A first cohort of 58 ALS patients was analyzed using a HaloPlex target enrichment system (Agilent Technologies, Santa Clara, CA, USA). A second cohort of 118 ALS patients (FLS and SLAS) was analyzed using a Twist target enrichment system (Twist Bioscience, CA, USA). The coding regions and intron–exon boundaries of the genes were analyzed. The following genes, known to be involved in ALS, were present in both designs: ALS2, ANG, CHCHD10, CHMP2B, DAO, DCTN1, DPYSL3, FIG4, FUS, GRN, MAPT, MATR3, NEFH, OPTN, PRPH, PSEN1, PSEN2, SETX, SIGMAR1, SOD1, SPG11, QSTM1, TAF15, TARDBP, TBK1, TREM2, TUBA4A, UBQLN2, VAPB and VCP. The FUS gene, listed as a causative gene for ALS, encodes an E3 enzyme of the ubiquitin pathway. Other ubiquitin pathway genes that have been studied were the following: in the first design (cohort 1) they were FBXO32, HECW1, MARCH5, RBX1, RNF19A, TRIM63, UBE2D2, UBED2D3, UHRF2; in the second design (cohort 2) they were HECW1, CCNF, UBQLN2. Libraries were sequenced using a MiSeq sequencer (Illumina, San Diego, CA, USA) with 150 bp paired-end sequencing (MiSeqReagent Kit V2; 300cycles) The sequences were analyzed using the bioinformatics pipeline of the ALS Center of the Hospital of Tours and the human reference genome UCSC hg19. Reads were aligned with the BWA algorithm (v.0.7.17), and variants were named using GATK tools (v.3.4) and annotated using the ANNOVAR software. Coverage was analyzed with samtools (v.1.8); variants were selected with minimum 30x coverage. Allelic frequencies in Exact databases and 1000 Genomes Projects were below 0.01% for all populations. Variants identified by NGS were validated by Sanger sequencing using a 3130xl genetic Analyzer (ThermoFisher) and classified according to ACMG [36 (link)].
Free full text: Click here
