Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of Fragile X Mental Retardation 1–related disorders

Abstract

Aim: Molecular diagnosis of fragile X syndrome demands assessment of fragile X mental retardation 1 (FMR1) CGG repeat size and methylation status, while predicting disease transmission risk requires determination of AGG interruption pattern. There is currently no single assay that provides all three categories of information. We describe a single-tube methylation-specific triplet-primed PCR assay for concurrently assessing methylation state, repeat size and structure of CGG repeat(s). Methods: Differentially labeled primers specific for methylated and unmethylated FMR1 alleles were used to amplify bisulfite-modified DNA, followed by capillary electrophoresis. Twenty-four reference DNAs and 107 patient samples were analyzed to evaluate assay performance. Results: Repeat size, AGG interruption pattern and methylation state were correctly identified in all tested samples. The assay also detected skewed X-inactivation when present in females, and somatic mosaicism in fragile X males. Conclusion: When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.

Keywords:

• AGG interruption
• CGG repeat
• FMR1
• methylation
• triplet-primed PCR

Acknowledgements

We thank CS Yoon for assistance with Southern blots of clinical samples.

Financial & competing interests disclosure

This work was supported by a grant (NMRC/1079/2006) from the National Medical Research Council of Singapore. SS Chong, CRL Teo, and CG Lee are the inventors of the diagnostic procedure described in this manuscript and have applied for a patent for the method. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• Hyperexpansion of a CGG repeat within the fragile X mental retardation 1 (FMR1) gene and its consequent methylation-mediated silencing leads to fragile X syndrome (FXS). Comprehensive assessment of CGG repeat size and methylation status is therefore crucial for accurate diagnosis of FXS, while information on the number of AGG interruptions at the 5′ end of the CGG repeat tract is essential to predict the risk of transmitting fully expanded FMR1 allele during maternal transmission.

• Southern blot analysis, the gold standard FXS diagnostic method is tedious, time-consuming, requires large amounts of DNA and does not facilitate accurate sizing, determination of AGG interruption pattern and discrimination between different FMR1 allelic classes.

• Traditional repeat-spanning PCRs that query CGG repeat size and/or methylation status do not provide AGG interruption information and are susceptible to amplification failure as the upper limit of full-mutation (FM, >200 repeats) detection ability of these assays is uncertain.

• In this study, we describe a simple methylation-specific method based on triplet-primed PCR (TP-PCR), a strategy involving repeat-annealing primers that results in the generation of heterogeneous amplicons differing by approximately 3 bp from each other. Unlike repeat-spanning PCR, this approach amplifies >200 CGG repeats and enables detection of all FM FMR1 alleles regardless of their actual repeat size. By assigning different fluorescent-tags to primers specific for the bisulfite-modified unmethylated and methylated FMR1 alleles, we demonstrate that both alleles can be amplified in a single-tube methylation-specific duplex TP-PCR (msTP-PCR) reaction.

• We tested 24 genotype-known reference DNAs from Coriell Cell Repositories (CCR) and 107 archived and previously characterized patient DNA samples. All the samples were correctly genotyped, and the methylation status was accurately determined.

• Skewed X-inactivation in females and the presence of low-level somatic mosaicism among FM males were detected. In addition, msTP-PCR also accurately determined the AGG interruption patterns in a panel of CCR and clinical samples verified by Sanger sequencing.

• This rapid high-throughput assay has potential applications in the molecular diagnosis of FXS and PM-related conditions, FXTAS and FXPOI, and could potentially reduce the number of samples that need to be reflexed for Southern blot analysis.