Molecular techniques in red cell disorders

Question 1

What is the location of the beta globin gene complex?

Answer 1

-Chromosome 11p15

Question 2

What is the location of the alpha globin gene complex?

Answer 2

-Chromosome 16p13.3

Question 3

-What genes are involved in embryonic Hb production?

Answer 3

Epsilon gene on the beta globin complex and the zeta 2 gene on the alpha globin complex

Question 4

What genes are involved in foetal Hb production?

Answer 4

Ggamma and Agamma genes on the beta globin complex and the alpha 1 and 2 genes on the alpha globin complex

Question 5

What are the normal ranges of HbA, HbA2 and HbF on High performance liquid chromatography (HPLC) in adults?

Answer 5

• HbA: 95.5-96.5%
  
  • HbA2: 2.5-3.5%
  • HbF: <1%

Question 6

-What are the most common kinds of mutations associated with the alpha genes and the beta genes?

Answer 6

• Alpha genes: deletions

- beta genes: point mutations

Question 7

What are the 4 most common alpha thalassaemia mutations?

Answer 7

• -alpha3.7
  - -alpha4.2
  - __SEA
  - __FIL

Question 8

What variant haemoglobin is associated with delta-beta gene fusion?

Answer 8

-Hb Lepore

Question 9

Once a sample has been taken for genetic testing, what methods can be used to analyse the DNA?

Answer 9

• GAP-PCR
  - MLPA (Multiplex ligation-dependent probe amplification)
  - Real time PCR

Question 10

What is thew structural change in Hb that causes HbS?

Answer 10

Glutamic acid is substituted for Valine at amino acid 7

Question 11

What is the structural change in Hb that causes HbC?

Answer 11

Glutamic acid is substituted for Lysine at amino acid 7

Question 12

What is the structural change in Hb that causes HbD?

Answer 12

Glutamic acid is substituted for Glysine at amino acid 122

Question 13

What is the structural change in Hb that causes HbE?

Answer 13

Glutamic acid is substituted for Lysine at amino acid 27

Question 14

What is the characteristic finding of Beta thalassaemia on HPLC?

Answer 14

-Raised HbA2, (and sometimes HbF)

Question 15

What is the characteristic finding of Alpha thalassaemia on HPLC?

Answer 15

• Normal HbA2 and HbF

- HbH peak

Question 16

Outline how a GAP PCR is performed (In the context of alpha thalassaemia)

Answer 16

-Primers are set up for a specific gene: one forward primer, one close reverse primer and one very distant reverse primer
-In a normal gene, with no deletion, the forward primer and distant reverse primer form a product that relates to the size of the specific gene
-A deletion brings the distant primer closer to form a smaller product
-Visualising the product shows the specific size of a deletion when compared to a control/ normal gene.
-The size of the deletion is used to identify the mutation
(-e.g allows comparison of –alpha3.7kb and –alpha4.2kb deletions)

Question 17

Give three examples of different types of mutation that can cause thalassaemia

Answer 17

• Mutations affecting transcriptional elements (e.g A mutation in the promoter regulatory elements)
  
  • Mutations affecting RNA processing (e.g A mutation in the splice junction)
  • Mutations affecting RNA translation (e.g A mutation in the initiation codon)

Question 18

-What molecular techniques are employed to detect point mutations (Such as in Beta thalassaemia)?

Answer 18

• Sanger sequencing
• RFLP (Restriction fragment length polymorphism)
• Allele specific/ARMS (Amplification-refractory mutation system)

Question 19

-What molecular techniques are employed to detect point mutations (Such as in Alpha thalassaemia)?

Answer 19

• GAP-PCR

- MPLA (Multiplex ligation-dependent probe amplification)

Question 20

What molecular technique is employed in analysing unknown mutations?

Answer 20

Next generation sequencing (NGS)