Detection Of NF1Gene Mutations By Ion Torrent Sequencing Technology In Patients With Neurofibromatosis Type Ⅰ

Objective:Use Ion Torrent personal genome machine to detect NFl gene of patients with neurofibromatosis type I, Explore the feasibility of NF1gene mutation by Ion Torrent sequencing technology, Evaluate the coherency of results in same site by Ion Torrent sequencing and Sanger sequencing, comprehend the mutation type of NF1gene in patients with neurofibromatosis type I.Method:Collected blood anticoagulanted by EDTA-Na2of14patients with neurofibromatosis type I, which were submitted to Guangzhou Kingmed Center for Clinical Laboratory during november2012to november2013. Extracted genome DNA, first all exons of NF1gene were amlified by two-tube multiplex PCR, after multiplex PCR products were purified, Ion Torrent personal genome machine(PGM) was used to detect the sequence of NF1gene of the patients. After pathogenic mutations were detected, designed PCR primers and amplifed corresponding exon, Sanger sequencing was used to verify the corresponding sites of NF1gene, exon which incompletely covered by Ion Torrent sequencing should be detected by Sanger sequencing again.Result:11patients of the14cases were detected pathogenic mutation of NF1gene,all mutations were located in different exons,no significant mutation hotpot was found,in all the mutations detected,two were nonsense mutation, five were small insertion or deletion mutation, two were missense mutation, two were splicing mutation, one patient was detected mutation which located in the missed area of5th exon,three patients were detected no pathogenic mutation. Ion Torrent coverage rate of NF1gene in all patients were greater than97%, average sequencing depth located between1500to2100X.×Sequencing results detected by Ion Torrent PGM were consistent with Sanger sequencing.Conclusion:1、Genes with large numbers of exon such as NF1detected by Ion Torrent sequencing technology is rapid and accurate, compare to traditional Sanger sequencing, operation of Ion Torrent sequencing is more simple, result is easier to read, time and cost of detection is saved, which have great application prospect and value;2、mutation type of NF1gene is diverse, no mutation hotpot was found, most of the mutations result in early termination of amino acid codon, encoded protein was truncated. In the11patients which were detected pathogenic mutation,4cases were new mutation.