Label Name: FMR1 FRAG X
Lab Discipline: Molecular Diagnostics
Subdiscipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6344 
Last Review:  3/16/2017 2:51:14 PM
Specimen Type
  Whole Blood (EDTA)
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Collection Notes
  • Peripheral Blood: TWO lavender-top EDTA tubes is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Amniocytes: 2-4ml of amniotic fluid is required. Forward promptly at ambient temperature only. SPECIMEN CANNOT BE FROZEN.
  Deliver peripheral blood and buccal swab specimens to laboratory at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.
Turn Around Time -  Routine: 10 days   Stat: N/A
Reference Values
FMR1 gene CGG repeat expansion not detected
  This assay determines the number of CGG repeats in the 5' UTR region of the fragile X gene (FMR1) using PCR amplification and subsequent size analysis by capillary gel electrophoresis. Genomic DNA is first extracted from a whole blood specimen. This DNA is then used in a polymerase chain reaction (PCR) amplification containing a forward oligonucleotide primer, a FAM-labeled reverse oligonucleotide primer (both flanking the CGG repeat region within the FMR1 gene) and a third internal CGG repeat primer. Following PCR amplification, the fluorescently labeled PCR products are resolved by capillary electrophoresis on the ABI 3130xl Genetic Analyzer. GeneMapper software (ABI) is used to resolve the number of repeats. CGG repeat lengths of less than 45 are interpreted as normal, between 45 and 54 are interpreted asindeterminate, between 55 and 200 are interpreted as premutation and greater than 200 are interpreted as a full mutation.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostic Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Special Information
  • Additional Patient Information Required: Due to the unique nature of genetic testing, patients offered this test should receive pre-test and post-test genetic counseling. Counseling should help the patient understand the strengths and limitations of DNA testing and the medical implications for the patient as well as for other family members. Patients are also required to give consent for testing.
Clinical Significance and Interpretive Data

The X-linked recessive disorder Fragile X syndrome (FXS) is the most common cause of inherited mental retardation, affecting approximately 1/4000 males. The syndrome is caused by mutations in the FMR1 gene located on the long arm of the X chromosome (Xq27.3). FMR1 mutations result in a loss or severe reduction in FMRP, the protein encoded by the FMR1 gene which is thought to play a crucial role in learning, memory, and synaptic plasticity. Clinical manifestations of FXS include mental retardation of variable severity, developmental and speech delay, behavior problems, and characteristic physical findings (a long face, large ears and post-pubertal macro-orchidism).

Fragile X syndrome is one of the few inherited disorders caused by a trinucleotide repeat expansion. A sequence of CGG trinucleotide repeats is located in the untranslated portion of exon 1 of the FMR1 gene. In the normal FMR1 allele, approximately 5 to 44 CGG repeats are present. Instability of the trinucleotide repeat can result in expansion of the repeat segment. The number of CGG repeats can be used to categorize FMR1 alleles as normal (5-44 repeats), intermediate (45-54 repeats), premutation (55-~200 repeats), or full mutation (> 200 repeats). Trinucleotide expansion usually leads to hypermethylation of the FMR1 gene which results in decreased or absent gene transcription. In patients with FXS, FMR1 often exhibits >200 CGG repeats as well as hypermethylation of the FMR1 locus. Rarely, a full mutation results in only partial methylation of the FMR1 locus (methylation mosaics). Patients with a mixture of cells having either a premutation or full mutation (premutation/full mutation mosaics) have also been described. These mosaic patients may express variable levels of FRMP protein with a corresponding variable degree of symptoms.

Inheritance of FXS full mutations usually occurs through the mother. In mothers with a premutation allele (55-~200 CGG repeats), repeat instability can occur leading to extensive repeat expansion. The fully mutated allele can then be passed on to offspring. In fathers, premutations are stable, and thus a newly expanded full mutation will not be passed on to a daughter.

Changes in the FMR1 gene are associated with two other diseases; premature ovarian failure and fragile X-associated tremor/ataxia syndrome. Women who carry the FMR1 premutation allele are at increased risk for premature ovarian failure (POF), defined as the cessation of menses before age 40. Approximately 21% of women carrying a premutation allele have POF, whereas 1% of the general population experiences POF. The biological mechanism linking FMR1 and POF has yet to be well characterized. Fragile X-associated tremor/ataxia syndrome principally affects male with FMR1 premutation alleles. This neurological disorder is characterized by gait ataxia, intention tremor, cognitive decline, and parkinsonian symptoms. Approximately 1/813 males carry an FMR1 premutation allele. Since fragile X-associated tremor/ataxia syndrome is a newly-defined disorder, the exact prevalence is uncertain. It is believed disease penetrance increases with age and may eventually affect a large number of premutation carriers. Prevalence may approach 1/3000 in the general population.

Test Interpretation:

Normal: <45 repeats
Grey zone: 45-54 repeats
Premutation: 55-200 repeats
Full mutation: >200 repeats

•All female cases that are homozygous for normal repeat alleles are tested with Southern blot before issuing a final report.

•All cases tested containing full mutations will be tested by Southern blot before issuing a final report.

• In consultation with the ordering clinician and genetic counselor, Southern blotting may be considered when PCR testing reveals a repeat size in the permutation range.


Chen L et al. An information-rich CGG repeat primed PCR that detects the fullrange of fragile X expanded alleles and minimizes the need for southern blot. Journal of Molecular Diagnostics. 2010; 12 (5):589-600.

Filipovic-Sadic S et al. A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome.
Clinical Chemistry. 2010; 56(3): 399-408.

Garber K, Smith K, et al. Transcription, translation and fragile X syndrome. Current Opinion in Genetics & Development 2006;16:270-275.

Jacquemont S, Hagerman RJ, et al. Penetrance of the Fragile X-Associated Tremor/Ataxia Syndrome in a Premutation Carrier Population. JAMA 2004;291:460-469.

Kronquist KE et al. Clinical significance of tri-nucleotide repeats in Fragile X testing: a clarification of American College of Medical Genetics guidelines.Genet Med. 2008; 10: 845-7.

Sherman S, Pletcher B, Driscoll D. Fragile X syndrome: Diagnostic and carrier testing. Genetics in Medicine 2005;7:584-587.

Technical standards and guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for ClinicalGenetics Laboratories of the American College of Medical Genetics, 2006.
    • According to the American College of Medical Genetics (ACMG) 2005 Practice Guideline (1)Molecular testing for FXS should be considered for the following populations:

1) Patients of either sex with unexplained mental retardation, developmental delay, or autism (especially individuals with clinical or physical characteristics of FXS, a family history of FXS, or relatives with mental retardation of undiagnosed etiology).

2) Individuals seeking reproductive counseling who have a family history of FXS or relatives with mental retardation of undiagnosed etiology.

3) Fetuses of a full mutation or premutation carrier mother.

• Evaluation of the FMR1 gene repeat region may also be useful in the setting of ovarian dysfunction. The ACMG recommends testing for "Women who are experiencing reproductive or fertility problems associated with elevated follicle stimulating hormone (FSH) levels, especially if they have (a) a family history of premature ovarian failure, (b) a family history of fragile X syndrome, or (c) male or female relatives with undiagnosed mental retardation (1)."

• Additionally, testing the FMR1 gene repeat region can be used to evaluate patients for fragile-X associated tremor/ataxia syndrome. The ACMG recommends testing "Men and women who are experiencing late onset intention tremor and cerebellar ataxia of unknown origin, especially if they have (a) a family history of movement disorders, (b) a family history of fragile X syndrome, or (c) male or female relatives with undiagnosed mental retardation."


Sherman S, Pletcher B, Driscoll D. Fragile X syndrome: Diagnostic and carrier testing. Genetics in Medicine 2005;7:584-587.
    • This assay detects all disease causing CGG repeat expansions in the FMR1 gene. These expansions cause >99% of all cases of Fragile X Syndrome.

• This assay will not detect rare causes of Fragile X Syndrome that do not involve repeat expansions (e.g. point mutations and deletions). It also does not detect mutations in other genes that may cause mental retardation or other environmental or non-inherited causes of mental retardation or developmental delay.

• Molecular analysis may detect FMR1 cellular mosaicism in either repeats or methylation. Individuals with cellular mosaicism may express variable amounts of FRMP protein and therefore exhibit higher functioning than expected. In cases of cellular mosaicism detected in young children, prognosticating severity of mental retardation is problematic and should be approached with caution. Further studies must be conducted to assess the development of these individuals.

• Mutations or polymorphisms in the DNA oligonucleotide primer binding regions, poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable or (rarely) inaccurate results.
Test Synonyms
Synonym(s):  FMR1
Synonym(s):  mental retardation
Synonym(s): Fragile X
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: