Hemoglobinopathies and Globin Genes

Hemoglobinopathies

• Encompasses disease states involving the hemoglobin molecule
• Arises from a genetic mutation that impacts hemoglobin synthesis
• Mutations affect either the quality or quantity of hemoglobin production
• Structural defects result in qualitative variations.
• Thalassemia results in quantitative variations.

Globin Genes

• Six functional human globin genes are located on two chromosomes
• Alpha and zeta genes are on chromosome 16 and are designated α-like genes
• Each chromosome contains two alpha and one zeta globin gene.
• Beta, gamma, delta, and epsilon genes are on chromosome 11 and are designated β-like genes
• Each chromosome contains two gamma and one beta, delta, and epsilon globin genes.

Globin Chain Production Timeline

• In the intrauterine stage:
• ζ2 + ε2 forms Gower-1.
• α2 + ε2 forms Gower-2.
• ζ2 + γ2 forms Portland.
• α2 + γ2 forms F.
• At birth:
• α2 + γ2 forms F, accounting for 60-90%.
• α2 + β2 forms A, accounting for 10-40%.
• In adulthood:
• α2 + γ2 forms F, accounting for less than 1-2%.
• α2 + δ2 forms A2, accounting for less than 3.5%.
• α2 + β2 forms A, accounting for >95%.

Genetic Mutations

• Over 1000 structural hemoglobin variants are known
• Point mutations involve replacing one original nucleotide in the normal gene with a different nucleotide
• A codon triplet remains intact, and this is the most common mutation type at 90%
• Deletions remove one or more nucleotides
• codon disruptions can nullify protein production
• Insertions add one or more nucleotides
• codon disruptions can nullify protein production
• Chain extensions occur when the stop codon is mutated
• this leads to translation continuing, resulting in abnormally long globin chains affecting function and structure
• Gene fusions arise when two normal genes break, switch, and realign with opposite genes
• this leads to abnormal chain folding and affects hemoglobin function
• Hemoglobinopathies are inherited as codominant traits.

Lab Testing

• Traditional hematology involves Complete Blood Count (CBC) and microscopic examination
• Hemoglobin carries an electrical charge based on amino acid sequence and pH
• This allows for hemoglobin electrophoresis testing using cellulose acetate (pH 8.4) and citrate agar gel (pH 6.2).
• High-performance liquid chromatography (HPLC) and PCR (Polymerase Chain Reaction) can be used

Beta (β)-Hemoglobinopathies

• Homozygous β-hemoglobinopathies
• Both beta genes are mutated and Hgb A is absent
• Sickle cell disease (Hb SS) and Hgb C disease are examples (Hb CC)
• Heterozygous β-hemoglobinopathies
• Only one gene is mutated, therefore one is normal
• The body attempts to minimize the impact of abnormal hemoglobin by having the variant hemoglobin in lesser amounts than Hgb A
• Hgb S trait (HB AS) and Hgb C trait (Hb AC) are examples

Hemoglobin S

• Sickle cell diseases (SCD) is used to describe a group of symptomatic hemoglobinopathies
• Patients express either Hb SS, or Hb S in combination with another Hgb β-chain mutation
• It’s the most frequent and severe of the abnormal hemoglobins
• Sickle cell disease is found worldwide, but endemic in Africa.
• SS is the Sickle Cell Anemia genotype.
• AS is the Sickle Cell Trait genotype
• By nomenclature, if a normal allele is present, it is listed first
• Caused by a point mutation at the 6th amino acid in the beta chain
• A nucleotide base substitutes for another
• Valine substitutes for glutamic acid
• “Val for glu = Hgb S"
• Presence of one sickle cell gene gives resistance to infection by Plasmodium falciparum
• Occurs because when a malaria-infected RBC sickles, the parasite dies.
• Common in African populations where 8-9% of African-Americans have sickle cell trait
• One in 300-600 are affected by SCD.
• Severe, moderate, & mild forms of SCD exist
• The variance in severity has an unknown etiology.

SCD Pathophysiology

• Deoxygenation in capillaries is normal, causing Hgb to switch from Relaxed to Tense as it offloads O2.
• This causes hydrophobic mutated valines to contact each other changing Hgb S structure, resulting in:
• Polymerization of the Hgb
• "Locking up" of spectrin's flexibility
• Bizarre RBC deformation
• This is usually reversible, but relies on time, temperature, pH, and oxygen tension
• RBC loses K+ & dehydration occurs, from the deformation of K+ channels.
• This leads to formation of intracellular crystals & further deformation as Hgb molecules stack up "like firewood".
• This makes RBCs more fragile, causing them to lyse more easily
• Sickle cells lead to increased blood viscosity and slow blood flow
• Hypoxic environment and lower tissue pH, promote sickling, due to prolonged reduced blood flow
• Some RBCs sickle reversibly, and some irreversibly
• The spleen removes irreversible sickle cells, though reversible cells are responsible for vaso-occlusive complications
• Chronic injury response equals "sickle cell crisis”
• Circulating endothelial cells increase during crises
• This triggers the extrinsic coagulation pathway & promotes microthrombi, which causes further necrosis and a vicious cycle.
• Risk factors for vaso-occlusion includes infection and inflammation
• Hgb S RBCs are prematurely destroyed by the spleen (extravascular hemolysis)
• The bone marrow responds with erythropoiesis
• In severe anemia, marrow spaces widen, bone cortex thins, & extramedullary sites for hematopoiesis are recruited
• Over time the spleen quits effectiveness causing SCD to causes a chronic, low-level, intravascular hemolytic anemia.
• RBC lifespan drops to 10-20 days
• Sickle Cell Crises occur on top of ongoing low-level hemolysis in SCD
• They’re caused by excessive deoxygenation from hyperthermia/hypothermia, dehydration, infection, violent exercise, labor, high altitude transitions
• About 80% of SCD patients have only a rare crisis.

Types of Sickle Cell Crises

• They are classified by symptoms or the situation that triggered them
• Vaso-Occlusive or Pain Crisis: rigidity, vascular occlusions & micro-thrombi (ex., strokes) results in tissue necrosis.
• It’s the #1 cause of death in adult SS patients.
• Acute Chest Syndrome (ACS) is when it predominantly affects lungs
• Infectious Crisis results from infection and is the primary cause of death in SCD within children
• S. aureus, S. pneumoniae, and H. flu are known causes
• Bone and Joint Crisis - Pain occurs in bones & joints ("sympathy response") as sickled cells accumulate in bone shafts infracting the bone marrow
• Splenic Sequestration Crisis - Sickled cells are trapped in the hypoxic, convoluted microcirculation of the spleen enlarging the organ
• This traps more cells creating a cycle.
• Hypovolemic shock can ensue and can be monitored by "pit count" in RBC membrane.
• Increased pits equals worsening splenic function
• Aplastic Crisis - Decreased bone marrow hematopoiesis is thought to be triggered by infection/fever from Parvovirus B19
• Results in temporarily (5-10 days) ↓ RBC count, Hgb, Hct & RPI.

Lab Findings: Peripheral Blood Smear

• It is classified as normocytic and normochromic anemia
• Cells are normal when produced in the bone marrow
• Moderate to marked:
• Anisocytosis (sickled cells, target cells)
• Polychromasia (due to reticulocytosis
• Basophilic stippling
• The above are all seen when a patient is in crisis, though less so when not in crisis
• The Red Cell Distribution Width (RDW) is increased
• Howell-Jolly & Pappenheimer bodies will be present indicating an overwhelmed or nonfunctional spleen

Other Lab Findings in SCD

• Hemoglobin Solubility Test - This the most common screening test. Where,
• Blood is combined with a reducing agent (sodium dithionite) and lysing agent
• Deoxygenated Hb S is insoluble and precipitates in solution resulting in a turbid result
• Solutions with non-sickling hemoglobins remain clear
• False positive results occur with hyperlipidemia
• False negative results occur with low Hct or low RBC counts
• All hemoglobins with the amino acid substitution found in Hb S will result in a positive test
• Hgb electrophoresis will identify alkaline electrophoresis
• 80-90% Hgb S levels will be shown with the remainder typically being F & A₂
• HPLC (high performance liquid chromatography) can quantify low amounts of A₂ and F
• DNA sequencing is a definitive test
• Usable samples include peripheral blood, CVS [chorionic villus sampling], amniotic fluid, or even IVF-derived 8-cell stage blastomeres sampled before implantation

Hemoglobin Electrophoresis

• Hemoglobin carries an electrical charge based upon the amino acid sequence of globin chains and the pH resulting in the surrounding environment. Cellulose acetate and citrate agar are used for it
• Treatment involves preventing crises

Strategies for Sickle Cell Disease Treatment

• Prevent crises by avoiding precipitating situations like lack of sleep and poor nutrition
• Hydroxyurea is used for ages 3 years and up
• It’s an antineoplastic drug that induces increased Hgb F production reducing sickling & RBC stickiness
• It reduces the WBC count inhibiting the overall inflammatory response.
• A side effect is macrocytosis.
• Keep patient hydrated to treat crises & provide pain relief with opiates
• Exchange transfusions are needed to prevent iron overload and alloimmunization
• SS patients with frequent crises that receives about 1 transfusion each month
• Bone marrow transplant is the only cure, though requires HLA-matched sibling without SCD and approximately 200 patients world wide have been treated this way so far
• Gene therapy is predicted to be useable in 10-20 years and has cured the disease in mice in 2001
• New anti-sickling agents such as NAC (N-acetylcysteine) which reduces strong reducing agent that are still in clinical trials
• Sickled cells produce increased free radicals, but have less GSH to mop them up
• NAC actually reverses irreversibly sickled cells in vitro
• Prophylactic penicillin for children starting around at at 3 months
• Pneumococcal vaccines
• Stem cell transplants from cord blood

Prognosis in SCD

• Average life expectancy with appropriate treatment is 45 - 50 years compared to 21 years in the 1960's

Sickle Cell Trait (AS)

• caused by heterozygous state, AS
• Often patients have no symptoms because Hgb A helps abnormal S
• Rare occasions include experiencing crises while encountering extreme tissue hypoxia like a severe case of hypothermia, acute respiratory infection, and/or pulmonary embolisms
• Red blood cell morphology is often normal with rare sickled cells
• A positive solubility test
• On Hemoglobin electrophoresis, 30-45% Hgb S is shown, (~60% Hgb A)
• No treatment is usually required
• Typically the life span and quality of life is normal
• Also often provides an increased resistance to malaria

Hemoglobin C Disease (CC)

• Involves the amino acid point substitution of lysine for glutamic acid at 6th position of the beta chain
• "Lysine = C"
• Second most common Hgb variant worldwide, after S, C, then E ("SCE")
• Considered to be more mild in nature than Hgb S
• Particularly common in Black populations
• Hgb C polymerizes under low oxygen tension dissimilar to Hgb S structure
• Hgb S polymers are long and thin, while Hgb C polymers form a short, thick crystal within the RBC
• Hgb C crystals don’t change the shape of the red cell
• Vaso-occlusion doesn’t occur
• Spleen typically removes crystals
• Crystals will appear in the peripheral blood if nonfunctional
• Presents with splenomegaly & abdominal pain while usually requiring no other clinical symptoms & treatment

Hemoglobin C Lab Findings

• Mild to moderate normocytic, normochromic anemia
• Some microcytosis and mild hypochromia
• Presents with numerous target cells and few spherocytes
• Increased retic count (4-8%) and decreased RBC survival
• Hgb C crystals shaped like thick or elongated hexagons, may appear free
• Negative hemoglobin solubility test

Lab Findings with Electrophoresis

• With Hgb CC, cellulose acetate electrophoresis results will yield no Hgb A and show Hgb C (co-migrating with A2, E, and O.)
• Hgb AC electrophoresis results will yield 60% Hgb A and 30% Hgb C
• C will migrate as a separate band on citrate agar at an acid pH
• Remember: C, E, O, A2 comigrate on cellulose acetate

Hemoglobin C Trait (AC)

• No symptoms or anemia as 40% of their cells are target cells
• The result is slight when hypochromasia % of Hgb A>% of Hgb C

Hemoglobin SC Disease

• Most common heterozygous syndrome resulting in a structural defect of Hgb
• Two amino acid substitutions on each of two β-globin chains
• Has milder clinical symptoms than Sickle Cell Disease w/ less frequent and disabling vaso-occlusive complications
• Presents a normo-, normo- anemia
• Typically see numerous target cells & folded or "pocketbook” cells
• The SC crystals that are seen have fingerlike projections, producing "Washington monument" or mitten cells
• Solubility test results are positive
• There are equal amounts of Hgb C & Hgb S when seeing from cellulose acetate electrophoresis.
• Supportive therapy is similar when sickle cell crises occurs as it would for SS patients
• Life span is relatively normal from (60-70 yrs), but quality of life reduced from retinal lesions

Hemoglobin E

• Third most common Hgb variant in the world, mainly within Southeast Asian populations at 13% prevalence
• Hemoglobin E occurs in Laos, Thailand, and Cambodia
• Involves a substitution of lysine for glutamic acid in the 26th position, like hemoglobin C
• The homozygous state with >90% Hgb E presents as a microcytic and normochromic anemia with numerous target cells
• Hgb AE state occurs
• Also occurs in combination with beta-thalassemia

Hemoglobin D and G

• These variants move in an alkaline pH at the electrophoretic position of Hgb S by their charge
• Causes mild hemolytic anemia as seen in CC & AC
• They have a negative solubility test with no sickling
• Cellulose acetate electrophoresis reveals 95% Hgb D to migrate towards the S position sharing the cellulose acetate and co-migrating with Hgb A1 & A2 on citrate agar
• Can be clinically normal yet may have some extent of splenomegaly

Hemoglobin O (OArab)

• Common in Arabic populations
• Presents with mild hemolytic anemia and numerous target cells
• Differentiated amongst Hgb C with the implementation of electrophoresis for diagnosis
• Characterized by a 121st amino acid substitution of lysine for glutamic acid

Unstable Hemoglobin

• Involves amino acid substitutions that disrupt physical contact between heme & globin
• Results in Hgb denaturation following precipitation of globin chains and the formation of Heinz bodies
• Predisposes red blood cells to develop hemolytic anemia
  • Heinz bodies are made of denatured Hgb
• Symptoms range from mild to severe, with episodes of hemolysis and subsequent jaundice
• Most common example is Hgb Koln (Congenital Heinz Body Hemolytic Anemia)
  • The hemoglobin Koln is unstable
  • All the patients are heterozygous.
• Lab findings present in unstable hemoglobin are decreased MCV, MCH, & MCHC that occurs from Hgb lost when Heinz bodies are taken out by spleen
• Presents with Anisocytosis, poikilocytosis, polychromasia, basophilic stippling, & "bite cells"

Hemoglobin Altered by O2 Affinity

• The first one to be described was Hgb Chesapeake

• Exhibits increase affinity to O2 with a shift to the left

• the Alpha chain variant is also worth noting here

• Whereas Hgb Kansas experiences O2 affinity with a shift to the right

• Occurs due to substitution mutations can disrupt the 2,3-DPG binding sites, binding of heme globin, as well as stabilize the iron into a oxidized Fe3+ state

Methemoglobin (Hgb M)

• Known as the first to be described was Hgb Chesapeake
• Exhibits and increase affinity to O2 with a shift to the left that is also found within the Alpha chain variant
• The alteration of Pathophysiology involves substitution mutations that can disrupt the 2,3-DPG binding sites in addition to the binding of heme globin as well as stabilizing oxidized iron within the Fe3+ state that can alter the delivery of oxygen
• Caused by Inherited MetHgb reductase deficiency as well as from toxin exposure specifically in newborns overwhelmed by GHS’s reducing ability in regards with genetic mutation solely calling the Hgb M
• Characterized by five genetic variants known that can affect α or β chain which presents and the most common kind occurs within Japanese populations

Hemoglobin M Pathophysiology and Symptoms

• Amino acid substitution is known prevent the reduction of Fe3+ state, so Fe remains in ferric state
• This form for oxidation denatures globin chains enabling those chains to percipitate
• Only Heterozygous patient is 20 to355 of M, but the homozygous state is not consistent with life
• Presents a characteristic blood in brownish colour and cyanosis that often reveals lavender/blue/gray coloring as it develops for delivery of O2

Kleihauer Betke Test

• Used for pregnant women
• The Kleihauer Betke test applies and an acid elution cytochemical method in the determination fetomaternal hemorrhage where identification for cells contains within hemoglobin F
• This percentage often delineates maternal and fetal blood that may come from laboratory reports
• If the fetal cells is elevated, it is then multiplied by using number of RBC count, by 100% to receive the total numbers in that RBC
• Absence is often regarded with a range from 0 to 0.1%