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 metho d constitutes a significant workload and requires large samples. There fore, for large research or screening projects in which a large majori ty of the samples will be normal, a more rapid and less expensive meth od is needed. We present a method for accurate, high-throughput analys is of the FRAXA (CGG)(n) region in the normal and premutation range. T he method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on fi lter-paper followed by automated capillary electrophoresis and detecti on by multicolour fluorescence. This method allows a throughput of 144 samples in 48 h, with an intra-assay accuracy in size determination o f 0.2-1.8 bp. We performed a blind reanalysis of samples from 30 patie nts, 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 num ber of (CGG)(n) repeats. All full-mutation alleles and one large premu tation allele in a sample of a heterozygote failed to amplify. The met hod was used to determine the distribution of FRAXA (CGG)(n) repeats i n the Danish population.