Fragile X

Question 1

In the context of prenatal diagnosis, what is important to remember about female fetus with full mutation?

Answer 1

The phenotype varies and is impossible to predict

Question 2

Referral reasons for FRAX testing?

Answer 2

Children with developmental delay, speech and language difficulties, autism spectrum disorders
Neurology- ataxia, intention tremor
Gynae- infertility
Genetics- family history

Question 3

Why is methylation status used in FRAX testing?

Answer 3

To distinguish between full and pre-mutation; full mutation is hypermethylated

Question 4

What tests can be used to determine methylation status?

Answer 4

Methylation-sensitive Southern blot

PCR (MS-MLPA)

Question 5

How does Methylation sensitive Southern blot work?

Answer 5

DNA cut with two restriction enzymes, one of which is methylation-sensitive (only cuts when methylation NOT present). When both enzymes cut and probe is hybridized this produces two clear bands (in a female) or one clear band (in a male). When methylation is present this produces a smear of DNA

Question 6

Potential problems with methylation-sensitive Southern blot

Answer 6

X chromosome aneuploidy
Skewed X inactivation
Incomplete digestion
Insufficient DNA loading
Poor probe hybridization