192 control chromosomes were screened for the presence of the mutations identified in the 10 patients using HRM. Primers were designed using the HRMA Assay Design module of Beacon Designer™ 8.10 (Premier Biosoft, CA, USA). HRM was performed on a LightCycler 480 (Roche, Penzberg, Germany) with the LCGreen+ incorporating dye (Idaho Technology Inc., Salt Lake City, UT, USA). Meltcurve analysis performed by the Gene Scanning module of the LightCycler software. Samples with deviating curves were analysed by Sanger sequencing. The mutation in patient 6 was identified using the same protocol, as part of the cohort of 148 probands with idiopathic ASD, for which microarray analysis did not reveal any abnormalities.
