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 (ms-PCR) and GeneScan 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.
Zhou
Youyou
Y
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, and Children's Medical Institute, National University Hospital, Singapore. zhouyouyou@fudan.edu.cn
Lum
Josephine M S
JM
Yeo
Gare-Hoon
GH
Kiing
Jennifer
J
Tay
Stacey K H
SK
Chong
Samuel S
SS
D019175:DNA Methylation; D019075:Electrophoresis, Capillary; D005260:Female; D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D016133:Polymerase Chain Reaction; D013447:Sulfites
D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1; D007202:Indicators and Reagents; D013447:Sulfites; C497683:FMR1 protein, human; C009279:sodium bisulfite