[Circumscribed choroidal hemangioma and non-pigmented choroidal melanoma: clinical, instrumental and molecular genetic features]. / Otgranichennaya gemangioma i bespigmentnaya melanoma khorioidei: kliniko-instrumental'nye i molekulyarno-geneticheskie osobennosti.

Circumscribed choroidal hemangioma (CCH) and early non-pigmented choroidal melanoma (CM) have similar clinical, ultrasound and morphometric features, which in some cases makes their differential diagnosis difficult. There are few studies in the literature devoted to a comparative analysis of the molecular genetic features of CCH and non-pigmented CM, and the results of those studies are contradictory. PURPOSE: This study attempts to develop a method of non-invasive molecular genetic differential diagnostics of CCH and non-pigmented CM. MATERIAL AND METHODS: Based on the results of clinical and instrumental examination methods, 60 patients (60 eyes) with CCH (n=30) and non-pigmented CM (n=30) were included in this prospective study. The control group consisted of 30 individuals without intraocular tumors. Mutations in the GNAQ/GNA11 genes were determined by real-time PCR using the analysis of genomic circulating tumor DNA isolated from peripheral blood plasma. The average follow-up period was 12.1±1.8 months. RESULTS: The study revealed a significant association of mutations in exons 4 and 5 of the GNAQ/GNA11 genes with the presence of non-pigmented CM (27/30; 90%). These mutations were not detected in the group of patients with CCH. Mutations in exons 4 and 5 of the GNAQ/GNA11 genes were also not detected in the control group of healthy individuals. CONCLUSION: This study proposes a method of non-invasive and low-cost differential diagnostics based on molecular genetic analysis and detection of mutations in exons 4 and 5 of the GNAQ and GNA11 genes, which are specific for CM (90%).

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

The M3 Muscarinic Acetylcholine Receptor Can Signal through Multiple G Protein Families.

The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.

MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome.

Sturge-Weber syndrome (SWS), a neurocutaneous disorder, is characterized by capillary malformations (CM) in the skin, brain, and eyes. Patients may suffer from seizures, strokes, and glaucoma, and only symptomatic treatment is available. CM are comprised of enlarged vessels with endothelial cells (ECs) and disorganized mural cells. Our recent finding indicated that the R183Q mutation in ECs leads to heightened signaling through phospholipase Cß3 and protein kinase C, leading to increased angiopoietin-2 (ANGPT2). Furthermore, knockdown of ANGPT2, a crucial mediator of pro-angiogenic signaling, inflammation, and vascular remodeling, in EC-R183Q rescued the enlarged vessel phenotype in vivo. This prompted us to look closer at the microenvironment in CM-affected vascular beds. We analyzed multiple brain histological sections from patients with GNAQ-R183Q CM and found enlarged vessels devoid of mural cells along with increased macrophage-like cells co-expressing MRC1 (CD206, a mannose receptor), CD163 (a scavenger receptor and marker of the monocyte/macrophage lineage), CD68 (a pan macrophage marker), and LYVE1 (a lymphatic marker expressed by some macrophages). These macrophages were not found in non-SWS control brain sections. To investigate the mechanism of increased macrophages in the perivascular environment, we examined THP1 (monocytic/macrophage cell line) cell adhesion to EC-R183Q versus EC-WT under static and laminar flow conditions. First, we observed increased THP1 cell adhesion to EC-R183Q compared to EC-WT under static conditions. Next, using live cell imaging, we found THP1 cell adhesion to EC-R183Q was dramatically increased under laminar flow conditions and could be inhibited by anti-ICAM1. ICAM1, an endothelial cell adhesion molecule required for leukocyte adhesion, was strongly expressed in the endothelium in SWS brain histological sections, suggesting a mechanism for recruitment of macrophages. In conclusion, our findings demonstrate that macrophages are an important component of the perivascular environment in CM suggesting they may contribute to the CM formation and SWS disease progression.

Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq.

Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.

Gα11 deficiency increases fibroblast growth factor 23 levels in a mouse model of familial hypocalciuric hypercalcemia.

Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 deficiency on FGF23 production in mice with heterozygous (Gna11+/-) or homozygous (Gna11-/-) ablation of Gna11. Both Gna11+/- and Gna11-/- mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11-/- mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and also showed evidence of systemic inflammation with elevated serum IL-1ß levels. Furin gene expression was significantly increased in the Gna11-/- liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating intact FGF23 levels. Gna11-/- mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH - at least those with GNA11 mutations - may be at risk for these complications.

Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

In Vitro Module Editing Of NRPS Enables Production Of Highly Potent Gq -Signaling Inhibitor FR900359 Derived From Unculturable Plant Symbiont.

Heterotrimeric G proteins are key mediators in the signaling of G protein-coupled receptors (GPCR) that are involved in a plethora of important physiological processes and thus major targets of pharmaceutical drugs. The cyclic depsipeptides YM-254890 and FR900359 are strong and selective inhibitors of the Gq subfamily of G proteins. FR900359 was first reported to be produced by unculturable plant symbiont, however, a culturable FR900359 producer was discovered recently by the standard strategy, screening of the producing strain from the environment. As another strategy, we introduce herein the different way to supply natural compounds of unculturable microorganism origin. We therefore embarked on constructing an artificial biosynthetic gene cluster (BGC) for FR900359 with YM-254890 BGC as a template using "in vitro module editing" technology, first developed for the modification of type-I PKS BGCs, to edit YM-254890 BGC. The resulting artificial BGCs coding FR900359 were heterologously expressed in the Pseudomonas putida KT2440 host strain.

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

Clinical and pathologic features of Sturge-Weber syndrome in patients with refractory epilepsy.

OBJECTIVES: We aimed to investigate the clinicopathologic features of and genetic changes in Sturge-Weber syndrome (SWS) in patients with refractory epilepsy. METHODS: Clinical data were retrospectively analyzed. H&E and immunohistochemistry were performed to assess pathologic changes. Targeted amplicon sequencing was applied to investigate the somatic GNAQ (c.548G>A) mutation. The potential predictors of seizure outcomes were estimated by univariate and multivariate statistical analyses. RESULTS: Forty-eight patients with SWS and refractory epilepsy were enrolled. According to the imaging data and pathologic examination, ipsilateral hippocampal sclerosis (HS), calcification of leptomeningeal arteries, and focal cortical dysplasia were found in 14 (29.2%), 31 (64.6%), and 37 (77.1%) patients, respectively. A high frequency of GNAQ alteration was detected in both cerebral cortex (57.7%) and ipsilateral hippocampus (50.0%) from patients with SWS. During follow-up, 43 of 48 patients (85.4%) had achieved seizure control (Engel class I). Statistically, HS signs on imaging were found to be independent predictors of unfavorable seizure outcomes (P = .015). CONCLUSIONS: Calcification of leptomeningeal arteries, focal cortical dysplasia, and GNAQ alteration are common features in SWS pathology. Patients with refractory epilepsy caused by SWS can achieve satisfactory seizure control after surgery, but seizure control was compromised in patients with comorbid HS.

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).

Expression of GNAQ, BAP1, SF3B1, and EIF1AX Proteins in the Aqueous Humor of Eyes Affected by Uveal Melanoma.

Purpose: The purpose of this study was to quantify specific aqueous humor (AH) proteins in eyes affected by posterior uveal melanoma (UM). Methods: Thirty-six eyes affected by primary UM were included. Tumor thickness and largest basal diameter were specific clinical characteristics. Tumors were staged with the American Joint Commission on Cancer Eighth Edition (AJCC) classification. During the brachytherapy (Iodine-125) surgical procedure, both the AH sample collection and the 25-gauge transscleral fine needle aspiration biopsy (FNAB) were performed. AH samples were analyzed by immunoprecipitation and SDS PAGE techniques to quantify GNAQ, BAP1, SF3B1, and EIF1AX proteins. Cytologic material underwent fluorescence in situ hybridization for chromosome 3. The AH of 36 healthy eyes was used as the control group. Cluster analysis of groups was also performed. Results: Compared with the control group, significantly higher protein levels of: GNAQ (P = 0.02), BAP1 (P = 0.01), and SF3B1 (P = 0.02) were detected in eyes with UM. Cluster analysis of UM group revealed 2 clusters, one showing higher expression of GNAQ and BAP1 protein and one of EIF1AX protein. Moreover, the 2 clusters corresponded with the chromosome 3 status of UM. Conclusions: Specific and selected proteins may be detected in the AH of eyes affected by UM. These findings confirm the possibilities provided by AH analysis in UM.

Design, synthesis and evaluation of quinazoline derivatives as Gαq/11 proteins inhibitors against uveal melanoma.

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC50 = 9.4 µM) and two UM cell lines MP41 (IC50 = 6.7 µM) and 92.1 (IC50 = 3.7 µM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

GNAQ/GNA11 Mosaicism Is Associated with Abnormal Serum Calcium Indices and Microvascular Neurocalcification.

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum.

Driver mutations in GNAQ and GNA11 genes as potential targets for precision immunotherapy in uveal melanoma patients.

Uveal melanoma (UM) is the most common ocular malignancy in adults. Nearly 95% of UM patients carry the mutually exclusive mutations in the homologous genes GNAQ (amino acid change Q209L/Q209P) and GNA11 (aminoacid change Q209L). UM is located in an immunosuppressed organ and does not suffer immunoediting. Therefore, we hypothesize that driver mutations in GNAQ/11 genes could be recognized by the immune system. Genomic and transcriptomic data from primary uveal tumors were collected from the TCGA-UM dataset (n = 80) and used to assess the immunogenic potential for GNAQ/GNA11 Q209L/Q209P mutations using a variety of tools and HLA type information. All prediction tools showed stronger GNAQ/11 Q209L binding to HLA than GNAQ/11 Q209P. The immunogenicity analysis revealed that Q209L is likely to be presented by more than 73% of individuals in 1000 G databases whereas Q209P is only predicted to be presented in 24% of individuals. GNAQ/11 Q209L showed a higher likelihood to be presented by HLA-I molecules than almost all driver mutations analyzed. Finally, samples carrying Q209L had a higher immune-reactive phenotype. Regarding cancer risk, seven HLA genotypes with low Q209L affinity show higher frequency in uveal melanoma patients than in the general population. However, no clear association was found between any HLA genotype and survival. Results suggest a high potential immunogenicity of the GNAQ/11 Q209L variant that could allow the generation of novel therapeutic tools to treat UM like neoantigen vaccinations.

Photoperiod Induces the Epigenetic Change of the GNAQ Gene in OVX+E2 Ewes.

GNAQ, a member of the alpha subunit encoding the q-like G protein, is a critical gene in cell signaling, and multiple studies have shown that upregulation of GNAQ gene expression ultimately inhibits the proliferation of gonadotropin-releasing hormone (GnRH) neurons and GnRH secretion, and ultimately affects mammalian reproduction. Photoperiod is a key inducer which plays an important role in gene expression regulation by affecting epigenetic modification. However, fewer studies have confirmed how photoperiod induces epigenetic modifications of the GNAQ gene. In this study, we examined the expression and epigenetic changes of GNAQ in the hypothalamus in ovariectomized and estradiol-treated (OVX+E2) sheep under three photoperiod treatments (short photoperiod treatment for 42 days, SP42; long photoperiod treatment for 42 days, LP42; 42 days of short photoperiod followed by 42 days of long photoperiod, SP-LP42). The results showed that the expression of GNAQ was significantly higher in SP-LP42 than in SP42 and LP42 (p < 0.05). Whole genome methylation sequencing (WGBS) results showed that there are multiple differentially methylated regions (DMRs) and loci between different groups of GNAQ. Among them, the DNA methylation level of DMRs at the CpG1 locus in SP42 was significantly higher than that of SP-LP42 (p < 0.01). Subsequently, we confirmed that the core promoter region of the GNAQ gene was located with 1100 to 1500 bp upstream, and the DNA methylation level of all eight CpG sites in SP42 was significantly higher than those in LP42 (p < 0.01), and significantly higher than those in SP-LP42 (p < 0.01), except site 2 and site 4 in the first sequencing fragment (p < 0.05) in the core promoter region. The expression of acetylated GNAQ histone H3 was significantly higher than that of the control group under three different photoperiods (p < 0.01); the acetylation level of sheep hypothalamic GNAQ genomic protein H3 was significantly lower under SP42 than under SP-LP42 (p < 0.05). This suggests that acetylated histone H3 binds to the core promoter region of the GNAQ gene, implying that GNAQ is epigenetically regulated by photoperiod through histone acetylation. In summary, the results suggest that photoperiod can induce DNA methylation in the core promoter region and histone acetylation in the promoter region of the GNAQ gene, and hypothesize that the two may be key factors in regulating the differential expression of GNAQ under different photoperiods, thus regulating the hypothalamus-pituitary-gonadal axis (HPGA) through the seasonal estrus in sheep. The results of this study will provide some new information to understand the function of epigenetic modifications in reproduction in sheep.

Detection of the Uveal Melanoma-Associated Mutation GNAQ Q209P from Liquid Biopsy Using CRISPR/Cas12a Technology.

Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The in-depth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.