Mutational Profile of GNAQ Q209 in Human Tumors

Background Frequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein α-subunit (GNAQ) in blue naevi 83%, malignant blue naevi (50%) and ocular melanoma of the uvea (46%). The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene. Methodology To assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas. Principal Findings We detected the previously reported mutations in 6/13 (46%) blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study.


Introduction
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 a-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 raslike 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 Gprotein-coupled receptors (GPCRs) and stimulates all four isoforms of b phospholipase C (PLCb) that catalyzes the hydrolysis of phosphatidylinositol biphosphate (PIP2). Nearly 40% of GPCRs rely upon Gqa 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, Ga, Gb and Gc, that cycle between inactive and active signalfling states in response to guanine nucleotides. Gqa (GNAQ), G11a (GNA11), G14a (GNA14) and G15a (GNA15) each have very different tissue and cell expression patterns. Gqa and G11a mRNA and protein are ubiquitously distributed across tissues [4]. Compared with Gqa human, G11a, G14a, and G15a 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.

Materials and Methods
Tumor sample and Ethics Statement Individual consent for this specific project was waivered by the Academic Medical Center (Amsterdam, The Netherlands) ethics committee because the research was performed on 'waste' material, stored in a coded fashion. The entire tumor database is described in Table 2.
PCRs were performed in 10-uL reaction volumes in 96-well format containing 0.25 mmol/L deoxynucleotide triphosphates, 1 umol/L each of the forward and reverse primers, 6% DMSO, 16PCR buffer, 1 ng/uL DNA, and 0.05 unit/uL AmpliTaq Gold DNA polymerase (Applied Byosystems, Foster City, CA) A touchdown PCR program was used for PCR amplification (Peltier Thermocycler, PTC-200, MJ Research, Bio-Rad Laboratories, Inc., Italy). PCR products were purified using AMPure (Agencourt Bioscience Corp., Beckman Coulter S.p.A, Milan, Italy). Cycle sequencing was carried out using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) with an initial denaturation at 97uC for 3 min, followed by 28 cycles of 97uC for 10 s, 50uC for 20 s, and 60uC for 2 min. Sequencing products were purified using CleanSeq (Agencourt Bioscience, Beckman Coulter) and analyzed on a 3730 DNA Analyzer, ABI capillary electrophoresis system (Applied Biosystems). Sequence traces were analyzed using the Mutation Surveyor software package (SoftGenetics, State College, PA). Only amplicons meeting quality criteria were analyzed: tumor samples had Phred quality scores of $20.
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 tumors as compared to the other tumor types.

Results and Discussion
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).