
• HbC Disease (HbCC): Mild to moderate chronic hemolytic anemia, splenomegaly, numerous target cells, and characteristic dense, block-like intraerythrocytic "bar of gold" HbC crystals on the peripheral smear.
• Hemoglobin SC Disease: A compound heterozygous condition where the patient inherits one HbS gene and one HbC gene. It presents as a milder form of sickle cell disease but with a higher propensity for retinopathy and avascular necrosis. The smear shows target cells, rare sickle cells, and unique "Washington Monument" or "gloved hand" crystals.

Laboratory Diagnosis of Hemoglobin Variants

Hemoglobin electrophoresis is the standard screening method for identifying hemoglobin variants. The migration of hemoglobin molecules depends on their net electrical charge.

1. Alkaline Electrophoresis (Cellulose Acetate, pH 8.4-8.6) At an alkaline pH, hemoglobin has a net negative charge and migrates from the negative electrode (cathode) toward the positive electrode (anode). The order of migration, starting from the cathode (slowest) to the anode (fastest), is C, S, F, A.

• A useful mnemonic is Crawls, Slow, Fast, Accelerates.
• Note that several variants co-migrate at an alkaline pH:
  • Hb C migrates with Hb A2, E, and O-Arab.
  • Hb S migrates with Hb D and G.

2. Acid Electrophoresis (Citrate Agar, pH 6.0-6.2) Because of the co-migration on cellulose acetate, an abnormal band must be confirmed using citrate agar at an acidic pH. This method relies on the interaction of the hemoglobin variant with the agar gel.

• It separates Hb C from A2, E, and O-Arab.
• It separates Hb S from D and G.
• The order of migration from cathode to anode is C, S, A, F.

Today, many laboratories also use high-performance liquid chromatography (HPLC) or capillary electrophoresis for faster, automated, and more precise quantification of hemoglobins A2 and F, and for the tentative identification of variants.

Quantitative Defects: The Thalassemias

Unlike hemoglobinopathies (which are structural/qualitative defects), thalassemias are quantitative defects. They are characterized by the reduced or absent synthesis of one or more normal globin chains. This imbalance in globin chain synthesis leads to the accumulation of the unpaired chains, which precipitate and damage the RBC membrane, causing premature destruction (ineffective erythropoiesis) and hemolysis.

Alpha Thalassemia

Alpha thalassemia is primarily caused by gene deletions. Because there are four alpha genes (two on each chromosome 16), the severity of the disease is directly proportional to the number of deleted genes.

• Silent Carrier (1 gene deleted, $-\alpha/\alpha\alpha$): Asymptomatic, normal CBC.
• Alpha Thalassemia Trait (2 genes deleted, $--/\alpha\alpha$ or $-\alpha/-\alpha$): Mild microcytic, hypochromic anemia. Often mistaken for iron deficiency, but the RBC count is typically normal or elevated, and the Mentzer index (MCV/RBC) is usually < 13.
• Hemoglobin H Disease (3 genes deleted, $--/-\alpha$): Severe imbalance. Excess beta chains form tetramers ($\beta_4$) known as Hemoglobin H. These precipitate as Heinz bodies (demonstrated with supravital stains like brilliant cresyl blue) causing moderate to severe hemolytic anemia.
• Hydrops Fetalis (4 genes deleted, $--/--$): Incompatible with life. The fetus produces predominantly Hemoglobin Barts ($\gamma_4$), which has such a high oxygen affinity that it cannot deliver oxygen to tissues, leading to severe hypoxia, heart failure, and death in utero.

Beta Thalassemia

Beta thalassemia is predominantly caused by point mutations (over 200 different mutations identified) rather than large deletions. These mutations either completely abolish beta chain production ($\beta^0$) or severely reduce it ($\beta^+$). Because there are only two beta genes, the clinical syndromes are broadly classified into three categories:

• Beta Thalassemia Minor/Trait ($\beta/\beta^0$ or $\beta/\beta^+$): The heterozygous state. Patients have a mild, asymptomatic microcytic hypochromic anemia. The hallmark laboratory finding is an elevated HbA2 (>3.5%, often 4-8%) on electrophoresis or HPLC. The peripheral smear shows striking microcytosis, hypochromia, target cells, and basophilic stippling.
• Beta Thalassemia Intermedia: Variable clinical severity, moderate anemia. Patients typically do not require regular transfusions but may have splenomegaly and iron overload.
• Beta Thalassemia Major (Cooley's Anemia, $\beta^0/\beta^0$): The homozygous state with severe absence of beta chains. It presents in infancy (usually around 6 months when the switch from gamma to beta chains fails). Patients suffer from severe, transfusion-dependent hemolytic anemia. Extramedullary hematopoiesis leads to hepatosplenomegaly and bone deformities (e.g., "crew-cut" skull x-ray, chipmunk facies). Electrophoresis shows little to no HbA and vastly increased HbF (often >90%).

Key Laboratory Distinctions

In evaluating a microcytic anemia, differentiating between iron deficiency anemia (IDA) and thalassemia trait is critical. In thalassemia trait, the microcytosis is out of proportion to the degree of anemia (e.g., hemoglobin 10.5 g/dL with an MCV of 62 fL). The RBC count is normal or elevated (>5.0 x $10^{12}$/L), while in IDA it is decreased. The RDW is usually normal in thalassemia trait but elevated in IDA. Iron studies are definitive: normal or elevated ferritin and serum iron in thalassemia, versus low ferritin and iron in IDA.