Polymerase Chain Reaction/Capillary Electrophoresis
- Preferred test to diagnose FXS, FXTAS, and for carrier screening in individuals with a positive family history
- Test Description
- Triplet repeat-primed polymerase chain reaction (PCR) followed by size analysis using capillary electrophoresis to determine FMR1 CGG repeat length
- Methylation-specific PCR is performed for >100 CGG repeats to distinguish between premutation and full mutation alleles
Polymerase Chain Reaction (PCR)/Capillary Electrophoresis
- Prenatal test for women with fragile X premutations or full mutations.
- Contraindications
- Prenatal testing for women with normal or intermediate allele sizes
- Testing of chorionic villus (CVS) samples, because methylation patterns are not fully established in the first trimester of pregnancy
Disease Overview
Fragile X syndrome (FXS), the most common heritable form of intellectual disability (ID) and autism, is caused by full FMR1 gene mutations. Individuals with an FMR1 gene premutation may develop fragile X-associated tremor/ataxia syndrome (FXTAS), FX-associated neuropsychiatric disorder, or primary ovarian insufficiency (POI).
Diagnostic testing should be offered to individuals with unexplained intellectual disability (ID), developmental delay, autism spectrum disorder, late-onset cerebellar ataxia and intention tremor, POI, or infertility associated with elevated follicle-stimulating hormone (FSH) levels.
Screening for FXS should be offered to women with a positive family history of FXS, FXTAS, unexplained ID, or autism. Fetal testing should be offered to women who carry a fragile X premutation or full mutation.
Symptoms of FXS
- Moderate ID (median IQ of 40-45 but ranges from <10 to normal range)
- Autism in 50-70%
- Perseverative speech
- Behavioral issues: hand flapping/biting, attention deficit hyperactivity disorder (ADHD), social anxiety, poor eye contact, tactile defensiveness, aggressiveness, and irritability
- Large ears, long face, large jaw, prominent forehead, and large testes
Symptoms of FXTAS
- Cerebellar gait ataxia
- Intention tremor
- Parkinsonism; muscle rigidity, unbalanced shuffling gait, slowed movement and speech
- Moderate to severe short-term memory loss
- Executive function deficit
- Incontinence/ impotence
Prevalence
FXS:
- 1/4,000 males
- 1/8,000 females
Premutation allele in U.S.:
- 1/1,000 males
- 1/350 females
Genetics
Gene
FMR1
Inheritance
X-linked
Structure/Function
FMR1 codes for fragile X mental retardation protein (FMRP), an RNA-binding protein expressed in many tissues
Mutations
Caused by FMR1 variants
- 99% caused by expansion of the FMR1 gene CGG repeat​
- Less than 1% caused by FMR1 sequence variants or partial/full FMR1 gene deletions
- Risk for CGG repeat expansion is dependent on sex of transmitting parent and size of repeat
CGG repeat sizes:
- Normal alleles: 5-44 CGG repeats (unmethylated)
- Stably transmitted
- Intermediate alleles: 45-54 CGG repeats (unmethylated)
- Unstable but will not expand to a full mutation in one generation
- Premutations: 55 to ~200 CGG repeats (unmethylated)
- Females:
- At risk for having offspring with FXS-transmission of CGG repeats to offspring is unstable
- 21% risk for POI (before age 40)
- At risk for fragile X-associated neuropsychiatric disorders
- 17% risk for FXTAS in women>50 years of age
- Males:
- Transmission of CGG repeats is stable
- All of their daughters and none of their sons will inherit the premutation
- 47% risk for FXTAS in men >50 years of age
- Females:
- Full mutations: typically >200 CGG repeats (methylated)
- Males are affected with FXS
- 50% of females have moderate ID
- Disease symptom severity cannot be predicted based on:
- Size of CGG repeat
- Degree of methylation
- Pattern of X-inactivation (in females)
Test Interpretation
Sensitivity/Specificity
Results
Result | Number of CGG Repeats | Clinical Significance |
---|---|---|
Full mutation |
>~200 (methylated) |
Male: FXS with ID |
Female: Variable expression of FXS; 50% have ID |
||
Premutation |
55 to ~200 (unmethylated) |
Male: 47% risk for FXTAS in men >50 years of age |
Female: At risk for POI At risk for FX-associated neuropsychiatric disorders 17% risk for FXTAS in women >50 years of age |
||
Indeterminate |
45 to ~54 |
Offspring at risk for inheriting premutation |
Negative |
5 to ~44 |
Normal; not affected with nor a carrier of FXS |
Limitations
- Estimated CGG repeat number is not provided for full mutations (alleles with >200 repeats)
- Sizing precision of CGG premutation alleles is within two-three CGG repeats
- Methylation patterns are not fully established in the first trimester of pregnancy; thus, CVS is not recommended for prenatal diagnosis. A small, full mutation may be distinguished from a large premutation in amniocytes
- Rare FMR1 variants unrelated to trinucleotide expansion will not be detected
- Diagnostic errors can occur due to rare sequence variations
- AGG trinucleotide interruptions within the FMR1 CGG repeat tract are not assessed
References
-
25210937
Nolin SL, Glicksman A, Ersalesi N, et al. Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers. Genet Med. 2015;17(5):358-364.
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31050164
Nolin SL, Glicksman A, Tortora N, et al. Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal, intermediate, and premutation alleles. Am J Med Genet A. 2019;179(7):1148-1156.
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GeneReviews - FMR1 Disorders
Hunter JE, Berry-Kravis E, Hipp H, et al. FMR1 disorders. In: Adam MP, Ardinger HH, Pagon RA, et al, editors. GeneReviews, University of Washington; 1993-2020. [Last Update: Nov 2019; Accessed: Feb 2020]
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20431035
Lyon E, Laver T, Yu P, et al. A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis. J Mol Diagn. 2010;12(4):505-511.
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24177047
Grasso M, Boon EMJ, Filipovic-Sadic S, et al. A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis. J Mol Diagn. 2014;16(1):23-31.
23765048
Monaghan KG, Lyon E, Spector EB, et al. ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics. Genet Med. 2013;15(7):575-586.