Identification of putative driver mutations in neurofibromatosis type 1 (NF1)-associated plexiform neurofibromas. A. Pemov1, H. Li2, N. F. Hansen3, J. C. Mullikin3,4, M. Wallace2, D. R. Stewart1 1) Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD; 2) University of Florida, Gainesville, FL; 3) Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; 4) NIH Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD.

   BACKGROUND. NF1 is an autosomal dominant tumor pre-disposition genetic disorder, caused by constitutive inactivation of one of two copies of the tumor suppressor NF1. Individuals with NF1 are prone to the development of both benign and malignant tumors. Plexiform neurofibromas (PNF) are benign tumors; however, the tumors are associated with high morbidity. In addition, 8-12% of people with NF1 develop malignant peripheral nerve sheath tumor (most originating from PNFs). Although it is accepted that somatic inactivation of NF1 is an early necessary step in development of PNF, little is known regarding which other genes are affected in the tumor. In this study, we performed whole-exome sequencing (WES) of 24 tumor samples matched with germline DNA obtained from 22 unrelated NF1 patients. METHODS. Germline DNA was extracted from peripheral white blood cells and tumor DNA was obtained from primary Schwann cell (SC) cultures established from dissected plexiform neurofibromas. All cell cultures were of low passage and contained at least 70% SC. We used the Illumina TruSeq Exome Enrichment Kit that targets 62 Mb of exonic sequence, including 5 UTR, 3 UTR, microRNA, and other non-coding RNA. After WES, aligning, genotype calling and filtering, we analyzed the data using statistical software (Youn & Simon, Bioinformatics, 2011, v.11, p.175) designed to identify driver genes from passengers. In addition to WES, the DNA samples were analyzed on Illumina 2.5M SNP-arrays to detect LOH regions. RESULTS. First, we analyzed NF1 locus in both germline and tumor DNA. We identified pathogenic mutations in the NF1 gene in all germline samples. In addition, we identified somatic inactivation of NF1 in 15 out of 24 tumors. Second, we estimated the mutation burden in individual samples. There were on average 10 (range 1-55) somatic mutations per tumor. Finally, we analyzed the data to distinguish driver mutations. After correcting P-values for multiple testing we identified 9 frequently-mutated statistically significant genes. Evaluation of biological functions of the genes revealed that such processes as chromosome partition, nonsense-mediated mRNA decay and RNA-mediated silencing might play a role in PNF progression. CONCLUSIONS. To our knowledge, this is the first attempt to identify somatic mutations in NF1-associated PNF via WES. PNFs accumulate modest number of mutations. We identified 9 putative driver genes. The majority of the tumors contained 2-3 driver mutations.

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