Genetic Mutation Analysis of Parkinson's Disease Patients Using Multigene Next-Generation Sequencing Panels.
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 new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent platform, make multigene sequencing cheaper, faster, and more reliable.



Our objective was to test the power of this next-generation sequencing technology to analyze large samples by screening the majority of the most relevant PD-related genes known for single and compound mutations.



To archive a rapid, robust, and cost-effective genetic analysis of a PD cohort, we designed a multiplex, polymerase chain reaction (PCR)-based primer panel to amplify and sequence coding exons of 15 PD-associated genes (SNCA, LRRK2, PARK2, PINK1, PARK7, GIGYF2, ATP13A2, UCHL1, PLA2G6, FBXO7, EIF4G1, VPS35, ACMSD, APOE, and GBA). We conducted parallel sequencing using the Ion Torrent Personal Genome Machine(®) system to detect mutations in 92 blood DNA samples from PD patients.



After bioinformatics analysis and filtering, 95.13 % coverage of the targeted region was obtained at >40-fold mean coverage. The results revealed 44 previously documented variants in these 15 genes, with five revealed as pathogenic. We also discovered six novel variants, five of which had an in silico prediction of being pathogenic.



Benchtop next-generation sequencing is a powerful method for genetic screening for PD. Our results indicated that it yielded a high frequency of discovery (66 %; n = 92) of variants in carriers from an enriched Spanish PD sample.
Genomics Platform, Biodonostia Research Institute, Dr. Begiristain s/n, San Sebastian, 20014, Gipuzkoa, Spain. ana.gorostidi@biodonostia.org.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain. ana.gorostidi@biodonostia.org.
Ana
A
Gorostidi
Neurology Service, Hospital Universitario Donostia, San Sebastian, Spain.|Ilundain Fundazioa, San Sebastian, Spain.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain.|Centro de Investigación Biomédicas en Red Enfermedades Neurodegenerativas (CIBERNED), Institute Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain.|Department of Neuroscience, University of the Basque Country UPV/EHU, Leioa, Spain.
José Félix
JF
Martí-Massó
Neurology Service, Hospital Universitario Donostia, San Sebastian, Spain.|Ilundain Fundazioa, San Sebastian, Spain.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain.|Centro de Investigación Biomédicas en Red Enfermedades Neurodegenerativas (CIBERNED), Institute Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain.
Alberto
A
Bergareche
Neurology Service, Hospital Universitario Donostia, San Sebastian, Spain.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain.|Centro de Investigación Biomédicas en Red Enfermedades Neurodegenerativas (CIBERNED), Institute Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain.|Ikerbasque. Basque Foundation for Science, Bilbao, Spain.
Mari Cruz
MC
Rodríguez-Oroz
Neurology Service, Hospital Universitario Donostia, San Sebastian, Spain.|Ilundain Fundazioa, San Sebastian, Spain.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain.|Centro de Investigación Biomédicas en Red Enfermedades Neurodegenerativas (CIBERNED), Institute Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain.|Department of Neuroscience, University of the Basque Country UPV/EHU, Leioa, Spain.
Adolfo
A
López de Munain
Neurology Service, Hospital Universitario Donostia, San Sebastian, Spain.|Ilundain Fundazioa, San Sebastian, Spain.|Neuroscience Area, Biodonostia Research Institute, San Sebastian, Spain.|Centro de Investigación Biomédicas en Red Enfermedades Neurodegenerativas (CIBERNED), Institute Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain.
Javier
J
Ruiz-Martínez
D000328:Adult; D000368:Aged; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010300:Parkinson Disease / Q000175:diagnosis / Q000235:genetics*; D040641:Quantitative Trait Loci; D012720:Severity of Illness Index