In this report, we present the first genetic and molecular proof of an association of glomus tumors of the fingers and toes with NF1. The mean age of adult
participants in our study (40 years) is comparable to that of the sporadic glomus tumor population (9
). However, one participant in our study (NF1-G7) was only 11 years old. We also observed multiple glomus tumors in 45% of our participants, a feature not observed in sporadic glomus tumors. In tumor predisposition syndromes like NF1, an early age of onset and the presence of multi-focal tumors are evidence of an association.
Glomus tumors are small (typically < 5 mm). We used two techniques to obtain tumor DNA. We identified both germline and somatic mutations in NF1
in six tumors; in a seventh tumor we found an NF1
germline mutation plus LOH (1/7 = 14%; 95% confidence interval: 0.003 - 0.578). The rate of LOH we detected in glomus tumors is not significantly different from the expected 25% observed in neurofibromas (binomial distribution) (31
) due to our modest sample size. In two tumors we showed that the wild-type chromosome harbored the somatic mutation (the “second hit” of Knudson’s two-hit hypothesis). Bi-allelic inactivation of NF1
is a common pathogenic mechanism of NF1-associated tumors. In four tumors from two individuals, four different somatic NF1
mutations were identified, suggesting that the multi-focal NF1-associated glomus tumors arise from independent events. Presumably NF1
-nullizygosity arises in glomus cells secondary to mitotic DNA replication errors in NF1
; this matters since glomus cells rely on neurofibromin-dependent RAS-MAPK-related growth factor cascades.
We also sought evidence of the functional consequences of the inactivation of NF1
. Biallelic inactivation of NF1
in NF1-associated glomus tumor-derived glomus cells results in an increased activation of the MAPK pathway, as observed in other tumor cells with bi-allelic inactivation of NF1
). Biochemical analysis of NF1-associated tumor-derived glomus cells (with germline and somatic NF1
mutations) showed stronger and longer activation of the MAPK pathway after stimulation with aFGF when compared to NF1-associated tumor derived fibroblasts (with a germline NF1
mutation only), sporadic tumor derived glomus cells (no NF1
mutations) and normal skin fibroblasts (no NF1
mutations). In three (of three) glomus tumors from three different fingertips from a single female, X-inactivation as detected by the HUMARA-MSP assay was consistent with monoclonal expansion of the glomus tumors. Such an expansion is compatible with the consequences of bi-allelic inactivation of a tumor suppressor gene (such as NF1
) in a single cell (32
We performed a genome-wide search for copy number changes in both cultured and paraffin-embedded tumor cells. In participant NF1-G10 tumor #1 (no somatic NF1
mutation identified) and tumor #3 (bi-allelic NF1
inactivation), qPCR was consistent with a partial deletion of a portion of the 5′-untranslated region of WASF1
(). Interestingly, WASF1
forms a bi-directional gene pair with the 5′ CDC40
. The bi-directional promoter of WASF1
is located within the putative deletions of both tumors from NF1-G10. Deletion of the bidirectional promoter may plausibly affect expression of both WASF1
. There is significant over-representation of bidirectional promoters associated with cancer-related genes (33
); their role in benign tumors is unknown. WASF1
is down-regulated in ovarian cancer (34
). There are no reported mutations in human CDC40,
an orthologue of yeast CDC40
, which is a controller of cell cycle arrest (35
). Both WASF1
are candidates for further investigation in glomus tumors.
A ~50 kb amplification within CRTAC1
was also observed in both glomus tumors from NF1-G10. CRTAC1
encodes human cartilage acidic protein 1 and is useful in distinguishing chondrocyte-like, osteoblast-like and mesenchymal stem cells in culture (36
). The PRINTS database1
predicts a C-terminal antifreeze type I domain in CRTAC1
. Antifreeze proteins (AFPs) were identified in polar fish as an adaptation to survive in hypothermic conditions preventing cell damage (37
). In prolonged sub-zero cryopreservation, AFPs protect the heart from freezing, improve survival and reduce apoptosis (38
). Antifreeze domains are rare in the human genome. Their role in the pathogenesis of glomus tumors, derived from cold-responsive contractile glomus cells, is unknown.
Analysis of the cultured NF1 tumor derived glomus cells by array-CGH did not show copy number alterations. It is possible that the cell culture procedure selected for glomus cells without copy number alterations.
Lastly, many neural crest-derived cell types are involved in NF1 (39
). Three observations from our data support a neural crest origin for glomus cells and their cognate tumors. First, glomus cells are αSMA-positive; progenitor cells cultured from rat sciatic nerve suggest that neural crest stem cells generate αSMA-positive myofibroblasts (40
). Second, the five NF1-associated tumor-derived cell cultures with a somatic NF1
mutation showed that only αSMA-positive glomus cells, and not tumor-derived fibroblasts, harbored somatic and germline NF1
mutations. Third, the unusual phenotype of participant NF1-G6 is consistent with somatic mosaicism mainly confined to cells of neural crest origin (28
). She had a mosaic NF1 phenotype, confirmed by molecular analysis: she presented with a glomus tumor, neurofibromas on the back and an intestinal ganglioneuroma but no freckling, learning disabilities, Lisch nodules or localized hyperpigmentation. Mosaicism in NF1 arises from a post-zygotic mutation of NF1
). In the case of NF1-G6, that mutation event likely occurred in the neural crest or a neural crest-derived cell, since both Schwann cells (neurofibroma) and intestinal ganglion cells (ganglioneuroma) are of neural crest origin. Accordingly, we hypothesize that glomus cells (glomus tumor) arise from myofibroblasts derived from neural crest stem cells.
In summary, we show that glomus tumors in NF1 arise secondary to bi-allelic inactivation of the tumor suppressor gene NF1 in αSMA-positive glomus cells. We observed that NF1-inactivated glomus cells show increased MAPK signaling. Taken together, these data prove that glomus tumors of the fingers are an integral part of the tumor spectrum of NF1. We hope that an increased awareness of these tumors will improve their early diagnosis and treatment in individuals with NF1.