Objectives: The aim of this study was to develop a rapid, non-radioactive and effective method for the molecular diagnosis of fragile X syndrome (FXS) by the polymerase chain reaction (PCR) of the CGG repeat and to establish a protocol to be used in: a)ruling out FXS in patients with non-specific mental retardation; b)determining the exact genotype of affected individuals; c)studying all at-risk individuals from families with FXS and identifying asymptomatic carriers, and d)offering accurate genetic and reproductive counselling to families with FXS.
Materials and methods: Samples from 438 individuals from 50 families with FXS were studied using three different PCR tests: the first to detect ethidium bromide through ultraviolet light, the second to detect digoxigenin and CSPD after blotting and hybridisation with the (CGG)5 oligoprobe, and the third to amplify and detect the DXS548 microsatellite.
Results: Of the 438 individuals studied, 121 had full mutations (60 males and 61 females), 86 had pre-mutations (7 males and 79 females), 16 showed mosaic patterns and 215 had no mutations. PCR techniques amplified up to 120-150 repeats, and direct study with probes was required when no bands or only one band was detected in females. PCR was more accurate than genomic DNA Southern blot analysis in pre-mutated carriers. In one family, recombination between the FRAXA locus and the DXS548 microsatellite was found.
Conclusions: These non-radioactive PCR protocols permit rapid and accurate diagnosis of FXS. They and are especially useful in prenatal diagnosis and in the identification of carriers.