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Simplified Molecular Diagnosis of Fragile X Syndrome by Fluorescent Methylation-Specific PCR and GeneScan Analysis

¹ Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore.
² Children’s Medical Institute and ³ Department of Laboratory Medicine, National University Hospital, Singapore.

^aAddress correspondence to this author at: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Level 4, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore. Phone 65-6772-4386; Fax 65-6779-7486; email paecs{at}nus.edu.sg

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 (ms-PCR) 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 fluorescently-labeled 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 electropherogram 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.

The following articles in journals at HighWire Press have cited this article:

				C. Dahl, K. Gronskov, L. A. Larsen, P. Guldberg, and K. Brondum-Nielsen A Homogeneous Assay for Analysis of FMR1 Promoter Methylation in Patients with Fragile X Syndrome Clin. Chem., April 1, 2007; 53(4): 790 - 793. [Abstract] [Full Text] [PDF]