Chapter 21: Thalassemias – Comprehensive Study Notes

Historical Context and Over-Arching Themes

The thalassemias illustrate, in a single disease family, every principle that modern molecular genetics has unraveled about eukaryotic gene structure, regulation, mutation and population genetics. They were the first Mendelian disorders in which (1) introns, (2) RNA-splicing, (3) locus-control regions, (4) naturally occurring promoter / enhancer variants, and (5) human “experiments of nature” (large deletions, unequal cross-overs, triplications, point mutations that generate new promoters, etc.) were systematically mapped. Clinically they constitute a spectrum ranging from the totally silent carrier to fatal hydrops fetalis, with severity determined by the degree of imbalance between the rate of α-chain synthesis and the aggregate synthesis of β-like chains.

Normal Globin Biology

Normal adult erythrocytes contain three tetramers: HbA ($\alpha<em>2\beta</em>2$, ≈95 %), HbA₂ ($\alpha<em>2\delta</em>2$, 2 %–3.5 %), and residual HbF ($\alpha<em>2\gamma</em>2$, 0.3 %–1.2 %, confined to 0.2 %–7 % of all cells – “F-cells”). In the fetus HbF predominates because its failure to bind $2,3$-DPG grants it a higher $O_2$ affinity that facilitates placental extraction.

Two linked gene clusters encode these chains:
• Chromosome 16p13.3 ( ζ – α₂ – α₁ ) – the α-like genes.
• Chromosome 11p15.5 ( ε – Gγ – Aγ – δ – β ) – the β-like genes.
Gene order mirrors developmental expression (“embryo → fetus → adult”). Each functional gene is interrupted by two introns; precise GT-AG splicing is mandatory because frame-shifts downstream are lethal to translation.

Developmental Switching

Hb Gower-1 ($ζ<em>2ε</em>2$), Gower-2 ($α<em>2ε</em>2$) and Portland ($ζ<em>2γ</em>2$) characterize yolk-sac erythropoiesis. Between weeks 6 and 10 the embryonic → fetal switch occurs; the fetal → adult switch coincides roughly with birth. Tissue location (yolk sac → liver → marrow) and switch timing are coordinated but not causal – ectopic expression studies prove time, not site, governs switching.

Cis-Acting Control Elements

Every globin promoter contains a TATA box (–25 bp), CCAAT, one or two CACC motifs, and one or more GATA sites. Major trans-acting factors:
• GATA-1 – X-linked zinc finger, essential; hypomorphic human alleles produce β-thalassaemia ± dyserythropoiesis.
• KLF1/EKLF – binds CCACACCCT; homozygous null mice die with lethal fetal β-thalassaemia; rare human loss-of-function elevates HbF.
• NF-E2 – heterodimeric bZIP, binds TGCTGATCA motif within the β-LCR.

The β-globin LCR (HS-1→HS-4 ≈30–50 kb 5′ to ε) and the α-cluster HS-40 play indispensable, copy-number–dependent “domain opening” roles. Long-range looping brings LCR cores into physical contact with active promoters; competition among promoters (γ vs β) plus stage-specific repressors (BCL11A) orchestrate the developmental switch.

Recent GWAS pinpointed three quantitative-trait loci that modulate HbF post-natally: the BCL11A enhancer on 2p, polymorphisms upstream of MYB on 6q, and intronic variants in HBS1L-MYB intergenic region. BCL11A knockdown re-activates $\gamma$-globin in adult erythroblasts and rescues murine sickle disease – the most compelling current pharmacologic target.

Classes of Mutations Producing Thalassaemia

1. Promoter/initiator changes – e.g.
   • –101 C→T (β silencing yet “silent carrier”).
   • –87 C→G (reduces TBP binding).
2. Splice-junction or cryptic splice-site creation – IVS-1 nt 1 G→A (β⁰), IVS-1 nt 5 G→C/T/A (β⁺ gradient), exon 1 codon 26 G→A (HbE) activates GTGGTGAGG cryptic donor.
3. Intron “new acceptor/donor” – IVS-2 654 C→T inserts pseudo-exon; IVS-1 110 G→A adds 19 nt, frameshift, β⁺.
4. Poly-A signal defects – AATAAA→AATGAA (β⁺) produce run-on transcripts.
5. Translation start-site or Kozak variants – ATG→ACG (β⁰) or CCACC→CCC- – (α⁺).
6. Nonsense or frameshift – classic β39 C→T, β41/42 –CTTT, many others.
7. Chain-instability (dominant) – Hb Evanston (α Trp14→Arg), β Showa-Yakushiji (β Leu110→Pro).
8. Large deletions –
   • α-cluster: −α3.7, −α4.2 (one-gene), −SEA, −MED (both genes, α⁰).
   • β-cluster: Sicilian (δβ⁰), HPFH-1 (≈106 kb), γδβ⁰ (Hispanic 39.5 kb) proving LCR essential.
9. Unequal crossover chimaeras – Hb Lepore (δβ fusion), Hb Kenya (Aγβ fusion).
10. Trans-acting factor defects – ATRX helicase (α-thal X-linked mental retardation), GATA-1 amino-finger (dominant β-thal + thrombocytopenia), TFIIH-XPD in trichothiodystrophy.

Population Genetics & Malaria

Both α- and β-thalassaemia heterozygosity confer an ≈50 % survival advantage against severe Plasmodium falciparum. Allele frequencies mirror historical endemicity (β-thal ≤20 % in coastal Greek villages; α⁺ as high as 68 % in Papua). Mechanisms include delayed HbF switch (β trait), reduced rosetting (α trait microcytosis), enhanced immune clearance and limited parasite iron supply.

Clinical Categorization

α-Thalassemia

Silent carrier (−α/αα) → Trait (−α/−α or −/αα) → HbH (−/−α) → Hydrops fetalis (−/−). Severity tracks the number of functional genes (4→0). HbH disease shows $\beta<em>4$ inclusions that precipitate with brilliant-cresyl-blue, chronic hemolysis, splenomegaly. Hydrops fetalis foetuses survive in utero on Hb Portland ($ζ</em>2γ_2$) but die neonatally.

β-Thalassemia

Silent (high-prob −101 C→T) → Trait (β ⁺/β , HbA₂ > $3.5\%$, MCV ≲ $70\,\text{fL}$) → Intermedia (transfusion independent, Hb ≈ $7–10\;\text{g/dL}$) → Major (Cooley). Key modifiers:
• Mutation severity (β⁰ vs β⁺).
• Co-inheritance of α-thal (
−α lowers chain excess → milder).
• Genetic HbF capacity (BCL11A, HBS1L-MYB, γ-promoter UP variants, HPFH deletions).

Compound States

HbS/β-thal, HbE/β-thal (prominent across South-East Asia), HbC/β-thal. Phenotype blends sickling or structural variant properties with chain imbalance.

Laboratory Hallmarks

• Peripheral smear: extreme micro-hypochromia, target cells, nucleated RBCs if marrow not fully suppressed.
• $[\alpha]/[\text{non}\,\alpha]$ chain biosynthesis by $^{3}$H-leucine:

Management Principles

1. Transfuse to maintain pre-transfusion Hb $\ge9.5$ g/dL (suppresses erythropoiesis, prevents bone deformity).
2. Iron chelation:
   • Deferoxamine $20–40\;\text{mg/kg}$ SC 5×/wk (binds NTBI).
   • Deferiprone $75–100\;\text{mg/kg/day}$ PO (beware agranulocytosis).
   • Deferasirox $20–40\;\text{mg/kg/day}$ PO (once daily, renal/hepatic monitoring).
3. Splenectomy if transfusion need >$200\;\text{mL}$ RBC/kg/yr or hypersplenism; vaccinate and give lifelong penicillin.
4. Allogeneic HLA-matched sibling stem-cell transplant (Lucarelli protocol) curative in >$90\%$ of class 1/2 children.
5. Pharmacologic HbF induction – hydroxyurea, butyrates, emerging BCL11A/KLF1 targeted approaches.
6. Gene therapy – lentiviral β-globin vectors now in early trials, CRISPR editing of $\gamma$ repressors under investigation.

Prenatal & Public-Health Impact

CVS‐PCR detection of parental mutations at 10 weeks is standard; non-invasive cell-free fetal DNA assays are emerging. Population screening + selective termination cut β-thal births by >$90\%$ in Sardinia, Greece & Cyprus (Fig 21-32).