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CHAPTER 18: Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency – Comprehensive Bullet Notes

G6PD in Red Blood Cell (RBC) Metabolism

  • G6PD = first enzyme of the pentose-phosphate pathway (hexose-monophosphate shunt).
  • Prime physiologic role ➜ generate NADPH (only ~10 % of glucose flux goes through pathway even under maximal oxidative stress).
  • NADPH uses:
    • Reductive potential for detoxifying H2O2 via catalase & glutathione peroxidase (GSHPX).
    • Regenerates GSH through glutathione reductase (GSSGR).
    • Structural cofactor for catalase.
  • Continuous production of oxygen radicals in Hb auto-oxidation makes RBCs highly dependent on G6PD.
  • Normal RBC G6PD decays exponentially with age; t_{1/2}\approx 50\text{–}60\,\text{days}.

Structure & Biochemistry of G6PD

  • Ubiquitous, ancient housekeeping enzyme (found from prokaryotes → mammals).
  • Active form: homodimer or homotetramer of 59 kDa subunits.
  • Domains (per monomer):
    • Coenzyme domain (aa 1–198).
    • Large \beta+\alpha domain (aa 199–514).
  • Key active-site residues:
    • K205 near glucose-6-phosphate (G6P) binding.
    • NADP binding motif: G\text{-}X!X!-G!X!X (aa 38–43).
    • Structural NADP molecule confirmed crystallographically.
  • Kinetics:
    • Very high specificity for NADP^+ over NAD^+.
    • Affinity (Km) for NADP^+ ≈ 10× higher than for G6P.
    • Product NADPH = potent quasi-competitive inhibitor.
  • Regulation:
    • Oxidative stress lowers \frac{NADPH}{NADP^+} ➜ relieves inhibition ➜ increases flux.
    • microRNA miR-1 down-regulates G6PD in nucleated cells.

Genetics of G6PD (Gd Gene)

  • Location: Xq28 (telomeric long arm of X chromosome).
  • Gene structure: 13 exons (exon 1 non-coding), ~18.5 kb; intron 2 ≈12 kb.
  • Promoter: GC-rich, 2 essential Sp1 sites; core ≈150 bp.
  • Inheritance consequences:
    1. Classical X-linked pattern.
    2. Severe deficiency more common in hemizygous males.
    3. Female heterozygotes ➜ mosaicism via random X-inactivation; predicted binomial distribution (32–64 embryonic precursor cells → ~2 % extreme skewing).
  • Somatic selection can skew ratios (e.g., favoring G6PD(+) cells in hematopoiesis).
  • Overlap with IKBKG/NEMO gene; large deletions causing incontinentia pigmenti remove G6PD exons but heterozygous females remain phenotypically G6PD(+).

Terminology & WHO Classification (1966)

  • Class I <10 % activity ➜ congenital nonspherocytic hemolytic anemia (CNSHA).
  • Class II/III <30 % activity ➜ no chronic hemolysis but risk of acute hemolytic anemia (AHA), favism, neonatal jaundice (NNJ).
  • Class IV normal; Class V increased (rare ‑ G6PD Hektoen).
  • Deficient males = Gd^-; females: Gd^-!/Gd^- homozygotes (deficient), Gd^+!/Gd^- heterozygotes (intermediate).

Molecular Basis of Deficiency

  • 186 mutant alleles catalogued:
    • 159 single missense, 13 double, 2 triple mutations.
    • 10 in-frame deletions, 2 splice defects.
    • Null (frameshift, nonsense) mutations embryonically lethal (shown in mouse model; hemizygous males non-viable).
  • Pathogenic mechanisms:
    1. In vivo protein instability (most common) ➜ accelerated exponential decay (Fig.-like). Severe when dimer interface or structural NADP binding disturbed (exons 10–11 hotspot).
    2. Altered catalysis (e.g., G6PD Orissa, Mahidol).
  • Polymorphic variants often compound (e.g., African G6PD A⁻ = N126D + V68M).

Epidemiology

  • >500 million affected worldwide.
  • High prevalence in tropical/sub-tropical belts; absent in Amerindians.
  • New high-resolution prevalence map (Howes et al. 2012) correlates with malaria endemicity.

Clinical Manifestations

Acute Hemolytic Anemia (AHA)

  • Triggered by oxidant stress: fava beans (favism), infections, drugs/chemicals (Table-type list).
  • Presentation (6–48 h post-exposure):
    • Dark “Coca-Cola” urine (hemoglobinuria), jaundice, pallor, tachycardia, splenomegaly.
    • Lab: normocytic anemia, marked anisopoikilocytosis, bite cells, Heinz bodies, retics ↑ (up to 30 %), haptoglobin 0, indirect bilirubin ↑, DAT –.
  • Course: self-limited; Hb normalizes in 3–6 wk. Transfusion needed if Hb <7\,\text{g/dL} or ongoing hemolysis.
  • Pathophysiology sequence:
    1. Oxidant → NADPH↓ → GSH↓.
    2. Hb –SH oxidation → Heinz body formation.
    3. Membrane cross-linking ➜ intravascular & splenic extravascular hemolysis.
  • Favism specifics:
    • Toxins: vicine & convicine → aglycones (divicine, isouramil) generate ROS.
    • Severity depends on bean maturity, quantity, body mass.
  • Key hemolytic drugs (definite): dapsone, primaquine, methylene blue, phenazopyridine, sulfonamides; others possible (high-dose aspirin, quinolones, nitrofurantoin, etc.).

Neonatal Jaundice (NNJ)

  • Peak day 2–3; anemia rare/severe only in minority.
  • Incidence higher in populations where G6PD deficiency common (e.g., Greece, Nigeria, Taiwan, USA recent data).
  • Mechanisms:
    • Inefficient bilirubin conjugation (UGT1A variant/Gilbert) + mild hemolysis.
    • Exacerbating factors: prematurity, breastfeeding, acidosis, infection, naphthalene, oxidant drugs, maternal fava ingestion.
  • Management per AAP: treat as high-risk group; earlier phototherapy, exchange transfusion if bilirubin >15\,\text{mg/dL} (\≤48 h) or >19\,\text{mg/dL} any time first week.

Congenital Nonspherocytic Hemolytic Anemia (CNSHA)

  • Caused by rare class I mutations (e.g., G6PD Harilaou, Nara, Guadalajara).
  • Features:
    • Persistent hemolysis from birth, variable transfusion need.
    • Retics up to 20–50 %, mild macrocytosis, splenomegaly, gallstones.
    • Membrane spectrin aggregates ➜ shear fragmentation.
  • Treatment: folate, avoid oxidants, transfuse as needed, consider splenectomy if hypersplenism or to convert transfusion-dependent → independent; iron chelation if chronic transfusion.

Laboratory Diagnosis

  • Quantitative spectrophotometric assay (absorbance 340\,\text{nm}): normal 7\text{–}10\,\text{IU/g Hb (30°C)}.
  • Screening spot tests: fluorescence spot, formazan color reduction; classify as normal/deficient (<30 %).
  • Pitfalls:
    • Post-hemolytic state & reticulocytosis elevate measured activity ➜ possible false-normal; repeat after 4–6 wk.
    • Transfused patients: donor RBCs mask deficiency.
  • Heterozygotes: cytochemical or flow-cytometric single-cell assays (metHb reduction) needed; DNA mutation analysis if extreme lyonization.

Genotype–Phenotype Relationships

  • 26 polymorphic alleles ≥1 % in some population; prototypic: Mediterranean (Ser188Phe), A⁻ (N126D+V68M), Canton (R459L), Mahidol (G163S), Viangchan (T383L).
  • Class I mutations cluster in exon 10–11 (dimer interface) ➜ severe instability.
  • Km(G6P) lower in class II/III vs. class I.
  • Clinical severity influenced more by trigger dose/exposure than precise variant within class II/III.

Preventive Medicine

  • Newborn screening advisable where prevalence high; cord blood best sample.
  • Education & avoidance lists prevent favism/drug AHA (Sardinia program ↓ admissions >80 %).
  • P. vivax eradication with primaquine in G6PD-deficient patients: use lower dose, prolonged regimen with monitoring.
  • Evaluate hemolytic potential of new drugs (in vitro RBC NADPH/GSH assays) – often lacking in development.

G6PD Deficiency in Non-Erythroid Cells

  • Nucleated cells synthesize G6PD; residual levels: Mediterranean ≈30 % in neutrophils; A⁻ nearly normal.
  • Rare class I variants (e.g., G6PD Barcelona) impair neutrophil oxidative burst ➜ staphylococcal infections.
  • Experimental data: G6PD-deficient mice macrophages show impaired ROS handling.
  • Possible associations: early cataracts, pterygium (eye lens = anuclear).

Coexisting Disorders

  • Hemoglobinopathies: no major effect on steady-state sickle cell anemia; additive risk during oxidative crises; β-thalassemia trait → MCV slightly ↑.
  • Non-hematologic:
    • Diabetes mellitus: conflicting epidemiology; AHA during ketoacidosis; altered retinal outcomes.
    • Viral hepatitis C therapy with ribavirin generally safe with monitoring.
  • Trauma: higher infection rate & anemia severity in G6PD-deficient poly-trauma patients.
  • Blood donors: G6PD-deficient units safe for most, avoid for NNJ exchange.

G6PD Polymorphism & Malaria Selection

  • Macro-geographic overlap of deficiency with Plasmodium\ falciparum belt (absent in Amerindians).
  • Field studies (Africa, SE Asia) show ~50 % reduction in severe malaria, variably in males/heterozygous females.
  • Mechanisms:
    • Parasite growth impairment after passage through G6PD-deficient cells.
    • Enhanced macrophage clearance of infected RBCs ("suicidal infection").
  • Convergent evolution: multiple independent deficient alleles arose & spread under selective pressure.

Ethical, Practical & Philosophical Considerations

  • Balancing antimalarial drug policy vs. hemolysis risk in endemic areas.
  • Genetic counseling: prenatal DNA testing for class I mutations; education of carriers.
  • Disparities: screening under-performs in heterozygous females ➜ continued favism cases in girls (Sardinia data).
  • Research frontier: potential gene therapy using lentiviral correction in hematopoietic stem cells (successful in mice & rhesus pre-clinical).

Key Numerical / Statistical References

  • >500\,000,000 people estimated G6PD-deficient globally.
  • RBC G6PD exponential decay: t_{1/2}\approx 60\,\text{days}; reticulocyte activity ≈5× oldest RBCs.
  • Drug-induced anemia: dapsone 7.5 mg/kg over 3 days in African children → mean Hb drop \approx 2\,\text{g/dL} (12 % required transfusion).
  • NNJ risk: African-American cohort – phototherapy need 20.3 % in deficient vs 5.7 % normals.
  • Favism prevention (Sassari): male admissions from 60 → <10 per year after screening/education.