A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

New strategies in pleural mesothelioma: BAP1 and NF2 as novel targets for therapeutic development and risk assessment.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer with limited therapeutic options. Recent work has focused on the frequent somatic inactivation of two tumor suppressor genes in MPM-NF2 (Neurofibromatosis type 2) and the recently identified BAP1 (BRCA associated protein 1). In addition, germline mutations in BAP1 have been identified that define a new familial cancer syndrome, which includes MPM, ocular melanoma, and other cancers. These recent advances may allow screening of high-risk individuals and the development of new therapies that target key pathways in MPM.

Neurofibromatosis.

Neurofibromatosis is a heritable disease characterized by disordered growth of ectodermal tissues.

Management of the patient and family with neurofibromatosis 2: a consensus conference statement.

A consensus conference on neurofibromatosis 2 (NF2) was held in 2002 at the request of the United Kingdom (UK) Neurofibromatosis Association, with particular emphasis on vestibular schwannoma (VS) surgery. NF2 patients should be managed at specialty treatment centres, whose staff has extensive experience with the disease. All NF2 patients and their families should have access to genetic testing because presymptomatic diagnosis improves the clinical management of the disease. Some clinical manifestations of NF2, such as ocular abnormalities, can be detected in infancy; therefore, clinical screening for at-risk members of NF2 families can start at birth, with the first magnetic resonance (MRI) scan at 10-12 years of age. Minimal interference, maintenance of quality of life, and conservation of function or auditory rehabilitation are the cornerstones of NF2 management, and the decision points to achieve these goals for patients with different clinical presentations are discussed.

Mutation scanning of the NF2 gene: an improved service based on meta-PCR/sequencing, dosage analysis, and loss of heterozygosity analysis.

We describe the development and implementation of a neurofibromatosis type 2 (NF2) mutation scanning service based on novel techniques. All 17 exons of the NF2 gene are amplified in four polymerase chain reaction (PCR) reactions, using the meta-PCR technique to link the NF2 exons into chimeric concatamers. The meta-PCR products are then scanned for point mutations by direct sequencing. A four-exon dosage assay is used to test for large deletion/duplication mutations. In certain cases when tumour studies are necessary, these techniques are also combined with loss of heterozygosity analysis with three highly polymorphic microsatellite markers located within or close to the NF2 gene. Over a period of 2 years, we have applied these techniques in a service setting to the analysis of 271 patient samples (245 lymphocyte DNA; 26 schwannoma DNA). Meta-PCR and sequencing identified 90 point mutations in the 271 blood and tumor samples, 48 of which have not been reported previously. Dosage analysis identified large deletions in 12 of the lymphocyte DNA samples. In addition, over 84% of mutations were identified in 23 schwannoma DNA samples in which complete analysis was possible. Adoption of this novel strategy has increased the overall mutation detection rate in familial NF2 cases to 88% and sporadic NF2 cases to 59%. It has also allowed us to decrease our reporting turnaround times, and because of a low overall failure rate, permitted the running of an efficient and cost-effective service.