L08 – Molecular Genetics of Thalassemia

Question 1

Heterozygotes for thalassemia is protected against what disease?

Answer 1

protected from severe effects of malaria

Question 2

Describe the globin genes for Hb synthesis. Which chromosome, what related genes/ transcription sites?

Answer 2

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

Question 3

Give the globin composition in normal fetal and normal adult Hb?

Answer 3

fetal= Hb F (α2γ2)

adult = Hb A (α2β2)

Question 4

Describe the changes in expression of globin genes from fetus to birth?

Answer 4

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

Question 5

Describe change in site of erythropoiesis from fetus to birth?

Answer 5

yolk sac > liver > spleen > bone marrow

Question 6

Explain why α- thalassemia causes inclusion bodies, but β- thal cause precipitation?

Answer 6

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

Question 7

Compare α and β thalassaemia in the location of hemolysis?

Answer 7

α- thal = peripheral haemolysis

β - thal = Destruction of RBCs in marrow, spleen&raquo_space; Ineffective erythropoiesis

Question 8

Genotype and status spectrum of a- thalassaemia?

Answer 8

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

Question 9

Genotype and phenotype spectrum of a- thalassaemia?

Answer 9

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

Question 10

List the different zygosity of a-thalassaemia?

Answer 10

Heterozygous = aa/a- or –/aa

Homozygous = aa/aa or a-/a- or –/–

Question 11

What is the risk of –/– a-thalassaemia if both parents are –/aa ? check

Answer 11

25% –/–

Hb H disease (a-/–) or heterozygous a- thal trait (–/aa)

Question 12

List the possible combinations of a-thal genes in offsprings made by aa/– and aa/a- parents

Answer 12

aa/aa = 25% = normal

aa/a- = 25% = silent carrier

aa/– = 25% = thalassaemia trait

a-/– = 25% = HbH

Question 13

Genetic pathogenesis of a- thalassaemia?

Answer 13

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

Question 14

Spectrum of B- thalassaemia?

Answer 14

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)

Question 15

Inheritance pattern of B- thal?

Answer 15

Autosomal recessive inheritance (large majority)

Question 16

The zygosity of B-thal is the same as a-thal. T or F?

Answer 16

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).

Question 17

What type of mutation is most likely in B-thalassaemia?

Answer 17

single nucleotide mutations

(point mutations, or mutations involving insertion / deletion of a few nucleotides

Question 18

Presence of concurrent α-thalassemia is an ameliorating genetic factor to which types of B-thal?

Answer 18

Ameliorating genetic factors (reduce severity, selective survival)

All B-thal except B-thal major

Question 19

Elevated hemoglobin F is an ameliorating factor to which types of B-thal?

Answer 19

All types of B-thal except B-thal trait

Question 20

Describe the most common genetic pathogenesis for B-thal?

Answer 20

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

Question 21

List the possible combinations of offspring genotype made by 2 parents with B- thalassaemia trait?

Answer 21

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

Question 22

Apart from RNA splicing mistake, list some other mutations that occur in B-thal?

Answer 22

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

Question 23

Examples of secondary modifiers of B-thal?

Answer 23

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

Question 24

What is the normal mechanism of stopping non-sense mutation at the mRNA level?

Answer 24

Prematurely terminated mRNAs are degraded by nonsense-mediated decay (NMD)

Question 25

What is the role of consensus nucleotide sequences in an RNA molecule? How does it relate to B-thal?

Answer 25

Signal beginning and end of most introns to be removed Mutations in these sites causes number of splicing mistakes >> B-thal

Question 26

Genetic pathology of β-Thalassemia trait?

Answer 26

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+

Question 27

Genetic pathology of β thalassaemia intermedia?

Answer 27

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

Question 28

Genetic pathology of β Thalassemia major?

Answer 28

2 β-globin genes carrying severe thalassemia mutation: i) β0 or β+ homozygote (β0/β0, β+/β+); or ii) Compound heterozygote (β0/β+)

Question 29

Genetic pathology of Hb E thalassaemia?

Answer 29

1 β-globin gene carrying thalassemia mutation (mild / severe) + 1 β-globin gene carrying point mutation encoding Hb E

Question 30

Compare the severity of anemia in the spectrum of B-thal?

Answer 30

B-thal trait = mild/ no anemia B- thal intermedia = mild to moderate B- thal major = severe Hb E = mild to severe

Question 31

Compare transfusion dependency between the spectrum of B-thal?

Answer 31

B- thal trait = not dependent B- thal intermedia = Relative independent B - thal major = Transfusion dependent**, begin in infancy HbE = Relative dependence

Question 32

Compare splenomegaly and bone deformities severity between spectrum of B-thal?

Answer 32

Splenomegaly and bone deformities: B- thal trait = none B- thal intermedia = severe *** B - thal major = depends on efficacy of transfusion therapy HbE = severe ***

Question 33

Compare severity of iron overload in spectrum of B-thal?

Answer 33

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

Question 34

Compare the severity of disease in spectrum of B- thal?

Answer 34

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

Question 35

List the ameliorating and exacerbating factors for: B-thal trait?

Answer 35

``` Ameliorating = concurrent a- thal Exacerbating = excess a-globin genes ```

Question 36

List the ameliorating and exacerbating factors for B-thal intermedia?

Answer 36

``` Ameliorating = concurrent a-thal, elevated HbF Exacerbating = >5 excess a-globin gene ```

Question 37

List the ameliorating and exacerbating factors for B-thal major?

Answer 37

``` Ameliorating = concurrent a- thal, elevated HbF (induced by drugs) Exacerbating = none ```

Question 38

List the ameliorating and exacerbating factors for HbE thal.?

Answer 38

``` Ameliorating = mild b- thal, concurrent a- thal, elevated HbF Exacerbating = severe b- thal mutaton ```

Question 39

what are the 3 targets of therapy against thalassaemia?

Answer 39

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

Question 40

List the steps in gene therapy of B-thalassaemia?

Answer 40

1. Collect hematopoietic stem cells (HSCs) from bone marrow and culture 2. Genetic manipulation by CRISPR: Introduce lentiviral particles with normal B-globin gene into cells > express 3. Chemotherapy to eradicate defective HSCs (conditioning) 4. Transplant/ infuse genetically modified HSCs into patient

Question 41

Role of BCL11A in B- thalassaemia?

Answer 41

transcriptional repressor of fetal γ hemoglobin | inhibition can be potential therapy for adult hemoglobinopathies

Question 42

List some drugs used to overcome ineffective erythropoiesis in thalassaemia?

Answer 42

 Ligand traps: sotatercept, luspatercept: Increase growth differentiation factor 11 (GDF11)  Janus kinase 2 (JAK2) inhibitor: increase proliferation of RBC  Long-acting hepcidin analogues  Erythropoietin

Question 43

List some drugs used to improve iron overload in thalassaemia?

Answer 43

 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

Question 44

What is the locus for hereditary hemochromatosis?

Answer 44

HFE

Question 45

What is the hepcidin hormone gene?

Answer 45

Hepcidin antimicrobial peptide (HAMP encodes for hepcidin hormone: master regulator of iron homeostasis)