Benchmarking of human Y-chromosomal haplogroup classifiers with whole-genome and whole-exome sequence data.

In anthropological, medical, and forensic studies, the nonrecombinant region of the human Y chromosome (NRY) enables accurate reconstruction of pedigree relationships and retrieval of ancestral information. Using high-throughput sequencing (HTS) data, we present a benchmarking analysis of command-line tools for NRY haplogroup classification. The evaluation was performed using paired Illumina data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments from 50 unrelated donors. Additionally, as a validation, we also used paired WGS/WES datasets of 54 individuals from the 1000 Genomes Project. Finally, we evaluated the tools on data from third-generation HTS obtained from a subset of donors and one reference sample. Our results show that WES, despite typically offering less genealogical resolution than WGS, is an effective method for determining the NRY haplogroup. Y-LineageTracker and Yleaf showed the highest accuracy for WGS data, classifying precisely 98% and 96% of the samples, respectively. Yleaf outperforms all benchmarked tools in the WES data, classifying approximately 90% of the samples. Yleaf, Y-LineageTracker, and pathPhynder can correctly classify most samples (88%) sequenced with third-generation HTS. As a result, Yleaf provides the best performance for applications that use WGS and WES. Overall, our study offers researchers with a guide that allows them to select the most appropriate tool to analyze the NRY region using both second- and third-generation HTS data.

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression.

Originally considered to act as a transcriptional co-factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient's clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow-cycling JARID1B gene.

Computational and Experimental Evaluation of the Immune Response of Neoantigens for Personalized Vaccine Design.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

Melanoma Clinical Decision Support System: An Artificial Intelligence-Based Tool to Diagnose and Predict Disease Outcome in Early-Stage Melanoma Patients.

This study set out to assess the performance of an artificial intelligence (AI) algorithm based on clinical data and dermatoscopic imaging for the early diagnosis of melanoma, and its capacity to define the metastatic progression of melanoma through serological and histopathological biomarkers, enabling dermatologists to make more informed decisions about patient management. Integrated analysis of demographic data, images of the skin lesions, and serum and histopathological markers were analyzed in a group of 196 patients with melanoma. The interleukins (ILs) IL-4, IL-6, IL-10, and IL-17A as well as IFNγ (interferon), GM-CSF (granulocyte and macrophage colony-stimulating factor), TGFß (transforming growth factor), and the protein DCD (dermcidin) were quantified in the serum of melanoma patients at the time of diagnosis, and the expression of the RKIP, PIRIN, BCL2, BCL3, MITF, and ANXA5 proteins was detected by immunohistochemistry (IHC) in melanoma biopsies. An AI algorithm was used to improve the early diagnosis of melanoma and to predict the risk of metastasis and of disease-free survival. Two models were obtained to predict metastasis (including "all patients" or only patients "at early stages of melanoma"), and a series of attributes were seen to predict the progression of metastasis: Breslow thickness, infiltrating BCL-2 expressing lymphocytes, and IL-4 and IL-6 serum levels. Importantly, a decrease in serum GM-CSF seems to be a marker of poor prognosis in patients with early-stage melanomas.

A Universal Antigen-Ranking Method to Design Personalized Vaccines Targeting Neoantigens against Melanoma.

Background: The main purpose of this article is to introduce a universal mathematics-aided vaccine design method against malignant melanoma based on neoantigens. The universal method can be adapted to the mutanome of each patient so that a specific candidate vaccine can be tailored for the corresponding patient. Methods: We extracted the 1134 most frequent mutations in melanoma, and we associated each of them to a vector with 10 components estimated with different bioinformatics tools, for which we found an aggregated value according to a set of weights, and then we ordered them in decreasing order of the scores. Results: We prepared a universal table of the most frequent mutations in melanoma ordered in decreasing order of viability to be used as candidate vaccines, so that the selection of a set of appropriate peptides for each particular patient can be easily and quickly implemented according to their specific mutanome and transcription profile. Conclusions: We have shown that the techniques that are commonly used for the design of personalized anti-tumor vaccines against malignant melanoma can be adapted for the design of universal rankings of neoantigens that originate personalized vaccines when the mutanome and transcription profile of specific patients is considered, with the consequent savings in time and money, shortening the design and production time.

Association of TYR SNP rs1042602 with Melanoma Risk and Prognosis.

Cutaneous melanoma is the most aggressive of skin tumors. In order to discover new biomarkers that could help us improve prognostic prediction in melanoma patients, we have searched for germline DNA variants associated with melanoma progression. Thus, after exome sequencing of a set of melanoma patients and healthy control individuals, we identified rs1042602, an SNP within TYR, as a good candidate. After genotyping rs1042602 in 1025 patients and 773 healthy donors, we found that the rs1042602-A allele was significantly associated with susceptibility to melanoma (CATT test: p = 0.0035). Interestingly, we also observed significant differences between patients with good and bad prognosis (5 years of follow-up) (n = 664) (CATT test for all samples p = 0.0384 and for men alone p = 0.0054). Disease-free-survival (DFS) analyses also showed that patients with the A allele had shorter DFS periods. In men, the association remained significant even in a multivariate Cox Proportional-hazards model, which was adjusted for age at diagnosis, Breslow thickness, ulceration and melanoma subtype (HR 0.4; 95% confidence interval (CI) 0.20-0.83; p = 0.0139). Based on our results, we propose that rs1042602-A is a risk allele for melanoma, which also seems to be responsible for a poorer prognosis of the disease, particularly in men.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

First computational design using lambda-superstrings and in vivo validation of SARS-CoV-2 vaccine.

Coronavirus disease 2019 (COVID-19) is the greatest threat to global health at the present time, and considerable public and private effort is being devoted to fighting this recently emerged disease. Despite the undoubted advances in the development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uncertainty remains about their future efficacy and the duration of the immunity induced. It is therefore prudent to continue designing and testing vaccines against this pathogen. In this article we computationally designed two candidate vaccines, one monopeptide and one multipeptide, using a technique involving optimizing lambda-superstrings, which was introduced and developed by our research group. We tested the monopeptide vaccine, thus establishing a proof of concept for the validity of the technique. We synthesized a peptide of 22 amino acids in length, corresponding to one of the candidate vaccines, and prepared a dendritic cell (DC) vaccine vector loaded with the 22 amino acids SARS-CoV-2 peptide (positions 50-71) contained in the NTD domain (DC-CoVPSA) of the Spike protein. Next, we tested the immunogenicity, the type of immune response elicited, and the cytokine profile induced by the vaccine, using a non-related bacterial peptide as negative control. Our results indicated that the CoVPSA peptide of the Spike protein elicits noticeable immunogenicity in vivo using a DC vaccine vector and remarkable cellular and humoral immune responses. This DC vaccine vector loaded with the NTD peptide of the Spike protein elicited a predominant Th1-Th17 cytokine profile, indicative of an effective anti-viral response. Finally, we performed a proof of concept experiment in humans that included the following groups: asymptomatic non-active COVID-19 patients, vaccinated volunteers, and control donors that tested negative for SARS-CoV-2. The positive control was the current receptor binding domain epitope of COVID-19 RNA-vaccines. We successfully developed a vaccine candidate technique involving optimizing lambda-superstrings and provided proof of concept in human subjects. We conclude that it is a valid method to decipher the best epitopes of the Spike protein of SARS-CoV-2 to prepare peptide-based vaccines for different vector platforms, including DC vaccines.

Evolution: On the origin of Basques.

As the only speakers of a non-Indo-European language lacking connections to any other language, the origin and evolution of the Basque population have been topics of interest. A new genomic analysis of Basques shows that they are not a relict Paleolithic population as once thought.

RKIP Regulates Differentiation-Related Features in Melanocytic Cells.

Raf Kinase Inhibitor Protein (RKIP) has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancers. However, little is known about RKIP in melanoma or regarding its function in normal cells. We examined the role of RKIP in both primary melanocytes and malignant melanoma cells and evaluated its diagnostic and prognostic value. IHC analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. As a whole, our data support the utility of RKIP as a diagnostic marker for early-stage melanomas. In addition, these findings indicate its participation in the maintenance of a differentiated state of melanocytic cells by modulating genes intimately linked to the cellular motility and explain the progressive decrease of RKIP often described in tumors.

BRAF V600E mutational load as a prognosis biomarker in malignant melanoma.

Analyzing the mutational load of driver mutations in melanoma could provide valuable information regarding its progression. We aimed at analyzing the heterogeneity of mutational load of BRAF V600E in biopsies of melanoma patients of different stages, and investigating its potential as a prognosis factor. Mutational load of BRAF V600E was analyzed by digital PCR in 78 biopsies of melanoma patients of different stages and 10 nevi. The BRAF V600E load was compared among biopsies of different stages. Results showed a great variability in the load of V600E (0%-81%). Interestingly, we observed a significant difference in the load of V600E between the early and late melanoma stages, in the sense of an inverse correlation between BRAF V600E mutational load and melanoma progression. In addition, a machine learning approach showed that the mutational load of BRAF V600E could be a good predictor of metastasis in stage II patients. Our results suggest that BRAF V600E is a promising biomarker of prognosis in stage II patients.

Forensically relevant phylogeographic evaluation of mitogenome variation in the Basque Country.

The Basque Country has been the focus of population (genetic) and evolutionary studies for decades, as it represents an interesting evolutionary feature: it is the only European country where a non-Indo-European language is still spoken today and, for which there are no known living or extinct relatives. Early studies that were based on anatomical and serological methods, along with subsequent molecular genetic investigations, contain controversial interpretations of their data. Additionally, the analysis of mitochondrial DNA, which is maternally inherited and thus suitable for the examination of the maternal phylogeny of the population, was the focus of some studies. Early mtDNA studies were however restricted to the information provided by the control region or its hypervariable segments only. These are known to harbour little phylogenetic information, particularly for haplogroup H that is dominant in Westeurasian populations including the Basques. Later studies analysed complete mitogenome sequences. Their information content is however limited, either because the number of samples was low, or because these studies only considered particular haplogroups. In this study we present the full mitogenome sequences of 178 autochthonous Basque individuals that were carefully selected based on their familial descent and discuss the observed phylogenetic signals in the light of earlier published findings. We confirm the presence of Basque-specific mtDNA lineages and extend the knowledge of these lineages by providing data on their distribution in comparison to other Basque and non-Basque populations. This dataset improves our understanding of the Basque mtDNA phylogeny and serves as a high-quality dataset that is provided via EMPOP for forensic genetic purposes.

Genomic Analyses of Human European Diversity at the Southwestern Edge: Isolation, African Influence and Disease Associations in the Canary Islands.

Despite the genetic resemblance of Canary Islanders to other southern European populations, their geographical isolation and the historical admixture of aborigines (from North Africa) with sub-Saharan Africans and Europeans have shaped a distinctive genetic makeup that likely affects disease susceptibility and health disparities. Based on single nucleotide polymorphism array data and whole genome sequencing (30×), we inferred that the last African admixture took place ∼14 generations ago and estimated that up to 34% of the Canary Islander genome is of recent African descent. The length of regions in homozygosis and the ancestry-related mosaic organization of the Canary Islander genome support the view that isolation has been strongest on the two smallest islands. Furthermore, several genomic regions showed significant and large deviations in African or European ancestry and were significantly enriched in genes involved in prevalent diseases in this community, such as diabetes, asthma, and allergy. The most prominent of these regions were located near LCT and the HLA, two well-known targets of selection, at which 40‒50% of the Canarian genome is of recent African descent according to our estimates. Putative selective signals were also identified in these regions near the SLC6A11-SLC6A1, KCNMB2, and PCDH20-PCDH9 genes. Taken together, our findings provide solid evidence of a significant recent African admixture, population isolation, and adaptation in this part of Europe, with the favoring of African alleles in some chromosome regions. These findings may have medical implications for populations of recent African ancestry.

Identification of a novel locus associated with skin colour in African-admixed populations.

Skin pigmentation is a complex trait that varies largely among populations. Most genome-wide association studies of this trait have been performed in Europeans and Asians. We aimed to uncover genes influencing skin colour in African-admixed individuals. We performed a genome-wide association study of melanin levels in 285 Hispanic/Latino individuals from Puerto Rico, analyzing 14 million genetic variants. A total of 82 variants with p-value ≤1 × 10-5 were followed up in 373 African Americans. Fourteen single nucleotide polymorphisms were replicated, of which nine were associated with skin colour at genome-wide significance in a meta-analysis across the two studies. These results validated the association of two previously known skin pigmentation genes, SLC24A5 (minimum p = 2.62 × 10-14, rs1426654) and SLC45A2 (minimum p = 9.71 × 10-10, rs16891982), and revealed the intergenic region of BEND7 and PRPF18 as a novel locus associated with this trait (minimum p = 4.58 × 10-9, rs6602666). The most significant variant within this region is common among African-descent populations but not among Europeans or Native Americans. Our findings support the advantages of analyzing African-admixed populations to discover new genes influencing skin pigmentation.

Sex-specific genetic effects associated with pigmentation, sensitivity to sunlight, and melanoma in a population of Spanish origin.

BACKGROUND: Human pigmentation is a polygenic quantitative trait with high heritability. In addition to genetic factors, it has been shown that pigmentation can be modulated by oestrogens and androgens via up- or down-regulation of melanin synthesis. Our aim was to identify possible sex differences in pigmentation phenotype as well as in melanoma association in a melanoma case-control population of Spanish origin. METHODS: Five hundred and ninety-nine females (316 melanoma cases and 283 controls) and 458 males (234 melanoma cases and 224 controls) were analysed. We genotyped 363 polymorphisms (single nucleotide polymorphisms (SNPs)) from 65 pigmentation gene regions. RESULTS: When samples were stratified by sex, we observed more SNPs associated with dark pigmentation and good sun tolerance in females than in males (107 versus 75; P = 2.32 × 10(-6)), who were instead associated with light pigmentation and poor sun tolerance. Furthermore, six SNPs in TYR, SILV/CDK2, GPR143, and F2RL1 showed strong differences in melanoma risk by sex (P < 0.01). CONCLUSIONS: We demonstrate that these genetic variants are important for pigmentation as well as for melanoma risk, and also provide suggestive evidence for potential differences in genetic effects by sex.

Expression profiling of human melanocytes in response to UV-B irradiation.

A comprehensive gene expression analysis of human melanocytes was performed assessing the transcriptional profile of dark melanocytes (DM) and light melanocytes (LM) at basal conditions and after UV-B irradiation at different time points (6, 12 and 24 h), and in culture with different keratinocyte-conditioned media (KCM + and KCM -). The data, previously published in [1], have been deposited in NCBI's Gene Expression Omnibus (GEO accession number: GSE70280).

Comparison of the Transcriptional Profiles of Melanocytes from Dark and Light Skinned Individuals under Basal Conditions and Following Ultraviolet-B Irradiation.

We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.