L08 – Molecular Genetics of Thalassemia Flashcards
Heterozygotes for thalassemia is protected against what disease?
protected from severe effects of malaria
Describe the globin genes for Hb synthesis. Which chromosome, what related genes/ transcription sites?
α- and β-globin gene clusters on human chromosomes:
1) α-like genes (chromosome 16):
- 2 functional alleles: α1, α2, ζ
- Pseudogenes
- Hypersensitive site (HS)-40
2) β-like genes (chromosome 11):
- Functional:
Embryonic: ε
Fetal: Gγ, Aγ
Adult: β, δ
- Locus control region (LCR): where transcription factors bind
Give the globin composition in normal fetal and normal adult Hb?
fetal= Hb F (α2γ2)
adult = Hb A (α2β2)
Describe the changes in expression of globin genes from fetus to birth?
Hemoglobin switching:
Switch from γ-globin to β-globin expression
> > begins before birth, completes by 6 months of age
α: constant
β: increase
γ: decrease
Describe change in site of erythropoiesis from fetus to birth?
yolk sac > liver > spleen > bone marrow
Explain why α- thalassemia causes inclusion bodies, but β- thal cause precipitation?
α- Thal: defect in making α globin, excess β globins
β - Thal: defect in making β globins, excess α globins
α globins are LESS soluble than β, thus excess = precipitation
Compare α and β thalassaemia in the location of hemolysis?
α- thal = peripheral haemolysis
β - thal = Destruction of RBCs in marrow, spleen»_space; Ineffective erythropoiesis
Genotype and status spectrum of a- thalassaemia?
No a-globin gene deletion = normal
1 deletion = silent carrier (asymptomatic)
2 deletions (cis or trans) = a- thalassaemia minor/ trat or carriers
3 deletions or 2 deletion + 1 nondeletional mutation = HbH disease
4 deleted = Hb Bart’s hydrops detalis/ a- thalassaemia major
Genotype and phenotype spectrum of a- thalassaemia?
Gene deletion = dosage effect:
No a-globin gene deletion = normal
1 deletion = silent carrier = asymptomatic
2 deletions (cis or trans) = a- thalassaemia minor/ trat or carriers = asymptomatic or mild anaemia
3 deletions or 2 deletion + 1 nondeletional mutation = HbH disease = symptomatic, moderate to sever anaemia
4 deleted = Hb Bart’s hydrops detalis/ a- thalassaemia major = Lethal intrauterine hemolytic anaemia death before or right after birth
List the different zygosity of a-thalassaemia?
Heterozygous = aa/a- or –/aa
Homozygous = aa/aa or a-/a- or –/–
What is the risk of –/– a-thalassaemia if both parents are –/aa ? check
25% –/–
Hb H disease (a-/–) or heterozygous a- thal trait (–/aa)
List the possible combinations of a-thal genes in offsprings made by aa/– and aa/a- parents
aa/aa = 25% = normal
aa/a- = 25% = silent carrier
aa/– = 25% = thalassaemia trait
a-/– = 25% = HbH
Genetic pathogenesis of a- thalassaemia?
unequal DNA crossover during meiosis
Normal = 2 homologous pairs of sister chromatids align side by side > connected at chiasma > 2 homologs pairs exchange DNA
a- thal = High homology of α-globin genes (incl. similar non-expressing pseudogenes)
» misaligned recombination between α1, α2 (unequal meiotic crossover i.e a2 align to a1, a1 align to pseudogene)
» generate de novo mutation and deletion
Spectrum of B- thalassaemia?
B-thal major (Cooley’s anemia), B-thal intermedia, B-thal trait, Silent B-thal
Decreasing severity:
b0 (little or no production of b chain);
b+ (b chain and Hb A are detectable);
b++ (mild defect in b chain production)
Inheritance pattern of B- thal?
Autosomal recessive inheritance (large majority)
The zygosity of B-thal is the same as a-thal. T or F?
False
many different mutations (>100)
> most patients with B- thal major are compound heterozygotes (i.e. having two different mutations), not true homozygotes (i.e. having two same mutations).
What type of mutation is most likely in B-thalassaemia?
single nucleotide mutations
(point mutations, or mutations involving insertion / deletion of a few nucleotides
Presence of concurrent α-thalassemia is an ameliorating genetic factor to which types of B-thal?
Ameliorating genetic factors (reduce severity, selective survival)
All B-thal except B-thal major
Elevated hemoglobin F is an ameliorating factor to which types of B-thal?
All types of B-thal except B-thal trait
Describe the most common genetic pathogenesis for B-thal?
Loss of function mutations of β-globin gene via RNA splicing mistake
SNP at Consensus nucleotide sequences of splice sites=
> > cause skip exons, activate cryptic splice sites, create new splice sites, incorporate new exons and cause frameshift
List the possible combinations of offspring genotype made by 2 parents with B- thalassaemia trait?
Thalassaemia trait = one B-globin gene has mutation: heterozygotes of B0 or B+
BNormal/B0 or BNormal/B+
25% = Bn/Bn = normal
50 % = Bn/B0 or Bn/B+ = thalassaemia trait
25% = B0/B0 or B+/B+ = severe or moderate B-thal
Apart from RNA splicing mistake, list some other mutations that occur in B-thal?
All lead to loss of function mutations of b globin gene (b0 and b+)
Nonsense mutation: premature stop codon, truncated
Missense mutation (including initiation codon mutation)
Frameshift (caused by insertion, deletion)
Cap site (5′ 7- methylguanylate cap (m7Gppp))
Poly (A) site
RNA stability or abundance
Insertion or deletion
Examples of secondary modifiers of B-thal?
Secondary
i) AHSP (α hemoglobin stabilizing protein; a chaperone that binds and stabilizes α globin, thereby reducing α4 precipitation)
ii) HPFH: hereditary persistence of fetal hemoglobin
What is the normal mechanism of stopping non-sense mutation at the mRNA level?
Prematurely terminated mRNAs are degraded by nonsense-mediated decay (NMD)
What is the role of consensus nucleotide sequences in an RNA molecule? How does it relate to B-thal?
Signal beginning and end of most introns to be removed
Mutations in these sites causes number of splicing mistakes»_space; B-thal
Genetic pathology of β-Thalassemia trait?
1 β-globin gene carrying thalassemia mutation – heterozygotes of:
- β0 (little / no production of β chain);
- β+ (β chain, Hb A = detectable: some production)
> > i.e. βNormal/β0 or βNormal/β+
Genetic pathology of β thalassaemia intermedia?
2 ways:
1) 2 β-globin genes carrying thalassemia mutation, at least 1 mild (i.e. B++/B+ or B++/B0)
2) 1 β-globin thalassemia mutation + excess α-globin genes
Genetic pathology of β Thalassemia major?
2 β-globin genes carrying severe thalassemia mutation:
i) β0 or β+ homozygote (β0/β0, β+/β+); or
ii) Compound heterozygote (β0/β+)
Genetic pathology of Hb E thalassaemia?
1 β-globin gene carrying thalassemia mutation (mild / severe) + 1 β-globin gene carrying point mutation encoding Hb E
Compare the severity of anemia in the spectrum of B-thal?
B-thal trait = mild/ no anemia
B- thal intermedia = mild to moderate
B- thal major = severe
Hb E = mild to severe
Compare transfusion dependency between the spectrum of B-thal?
B- thal trait = not dependent
B- thal intermedia = Relative independent
B - thal major = Transfusion dependent**, begin in infancy
HbE = Relative dependence
Compare splenomegaly and bone deformities severity between spectrum of B-thal?
Splenomegaly and bone deformities:
B- thal trait = none
B- thal intermedia = severe ***
B - thal major = depends on efficacy of transfusion therapy
HbE = severe ***
Compare severity of iron overload in spectrum of B-thal?
B- thal trait = none
B- thal intermedia = depends on severity of anemia and transfusion or not
B - thal major = Severe iron overload ***
HbE = depends on severity of anemia and transfusion or not
Compare the severity of disease in spectrum of B- thal?
B- thal trait = asymptomatic
B - thal intermedia = range from asymptomatic to severe
B - thal major = severe, require lifelong support
Hb E = range from asymptomatic to severe
List the ameliorating and exacerbating factors for: B-thal trait?
Ameliorating = concurrent a- thal Exacerbating = excess a-globin genes
List the ameliorating and exacerbating factors for B-thal intermedia?
Ameliorating = concurrent a-thal, elevated HbF Exacerbating = >5 excess a-globin gene
List the ameliorating and exacerbating factors for B-thal major?
Ameliorating = concurrent a- thal, elevated HbF (induced by drugs) Exacerbating = none
List the ameliorating and exacerbating factors for HbE thal.?
Ameliorating = mild b- thal, concurrent a- thal, elevated HbF Exacerbating = severe b- thal mutaton
what are the 3 targets of therapy against thalassaemia?
Primary, secondary and tertiary targets:
1) Target defective genes ( γ genes, α genes, β genes)
2) Target ineffective erythropoiesis/ anaemia
3) Improve iron overload, jaundice and gallstones, oxidative damage
List the steps in gene therapy of B-thalassaemia?
- Collect hematopoietic stem cells (HSCs) from bone marrow and culture
- Genetic manipulation by CRISPR: Introduce lentiviral particles with normal B-globin gene into cells > express
- Chemotherapy to eradicate defective HSCs (conditioning)
- Transplant/ infuse genetically modified HSCs into patient
Role of BCL11A in B- thalassaemia?
transcriptional repressor of fetal γ hemoglobin
inhibition can be potential therapy for adult hemoglobinopathies
List some drugs used to overcome ineffective erythropoiesis in thalassaemia?
Ligand traps: sotatercept, luspatercept: Increase growth differentiation factor 11 (GDF11)
Janus kinase 2 (JAK2) inhibitor: increase proliferation of RBC
Long-acting hepcidin analogues
Erythropoietin
List some drugs used to improve iron overload in thalassaemia?
Iron chelators
Long-acting hepcidin analogues (minihepcidins): restrict iron absorption in iron overload
Antisense oligonucleotides, siRNAs: stimulate endogenous hepcidin production
+ drugs that target hepcidin hormone expression: TMPRSS inhibitor, Minihepcidins
What is the locus for hereditary hemochromatosis?
HFE
What is the hepcidin hormone gene?
Hepcidin antimicrobial peptide (HAMP encodes for hepcidin hormone: master regulator of iron homeostasis)
