Flexigene

DNA was extracted from peripheral blood or frozen brain tissue following the manufacturer’s protocols (Flexigene (Qiagen) or QuickGene DNA whole blood kit (Autogen) for blood, and QIAsymphony DNA Mini Kit (Qiagen) for brain tissue). All patients were screened for C9orf72 hexanucleotide repeat expansions using a modified repeat-primed polymerase-chain reaction (PCR) as previously described(Suh et al., 2015 ), and we excluded any patient with >=30 hexanucleotide repeats. Of the remaining individuals, we evaluated family history using a three-generation pedigree history as previously reported(Wood et al., 2013 (link)). For cases that had a family history of the same disease we sequenced 45 genes previously associated with neurodegenerative disease(Toledo et al., 2014 (link)), including four genes known to be associated with ALS (e.g. SOD1(Rosen, 1993 (link)), TARDBP(Kabashi et al., 2008 (link)), FUS(Kwiatkowski et al., 2009 (link)) (Vance et al., 2009 (link)), and VCP(Johnson et al., 2010 (link))). Sequencing was performed using a custom-targeted next-generation sequencing panel (MiND-Seq)(Toledo et al., 2014 (link)), and analyzed using Mutation Surveyor software (Soft Genetics, State College, PA). We excluded any individuals identified as having a pathogenic mutation.

Once final consent was obtained, subjects provided a 10 ml sample of blood, which was labeled with their name, date of birth, and medical record number and then sent to a processing laboratory. In the processing laboratory, blood samples were verified, coded, entered into the Freezerworks^© specimen management software system and stored at 4°C for no more than 10 days. DNA was then extracted using Autogen FlexSTAR^© and Qiagen Flexigene^© technology, placed in pre-labeled 2 ml rubber-gasketed cryovials and stored at -80°C. The same basic procedure for collection and processing was used throughout the study on all samples.