Background:
Fragile X syndrome (FXS) is the most frequent inherited cause of mild to severe intellectual disability and the most common monogenic cause of autism spectrum disorder (Stone et al. 2023). FXS is an X-linked disorder with variable expression in males and females. It is caused by an expansion of the CGG trinucleotide repeat in the 5’ UTR (untranslated region) of the FMR1 gene. This trinucleotide repeat region is highly variable in the general population with the number of repeats ranging from 5 to 44. These normal alleles are passed from one generation to the next. Small expansions of this region range from 55-200 repeats and are called premutations. Individuals with premutations do not exhibit features consistent with FXS but are at an increased risk for other FMR1-related disorders including Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and FMR1-associated primary ovarian insufficiency (POI). FXTAS affects approximately in 40-45% of male FMR1 premutation carriers over the age of 50 and is characterized by intention tremor and/or ataxia; peripheral neuropathy, autonomic dysfunction, gradual cognitive decline and psychiatric features such as anxiety, dysinhibition, depression, and apathy. FMR1-associated POI is observed in 20% of women with a premutation allele and defined as hypergonadotropic hypogonadism before age 40 (Hunter et al. 2024).
Repeat instability during transmission differs by parent of origin. Premutation alleles passed from fathers to daughters generally show little change in repeat number, whereas premutations transmitted by mothers are more likely to undergo further expansion. Full size repeat expansions are typically greater than 200 CGG repeats and result in abnormal methylation of a region adjacent to the FMR1 gene interfering with normal FMR1 gene expression.
Please note that expansion size mosaics and methylation mosaics have been previously described and may affect the severity of the clinical presentation (Monaghan et al. 2013).
Genetic counseling is recommended for all premutation and full mutation carriers.
Reason for Referral
Methodology:
PCR and capillary electrophoresis analysis is used to measure the number of CGG trinucleotide repeats in the 5’ untranslated region of the FMR1 gene at Xq27.3; additionally, CGG repeat size and methylation status is determined by Southern blot for large premutations (>CGG150) and for fully expanded alleles.
Allele Types #CGG Repeats:
Normal: 5 - 44
Intermediate: 45 - 54
Premutation: 55 – 200
Full mutation (i.e. disease-causing): >200
Specimen Requirements:
Blood: EDTA (purple-top) tube or ACD (yellow-top) tube
- Adult: 5 mL
- Child: 5 mL
- Infant: 2-3 mL
DNA: 20µg at a minimum of 100ng/µL(DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).
Saliva:2 ORAgene™ Saliva Collection Kits (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Assisted Saliva: 4 ORAgene™ Assisted Saliva Collection Kits (OGR-575) used according to manufacturer instructions. Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.
Buccal Swab: Please contact KDL Client Services for Buccal Swab Collection Kit(s) for patients that cannot provide a blood sample.
Notice Regarding Saliva and Buccal specimens: If expansion is seen, Saliva and Buccal swabs will not yield enough DNA to size the expansion, a blood draw will be required.
Prenatal testing:
- Direct Amniotic Fluid (10-20mL)
- Direct CVS*
- Direct POC
- Cultured Amniocytes (2-T25 flasks)
- Cultured CVS (2-T25 flasks)*
- Cultured Fetal Tissue: Product of Conception (2 T25 flasks)
- Cord Blood (1-2mL)
*When testing CVS, sizing can be performed and expansions will be detected but methylation status may not be reliably determined. If methylation status is requested or required by the referring provider, testing of CVS is not recommended.
Notice Regarding Molecular Genetic Testing on Prenatal Specimens:Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood (or saliva) in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.
For routine testing of blood, saliva and buccal swabs, KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed within the above windows, please contact our lab.
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including ethnicity, clinical history, and family history.
Test Performed (Days):
Weekly
Turn Around Time:
7-10 days
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold. but not frozen.
- Contact Client Services for shipping kits and instructions.
- Please use the cold pack provided in the KDL shipping kit.
- Ship the specimen overnight express, using the FedEx priority overnight label provided.
References:
- Hunter JE, Berry-Kravis E, Hipp H, et al. FMR1 Disorders. 1998 Jun 16 [Updated 2024 May 16]. In: Adam MP, Bick S, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2025. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1384/
- Stone WL, Basit H, Shah M, et al. Fragile X Syndrome. [Updated 2023 Oct 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK459243/
- Monaghan et al. Genet Med. 2013 Jul;15(7):575-86. PMID: 23765048
Additional Info:
Prior to any genetic testing we recommend genetic counseling. To receive forms and information about prenatal diagnostic testing, please contact Client Services at (855) 535-1522.