HIS08 Molecular Genetics Of Thalassaemia Flashcards
Thalassaemia
- Most common genetic disease worldwide
- Anaemia with smaller and paler RBC
- Either α / β globin chain is structurally normal but ***reduced in quantity —> Imbalance (Haemoglobin: 2α, 2β)
- Heterozygotes for Thalassaemia are protected from severe effects of malaria
α and β globin gene cluster on human chromosomes
α and β globin gene:
- not single gene
- gene clusters (duplication of single gene during evolution)
α-like genes:
- Chromosome ***16
- 5’ end —> HS-40 (hypersensitive site) —> regulatory sequences
β-like genes:
- Chromosome ***11
- 5’ end —> LCR: locus control region (contains enhancers, promoters) —> regulatory sequences
- 3’ end —> HS-1 —> regulatory sequences
***Haemoglobin proteins
- Fetal Hb: ***HbF (2α2γ)
- Adult Hb: ***HbA (2α2β) + HbA2 (2α2δ)
—> β, γ globin —> fully functional
Developmental switches in globin expression:
A switch from γ-globin to β-globin begins **before birth and complete by **6 months of age —> over 95% 2α2β
(Embryonic: Hb Gower 1 (2ζ2ε), Hb Gower 2 (2α2ε), Hb Portland (2ζ2γ))
Site of Erythropoiesis
Yolk sac —> Liver + Spleen —> Red bone marrow
***Pathophysiology of Thalassaemia
Normal:
- 2α2β tetramers —> healthy RBC
α-Thalassaemia: - defect in α chain production —> severely reduced α chain —> excess β chains —> β4 tetramers (***HbH) —> ***inclusion bodies of β4 in RBC —> ***Peripheral haemolysis
β-Thalassaemia:
- defect in β chain production
—> severely reduced β chain
—> excess α chains
—> α4 tetramers (inviable tetramer, very **insoluble)
—> **precipitation of α4 (even worse)
—> ***Destruction of RBC in marrow, spleen (early in fetal life)
Excessive globin chains precipitates
- Haemolysis
- Ineffective erythropoiesis
- Impaired oxygen transport
- Anaemia (Hypochromic Microcytic)
Severity depends on:
- Degree of α/β chain imbalance
- Type of globin allele present
- Other type of thalassaemia present
***α-Thalassaemia: Genotypes and Phenotypes
- Normal:
- 4 functional α alleles (1 mother, 1 father —> code for 1 α chain, total 2 α chains) - Carrier (asymptomatic):
- 1 functional α allele deleted
- α-/αα
- silent carriers - α-Thalassaemia Trait / Minor (mild / asymptomatic):
- 2 functional α alleles deleted (cis: on same chromosome, trans: on different chromosomes)
- α-/α- or –/αα
- mild anaemia, considered carriers - HbH disease (symptomatic)
- 3 functional α alleles deleted / 2 deleted, 1 disrupted (i.e. nondeletional mutation)
- HbH: ***β4; α-/–
- moderate to severe anaemia - Haemoglobin Bart’s hydrops fetalis (incompatible with life)
- all 4 genes affected
- α-Thalassaemia Major; hydrops fetalis with Hb Bart’s i.e. ***γ4; –/–
- could not survive and usually die before/shortly after birth due to lethal intrauterine haemolytic anaemia caused by precipitation of γ4
- rarely can be homozygous for non-deletional forms of α-Thalassaemia
- if there is persistence of intact embryonic ζ-globin gene —> neonates may survive
α-Thalassaemia inheritance
Inheritance pattern for α-Thalassaemia: ***Mendelian
Gene mutation arise de-novo from:
- ***misaligned DNA crossover between homologous chromosomes during meiosis —> misaligned homologous recombination
Production of α chain: 0-100% (–/– to αα/αα)
Zygosity:
- Heterozygous (αα/α-, –/αα, –/-α) —> 2 different chromosomes
- Homozygous (αα/αα, α-/α-, –/–)
Genetics:
–/– can only arise at risk of 25% when each parent caries a – chromosome
—> either HbH disease / Heterozygous α thal trait (–/αα)
Molecular epidemiology:
- –/αα more common (up to 15% in SE Asians)
- α- and -α found frequently in African Americans (30%)
Summary
Haemoglobin switching (during course of development):
- γ —> β
- HbF —> HbA
α-Thalassaemia:
- Gene deletion (dosage effect)
- Molecular genetics
***β-Thalassaemia: Genotypes and Phenotypes
There is ONLY 1 β gene on each chromosome!!! (i.e. 2 β genes in body vs 4 α genes in body)
Severity of β-Thalassaemia:
- Major (Cooley’s anaemia)
- Intermedia
- Minor
βo (little / no production of β chain)
β+ (β chain and HbA are detectable)
β++ (defect in β chain production very mild)
βsilent (minimal effect on β globin production)
βN (normal)
- β-Thalassaemia minor / trait (e.g. β++/βN; βsilent/βN):
- **βo / β+ heterozygous (i.e. βo/βN, β+/βN)
- **mild / no anaemia
- Ameliorating factors: Concurrent α-Thalassaemia
- Exacerbating factors: Excess α-globin genes - β-Thalassaemia intermedia (e.g. β++/β++):
- 2 β-globin genes carrying a thalassaemia mutation (at least 1 mild) OR
- 1 β-globin Thalassaemia mutation in combination with **excess α globin genes (less common)
- **mild to moderate anaemia
- Ameliorating factors: Concurrent α-Thalassaemia, Elevated HbF
- Exacerbating factors: Excess α-globin genes (>=5) - β-Thalassaemia major (e.g. βo/βo):
- **2 β-globin genes carrying a **severe thalassaemia mutation (βo / β+ **homozygous OR **compound heterozygous e.g. βo/β+)
- severe anaemia requiring ***regular transfusion
- Ameliorating factors: Concurrent α-Thalassaemia, Elevated HbF
- Exacerbating factors: N/A - Haemoglobin E Thalassaemia:
- 1 β-globin gene carrying a Thalassaemia mutation (mild/severe) in combination with 1 β-globin gene carrying point mutation encoding HbE
- mild to severe anaemia
- Ameliorating factors: Mild β-Thalassaemia mutation, Concurrent α-Thalassaemia, Elevated HbF
- Exacerbating factors: Severe β-Thalassaemia mutation
Primary and secondary modifiers of β-Thalassaemia phenotype includes variable output from:
1. **β-globin gene
2. **α-globin gene
3. ***HbF response
—> determine degree of chain imbalance (α / non-α globin ratio) + severity of ineffective erythropoiesis
Molecular pathology of β-Thalassaemia
Primary cause:
- α-globin, β-globin, γ-globin gene —> affect magnitude of ***excess α-chains
Secondary modifiers (treat products of β-Thalassaemia):
- AHSP (α haemoglobin stabilising protein —> reduce α4 precipitation)
- HPFH
Tertiary modifiers (treat end results of β-Thalassaemia): - VDR, ESR1, HFE, HAMP, UGT1, HLA-DR, TNF, ICAM1, GDF11, JAK2, TMPRSS6
Result:
**Excess α globin chains (Secondary target)
—> **Inclusion bodies + **Ineffective erythropoiesis + **Anaemia + **Haemolysis + Proteolysis
—> **Jaundice + gallstones, **Iron overload (Hepcidin suppressed), **Oxidative damage, Bone disease, Infection (Tertiary target)
β-Thalassaemia mutations
Due to **single nucleotide mutations (or insertion/deletion of a few nucleotides rather than deletions of whole gene)
—> result: **Loss-of-function of β globin gene (βo and β+) (vs α-Thalassaemia: ***Deletion of α gene)
So many different mutations that most patients with β-Thalassaemia Major are compound heterozygotes (having 2 different mutations) and NOT true homozygotes (having 2 same mutations)
Inheritance: ***Autosomal recessive (however dominant β-Thalassaemia also found)
- **Loss-of-function mutations of β globin gene (βo and β+)
- transcription (promoter)
- ***RNA splicing (most common, >24, including synonymous mutation that affect splicing) (切錯野 e.g. 切多左/切少左)
- cap site
- poly(A) site
- RNA stability / abundance
- insertion/deletion
- **nonsense/missense —> **Premature stop codon / Switch in a.a.
- frameshift (by addition / deletion of nucleotide)
Prematurely terminated mRNAs are degraded by nonsense-mediated decay (NMD)
—> no protein produced
Consensus sequences of splice sites:
—> GU at start of intron, AG at end of intron are invariant nucleotides
—> mutation at splice sites
—> wrong splicing
β-Thalassaemia splicing mutations
- Normal adult β globin primary RNA transcript:
- normal mRNA formed from ***3 exons -
**Exon skipping: Normal splice site destroyed due to single nucleotide changes
- mRNA with **exons missing (切多左) -
**Cryptic splice sites activated: Normal splice site destroyed due to single nucleotide changes
- mRNA with **extended exon (切少左) -
**New exons to be incorporated: New splice sites created due to single nucleotide
- mRNA with **extra exons (切少左)
—> ALL change protein sequence encoded —> destroyed by NMD
Common β-Thalassaemia mutations in China
- ***Codons 41/42 (4 nucleotide deletion frameshift) (40%) —> βo
- Intron 2-654 (extra intron) (15.7%) —> βo
- Codons 71/72 (+1 )(12.4%) —> βo
- -28 (A—>G, TATA box: consensus sequence of promoter) (11.6%) —> β+ (additional production)
- Codon 17 (nonsense A—>T) (10.5%) —> βo
Novel targets for treatment of β-Thalassaemia
- α/β chain imbalance
- Gene therapy / Gene editing - Increased proliferation (lead to ineffective erythropoiesis)
- JAK2 inhibitors - Increased GDF11 (lead to decreased differentiation —> ineffective erythropoiesis)
- Sotatercept
- Luspatercept - Decreased Hepcidin (lead to Iron overload)
- Iron chelators for Iron overload currently
- Minihepcidins
- TMPRSS inhibitors (downregulate metalloprotease TMPRSS6: control Hepcidin expression) - Cellular and molecular modifiers (5-azacytidine, hydroxyurea etc.)
- Anti-oxidants
- CRISPR/Cas9 (correcting mutations by targeted genome editing)
- HSC transplantation
***Gene therapy of β-Thalassaemia
- Haematopoietic stem cells collected from bone marrow of patient
—> **Lentiviral-vector particles containing a **functional β-globin gene introduced into HSC
—> expand further in culture
—> **chemotherapy for patient to eradicate patient’s remaining HSC to make room for genetically modified cells
—> **genetically modified HSC transplanted back to patient - Activate transcriptional repressor (inverse collection between BCL11A gene and HbF expression)
—> ***suppress BCL11A gene
—> keep γ expression switched on during adult life
—> HbF ↑ —> production ↑ —> selective survival
