Molecular basis of hemoglobin disorders

Question 1

How many O2 molecules can be carried by one Hb molecule?

Answer 1

4

Question 2

What is a globin gene?

Answer 2

Globin gene are families of proteins that are clustered with structural and functional similarities

Question 3

What are the different types of globins?

Answer 3

1) Embryonic hemoglobin (Hb):
- Hb Gower-1 “z2e2”
- Hb Gower-2 “a2e2”

2) Fetal hemoglobin (HbF) “a2y2”

3) Minor hemoglobin (HbA2) “a2d2”

4) Adult hemoglobin (HbA) “a2b2”

Question 4

What are the globin proteins that are found on chromosome 16?

Answer 4

1) 2 Alpha globin genes

2) Zeta globin

Question 5

What are the globin genes that are found on chromosome 11?

Answer 5

1) Epsilon

2) 2 Gamma

3) Delta

4) Beta

Question 6

What is meant by the alpha-globin gene family and the beta-globin gene family?

Answer 6

• The families are similar in structure and function (a & z, and epsilon, gamma, delta, and beta are similar)

Question 7

What is meant by globin switching?

Answer 7

It is the change in the expression of various globin genes during development

• epsilon globin switches to G-and A- globins
• G-globins then switch to B-globin and some a-globin switched to delta

Question 8

What are the different globin genes expressed during development?

Answer 8

1) 5-6 weeks Gower-1, z2e2,

2) after the 6th week alpha globin is expressed and increases throughout life

3) At the 7th week gamma-globin gene is up and beta is still low which is exactly opposite to what happens after birth

4) Delta globin is expressed in low quantities after the 6th week postnatally

Question 9

What are the different organs that produce RBC throughout life?

Answer 9

1) Yolk sac: till the 6th week

2) Liver: from week 6 till the 6th week postnatally

3) Spleen: from week 14 till week 6 postnetally

4) Bone marrow: from week 15 till the end of life

Question 10

What is the normal variation in the expression of globin genes?

Answer 10

1) HbF (a2y2) 75% in newborns and <1-5% in adults

2) 2) HbA2 (a2d2) <1% in newborns and <3% in adults

3) HbA (a2b2) 25% in newborns and >97% in adults

Question 11

What are the components involved in B-globin gene family expression?

Answer 11

• They are partially controlled by the promotor & mostly controlled by the locus control region (LCR)
• It is not a part of a promoter region
• First identified in εγδβ-thalassemia patients, who were found to have no expression of β-globin cluster genes (εγδβ), even though the genes themselves (including their promoter) were intact, Patients identified to carry deletion of LCR region

Question 12

How does the LCR (locus control region) code for the beta gene cluster?

Answer 12

1) LCRs are found in a highly condensed chromatin region

2) It has 5 DNase (enzymes that break DNA) sites

3) The DNase sites will help in configuring/opening the chromatin site making it accessible to transcription factors (TFs)

4) LCR with TF (format an active chromatin hub) helps in the expression of globin genes

Question 13

What is meant by hemoglobinopathy?

Answer 13

It is the loss of hemoglobin that occurs due to a mutation to the alpha or beta globin gene

Question 14

What are the different types of hemoglobinopathy?

Answer 14

1) qualitative defects (structural)

2) Quantitative defects (synthesis)

3) Defective globin switching

Question 15

What is meant by a qualitative defect?

Answer 15

It is a structural “conformational” defect of the Hb without affecting the quantity, (there are more than >400 variants) including HbS

Question 16

What is meant by a quantitative defect?

Answer 16

It is a defect in the synthesis of one or more of the globin chains like in a- & b-thalassemia

Question 17

What is meant by a defective globin switching?

Answer 17

The problem is when switching between the globin genes, where a mutation in the gamma gene or in the promoter region will impair the switch between the game globin to the beta-globin resulting in (hereditary persistence of fetal hemoglobin)

Question 18

What is a mutation?

Answer 18

• It is any pathological change in the DNA sequence
• They exist in two forms heterozygous (only one allele is affected), and homozygous (both alleles are affected in the same nucleotide)

Question 19

What are the different types of mutations?

Answer 19

1) Based on the molecular event in the DNA

• Single nucleotide substitution mutation (point mutation)
• Frameshift mutation (nucleotide deletion or insertion)

2) Based on the effect on the protein

• Missense mutations (Amino acid substitution)
• Nonsense mutations (Generation of STOP codon)
• Splice site mutations (Aberrant mRNA splicing)
• Silent mutations (Generally no effect)

Question 20

What are the different forms of heterozygote mutations?

Answer 20

1) Cis compound heterozygous: two different heterozygous mutations on the same allele

2) Trans compound heterozygous: two different heterozygous mutations on two different alleles (each allele is affected by one mutation)

3) Double heterozygous: 2 gene mutations found in two different genes (problem in thalassemia)

Question 21

What are the abnormal hemoglobin variants?

Answer 21

1) Hb-S, glutamate to valine, under deoxygenation the RBC might sickle and occlude the vascular supply

2)Hb-C, switching of glutamate to lysine, the RBC will crystalize

these are mild sickle cell disease

Question 22

What is sickle cell anemia?

Answer 22

A condition caused by a missense mutation (Glu6Val) in the beta-globin gene, which forms sickle hemoglobin (HbS), aggregating the hemoglobin under low oxygen levels

• In heterozygous sickle cell anemia people are asymptomatic, protecting those individuals from malaria
• The normal codon GAG will be mutated to the sickle cell codon GTG which will lead to the polymerization of RBCs under deoxygenated conditions

Question 23

What are the clinical presentations of sickle cell anemia?

Answer 23

1) Fatigue, dizziness, and headaches (all due to oxygen insufficiency which is due to abnormal hemoglobin)

• Dut to the fact that Sickled RBCs cannot move through the capillaries as smoothly as RBCs

2) Chronic pain especially in joints

3) Reduced immune response to infection

• Especially in homozygous conditions:

4) Myocardial infarction

5) Strokes

Question 24

What is the cause of thalassemia?

Answer 24

It is caused by the decreased or absence of globin chain synthesis, where there is a relative/total absence of a or b-globin chains in the RBCs causing an imbalance between the a- and b- chains

Question 25

What are the mutation patterns that will affect people with thalassemia?

Answer 25

Homozygous or compound heterozygous

Question 26

What are the types of thalassemia?

Answer 26

1) a-thalassemia 2) b-thalassemia

Question 27

Describe a-thalassemia

Answer 27

It is the absence of the alpha globin in the hemoglobin - It will affect both the synthesis of fetal and adult hemoglobin - The absence of the a-chains will increase the amount of y-globin during fetal life and beta-globin during adult life - A defect in the alpha globin will cause both intrauterine and postnatal diseases, but the severity depends on the number of genes affected

Question 28

What is the cause of a-thalassemia?

Answer 28

1) The most common cause is a a-gene deletion which is likely due to the homologous recombination during crossing over 2) Deletion in the locus control region (LCR) 3) Chain termination (nonsense) mutation

Question 29

What is the mechanism of a-thalassemia?

Answer 29

The chromosomes are not aligned on top of each other properly so alpha-1 will end up crossing over with alpha-2 The result of this miscommunication is that there will be a defective gene produced (in red circles) 2 alleles will be produced after crossing over: In possible defective allele 1: there’s no intact alpha-globin gene (deletion) In possible defective allele 2: there are 2 intact alpha globin genes (in green circles)

Question 30

What are thedifferent types of a-thalassemia?

Answer 30

1) Silent carries (one alpha gene deletion) 2) a-Thal trait (deletion of two alpha genes, mild hypochromic microcytic anemia) 3) Hb-H disease (deletion of three alpha genes, mild-moderate anemia, hepato-splenomegaly, and yellowing of the eyes and skin) 4) Barts Hb/Hydrops fetalis (deletion of all a gene, which will result in not compatible with life)

Question 31

What is b-thalassemia and what are its characteristics?

Answer 31

- Autosomal recessive disorders that are caused by several types of mutations in the B-globin gene - Splice site mutation that causes a Reduced/absent b-globin chain synthesis - Its severity depends on the location of the mutation (b0 = no b-globin synthesis as the mutations are on the exons, while b+ - reduced b-globin synthesis as the mutations are on the introns and regulatory region) - b-globin deficiency will result in an over-production of globin chains which will precipitate in the cells and cause splenic destruction of RBCs, increasing erythropoiesis

Question 32

What are the characteristics & clinical features of B-thalassemia?

Answer 32

- characterized by: 1) Hypochromic (low Hb conc) & microcytic anemia 2) Increased a-hemoglobin chain synthesis 3) a-globin precipitation 4) poor growth, and skeletal abnormalities 5) Liver and heart failure 1) Prominent cheekbones 2) Extension of the upper jaw (due to the expansion of the marrow cavity in the bones of the skull and face, as the bodies try to compensate for the lysed RBCs) 3) Poor oxygen-carrying capacity of RBCs 4) Increased a-globin production and precipitation 5) Increased splenic destruction of nonfunctional RBCs 6) Anemia, jaundice, splenomegaly

Question 33

What are the types of b-thalassemia?

Answer 33

1) b-thalassemia minor (b0/b or b+/b) - b-thalassemia trait - Minor point mutation - Minimal anemia and it doesn't require a treatment 2) b-thalassemia intermediate (b0/b+ or b+/b+) - b-chain is produced in lower quantities than the minor one, but not absent - the patient won't be requiring a chronic blood transfusion 3) b-thalassemia major (cooley's anemia b0/b0) - Absence of b-globin chain production - It is mainly caused by splice site, frameshift, and nonsense mutations - The patient will require regular blood transfusions

Question 34

Describe the b-globin gene mutation

Answer 34

-The mutations are found all over the gene and they might impact: 1) Defective mRNA synthesis 2) Non-functional mRNA 3) Coding region mutations that alter the splicing - Splice site mutations are the most common cause of beta-thalassemia (3 types, splice junction mutation, intron mutation, and coding (exon) sequence mutation), normally the splice sites are found on the boundaries of the intronic and exonic regions but in b-thalassemia patients the splice sites in found within them

Question 35

What are the complex thalassemias?

Answer 35

1) Due to large deletions - β-globin gene plus one or more other genes in cluster - LCR region from the β-globin cluster 2) Hereditary persistence of fetal hemoglobin - Abnormal switch from y-globin to b-globin synthesis - Some patients can live normally while others cannot due to the difference un the location of the mutation in the beta cluster