Fast and accurate SNVs and CNVs screening in Parkinson's Disease patients using Next-Generation approach

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting millions of people. Genome-wide association studies (GWAS) have found >25 genetic risk factors and at least 15 loci directly associated with PD. Recent advances in Next Generation DNA Sequencing technologies, such as the semiconductor-based Ion Torrent platform, make multigene sequencing cheaper, faster, and more reliable. Objectives: Our objective was to test the power of the Next Generation Sequencing technology to analyze a large cohort samples of PD patients from Southern Italy by screening the majority of the most relevant PD-related genes known for single and compound mutations. Methods: To achive a rapid,robust, and cost-effective genetic analysis of a PD cohort, we designed a multiplex, amplicon based gene panel made by 42 genes. We conducted parallel sequencing using the Ion Torrent Personal Genome Machine (PGM)(®) system to detect mutations in 42 blood DNA samples from PD patients. To process data from PGM Ion Torrent runs, Ion Torrent Suite (TS) software v. 5.10 was used. Annotation was made using ANNOVAR and the variants where priorized using a standard filtering pipeline. Results: After bioinformatics analysis and filtering, 98% coverage of the targeted regions was obtained with at least >200-fold mean depth. We detected 50 coding nonsynonymous variants (indels, single-nucleotide variations (SNVs) and frameshift variants with a MAF<0.01. Of these 50 variations, 9 were identified in PARK2, 12 in LRRK2 and 1 in PINK1. The remaining variations were found in the other genes sequenced, most of which are strongly involved in the pathogenesis of PD. We are able to find a total of 154 CNVs (52 amplifications and 102 deletions). Out of these SNCA1 amplification, PRKN 1 deletion, PARK7 2 deletions, LRRK2 1 amplification and 7 deletions.Conclusions: Benchtop next-generation sequencing is a powerful method for genetic screening for PD. Our results indicated that NGS analysis yielded a high frequency of discovery SNVs e CNVs variants in carriers from an enriched Southern-Italy PD sample.