Introduction

Hemoglobin is an oxygen-binding protein found in erythrocytes that transports oxygen from the lungs to tissues. Each hemoglobin molecule is a tetramer made of four polypeptide globin chains. Each globin subunit contains a heme moiety formed of an organic protoporphyrin ring and a central iron ion in the ferrous state (Fe2+). The iron molecule in each heme moiety can bind and unbind oxygen, allowing for oxygen transport in the body. The most common type of hemoglobin in the adult is HbA, which comprises two alpha-globin and two beta-globin subunits. Different globin genes encode each type of globin subunit.[1]

The two main components of hemoglobin synthesis are globin production and heme synthesis. Globin chain production occurs in the cytosol of erythrocyte precursors and occurs by genetic transcription and translation. Many studies have shown that the presence of heme induces globin gene transcription. Genes for the alpha chain are on chromosome 16, and genes for the beta chain are on chromosome 11. Heme synthesis occurs in both the cytosol and the mitochondria of erythrocytes precursors. It begins with glycine and succinyl coenzyme A and ends with the production of a protoporphyrin IX ring. The binding of the protoporphyrin to a Fe2+ ion forms the final heme molecule.[2]

Fundamentals

There are a few different forms of normal hemoglobin in human blood. The percent prevalence of each hemoglobin type depends on the stage of development.

During pregnancy, the fetus primarily produces fetal hemoglobin (HbF). HbF comprises two a and two gamma-globin subunits. HbF has a stronger oxygen affinity than HbA, allowing oxygen to flow from maternal to fetal circulation through the placenta. Production of HbF drops significantly after birth, reaches low, near-adult levels by two years, and ultimately makes up 2 to 3% of hemoglobin in adults.

HbA, the most common adult form of hemoglobin, comprises two alpha and two beta-globin subunits. Inversely to HbF, HbA production explodes after birth and ultimately makes up 95-98% of hemoglobin in adults.

HbA2 is a less common adult form of hemoglobin. It comprises two alpha and two delta-globin subunits and makes up 1 to 3% of hemoglobin in adults.[3]

Issues of Concern

In hemoglobin synthesis, there are numerous steps and processes where errors can arise. For example, heme synthesis involves multiple enzymes (described below). Issues arise when one of these enzymes is deficient or inadequately functioning. During globin subunit production, potentially serious consequences can ensue if there are mutations or deletions in genes coding for globin chains; this relates to disorders in which abnormal hemoglobin predominates in the blood.

Molecular Level

There are multiple steps involved in heme synthesis. Eight enzymes accomplish this process, four working in the mitochondria and four in the cytosol. The process starts in the mitochondria, where  ALA (aminolevulinic acid) synthase links glycine and succinyl coenzyme A to form ALA. Steps 2 through 5 occur in the cytosol. Next, ALA dehydratase takes two molecules of ALA and produces porphobilinogen (PBG). In the third step, porphobilinogen deaminase takes four molecules of PBG and produces hydroxymethylbilane. Next, uroporphyrinogen III cosynthase takes hydroxymethylbilane and produces uroporphyrinogen III. In the fifth step, uroporphyrinogen decarboxylase takes uroporphyrinogen III and produces coproporphyrinogen III. The final three steps of heme synthesis occur in the mitochondria. Coproporphyrinogen III is then transformed to protoporphyrinogen IX by coproporphyrinogen oxidase. The seventh step occurs when protoporphyrinogen oxidase converts protoporphyrinogen IX to protoporphyrin IX. The eighth and final step of heme synthesis is the addition of Fe to protoporphyrin IX by ferrochelatase, producing a heme molecule.[2][4]

Testing

In patients with an abnormal complete blood count (CBC), signs and symptoms of hemolytic anemia (increase in unconjugated bilirubin, a decrease in hemoglobin levels, weakness, fatigue, jaundice, hemoglobinuria), or a family history of hemoglobinopathy, further testing screens for and diagnoses hematologic disorders.

CBC includes measurement of hemoglobin level in the blood. Normal hemoglobin concentrations are approximately 13.5 to 18.0 grams per deciliter in men and 11.5 to 16.0 grams per deciliter in women. CBC also measures the size of erythrocytes through the mean corpuscular volume (MCV). Low MCV, also known as microcytosis, is often the first indicator of thalassemia or thalassemia trait. A peripheral blood smear can also detect suspected hematologic disorders. Providers can see sizes, colors, and variations in the shape of erythrocytes. Peripheral smears can detect nuclei in erythrocytes, which are abnormal and signify pathology.

Hemoglobin variant testing measures by percentage of the relative hemoglobin types present in erythrocytes. This testing allows for the detection of hemoglobin variants and thalassemic disorders. DNA and genetic testing help identify deletions and mutations in the alpha and beta-globin-producing genes, which has become a standard part of newborn screening to improve early detection and treatment (if applicable) of hemoglobin variants, including sickle cell disease/carrier status and thalassemia disorders.

Clinical Significance

Thalassemia

Thalassemias are disorders caused by reductions or absence of globin chain synthesis. As the globin subunit synthesis is a critical portion of hemoglobin synthesis, thalassemias are relevant and clinically significant hematologic disorders. Alpha-thalassemia occurs with decreased or absent production of alpha-globin subunits. Beta-thalassemia occurs with reduced or absent production of beta-globin subunits. 

Alpha-thalassemia comprises four subtypes based on the severity of the disease. They are all caused by alpha-globin gene deletions that negatively impact alpha-globin subunit synthesis. The difference in a subtype is the number of alpha-globin gene deletions. One gene deletion results in alpha-thalassemia (also known as alpha-thalassemia minima or a silent carrier), which has no significant hematologic consequences. Two gene deletions result in alpha-thalassemia (also known as alpha-thalassemia minor), which causes mild microcytic, hypochromic anemias. Two deletions on the same chromosome 16 (cis deletion) are prevalent in Asian populations, whereas one deletion on each chromosome 16 (trans deletion) is prevalent in African American populations. Three gene deletions result in Hemoglobin H (HbH) disease, which causes moderate to severe microcytic, hypochromic anemia, causing the accumulation of beta-globin subunits to combine and form beta tetramers (HbH). HbH is an unstable form of hemoglobin that precipitates and causes damage to erythrocytes as they age. Four gene deletions result in Hemoglobin Bart’s disease (Hb Bart’s), which is incompatible with life. The absence of alpha-globin subunits allows gamma-globin subunits in utero to combine and form gamma tetramers. Hb Bart’s has a high oxygen affinity and does not allow the release of oxygen to body tissues, leading to severe hypoxia of the infant and, ultimately, a lethal condition known as hydrops fetalis.[5][6]

Beta-thalassemia comprises two major subtypes based on the severity of the disease. Beta-globin gene mutations that negatively impact beta-globin subunit synthesis cause both. Heterozygotes with only one gene mutation have beta-thalassemia minor, which causes diminished production of beta-globin subunits. Although some patients may develop mild microcytic anemia, most are asymptomatic. There is typically no evidence of hemolysis. Homozygotes with two gene mutations have beta-thalassemia major, which causes absent production of beta-globin subunits—the lack of beta-globin results in the accumulation of alpha-globin subunits and alpha tetramers which damage erythrocytes. Ultimately, ineffective erythropoiesis and extravascular hemolysis cause severe microcytic, hypochromic anemia. These patients require chronic blood transfusions.[6][7]

Porphyria

The porphyrias are a group of hereditary or acquired disorders caused by defective heme synthesis. Ineffective enzymes in the heme synthesis pathway result in a buildup of potentially toxic heme precursors. There are nine different porphyrias. Porphyria cutanea tarda (PCT) is the most common, and acute intermittent porphyria (AIP) is second.

PCT is a chronic hepatic porphyria caused by deficient activity of uroporphyrinogen decarboxylase (UROD). The result is the accumulation of porphyrinogens (such as uroporphyrinogen III) within hepatocytes. Clinically, cutaneous photosensitivity and hyperpigmentation are characteristic. The gradual formation of vesicles, bullae, blisters, and sores occurs in sun-exposed areas, especially the hands. Common associations with PCT include excessive alcohol consumption, hepatitis C, and human immunodeficiency virus (HIV).

AIP is an acute hepatic porphyria causing deficient activity of porphobilinogen deaminase. The result is the accumulation of neurotoxic metabolites, including ALA and porphobilinogen. Caloric deprivation, medications that induce cytochrome P-450, and hepatic ALA synthase precipitate this disease. Abdominal pain occurs in up to 90% of patients with AIP, making it the hallmark of an acute attack. Patients can also present with nausea, vomiting, constipation, fever, tachycardia, and hypertension. Neurotoxic effects of the metabolite buildup include autonomic instability, peripheral neuropathy, neuropathic pain, and psychological disturbances such as anxiety and hallucinations.[4]

Sickle Cell Trait and Disease

The most common abnormal hemoglobin variant is HbS (sickle cell hemoglobin). HbS results from a substitution of the sixth amino acid in the beta-globin subunits. The genetic mutation results in the replacement of glutamic acid with valine and occurs most frequently in African Americans. Heterozygous individuals have a mutation in only one of the two beta chains, resulting in sickle cell trait. Resistance to falciparum malaria infection and complications are benefits of a sickle cell trait. Homozygous individuals have mutations in both beta chains, resulting in sickle cell disease. When deoxygenated, HbS causes the deformation of erythrocytes from a biconcave disc to a crescent or “sickle” shape. This change in shape causes damage to erythrocyte membranes, premature destruction of erythrocytes, and chronic hemolytic anemia. Sickled erythrocytes can obstruct blood flow and cause tissue hypoxia, which can cause severe ischemic pain or even stroke. These patients also have functional asplenia and are at risk for infections with encapsulated organisms.[6][8]