A homogeneous assay for analysis of FMR1 promoter methylation in patients with fragile X syndrome

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 1, using primers that do not differentiate between methylated and unmethylated FMR1 molecules. After amplification, the samples were melted at 0.05 degrees C/s, and fluorescence melting curves were recorded. We studied samples, previously characterized by Southern blot analyses, from 10 female and 10 male donors with normal numbers of CGG trinucleotide repeats, 9 male donors who were premutation carriers, 4 male donors who carried both a premutation and a full mutation, and 25 patients with fragile X syndrome. Results: Samples from all 20 male patients 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. Of 24 samples from affected males, 9 (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 fragile X syndrome in male patients. (c) 2007 American Association for Clinical Chemistry.