Increased copy-number variant load of associated risk genes in sporadic cases of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-related neurodegenerative disease characterized by selective loss of motor neurons in the brainstem and spinal cord. Several genetic factors have been associated to ALS, ranging from causal genes and potential risk factors to disease modifiers. Here, we applied an exon-centric aCGH method to investigate, in sporadic ALS patients, the load of CNVs in 131 genes previously associated to ALS. Our approach revealed that CNV load, defined as the total number of CNVs or their length, was significantly higher in ALS cases. A total of 32 Southern Italian patients (17 males and 15 females), with a diagnosis of sALS according to the EL Escorial criteria (21), and 20 controls of Southern Italian patients affected by neurological disorders without diagnosis of ALS, were analysed by using an 8x60K custom exon-centric NeuroArray platform v. 2.0 (Agilent Technologies, Santa Clara, CA), tailored to detect single/multi-exon deletions and duplications in a large panel of ALS-related genes. DNA test and a reference of the same sex (Euro Reference, Agilent Technologies, Santa Clara, CA) were processed according to the manufacturer’s protocol (Agilent Technologies, Santa Clara, CA). sALS patient DNAs were labeled with Cy5-dUTP and reference DNAs for reference DNAs. Arrays were scanned at 3 µm resolution using an Agilent G4900DA SureScan Microarray Scanner System and aCGH image data were processed using Agilent’s Feature Extraction software. Feature extracted raw data was normalized, analyzed and visualized using Agilent CytoGenomics v. 4.0.3.12 and Genomic Workbench v. 7.0.4.0 software (Agilent Technologies, Santa Clara, CA, USA). GC correction with a window size of 2 kb and Diploid Peak Centralization were used for normalization. The Centralization Normalization Algorithm with a threshold of 6.0 and a bin size of 10 was also used for detecting aberrant regions or regions of constant CNVs. Aberrations were detected by the Aberration Detection Method II algorithm (ADM-2), with a sensitivity threshold of 6.0 and moving average window of 2Mb. Human reference sequence hg19 assembly was used to define the genomic coordinates of detected CNVs.