FMR1 genes with >200 CGG repeats are usually hypermethylated and inactivated
resulting in the loss of FMRP, an important protein involved in microRNA and mRNA regulation and
neural development. The consequences of this gene inactivation were revealed in a seminal 14 center
study by Rousseau et al in 1994 that demonstrated that the prediction of intellectual disability was
improved by considering both the CGG expansion size and the gene methylation status . In some
cases, individuals with greater than 200 CGG but an unmethylated or mosaic allele had a normal
cognitive phenotype or less severe phenotype . The severity of fragile X syndrome symptoms in
females can also vary based on methylation status of the normal allele . Moreover, the response
to emerging therapies for fragile X is influenced by the degree of methylation . Since 1991, the
primary method used to determine methylation status has been Southern blot analysis. Yet, Southern
blot analysis is laborious, requires large quantities of high-quality DNA for analysis and has low
resolution and sensitivity compared to PCR-based methods. Methods to access methylation status without
Southern blot have typically relied on either chemical or enzymatic pretreatment of the DNA prior to
PCR amplification. Chemical treatments require sodium bisulfite to convert methylated cytosine to uracil.
These changes may be recognized through various techniques including DNA sequencing, PCR and melting
curve analysis [5-7]. Such methods are more suitable to the analysis of male samples because the
presence of two X chromosomes in female samples can confound the analysis. Enzyme-based methods
use methylation sensitive restriction enzymes to digest unmethylated DNA. Multiple techniques, including
real-time PCR, probe ligation and/or capillary electrophoresis may then be used to determine the
methylation status by comparing amplicons generated from digested and undigested DNA [8, 9]. Recent
methods have shown that enzymatic pretreatment and full-length amplification of the FMR1 CGG repeats
can yield repeat length and methylation status in a high throughput workflow that is accurate for both
male and female samples . Emerging markers in the FMR1 gene region may also be predictive of the
fragile X phenotype . In total these novel capabilities have the potential to improve methylation
assessment and to eliminate the need for Southern blot analysis.
- • To understand the diagnostic value of determining FMR1 methylation
- • Review of Southern blot challenges and limitations
- • Learn about new approaches with PCR based assays, capable of improving the throughput, resolution, FMR1 molecular diagnostics
1. A multicenter study on genotype-phenotype correlations in the fragile X syndrome, using direct diagnosis with probe StB12.3: the first 2,253 cases
Rousseau, F et al, Am J Hum Genet, 1994. 55(2): 225-37
**This multi-center study established the clinical validity of Southern blot analysis by linking phenotype with methylation status. Notably, the magnitude of the repeat length above 200 CGG was less associated with phenotype than methylation status making the reporting of repeat length beyond 200 CGG unnecessary.
2. Normal phenotype in two brothers with a full FMR1 mutation
Smeets, H.J. et al., Hum Mol Genet, 1995. 4(11): 2103-8.
3. Molecular predictors of cognitive involvement in female carriers of fragile X syndrome
Taylor, A.K. et al., J Am Med Assoc, 1994. 271(7): 507-14.
4. Epigenetic modification of the FMR1 gene in fragile X syndrome is associated with differential response to the mGluR5 antagonist AFQ056.
Jacquemont, S. et al., Sci Transl Med, 2011. 3(64): 64ra1.
5. Methylation-specific PCR
Coffee, B., Curr Protoc Hum Genet, 2009. Chapter 10: Unit 10 6.
*The authors describe bisulfite treatment and quantitative PCR of CpG islands in the FMR1 gene as an effective surrogate marker of CGG repeat length. The method was effective for male samples and applicable to broad screening for identification of fully methylated alleles in large populations.
6. A ligation assay for multiplex analysis of CpG methylation using bisulfite-treated DNA
Dahl, C. and P. Guldberg, Nucleic Acids Res, 2007. 35(21): e144
*The authors present a closed-tube format based on melting temperature (Tm) of probes targeting bisulfite-treated DNA. The method streamlined the identification of fully methylated full mutation alleles in male samples, and could be useful for a lower-cost screening method in large sample pools.
7. Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis
Zhou, Y., et al., Clin Chem, 2006. 52(8): 1492-500.
8. Methylation-specific multiplex ligation-dependent probe amplification enables a rapid and reliable distinction between male FMR1 premutation and full-mutation alleles.
Nygren, A.O. et al., J Mol Diagn, 2008. 10(6): 496-501.
*To overcome limitations of sizing longer CGG repeats using PCR, the authors present an enzymatic pre-treatment of DNA using HhaI and multiplex ligation dependent probe amplification. Amplified fragments were resolved using capillary electrophoresis yielding peak patterns reflective of methylation status in male samples.
9. An assay for X inactivation based on differential methylation at the fragile X locus, FMR1.
Carrel, L. and H.F. Willard, Am J Med Genet, 1996. 64(1): 27-30.
10. High-resolution methylation polymerase chain reaction for fragile X analysis: evidence for novel FMR1 methylation patterns undetected in Southern blot analyses.
Chen, L., et al., Genet Med, 2011. 13(6): 528-38.
**The authors describe the first accurate and comprehensive PCR only workflow that accurately assesses methylation status in both male and female samples. The method combines efficient FMR1 PCR with pretreatment of the DNA using a methylation sensitive restriction digestion. The methylation status of all expanded alleles were in agreement with Southern blot analysis and novel methylation patterns in female premutation samples were identified.
11. Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.
Godler DE et al., Clin Chem, 2012. 58(3): 590-8.
*This preliminary study demonstrates a correlation between the methylation status of novel FMR1 epigenetic markers and the cognitive phenotype of female subjects with a full mutation expansion using a novel, mass spectrometry-based assay.