Received on February 9, 2006
Accepted on May 31, 2006

Molecular Diagnostics and Genetics

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, National University Hospital, Singapore
³ Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, and Children's Medical Institute, National University Hospital, Singapore
⁴ Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Children's Medical Institute and Department of Laboratory Medicine, National University Hospital, Singapore

^* To whom correspondence should be addressed. E-mail: zhouyouyou{at}fudan.edu.cn.

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 NL 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 variational 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, prevariation, and full-variation FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various genotypes. The number of CGG repeats in all NL and prevariation 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.