Chapter 24: Hemoglobinopathies

Compound Heterozygosity with Hemoglobin S and another B Globin Gene Mutation (Hb S-Korle Bu)

Rare hb variant with substitution of aspartic acid for asparagine at position 73 of beta chain

Deletion Mutations

removal of one or more nucleotides. (happens in thalassemias)

Insertion mutations

results in insertions of addition of one or more nucleotides

Nonmeclature

the naming of the normal hemoglobin and the abnormal hemoglobin

Hemoglobin with Increased and Decreased Oxygen Affinity

* High-affinity variants and usually associated with familial erythrocytosis. The rest are variants associated with low oxygen affinity. *Increased-some Hb fails to release oxygen and it causes hypoxia. Kidneys release EPO in response and leads to erythrocytosis. The unstable Hb can have abnormal oxygen affinity. People are usually asymptomatic but the RBC count is elevated, as well as H&H. Everything else is normal. ID is by hb electrophoresis. No treatment *Decreased-quick release of oxygen to the tissues which leads to normal or decreased Hb concentration,mild anemia, and cyanosis.

Hemoglobin C-Harlem

*Alternate name: Hb C-Georgetown *Amino acid substitutions-double substitution on the B chain. Valine is subbed for glutamic acid at position 6 of the beta chain AND aspartic acid for asparagine *Clinical symptoms: Heterozygous-asymptomatic If they have compound heterozygosity which is HbS and HbC-Harlem then they will have similar symptoms as seen in sickle cell disease HbSS. *Solubility test results-positive *HgB electrophoresis or HPLC- Confirms by Hb C-Harlem migrating to the C position.

Zygosity

*Association between the number of gene mutations and the level of severity of the genetic defect. *Because the dominant hemoglobin in adults, Hb A is made up of alpha and beta chains, beta gene mutations affect overall hb function more than alpha chain mutations. *B chain variants is referred to as heterozygous (trait)when only one B gene is mutated. It is considered homozygous (disease) when both beta genes are mutated.

Concomitant CIS Mutations with Hemoglobin S

*Cis Mutation-So on the already mutated HbS there is a second mutation on the same gene. *Hb C Harlem-two subs on Beta chain, patients heterozygous for only HbC Harlem are asymptomatic. Compound heterozygous HbSC Harlem have symptoms like Hb SS. *Hemoglobin S-Antilles and Hemoglobin S-Oman: Antillles has the S mutation and a substitute of isoleucine for valine at position 23. OMAN-has the Hb S mutation and a substitution of lysine for glutamic acid at position 121. *The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells.

Hemoglobin S (Sickle Cell Trait)

*Definition-heterozygous, benign Hb AS *Ethnic distribution-Central America, Asia, and the Mediterranean *Clinical features-usually asymptomatic, under extreme hypoxia cells can sickle *Laboratory features-normal RBC morphology with a few target cells, no abnormal WBC and PLTs, solubility screening yields positive, and electrophoresis will detect Hb S and Hb A. *Treatment-no treatment

Compound Heterozygosity with Hemoglobin S and another B Globin Gene Mutation (Hb S-Beta-Thalassemia)

*Ethnic distribution-Mediterranean descent *Clinical syndrome-Compount heterozygosity for Hb S and B-thalassemia is most common cause of sickle cell syndrome *Severity-milder than HbSC *Clinical course-microcytosis, hemolytic anemia, abnormal peripheral blood and splenomegaly

Hemoglobin O-Arab

*Features of hemoglobin: Amino acid substitution-lysine for glutamic acid at position 121 *Ethnic distribution-rare disorder, Kenya, Israel, Egypt, Bulgaria *Clinical features: Along with Hb S results in severe symptoms *Laboratory features: Blood smear-homozygous has mild anemia with many target cells and negative solubility test. Electrophoresis or HPLC-confirmed *Treatment-no treatment

Hb C Harlem

*Hb C-Harlem is like Hb C, but unlike Hb C, and like Hb S, Hb C-Harlem can polymerize when deoxygenated because it has the sickle cell β6 Glu-Val mutation. *When it is heterozygous by itself Hb C-Harlem is benign, as is sickle cell trait. *But if it is part of a compound heterozygote with Hb S then it results in severe sickle cell disease.

Hb M

*Hb M is caused by a variety of mutations in the α-, β-, and γ-globin genes, all of which result in the production of methemoglobin, hence the Hb M designation. *Remember methemoglobin is a dyshemoglobin.

Hemoglobin Development

*Hb molecule is made up of four globin chains. 2 alpha-like and 2 beta-like. *Embryo: The zeta and epsilon pair to form Gower-1 as the embryo develops into Hb Gower 2 with the expression of alpha and epsilon and finally Hb Portland with zeta and gamma *Fetus: Alpha and gamma is left as the fetus develops and zeta and epsilon chain formation ceases. The gamma will begin to cease during the 6 months after birth and the Beta chain takes over. The gamma gene is silenced by repressors as the baby develops. *Adult: 2 alpha and 2 beta chains.

Concomitant Cis Mutations with Hb S

*Hemoglobin C-Harlem (benign) and Hb S-Antilles and Hb S-Oman (The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. *In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells.)

Hb D and G

*Hemoglobin D or G trait is not disease and is not associated with anemia or other disease medical issues. *Although hemoglobin D trait and hemoglobin G trait have no immediate clinical significance, this information is important for future reproductive decisions of the child and other family members.

Unstable Hemoglobin Variants

*Hemoglobin Features: globin chain most affected-beta, altered oxygen affinity-increased in most, inheritance-appears after birth, patients are heterozygous/homozygous incompatible with life *clinical features: jaundice and splenomegaly *Laboratory features: Heinz bodies, slight hypochromia, decreased Hb, retics *treatment: splenomegaly, avoid certain medications

Genetic Mutations

*Most current data through March 2021: It has been estimated that in excess of 300,000 children are born in the world each year with a severe inherited disorder of hemoglobin (eg, the thalassemic and sickle cell disorders), and that approximately 80 percent of these births occur in low- or middle-income countries [3]. *Mutations are caused by an alteration of the amino acid sequence of Hb.

Nomenclature

*Normal Adult Hemoglobin is HbA and fetal hemoglobin is HbF. *Abnormal Hemoglobin is typically named by the person who discovered it or a special characteristic, or designation of the variant chain.

Hemoglobin M

*Hemoglobin M (HbM) refers to group of autosomal dominant methemoglobinemias that are caused by heterozygous mutations in either the α- or β-globin genes. These mutations result in the production of what is referred to as M hemoglobin (HbM) which is a form of hemoglobin that stabilizes the heme iron in the ferric (Fe3+) state and which is not amenable to reduction. The designation HbM also can refer to a form of hemoglobin that exhibits an unusual susceptibility to oxidizing agents. *At least five different forms of hemoglobin M have been characterized. Four of these, HbM (Boston), HbM (Hyde Park), HbM (Iwate), and HbM (Saskatoon) are due to mutations that alter the critical His residues to Tyr in the heme binding pocket of either α-globin or β-globin proteins. The fifth form of HbM (Milwaukee-1) results from a Glu for Val substitution at a position four amino acid residues from the distal His of the heme binding pocket. These mutations all stabilize the heme iron in the ferric (oxidized) state. *The primary pathology in patients harboring HbM mutations is cyanosis but otherwise they are asymptomatic. If the HbM mutation is in the α-globin gene the cyanosis is apparent at birth. If the HbM mutation is in the β-globin gene then cyanosis appears later or intensifies when β-subunit production increases. Neonates that harbor mutations in the γ-globin gene will exhibit cyanosis at birth but it will disappear when the complete γ-globin to β-globin gene expression switch occurs. *The blood specimen will appear brown. Heinz bodies. Hb is converted to methemoglobin by adding potassium cyanide to the specimen before electrophoresis. *HPLC may be used to confirm. *No treatment necessary

Compound Heterozygosity with Hemoglobin S and another B Globin Gene Mutation (Hb SD and SG-Philadelphia)

*Hemoglobin features: Compound heterozygous and Hb SG-Philadelpha is DOUBLE heterozygous. HbSG asymptomatic, HbSD may cause miled to severe hemolytic anemia because of Vasoocclusive complications. *Clinical features-mild to severe Hemolytic anemia Laboratory features *Blood smear-similar to SS disease *Treatment is similar to SCD

Hemoglobin C

*History: This was the next hemoglobinopathy to be described after Hb S. *Prevalence: 17-28% of people of West African and 2-3% African Americans. *Etiology: This common mutation at codon 6 is the conversion to a Lys codon (AAG) (Lysine replaces glutamic acid) which results in the generation of Hb C. Like sickle cell anemia, HbC disease is inherited as an autosomal recessive condition. *Pathophysiology: The Hb C polymers (a chain-like molecule made up of smaller molecules) are short, thick crystals within the rbc. It is less soluble in HbA and will crystalize in the oxygenated state IT does not alter the shape to the same extent of HbS. *Clinical features: Homozygous HbCD is milder than SCD. Mild splenomegaly, hemolysis (NO VASOOCCLUSION), The heterozygous form is asymptomatic. *Laboratory diagnosis: Type of anemia-mild to moderate normocytic, normochromic *Blood smear evaluation-some microcytosis and hypochromia. Increased target cells, slight to moderate increase in retics and NRBC Hexagonal crystals-Crystals that form extracellularly, oblong and pyramid or pointed ends. *Hb electrophoresis or HPLC-No Hb A present in Hb CC disease. *Treatment: None required in most cases and genetic counseling recommended

Structure of Globin Genes

*In adult hemoglobin, the hemoglobin molecule is made up of four protein sub-units. Typically, there are 2 subunits of alpha-globin and 2 subunits of beta-globin chains. *Number of functional globin genes: six located on two chromosomes. Chromosome 16=alpha and zeta. Chromosome 11=beta, gamma, delta, epsilon *Each globin gene codes for a chain: Alpha globin genes are HBA1 and HBA2: The HBA1 gene provides instructions for making a protein called alpha-globin. This protein is also produced from a nearly identical gene called HBA2. These two alpha-globin genes are located close together in a region of chromosome 16 known as the alpha-globin locus.

Hemoglobin S (Incidence with Malaria)

*It is thought that Sickle Cell trait offers some protection against Malaria caused by Plasmodium falciparum. *The Malaria parasite requires oxygen to live and because the sickle cell mutation reduces oxygen tension , it decreases the number of malarial organisms able to survive. *One explanation for this is that the infected cell which is already being recognized by spleen and liver as not healthy, is destroyed.

Hemoglobin S (Coarse and Prognosis)

*Median life expectancy: Sickle cell anemia (Hb SS)-Some greater than 50 years. Hb SC-60-68. *Occupational limitations-discouraged strenuous physical exertion and exposure to high altitudes, or extreme environmental temperature variations. *Sickle cell disease and pregnancy: Preconception-increased risk, suggest genetic counseling. Prenatal (antenatal)-greater risk of maternal and fetal death. Delivery (intrapartum)-patient to be kept warm, oxygenated, hydrated. Postpartum-watch for clots, monitor fluids, stay in hospital for 3 days

Hemoglobin E

*Prevalence: 20% in Southeast Asia and 50% in areas around Cambodia, Laos, and Thailand *Etiology: Lysine is substituted for glutamic acid in position 26 *Pathophysiology: Qualitative defect and a quantitative defect. Coinherited with either alpha or beta thalassemia. *Clinical features: Diseases Hb EE Mild anemia with microcytes and target cells, short life span. Beta-thalassemia: Differentiating it from iron deficiency B-thalassemia trait. Malaria-Possibly offers protection from Plasmodium falciparum *Laboratory features: negative hb solubility, confirm with electrophoresis, mild anemia, low MCV, Few target cells, normal retics *Treatment and prognosis: no therapy required for Hb E disease and trait

Compound Heterozygosity with Hemoglobin S and another B Globin Gene Mutation (Hb SO-Arab and HB SD-Punjab)

*Rare heterozygous that causes severe chronic hemolytic anemia with vasoocclusion. *Mutations: O-Arab subs lysine at position 121 and Punjab replaces glutamine. *Neither one are clinically significant

What do those letters stand for again?

*Remember RNA is single-stranded. Attached to each one of the sugars is a base. See the bases A, U, G, and C. DNA → RNA → ProteinRNA is produced when DNA is transcribed. *It is structurally similar to DNA, with the following major differences: 1. The nucleotide uracil (U) is used instead of DNA's thymine (T). 2. RNA contains ribose instead of deoxyribose (deoxyribose lacks the oxygen molecule on the 2' position found in ribose). 3. RNA is single-stranded, whereas DNA is double-stranded. *So, DNA is transcribed to RNA, then RNA is translated into a protein. RNA is translated into protein, the primary structure of the protein is determined by the sequence of amino acids of which it is composed. *Protein is the molecule responsible for carrying out most of the tasks of the cell and can have many functions, such as enzymatic, contractile, transport, immune system, signal, and receptor to name a few.

Hemoglobin S (Sickle Cell Anemia)

*Reported first by a cardiologist in a student with severe anemia. Recorded the sickling. They found the RBCs were deficient in oxygen. Researchers also found that individuals with sickle cell disease were resistant to Malaria caused by plasmodium falciparum. *Hb S is caused by an amino acid substitution in the globin chain. Heterozygous sickle trait is Hb AS and the homozygous sickle cell disease is Hb SS. (sickle cell disease can also be seen with combinations of B-chain mutations like Hb C or Beta-thalassemia. Inheritance: For sickle cell disease, the patient has a sickle gene from one parent and either another S, C, or B-thalassemia gene from the other. The people with Hb SS is more severe. *Prevalence: Found in sub-Saharan Africa most frequently, then Ara—India, Americas, Eurasia, and southeast Asia.

Hemoglobin S (Pathophysiology)

*Shape of RBCs: Normal red blood cells are disc-shaped; normal blood oxygen levels should be well above 90%. Typically, the rage for healthy individuals is 95-100% oxygen saturation. *In the homozygotes, the sickling begins when oxygen saturation decreases to less than 85%. This means when the oxygen begins dropping the stronger homozygotes will react quickly. Remember homozygotes mean Hb SS, Hb CC, Hb DD, Hb EE In heterozygotes, the sickling doesn't occur until oxygen is less than 40%. Because the mutation is only on one of the globin chains, it takes a much steeper drop in oxygen before the cells become affected. *Two forms: Reversible and irreversible Reversible sickle cells change shape in response to oxygen tension. They circulate as normal biconcave discs when fully oxygenated and change shapes when deoxygenated. *Irreversible sickle cells do not change their shape with oxygen fluctuations and appear elongated on the blood smear. These will likely be removed by the spleen and removed from circulation. These abnormally shaped cells can cause occlusion of the vessel.

Hemoglobin S (Etiology)

*Structural formula: two alpha chains, 2 beta chains where glutamic acid is replaced by valine. This affects the charges of the Hb molecule, and an amino acid substitution occurs. *About 1 out of every 12 African-Americans has sickle cell trait, and about 1 out of every 100 Latinos has sickle cell trait. Sickle cell trait also affects many people whose ancestors came from Africa, Latin America, Asia, India, and the Mediterranean region. However, it is possible for a person of any race or nationality to have sickle cell trait.

Hemoglobin S (Treatment)

*Supportive: Hydration, vitamin therapy, avoid low oxygen environments, analgesics for pain, antibiotics at the first sign of infection *Hydroxycarbamide therapy: The hydroxyurea increases the amount of HbF *Hand and foot syndrome: pain and swelling in the hands and feet *Infections *Blood transfusions-can cause transfusion reactions, transfusion-related infections, and iron overload. *Bone marrow transplants—can be curative

Quantitative (Thalassemias)

*There is a reduced rate of hb synthesis but doesn't affect the amino acid sequence. *This can produce an anemia and stimulate the production of other hb that isn't affected by the mutation. *This is your bodies way of making up for the anemia.

Compound Heterozygosity with Hemoglobin S and another B Globin Gene Mutation (Hb S)

*This occurs when you inherit one mutation from one parent and another mutation from the other parent. *Hb SC is the most common compound heterozygous syndrome. There are 2 different amino acid substitutions on each of the beta globin chains. Position 6 glutamic acid replaced by valine on one of the beta chains and on the other is by lysine on the other beta chain. *Prevalence 25% in West Africa, US 1 in 833 births Clinical features: Milder form of SCD, growth and development delay. Mild until teen. Vasoocclusive complications, moderate hemolytic anemia and splenomegaly. *Retinopathy and respiratory tract infections common. Live longer than Hb SS patients. Lab: normocytic normochromic, Hb 11-13 g/dL reticulocytosis, crystalline aggregates present, solubility positive, positive on electrophoresis Treatment: similar to SCD

Hemoglobin S (Laboratory Testing)

*Tube solubility test Principle-Screening test for HbS. Blood is added to a salt solution with a reducing agent and detergent if it is positive the solution will be cloudy, if clear then it is negative. *False positives-hyperlipidemia, too much blood is added, and in some hemoglobinopathies *False negatives-infants younger than 6 months and patients with low Hct *Hemoglobin electrophoresis-Separation of hb molecules in an electrical field that results in various molecular charges. *Other tests may include: HPLC, Capillary electrophoresis, and Isoelectric focusing (IEF)

Hemoglobin D and G

*Types of variants 16 types *Amino acid substitutions- Hb D-Punjab is a variant derived from a point mutation in the beta-globin gene (HBB) in the first base of the 121 codon (GAA→CAA) with the substitution of glutamine for glutamic acid (Glu>Gln) in the beta globin chain Hb G-most common G variant where lysine replaces asparagine at position 68. *Ethnic distribution: Hb D prevalence in India and African Americans at greater frequency and Hb G is also found in Ghana *Treatment-none

Hemoglobin S (Clinical Features)

*Up until 6 months Hb F is protecting by making up for the mutation. But remember it starts to decrease and the mutated chain takes over. Symptoms vary from mild to life-threatening. *Types of crises: 1-Vasoocclusive: when the small vessels are blocked by sickled cells, then the tissue becomes ischemic (lacking oxygen) and can result in damaging the organ and causing pain. 2-Splenic sequestration and infarcts-the spleen traps the blood and Hb drops below 6g/dL. This can make the spleen enlarge and eventually, autosplectomy occurs where the spleen fails to function properly. 3-Acute chest syndrome-acute illness with fever and/or respiratory symptoms that displays pulmonary infiltrates on the chest x-ray. This is the most common cause of hospitalizations and the third most common cause of SCD patients. 4-Fat embolism and bone marrow necrosis-Necrotic (dead) pieces of bone marrow and fat particles are released into circulation and make it to the lungs where they break apart and reenter the circulation traveling to various organs causing ischemia. 5-Bacterial infections-Most common cause of death in SCD, bacterial infection. 6-Chronic hemolysis-shorted RBC lifespan to 16-20 days, decreased Hb/Hct, increased retic and jaundice. 7-Megaloblastic episodes, Folate depletion causes cessation of erythropoiesis. 8-Aplastic episodes-infection (usually parvovirus) 9-Cardiac abnormalities-cardiomegaly because the heart is having to work harder. 10-bone and skin abnormalities-decreased blood supply to the head of femur and humerus 11-Retinopathy-increased retinal ischemia and neovascularization (new vessels) *Clinically significant variants: Hb SS, Hb S-B-thal, Severe Hb S B-Thal, Hb SD-Punjab, Hb SO-Arab, Hb SC-Harlem, Hb CS-Antilles, and Hb S Quebec-chori *Vasoocclusive of painful splenic sequestration, chronic hemolytic anemia, megaloblastic anemia, and aplastic anemia. *Risk factors: polymerization, decreased deformability, sickle cell-endothelial cell adherence, endothelial cell activation, WBC and PLT activation, hemostatic activation and altered vascular tone.

Hemoglobin S (Laboratory Diagnosis)

*chronic hemolytic anemia that is normocytic, normochromic *on wright stain the sickle cell is a long, curved cell with point at each end, poikilocytosis, anisocytosis, sickle cells, target cells, asophilic stippling, pappenheimer and Howell-jolly bodies present. *SICKLE CELLS AND TARGET CELLS are signs of SCD. *Reticulocyte count-elevated *Mean cell volume (MCV) and RBC distribution width (RDW) indices-MCV normal to slightly elevated, RDW elevated. *Cell counts-moderate leukocytosis 40-50x10^9 WBC/L *Leukocyte alkaline phosphatase (LAP) score-not elevated *Bone marrow studies-erythroid hyperplasia, increased retic, increased nrbc

Hb E

*disease is a mild, inherited blood disorder characterized by an abnormal form of hemoglobin, called hemoglobin E. *People with this condition may have very mild anemia , but the condition typically does not cause any symptoms caused by a mutation of the Hb B gene. *It is an autosomal recessive disorder.

Qualitative (Structural)

*hb synthesis occurs at a normal or near-normal rate, but the hb molecule has an altered AMINO ACID sequence within the globin chain. *This changes the amino acid sequence which causes a change in the structure of the Hb molecule and its function. *Examples of this is Sickle Cell Anemia for example.

Hb O-Arab

*is a stable, minimally increased oxygen affinity variant hemoglobin, and O-Arab trait (1 mutated gene) is completely silent. *Homozygous hemoglobin O-Arab (2 mutated genes) is rare and is only occasionally reported to cause a very mild hemolytic anemia with some splenomegaly.

Hemoglobinopathy

*refers to a disease state that involves Hb molecule. *They are the most common genetic diseases. *ALL hemoglobinopathies result from a gene mutation. *There are two ways the mutations affect hemoglobin synthesis which is qualitative or quantitative.

Pathophysiology

*the manner a disorder translates into clinical symptoms. *Changes in Hb structure can affect the function of the Hb. The mutation causing sickle cell anemia is a single nucleotide substitution (A to T) in the codon for amino acid 6. The change converts a GLU codon (GAG) to a VAL codon (GTG). *Example: sickle cells have one amino acid substitution which causes long hemoglobin crystal formation and stretches the RBC like a sickle. *In B-hemoglobinopathies the homozygous pattern results in both B genes being mutated and the variant hb becomes dominant instead of normal Hb A. *Hertozygous B-hemoglobinopathies, only one B gene is mutated and thus might have lessened symptoms. *Abnormal Hbs that result in hemolytic anemia, abnormal hemoglobins that result in methemoglobinemia, hemoglobin with either increased or decreased oxygen affinity, abnormal Hb with no clinical or functional effect. *Many variants are clinically insignificant.

Global Burden of Hemoglobinopathies

As other health care issues are resolved, patients will live longer with hemoglobinopathies so more treatment options must be made available. Also, there is a need for increased pregnancy education including genetic counseling.

Compound heterozygosity with Hb S

Patient demonstrates Hb S and Hb C which manifests as a milder form of sickle cell disease.

Hb S

causes red blood cells to become stiff and abnormally shaped like a sickle or crescent moon.

Point Mutations

most common. Replacement of one original nucleotide in the normal gene with a different nucleotide which results in an amino acid substitution.

Hb C

produces sickle cell trait but not the disease, it causes only mild sickling of the RBCs

Fusion mutations

two normal genes break between nucleotides, switch positions and change to the opposite gene. This causes globin chains to fold differently and affects the function of the hb.