Rol del gen de pigmentació MC1R en la susceptibilitat a malalties neurodegeneratives

[cat] El gen MC1R (codi OMIM: #155555) codifica pel receptor de la melanocortina 1 (MC1R) que s’expressa majoritàriament a la membrana dels melanòcits i està implicat en la regulació de la síntesi de melanina. El gen MC1R és clau en la regulació del color de la pell i el cabell i és el responsable de controlar la quantitat relativa de síntesi d’eumelanina i feomelanina. Aquest gen és altament polimòrfic, s’hi han identificat més de 100 variants no sinònimes, però l’alteració funcional que provoquen en la proteïna la majoria d’elles és desconeguda. No obstant, hi ha 9 d’aquestes variants: p.V60L, p.D84E, p.V92M, p.R142H, p.R151C, p.I155T, p.R160W, p.R163Q i p.D294H que presenten una freqüència al·lèlica (MAF) > 0,01 en diferents poblacions caucàsiques d’ètnia blanca i que han estat àmpliament estudiades tant a nivell epidemiològic com a nivell funcional. Entre aquestes, hi ha 6 variants al·lèliques que s’han definit com a variants de pèrdua de funció (LOF) (p.D84E, p.R142H, p.R151C, p.I155T, p.R160W i p.D294H), ja que disminueixen notablement la capacitat de senyalització del MC1R a través de la via de l’AMPc i impedeixen la síntesi d’eumelanina. Els individus portadors d’aquestes variants LOF presenten una forta associació amb el fenotip “color de cabell vermell” (cabell pèl-roig, un elevat nombre de pigues, una baixa capacitat per a bronzejar-se i una elevada sensibilitat a la RUV) i un augment del risc a desenvolupar càncer cutani. L’anàlisi del transcriptoma de les cèl·lules cutànies portadores de variants LOF en MC1R va mostrar una desregulació constitutiva de gens implicats en les vies de senyalització de malalties neurodegeneratives i un augment de l’estrès oxidatiu. Donat que l’expressió del MC1R s’ha identificat també en les cèl·lules del sistema nerviós central i que un elevat nivell de dany oxidatiu s’ha observat en les àrees del cervell on hi ha neurodegeneració, varem hipotetitzar que el gen MC1R podria estar implicat en la patogènesi de les malalties neurodegeneratives. Es van dissenyar dos estudis cas-control que incloïen 870 pacients de Parkinson, 525 pacients amb Alzheimer i 736 controls d’origen espanyol. A més, es va avaluar el gen MC1R com a factor modificador de l’edat d’aparició de la malaltia de Huntington, seqüenciant el gen en 600 pacients. Tant els anàlisis d’associació genotípica cas –control com els estudis de regressió múltiple es van dur a terme amb totes aquelles variants genètiques no sinònimes que presentaven una freqüència al·lèlica > 0,01 en els casos avaluats. Complementàriament, amb l’objectiu de posar en context biològic la signatura gènica associada a la pèrdua de funció del MC1R, es va realitzar un anàlisi de xarxes d’interacció proteïna-proteïna (PPI). Els individus portadors de la variant LOF p.R160W presenten un augment del risc a desenvolupar Parkinson (OR=2,10; 95% IC: 1,18-3,73; p-valor ajustat = 0,009) i l’al·lel p.V92M de MC1R s’associa amb un augment del risc a desenvolupar la malaltia d’Alzheimer, especialment en aquells individus on el risc no pot atribuir-se a l’al·lel 4 de l’APOE. A més, el gen MC1R s’ha identificat com un factor modificador de l’edat d’inici de la malaltia de Huntington, ja que els casos portadors de l’al·lel p.R151C presenten una disminució de 5,13 anys respecte a l’edat mitjana d’aparició del Huntington. Concretament, el percentatge de variació en l’edat de debut atribuït a l’al·lel p.R151C és del 1,42%. Els resultats de la present tesi indiquen que el gen MC1R està implicat en la patogènesi de les malalties neurodegeneratives, i que la pèrdua de funció del MC1R pot contribuir a la patogènesi d’aquestes a través d’un augment del dany oxidatiu, una disminució de la resposta antiinflamatòria i una desregulació de l’autofàgia.[eng] The melanocortin 1 receptor (MC1R) gene is a key regulator of skin and hair colour. Certain MC1R polymorphisms, which cause a loss of protein function, have been associated with red hair color (RHC) phenotype (red hair, fair skin, high UV sensitivity and low tanning capacity) and a higher risk of developing skin cancer. Although the MC1R is mainly expressed in melanocytes, it is also detected in several types of brain cells. We have identified that skin cells harbouring loss-of-function MC1R polymorphisms show a deregulation of genes involved in pathways related to neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and Huntington’s disease (HD). It has also been shown that skin cells with loss-of-function MC1R polymorphisms show a significantly greater oxidative damage, which is a physiological condition found in areas with neuronal degeneration. Based on these evidences, we hypothesize that MC1R gene could be involved in neurodegenerative diseases pathogenesis. We performed two case-control studies to elucidate the role of MC1R in PD and AD. We sequenced the MC1R gene in 870 PD patients, 525 AD patients and 736 controls from Spain. We assessed the role of MC1R as a modifier factor of age of onset (AOO) in HD, sequencing MC1R in 600 HD patients. We analyzed all non-synonymous MC1R variants with a minor allele frequency of at least 0.01. Additionally, we analyzed the gene signature identified in cells harbouring loss-of-function MC1R polymorphisms by protein-protein interaction network analysis (PPI) to identify biological processes related with loss of MC1R function. We found that the allele p.R160W of MC1R increases risk of PD (OR=2.10, 95% CI: 1.18-3.73, adjusted P = 0.009) and that the p.V92M MC1R allele is associated with AD (OR=1.99, 95% CI: 1.08-3.64, adjusted P = 0 026). Furthermore, the p.R151C MC1R allele modifies AOO in HD (Bonferroni-corrected P = 0.032), its effect explains 1.42% of the variance in AOO that cannot be accounted for by the expanded HD allele. Our results suggest that loss of MC1R function may be involved in neurodegenerative diseases etiology through an increased of oxidative damage, autophagy deregulation and reduced anti-inflammatory response

Inherited MC1R variants in patients with melanoma are associated with better survival in women

Background: Women have a better melanoma prognosis, and fairer skin/hair colour. The presence of inherited MC1R variants has been associated with a better melanoma prognosis, but its interaction with sex is unknown. Objectives: To evaluate the relationship between germline MC1R status and survival, and determine any association with sex. Methods: This was a cohort study including 1341 patients with melanoma from the Melanoma Unit of the Hospital Clinic of Barcelona, between January 1996 and April 2018. We examined known sex‐related prognosis factors as they relate to features of melanoma and evaluated the sex‐specific role of MC1R in overall and melanoma‐specific survival. Hazard ratios (HRs) were calculated using univariate and multivariate Cox logistic regression. Results: Men showed lower overall survival than women (P < 0·001) and the presence of inherited MC1R variants was not associated with better survival in our cohort. However, in women the presence of MC1R variants was associated with better overall survival in the multivariate analysis [HR 0·57, 95% confidence interval (CI) 0·38-0·85; P = 0·006] but not in men [HR 1·26, 95% CI 0·89-1·79; P = 0·185 (P‐value for interaction 0·004)]. Analysis performed for melanoma‐specific survival showed the same level of significance. Conclusions: Inherited MC1R variants are associated with improved overall survival in women with melanoma but not in men. Intrinsic sex‐dependent features can modify the role of specific genes in melanoma prognosis. We believe that survival studies of patients with melanoma should include analysis by sex and MC1R genotype

Prevalence of MITF p.E318K in patients with melanoma independent of the presence of CDKN2A causative mutations

Importance The main high-penetrance melanoma susceptibility gene is CDKN2A, encoding p16INK4A and p14ARF. The gene MITF variant p.E318K also predisposes to melanoma and renal cell carcinoma. To date, the prevalence of MITF p.E318K and its clinical and phenotypical implications has not been previously assessed in a single cohort of Spanish patients with melanoma or in p16INK4A mutation carriers.Objectives To evaluate the prevalence of MITF p.E318K in Spanish patients with melanoma and assess the association with clinical and phenotypic features.Design, Setting, and Participants A hospital-based, case-control study was conducted at the Melanoma Unit of Hospital Clinic of Barcelona, with MITF p.E318K genotyped in all patients using TaqMan probes. We included 531 patients: 271 patients with multiple primary melanoma (MPM) without mutations affecting p16INK4A (wild-type p16INK4A); 191 probands from melanoma-prone families with a single melanoma diagnosis and without mutations affecting p16INK4A, and 69 probands from different families carrying CDKN2A mutations affecting p16INK4A. A population-based series of 499 age- and sex-matched cancer-free individuals from the Spanish National Bank of DNA were included as controls. Patients were recruited between January 1, 1992, and June 30, 2014; data analysis was conducted from September 1 to November 30, 2014.Main Outcomes and Measures The genetic results of the MITF p.E318K variant were correlated with clinical and phenotypic features.Results Among the 531 patients, the prevalence of the MITF p.E318K variant was calculated among the different subsets of patients included and was 1.9% (9 of 462) in all melanoma patients with wild-type p16INK4A, 2.6% (7 of 271) in those with MPM, and 2.9% (2 of 69) in the probands of families with p16INK4A mutations. With results reported as odds ratio (95% CI), the MITF p.E318K was associated with an increased melanoma risk (3.3 [1.43-7.43]; P 200 nevi) (8.4 [2.14-33.19]; P < .01). Two fast-growing melanomas were detected among 2 MITF p.E318K carriers during dermatologic digital follow-up.Conclusions and Relevance In addition to melanoma risk, MITF p.E318K is associated with a high nevi count and could play a role in fast-growing melanomas. Testing for MITF p.E318K should not exclude patients with known mutations in p16INK4A. Strict dermatologic surveillance, periodic self-examination, and renal cell carcinoma surveillance should be encouraged in this context

Capturing the biological impact of CDKN2A and MC1R genes as an early predisposing event in melanoma and non melanoma skin cancer

Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson's, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development

Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas.

BACKGROUND: BRAF and NRAS mutation detection is crucial for advanced melanoma treatment. Our aim was to evaluate how different characteristics from formalin-fixed paraffin-embedded (FFPE) samples, age of the block or DNA concentration could influence the success of BRAF and NRAS mutational screening. METHODS: DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis). BRAF and NRAS were sequenced by Sanger sequencing. RESULTS: Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them. BRAF was mutated in 55% (29/53) and NRAS in 11% (5/45) of the primary melanomas sequenced. DNA concentration correlated with the tumor area used for DNA extraction (mm2) (adj p-value<0.01, r=0.73). The age of the block did not affect sequencing success. In 60% of samples kept for more than 10 years, both BRAF and NRAS were successfully sequenced. CONCLUSIONS: Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient

Multiple BRAF Wild-Type Melanomas During Dabrafenib Treatment for Metastatic BRAF-Mutant Melanoma.

IMPORTANCE: BRAF inhibitors have become the standard of care in metastatic BRAF-mutant melanomas. Compared with chemotherapies, BRAF inhibitors improve overall and disease-free survival and speed the recovery of symptomatic patients with metastatic disease. The most worrisome finding is the possible development of resistance to new malignant tumors. OBSERVATIONS: A patient in her 30s developed massive BRAFV600E melanoma metastasis during her 30th week of pregnancy. After emergency cesarean delivery, oral dabrafenib treatment was initiated, and a partial radiologic response was confirmed within 1 month. At dermatologic digital follow-up aided by confocal microscopy 8 weeks after initiation of dabrafenib treatment, 4 melanomas were detected. Unfortunately, within the next month, the melanoma rapidly progressed. The 4 new melanomas were wild-type BRAFmelanomas, whereas the new metastasis carried a different BRAF mutation (S467L). CONCLUSIONS AND RELEVANCE: Cutaneous malignant tumors are the most frequent adverse events of BRAF inhibitors; therefore, strict dermatologic surveillance in a referral center aided by digital follow-up is mandatory, especially when multiple nevi are present and these drugs are used in an adjuvant setting. In view of our findings, the pathogenesis of the development of new melanomas seems to be different from therapy resistance. Whether paradoxical RAF activation could explain these BRAF wild-type secondary malignant tumors is still unknown

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma

Melanocortin-1 receptor (MC1R) genotypes do not correlate with size in two cohorts of medium-to-giant congenital melanocytic nevi

Congenital melanocytic nevi (CMN) are cutaneous malformations whose prevalence is inversely correlated with projected adult size. CMN are caused by somatic mutations, but epidemiological studies suggest that germline genetic factors may influence CMN development. In CMN patients from the U.K., genetic variants in MC1R, such as p.V92M and loss-of-function variants, have been previously associated with larger CMN. We analyzed the association of MC1R variants with CMN characteristics in two distinct cohorts of medium-to-giant CMN patients from Spain (N = 113) and from France, Norway, Canada, and the United States (N = 53), similar at the clinical and phenotypical level except for the number of nevi per patient. We found that the p.V92M or loss-of-function MC1R variants either alone or in combination did not correlate with CMN size, in contrast to the U.K. CMN patients. An additional case-control analysis with 259 unaffected Spanish individuals showed a higher frequency of MC1R compound heterozygous or homozygous variant genotypes in Spanish CMN patients compared to the control population (15.9% vs. 9.3%; p = .075). Altogether, this study suggests that MC1R variants are not associated with CMN size in these non-UK cohorts. Additional studies are required to define the potential role of MC1R as a risk factor in CMN development.© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

A Common Variant in the MC1R Gene (p.V92M) is associated with Alzheimer's Disease Risk.

Despite the recent identification of some novel risk genes for Alzheimer's disease (AD), the genetic etiology of late-onset Alzheimer's disease (LOAD) remains largely unknown. The inclusion of these novel risk genes to the risk attributable to the APOE gene accounts for roughly half of the total genetic variance in LOAD. The evidence indicates that undiscovered genetic factors may contribute to AD susceptibility. In the present study, we sequenced the MC1R gene in 525 Spanish LOAD patients and in 160 controls. We observed that a common MC1R variant p.V92M (rs2228479), not related to pigmentation traits, was present in 72 (14%) patients and 15 (9%) controls and confers increased risk of developing LOAD (OR: 1.99, 95% CI: 1.08-3.64, p = 0.026), especially in those patients whose genetic risk could not be explained by APOE genotype. This association remains and even increased in the subset of 69 patients with typical AD cerebrospinal fluid profile (OR: 3.40 95% CI: 1.40-8.27, p = 0.007). We did not find an association between p.V92M and age of onset of AD. Further studies are necessary to elucidate the role of MC1R in brain cells through the different MC1R pathways

T-type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E)