Fast and Robust Next-Generation Sequencing Technique Using Ion Torrent Personal Genome Machine for the Screening of Neurofibromatosis Type 1 (NF1) Gene

Abstract

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational "hot spots." There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.

Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?

“…All previously known pathogenic variants in the 30 samples studied were identified (including point mutations, short insertions and/or deletions, and exon CNAs; Supplementary Table S2), highlighting the accuracy of this strategy as a molecular diagnostic tool for NF1, as previously described. 27 As 48 samples were multiplexed on one chip, genotyping throughput was increased over 10 times, as compared with Sanger sequence genotyping. In our hands, the NF1 and SPRED1 diagnostic NGS analysis cost approximately h90 for consumables per sample (Supplementary Figure S5).…”

“…This represents a mutation detection rate of 100%, with zero false-positive calls: all of the called variants were validated by Sanger sequencing, as described in a previous publication using the same amplification-based approach on a PGM instrument (Life Technologies). 27 In our validation sample (N ¼ 30) with a 100% experimental sensitivity, the probability of a false-negative event can be estimated at 3/N ( ¼ 10% with N ¼ 30) corresponding to an overall sensitivity Z90%, calculated using the 'rule of 3' estimate of power according to sample size. 28 Diagnostic routine application: NF1 and SPRED1 point mutations, and large complete or partial deletions identified in the prospective cohort A prospective mutation screening of the NF1 and SPRED1 genes was performed in 279 probands of the validation cohort.…”

A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor

“…A preliminary report applied next-generation sequencing to samples from patients with NF1 (REF. 41), and DNA-based sequencing is now being offered as a clinical test for diagnostic purposes (see the website of University of Alabama at Birmingham Medical Genomics Laboratory ). Thus, NF1 mutation analysis can assist in diagnosing cases of NF1 in which a clinical diagnosis cannot be established with certainty.…”

Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1

“…In this study, 52 NF1 mutations were identified, including 30 single base substitutions (12 missense and 18 nonsense), 11 splicing mutations, 7 small insertion or deletions, and 4 gross deletions [14]. As a new trend, next generation sequencing (NGS), is beginning to be used in NF1 mutation analysis [15]. Although, this new technology is robust and highly effective in screening large numbers of DNA sequences, the running cost remains expensive even if multiple samples are processed simultaneously.…”