Burden of rare coding variants in an Italian cohort of familial multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system. It is a complex and heterogeneous disease caused by a combination of genetic and environmental factors, and it can cluster in families. OBJECTIVE: to evaluate at gene-level the aggregate contribution of predicted damaging low-frequency and rare variants to MS risk in multiplex families. METHODS: We performed whole exome sequencing (WES) in 28 multiplex MS families with at least 3 MS cases (81 affected and 42 unaffected relatives) and 38 unrelated healthy controls. A gene-based burden test was then performed, focusing on two sets of candidate genes: i) literature-driven selection and ii) data-driven selection. RESULTS: We identified 11 genes enriched with predicted damaging low-frequency and rare variants in MS compared to healthy individuals. Among them, UBR2 and DST were the two genes with the strongest enrichment (p = 5 × 10-4 and 3 × 10-4, respectively); interestingly enough the association signal in UBR2 is driven by rs62414610, which was present in 25% of analysed families. CONCLUSION: Despite limitations, this is one of the first studies evaluating the aggregate contribution of predicted damaging low-frequency and rare variants in MS families using WES data. A replication effort in independent cohorts is warranted to validate our findings and to evaluate the role of identified genes in MS pathogenesis.

Transcriptomic Analysis of Peripheral Monocytes upon Fingolimod Treatment in Relapsing Remitting Multiple Sclerosis Patients.

Fingolimod (FTY), a second-line oral drug approved for relapsing remitting Multiple Sclerosis (RRMS) acts in preventing lymphocyte migration outside lymph nodes; moreover, several lines of evidence suggest that it also inhibits myeloid cell activation. In this study, we investigated the transcriptional changes induced by FTY in monocytes in order to better elucidate its mechanism of action. CD14+ monocytes were collected from 24 RRMS patients sampled at baseline and after 6 months of treatment and RNA profiles were obtained through next-generation sequencing. We conducted pathway and sub-paths analysis, followed by centrality analysis of cell-specific interactomes on differentially expressed genes (DEGs). We investigated also the predictive role of baseline monocyte transcription profile in influencing the response to FTY therapy. We observed a marked down-regulation effect (60 down-regulated vs. 0 up-regulated genes). Most of the down-regulated DEGs resulted related with monocyte activation and migration like IL7R, CCR7 and the Wnt signaling mediators LEF1 and TCF7. The involvement of Wnt signaling was also confirmed by subpaths analyses. Furthermore, pathway and network analyses showed an involvement of processes related to immune function and cell migration. Baseline transcriptional profile of the HLA class II gene HLA-DQA1 and HLA-DPA1 were associated with evidence of disease activity after 2 years of treatment. Our data support the evidence that FTY induces major transcriptional changes in monocytes, mainly regarding genes involved in cell trafficking and immune cell activation. The baseline transcriptional levels of genes associated with antigen presenting function were associated with disease activity after 2 years of FTY treatment.

Identification of differential DNA methylation associated with multiple sclerosis: A family-based study.

Multiple Sclerosis (MS) is caused by a still unknown interplay between genetic and environmental factors. Epigenetics, including DNA methylation, represents a model for environmental factors to influence MS risk. Twenty-six affected and 26 unaffected relatives from 8 MS multiplex families were analysed in a multicentric Italian study using MeDIP-Seq, followed by technical validation and biological replication in two additional families of differentially methylated regions (DMRs) using SeqCap Epi Choice Enrichment kit (Roche®). Associations from MeDIP-Seq across families were combined with aggregation statistics, yielding 162 DMRs at FDR ≤ 0.1. Technical validation and biological replication led to 2 hypo-methylated regions, which point to NTM and BAI3 genes, and to 2 hyper-methylated regions in PIK3R1 and CAPN13. These 4 novel regions contain genes of potential interest that need to be tested in larger cohorts of patients.

Basal vitamin D levels and disease activity in multiple sclerosis patients treated with fingolimod.

BACKGROUND: Several studies have shown an association between 25-hydroxyvitamin D (25[OH]D) levels and multiple sclerosis (MS) susceptibility and/or level of disease activity in patients treated with first line drugs. AIMS: To investigate whether baseline 25[OH]D values could influence disease activity also during treatment with the second-line drug fingolimod (FTY). PATIENTS AND METHODS: We enrolled 176 MS patients who started FTY at the San Raffaele Hospital (OSR) MS center with available 25[OH]D measurement at the time of treatment start. We then prospectively followed them for 2 years with periodic clinical examinations and MRI scans. RESULTS: We found no linear correlation between baseline 25[OH]D levels and annualized relapse rate (ARR) or time to first relapse. However, we observed that patients with serum 25[OH]D ≥ 100 nmol/l showed a lower number of Gd+ and combined unique activity (CUA) lesions at baseline compared to patients with the lowest 25[OH]D levels (less than 50 nmol/l, p value < 0.05). Moreover, they showed fewer CUA lesions at 2-year follow-up also when accounting for baseline level of disease activity (p value < 0.05). CONCLUSIONS: In patients treated with FTY, those with the highest baseline 25(OH)D levels had a significantly lower number of active lesions at baseline; the same effect, even if weaker, was observed also at 2-year follow-up when adjusting for baseline disease activity. Given Vitamin D supplementation safety profile, also if a causal effect has not yet been shown, most of MS patients could probably benefit from 25[OH]D levels above those currently considered to be sufficient.

Clinical response to Nabiximols correlates with the downregulation of immune pathways in multiple sclerosis.

BACKGROUND AND PURPOSE: Nabiximols (Sativex® ) is a cannabinoid-based compound used for the treatment of moderate to severe spasticity in multiple sclerosis (MS). The aim of the study was to investigate the effect of the administration of Nabiximols on blood transcriptome profile of patients with MS and to interpret it in the context of pathways and networks. METHODS: Whole-genome expression profiling was performed in whole blood of 33 subjects with MS at baseline and after 4 weeks of drug treatment. Patients were classified as responders (n = 19) and non-responders (n = 14). Pathway and network analyses on genes modulated by the drug were performed, followed by in vitro stimulation of peripheral blood mononuclear cells with pro-inflammatory agents to support the immunomodulatory properties of the drug. RESULTS: Individual effect size was modest; however, we observed a downregulation of several immune-related pathways after 4 weeks of treatment, which was more pronounced when restricting analyses to responders. Interesting hub molecules functionally related to the immune system emerged from network analysis, including NFKB1, FYN, MAP14 and TP53. The immunomodulatory properties of the drug were confirmed through in vitro assays in peripheral blood mononuclear cells collected from patients with MS. CONCLUSIONS: Our findings support the immunomodulatory activity of cannabinoids in patients with MS. Further studies in more specific cell types are needed to refine these results.

Pharmacogenetic study of long-term response to interferon-ß treatment in multiple sclerosis.

The aim of the study is the identification of genetic factors that influence the long-term response to interferon-ß (IFNß) (4-year follow-up). We performed a genome-wide association study in 337 IFNß-treated Italian multiple sclerosis patients at the extreme of treatment response, and we meta-analyzed association effects, integrating results with pathway analysis, gene-expression profiling of IFNß-stimulated peripheral blood mononuclear cells from 20 healthy controls (HC) and expression quantitative locus (eQTL) analyses. From meta-analysis, 43 markers were associated at P<10-4, and two of them (rs7298096 and rs4726460) pointed to two genes, NINJ2 and TBXAS1, that were significantly downregulated after IFNß stimulation in HC (P=3.1 × 10-9 and 5.6 × 10-10). We also observed an eQTL effect for the allele associated with favorable treatment response (rs4726460A); moreover, TBXAS1 appeared downregulated upon IFNß administration (ß=-0.39; P=0.02). Finally, we found an enrichment of pathways related to inflammatory processes and presynaptic membrane, the latter with involvement of genes related to glutamatergic system (GRM3 and GRIK2), confirming its potential role in the response to IFNß.

[Algorithm for assessment of exposure to asbestos]. / Proposta di algoritmo per la valutazione stimata dell'esposizione lavorativa ad amianto.

There is no universally approved method in the scientific literature to identify subjects exposed to asbestos and divide them in classes according to intensity of exposure. The aim of our work is to study and develope an algorithm based on the findings of occupational anamnestical information provided by a large group of workers. The algorithm allows to discriminate, in a probabilistic way, the risk of exposure by the attribution of a code for each worker (ELSA Code--work estimated exposure to asbestos). The ELSA code has been obtained through a synthesis of information that the international scientific literature identifies as the most predictive for the onset of asbestos-related abnormalities. Four dimensions are analyzed and described: 1) present and/or past occupation; 2) type of materials and equipment used in performing working activity; 3) environment where these activities are carried out; 4) period of time when activities are performed. Although it is possible to have informations in a subjective manner, the decisional procedure is objective and is based on the systematic evaluation of asbestos exposure. From the combination of the four identified dimensions it is possible to have 108 ELSA codes divided in three typological profiles of estimated risk of exposure. The application of the algorithm offers some advantages compared to other methods used for identifying individuals exposed to asbestos: 1) it can be computed both in case of present and past exposure to asbestos; 2) the classification of workers exposed to asbestos using ELSA code is more detailed than the one we have obtained with Job Exposure Matrix (JEM) because the ELSA Code takes in account other indicators of risk besides those considered in the JEM. This algorithm was developed for a project sponsored by the Italian Armed Forces and is also adaptable to other work conditions for in which it could be necessary to assess risk for asbestos exposure.

[Meta-analysis of respiratory function of workers exposed to asbestos]. / Meta-analisi sulla funzionalità respiratoria nei lavoratori esposti ad amianto.

The monitoring of health conditions in workers exposed to asbestos for prolonged periods is a problematic issue. The Occupational Health physician being required to carry out his activity of prevention of the medical consequences and protection of the workers can use various instruments among which there is the evaluation of lung function. We have performed a meta-analysis on the scientific evidence on this matter in order to identify significant alterations of the lung function parameters in subjects professionally exposed to asbestos compared with controls. By the means of a bibliographic search and the application of selected inclusion and exclusion criteria, we identified 21 articles presenting data from controlled studies. Our results show that the number of controlled trials is very small compared to the totality of studies performed on this matter (1 to 70 ratio) and that the number of the subjects included in the majority of the analyzed studies is oddly divided in the experimental and control group. For this and other reasons the analyzed studies are very heterogeneous. Despite these difficulties, the meta-analysis evidences that some parameters of respiratory functioning are more likely to be influenced by asbestos exposure taking into account the smoking habit and that other parameters are sinergically influenced by both asbestos exposure and smoking. Our meta-analysis points out the necessity to define a common methodology about the technical and operatives procedures in the evaluation of the effects on lung function measures determined by an occupational exposure to asbestos and a thorough examination of the intensity of abnormality levels that could allow using the values obtained from the above mentioned measures in a discriminating diagnostic way.

[Prognostic, clinical and anatomo-pathological aspects of gastric lymphoma]. / Aspetti prognostici, clinici ed anatomopatologici del linfoma gastrico.

The authors refer about the results of a study made on 15 cases of gastric lymphoma with a mean follow-up of 6 years. Among the examined features they underlie the clinical appearance of each case according to histologic cell-type, stage of disease and performed treatment with relation to the survival rate. According to the Rappaport classification, diffuse histiocytic lymphoma was the most frequent histologic type of disease: the prognosis turned out to be significantly favorable in case of stage I, while no correlation was observed between different surgical procedures and cell-type. The overall 5 years survival rate was 33.3% with a strong suggestion of better survival in case of early diagnosis.