Hot Topics

Question 1

Aminoglycosides ototoxicity

Answer 1

• m.1555A>G MT- RNR1
• Hearing loss occurs days to weeks after treatment with antybiotics eg gentamicin (even single dose)

Question 2

Aminoglycosides ototoxicity - testing

Answer 2

Genedrive® MT-RNR1 ID Kit
- rapid bed side testing - portable, quick (26 mins), buccal swab
- Loop-mediated isothermal amplification (LAMP) + melt-curve analysis
- Detects m.1555A and m.1555G
- introduced in Manchester in 2022, extended to Greater Manchester in March 2023

Question 3

Where did the CRIPSR/Cas9 system originate?

Answer 3

It is a prokaryotic immune defence response against invading bacteriaphage

Question 4

How does the CRIPSR/Cas9 system work?

Answer 4

The CRISPR system is modified for genome editing in humans.
Cas9 is bound to a RNA targeting sequence, used to direct the nuclease activity of the Cas9 to the site of interest.
Cas9 and RNA are incorporated into a plasmid vector and delivered to the site of action.
Cas9 nicks the DNA (depending on the Cas9 used, this may be dsDNA or ssDNA nick) ready for a gene to be removed or added.
Subsequent repair of the site is carried out by NHEJ or HRR.
dead Caspases, without nuclease activity, can also be used to target sequence or epigenetic modification; promoters can be targeted to turn genes on/off.

Question 5

Why is it controversial?

Answer 5

Any changes may be introduced into the germline and become heritable.
There is a current moratorium preventing the use of this system for germline editing.

Question 6

Give examples of how this system is showing promise for treatment of rare disease.

Answer 6

In cell lines it has been demonstrated to turn methylated genes on. This may be useful for FrAX treatment or Angelman syndrome to increase expression of UBE3A from the paternal allele.
It has been shown to correct a specific DMD mutation and restore function
It has also been shown to raise FXN levels in Friedriech’s Ataxia patients.

Question 7

What is the main issue with CRISPR?

Answer 7

Delivery of gene editing components to target sites is still difficult, and studies have demonstrated off-target effects (Cas9 nuclease enzyme’s low specificity).

Question 8

DPYD deficiency - why important

Answer 8

fluoropyrimidine toxicity related to chemotherapy with fluoropyrimidines
e.g. 5-fluorouracil, capecitabine

Question 9

DPYD testing strategies

Answer 9

1. ARMS followed by capillary electrophoresis - fast, cheap, targeted testing
2. Sanger seq - slower, more expensive, risk of VUSs
3. NGS panels - expensive, not suitable for quick TAT
4. LAMP (Loop-Mediated Isothermal Amplification ) + melt curve analysis; commercial kit restricted to 4 common variants

Question 10

DPYD Analysis

Answer 10

Common variant assign DPD activity scores; 0- no activity, 0.5 - decreased, 1 - normal
Final result= sum of scores, chemo treatment or dosage adopted, if poorly tolerated

Question 11

PKU - gene, location, symptopms

Answer 11

PAH
12q23.2

intellectual disability, microcephaly, motor deficits, autism, seizures, developmental problems, neuropsychological symptoms, and skin problems (eczema)
related to increased level of Phe in blood > irreversible damage during brain development

Question 12

PKU Treatment

Answer 12

Sapropterin dihydrochloride - synthetic BH4 - excess activates residual PAH enzyme improving Phe metabolisim – reduces blood Phe concentration and increasing dietary Phe tolerance