RAPID IDENTIFICATION OF PATHOGENIC VARIANTS IN NEUROFIBROMATOSIS DISEASE BY GENE-PANEL SEQUENCING.

INTRODUCTION: Neurofibromatosis (NF) is a group of three conditions characterized by tumors grow in the nervous system: neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis. In NF1 the tumors are neurofibromas, while in NF2 and schwannomatosis tumors of Schwann cells are more common. All types are autosomal dominant disorders. AIM: Since the actual methods (Sanger sequencing, DHPLC) to diagnose NF disease is time and money consuming, especially for large genes such as NF1, we have developed a genetic panel to study either the genes responsible for the different types of the disease or genes associated with the disease. 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 the following genes associated with NF phenotype: NF1, NF2, SMARCB1, LZRT1, COQ6 ad SUZ, reaching a target coverage of 99,8%. We analysed 25 patients, three of whom were NF1 positive controls and two negative controls. The amplicon libraries obtained by the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific), were subsequently barcoded, pooled together in equimolar concentrations and enriched using the One Touch 2 instrument. Sequencing was perfomed on an Ion PGM machine using an Ion 318 sequencing chip. The obtained raw data were analysed with the Ion Torrent Suite 5.2 for the alignment and variants caller. To annotate the variants ANNOVAR tool was used. RESULTS: We were able to obtain an output of about 6 million of reads which were aligned to 99,6% with the Human genome 19 (GRCh37/hg19), returning a mean depth of 1500X per samples. In addition to confirming the variants identified in the three controls, 11 variations were identified in the NF1 gene, all confirmed by Sanger sequencing. Interestingly, using such depth, we are also able to identify in a patient a NF1 mosaic variation. CONCLUSIONS: Using the NGS sequencing with an Ampliseq approach, we were able to detect pathogenic variations in the NF1 gene. The NGS sequencing, respect to the standard technology such as Sanger sequencing, is less expensive and faster to analyze multiple genes in different patients at the same time. Furthermore, the NGS can used to take over mosaic variations, difficult to find out by the standard methods.