Hemoglobin variants including Hb C, Hb D, and Hb E

INTRODUCTION — This topic discusses genetic variants in globin genes. This includes especially common variants that produce hemoglobin (Hb) C, D, E, and others. An approach to Hb C genetic test results is presented separately. (See "Gene test interpretation: Hemoglobin C (Hb C) variant in the hemoglobin beta locus (HBB)".)

Separate topic reviews discuss the sickle cell variant that causes sickle cell disease (SCD) and variants affecting alpha globin and beta globin production that cause thalassemias.

●SCD – (See "Gene test interpretation: Sickle cell variant in the hemoglobin beta locus (HBB)" and "Pathophysiology of sickle cell disease".)

●Thalassemias – (See "Molecular genetics of the thalassemia syndromes" and "Pathophysiology of thalassemia", section on 'Terminology and disease classification'.)

NORMAL HEMOGLOBINS — A series of normal hemoglobins (Hbs) are produced during embryonic, fetal, and postnatal life.

●Embryonic – Embryonic Hbs are primarily the product of yolk sac erythroblasts and are detectable only during the very earliest stages of embryogenesis (except for trace amounts in severe alpha thalassemia). They consist of embryonic globin chains including zeta and epsilon and are named Gower I, Portland, and Gower II. (See "Structure and function of normal hemoglobins".)

●Fetal – By approximately the 14^th week of gestation, embryonic Hbs are completely replaced by fetal Hb (Hb F; a tetramer of two alpha globin chains and two gamma globin chains [alpha2gamma2]). Hb F has a higher oxygen affinity than adult Hb (Hb A; alpha2beta2), which allows preferential transfer of oxygen from the maternal circulation to the fetal circulation. (See "Structure and function of normal hemoglobins".)

●Adult – Adult Hbs are produced starting in the first year of life. The predominant form is Hb A, consisting of two alpha chains and two beta chains (alpha2beta2). Red blood cells also contain a small portion of Hb A₂ (alpha2delta2) and Hb F, in the following percentages:

•Hb A – 95 to 98 percent

•Hb A₂ – 2 to 3 percent

•Hb F – <2 percent

Details of globin gene switching, other aspects of regulation, and functional properties of the different Hbs are discussed separately. (See "Structure and function of normal hemoglobins" and "Fetal hemoglobin (Hb F) in health and disease", section on 'Biology of Hb F' and "Fetal hemoglobin (Hb F) in health and disease", section on 'Hemoglobin switching and downregulation of Hb F expression'.)

OVERVIEW OF HEMOGLOBIN VARIANTS

Scope — Over 1000 different variants (genetic changes) have been described in the genes that encode the different globin chains [1,2]. Variants can be classified into the clinical phenotype (anemia, polycythemia, cyanosis, clinically silent) by the type of hematologic changes they produce (hemolysis, reduced expression, altered oxygen affinity, sickling), by which globin chain is affected (alpha, beta, or gamma), and by the type of genetic variation (point mutation causing a single amino acid change, insertion or deletion affecting protein sequence or abundance). Many clinically silent variants are identified with newborn screening for hemoglobinopathy. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Population screening (eg, routine newborn screen)'.)

The tables summarize selected clinical disorders (table 1) and selected classes of DNA changes (table 2).

Protection against malaria — Some of the higher prevalence variants are thought to have evolved because the heterozygous states offer partial protection against malaria and generally are not associated with significant adverse clinical complications. Thus, evolutionary pressures favored their selection and/or did not favor their elimination. (See "Protection against malaria by variants in red blood cell (RBC) genes".)

Examples of common variants that are highly represented in regions with a high malaria burden include:

●Sickle hemoglobin – Most common in Nigeria, Democratic Republic of Congo, and India (figure 1), as well as certain Mediterranean regions and other countries in sub-Saharan Africa [3].

●Alpha thalassemia – Most common in Southern China, India, and Southeast Asia. Heterozygous alpha thalassemia is present at high prevalence throughout all tropical and subtropical regions, including most of Southeast Asia, the Mediterranean area, the Indian subcontinent, the Middle East, and Africa. Overall, alpha thalassemia variants have reached frequencies of approximately 5 percent worldwide. Frequencies of 70 percent and up to 90 percent have been reported in Melanesia and in parts of Nepal [4].

●Beta thalassemia – Most common in Africa, Southeast Asia, Italy, Iran, and Greece.

●Hb C – Most common in Burkina Faso; also in Mali, Ghana, Togo, and Benin [5].

●Hb E – The highest frequencies are observed in India, Bangladesh, and throughout Southeast Asia; it is particularly high in Bangladesh, Cambodia, and Southern China. Demographic mobility has resulted in high frequencies in California and other coastal regions [6,7].

Increased migration around the world has expanded the distribution of all of these variants. Race, ethnicity, or country of origin cannot be used to diagnose or exclude the presence of a variant.

Other variants that have prevalences of approximately 1 to 2 percent in the population may be the result of a founder effect, but they do not have a clear association with evolutionary selective pressures. Examples include Hb D-Punjab (also called Hb D-Los Angeles), Hb G-Philadelphia, and Hb Hasharon.

Age at presentation — Individuals with variants affecting globin chains may come to medical attention for different reasons. Variants affecting beta globin present later in infancy, after the switch from fetal to adult hemoglobin. (See 'Presenting findings' below.)

●Newborn screening – Newborn screening typically includes hemoglobin analysis. Newborn screening has been implemented to identify individuals with sickle cell disease (SCD) for early intervention to reduce morbidity and mortality. However, other symptomatic and asymptomatic variants are detected with screening. This evaluation may be done using a protein-based method or a DNA-based method. The method determines which variants will be identified. (See "Overview of newborn screening" and "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Common clinical scenarios'.)

●Symptomatic presentations – Many individuals are symptomatic in childhood, but in some cases the initial presentation may occur later in adolescence or in adulthood. Symptomatic individuals are typically homozygous or compound heterozygous for a clinically significant variant(s), but some are heterozygous. Examples are summarized in the table (table 1) and include:

•Anemia

•Microcytosis

•Hemolytic anemia

•Erythrocytosis

•Cyanosis

•Jaundice

•Sickling complications

●Incidental finding – Some individuals may become aware of a variant when a complete blood count (CBC), hemoglobin analysis, or genetic testing is done for a reason other than clinical symptoms (evaluation for another condition, reproductive testing and counseling). These individuals are typically heterozygous for an autosomal recessive variant.

●Family history – Some hemoglobin variants are inherited in a recessive fashion (eg, SCD, thalassemia). However, some variants, such as those that create an unstable hemoglobin, are autosomal dominant, and the family history may be positive.

Presenting findings — Presenting findings differ depending on which globin chain is affected and how the variant affects hemoglobin production and properties. General findings include:

●Age of onset – Variants affecting beta globin chains that are symptomatic will present with clinical symptoms after four to six months of age. Variants affecting alpha and gamma globin chains that are symptomatic will present with symptoms at (or before) birth. (See 'Age at presentation' above.)

●Sickle cell syndromes – SCD due to homozygous Hb SS or Hb S beta⁰ thalassemia typically presents in infancy (age six months to one year, as beta globin production increases) with vaso-occlusive pain (dactylitis) or hemolytic anemia. Before the era of early disease identification, patients presented with acute splenic sequestration crisis or pneumococcal sepsis. Other compound sickle cell syndromes (sickle-beta⁺ thalassemia or Hb SC disease) may have milder presentations or present at a later age, usually after six months of age or older. Hb SE disease is usually asymptomatic, but over time at least one-third experience vaso-occlusive events [8]. (See "Overview of compound sickle cell syndromes".)

In SCD, there is reticulocytosis and findings of hemolysis. The blood smear in Hb SS often demonstrates red blood cells (RBCs) with Howell-Jolly bodies, target cells, nucleated RBCs, and irreversibly sickled cells. Many of these characteristic changes are less prominent in the less anemic sickle variants. In Hb SC disease, the RBCs demonstrate a predominance of target cells, microcytosis, and elevated mean corpuscular hemoglobin concentration (MCHC), but irreversibly sickled cells are not often seen. The Hb S-beta thalassemia syndromes produce a microcytic, hypochromic anemia with target cells, basophilic stippling, and Pappenheimer bodies.

Sickle cell trait (heterozygosity for the sickle variant with a normal sequence at the other HBB allele) is essentially an asymptomatic carrier state with minimal clinical implications for most individuals. However, clinically significant complications can occur. The CBC and blood smear in sickle cell trait are usually normal. (See "Overview of the clinical manifestations of sickle cell disease" and "Overview of compound sickle cell syndromes" and "Sickle cell trait".)

Conditions that can result in symptoms of SCD include:

-Hb SS disease

-Hb SC disease

-Hb SE disease

-Hb S beta thalassemia

-Hb SD-Punjab/Los Angeles

-Hb S plus Hb O Arab

-Hb C plus Hb C-Harlem or Hb S plus Hb C-Harlem

Other conditions with the sickle variant cause minimal or no sickling symptoms:

•Hb S with hereditary persistence of fetal hemoglobin (HPFH)

●Thalassemias – Genetic variants associated with alpha or beta thalassemia cause reduced hemoglobin expression; they do not change the amino acid composition of hemoglobin. An exception is Hb Constant Spring, an alpha thalassemic variant that produces an elongated alpha chain and a hemoglobin tetramer with abnormal migration on electrophoresis. (See 'Hb Constant Spring' below.)

•Alpha thalassemia – The age of presentation and clinical severity depend on the number and type of alpha chains affected (of four total), ranging from hydrops fetalis or severe anemia in early infancy to mild anemia or isolated microcytosis discovered incidentally during adulthood. (See "Diagnosis of thalassemia (adults and children)".)

Key aspects of the clinical presentation are:

-Microcytic anemia present at birth

-Hb Barts on the newborn screen

Other findings include an increased RBC count, decreased mean corpuscular volume (MCV), and decreased mean corpuscular hemoglobin (MCH). The blood smear shows microcytosis and hypochromia. Hb Barts (a tetramer of gamma globin chains) is detected in newborn screening. When the individual has a three gene deletion, Hb H may appear on Hb electrophoresis. Hb Constant Spring (which accounts for 20 percent of alpha thalassemia variants in Southeast Asia), while more severe than deletional alpha thalassemia in general, does not cause significant microcytosis.

•Beta thalassemia – The age of presentation and severity depend on whether one or both beta chains are affected and whether the variant(s) reduce beta globin production (beta⁺) or abolish it completely (beta⁰). Heterozygosity for a beta thalassemia variant may cause mild anemia, microcytosis, and occasionally splenomegaly. Individuals with beta⁰ thalassemia are homozygous or compound heterozygous for beta globin gene variants that produce no Hb A. This condition presents in infancy as beta globin production increases and gamma globin production decreases. Usually, severe transfusion-dependent anemia and signs of extramedullary hematopoiesis occur, and transfusion therapy is required. In general, individuals who are double heterozygotes with beta⁰/beta⁺ or beta⁺/beta⁺ thalassemia have a less severe phenotype, but variability exists. (See "Diagnosis of thalassemia (adults and children)".)

Key aspects of the clinical presentation are:

-Microcytic anemia after six months of age

-Increased hemoglobin A₂ after six months of age

●Hb C – Homozygosity for Hb C (Hb CC disease) can present in infancy, older childhood, or adulthood, depending on other factors that affect clinical severity. Hb CC disease causes mild hemolytic anemia and often splenomegaly without vaso-occlusive manifestations. More severe disease can occur when Hb C is combined with a severe beta globin variant. Heterozygosity for Hb C is an asymptomatic carrier state. (See 'Hb C' below.)

In suspected Hb C disease, the blood smear may show cells with Hb C crystals inside contracted cells and classic RBC shape changes that suggest the likely diagnosis (picture 1).

Hb C in combination with beta thalassemia may result in a mild microcytic anemia. More severe disease can occur when Hb C is combined with a severe beta mutation (sickle variant or a severe beta thalassemia variant).

●Hb E – Individuals who are homozygous for Hb E have mild microcytic anemia. Hb E in combination with beta thalassemia can range from mild to moderate to severe depending on the other beta globin variant. Over half of individuals with Hb E plus beta⁰ thalassemia are transfusion dependent. Heterozygosity for Hb E is an asymptomatic carrier state.

●Hb M and low oxygen affinity variants – M Hbs, which cause congenital methemoglobinemia, and low oxygen affinity Hb variants can cause cyanosis in infancy or early childhood. They are symptomatic in the heterozygous state. (See "Methemoglobinemia", section on 'Congenital methemoglobinemia' and "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Low oxygen affinity hemoglobin variants: Cyanosis'.)

●High oxygen affinity variants – High oxygen affinity variants can cause polycythemia. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'High oxygen affinity hemoglobins: Erythrocytosis'.)

How to evaluate — The evaluation starts with review of the CBC, RBC indices, reticulocyte count, and blood smear.

Diagnostic tools for detecting and characterizing variants in globin chains have evolved over time. Electrophoresis was used in early studies as a way to separate proteins based on their electrical charge. Other methods such as high-performance liquid chromatography (HPLC) and capillary electrophoresis are increasingly used. Molecular methods such as next-generation DNA sequencing can also be diagnostic, especially for suspected alpha thalassemia. Specialized diagnostic reference laboratories can provide assistance in selecting the method, performing the testing, and interpreting results. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Referral to a specialized laboratory' and 'Resources' below.)

For unstable variants, Heinz body preps and heat stability assays can be informative. (See "Unstable hemoglobin variants", section on 'Diagnostic evaluation'.)

CBC and blood smear findings that suggest an Hb variant

●Anemia with an elevated reticulocyte count – Expected with an Hb variant associated with hemolysis such as a sickling syndrome or an unstable Hb variant.

●Microcytosis – Typical of both alpha and beta thalassemia; also characteristic of Hb E and C disease.

●Sickle forms – Seen with a sickle syndrome, particularly with Hb SS or Hb S-beta⁰ thalassemia. Sickle forms may be absent in Hb SC disease or H S-beta⁺ thalassemia.

●Target cells – Commonly seen in Hb E disease, Hb C disease, and thalassemia.

Which method to use to narrow the diagnosis — For many Hb disorders, a protein-based method (electrophoresis, HPLC, capillary electrophoresis) is the appropriate first step. This includes Hb S, Hb C, and other structural variants. (See "Diagnosis of sickle cell disorders" and "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Patient with suspected sickle cell disorder'.)

In suspected thalassemia, iron deficiency should be tested first using iron studies (ferritin, transferrin, iron, transferrin saturation [TSAT]) and corrected if present. This is because iron deficiency is in the differential diagnosis of thalassemia (both cause microcytic anemia), and, if both iron deficiency and thalassemia are present, the results of protein-based hemoglobin analysis will be skewed by the concomitant iron deficiency, making diagnosis more challenging. (See "Microcytosis/Microcytic anemia".)

Alpha globin variants usually require DNA testing for definitive diagnosis; protein-based analysis methods are not reliable. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)

DNA-based methods are most useful for confirming alpha thalassemias or detecting a specific (often rare) familial variant. In some jurisdictions, DNA-based methods are also being incorporated into newborn screening.

Often the choice of a specific protein-based or DNA-based method will be determined by the institutional laboratory and their interpretation of the most likely diagnosis. Details of the strengths and weaknesses of different methods are presented separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)

Interpretation of results — The two major abnormal findings on protein-based methods are presence of an abnormal Hb and/or an altered ratio of normal hemoglobins. General aspects of interpretation of hemoglobin include:

●Heterozygous beta chain variants will result in 40 to 60 percent abnormal hemoglobin by electrophoresis or HPLC.

●Positively charged heterozygous beta chain variants will result in approximately 40 percent abnormal hemoglobin (eg, Hb S, Hb C, Hb E). (See 'Abnormal hemoglobins' below.)

●Alpha chain deletions combined with positively charged beta chain variants will further reduce the abnormal hemoglobin below 40 percent. (See 'Altered ratios' below.)

●Each alpha thalassemia variant will result in approximately 25 percent of the abnormal hemoglobin variant (as there are four alpha chains).

For unknown variants that present on a newborn screen, it may be reasonable to repeat the screen, perform DNA sequencing, and/or conduct family studies. Reference laboratories may help with interpretation. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Referral to a specialized laboratory' and 'Resources' below.)

Abnormal hemoglobins — Hemoglobinopathies that produce an abnormal hemoglobin (Hb S, Hb C) are relatively easy to detect on most protein-based methods because they have altered properties (charge, mass) that affect their migration on electrophoresis or produce abnormal peptides on HPLC. DNA testing is also relatively straightforward, as a single point mutation can be identified. In the simplest of cases, heterozygotes will have 35 to 55 percent of the abnormal Hb, with the remainder being Hb A; compound heterozygotes (eg, Hb SC disease) will have two abnormal Hbs in approximately equal ratios with no Hb A; homozygotes will have only a single mutant Hb and no Hb A (figure 2) [9]. In more complex situations such as concomitant alpha thalassemia, more extensive molecular studies may be required.

Altered ratios — In some cases, normal Hbs including Hb A, Hb A₂, and Hb F are present but at a different ratio than is typically seen. These patterns are summarized in the table (table 3).

●Causes of increased Hb F levels are discussed separately. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Conditions causing increased Hb F'.)

●Causes of increased or decreased Hb A₂ are listed in the table (table 4) and discussed separately. (See "Structure and function of normal hemoglobins", section on 'Hb A2'.)

The percentage of different abnormal Hbs also depends on various factors including electrostatic charge, protein stability, and changes in other globin chains. In individuals heterozygous for a variant, the proportion of the variant is charge dependent, generally <50 percent for positively charged variants and >50 percent for negatively charged variants, as illustrated in the figure (figure 3) [10,11].

●Positively charged beta chain variants such as Hbs S, C, D-Punjab, and E constitute significantly less than one-half of the total Hb in heterozygotes, and their levels are reduced further in the presence of alpha thalassemia [9,12,13]. As an example, in individuals with sickle cell trait, the percent Hb S can be affected by concomitant alpha thalassemia, as summarized in the table (table 5) [14].

●Many of the negatively charged beta chain variants in heterozygotes constitute more than half of the total Hb. In two heterozygotes who had a negatively charged Hb variant (Hb Baltimore or Hb Iran) in conjunction with alpha thalassemia, the proportion of the abnormal Hb was found to be even further increased [9,12].

Resources — The Globin Gene Server (globin.bx.psu.edu) provides a link to the HbVar database of hemoglobin variants and additional educational resources [15,16].

Specialized laboratories can assist with testing and interpretation; specific laboratories and contact information are listed. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Referral to a specialized laboratory'.)

BETA GLOBIN VARIANTS — Variants in the hemoglobin beta locus (HBB) generally present at approximately six months of age, as the shift from gamma globin to beta globin production occurs after birth. Because there are two HBB alleles (figure 4), each allele contributes approximately 50 percent of the beta globin in the red blood cell (RBC).

Beta thalassemia and sickle cell disease (SCD) are the most common genetic disorders of beta globin.

●There are numerous HBB variants that reduce or abolish beta globin chain production, leading to beta thalassemia. (See "Molecular genetics of the thalassemia syndromes" and "Pathophysiology of thalassemia".)

●The sickle cell variant is a single point mutation that causes a single amino acid change (p.Glu7Val). Deoxygenated sickle hemoglobin (Hb S) forms polymers that affect RBC morphology and other properties. Heterozygosity for the variant causes sickle cell trait; homozygosity or compound heterozygosity with a thalassemia variant or hemoglobin C causes SCD. (See "Diagnosis of sickle cell disorders" and "Pathophysiology of sickle cell disease", section on 'Genetics'.)

Hb C

Hb C genetics — Hb C results from a point mutation in HBB that changes glutamic acid at amino acid 7 to a lysine (p.Glu7Lys; c.19G>A); this was referred to as amino acid 6 in a previous numbering system. The point mutation affects the same DNA codon as the sickle mutation (Hb S; p.Glu7Val), as illustrated in the figure (figure 5). An approach to genetic test results showing this variant are presented separately. (See "Gene test interpretation: Hemoglobin C (Hb C) variant in the hemoglobin beta locus (HBB)".)

●Hb C trait – Heterozygosity for Hb C causes Hb C trait, a carrier state that is essentially asymptomatic. However, preconception counseling and partner testing is important; if the partner also carries any HBB variant, the children may be more severely affected. (See "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis".)

●Hb C disease – Biallelic Hb C variants or Hb C plus a beta thalassemia variant cause Hb C disease. (See 'Hb C evaluation' below.)

●Hb SC disease – Compound heterozygosity for Hb C and Hb S causes Hb SC disease, a form of SCD. The clinical severity is usually less than that in individuals with homozygous Hb SS or Hb S-beta⁰ thalassemia. (See "Overview of compound sickle cell syndromes", section on 'Hb SC disease'.)

The prominent clinical manifestations of Hb SC disease versus sickle cell trait, both of which have Hb S at one HBB allele, can be attributed in part to differences in the intracellular content of Hb S in Hb SC disease compared with sickle cell trait [12]. This is due to the higher affinity of alpha globin chains for Hb S beta chains than for Hb C beta chains [12,17,18].

Hb C evaluation — Hb C disease may be suspected in an individual with one or more of the following [19-22]:

●A parent (or sibling) with Hb C disease, Hb SC disease, or Hb C trait.

●Mild hemolytic anemia with a high mean corpuscular hemoglobin concentration (MCHC), reflecting RBC dehydration. Individuals with Hb C trait also appear to have mildly increased MCHC [23]. The mean corpuscular volume (MCV) can be markedly decreased in Hb C disease and mildly decreased in Hb C trait. The cause of the microcytosis and increased MCHC involves activation of the potassium chloride (KCl) co-transporter, which leads to the loss of cellular water, producing smaller, denser cells.

●Hexagonal crystals on the peripheral blood smear (picture 1).

●Clinical findings related to chronic hemolysis including jaundice, pigment gallstones, or splenomegaly.

Hb C does not cause vaso-occlusive events. If an individual with apparent Hb C disease has vaso-occlusive complications, they should be evaluated for possible Hb C-Harlem and/or SCD [24]. (See 'Hb C-Harlem' below.)

Hb C is detected using a protein-based hemoglobin analysis method. On alkaline electrophoresis (pH 8.6 to 8.8), Hb C has the slowest migration and Hb A the fastest migration (figure 2). In Hb C disease, Hb electrophoresis indicates that Hb C comprises almost all the Hb detected. There may be slight elevations in Hb F. Acid pH can be used to separate Hb C from other Hbs that co-migrate at alkaline pH such as Hb A₂. Alternatively, methods such as high-performance liquid chromatography (HPLC) or capillary electrophoresis can be used. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Protein chemistry methods'.)

Hb C management — Hb C disease (Hb CC) causes mild chronic hemolytic anemia; Hb C trait (Hb AC) is an asymptomatic carrier state.

●Hb C disease – Hb C disease carries an increased risk of gallstones and splenomegaly. The anemia is mild, and the clinical course is usually benign, but the patient should be seen annually for monitoring of spleen enlargement and gallstone development.

The mild hemolysis of Hb C disease (and all hemolytic conditions) falsely lowers the HbA1C. If diabetes is suspected, another method for assessment other than HbA1C should be used, such as serum fructosamine. (See "Screening for type 2 diabetes mellitus", section on 'Glycated hemoglobin (A1C)' and "Measurements of chronic glycemia in diabetes mellitus", section on 'Other biomarkers'.)

Preconception counseling should be provided, with partner testing for hemoglobinopathies to determine the risk of hemoglobinopathy in children.

●Hb C trait – Individuals with Hb C trait should receive preconception counseling and partner testing for hemoglobinopathies to determine the risk of hemoglobinopathy in children.

●Hb SC disease – Hb SC disease is a form of SCD with a milder phenotype than sickle cell anemia (Hb SS) or sickle-beta⁰ thalassemia. However, individuals with Hb SC disease are at risk for all of the complications of SCD. Management of Hb SC disease is discussed in detail separately.(See "Overview of the management and prognosis of sickle cell disease" and "Overview of compound sickle cell syndromes", section on 'Hb SC disease'.)

Hb C-Harlem — Hb C-Harlem is an HBB variant that is genetically and clinically distinct from Hb C; it was initially labeled as a type of Hb C because it co-migrates with Hb C on alkaline gel electrophoresis.

Hb C-Harlem has two point mutations in the same HBB gene, the sickle mutation (p.Glu7Val; c.20A>T) and an additional mutation at amino acid 73 (p.Asp73Asn; c.220G>A) [25]. Like Hb S, Hb C-Harlem can polymerize when deoxygenated [24,25]:

●Heterozygosity for Hb C-Harlem is a benign condition, similar to sickle cell trait.

●Compound heterozygosity of Hb C-Harlem with Hb C resembles Hb SC disease.

●Homozygosity for Hb C-Harlem or compound heterozygosity with Hb S causes clinically severe SCD.

Management is similar to the related sickle cell syndrome. (See "Sickle cell trait", section on 'Management and preventive care' and "Overview of the management and prognosis of sickle cell disease".)

Hb D — There are several variants that can produce Hb D. Hb D-Punjab (also called Hb D-Los Angeles) is caused by a glutamic acid to glutamine substitution at codon 121 of HBB (Glu121Gln) [26]. Hb D-Punjab is one of the commonly observed abnormal Hbs worldwide and is seen in many genetic backgrounds.

Compound heterozygosity for Hb D-Punjab and Hb S causes an SCD syndrome. Other rare Hb D variants that result from different amino acid substitutions, such as Hb D-Ibadan (Thr88Lys) and Hb D-Iran (Glu23Gln), do not cause sickling when combined with Hb S.

Hb D-Punjab has an electrophoretic mobility similar to Hb S at alkaline pH. It can be distinguished from Hb S by HPLC or by the fact that it has the same migration at acid pH as Hb A and does not undergo the sickling phenomenon in vitro.

●Individuals with Hb D trait (Hb AD) are asymptomatic, are not anemic, and have normal RBC indices.

●Homozygosity for Hb D (Hb D disease, Hb DD) is rare and usually presents as mild hemolytic anemia and mild to moderate splenomegaly [27,28].

●Coinheritance of Hb D together with Hb S (Hb SD disease) can result in severe disease with clinical manifestations similar to homozygous sickle cell anemia [29]. (See "Overview of compound sickle cell syndromes", section on 'Sickle-Hb D disease'.)

Preconception counseling and partner testing for hemoglobinopathy is important for all of these individuals.

Hb E — Hb E is an HBB variant that causes reduced expression of beta globin (a beta thalassemic phenotype) and is mildly unstable to oxidative damage [30,31]. The Hb E variant is due to a mutation that substitutes lysine for glutamic acid at amino acid 26 of the beta globin chain (Glu26Lys). This beta chain is produced at a reduced rate relative to alpha chains and therefore clinically functions as a thalassemia variant.

The Hb E variant is most common in the Indian subcontinent and Southeast Asia, where the frequency of the Hb E carrier state approaches 60 percent. (See "Protection against malaria by variants in red blood cell (RBC) genes", section on 'Hemoglobin E'.)

Hb E has an electrophoretic mobility similar to that of Hb C, Hb A₂, and Hb O-Arab on alkaline electrophoresis. It can be distinguished from Hb C as it has the same migration at acid pH as Hb A. It can be distinguished from Hb O-Arab, which migrates closer to Hb S on acid pH, or by HPLC [32]. The high amount of Hb E in heterozygotes (30 percent) or homozygotes (>90 percent) distinguishes Hb E from Hb A₂, since the latter is a minor hemoglobin usually constituting less than 5 percent of total hemoglobin.

Clinically, Hb E causes few abnormalities other than microcytosis, target cells, and hypochromia when present in a heterozygous or homozygous state [6]:

●Heterozygous individuals (Hb E trait, Hb AE) are not usually anemic but may have minimal degrees of microcytosis and hypochromia. Hb analysis shows approximately 30 percent Hb E, 1 percent Hb F, and 70 percent Hb A.

●Homozygous individuals (Hb E disease, Hb EE ) have minimal anemia along with hypochromia, target cells, and prominent microcytosis [33]. Hb analysis shows >90 percent Hb E, with the remainder Hb F. There is no Hb A.

Hb E-beta⁰ thalassemia is a moderate to severe disorder, ranging from a transfusion-dependent, severe beta thalassemia to a beta thalassemia intermedia phenotype [34,35]. In general, Hb E-beta thalassemic disorders will be milder when there is concomitant alpha thalassemia trait, because there will be less globin chain imbalance. (See "Overview of compound sickle cell syndromes", section on 'Sickle-Hb E disease' and "Molecular genetics of the thalassemia syndromes", section on 'Hb E: A special case' and "Diagnosis of thalassemia (adults and children)", section on 'Overview of subtypes and disease severity'.)

In the newborn, Hb E-beta⁰ thalassemia has a similar appearance on electrophoresis as Hb EE, a benign condition. Further diagnostic testing using DNA analysis is required, unless the genotype of both parents is known. After early infancy, Hb F levels remain high in Hb E-beta⁰ thalassemia but are low in Hb EE disease.

Hb Lepore — Hb Lepore arises from an unequal crossing over and recombination event between adjacent genes that encode delta globin (HBD) and beta globin (HBB) to form a novel fusion gene. The fusion gene produces a functional and stable Hb with the mobility of Hb S on alkaline electrophoresis and Hb A on acid electrophoresis. Hb Lepore does not undergo the sickling phenomenon, but it may be confused with Hb S due to its position on alkaline electrophoresis (figure 2).

Because the production of the abnormal globin is under the control of the HBD promoter, which is only 2 to 3 percent as active as the HBB promoter, there is severe underproduction of this abnormal globin chain, such that Hb Lepore represents only approximately 3 to 20 percent of total Hb in the affected individual. Since no functional delta chain can be produced from the involved allele, the concentration of Hb A₂ is decreased to approximately 50 percent of normal (from 2 to 3 percent reduced to 1 to 1.5 percent). Hb F levels are usually slightly increased.

Hb Lepore also causes an imbalance in the ratio of alpha- and beta-like chains, producing a form of beta thalassemia of moderate-to-high severity in homozygotes, often requiring transfusion therapy; the phenotype is mild in heterozygotes [36]. (See "Molecular genetics of the thalassemia syndromes", section on 'Hb Lepore'.)

Hb O-Arab — Hb O-Arab is a beta globin gene (HBB) variant (HBB Glu121Lys) originally described in an Israeli Arab family, but its distribution is widespread. Hb O-Arab migrates with the same mobility as Hb C on alkaline electrophoresis and migrates between Hb A and Hb S on acid electrophoresis [32]. Hb O-Arab can also be distinguished from Hb C or Hb E by HPLC or genetic testing.

Hb O-Arab heterozygotes are clinically asymptomatic with no hematologic abnormalities, while homozygotes may have a mild, asymptomatic, compensated hemolytic anemia [32].

Compound heterozygosity for Hb O-Arab and Hb S causes severe SCD (similar in severity to Hb SS). (See "Overview of compound sickle cell syndromes", section on 'Sickle-Hb O Arab disease'.)

ALPHA GLOBIN VARIANTS — There are two alpha globin loci, HBA1 and HBA2 (figure 4). Thus, alpha globin is produced from four separate genes (two from each parent). Alpha globin variants begin to generate the abnormal Hb in utero, since alpha globin is a component of fetal Hb (Hb F; alpha2gamma2).

Alpha thalassemia variants — There are over 70 non-deletional variants that can cause alpha thalassemia, including point mutations that affect genomic regions critical for the normal expression of alpha globin genes. Point mutations that affect HBA2 have a more significant effect on alpha chain production [37]. An approach to genetic testing showing alpha thalassemia variants is presented separately. (See "Gene test interpretation: HBA1 and HBA2 (alpha globin genes)".)

General information about the molecular pathogenesis and pathophysiology of alpha thalassemia is presented separately. (See "Molecular genetics of the thalassemia syndromes" and "Pathophysiology of thalassemia", section on 'Alpha thalassemia'.)

Hb Constant Spring — Hb Constant Spring is a structural alpha globin chain termination variant that is quite common in Southeast Asia. It is associated with the presence of a minor, very slowly migrating abnormal Hb on electrophoresis. (See "Molecular genetics of the thalassemia syndromes", section on 'Failed translation termination: Hb constant spring'.)

The phenotype depends on whether there are other alpha chain deletions. Clinically, it is more severe than deletional Hb H and is associated with moderate to severe anemia with less microcytosis. (See "Pathophysiology of thalassemia", section on 'Alpha thalassemia' and "Diagnosis of thalassemia (adults and children)", section on 'Anemia'.)

Hb Quong Sze (Hb QS) — Hb QS is a type of non-deletional mutation affecting HBA2 in which the amino acid leucine replaces proline at codon 126 (Leu126Pro) and an extended alpha globin chain is produced. The abnormal Hb causes membrane dysfunction and hemolysis. Hb QS is one of the major alleles that causes non-deletional Hb H disease in Chinese populations [38].

Hb Barts and Hb H — Hb Barts and Hb H are abnormal Hbs that only form when alpha chain production is significantly reduced, allowing beta-like chains to form homotetramers rather than binding to alpha globin. Thus, their primary cause is alpha thalassemia, and severe alpha thalassemia is also called Hb H disease.

Hb Barts is composed of tetramers of gamma globin (gamma4); gamma globin is the beta globin-like chain used to make fetal hemoglobin (Hb F). Small fractions of Hb Barts (2 to 5 percent) on a newborn screen are seen with alpha thalassemia trait (two alpha gene abnormalities), and higher fractions are seen with severe alpha thalassemia. (See "Diagnosis of thalassemia (adults and children)", section on 'Hemoglobin analysis and genetic testing'.)

Hb H is composed of tetramers of beta globin (beta4). These form when alpha globin chain production is substantially reduced (see "Pathophysiology of thalassemia", section on 'Globin chain imbalance'). It is unstable and therefore not detected in alpha thalassemia trait.

Hb Barts and Hb H have extremely high oxygen affinity and therefore do not function in oxygen delivery.

●Hydrops fetalis and Hb Barts – Individuals with deletion of all four alpha globin genes (--/--) produce Hb Barts in utero and develop hydrops fetalis that usually results in fetal death unless fetal intrauterine transfusions are performed. (See "Nonimmune hydrops fetalis", section on 'Anemia' and "Diagnosis of thalassemia (adults and children)", section on 'Alpha thalassemias'.)

●Hb H disease – Hb H disease most often occurs in individuals with alpha thalassemia who have variants or deletions affecting three of the four alpha globin genes. It less commonly occurs when non-deletional and hyper-unstable mutations are involved. Hydrops fetalis can sometimes occur when a non-deletional mutation is involved. In deletional Hb H disease (--/a-), Hb H comprises 5 to 30 percent of total Hb. In non-deletional Hb H disease (--/aa^CS), Hb Constant Spring is present along with Hb H. (See "Pathophysiology of thalassemia", section on 'Terminology and disease classification' and 'Hb Constant Spring' above.)

●Acquired Hb H disease – Acquired Hb H disease, also called acquired alpha thalassemia, may be seen in patients with myelodysplastic syndromes (MDS) or other hematologic malignancies (figure 6). An acquired variant in the ATRX gene, an X-linked gene encoding a chromatin-associated protein, has been linked to acquired Hb H disease. (See "Molecular genetics of the thalassemia syndromes", section on 'ATRX variants and alpha thalassemia' and "Clinical manifestations, diagnosis, and classification of myelodysplastic syndromes (MDS)", section on 'Acquired hemoglobin H disease'.)

Hb H forms multiple small inclusions that can be visualized when RBCs are incubated in the presence of a redox dye such as brilliant cresyl blue (picture 2). (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Hb H staining'.)

Hb H is fast moving on gel electrophoresis, with a mobility at alkaline pH significantly faster than Hb A (figure 6). If Hb H is suspected and gel electrophoresis is being used, it is reasonable to reduce the length of time that electrophoresis is carried out in order to prevent Hb H, if present, from completely running off the gel and being considered absent. Hb H is unstable, and its abundance (and ability to be detected) will decrease over time.

VARIANTS THAT CAN AFFECT ALPHA OR BETA GLOBIN

Unstable Hbs — Unstable hemoglobin variants produce Hbs that are relatively insoluble. Rather than forming ordered polymers (as with Hb S) or crystals (as with Hb C), the resulting Hbs aggregate into amorphous precipitates (Heinz bodies), which cause hemolysis because they attach to the red blood cell (RBC) membrane and impair RBC deformability.

These variants are relatively uncommon. Most are private (they affect only a single family/pedigree). Hb Köln and Hb Zurich have been reported in several families, and Hb Hasharon is seen in individuals with Ashkenazi Jewish ancestry. Transmission is generally autosomal dominant.

Hemoglobin Köln is the most common unstable hemoglobin variant worldwide. Patients with Hb Köln often have mild hemolytic anemia characterized by reticulocytosis, splenomegaly, and elevated bilirubin and lactate dehydrogenase (LDH). In addition, some case reports describe patients with Hb Köln having priapism and falsely low SpO₂ on pulse oximetry. (See "Unstable hemoglobin variants", section on 'Hemoglobin Köln'.)

Clinical manifestations include varying degrees of hemolysis and associated complications (pigment gallstones, splenomegaly). Details are presented separately. (See "Unstable hemoglobin variants".)

M Hbs — These variants produce Hb M (for methemoglobin), in which the heme iron is locked in the ferric (Fe⁺⁺⁺) form, which cannot bind oxygen. M Hbs can occur due to changes in alpha globin, beta globin, or (rarely) gamma globin; generally, the variants substitute an amino acid in the heme pocket that prevent reduction of the iron from the ferric to the ferrous state. Affected individuals may have cyanosis. (See "Methemoglobinemia", section on 'Hemoglobin M disease and cytochrome b5 deficiency'.)

High oxygen affinity variants — Hb variants with abnormally high oxygen affinity bind oxygen tightly and fail to release it to the tissues (figure 7). As a result of impaired oxygen delivery, affected individuals develop erythrocytosis. Examples of high oxygen affinity variants include Hb Chesapeake, Hb Montefiore, Hb Tarrant, Hb Fukotomi, Hb Crete, and Hb Malmö. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Selected high oxygen affinity Hbs'.)

Many of these variants can be identified using protein-based methods, although some are electrophoretically silent. Diagnosis is made by demonstrating that the partial pressure of oxygen at which they become 50 percent saturated with oxygen (p50) is decreased and/or by genetic testing. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'High oxygen affinity hemoglobins: Erythrocytosis'.)

Low oxygen affinity variants — Hb variants with abnormally low oxygen affinity, which are very rare, do not bind oxygen very tightly (figure 7) and as a result can cause cyanosis. Oxygen delivery to the tissues is usually adequate, and most individuals are asymptomatic except for the cosmetic effects, although some of the low oxygen affinity variants are also unstable and can cause anemia. Examples of low oxygen affinity variants include Hb Beth Israel, Hb Kansas, Hb Saint Mande, Hb Bruxelles, and Hb Sarajevo. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Selected low oxygen affinity Hbs'.)

Many of these variants can be identified using protein-based methods. Diagnostic confirmation can be done by measuring the p50 and/or by genetic testing. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Low oxygen affinity hemoglobin variants: Cyanosis'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Sickle cell disease and thalassemias".)

SUMMARY AND RECOMMENDATIONS

●Overview – Adult hemoglobin (Hb A) is a tetramer of two alpha globin and two beta globin chains. Numerous variants (genetic changes) affecting globin genes have arisen. The tables summarize selected clinical disorders (table 1), patterns on protein-based analysis (table 3), and DNA changes (table 2). (See 'Normal hemoglobins' above and 'Scope' above.)

●Evaluation – Hb variants may be suspected when a parent has a known variant or when an individual has non-immune hemolytic anemia. Newborn screening programs will identify many individuals at birth. Selected resources for testing and interpretation are listed above. Testing methods are discussed separately. (See 'Age at presentation' above and 'How to evaluate' above and 'Resources' above and "Methods for hemoglobin analysis and hemoglobinopathy testing".)

●Beta globin – Variants in the human beta globin locus (HBB) can affect beta globin structure, function, or abundance. Heterozygosity is usually an asymptomatic carrier state.

•Hb S – The sickle mutation causes sickle cell disease (SCD) when homozygous or coinherited with another beta globin variant. Sickle hemoglobin polymerizes when deoxygenated, leading to hemolysis, vascular occlusion, repeated pain episodes, and chronic organ damage. (See "Overview of the clinical manifestations of sickle cell disease" and "Sickle cell trait".)

•Beta thalassemia – Beta thalassemia variants reduce beta globin production, causing microcytic anemia and ineffective erythropoiesis. There is marked variation in genotypes and phenotypes. (See "Diagnosis of thalassemia (adults and children)".)

•Hb C – Hb C disease causes mild hemolytic anemia, jaundice, and splenomegaly, with a high mean corpuscular hemoglobin concentration (MCHC), reflecting red blood cell (RBC) dehydration, a low mean corpuscular volume (MCV), and hexagonal crystals inside RBCs. Hb C disorders do not cause sickling unless coinherited with Hb S. (See 'Hb C' above and "Gene test interpretation: Hemoglobin C (Hb C) variant in the hemoglobin beta locus (HBB)".)

•Hb C-Harlem – Hb C-Harlem consists of the sickle mutation plus a second point mutation (p.Asp73Asn; c.220G>A). Hb C-Harlem can polymerize when deoxygenated, and it causes SCD when coinherited with another beta globin variant. (See 'Hb C-Harlem' above.)

•Hb D – The most common Hb D variant, Hb D-Punjab (also called Hb D-Los Angeles) is distributed worldwide. Hb D-Punjab causes SCD when combined with Hb S; Hb D-Abadan and Hb D-Iran do not. Homozygosity for Hb D is rare and causes only mild laboratory abnormalities. (See 'Hb D' above.)

•Hb E – Hb E causes a beta thalassemic phenotype and is mildly unstable to oxidative damage. Homozygotes have minimal anemia with hypochromia, target cells, and prominent microcytosis. (See 'Hb E' above.)

•Hb Lepore – Hb Lepore arises from a recombination event between adjacent delta globin and beta globin genes to form a fusion gene with reduced production, causing a beta thalassemia phenotype. (See 'Hb Lepore' above.)

•Hb O-Arab – Hb O-Arab causes mild, compensated hemolytic anemia in homozygotes. Compound heterozygosity for Hb O-Arab and Hb S causes severe SCD. (See 'Hb O-Arab' above.)

●Alpha globin – Variants in the alpha globin loci (HBA1 and HBA2) typically reduce alpha globin production, causing alpha thalassemia. There are four alpha genes. Deletion or mutation of three cause Hb H disease; deletion of all four causes a fatal intrauterine condition without fetal transfusion. Hb Barts (gamma tetramers) and Hb H (beta tetramers) form when there are very few alpha chains available; these Hbs are functionally useless. Hb Constant Spring is a structural alpha globin chain termination variant common in Southeast Asia. (See 'Alpha globin variants' above and "Gene test interpretation: HBA1 and HBA2 (alpha globin genes)".)

●Either beta or alpha globin – Variants in either globin gene can cause unstable Hbs, congenital methemoglobinemia (Hb M), or high or low oxygen affinity Hbs. (See 'Variants that can affect alpha or beta globin' above and "Unstable hemoglobin variants" and "Methemoglobinemia" and "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

ACKNOWLEDGMENTS — UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.

The UpToDate editorial staff acknowledges the extensive contributions of Donald H Mahoney, Jr, MD and William C Mentzer, MD, to earlier versions of this and many other topic reviews.