title: A new method for FMR1 gene methylation screening by multiplex methylation-specific real-time polymerase chain reaction.
abstract: Fragile X Syndrome (FXS) is the most common form of inherited mental retardation in men. It is caused by abnormalities in the FMR1 gene that are associated with CGG repeat expansion and the hypermethylation status of its promoter. Methylated alleles lead to transcriptional inhibition and consequent loss of Fragile X Mental Retardation Protein. Chemical modification of cytosine to uracil by bisulfite treatment has proved to be an attractive method for DNA methylation studies that precludes labor-intensive Southern blot analysis, which is the gold standard test for FXS. In this report, bisulfite-treated DNA samples were amplified using real-time multiplex methylation-specific polymerase chain reaction followed by melting curve analysis. Our results show that all control samples with known CGG repeat numbers and methylation statuses were correctly diagnosed. The samples from 43 male patients were also successfully diagnosed, which were in complete agreement with the results of Southern blotting. By such means, 39 patients were found to have an unmethylated allele; 3, a fully mutated allele; and 1, both methylated and unmethylated alleles, thus implying a diagnosis of mosaicism. In conclusion, we find our method to be convenient for screening a large number of male patients with FXS, because it is rapid and easy to perform, especially when there is a low quantity of DNA that must be sensitively and accurately assayed.
authors.affiliation: Human Genome Center, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
authors.forename: Marjanu Hikmah
authors.initials: MH
authors.lastname: Elias
authors.forename: Ravindran
authors.initials: R
authors.lastname: Ankathil
authors.forename: Abdul Razak
authors.initials: AR
authors.lastname: Salmi
authors.forename: Wanna
authors.initials: W
authors.lastname: Sudhikaran
authors.forename: Pornprot
authors.initials: P
authors.lastname: Limprasert
authors.forename: Bin Alwi
authors.initials: BA
authors.lastname: Zilfalil
mesh_terms: D000483:Alleles; D019175:DNA Methylation; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D013447:Sulfites; C497683:FMR1 protein, human; C042345:hydrogen sulfite