A Highly Sensitive Genetic Protocol to Detect NF1 Mutations

Valero, M. Carmen; Martín, Yolanda; Hernández-Imaz, Elisabete; Hernández, Alba; Melean, Germán; Valero, Ana; Rodríguez-Álvarez, Francisco J.; Tellerı́a, Dolores; Hernández-Chico, Concepción

doi:10.1016/j.jmoldx.2010.09.002

Cited by 95 publications

(49 citation statements)

References 44 publications

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“…8,9,23 This is consistent with the results of our study, in which patients with large deletions had severe clinical phenotypes. In particular, gross generalized cutaneous neurofibromas were observed in patient 29, and these were more severe than those of other patients with large deletions.…”

Section: Discussionsupporting

confidence: 92%

Clinical and Molecular Characterization of NF1 Patients

et al. 2016

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Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, and the presence of pseudogenes.Our goal was to establish a sensitive, feasible, and comparatively economical protocol to detect NF1 mutations using blood samples.We developed a method to screen patients for mutations. Thirty-two NF1 patients from 32 unrelated families and 120 unrelated population-match controls were investigated in this study. Specific primers were designed for NF1 to avoid pseudogenes. NF1 mutations were detected by sequencing at the deoxyribonucleic acid (DNA) and complementary DNA (cDNA) levels, and multiplex ligation-dependent probe amplification (MLPA) and familial segregation analyses were used.Forty-four specific primers designed according to the NF1 structure were successfully used for polymerase chain reaction (PCR) and DNA sequencing, which was more feasible and useful than cDNA sequencing. Thirty distinct NF1 mutations were identified in 32 patients. Thirteen mutations were novel and most were frameshift mutations (33.3%). Mutations were detected at a rate of 93.8%.Our study suggests that this sensitive, feasible, and comparatively economical protocol is effective for the detection of NF1 mutations.

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Section: Discussionsupporting

confidence: 92%

Clinical and Molecular Characterization of NF1 Patients

et al. 2016

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“…We identified 10 different pathogenic variations in 11 unrelated patients with clinical diagnosis of NF1, which represents a mutation detection rate of 91%. This high mutation rate is similar to the results of other studies that had used an exhaustive multistep methodology [9,24]. …”

Section: Discussionsupporting

confidence: 87%

“…The latter is a property of the capture probes, and therefore with the advance of technologies, it is expected that, to some extent, this limitation is going to be compensated by a better probe set design [26], which would substantially improve the performance of our approach. Sequencing the exon 1 of the NF1 gene is also a challenge in cDNA-based protocols with Sanger methodology [24]. …”

Section: Discussionmentioning

confidence: 99%

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

Cunha

Oliveira

Silva

et al. 2016

Genes

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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.

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“…A multi-step protocol involving analysis of genomic DNA and mRNA with RT-PCR, direct sequencing, multiplex ligation-dependent probe amplification (MLPA), and previously using also microsatellite marker analysis and FISH, was required to identify up to 95% of pathogenic mutations in individuals fulfilling the clinical NIH diagnostic criteria [148-150]. Analysis of RNA is essential as splicing mutations may be present in more than 20% of individuals with NF1 syndrome [144, 149, 150], and may be located deep in introns which may be missed when only exons are studied.…”

Section: Introductionmentioning

confidence: 99%

The NF1 gene revisited - from bench to bedside

et al. 2014

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Neurofibromatosis type 1 (NF1) is a relatively common tumour predisposition syndrome related to germline aberrations of NF1, a tumour suppressor gene. The gene product neurofibromin is a negative regulator of the Ras cellular proliferation pathway, and also exerts tumour suppression via other mechanisms.Recent next-generation sequencing projects have revealed somatic NF1 aberrations in various sporadic tumours. NF1 plays a critical role in a wide range of tumours. NF1 alterations appear to be associated with resistance to therapy and adverse outcomes in several tumour types.Identification of a patient's germline or somatic NF1 aberrations can be challenging, as NF1 is one of the largest human genes, with a myriad of possible mutations. Epigenetic factors may also contribute to inadequate levels of neurofibromin in cancer cells.Clinical trials of NF1-based therapeutic approaches are currently limited. Preclinical studies on neurofibromin-deficient malignancies have mainly been on malignant peripheral nerve sheath tumour cell lines or xenografts derived from NF1 patients. However, the emerging recognition of the role of NF1 in sporadic cancers may lead to the development of NF1-based treatments for other tumour types. Improved understanding of the implications of NF1 aberrations is critical for the development of novel therapeutic strategies.

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A Highly Sensitive Genetic Protocol to Detect NF1 Mutations

Cited by 95 publications

References 44 publications

Clinical and Molecular Characterization of NF1 Patients

Clinical and Molecular Characterization of NF1 Patients

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

The NF1 gene revisited - from bench to bedside

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