Targeted Next Generation Sequencing is a valuable diagnostic tool for Inherited Peripheral Neuropathies

Introduction:Inherited Peripheral Neuropathies.represent a large group of hereditary diseases that comprises the hereditary motor and sensory neuropathy, more known as Charcot-Marie-Tooth (CMT), hereditary sensory and autonomic neuropathy (HSNA) and distal motor neuropathy (dHMN). Typically IPNs are characterized by distal wasting and weakness, decreased deep tendon reflexes, contractures and skeletal deformities. More than 60 nuclear genes are implicated in the pathogenesis.In the last few years, the molecular diagnosis improved with the advent of NGS, which is now the strategy used in routine in many laboratories. Aim of the study We performed genetic tests for 49 genetically un-diagnosed IPNs patients who had been excluded 17p12 (PMP22) duplication by Real Time PCR and microsatellite markers. Materials and Methods: We designed a genes panel using the Ion Ampliseq Designer (Thermo Fisher Scientific) targeting the coding sequence comprising the intron/exon boundaries of 32 genes associated with IPNs phenotype, reaching a target coverage of 99,13%. The amplicon libraries obtained by the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific), were subsequently barcoded, pool together in equimolar concentrations and enriched using the One Touch 2 instrument. Sequencing was performed on an Ion PGM machine using an Ion 318 sequencing chip. The obtained raw data were analyzed with the Ion Torrent Suite 5.10 for the alignment and variants caller. To annotate the variants ANNOVAR tool was also used. The identified variants were confirmed by Sanger sequencing. Results: By using IPNs genes panel, we are able to detect in 19 out of 49 examined patients pathogenic variants in the following genes: KIF1B, MFN2, MPZ, SETX, SBF2, DCTN1, GARS, GAN, DNM2, WINK1 and FIG4. KIF1B and MFN2 genes result to be, in our study, the most frequent mutated genes. Conclusions: Targeted gene panel sequencing is a power tool to resolve uncertain cases with suspected IPN diagnosis. It strikingly reduces the cost and the time with better detection efficiency; but, because the number of causative genes for IPNs is still growing, it is necessary to have a continuous updating of the involved genes to facilitate the diagnosis of the unsolved cases.