Computational protocol: Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients

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Protocol publication

[…] Genomic DNA isolation was performed either from peripheral blood using the QIAamp Blood Midi Kit (Qiagen GmbH, Hilden, Germany) or by means of the phenol-chloroform extraction method as previously described with minor modifications or saliva as previously described []. Whole-genome sequencing was performed using Complete Genomics’ (CA, USA) DNA nanoarray platform []. DNA sequencing coverage was ×110. Only high-quality call variants were included in the downstream analysis (> 93%). Genomes were aligned with the hg19 reference genome. Next-generation sequencing data (Complete Genomics Inc., CA) were analyzed using Ingenuity Variant Analysis version 3.1.2 (Ingenuity® Systems, This is well-established software that identifies associations between phenotypes, defined by the user in terms of the classification of the tested individuals, and variants in the sequenced genome. Upon classification by phenotype (sALS vs. healthy individuals), a number of variants were listed; the output was filtered into smaller variant lists of (i) those found only in sALS patients (n = 174 single nucleotide polymorphisms (SNPs)), (ii) those not previously annotated or annotated only once, (iii) those with matching Human Gene Mutation Database (HGMD) entries, and (iv) whole-exome findings. All variants were filtered according to the analysis required, using custom scripts and Complete Genomics Analysis Tools (CGA™ Tools). [...] Pair-wise linkage disequilibrium (LD) calculations were based on phase genotype data (SNAP v2.2) [], utilizing the HapMap Phase II + III (release 28) [] and the 1000 Genomes Project ( dataset for Caucasians (CEU). Findings were visualized on HaploView 4.2 and by using the LDmatrix module on the LDlink web tool []. Τo further investigate the role of the selected FTO and TBC1D1 variants, we explored their effect on splicing motifs (including the “accept” and “donor” splice sites), the branch point, and auxiliary sequences that enhance (exonic splicing enhancers, ESE) or repress (exonic splicing silencers, ESS) splicing. To this end, in silico prediction was performed, using Human Splicing Finder ( This is a system that has rapidly become an international standard, as it combines 12 different algorithms []. Intronic variants, if “significant,” are often located within gene regions that are characterized by a reduced level of genetic variation. Conserved elements were therefore explored using Variant Effect Predictor []. […]

Pipeline specifications

Software tools Ingenuity Variant Analysis, Haploview, LDlink, HSF, VEP
Databases HGMD
Applications WGS analysis, GWAS
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Neoplasms, Motor Neuron Disease, Genetic Diseases, Inborn