Vitamin D status in children with congenital melanocytic nevi.

Congenital melanocytic nevi (CMN) are rare, pigmented birthmarks that can predispose patients to melanoma of the central nervous system and skin. Data from non-CMN melanoma cohorts suggest that vitamin D levels may be connected to outcome, prompting this study of 25-hydroxyvitamin D levels in plasma samples from 40 children with CMN. While 27% were insufficient and 13% deficient, this was representative of European populations, and UK supplementation guidelines are already in place. Our data support routine vitamin D supplementation for all CMN patients during winter months, without routine serum measurement.

Impact of public exhibition on the perception of birthmarks.

BACKGROUND: The importance of photographs in social media, the steep rise in popularity of tattoos, and the prominence of individuals with visibly different skin in fashion are likely to be changing the landscape of self- and public perception of birthmarks. Study objectives were to assess the impact of a photoshoot and public exhibition on the self-perception of individuals with extensive birthmarks, and to explore the viewing public's reactions. METHODS: Thirty individuals with congenital melanocytic nevi (CMN) were recruited internationally. Each had a professional photoshoot portrait with their skin exposed, resulting in a public exhibition in London entitled "How do you C Me Now?" Participants/parents completed pre- and post-questionnaires relating to self-perception and the impact of their birthmarks on behavior. Over 8000 members of the public viewed the exhibition, 464 completing an on-site questionnaire on its effects. RESULTS: All participants/parents rated the experience as positive, valuable and helpful. Scores on self-appreciation and self-confidence were significantly higher after the photo shoot. Members of the general public overwhelmingly reported the exhibition increased their positive feelings towards people with birthmarks. The majority of public respondents also reported that the exhibition made them feel better about their own skin and about their looks in general. CONCLUSION: This unique exhibition and the associated research has provided a striking new perspective on potential psychological interventions for individuals with birthmarks.

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.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

Misaligned foveal morphology and sector retinal dysfunction in AKT1-mosaic Proteus syndrome.

PURPOSE: Proteus syndrome arises as a result of a post-zygotic mosaic activating mutation in the AKT1 oncogene, causing a disproportionate overgrowth of affected tissues. A small number of ocular complications have been reported. We present the unique findings in a patient who had molecular confirmation of AKT1 mosaicism alongside fulfilling the clinical criteria for Proteus syndrome. METHODS: Pattern electroretinography, visual evoked potentials and multifocal electroretinography testing were performed alongside detailed retinal imaging and clinical examination to detail the ophthalmic characteristics. RESULTS: Electrophysiological findings characterised unilateral macular dysfunction alongside sector retinal dysfunction of the right eye. This was demonstrated through optical coherence tomography and ultra-wide-field imaging to be associated with a misaligned foveal morphology and sector retinal dysfunction extending into the temporal retina. CONCLUSION: We propose this patient has asymmetric foveal development and concomitant sector retinal dysfunction as the result of the mosaic AKT1 mutation, either through disruption in the retinal PI3K-AKT1 signalling pathway or through mechanical distortion of ocular growth, resulting in disproportionate inner retinal development. The findings expand the ocular phenotype of Proteus syndrome and encourage early assessment to identify any incipient ocular abnormalities.

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.

Striking contiguous depigmentation across the lower limbs in piebaldism and its implications for understanding melanocytic migration and development.

Piebaldism is a rare autosomal dominant disorder of pigmentation that is characterized by variable patches of depigmentation on the face, chest, abdomen, and extremities. We describe two cases of piebaldism, in whom the remarkable asymmetric distribution of the depigmented patches in a connected, contiguous pattern across the legs provides embryologic insights. This finding is not explained by the traditional theory that melanocytic migration only originates in the neural crest and progresses unilaterally down each leg. We propose that our cases, and other similar cases, can be explained by a recent theory of mesodermal melanocyte migration.

Aggressive melanoma in an infant with congenital melanocytic nevus syndrome and multiple, NRAS and BRAF mutation-negative nodules.

We report the case of a newborn boy with multinodular NRAS and BRAF mutation-negative congenital melanocytic nevi and cerebral lesions compatible with congenital intraparenchymal melanosis. Histopathology from skin lesions showed atypical nodular melanocytic proliferation with marked melanocytic atypia and a large number of mitoses and apoptosis, indicating aggressive proliferation. The child developed several new subcutaneous tumors and multiple internal lesions, which were confirmed to be metastases, and died at 5 months of age. This case may represent an infantile melanoma developing from a giant congenital melanocytic nevus or a congenital melanoma.

MEK inhibition appears to improve symptom control in primary NRAS-driven CNS melanoma in children.

BACKGROUND: Primary melanoma of the CNS in children is extremely rare, and usually linked to congenital melanocytic naevus syndrome, caused by mosaicism for oncogenic NRAS mutations. Outcome is fatal in all cases. Data from murine and in vitro studies suggest that MEK inhibition is a possible therapeutic option. METHODS: Four children with NRAS-mutated CNS melanoma were treated with Trametinib on a compassionate basis. RESULTS: All four had an improvement in symptoms and objectively in signs. These varied from mild improvement for 1 month, to a sustained symptom-free period of 9 months in one case. In all cases there was eventual disease progression through treatment, followed by rapid death after discontinuation. There were no clinically-significant side effects. CONCLUSIONS: Trametinib is the first therapy to show any objective or measurable effect in NRAS-mutated primary CNS melanoma, with few side effects in this small series. The role of this therapy should be explored further in this rare paediatric tumour.

The adverse effect profile of oral azathioprine in pediatric atopic dermatitis, and recommendations for monitoring.

BACKGROUND: Azathioprine is efficacious in the treatment of severe childhood atopic dermatitis; however, robust data on adverse effects in this population are lacking. OBJECTIVE: We sought to assess adverse effects of azathioprine treatment in a pediatric atopic dermatitis cohort, and make recommendations for monitoring based on these data. METHODS: Blood test results for all 82 children prescribed oral azathioprine for atopic dermatitis in our department between 2010 and 2012 were collated prospectively, and clinical notes were reviewed retrospectively. RESULTS: Mean age at commencing azathioprine was 8.3 years (SEM 0.4). Mean maximum doses were 2.4 mg/kg (SEM 0.1) and 1.5 mg/kg (SEM 0.1) for normal and reduced serum thiopurine-S-methyltransferase levels, respectively. Adverse effects on blood indices occurred in 34 of 82 patients (41%), with pronounced effects in 18 of 82 (22%) after a median time of 0.4 years. Two patients stopped therapy as a result of abnormal blood indices. Clinical adverse effects occurred in 16 of 82 (20%), two resulting in cessation of therapy. Incidence of adverse effects was unaffected by age, sex, thiopurine-S-methyltransferase level, and drug dose on multivariate regression. LIMITATIONS: Comparison with other studies is limited by varying definitions of adverse effects. CONCLUSION: Oral azathioprine was associated with few pronounced adverse effects for the duration of use and dosage in this cohort. Recommendations for monitoring are made.

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 Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 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 nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 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.

RNA therapy for oncogenic NRAS-driven naevi induces apoptosis.

RAS proteins regulate cell division, differentiation and apoptosis via multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers, however they also drive many benign lesions predisposing to malignancy, such as melanocytic naevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence, however the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. Here we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic naevi using siRNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of siRNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MEK inhibitor. Protective packaging of the targeted siRNA into lipid nanoparticles permits successful delivery into a humanised mouse model of melanocytic naevi, and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic naevi and anticipate that targeted siRNA could form the basis of clinical trials for RAS-driven benign tumours.

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.

PTPN11 Mosaicism Causes a Spectrum of Pigmentary and Vascular Neurocutaneous Disorders and Predisposes to Melanoma.

Phakomatosis pigmentovascularis is a diagnosis that denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement, including CNS disease, asymmetrical growth, and a predisposition to malignancy. Using a tight phenotypic 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 phakomatosis pigmentovascularis 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 show that the same variants can cause either the pigmentary or vascular phenotypes alone, and drive melanoma development within pigmentary lesions. Protein structure modeling highlights that although variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modeling of the missense variants confirms downstream MAPK pathway overactivation and widespread disruption of human endothelial cell angiogenesis. Importantly, patients with PTPN11 mosaicism 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 nevus spilus and capillary malformation syndromes, paving the way for better clinical management.