On some technical aspects of direct DNA diagnosis of the fragile X syndrome.

Direct DNA analysis of fragile X [Fra(X)] mutations has already shown its clear superiority for postnatal and prenatal diagnosis of the disorder and for carrier detection. However, it is of great importance to have conditions which guarantee optimal reliability and sensitivity. Some mutations may be more difficult to detect, especially in female carriers: this is the case for small amplifications of the CGG repeat (premutations) or for smears which can be generated by the instability of the full mutation in somatic tissues. We present the various alternatives (probe/enzymes combinations) for Southern blot based diagnosis, the possible artefacts and our detailed experimental protocol, which has given excellent results on a large number of families. While detection of amplification, using for instance EcoRI, appears sufficient for initial testing of mentally retarded patients, once the fra(X) diagnosis has been established, we favor the use of an EcoRI+EagI digest, which detects both amplification and abnormal methylation, for analysis of the family, including carrier detection and prenatal diagnosis. We discuss the place of proposed PCR based techniques for detection of mutations, or for indirect tracking using polymorphic microsatellites in the immediate vicinity of the fra(X) locus.