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.

Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

“…MCA has previously been reported for 2 methods (40,41 ) for evaluating the methylation status of the FMR1 promoter region; however, neither published method can be used to screen female patients. The dTP-PCR MCA assays we have described can reliably detect expansions in both males and females.…”

ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics

“…33 Using melt analysis, methylated and unmethylated alleles are amplified simultaneously but due to differences of GC content can be resolved by differences in melting temperature between methylated and unmethylated alleles. 93 These methods have high analytical sensitivity and specificity for detecting methylation in males but are less sensitive and specific in females. Note that it is not necessarily obvious that the borderline category refers to the border between normal and premutation and not to the border between premutation and full mutation.…”

Recent advances in assays for the fragile X-related disorders

“…Other assays using bisulfite modification followed by PCR and melt curve analysis (MCA)/high resolution melting (HRM) have also been described (Dahl et al 2007; Elias et al 2011; Inaba et al 2014; Rajan-Babu et al 2015). These assays exploit the fact that bisulfite conversion of unmethylated alleles results in templates that have a lower G+C content than methylated alleles, and thus a lower melting temperature.…”