Activation of the MAPK signaling pathway plays an important role in tumorigenesis. Multiple components of this pathway such as H, N, K-RAS and BRAF are often mutated in human cancer ^[1].

Most melanocytic neoplasms show oncogenic mutations in components of the MAPKinase cascade, particularly in BRAF and NRAS^[2]. A recent study has reported frequent somatic mutations in the heterotrimeric G protein ?-subunit (GNAQ) in a subset of melanocytic neoplasms which do not present alterations in the RAS or BRAF genes ^[3]. Genetic, biochemical and biological analysis has shown that GNAQ behaves as a bona fide human oncogene. The reported mutations occur exclusively in codon 209 in the ras-like domain and lead to constitutive activation ^[3]. The glutamine at codon 209 of GNAQ corresponding to residue 61 of RAS and is essential for GTP hydrolysis ^[3]. It has been previously shown that in other RAS family members, mutations at this site cause loss of GTPase activity with constitutive activation ^[3].

GNAQ encodes for alpha subunit of q class of heterotrimeric GTP binding protein (Gq) that mediates signals between G-protein-coupled receptors (GPCRs) and stimulates all four isoforms of ? phospholipase C (PLC?) that catalyzes the hydrolysis of phosphatidylinositol biphosphate (PIP2). Nearly 40% of GPCRs rely upon Gq? family members to stimulate inositol lipid signalling. These include more then 50 subtypes of receptor responsive to a range of hormone, neurotransmitters, neuropeptides, chemokines, autocrine and paracrine molecules ^[4]. The Gq family members, Gq, G11, G14, and G15/16, like all heterotrimeric G proteins, are composed of three subunits, G?, G? and G?, that cycle between inactive and active signalling states in response to guanine nucleotides. Gq? (GNAQ), G11? (GNA11), G14? (GNA14) and G15? (GNA15) each have very different tissue and cell expression patterns. Gq? and G11? mRNA and protein are ubiquitously distributed across tissues ^[4]. Compared with Gq? human, G11?, G14?, and G15? share 90%, 80%, and 57% amino acid sequence identity, respectively (Table 1). While GNAQ and GNA11 are ubiquitously expressed, other members of the family show a very restricted pattern of expression. For example GNA 15 is confined to tissues rich in cell types of hematopoietic origin and are enriched in cells in the earlier stages of differentiation ^[5]. GNA 14 has been demonstrated to be expressed mainly in kidney, liver, lung and pancreas ^[4]. Of note exon 5 of GNA11 contains an equivalent residue to Q209 of GNAQ. We hypothesized that mutations in GNAQ may also be present in tumor types from non melanocytic origin where they could represent alternative route to MAPKinase activation. To assess this hypothesis, we performed a systematic mutational profile of exon 5 of the GNAQ gene in a large panel of human tumors from different tissue types (Table 2). In light of its ubiquitous expression we also performed the analysis of the GNA11 gene (exon 5) in the same tumor set.

We sequenced exon 5 of the GNAQ and GNA11 genes in in a panel of 922 tumors, including glioblastoma, gastrointestinal stromal tumors, acute myeloid leukemia, blue naevi, melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas (Table 2). The samples included in the analysis have been previously used for mutational profiling of cancer genes and we have shown that common mutations can be identified in this tumors database ^[6], ^[7], ^[8]. A total of 1844 PCR products, spanning 423 kb of tumor genomic DNA, were generated and subjected to direct sequencing. Sequences analysis identified the presence of the Q209L (c.A627T) mutation in GNAQ in 6/13 (46%) of blue naevi tumors (Figure 1 and Table 2), thus confirming previous data ^[3]. Importantly, no mutations of GNAQ exon 5 were found in any tumor types, other than blue naevi (Table 2). Similarly, we did not detect mutations in exon 5 of GNA11.

To assess whether these results were statistically significant we performed the Fisher's exact test to determine the tissue specificity for GNAQ mutation in blue naevi tumor as compared to the other tumor types (Table 2).

Our data confirm that GNAQ is a pivotal cancer gene in blue naevi while at the same time unveils a striking tissue-specific pattern of the GNAQ^Q209 mutations in human cancer.

Blue naevi arise from intradermal melanocytic proliferations, which can be congenital or acquired, and present in diverse ways ranging from discrete bluish moles (blue naevi) to large blue?grey patches affecting the conjunctiva and periorbital skin (naevus of Ota), shoulders (naevus of Ito) and the lower back (Mongolian spot) ^[3]. Uveal melanomas are thought to originate from melanocytes within the choroidal plexus of the eye and are distinct from cutaneous melanoma by characteristic cytogenetic alterations. Of note, a potential connection between intradermal melanocytic neoplasms and uveal melanoma is suggested by the fact that naevus of Ota is a risk factor for uveal melanoma ^[3]. Oncogenic alterations in the RAS and BRAF genes are known to frequently affect cutaneous melanoma. Until the discovery of the GNAQ^Q209 mutations there were no oncogenes altered at high frequency in uveal melanomas and blue naevi. The tissue-specificity of GNAQ mutations may be linked to its involvement in endothelin signaling, which is important for development of melanocytes and also is required for the migration of melanoblasts ^[5]. The canonical downstream signaling pathways of GNAQ are the ?-isoforms of phospholipase C (PLC-?). Gq?, G11?, bind and stimulate PLC-? enzymes to initiate inositol lipid signalling. PLC-? enzymes catalyze the hydrolysis of the phospholipid phosphatidylinositol bisphosphate, (PIP₂), to release inositol trisphosphate (IP₃) and diacylglycerol (DAG). These second messengers propagate and amplify the G?-mediated signal trough stimulation of protein kinase C (PKC). ^[3] One hypothesis is that GNAQ regulates cell growth through a RAS dependent or RAS- independent signaling mechanism involving the PKC-dependent ERK pathway. ^[9]. Functional assays are now required to assess this possibility and to understand in details the oncogenic signaling mechanisms regulated by GNAQ mutant alleles. Targeting either the mutated GNAQ^Q209 protein or the oncogenic signaling pathway controlled by mutated GNAQ may open up new therapeutic strategies for melanocytic tumors.

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work was supported by grants from Italian Association for Cancer Research (AIRC), Italian Ministry of Health, Regione Piemonte, Italian Ministry of University and Research, CRT Progetto Alfieri, Fondazione Monte dei Paschi di Siena, Association for International Cancer Research (AICR-UK) , EU FP6 contract n 037297 (MCSC), EU FP7 Marie Curie, contract n 218071 (Cancer-Gene). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors thank Dr F. Di Nicolantonio for critical reading of the manuscript, Dr. S. Leenstra, Dr. T Hulsebos, and Professor Troost for making glioblastoma tumor samples available from the Brain Tumor Bank maintained by the Departments of Neurosurgery, Neuropathology, and Neurogenetics, Academic Medical Center, Amsterdam. Dr Aniello Cerrato and Dr Massimo Santoro for providing the DNA samples of thyroid carcinomas (medullary histotype).