• Detect fragile X syndrome (FXS) carriers

• Determine an individual’s risk of having a child with FXS

• Diagnose FXS postnatally

FXS is the most common inherited cause of developmental delay and intellectual disability, occurring in approximately 1 in 4000 males and 1 in 6000 to 8000 females.¹ The prevalence of carriers in the Caucasian population is an estimated 1 per 259 females and 1 per 813 males.^2,3

Affected males usually have moderate to severe intellectual disability, pervasive speech delay, and behavioral problems (eg, attention deficit hyperactivity disorder [ADHD]). Autism-spectrum disorders are frequently diagnosed in the 2^nd or 3^rd year of life.⁴ Affected females have a variable phenotype that can range from normal intelligence to severe intellectual disability, with or without learning disabilities or personality disorders.

In more than 99% of cases, FXS is caused by an expansion of a polymorphic CGG trinucleotide repeat in the 5´ untranslated region of the FMR1 gene, located on the X chromosome, resulting in hypermethylation of the FMR1 promoter.⁵ The extent of expansion and hypermethylation correlates negatively with the amount of a protein (absent in affected males and reduced in affected females) that plays a role in brain synaptic development. The severity of the phenotype is related to the extent of expansion (Table 1). Other rare mutations of FMR1 associated with FXS include large deletions, point mutations, and missense mutations.

FMR1-related disorders are inherited in an X-linked dominant manner with variable penetrance, and inheritance is affected by the number of CGG repeats present (Table 2).⁷ Individuals with CGG repeats in the intermediate and premutation range are carriers.

The molecular diagnosis of FXS is based on detecting the number of CGG repeats and methylation status of the FMR1 gene. Polymerase chain reaction (PCR) can detect and accurately measure repeat numbers in the normal and small premutation ranges; Southern blot is required to quantify larger CGG repeats. Southern blot, however, is a time-consuming, laborious process, which has limited the potential of carrier screening. Therefore, a new method called triplet-primed PCR has been developed.⁸ A unique amplicon containing stutter peaks is produced when the individual is at least a fragile X carrier. In these cases, a Southern blot will be performed to establish the exact size and methylation status of the expanded allele. The absence of stutter peaks indicates absence of an expanded allele.

• Individuals with a family history of FXS or undiagnosed intellectual disability, including those seeking reproductive counseling

• Symptomatic children and adults

• PCR and capillary electrophoresis to determine gender and number of CGG repeats

• Triplet-primed PCR and capillary electrophoresis to detect stutter peaks

• Southern blot confirmation of FMR1 expansions performed as reflex if PCR indicates an expanded allele

• Results reported: number of CGG repeats and methylation status of any expanded alleles

A negative result indicates a normal gene. When >44 CGG repeats are identified, an individual’s mutation status and phenotype are determined by the number of repeats present (see Table 1). The associated risk of having a child with FXS is explained in Table 2.

This assay does not detect other mutations (eg, deletions, point mutations, missense mutations) that disrupt the function of the FMR1 gene and/or protein. Results should be interpreted in conjunction with other laboratory and clinical findings. Additional assistance in interpretation of results is available from our Genetic Counselors by calling 1-866-GENE-INFO (1-866-436-3463).