Fragile X syndrome (FXS) is the most common cause of inherited mental disability and known
genetic cause of autism. It is associated with the expansion of the CGG trinucleotide repeat in
the fragile X mental retardation 1 (FMR1) gene on the X chromosome. Diagnosis of FXS includes
using a genetic test that determines the number of CGG repeats in the fragile X
gene. The patient is classified as normal, intermediate (or “gray zone”), premutation or full
mutation based on the number of CGG repeats. Patients with a full mutation are associated with
fragile X syndrome; those with a premutation are carriers and may have an FMR1-related disorder.
The problem in fragile X testing is that the high fraction of GC bases in the repeat region makes it extremely
difficult for standard PCR techniques to amplify beyond about 100-150 CGG. As a result, Southern blot analysis
is commonly used to determine the number of triplet repeats in FXS. Recent advances in PCR methodologies, however,
[1,2,3,4] have enabled laboratories to accurately detect even very long repeat
segments and reduce their Southern blot testing by as much as 50-fold. These PCR methodologies have demonstrated
the ability to amplify >1000 CGG repeats, and some designs are capable of detecting the presence of a full mutation
irrespective of size.[3,4,5]
- • To gain a comprehensive understanding of the existing approaches for FMR1 testing, including both lab developed tests (LDTs) and commercial options
- • Review the challenges and advantages that these offer to a clinical molecular diagnostic lab
1. An Enhanced Polymerase Chain Reaction Assay to Detect
Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene.
Saluto A et al. J Mol Diagn. 2005; 7 (5): 605-612
2. A Novel FMR1 PCR Method for the Routine Detection of Low Abundance Expanded Alleles and Full Mutations in Fragile X Syndrome.
Filipovic-Sadic S et al. Clin Chem 2010; 56 (3): 400-409
3. An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis.
Chen L et al. J Mol Diagn. 2010; 1 (5): 589-600
4. A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis. Lyon E et al.
J Mol Diagn. 2010; 12 (4): 505-511
5. Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening.
Hantash FM et al. Genet Med 2010; 12(3):162-173
6. Draft Best Practice Guidelines for Molecular Analysis in Fragile X Syndrome.
Biancalana B. et al. EMQN 2006
7. Molecular testing for Fragile X Syndrome: Lessons learned from 119,232 tests performed in a clinical laboratory.
Strom CM et al. Genet Med 2007; 9(1): 46-51
8. Testing for Fragile X Gene Mutations Throughout the Life Span.
Hagerman RJ et al. JAMA. 2008; 300(20):2419-2421
9. Consensus Characterization of 16 FMR1 Reference Materials: A Consortium Study.
Wilson JA et al. J Mol Diagn. 2008; 10 (1): 2-12
*A multi-center study of repeat length assessment of fragile X cell lines available from the Coriell Repository (ccr.coriell.org) The group reports consensus repeat lengths using in-house developed and a common FMR1 PCR protocol in comparison to DNA sequencing analysis for 16 cell lines.
10. Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome.
Hawkins M et al. Eur J Hum Genet 2010; 1-8
**A set of internationally certified reference standards are described and validated for repeat length in a multi-center evaluation of 3 female and 2 male cell line DNA samples representing different repeat lengths. These standards are accessible through the National Institute of Biological Standards. (www.nibsc.ac.uk)
11. A Novel Assay for Evaluating Fragile X Locus Repeats.
Adler K et al. J Mol Diagn. 2011; 13 (6): 614-620