Expanded Hexanucleotide Repeat Detection

The presence of the expanded hexanucleotide repeat and the number repeats for the longest allele was determined by previously reported methods for both a modified repeat-primed PCR and a fluorescence-based fragment size analysis as previously reported [80 (link),101 ,102 ]. Briefly, repeat-primed PCR was performed containing 100 ng genomic DNA, 1x FastStart PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA), 3.5% DMSO, 1x Q solution (Quiagen, Valencia, CA) and 0.18 mM of deazaGTP (NEB, Ipswich, MA). PCR products were run on a Genetic Analyzer 3500 (Applied Biosystems) and analyzed using GeneMapper. A sample was considered positive for a repeat expansion when assay replicates demonstrated >30 peaks and a decrementing saw-tooth pattern with 6 bp periodicity.