Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

Abstract

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P¼0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (Po0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.

Exhaustive non-synonymous variants functionality prediction enables high resolution characterization of the neurofibromin architecture

“…Although such misclassification of pathogenic variants as benign during training might lead to biased estimations, their overall frequency is expected to be low and therefore their relative impact on overall model performance is expected to be minor. Finally, we note that although gene-specific in-silico analysis such as the one described here may facilitate variant prioritization and functional assessment, functional characterization remains the gold standard for the definitive classification of variants [ 48 , 50 ].…”

Whole Exome Sequencing of Growing and Non-Growing Cutaneous Neurofibromas from a Single Patient with Neurofibromatosis Type 1

“…As such, NF1 is considered a classic tumor suppressor gene and its mechanism in NF1 is felt to be consistent with Knudson's two-hit hypothesis in which a patient carries a mutated germline NF1 gene copy and tumor development, including CN, then requires a second hit[14]. While the germline mutations in NF1 are well cataloged, with >1000 mutations identified to date, there is a paucity of information on the assumed acquired somatic mutations in CN[15]. This deficiency is likely related to both the difficulty of detecting somatic mutations in CN due to cellular heterogeneity within the tumors and also the small number of CN analyzed.…”

Mosaic Deletion of the NF1 Gene in a Patient With Cognitive Disability and Dysmorphic Features But Without Diagnostic Features of NF1

“…However, the condition is very variable, even within families (for a review see [Pasmant et al, ]), and it is therefore thought that modifier genes, environmental effects and random events may also be important. Schwann cells in neurofibromas ([Bottillo et al, ; Thomas et al, ]) and melanocytes in café‐au‐lait patches [Maertens et al, ; De Schepper et al, ]) have been shown to have a mutation in both NF1 alleles, representing germline and acquired somatic mutations.…”