A Novel Plant-Based Nutraceutical Combined with Exercise Can Revert Oxidative Status in Plasma and Liver in a Diet-Induced-Obesity Animal Model.

The prevalence of obesity increases alarmingly every year mostly due to external factors such as high-fat and high-refined sugar intake associated with a sedentary lifestyle. It triggers metabolic disorders such as insulin resistance, hyperlipemia, non-alcoholic fatty liver disease, chronic inflammation, oxidative stress, and gut microbiota dysbiosis. The aim of this study was to evaluate the beneficial effects of a combined intervention with caloric restriction, nutraceutical intake, and a mixed training protocol on oxidative stress, inflammation, and gut dysbiosis derived from the development of obesity in a C57BL6/J mouse experimental model of diet-induced obesity (4.6 Kcal/g diet, 45% Kcal as fat, and 20% fructose in the drinking fluid). The nutraceutical was formulated with ethanolic extracts of Argania spinosa pulp (10%) and Camelina sativa seeds (10%) and with protein hydrolysates from Psoralea corylifolia seeds (40%) and Spirodela polyrhiza whole plants (40%). The combination of nutraceutical and exercise decreased the animals' body weights and inflammatory markers (TNFα, IL-6, and resistin) in plasma, while increasing gene expression of cat, sod2, gsta2, and nqo1 in the liver. Obese animals showed lower ß-diversity of microbiota and a higher Firmicutes/Bacteroidetes ratio vs. normocaloric controls that were reversed by all interventions implemented. Dietary inclusion of a nutraceutical with high antioxidant potential combined with an exercise protocol can be beneficial for bodyweight control and improvement of metabolic status in patients undergoing obesity treatment.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

A holistic approach to understanding immune-mediated inflammatory diseases: bioinformatic tools to integrate omics data.

Immune-mediated inflammatory diseases (IMIDs) comprise a complex group of pathologies with diverse etiologies and clinical manifestations. In particular, omics technologies have remodeled our understanding of a set of IMIDs such as systemic autoimmune rheumatic diseases (SARDs), generating vast amounts of data on the genome, epigenome, transcriptome, proteome and metabolome of immune cells and SARDs patients. However, the integration of omics data to advance our knowledge of these diseases is challenging, requiring advanced bioinformatic tools. This review explores different multi-omic integrative tools for refining previous research, exploring the biological relevance of datasets within different contexts, or translating omics results into clinical advances. We also discuss relevant multi-omic studies in SARDs research and the potential of omics data from available repositories to complement ongoing investigation in this field.

Cortistatin deficiency reveals a dysfunctional brain endothelium with impaired gene pathways, exacerbated immune activation, and disrupted barrier integrity.

BACKGROUND: Brain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood-brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment. METHODS: Wild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS. RESULTS: The endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage. CONCLUSIONS: The neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Explainable artificial intelligence to predict and identify prostate cancer tissue by gene expression.

BACKGROUND AND OBJECTIVE: Prostate cancer is one of the most prevalent forms of cancer in men worldwide. Traditional screening strategies such as serum PSA levels, which are not necessarily cancer-specific, or digital rectal exams, which are often inconclusive, are still the screening methods used for the disease. Some studies have focused on identifying biomarkers of the disease but none have been reported for diagnosis in routine clinical practice and few studies have provided tools to assist the pathologist in the decision-making process when analyzing prostate tissue. Therefore, a classifier is proposed to predict the occurrence of PCa that provides physicians with accurate predictions and understandable explanations. METHODS: A selection of 47 genes was made based on differential expression between PCa and normal tissue, GO gene ontology as well as the literature to be used as input predictors for different machine learning methods based on eXplainable Artificial Intelligence. These methods were trained using different class-balancing strategies to build accurate classifiers using gene expression data from 550 samples from 'The Cancer Genome Atlas'. Our model was validated in four external cohorts with different ancestries, totaling 463 samples. In addition, a set of SHapley Additive exPlanations was provided to help clinicians understand the underlying reasons for each decision. RESULTS: An in-depth analysis showed that the Random Forest algorithm combined with majority class downsampling was the best performing approach with robust statistical significance. Our method achieved an average sensitivity and specificity of 0.90 and 0.8 with an AUC of 0.84 across all databases. The relevance of DLX1, MYL9 and FGFR genes for PCa screening was demonstrated in addition to the important role of novel genes such as CAV2 and MYLK. CONCLUSIONS: This model has shown good performance in 4 independent external cohorts of different ancestries and the explanations provided are consistent with each other and with the literature, opening a horizon for its application in clinical practice. In the near future, these genes, in combination with our model, could be applied to liquid biopsy to improve PCa screening.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

Exploring the Microbiome in Human Reproductive Tract: High-Throughput Methods for the Taxonomic Characterization of Microorganisms.

Microorganisms are important due to their widespread presence and multifaceted roles across various domains of life, ecology, and industries. In humans, they underlie the proper functioning of multiple systems crucial to well-being, including immunological and metabolic functions. Emerging research addressing the presence and roles of microorganisms within human reproduction is increasingly relevant. Studies implementing new methodologies (e.g., to investigate vaginal, uterine, and semen microenvironments) can now provide relevant insights into fertility, reproductive health, or pregnancy outcomes. In that sense, cutting-edge sequencing techniques, as well as others such as meta-metabolomics, culturomics, and meta-proteomics, are becoming more popular and accessible worldwide, allowing the characterization of microbiomes at unprecedented resolution. However, they frequently involve rather complex laboratory protocols and bioinformatics analyses, for which researchers may lack the required expertise. A suitable pipeline would successfully enable both taxonomic classification and functional profiling of the microbiome, providing easy-to-understand biological interpretations. However, the selection of an appropriate methodology would be crucial, as it directly impacts the reproducibility, accuracy, and quality of the results and observations. This review focuses on the different current microbiome-related techniques in the context of human reproduction, encompassing niches like vagina, endometrium, and seminal fluid. The most standard and reliable methods are 16S rRNA gene sequencing, metagenomics, and meta-transcriptomics, together with complementary approaches including meta-proteomics, meta-metabolomics, and culturomics. Finally, we also offer case examples and general recommendations about the most appropriate methods and workflows and discuss strengths and shortcomings for each technique.

Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice.

The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a "new" technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages.

Leishmaniasis is considered to be one of the most neglected tropical diseases affecting humans and animals around the world. Due to the absence of an effective vaccine, current treatment is based on chemotherapy. However, the continuous appearance of drug resistance and therapeutic failure (TF) lead to an early obsolescence of treatments. Identification of the factors that contribute to TF and drug resistance in leishmaniasis will constitute a useful tool for establishing future strategies to control this disease. In this manuscript, we evaluated the transcriptomic changes in the intracellular amastigotes of the Leishmania infantum parasites isolated from patients with leishmaniasis and TF at 96 h post-infection of THP-1 cells. The adaptation of the parasites to their new environment leads to expression alterations in the genes involved mainly in the transport through cell membranes, energy and redox metabolism, and detoxification. Specifically, the gene that codes for the prostaglandin f2α synthase seems to be relevant in the pathogenicity and TF since it appears substantially upregulated in all the L. infantum lines. Overall, our results show that at the late infection timepoint, the transcriptome of the parasites undergoes significant changes that probably improve the survival of the Leishmania lines in the host cells, contributing to the TF phenotype as well as drug therapy evasion.

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo.

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids.

OBJECTIVES: Giant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterisation of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis. METHODS: We performed an integrated epigenome-and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical statuses (active, in remission with or without glucocorticoid (GC) treatment), and 31 healthy controls. RESULTS: We identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more proinflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to IL-6 and IL-1, we identified response to IL-11 as a new pathway potentially implicated in GCA. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such as ITGA7 and CD63, as well as genes mediating the molecular response to GC, including FKBP5, ETS2, ZBTB16 and ADAMTS2. CONCLUSION: Our results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite's ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

RNA-Seq, Bioinformatic Identification of Potential MicroRNA-like Small RNAs in the Edible Mushroom Agaricus bisporus and Experimental Approach for Their Validation.

Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.

Single-cell Atlas of common variable immunodeficiency shows germinal center-associated epigenetic dysregulation in B-cell responses.

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.

Identification of Group II Intron RmInt1 Binding Sites in a Bacterial Genome.

RmInt1 is a group II intron encoding a reverse transcriptase protein (IEP) lacking the C-terminal endonuclease domain. RmInt1 is an efficient mobile retroelement that predominantly reverse splices into the transient single-stranded DNA at the template for lagging strand DNA synthesis during host replication, a process facilitated by the interaction of the RmInt1 IEP with DnaN at the replication fork. It has been suggested that group II intron ribonucleoprotein particles bind DNA nonspecifically, and then scan for their correct target site. In this study, we investigated RmInt1 binding sites throughout the Sinorhizobium meliloti genome, by chromatin-immunoprecipitation coupled with next-generation sequencing. We found that RmInt1 binding sites cluster around the bidirectional replication origin of each of the three replicons comprising the S. meliloti genome. Our results provide new evidence linking group II intron mobility to host DNA replication.

Selective histone methyltransferase G9a inhibition reduces metastatic development of Ewing sarcoma through the epigenetic regulation of NEU1.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.

Differently Regulated Gene-Specific Activity of Enhancers Located at the Boundary of Subtopologically Associated Domains: TCRα Enhancer.

Enhancers activate transcription through long-distance interactions with their cognate promoters within a particular subtopologically associated domain (sub-TAD). The TCRα enhancer (Eα) is located at the sub-TAD boundary between the TCRα and DAD1 genes and regulates transcription toward both sides in an ∼1-Mb region. Analysis of Eα activity in transcribing the unrearranged TCRα gene at the 5'-sub-TAD has defined Eα as inactive in CD4-CD8- thymocytes, active in CD4+CD8+ thymocytes, and strongly downregulated in CD4+ and CD8+ thymocytes and αß T lymphocytes. Despite its strongly reduced activity, Eα is still required for high TCRα transcription and expression of TCRαß in mouse and human T lymphocytes, requiring collaboration with distant sequences for such functions. Because VαJα rearrangements in T lymphocytes do not induce novel long-range interactions between Eα and other genomic regions that remain in cis after recombination, strong Eα connectivity with the 3'-sub-TAD might prevent reduced transcription of the rearranged TCRα gene. Our analyses of transcriptional enhancer dependence during T cell development and non-T lineage tissues at the 3'-sub-TAD revealed that Eα can activate the transcription of specific genes, even when it is inactive to transcribe the TCRα gene at the 5'-sub-TAD. Hence distinct requirements for Eα function are necessary at specific genes at both sub-TADs, implying that enhancers do not merely function as chromatin loop anchors that nucleate the formation of factor condensates to increase gene transcription initiated at their cognate promoters. The observed different regulated Eα activity for activating specific genes at its flanking sub-TADs may be a general feature for enhancers located at sub-TAD boundaries.

Identification of the genetic mechanism that associates L3MBTL3 to multiple sclerosis.

Multiple sclerosis (MS) is a complex and demyelinating disease of the central nervous system. One of the challenges of the post-genome-wide association studies (GWAS) era is to understand the molecular basis of statistical associations to reveal gene networks and potential therapeutic targets. The L3MBTL3 locus has been associated with MS risk by GWAS. To identify the causal variant of the locus, we performed fine mapping in a cohort of 3440 MS patients and 1688 healthy controls. The variant that best explained the association was rs6569648 (P = 4.13E-10, odds ratio = 0.71, 95% confidence interval (CI) = 0.64-0.79), which tagged rs7740107, located in intron 7 of L3MBTL3. The rs7740107 (A/T) variant has been reported to be the best expression and splice quantitative trait locus (eQTL and sQTL) of the region in up to 35 human genotype-tissue expression (GTEx) tissues. By sequencing RNA from blood of 17 MS patients and quantification by digital qPCR, we determined that this eQTL/sQTL originated from the expression of a novel short transcript starting in intron 7 near rs7740107. The short transcript was translated into three proteins starting at different translation initiation codons. These N-terminal truncated proteins lacked the region where L3MBTL3 interacts with the transcriptional regulator Recombination Signal Binding Protein for Immunoglobulin Kappa J Region which, in turn, regulates the Notch signalling pathway. Our data and other functional studies suggest that the genetic mechanism underlying the MS association of rs7740107 affects not only the expression of L3MBTL3 isoforms, but might also involve the Notch signalling pathway.