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What is being tested?

Fragile X CGG repeat sizing is routinely performed using a polymerase chain reaction (PCR) method with fluorescently labelled primers. The labelled pieces of DNA are then separated by size using capillary gel electrophoresis to determine the number of CGG repeats in the controls, standards and patients. The PCR technique cannot detect larger pre-mutations (great than 180 repeats) or full mutations so the Southern Blot analysis has been used for repeat length analysis in these situations.

In the Southern Blot analysis technique, genomic DNA is digested (i.e. cut up) into small pieces using two enzymes called restriction endonucleases, the resulting DNA fragments are then separated by size using agarose gel electrophoresis and then transferred onto a nylon membrane. A DNA labelled probe hybridises with the DNA on the membrane and its presence is detected by a sensitive technique called chemiluminesces. The difference in size, methylation and DNA copy number can be determined using this method.

How is it used?

Testing is performed when a person has intellectual disability, ataxia ( i.e. when you have trouble moving parts of your body the way you want, and your arms and legs may move when you don't want them to), neurodegeneration ( the progressive loss of neurones from the brain), or premature ovarian failure and infertility.

When is it requested?

Testing is requested by the doctor when it is suspected that the clinical signs are due to Fragile-X.

What does the result mean?

If the number of CGG repeats is in the range of 5 to 44 CGG, this is considered a normal result. If the repeats are in the size range of 55 to 200 CGG repeats this is a pre-mutation result and if the repeat size is greater than 200 CGG repeats, this is a full mutation and the person has Fragile-X.

Is there anything else I should know?

Although current guidelines still make reference to Southern blot analysis, the test is subjective, compared with more objective newer methods. Southern blotting is technically complex, expensive, laborious to perform and requires a large quantity of DNA for testing. Newer methods are therefore replacing Southern blotting, for example, a triplet repeat primed PCR assay followed by high-thoughput automated capillary electrophoresis to detect the number of CGG repeats is now widely used. As a result there has been a decline in the proportion of patients in whom Southern blot analyses is being performed.

Common questions

  • How common is Fragile- X syndrome?

Fragile -x syndrome is the most common known cause of inherited intellectual and development disability affecting about 1 in 4000 males and between 1 in 5000 and 1 in 8000 females.

  • If someone in the family has Fragile-X should other family members be tested?

People who have a relative with FXS or another Fragile X-associated Disorder have a higher risk of being a carrier of FX than most people. The cost of FX carrier screening by DNA testing is covered by Medicare for people with a family history of Fx-associated Disorders. 

Last Updated: Thursday, 1st June 2023

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