Abstract
We have developed a novel fragile X locus repeat assay that is a simple and high-throughput method that, with clinical validation, may be suitable for screening. It uses amplification of the FMR1 trinucleotide repeat region, followed by a hybridization assay to quantify the number of repeats in the amplicons. To our knowledge, this is the first repeat-counting assay that uses fluorescent signals rather than electrophoresis or mass spectrometry as the signaling mechanism. We also report the development of a simple microfluidic electrophoresis reflex test that uses the same amplicons and reduces the need for Southern blots to differentiate homozygous female normal samples from full mutations. The new assay, which is based on a suspension-array hybridization method, was tested on a series of male and female reference samples spanning the range from normal to full mutations. It was also tested on DNA from 1008 dried blood spot samples from pregnant women in their first trimester. The hybridization assay identified 51 of those as potentially expanded alleles of ≥45 repeats or as intermediate or higher in FMR1 repeat classification. Of these screen-positive samples, eight were confirmed by microfluidic electrophoresis as premutations consisting of ≥55 repeats. The FMR1 repeat assay is straightforward to run in high throughput, and the results are in the form of numerical ratios for ease of initial interpretation.