Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

Abstract

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

“…Thirty adults had both types of tumors (30/64 ≥19 years, 46.9%). Ten individuals ≥17 years had >100 cutaneous and/or subcutaneous nodules, including a 47-year-old man previously reported 45 with >1,400 neurofibromas (individual counts of externally visible neurofibromas; BRA-R38) and a 17-year-old woman (ROT-R1CMUL) with >500 cutaneous neurofibromas, >100 subcutaneous neurofibromas, and >100 intradermal neurofibromas. Nine out of ten individuals with a very high number of neurofibromas carried a missense mutation at codon 847: c.2540T>G (p.Leu847Arg) (2/9) or c.2540T>C (p.Leu847Pro) (7/9, including two individuals with metastasized MPNSTs).…”

Comprehensive targeted next‐generation sequencing approach in the molecular diagnosis of gastrointestinal stromal tumor

“…No second hit in NF1 could be found in this particular case but since no other diver could be identified, we assume that a second hit was most likely missed by targeted NGS analysis. Indeed, sequencing the NF1 gene is particularly challenging given its large size and the potential presence of deep intronic splicing variants, which are typically missed when targeted DNA‐based sequencing assays are used that are limited to the analysis of exonic regions and conserved splice sites 38 …”

Genetic diagnosis of neurofibromatosis type 1: targeted next- generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis

“…However, the procedures for molecular diagnosis of NF1 are usually expensive, time-consuming, and labor-intensive [ 15 – 21 , 26 – 28 ]. The development of next-generation sequencing (NGS) technologies which allows for rapid identification of disease-causing mutations and high-risk alleles has recently been introduced into NF1 diagnosis [ 29 – 34 ]. Owing to the complexity with some aspects of clinical diagnoses and the need for a better understanding of its molecular relationships, an extended genetic characterization of this disorder will be helpful in a clinical setting.…”