Evaluation of a CGG Repeat Primed PCR system, designed for detection of Fragile X expanded alleles, in clinical prenatal samples.

Abnormal expansion of the (CGG)n repeat in the 5'untranslated region (UTR) of the FMR1 gene is known to generate Fragile X syndrome (FXS), the most common form of cognitive impairment, and several other diseases in patients. The expansion reduces the expression of the FMR1 by promoting DNA hypermethylation of this region when exceeding over 200 repeats. Molecular diagnosis relies on determination of the number of allele repeats in the DNA template, and assessment of the methylation state of the FMR1 locus. Current conventional PCR amplification is only successful for normal and small permutation alleles, and is not informative for homozygous repeat alleles. Expanded alleles too large to amplify efficiently and female homozygous samples require additional Southern Blot (SB) analysis for categorization and sizing. Although regarded as gold standard, this technique is laborious, time consuming and involves (not always available) large amounts of DNA. However, accurate and efficient quantification of the number of repeats in the 5' UTR of FMR1 gene is essential as premutation alleles are common in the general population.
We evaluated a commercially available assay (Asuragen) using a large set of blinded archived prenatal samples that were previously analyzed for FXS with conventional PCR and SB analysis in our Center. The PCR reagentia were able to identify accurately and quickly FMR1 genotypes, ranging from normal over premutation to full mutation alleles, in patient and control samples. Exact sizing was possible for a spectrum of permutation alleles. The assay is also sensitive to size mosaicism and AGG interruptions.