NeuroArray: A Customized aCGH for the Analysis of Copy Number Variations in Neurological Disorders

Abstract

Background: Neurological disorders are a highly heterogeneous group of pathological conditions that affect both the peripheral and the central nervous system. These pathologies are characterized by a complex and multifactorial etiology involving numerous environmental agents and genetic susceptibility factors. For this reason, the investigation of their pathogenetic basis by means of traditional methodological approaches is rather arduous. High-throughput genotyping technologies, including the microarray-based comparative genomic hybridization (aCGH), are currently replacing classical detection methods, providing powerful molecular tools to identify genomic unbalanced structural rearrangements and explore their role in the pathogenesis of many complex human diseases.

Methods: In this report, we comprehensively describe the design method, the procedures, validation, and implementation of an exon-centric customized aCGH (NeuroArray 1.0), tailored to detect both single and multi-exon deletions or duplications in a large set of multi- and monogenic neurological diseases. This focused platform enables a targeted measurement of structural imbalances across the human genome, targeting the clinically relevant genes at exon-level resolution.

Conclusion: An increasing use of the NeuroArray platform may offer new insights in investigating potential overlapping gene signatures among neurological conditions and defining genotype-phenotype relationships.

Keywords: CNVs; Custom array; Genes; Methods; Neurological diseases; aCGH.

Fig. (1)

Clinically relevant genes selected for the NeuroArray customization. Graphical representation showing the number of clinically relevant genes involved in neurological diseases and included in the aCGH NeuroArray. The largest number of genes belongs to Alzheimer’s disease’s panel, followed by Parkinson’s disease and Epilepsy forms.

Fig. (2)

NeuroArray vs. commercial platforms. A: The human SPG7 gene is located on chromosome 16q24.3, spanning 49.3 Kb of genomic DNA. B: This gene produces two different transcripts, the longest of which encompasses 17 exonic regions. Both transcripts are illustrated in the figure and are indicated by the NCBI Accession Number on the right. SPG7 exons are represented in the figure by black boxes and are numbered consecutively. The dashed line represents intronic regions. C: Distribution of oligonucleotide probes on the commercially available whole-genome Agilent SurePrint G3 Human CGH Microarray 8x60K. D: Distribution of oligonucleotide probes in the entire exonic regions of SPG7 gene in the customized NeuroArray design. E: Detection of heterozygous exonic deletion in SPG7 gene in a patient with Hereditary Spastic Paraplegia 7. NeuroArray showed the deletion of the exon 2 and 3 of SPG7 gene, later confirmed by MLPA assay.

Fig. (3)

Detection of NF1 gene and neighboring genes deletion in a patient with Neurofibromatosis type I. NeuroArray confirmed the deletion of the tumor-suppressive NF1 gene, previously detected by two MLPA kits (SALSA MLPA P081/082 and P122-C1 by MRC-Holland, Amsterdam, The Netherlands). Furthermore, it revealed a larger deletion including further overlapping or neighboring genes. On the left is represented chromosome 17. NeuroArray aCGH data visualization and analysis obtained from CytoGenomics software are shown in the middle panel. The red area represents the deleted region. On top of the panel, the size of the deletion and the chromosomal locus are indicated. Red and blue dots represent the log2 ratios for the relative hybridization intensities of each spotted probe. The dots with an average log2 ratio around -1 indicate a heterozygous deletion. On the right, probe coverage of MLPA kits is graphically represented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (4)

Detection of three single-gene deletions (VPS54, SNC7A, and CHMP2B) in a patient with ALS. NeuroArray has revealed the heterozygous deletion of three genomic regions encompassing three known ALS-related genes in an ALS patient. In particular, the regions included: i) 64,184 Kb deleted on Chr.2 (cytoband 2p14) embracing VPS54 gene (Panel A); ii) 5,1 Kb in the cytoband 2q24 including the SCN7A gene (Panel B) and iii) a region on Chr.3 (2.5 Mb from cytoband 3p11.2 to 3p11.1) embracing CHMP2B and further overlapping and neighboring genes (Panel C). The qPCR assay, performed on exon 4 of VPS54, exon 3 of SCN7A and exon 5 CHMP2B, has confirmed NeuroArray findings. For each gene is reported the comparison between NeuroArray aCGH results (left) and the corresponding measurement by real-time quantitative qPCR (right). NeuroArray aCGH data visualization and analysis were performed by CytoGenomics software. Resultant Real-Time qPCR standard curves for detection of each gene and related calibrator controls are shown; cycle number (axis X) is blotted against fluorescent signal (axis Y) obtained in every cycle at the end of the annealing step.