High-throughput analysis of fragile X (CGG)(n) alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis

Fragile X syndrome is caused by expansion of a (CGG)(n) trinucleotide repeat within the 5' untranslated region of the FMR1 gene transcript. The disease is reliably diagnosed by Southern blotting, but this method constitutes a significant workload and requires large samples. Therefore, for large research or screening projects in which a large majority of the samples will be normal, a more rapid and less expensive method is needed. We present a method for accurate, high-throughput analysis of the FRAXA (CGG)(n) region in the normal and premutation range. The method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on filter-paper followed by automated capillary electrophoresis and detection by multicolour fluorescence. This method allows a throughput of 144 samples in 48 h, with an intra-assay accuracy in size determination of 0.2-1.8 bp. We performed a blind reanalysis of samples from 30 patients, previously analysed by Southern blotting or PCR with radioactive labelling. In this study normal and premutation alleles, ranging from 28-121 (CGG)(n) repeats, were correctly determined with respect to number of (CGG)(n) repeats. All full-mutation alleles and one large premutation allele in a sample of a heterozygote failed to amplify. The method was used to determine the distribution of FRAXA (CGG)(n) repeats in the Danish population.