Hemoglobinopathies

Overview Framework

• Two broad categories:
  • Structural hemoglobinopathies: an amino acid substitution changes the hemoglobin structure or function (HbS, HbC).
  • Thalassemic hemoglobinopathies: the variant is produced in reduced amount, giving a thalassemia-like (quantitative) picture (HbE, Hb Lepore, Hb Constant Spring).
• Chain composition: HbA = α2β2, HbA2 = α2δ2, HbF = α2γ2.
• Developmental mnemonic: "Alpha Always; Gamma Goes, Beta Becomes" (α is present throughout life; γ falls after birth as β rises).
• Sickle patterns for quick recall: sickle trait ~60% A / ~35 to 40% S (NOT microcytic); sickle β+ thalassemia ~60% S / ~35% A; sickle plus α-thal trait ~70% A / ~25 to 30% S (microcytic).

Normal Adult Hemoglobins

• HbA (α2β2): ~95 to 98% in adults.
• HbA2 (α2δ2): ~1.5 to 3.5%.
• HbF (α2γ2): <1% in adults; major fetal Hb (~80% at birth, decreases over first 6 mo).
• Embryonic Hb: Gower 1, Gower 2, Portland (first trimester).

Genetic Architecture

• α-globin: 4 genes total (2 each on chromosome 16), HBA1 and HBA2.
• β-globin: 2 genes total (1 each on chromosome 11), HBB. Cluster: ε, Gγ, Aγ, δ, β.
• Hb switch: γ → β occurs ~6 mo of life (clinical disease in β-globinopathies emerges then; α-globinopathies present at birth or in utero).
• BCL11A: master HbF silencer; target of fetal hemoglobin reactivation strategies (exa-cel CRISPR therapy edits the BCL11A erythroid enhancer).

Diagnostic Tests

• Hb electrophoresis (cellulose acetate at alkaline pH, citrate/agarose at acid pH): separates Hb variants by charge (HbA, HbS, HbC, HbE, HbA2, HbF).
• HPLC: more accurate quantification; current gold standard.
• Capillary electrophoresis: alternative to HPLC.
• Solubility / sickle prep: screening only; does NOT distinguish trait from disease or compound heterozygotes.
• DNA testing: confirms variants; needed for molecular prenatal diagnosis and to resolve certain genotypes (including some alpha-thal); genetic counseling can begin after carrier identification by Hb analysis and for distinguishing certain α-thal genotypes.
• Newborn screening: standard in US; detects SCD and thalassemias.

Hemoglobin Migration on Electrophoresis

• Key trap: a band running with HbS on alkaline gel that does NOT sickle can be Hb Lepore or Hb D; confirm with acid gel and a sickle solubility test.

Electrophoresis Interpretation by Genotype

Hemoglobin E

• Mutation: β-chain codon 26 Glu → Lys, which also decreases β-chain synthesis, so it behaves as a mild thalassemic variant.
• Epidemiology: very common on the Indian subcontinent and Southeast Asia (gene frequency up to 60%).
• Migration: with A2 and C on alkaline electrophoresis; with A and A2 on acid agarose. Migrates like HbA2 on gel but you never see 30% "HbA2".
• Heterozygote (HbAE, trait): usually not anemic; minimal microcytosis/hypochromia. Analysis ~70% HbA, ~30% HbE, ~1% HbF.
• Homozygote (HbEE, disease): minimal anemia with hypochromia, target cells, prominent microcytosis; >90% HbE, no HbA, remainder HbF; relatively asymptomatic.
• HbE plus other globin mutations: a variable and occasionally much more severe phenotype (e.g., HbE/β-thalassemia can mimic β-thal major).

Hemoglobin Lepore (α2(δβ)2)

• Origin: unequal crossover fusing adjacent δ and β genes into a single δβ fusion globin (stable, functional).
• Migration: runs with HbS on alkaline gel and with HbA on acid gel; can be reported as "HbS" but shows NO sickling on smear or sickle test.
• Quantity: production is driven by the weak δ promoter (only ~2 to 3% as active as the β promoter), so Hb Lepore is only ~3 to 20% of total Hb instead of the expected ~50%. HbA2 is decreased (no normal δ from the abnormal chromosome, so ~3% falls to ~1.5%); HbF is usually slightly increased.
• Phenotype: heterozygotes resemble β-thal minor; homozygotes give β-thal intermedia to major.
• Population: more common in people of Central European descent (Balkans).

Hemoglobin Constant Spring

• Mechanism: an α-globin chain-termination variant; instead of stopping, translation adds 31 extra amino acids, making an elongated α chain. Common in Southeast Asia.
• Electrophoresis: shows a minor, very slowly migrating abnormal component. Hb Constant Spring is itself unstable/toxic and results in a deficiency of normal α chains.
• Relation to HbH disease:
  • Deletional HbH (−α/−−): only one α allele, makes β4 tetramers.
  • Non-deletional HbH (αα^CS/−−^SEA): two α genes but one carries the Constant Spring mutation, which yields unstable mRNA and an elongated, unstable α chain (greatly reduced effective α output plus extra red-cell damage); more severe than deletional HbH disease.

Hereditary Persistence of Fetal Hemoglobin (HPFH)

• Mechanism: upregulated γ-globin synthesis with HbF persisting beyond infancy; behaves like an allele of the β-globin gene.
• Causes: deletional (large deletions of δ and β with intact γ) and non-deletional forms; deletional and non-deletional forms; HbA/HbA2 production depends on genotype (heterozygotes make HbA and HbA2 from the normal chromosome; homozygotes make 100% HbF)
• Population: prevalent in people of African ancestry.
• Homozygote (HPFH/HPFH): 100% HbF, no anemia.
• Compound heterozygote (HbS/HPFH): mild sickling disorder, HbS ~70%, HbF ~30% (high HbF prevents sickling).

• δβ-thalassemia (homozygous): both δ and β genes deleted, so only HbF can be made (HbF 100%, no HbA or HbA2), microcytic.

Unstable Hemoglobins

• Laboratory features: normocytic to microcytic anemia (constant or intermittent); smear shows polychromasia, basophilic stippling, and bite cells; new methylene blue reveals Heinz bodies; heat-stability and isopropanol-stability tests positive; electrophoresis/HPLC may be normal (many variants are charge neutral); diagnosis rests on stability tests, Heinz bodies, and molecular studies; molecular studies identify the mutation.
• Treatment: folic acid; avoid oxidant drugs if a sensitive variant; prompt treatment of infections; intermittent red cell transfusions; splenectomy in severe cases.
• Example, Hb Koln: the most common unstable variant; an unstable, high-affinity Hb (β98 Val → Met) that destabilizes the heme pocket, causing mild anemia.

Hemoglobin M Disorders and Methemoglobinemia

• HbM: hereditary methemoglobinemia and cyanosis. A group of α, β, and γ chain variants with an amino acid substitution in the heme pocket that favors oxidation of iron from ferrous (Fe2+) to ferric (Fe3+); the oxidized chain cannot carry oxygen. Autosomal dominant.
• Clinical: asymptomatic cyanosis, slate-gray/brownish skin, no dyspnea, normal life expectancy.
• Treatment: cyanosis is NOT reversible with vitamin C or methylene blue; no treatment needed.
• Labs: Hb normal or high; abnormal pulse oximeter saturation; electrophoresis/spectrometry abnormal; methemoglobin usually <30%. Distinguish from other congenital and acquired causes.
• Cytochrome b5 reductase (CYB5R3) deficiency:
  • Type 1: most common congenital methemoglobinemia; autosomal recessive; defective reduction of Fe3+ to Fe2+ in RBCs only; cyanosis/hypoxia; methemoglobin usually <30%; treat cyanosis with methylene blue or ascorbic acid.
  • Type 2: ~10 to 15% of cases; enzyme deficiency in all cells; mental deficiency and developmental delay; methylene blue improves cyanosis but not CNS symptoms.
• Acquired (drug/chemical) methemoglobinemia: oxidizers include dapsone, nitrites, chloroquine, lidocaine, naphthoquinone, sulfanilamide, phenylhydrazine, and others.
  • Clinical: cyanosis with lack of response to 100% oxygen; methemoglobin >30% symptomatic (dyspnea, nausea, tachycardia); >50% can be lethal (lethargy, stupor, altered consciousness, cardiac failure).
  • Emergency treatment: methylene blue 1 to 2 mg/kg as a 1% solution IV over 10 to 15 minutes.

High Oxygen Affinity Hemoglobins

• Mechanism: α or β chain variants (>120 described), generally autosomal dominant, with a left-shifted oxygen dissociation curve. Tissue hypoxia drives ↑ erythropoietin → secondary erythrocytosis.
• Presentation: familial polycythemia, autosomal dominant, no hepatosplenomegaly.
• Diagnosis: high Hb/Hct, low P50 (left-shifted curve), electrophoresis or HPLC, PCR/gene sequencing.
• Treatment: phlebotomy not needed for mild polycythemia.

Low Oxygen Affinity Hemoglobins

• Mechanism: α or β chain variants with high P50 (right-shifted oxygen dissociation curve).
• Presentation: asymptomatic anemia and cyanosis.
• Diagnosis: electrophoresis/HPLC and molecular studies; no treatment required.

Common Sickle and Variant Hemoglobinopathies

• HbS: β6 Glu → Val; deoxygenated HbS polymerizes → sickling.
• Sickle cell trait (HbAS): ~8% of African Americans; usually asymptomatic; rare complications, splenic infarct at altitude, exertional rhabdomyolysis, hyposthenuria, papillary necrosis, renal medullary carcinoma (associated; rare but aggressive).
• HbSS (sickle cell disease): see separate SCD note.
• HbSC: milder than SS; ~25 to 30% of SCD cases; retinal disease and AVN more common; less acute chest.
• HbS/β-thalassemia: SS-like (HbS/β0) or milder (β+).
• HbC disease (CC): target cells; mild hemolytic anemia; HbC crystals in cells.
• HbE: SE Asia; mild β-thal-like; HbE/β-thal can be severe.
• HbD-Punjab, HbO-Arab: variants.

Distinguishing Microcytic Anemias (boards classic)

• IDA: ferritin ↓, RDW ↑↑, Mentzer index (MCV/RBC count) >13, target cells uncommon.
• α/β-thal trait: ferritin normal, RDW normal, Mentzer index <13, target cells common, more pronounced microcytosis for the Hb level.
• ACD: ferritin normal/↑, Fe ↓, TIBC ↓.
• Lead: basophilic stippling, ↑ ZPP/EP, ↑ blood lead.
• Sideroblastic: ringed sideroblasts on iron stain; ↑ Fe, ↑ ferritin.

Curative and Disease-Modifying Therapies (cross-cutting)

• Allo-HSCT: curative for SCD and β-thal; best in young patients with a matched donor.
• Gene therapy / gene editing:
  • Exa-cel (Casgevy, exagamglogene autotemcel): CRISPR-Cas9 editing of the BCL11A erythroid enhancer to reactivate HbF. FDA Dec 2023 for SCD and Jan 2024 for transfusion-dependent β-thal (age ≥2 since July 2026) (CLIMB SCD-121, CLIMB THAL-111).
  • Lovo-cel (Lyfgenia): lentiviral βA-T87Q globin gene addition. FDA Dec 2023 for SCD.
  • Beti-cel (Zynteglo): lentiviral β-globin for transfusion-dependent β-thal. FDA Aug 2022.
• Hydroxyurea: HbF inducer; standard for SCD; some role in β-thal intermedia.
• Voxelotor: HbS polymerization inhibitor, withdrawn 2024 (mortality concern in HOPE-KIDS trial).
• Crizanlizumab (P-selectin inhibitor): FDA 2019 for SCD VOC reduction; withdrawn EU 2023 after STAND failed; US approval continues.
• Luspatercept: FDA Nov 2019 for transfusion-dependent β-thal; activin receptor ligand trap; reduces transfusion burden.
• Mitapivat (Aqvesme): oral pyruvate kinase activator; FDA Dec 2025 for anemia in adults with α- or β-thalassemia, TDT and NTDT (ENERGIZE, ENERGIZE-T); boxed warning for hepatocellular injury, REMS liver monitoring.

High-Yield Pearls

• Newborn screening: identifies SCD and thal; allows early prophylaxis (penicillin in SCD).
• Mentzer index <13 plus normal RDW plus microcytosis = thal trait.
• HbA2 >3.5% on HPLC supports β-thal trait when interpreted with the CBC and full Hb analysis (HbE and Hb Lepore coelute in the HbA2 window; not diagnostic alone)
• α-thal trait: HPLC may be normal; need DNA analysis.
• Cis vs trans α-thal trait: SE Asian (cis) confers offspring risk for Hb Barts.
• Solubility / sickle prep: screening only; cannot distinguish AS vs SS vs SC.
• Lepore and Hb D: run with HbS on alkaline gel but do NOT sickle.
• Exa-cel and lovo-cel: 2023 SCD curative gene therapies.
• Voxelotor: withdrawn 2024.
• Renal medullary carcinoma: associated with sickle cell trait (rare but classic).