A PCR-BASED TEST SUITABLE FOR SCREENING FOR FRAGILE-X SYNDROME AMONG MENTALLY-RETARDED MALES

Ever since the identification of the genetic cause of fragile X syndro me as the expansion of an unstable trinucleotide sequence, several dia gnostic strategies have evolved from molecular studies. However, we st ill lack a simple test suitable for population screening. We have ther efore developed a nonisotopic polymerase chain reaction (PCR)-based te chnique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacry lamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)(29) allele. Conditions were established such that full mutation s failed to amplify and were thus identified with 98% sensitivity comp ared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fra gment, allowing the multiplex amplification of a monomorphic band corr esponding to a CG-rich stretch 147 bp upstream of the polymorphic regi on. In trials involving 41 patients and 74 controls, the PCR-based tes t here described showed specificity of more than 98.6%, accuracy of 99 % and a sensitivity of 98%. Thus, although not suitable for medical di agnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.