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

Date Published: January 18, 2017

Publisher: Public Library of Science

Author(s): Daniel L. Faden, Saurabh Asthana, Tarik Tihan, Joseph DeRisi, Michel Kliot, Jian-Xin Gao.


The growth behaviors of cutaneous neurofibromas in patients with Neurofibromatosis type 1 are highly variable. The role of the germline NF1 mutation, somatic NF1 mutation and mutations at modifying loci, are poorly understood. We performed whole exome sequencing of three growing and three non-growing neurofibromas from a single individual to assess the role of acquired somatic mutations in neurofibroma growth behavior. 1–11 mutations were identified in each sample, including two deleterious NF1 mutations. No trends were present between the types of somatic mutations identified and growth behavior. Mutations in the HIPPO signaling pathway appeared to be overrepresented.

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Neurofibromatosis type 1 (NF1), is an autosomal dominant disorder that affects approximately one in 3500 people[1]. The underlying cause is a heterozygous mutation in the Neurofibromatosis type 1 gene (NF1). Cutaneous neurofibromas (CN) are one the most frequent manifestations and a key portion of the diagnostic criteria. NF1 has considerable variability in clinical presentation among affected individuals, within families, and even within an individual throughout life[2]. CN can vary from a few to thousands, develop throughout life at different rates and may or may not continue to grow once they have appeared. The genomic underpinnings of CN growth and development are poorly understood.

This project was approved by the IRB of UCSF and was given an exempt status an exempt status as all information was de-identified. Fresh-frozen tissue from surgically resected CNs and matched blood were obtained from a thirty-seven year old patient under Institutional Review Board approved protocols from University of California San Francisco. All tumor samples were snap-frozen at the time of surgery and stored at -80 until the time of processing. Six tumors from different body sites were selected for exome sequencing (3 growing, 3 non-growing) based on stringent quality assessment of normal and tumor DNA. Growing tumors demonstrated clinically apparent growth by serial physical exam and measurement by the senior author (MK) on 3–6 month serial exams, for greater than one year. Non-growing tumors were similarly evaluated. Before analysis, the diagnosis of each specimen underwent central pathological review and typical neurofibroma was confirmed. None of the tumors demonstrated atypical histology or evidence of malignant peripheral nerve sheath tumor. Snap frozen tumor tissue samples were analyzed by frozen section to assess neoplastic cellularity. Tumors were macrodissected to enhance tumor tissue, as confirmed by serial frozen sections. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according the manufacturers guidelines. Library preparation and whole exome sequencing was performed at Centrillion Technologies (Palo Alto, CA) using Agilent SureSelect Human All Exon v5 (Santa Clara, CA) on an Illumina HiSeq 2000 (San Diego, CA).

The mean depth of coverage across the seven samples was 40X-70X. Interrogation of the germline sample was performed and revealed a missense mutation in NF1, f2741v. Copy number analysis revealed no changes. Whole exome somatic mutation analysis identified 84 mutations total (15–26 mutations per sample) (S1 Table). After visualization in IGV this was narrowed to 32 high confidence mutations (1–11 mutations per sample). The average number of mutations per sample was five (Table 1). Two somatic mutations were found in NF1. Three mutations were cataloged in COSMIC ( CD5, NF1 and SFN. Additionally, a mutation in HLA-A was present in the COSMIC Cancer Gene Census ( as known to cause cancer. Canonical pathway overrepresentation analysis revealed the HIPPO pathway as significantly overrepresented (p value, 2.48E-4), which included the genes SFN, RASSF1 and DLG-4 (Fig 1). Biologic network overrepresentation analysis identified Nervous System Development and Function as the most significantly overrepresented (p value 4.98E-4), which included the genes: DLG4, HFE, HLA-A, and NF1.

Patients with NF1 characteristically develop CN. The number, age of occurrence, and the growth rates of these tumors are highly variable among individuals and even within the same individual. The NF1 gene encodes for the protein Neurofibromin. Neurofibromin is a negative regulator of the Ras/mitogen-activated protein kinase (MAPK) pathway[5]. 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. Most analyses of somatic mutations in CN identify high confidence NF1 mutations in only about half of the tumors[4,7].

CN growth behavior in NF1 is poorly understood due to the multitude of variables potentially effecting tumor development and growth. Here we performed WES on three growing and three non-growing CN from a single individual to test the hypothesis that somatic mutations in modifying loci could account for differences in growth behaviors between growing and non-growing CN. We identified between 1–11 mutations per samples with deleterious NF1 mutations in two samples. While provocative mutations were identified in each of the samples at potential modifying loci, no trends were identified between mutations and in growing and non-growing samples. Mutations in genes in the HIPPO pathway appeared to be over-represented. Additional studies of the exome and transcriptome, as well as epigenetic modifications, in larger cohorts of growing and non-growing CN, are needed.