Methyl-CpG-Binding PCR of Bloodspots for Confirmation of Fragile X Syndrome in Males

Abstract

This study demonstrates that methyl-CpG-binding PCR (MB-PCR) is a rapid and simple method for detecting fragile X syndrome (FXS) in males, which is performed by verifying the methylation status of theFMR1promoter in bloodspots. Proteins containing methyl-CpG-binding (MB) domains can be freeze-stored and used as stocks, and the entire test requires only a few hours. The minimum amount of DNA required for the test is 0.5 ng. At this amount, detection sensitivity is not hampered, even mixing with excess unmethylated alleles up to 320 folds. We examined bloodspots from 100 males, including 24 with FXS, in a blinded manner. The results revealed that the ability of MB-PCR to detectFMR1promoter methylation was the same as that of Southern blot hybridization. Since individuals with 2 or more X chromosomes generally have methylatedFMR1alleles, MB-PCR cannot be used to detect FXS in females.