Glomus tumors in neurofibromatosis type 1: genetic, functional, and clinical evidence of a novel association

Abstract

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1(-/-) glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3'-end of CRTAC1 and a deletion of the 5'-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1.

Figures

Figure 1

Pedigree of NF1-G10 with phased 11 SNP NF1 haplotypes spanning ∼ 203 kb (5′ end at top, 3′ end at bottom). The wild-type haplotype harbors the two somatic NF1 mutations identified in her glomus tumors #2 and #3, evidence of NF1 bi-allelic inactivation. SNP alleles sub-cloned with germline and somatic mutations are in bold. The maternal haplotype harboring the NF1 germline mutation (c.6789_6792 del TTAC, sub-cloned with SNP rs2525565, in black) co-segregates with the NF1 affectation status in the two affected sons (son 1 and son 2). The maternal haplotype harboring the two somatic NF1 mutations (light gray) is transmitted to the unaffected son (son 3).

Figure 2

A, photomicrograph of glomus tumor #1 from NF1-G7 showing a uniform population of tumor cells with rounded nuclei and eosinophilic cytoplasm. Note the perivascular arrangement of the tumor cells (inset). Hematoxylin & eosin stain, original magnification 250X, 400X (inset). Leica DMLB microscope. Bar = 15 microns. B, immunocytochemistry of glomus tumor-derived αSMA positive cells from tumor #3 of NF1-G3. Nuclei are stained with DAPI (blue). αSMA-positive structures are green. Zeiss Axiophot fluorescent microscope. Bar = 15 microns.

Figure 3

aFGF stimulation of the RAS-MAPK pathway in cultured cells. Comparison of NF1-associated glomus tumor-derived glomus cells, NF1-associated glomus tumor-derived fibroblasts, sporadic glomus tumor-derived glomus cells and control skin fibroblasts before and at different time points after stimulation with aFGF. A, cell extracts immunoblotted with the indicated antibodies. B, ratios of pMEK/MEK and pERK/ERK. All ratios were normalized to the ratio of the specific cell type before stimulation. Error bars represent standard deviation (SD). Average pMEK/MEK and pERK/ERK ratios of the four cell types were significantly different (P = <10-3). *Significant at the 5% level.

Figure 4

Comparison of log2 R ratio of SNP rs4945851 (WASF1) and nearby SNP loci in glomus tumors #1 (log2 R = -0.9) and #3 (log2 R = -2.0) and germline DNA (log2 R = -0.3, normal) from NF1-G10. A ∼0.45 Mb region (110,360,210 – 110,807,710 bp) surrounding rs4945851 (arrow; position 110,603,926 bp) harboring 82 SNPs on chromosome 6 is shown in panels A-C (glomus tumor #1, glomus tumor #3 and germline sample). Locus rs4945851 and 10 adjacent SNPs (5 upstream and 5 downstream) are shown in red. The vertical axis is the log2 R ratio of the intensity of the SNP-associated fluors. Panel D shows the genomic position in increments of 4,475 basepairs, cytoband (6q21) and surrounding genes.

Figure 5

Quantitative PCR of CRTAC1 and WASF1 loci in germline DNA (A), glomus tumor #1 (B), and glomus tumor #3 (C) from NF1-G10. Abundance of DNA at each locus was normalized to the quantity of DNA in the germline sample.