Sanger Sequencing for Disease Variant Confirmation

Most likely disease-causing variants were confirmed by Sanger sequencing in the patient and the available family members. Primers were designed using Primer3 algorithm[14 (link)] and purchased at Microsynth AG (Balgach, Switzerland). All target regions were amplified in duplicate from genomic DNA of the patients and available family members using Hot FirePol^® DNA Polymerase (Solis BioDyne, Tartu, Estonia). PCR products were purified by treating them with ExoSAP reagent (Affymetrix, Santa Clara, CA) and sequenced using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, California, USA) and ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA). Sanger sequencing data analysis was performed using the Sequencing Analysis Software v5.4, SeqScape v2.6 (Applied Biosystems, Carlsbad, California, USA), MutationSurveyorV5.0.0 (Soft Genetics, Pennsylvania, USA) and Chromas (Technelysium, South Brisbane, Australia) to identify the likely disease causing mutations. Mutation is defined as previously described[15 (link)].