Biochemistry 1: Thalassaemia

Question 1

Clinical diagnosis of thalassaemia

Answer 1

3-tier approach:

1. CBC, Red cell indices
2. Peripheral blood film
3. Haemoglobin electrophoresis / chromatography —> Quantitation of HbF, HbA2
4. Detection of HbH inclusions by Supravital stains
5. Genotyping
6. Fe studies

Question 2

DNA mutation analysis

Answer 2

Most effective way to detect influence on overall phenotype due to Primary gene mutations / gene-gene interactions
- but cannot serve as shortcut / primary test in detecting thalassaemia

PCR-based of detection:

• in-vitro amplification of specific DNA sequences (globin genes) from starting material (genomic DNA)
• can identify all molecular defects causing thalassaemias except a few rare deletions, rearrangements

Most commonly used PCR-based methods

1. Gap-PCR —> detect ***Deletions
2. Amplification Refractory Mutation System (ARMS) —> detect ***Mutations involving single-base changes / small deletions

Materials:

1. A pair of specific synthetic oligonucleotides (primers)
   - flank DNA region of interest
2. Deoxynucleotides (dNTPs)
3. DNA polymerase

How to visualise:

1. 1.5% agarose gel for separation
2. DNA markers of known sizes as reference
3. Stain with Ethidium bromide
4. Visualise under UV illumination

Question 3

α-Thalassaemia

Answer 3

Gene deletion types:
SEA type (90%): 2 α gene deletion
α3.7 type: single α gene deletion
α4.2 type: single α gene deletion

Question 4

Detection of α-thalassaemia

Answer 4

2. ***Gap-PCR:
   - detect large part of DNA deletion
   —> by primers that flank region of DNA deletion (normally too long for DNA amplification)
   —> 2 primers are brought into close proximity due to deletion
   —> can amplify DNA product

• Southeast Asia type (SEA) α-thalassaemia:
  —> gene deletion of ~20.5kb in α-globin gene cluster
  —> design 3 primers bridging the deletion breakpoint:
  —> 2 amplify **fragment specific for the deletion (primers 1 and 3) —> if deletion occur —> **can detect fragments by 1 and 3
  —> others (primers 1 and 2) amplify **normal specific sequence —> if deletion occur —> **cannot detect fragments 1 and 2
• Electrophorese, visualise amplified products under UV illumination after ethidium bromide staining

Historical interest only
2. Restriction fragment length polymorphism (RFLP) analysis
- α-thalassaemia arises most commonly from gene deletions
—> create / eliminate restriction enzyme sites

• digest with restriction enzymes
  —> generate DNA fragments with sizes different from wild type allele
  —> separated by gel electrophoresis
  —> immobilised onto a solid support (e.g. Nylon membrane)
  —> hybridise to a radiolabelled / chemically labelled probe
  —> diagnose different α-globin genotypes (type of deletion) from fragment length

Question 5

β-Thalassaemia

Answer 5

Point mutation:

1. Codons 41-42 (-CTTT) βo (46%)
2. IVS2-654 (C —> T) βo (28%)
3. nt -28 (A —> G) β+ (13%)
4. Codon 17 (A —> T) βo (6%)
5. Codon 71/72 (insA; +1 frameshift)

Question 6

Detection of β-thalassaemia

Answer 6

1. ***Amplification Refractory Mutation System (ARMS)
   - DNA amplified by allele specific primers
   - mismatch at 3’ end dramatically reduce annealing, amplification —> can identify point mutations / polymorphism
   - also able to identify whether change in DNA is heterozygous / homozygous
   - differentiated by using ARMS primers for mutant and normal alleles
   - 完全無amplification —> homozygous
   - 有少少 —> heterozygous
   - Sanger sequencing —> also confirm Homozygous / Heterozygous

For reference only:
2. Allele-specific oligonucleotide (ASO) hybridisation:
- β-thalassaemia arises usually from ***single nucleotide mutations
- Amplify β-globin gene from patient’s genomic DNA
—> dot onto a nylon membrane
—> hybridise with designed probes to detect presence of mutant and normal allele:
—> Oligonucleotide probes target known mutations in a gene (e.g. point mutation on β-globin gene): cannot hybridise to normal allele
—> another probe correspond to normal counterpart of the gene: cannot bind to mutated allele

Question 7

Multiplex ligation-dependent probe amplification (MLPA)

Answer 7

ONLY method to detect **unknown **large ***deletions

Probes are designed along DNA segment of interest

• MLPA probemix target a specific segment of sequence
• contain ***left + right probe oligonucletide (LPO + RPO)
• LPO / RPO contain:
  1. **PCR primer
  2. **DNA hybridisation sequences

When probe sequence match with genomic sequence of a segment of gene for interest
—> probe will hybridise at adjacent sequence
—> **ligation of probe by ligase when there is no mismatch
—> ligated probe act as primer of subsequent PCR reaction and gene products then amplified using ONLY 1 primer pair
—> **probes but not DNA of interest are amplified

Question 8

In order for PCR reaction to be valid

Answer 8

***Internal control must be present on electrophoresis gel

Question 9

***Summary

Answer 9

Detect known large deletion: Gap-PCR
Detect known point mutation: ARMS (Amplification Refractory Mutation System)

Detect unknown large deletion: MLPA (Multiplex Ligation-dependent Probe Amplification)
Detect unknown point mutation: Sanger sequencing