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Technical Briefs

A Homogeneous Assay for Analysis of FMR1 Promoter Methylation in Patients with Fragile X Syndrome,

¹ Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark
² The Kennedy Institute-National Eye Clinic, Glostrup, Denmark
³ Wilhelm Johannsen Centre for Functional Genome Research, Department of Medical Biochemistry and Genetics, University of Copenhagen, Denmark

Background: Fragile X syndrome is caused by the expansion of a CGG trinucleotide repeat at the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1). When expanded to >200 repeats (full mutation), the repeat region and the adjacent promoter CpG island become hypermethylated, rendering FMR1 transcriptionally inactive. Conventional molecular diagnosis of fragile X syndrome involves determination of the CGG repeat number by Southern blot analysis.

Methods: A homogeneous methylation-specific melting curve analysis (MS-MCA) assay for methylation status of the FMR1 promoter region was developed on the LightCycler platform. Genomic DNA was treated with sodium bisulfite, and a region containing 8 CpG sites was amplified in the presence of SYBR Green I, using primers that do not discriminate between methylated and unmethylated FMR1 molecules. After amplification, the samples were melted at 0.05 °C/s, and fluorescence melting curves were recorded. We studied samples, previously characterized by Southern blot analyses, from 10 females and 10 males with normal numbers of CGG trinucleotide repeats, 9 male premutation carriers, 4 mosaic males carrying both a premutation and a full mutation, and 25 patients with fragile X syndrome.

Results: Samples from all 20 males with fragile X syndrome showed a high-melting peak corresponding to fully methylated FMR1, whereas samples from healthy males showed a single low-melting peak corresponding to unmethylated FMR1. Nine of the 24 affected-male samples (38%) showed 2 melting peaks, suggesting that cellular methylation mosaicism is common in fragile X syndrome.

Conclusions: MS-MCA allows rapid and reliable identification of males with fragile X syndrome.