Exam

Question 1

Criteria for selecting disorders for newborn screening

Answer 1

1. The condition should be a serious health problem that leads to significant morbidity or mortality
2. Should be a benefit to conducting the screening in the newborn period
3. Should be a suitable test protocol to identify presence of condition
4. Test protocol should be ethical
5. Health care services for diagnosis and management should be available
6. Should be an accepted intervention

Question 2

Sanger technique / Dideoxy sequencing

Answer 2

• Uses dideoxynucleotides
• These are molecules that resemble normal nucleotides but lack the normal -OH group

Question 3

Dideoxy DNA sequencing steps 1-5

Answer 3

1. DNA template is denatured to single strands.
2. Single DNA primer (3’ end near sequence of interest) is annealed to template DNA and extended with DNA polymerase.
3. Four reactions are set up
4. A different labeled dideoxynucleotide is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs
5. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond.

Question 4

Dideoxy DNA sequencing steps 7-10

Answer 4

7. Whenever the labeled ddNTPs are incorporated in the chain, DNA synthesis terminates
8. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions.
9. Extension products in each of the four reaction mixtures also end with a different labeled ddNTP (depending on the base).
10. Gel electrophoresis is then conducted

Question 5

What do the four reactions of dideoxy DNA sequencing contain

Answer 5

1. DNA template
2. Primer annealed to template DNA
3. DNA polymerase
4. dNTPS

Question 6

Other name for dideoxy DNA sequencing

Answer 6

Dye terminator sequencing

Question 7

Breast cancer and T-DM1

Answer 7

• Some breast cancers make too many HER2 receptors, which helps the cancer develop and spread
• T-DM1 (antibody-drug conjugate) used to treat these cancers and works by attaching to HER2 receptors on cancerous cells and killing them
• Tumour tissue is tested to determine if T-DM1 is the right treatment
• If there is high number of HER2, T-DM1 can be used
• IF tumour is HER2 negative, T-DM1 will not work

Question 8

Statins and muscle problems

Answer 8

• Statins act in liver to lower cholesterol
• To work must first be taken into the liver cells.
• Statins are transported by a protein made by the SLCO1B1 gene.
• A DNA variant causes reduced simvastatin to be absorbed by cells.
• When taken at high doses, simvastatin can build up causing muscle weakness and pain.
• Genetic testing the SLCO1B1 gene can be done to determine if simvastatin is the best statin and what dose would work best.

Question 9

Depression and amitriptyline

Answer 9

• The breakdown of the antidepressant drug amitriptyline is influenced by two genes CYP2D6 and CYP2C19.
• Genetic testing for these genes can help decide what dose of the drug is needed.
• Fast metabolisers will need a higher dose, or a different drug.
• Slow metabolizers will need a smaller dose or a different drug to avoid a bad
  reaction.

Question 10

Steps of MLPA

Answer 10

1. Sample denaturation and probe hybridisation
2. Probe ligation
3. Probe amplification
4. Fragment separation by capillary electrophoresis
5. Data analysis

Question 11

Analysis by MALDI TOFF

Answer 11

1. Sample mixed with matrix, dried on plate
2. Plate placed in high vacuum of MS
3. Sample irradiated with laser, energy absorbed volatises
   sample/matrix, ionised matrix protonates oligonucleotides
4. Ions strike detector, time of flight determines mass

Question 12

ACTA1 gene

Answer 12

• ACTA1 mutations can result in different muscle pathologies
• ACTA1 mutations are responsible for 50% of the severe cases of nemaline myopathy
• Many ACTA1 patients are born almost completely paralysed and die within the first year of life
• Majority of patients have dominant de novo mutations