Neuroblastoma somatic mutations enriched in cis-regulatory elements collectively affect genes involved in embryonic development and immune system response

Background | The ever-growing interest towards noncoding cis-regulatory variants as cancer drivers is currently hampered by numerous challenges and limitations of variant prioritization and interpretation methods and tools. Methods | To overcome these limitations, I focused on active cis regulatory elements (aCREs) to design a customized panel for deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. CREs were defined by a reanalysis of H3K27ac ChiP-seq peaks of 25 neuroblastoma cell lines. aCREs were further identified by the presence of open chromatin as defined by DNase Hypersensitivity sites. This provided a small subset of genomic regions with evident regulatory functions in which to search for driver mutations. I tested these regions for an excess of somatic mutations and assessed the statistical significance with a global approach accounting for chromatin accessibility and replication timing. Additional validation was provided by analyzing whole genome sequences of 151 neuroblastomas. For the mutated regions, I determined their candidate target genes through HiC data analysis. Results | I identified a significant excess of somatic mutations in aCREs of diverse genes including IPO7, HAND2, and ARID3A. A gene expression signature built on basis of these three, and nearby, interacting genes strongly correlated with negative prognostic markers and low survival rates of patients affected by neuroblastoma. Moreover, I observed a convergence of biological functions of the target genes of mutated aCREs and transcription factors with binding motifs altered by mutations towards processes related to embryonic development and immune system response. Conclusion | My strategy led me to identify somatic mutations in regulatory elements that collectively can drive neuroblastoma onset

Six months of different exercise type in sedentary primary schoolchildren: impact on physical fitness and saliva microbiota composition

Introduction Lifestyle influences microbiota composition. We previously reported a healthier microbiota composition in saliva from active schoolchildren compared to sedentary. In the present study, we evaluated the effects of 6 months of different exercise types on physical fitness and saliva microbiota composition in 8-11-years-old sedentary schoolchildren.Methods Sixty-four sedentary children from five primary schools in Turin, Italy, were divided into three groups: one continued normal curricular activity while two underwent different exercise protocols for 6 months. The Structured Exercise (Sa) group did 2 h per week of muscle activation, strength and coordination exercises supervised by a kinesiologist. The Daily Mile (Dm) group did 1 h per week of Sa plus 15 min of walking/running outdoors four times a week, supervised by a class teacher; control group (Ct) did 2 h a week of curricular exercise supervised by a class teacher. Physical fitness was evaluated before and after the intervention. Saliva samples were collected post-intervention in all participants and analyzed using PCR amplification of 16S rRNA bacterial genes. The Amplicon Sequence Variants were filtered, decontaminated, and phylogenetically classified using DADA2 software. Differential abundance analysis of microbiome taxa and pathway data was conducted using the LEfSe algorithm and PICRUSt.Results The Sa group showed better performances in lower limb power and sprint performance while both the Sa and Dm groups improved in endurance and balance at the end of the intervention; only balance resulted slightly improved in the Ct group. Among the genera differently enriched in saliva after the training intervention, we found that the Prevotella, the Dubosiella and the Family XIII AD3011 group were the most abundant in the Sa group; differently, the Neisseria and the Abiotrophia in Ct group. Four species showed significant the Prevotella melaninogenica and the Prevotella nanceiensis were more abundant in the Sa, conversely, Gemella sanguinis was enriched in Dm and Abiotrophia defectiva in Ct saliva group.Conclusion We demonstrated that Sa and Dm, not curricular exercise, improve the physical fitness components in sedentary schoolchildren correlated to health and promote an enrichment in saliva microbiota species associated to a healthier profile

Impact of active lifestyle on the primary school children saliva microbiota composition

Unlabelled: The aim of the study was to evaluate the effects of Active or Sedentary lifestyle on saliva microbiota composition in Italian schoolchildren. Methods: Male (114) and female children (8-10 years) belonging to five primary schools in the neighborhoods of Turin were classified as active (A) or sedentary (S) based on PAQ-C-It questionnaire. PCR amplification of salivary DNA targeted the hypervariable V3-V4 regions of the 16S rRNA bacterial genes. DADA2 workflow was used to infer the Amplicon Sequence Variants and the taxonomic assignments; the beta-diversity was obtained by PCoA with the UniFrac method; LEfSe algorithm, threshold at 5%, and Log LDA cutoff at ±0.5 were used to identify differently abundant species in A compared to S saliva sample. Daily food intake was assessed by 3-Days food record. The metabolic potential of microbial communities was assessed by PICRUSt. Results: No significant differences were found in individual's gender distribution (p = 0.411), anthropometry, BMI (p > 0.05), and all diet composition between A and S groups (p > 0.05). Eight species were differently abundant: Prevotella nigrescens (LDA score = -3.76; FDR = 1.5×10-03), Collinsella aerofaciens (LDA score = -3.17; FDR = 7.45×10-03), Simonsiella muelleri (LDA score = -2.96; FDR = 2.76×10-05), Parabacteroides merdae (LDA score = -2.43; FDR = 1.3×10-02) are enriched in the A group; Gemella parahaemolysans, Prevotella aurantiaca (LDA score = -3.9; FDR = 5.27×10-04), Prevotella pallens (LDA score = 4.23; FDR = 1.93×10-02), Neisseria mucosa (LDA score = 4.43; FDR = 1.31×10-02; LDA score = 2.94; FDR = 7.45×10-03) are enriched in the S group. A prevalence of superpathway of fatty acid biosynthesis initiation (E. coli) and catechol degradation II (meta-cleavage pathway) was found in saliva from A compared to S children. Conclusion: Our results showed that active children had an enrichment of species and genera mainly associated with a healthier profile. By contrast, the genera and the species enriched in the sedentary group could be linked to human diseases

Common variants at 21q22.3 locus influence MX1 and TMPRSS2 gene expression and susceptibility to severe COVID-19

The established risk factors of coronavirus disease 2019 (COVID-19) are advanced age, male sex and comorbidities, but they do not fully explain the wide spectrum of disease manifestations. Genetic factors implicated in the host antiviral response provide for novel insights into its pathogenesis. We performed an in-depth genetic analysis of chromosome 21 exploiting the genome-wide association study data, including 6,406 individuals hospitalized for COVID-19 and 902,088 controls with European genetic ancestry from the COVID-19 Host Genetics Initiative. We found that five single nucleotide polymorphisms within TMPRSS2 and near MX1 gene show associations with severe COVID-19. The minor alleles of the five SNPs correlated with a reduced risk of developing severe COVID-19 and high level of MX1 expression in blood. Our findings demonstrate that host genetic factors can influence the different clinical presentations of COVID-19 and that MX1 could be a potential therapeutic target

Impact of active lifestyle on the primary school children saliva microbiota composition

The aim of the study was to evaluate the effects of Active or Sedentary lifestyle on saliva microbiota composition in Italian schoolchildren.MethodsMale (114) and female children (8–10 years) belonging to five primary schools in the neighborhoods of Turin were classified as active (A) or sedentary (S) based on PAQ-C-It questionnaire. PCR amplification of salivary DNA targeted the hypervariable V3–V4 regions of the 16S rRNA bacterial genes. DADA2 workflow was used to infer the Amplicon Sequence Variants and the taxonomic assignments; the beta-diversity was obtained by PCoA with the UniFrac method; LEfSe algorithm, threshold at 5%, and Log LDA cutoff at ±0.5 were used to identify differently abundant species in A compared to S saliva sample. Daily food intake was assessed by 3-Days food record. The metabolic potential of microbial communities was assessed by PICRUSt.ResultsNo significant differences were found in individual’s gender distribution (p = 0.411), anthropometry, BMI (p > 0.05), and all diet composition between A and S groups (p > 0.05). Eight species were differently abundant: Prevotella nigrescens (LDA score = −3.76; FDR = 1.5×10–03), Collinsella aerofaciens (LDA score = −3.17; FDR = 7.45×10–03), Simonsiella muelleri (LDA score = −2.96; FDR = 2.76×10–05), Parabacteroides merdae (LDA score = −2.43; FDR = 1.3×10–02) are enriched in the A group; Gemella parahaemolysans, Prevotella aurantiaca (LDA score = −3.9; FDR = 5.27×10–04), Prevotella pallens (LDA score = 4.23; FDR = 1.93×10–02), Neisseria mucosa (LDA score = 4.43; FDR = 1.31×10–02; LDA score = 2.94; FDR = 7.45×10–03) are enriched in the S group. A prevalence of superpathway of fatty acid biosynthesis initiation (E. coli) and catechol degradation II (meta-cleavage pathway) was found in saliva from A compared to S children.ConclusionOur results showed that active children had an enrichment of species and genera mainly associated with a healthier profile. By contrast, the genera and the species enriched in the sedentary group could be linked to human diseases

Single-cell transcriptomics of neuroblastoma identifies chemoresistance-associated genes and pathways

High-Risk neuroblastoma (NB) survival rate is still <50%, despite treatments being more and more aggressive. The biggest hurdle liable to cancer therapy failure is the drug resistance by tumor cells that is likely due to the intra-tumor heterogeneity (ITH). To investigate the link between ITH and therapy resistance in NB, we performed a single cell RNA sequencing (scRNAseq) of etoposide and cisplatin resistant NB and their parental cells. Our analysis showed a clear separation of resistant and parental cells for both conditions by identifying 8 distinct tumor clusters in etoposide-resistant/parental and 7 in cisplatin-resistant/parental cells. We discovered that drug resistance can affect NB cell identities; highlighting the bi-directional ability of adrenergic-to-mesenchymal transition of NB cells. The biological processes driving the identified resistant cell subpopulations revealed genes such as (BARD1, BRCA1, PARP1, HISTH1 axis, members of RPL family), suggesting a potential drug resistance due to the acquisition of DNA repair mechanisms and to the modification of the drug targets. Deconvolution analysis of bulk RNAseq data from 498 tumors with cell subpopulation signatures showed that the transcriptional heterogeneity of our cellular models reflected the ITH of NB tumors and allowed the identification of clusters associated with worse/better survival. Our study demonstrates the distinct cell populations characterized by genes involved in different biological processes can have a role in NB drug treatment failure. These findings evidence the importance of ITH in NB drug resistance studies and the chance that scRNA-seq analysis offers in the identification of genes and pathways liable for drug resistance

Somatic mutations enriched in cis-regulatory elements affect genes involved in embryonic development and immune system response in neuroblastoma

: Noncoding cis-regulatory variants have gained interest as cancer drivers, yet progress in understanding their significance is hindered by the numerous challenges and limitations of variant prioritization. To overcome these limitations, we focused on active cis-regulatory elements (aCRE) in order to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. In order to search for driver mutations, aCREs were defined by reanalysis of H3K27ac ChiP-seq peaks in 25 neuroblastoma cell lines. These regulatory genomic regions were tested for an excess of somatic mutations and assessed for statistical significance using a global approach that accounted for chromatin accessibility and replication timing. Additional validation was provided by whole genome sequence analysis of 151 neuroblastomas. Analysis of Hi-C data determined the presence of candidate target genes interacting with mutated regions. An excess of somatic mutations in aCREs of diverse genes were identified, including IPO7, HAND2, and ARID3A. CRISPR-Cas9 editing was utilized to assess the functional consequences of mutations in the IPO7 aCRE. Patients with noncoding mutations in aCREs showed inferior overall and event-free survival independent of age at diagnosis, stage, risk stratification, and MYCN status. Expression of aCRE-interacting genes correlated strongly with negative prognostic markers and low survival rates. Moreover, a convergence between the biological functions of aCRE target genes and transcription factors with mutated binding motifs was associated with embryonic development and immune system response. Overall, this strategy enabled the identification of somatic mutations in regulatory elements that collectively promote neuroblastoma tumorigenesis

Somatic mutations in specific and connected sub-pathways are associated to short neuroblastoma patients' survival and indicate proteins targetable at onset of disease

Neuroblastoma (NB) is an embryonic malignancy of the sympathetic nervous system with heterogeneous biological, morphological, genetic and clinical characteristics. Although genomic studies revealed the specific biological features of NB pathogenesis useful for new therapeutic approaches, the improvement of high-risk (HR)-NB patients overall survival is still unsatisfactory. To further clarify the biological basis of disease aggressiveness, we used whole exome sequencing to examine the genomic landscape of HR-NB patients at stage M with short survival (SS) and long survival (LS). Only a few genes, including SMARCA4, SMO, ZNF44 and CHD2, were recurrently and specifically mutated in the SS group, confirming the low recurrence of common mutations in this tumor. A systems biology approach revealed that in the two patient groups, mutations occurred in different pathways. Mutated genes (ARHGEF11, CACNA1G, FGF4, PTPRA, PTK2, ANK3, SMO, NTNG2, VCL and NID2) regulate the MAPK pathway associated with the organization of the extracellular matrix, cell motility through PTK2 signaling and matrix metalloproteinase activity. Moreover, we detected mutations in LAMA2, PTK2, LAMA4, and MMP14 genes, impairing MET signaling, in SFI1 and CHD2 involved in centrosome maturation and chromosome remodeling, in AK7 and SPTLC2, which regulate the metabolism of nucleotides and lipoproteins, and in NALCN, SLC12A1, SLC9A9, which are involved in the transport of small molecules. Notably, connected networks of somatically mutated genes specific for SS patients were identified. The detection of mutated genes present at the onset of disease may help to address an early treatment of HR-NB patients using FDA-approved compounds targeting the deregulated pathways. This article is protected by copyright. All rights reserved

FKBP51s signature in peripheral blood mononuclear cells of melanoma patients as a possible predictive factor for immunotherapy.

The inhibitory immune checkpoint PD-L1/PD1 promotes the alternative splicing of the FKBP5 gene, resulting in increased expression of its variant 4 in the peripheral blood mononuclear cells of melanoma patients. The variant 4 transcript is translated into the truncated FKBP51s protein. Given the importance of co-inhibitory signalling in tumour immune escape, here we tested the potential for using FKBP51s expression to predict immunotherapy outcomes. To do this, we immunophenotyped PBMCs from 118 melanoma patients and 77 age- and sex-matched healthy controls. Blood samples were collected before patients underwent ipilimumab treatment. In 64 of the 118 patients, FKBP51s expression was also assessed in regulatory T cells (Tregs). We found that each PBMC subset analysed contained an FKBP51s fraction, and that this fraction was greater in the melanoma patients than healthy controls. In CD4 T lymphocytes, the FKBP51s fraction was significantly impaired. Tregs count was increased in melanoma patients, which is in line with previous studies. Also, by analyses of FKBP51s in Tregs, we identified a subgroup of ipilimumab nonresponder patients (p = 0.002). In conclusion, FKBP51s-based immunophenotyping of melanoma patients revealed several profiles related to a negative immune regulatory control and identified an unknown Treg subset. These findings are likely to be useful in the selection of the patients that are candidate for immunotherapy