RESUMO
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.
Assuntos
Melanoma , Nevo , Neoplasias Cutâneas , Humanos , Imuno-Histoquímica , Melanoma/genética , Fenótipo , Neoplasias Cutâneas/genéticaRESUMO
BACKGROUND: Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS: To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS: We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION: Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING: This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L'Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).
Assuntos
Alelos , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/genética , Mutação , Fenótipo , Malformações Vasculares , Proteínas ras , Adolescente , Adulto , Animais , Criança , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Lactente , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Masculino , Malformações Vasculares/genética , Malformações Vasculares/metabolismo , Malformações Vasculares/patologia , Peixe-Zebra , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11(R183C) and GNA11(Q209L) in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11(R183C) under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mancha Mongólica/genética , Mutação , Síndromes Neurocutâneas/genética , Dermatopatias/genética , Alelos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Análise Mutacional de DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Células HEK293 , Humanos , Lactente , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fenótipo , Fosforilação , Transdução de Sinais , Peixe-ZebraRESUMO
Congenital melanocytic nevus (CMN) syndrome is the association of pigmented melanocytic nevi with extra-cutaneous features, classically melanotic cells within the central nervous system, most frequently caused by a mutation of NRAS codon 61. This condition is currently untreatable and carries a significant risk of melanoma within the skin, brain, or leptomeninges. We have previously proposed a key role for Wnt signaling in the formation of melanocytic nevi, suggesting that activated Wnt signaling may be synergistic with activated NRAS in the pathogenesis of CMN syndrome. Some familial pre-disposition suggests a germ-line contribution to CMN syndrome, as does variability of neurological phenotypes in individuals with similar cutaneous phenotypes. Accordingly, we performed exome sequencing of germ-line DNA from patients with CMN to reveal rare or undescribed Wnt-signaling alterations. A murine model harboring activated NRAS(Q61K) and Wnt signaling in melanocytes exhibited striking features of CMN syndrome, in particular neurological involvement. In the first model of treatment for this condition, these congenital, and previously assumed permanent, features were profoundly suppressed by acute post-natal treatment with a MEK inhibitor. These data suggest that activated NRAS and aberrant Wnt signaling conspire to drive CMN syndrome. Post-natal MEK inhibition is a potential candidate therapy for patients with this debilitating condition.
Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas de Membrana/fisiologia , Nevo Pigmentado/congênito , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/congênito , Proteínas Wnt/fisiologia , Animais , Criança , DNA/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Mutação/genética , Nevo Pigmentado/metabolismo , Nevo Pigmentado/fisiopatologia , Análise de Sequência de DNA , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/fisiopatologiaAssuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Genótipo , Mutação/genética , Domínios Proteicos/genética , Pele/metabolismo , Síndrome de Sturge-Weber/genética , Capilares/anormalidades , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Mosaicismo , Fenótipo , Estudos Prospectivos , Pele/patologia , Pigmentação da Pele , Síndrome de Sturge-Weber/diagnóstico , Malformações VascularesRESUMO
Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown-rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (-132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects.
Assuntos
Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Impressão Genômica/genética , Placenta/metabolismo , Peso ao Nascer/fisiologia , Proteínas de Ligação ao Cálcio , Vilosidades Coriônicas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Gravidez , Receptores de Somatomedina/metabolismoRESUMO
CONTEXT: Fetal growth involves highly complex molecular pathways. IGF2 is a key paternally expressed growth hormone that is critical for in utero growth in mice. Its role in human fetal growth has remained ambiguous, as it has only been studied in term tissues. Conversely the maternally expressed growth suppressor, PHLDA2, has a significant negative correlation between its term placental expression and birth weight. OBJECTIVE: The aim of this study is to address the role in early gestation of expression of IGF1, IGF2, their receptors IGF1R and IGF2R, and PHLDA2 on term birth weight. DESIGN: Real-time quantitative PCR was used to investigate mRNA expression of IGF1, IGF2, IGF1R, IGF2R and PHLDA2 in chorionic villus samples (CVS) (n = 260) collected at 11-13 weeks' gestation. Expression was correlated with term birth weight using statistical package R including correction for several confounding factors. RESULTS: Transcript levels of IGF2 and IGF2R revealed a significant positive correlation with birth weight (0.009 and 0.04, respectively). No effect was observed for IGF1, IGF1R or PHLDA2 and birth weight. Critically, small for gestational age (SGA) neonates had significantly lower IGF2 levels than appropriate for gestational age neonates (p = 3.6 × 10(-7)). INTERPRETATION: Our findings show that IGF2 mRNA levels at 12 weeks gestation could provide a useful predictor of future fetal growth to term, potentially predicting SGA babies. SGA babies are known to be at a higher risk for type 2 diabetes. This research reveals an imprinted, parentally driven rheostat for in utero growth.