Targeted NGS-panel can improve the simultaneous identification of CNVs and SNVs in Mendelian diseases

The current genetic molecular diagnosis has been strongly improved by the Next-Generation sequencing (NGS). The new technology has demonstrated a big power to identify single-nucleotide variations (SNVs), small insertions/deletions and copy number variants (CNVs) underpinning disease onset. We used a NGS approach, to detect simultaneously CNVs and SNVs in patients affected by Hereditary Spastic Paraplegia (HSP) and Neurofibromatosis type 1 (NF1). High coverage targeted NGS data were generated at the Next Generation Sequencing Core of the Institute of Neurological Sciences, CNR, Mangone (CS) by amplicon-based gene panels. We used a neurological panel containing 755 genes responsible for different neurological disease and a custom panel targeting the coding sequence of six genes associated with neurocutaneous syndromes. The enriched libraries were sequenced on the Personal Genome Machine (PGM) from ThermoFisher Scientific. Primary bioinformatic analisys was carried out using Ion Torrent suite vers. 5.6. To calculate the copy number variants we used the sequencing read depth (RD) approach, based on the assumption that the RD signal is proportional to the number of copies of the chromosomal segment present in the DNA samples. For the normalization we generated a baseline with ten normal controls. By using the neurological panel we detected in an individual affected by a recessive form of HSP the variation p.A510V and the deletion of the exons 1-3 in SPG7 gene. In the NF1 screening using the custom panel, we identified in one individual the already reported deletion of exons 6-8 and in another subject the novel SNV c.2850+2 T>C. The SNVs were validated by Sanger method, whereas the deletion in SPG7 gene was confirmed by MLPA and the deletion in NF1 gene by qPCR using Syber green and ??Ct method. In conclusion, unlike the methods already in use such as Sanger sequencing, MLPA, CGH array and qPCR, the data obtained by NGS can be integrated allowing, by a single experiment, to detect all types of the genetic variations, drastically reducing the costs and times of various genetic tests.