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

Mosaic BRAF fusions are a recurrent cause of congenital melanocytic naevi targetable by MEK inhibition

Among children with multiple congenital melanocytic naevi (CMN), 25% have no established genetic cause, of which many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of CMN. Here, we study 169 CMN patients, 38 of whom were double wild-type for NRAS/BRAF mutations. Nineteen of these 38 patients had sufficient tissue to undergo RNAseq, which revealed mosaic BRAF fusions in 11/19 patients and mosaic RAF1 fusions in 1/19. Recurrently, fusions involved the loss of the 5’ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate naevi in 5/12 patients, confirming clonality. The absence of the fusion in blood in 8/12 patients indicated mosaicism. Primary culture of BRAF-fusion naevus cells from 3/12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement, and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes

PTPN11 mosaicism causes a spectrum of pigmentary and vascular neurocutaneous disorders and predisposes to melanoma

Phakomatosis pigmentovascularis (PPV) is a diagnosis which denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement including central nervous system disease, asymmetrical growth and a predisposition to malignancy. Using a tightly phenotyped group and high depth next generation sequencing of affected tissues we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of PPV type III or spilorosea. Within an individual the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to demonstrate that the same variants can cause either the specific pigmentary or vascular phenotypes alone, as well as driving melanoma development within the pigmentary lesion. Protein conformational modelling highlights that while variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modelling of the missense variants confirms downstream MAPK pathway overactivation, and widespread disruption of human endothelial cell angiogenesis. Importantly, PTPN11-mosaic patients theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of naevus spilus and capillary malformation syndromes, paving the way for better clinical management

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex

Genetic studies in Congenital Melanocytic Naavi

Background: Congenital melanocytic naevi (CMN) are moles present at birth, can be associated with extracutaneous features and the most severely affected children have a 10-15% risk of development of fatal melanoma. The cause of multiple CMN was previously identified as somatic mosaicism for NRAS in 70% of cases. Although a somatic mosaic condition, up to 1/3 of patients have a family history of CMN, implying a role for Mendelian genetics in disease onset or presentation. Identification of novel somatic and germline variants in CMN will enable us to understand which effector pathways are important in pathogenesis, with a view to identifying novel therapeutic targets. Aims: To establish genotype-phenotype associations in CMN, to look for novel somatic genetic causes of CMN, to characterise the role and pathogenetic mechanism of novel germline duplications affecting the gene PPP2R3B in CMN and melanoma and to establish the natural colour history of CMN. Methods: Next generation sequencing (NGS) was used to identify novel somatic variants and copy number variants in a population of CMN and adult melanoma. Cellular modelling of PPP2R3B duplications was undertaken using stable, inducible PPP2R3B cell lines and proliferation and migration studies. A standardised measurement of colour of CMN was devised and lightening measured over time. Results: NRAS is the commonest cause of multiple CMN found in 68%. BRAF was identified as a new, rare cause of multiple CMN, affecting 7%. No novel somatic variants were identified following whole exome sequencing or targeted next generation sequencing. PPP2R3B duplications were identified in an adult melanoma cohort and overexpression of PPP2R3B leads to increased proliferation and decreased migration of melanoma cell lines via C21orf91. Final CMN colour is genetically-determined, related to inherited pigmentary phenotype, not related to colour in the first three months of life, and not affected by superficial removal techniques

Atypical dermal melanocytosis: diagnostic clue in constitutional mismatch repair deficiency syndrome.

International audienceA twenty-three month old female presented with abnormal pigmentation since birth. She had a past history of mediastinal non-Hodgkin's lymphoma diagnosed at the age of 9 months and treated with surgery and chemotherapy. Family history revealed that her grandfathers were brothers, and both died of colon cancer in their fifth decade. Parents were both fit and well in their early thirties

Keratinocytic epidermal nevi associated with localized fibro-osseous lesions without hypophosphatemia

Keratinocytic epidermal nevi (KEN) are characterized clinically by permanent hyperkeratosis in the distribution of Blaschko's lines and histologically by hyperplasia of epidermal keratinocytes. KEN with underlying RAS mutations have been associated with hypophosphatemic rickets and dysplastic bone lesions described as congenital cutaneous skeletal hypophosphatemia syndrome. Here, we describe two patients with keratinocytic epidermal nevi, in one associated with a papular nevus spilus, who presented with distinct localized congenital fibro-osseous lesions in the lower leg, diagnosed on both radiology and histology as osteofibrous dysplasia, in the absence of hypophosphatemia or rickets, or significantly raised FGF23 levels but with distinct mosaic HRAS mutations. This expands the spectrum of cutaneous/skeletal mosaic RASopathies and alerts clinicians to the importance of evaluating for bony disease even in the absence of bone profile abnormalities.status: publishe