Simple Sensitive Assays Used for Analyzing Fragile X Syndrome

Fragile X syndrome, the most common heritable cause of intellectual disability and a frequent cause of autism, is characterized by abnormalities of the fragile X mental retardation 1gene (FMR1) that are difficult to analyze.

Preclinical studies of Fragile X and the Fragile X related disorders are hampered by the lack of low-cost and sensitive yet simple methods, but a newly developed a set of assays that are robust, cheap enough for routine use may be suitable for initial patient screening.


Scientists at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD, USA) developed the assays which have the ability to amplify alleles with up to approximately 1,000 repeats, even in samples from patients who are mosaic, that is those who have a mixture of cells with different repeat numbers. The assays are sensitive enough to analyze saliva samples with minimal purification. Testing can be completed within a timeframe similar to that of recent commercial diagnostic assays in less than 24 hours to determine repeat size and/or methylation status and in less than 24 hours to determine the interruption status and percent methylation and are comparable in terms of hands-on time required.

These assays make it possible to detect even small changes in DNA methylation, making them useful in the hunt for new drugs to reverse the effects of repeat expansion. Genomic DNA from cell lines was prepared using standard procedures. Genomic DNA from human saliva was collected in an OGR-500 (Oragene, DNA Genotek, Kanata, ON, Canada) and purified using their prepIT-L2P reagent. DNA quantification was performed on a Denovix DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA). A quantitative FMR1 promoter methylation polymerase chain reaction assay (qMS-PCR) was performed using a StepOne Plus PCR machine (Thermo Fisher Scientific, Waltham, MA, USA).

Karen Usdin, PhD, the senior investigator and co-author of the study, said, “Careful analysis of the total number of repeats, the number of interruptions in the repeat tract, and the methylation status of the FMR1 gene is important for a proper understanding of an individual's risk of transmission of larger alleles to their offspring and to their personal risk of disease pathology. Without the ability to verify CGG-repeat number and methylation status, it is impossible to distinguish between bona fide developmentally-regulated changes and artifacts arising from the instability in repeat number and methylation commonly associated with these cells.” The study was published online on August 1, 2016, in The Journal of Molecular Diagnostics.

Related Links:
National Institute of Diabetes and Digestive and Kidney Diseases
DNA Genotek
DeNovix
Thermo Fisher Scientific