Fragile X Syndrome, PCR With Reflex to Southern Blot

Carrier screening for individuals who are pregnant or considering pregnancy and who have no family or personal history of fragile X syndrome, fragile X-related disorders (primary ovarian insufficiency, or late-onset ataxia) or unexplained intellectual disabilities, developmental delay, or autism.

Polymerase chain reaction (PCR) followed by capillary electrophoresis with reflex to Southern blot analysis for all positive samples.

Fragile X syndrome: DNA is amplified by the polymerase chain reaction (PCR) to determine the size of the CGG repeat region within the FMR1 gene. PCR products are generated using a fluorescence labeled primer and sized by capillary gel electrophoresis. If indicated, Southern blot analysis is performed by hybridizing the probe StB12.3 to EcoRI- and EagI- digested DNA. The analytical sensitivity of both Southern blot and PCR analyses is 99% for expansion mutations in the FMR1 gene. Reported CGG repeat sizes may vary as follows: +/- one for repeats less than 60, and +/- two to four for repeats in the 60 - 120 range. For repeats greater than 120, the accuracy is +/- 10%. If 55-90 trinucleotide repeats are detected in carrier screening females, a PCR assay targeting AGG sequences within the CGG repeats is performed to assess the number and position of AGG interruptions.

Fragile X syndrome is the leading cause of inherited intellectual disability, affecting approximately 1 in 4,000 males and 1 in 8,000 females. It is found in all ethnic groups, and can occur in families with no history of intellectual disability. Women may decide to have testing to find out if they are carriers, and therefore are at risk of having a child with fragile X syndrome. The carrier frequency in women is approximately 1 in 260. Fragile X test is commonly used for individuals at higher risk, including those that may be symptomatic or have a family history suggestive of fragile X syndrome.