Methylation-specific multiplex ligation-dependent probe amplification enables a rapid and reliable distinction between male FMR1 premutation and full-mutation alleles.

Fragile X syndrome is the most common cause of inherited mental retardation and the second most common cause of mental impairment after trisomy 21. It occurs because of a failure to express the fragile X mental retardation protein. The most common molecular basis for the disease is the abnormal expansion of the number of CGG repeats in the fragile X mental retardation 1 gene (FMR1). Based on the number of repeats, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54), premutation (55 to 200), and full mutation (>200). Today, the diagnosis of fragile X syndrome is performed through a combination of PCR to identify fewer than 100 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We have developed a methylation-specific multiplex ligation-dependent probe amplification assay to analyze male fragile X syndrome cases with long repeat tracts that are not amplifiable by PCR. This inexpensive, rapid and robust technique provides not only a clear distinction between male pre- and full-mutation FMR1 alleles, but also permits the identification of genomic deletions, a less frequent cause of fragile X syndrome.