Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis

Background: Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (msPCR) and GeneScan (TM) analysis for molecular diagnosis of fragile X syndrome. Methods: We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescentlylabeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram. Results: DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherograrn pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods. Conclusions: This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS. (c) 2006 American Association for Clinical Chemistry.