SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.

Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Landscape of pathogenic variations in a panel of 34 genes and cancer risk estimation from 5131 HBOC families.

PURPOSE: Integration of gene panels in the diagnosis of hereditary breast and ovarian cancer (HBOC) requires a careful evaluation of the risk associated with pathogenic or likely pathogenic variants (PVs) detected in each gene. Here we analyzed 34 genes in 5131 suspected HBOC index cases by next-generation sequencing. METHODS: Using the Exome Aggregation Consortium data sets plus 571 individuals from the French Exome Project, we simulated the probability that an individual from the Exome Aggregation Consortium carries a PV and compared it to the estimated frequency within the HBOC population. RESULTS: Odds ratio conferred by PVs within BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATM, BRIP1, CHEK2, and MSH6 were estimated at 13.22 [10.01-17.22], 8.61 [6.78-10.82], 8.22 [4.91-13.05], 4.54 [2.55-7.48], 5.23 [1.46-13.17], 3.20 [2.14-4.53], 2.49 [1.42-3.97], 1.67 [1.18-2.27], and 2.50 [1.12-4.67], respectively. PVs within RAD51C, RAD51D, and BRIP1 were associated with ovarian cancer family history (OR = 11.36 [5.78-19.59], 12.44 [2.94-33.30] and 3.82 [1.66-7.11]). PALB2 PVs were associated with bilateral breast cancer (OR = 16.17 [5.48-34.10]) and BARD1 PVs with triple-negative breast cancer (OR = 11.27 [3.37-25.01]). Burden tests performed in both patients and the French Exome Project population confirmed the association of PVs of BRCA1, BRCA2, PALB2, and RAD51C with HBOC. CONCLUSION: Our results validate the integration of PALB2, RAD51C, and RAD51D in the diagnosis of HBOC and suggest that the other genes are involved in an oligogenic determinism.

Mesalazine initiates an anti-oncogenic ß-catenin / MUCDHL negative feed-back loop in colon cancer cells by cell-specific mechanisms.

Chronic inflammation associated with intestinal architecture and barrier disruption puts patients with inflammatory bowel disease (IBD) at increased risk of developing colorectal cancer (CRC). Widely used to reduce flares of intestinal inflammation, 5-aminosalicylic acid derivatives (5-ASAs) such as mesalazine appear to also exert more direct mucosal healing and chemopreventive activities against CRC. The mechanisms underlying these activities are poorly understood and may involve the up-regulation of the cadherin-related gene MUCDHL (CDHR5). This atypical cadherin is emerging as a new actor of intestinal homeostasis and opposes colon tumorigenesis. Here, we showed that mesalazine increase mRNA levels of MUCDHL and of other genes involved in the intestinal barrier function in most intestinal cell lines. In addition, using gain / loss of function experiments (agonists, plasmid or siRNAs transfections), luciferase reporter genes and chromatin immunoprecipitation, we thoroughly investigated the molecular mechanisms triggered by mesalazine that lead to the up-regulation of MUCDHL expression. We found that basal transcription of MUCDHL in different CRC cell lines is regulated positively by CDX2 and negatively by ß-catenin through a negative feed-back loop. However, mesalazine-stimulation of MUCDHL transcription is controlled by cell-specific mechanisms, involving either enhanced activation of CDX2 and PPAR-γ or repression of the ß-catenin inhibitory effect. This work highlights the importance of the cellular and molecular context in the activity of mesalazine and suggests that its efficacy against CRC depends on the genetic alterations of transformed cells.

OutLyzer: software for extracting low-allele-frequency tumor mutations from sequencing background noise in clinical practice.

Highlighting tumoral mutations is a key step in oncology for personalizing care. Considering the genetic heterogeneity in a tumor, software used for detecting mutations should clearly distinguish real tumor events of interest that could be predictive markers for personalized medicine from false positives. OutLyzer is a new variant-caller designed for the specific and sensitive detection of mutations for research and diagnostic purposes. It is based on statistic and local evaluation of sequencing background noise to highlight potential true positive variants. 130 previously genotyped patients were sequenced after enrichment by capturing the exons of 22 genes. Sequencing data were analyzed by HaplotypeCaller, LofreqStar, Varscan2 and OutLyzer. OutLyzer had the best sensitivity and specificity with a fixed limit of detection for all tools of 1% for SNVs and 2% for Indels. OutLyzer is a useful tool for detecting mutations of interest in tumors including low allele-frequency mutations, and could be adopted in standard practice for delivering targeted therapies in cancer treatment.

Anti-inflammatory effects of an injectable copolymer of fatty acids (Ara 3000 beta®) in joint diseases.

BACKGROUND: In inflammatory joint disease, such as osteoarthritis or arthritis, there is an increased level of pro-inflammatory cytokines, such as interleukin-1ß. These cytokines stimulate the expression and release of matrix metalloproteases (MMP), leading to the degradation of cartilage extracellular matrix and subsequently mobility difficulty and suffering for patients. The aim of this study was to examine the therapeutic potential of a fatty acid copolymer in in vitro and in vivo models of cartilage inflammation. METHODS: Inflammation was mimicked in vitro by treatment of human articular chondrocytes with interleukin-1ß. Effects of a co-treatment with a copolymer of fatty acids (Ara 3000 beta®) were determined by evaluating MMP production by RT-PCR and ELISA, NO release by Griess assay, and PGE2 expression by ELISA. In addition, in vivo analysis (evolution of weight and edema) were also performed after injection of Freund adjuvant in rats treated or not with the copolymer of fatty acids. RESULTS: The copolymer of fatty acids clearly reduces inflammation in joint. In vitro, it impairs IL1 stimulated-MMP production and release, as well as the release of NO and PGE2 and the activation of NFκB. Furthermore, in vivo experiments using adjuvant induced-arthritis corroborates the anti-inflammatory effects of the copolymer of fatty acids, with a reduction of edemas, erythemas and ankylosis in arthritic rats. CONCLUSIONS: The results support the hypothesis that a copolymer of fatty acids, such as Ara 3000 beta®, is a powerful anti-inflammatory compounds, suggesting that it has a potential for preventing cartilage degradation associated with chronic inflammatory joint disease.

Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines.

We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116 whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity and that the α-hydroxyl group was found to be more beneficial at the 7-position in steroid skeleton.