A 15-year case-mix experience for fragile X syndrome molecular diagnosis and comparison between conventional and alternative techniques leading to a novel diagnostic procedure.

Fragile X syndrome (FXS) is the main cause of heritable mental retardation. In most patients, it is associated with an increased number of CGG repeats (>200) within the 5′-untranslated region of the FMR1 gene, and with methylation of the expanded repeats and of the promoter. FXS female carriers and transmitting males have expansions of between 55 and 200 repeats (premutated alleles). Alleles with premutations are unstable in female meioses. Normal and premutated repeats are unmethylated in males and subject to lyonization in females. Here, we report the postnatal and prenatal molecular diagnoses of FXS made with conventional PCR and Southern blotting in a cohort of Italian patients and their families over a period of 15 years. Moreover, we tested two novel high-performance PCR procedures (PCR with a chimeric primer, and the AmplideX™ FMR1 kit) in our patients and compared the results with our previous observations. We concluded that the high-performance PCR assays complement the results obtained by conventional methods, but they cannot replace the Southern blot procedure. Consequently, also based on cost–benefit considerations, our FXS diagnostic flowchart now consists of conventional PCR and Southern blotting plus the chimeric primer PCR procedure, whereas the AmplideX™ procedure is reserved for doubtful cases.