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.

No evidence for association between rs10191329 severity locus and longitudinal disease severity in 1813 relapse-onset multiple sclerosis patients from the MSBase registry.

BACKGROUND: The International Multiple Sclerosis Genetics Consortium and MultipleMS Consortium recently reported a genetic variant associated with multiple sclerosis (MS) severity. However, it remains unclear if these variants remain associated with more robust, longitudinal measures of disease severity. METHODS: We examined the top variant, rs10191329, from Harroud et al.'s study in 1813 relapse-onset MS patients from the MSBase Registry to assess association with longitudinal disease severity. RESULTS: Our analysis revealed no significant association between rs10191329 genotype and longitudinal binary disease severity (p > 0.05). CONCLUSION: These findings highlight the complexity of genetic factors mediating long-term MS outcomes and the need for further research.

Not all roads lead to the immune system: the genetic basis of multiple sclerosis severity.

Multiple sclerosis is a leading cause of neurological disability in adults. Heterogeneity in multiple sclerosis clinical presentation has posed a major challenge for identifying genetic variants associated with disease outcomes. To overcome this challenge, we used prospectively ascertained clinical outcomes data from the largest international multiple sclerosis registry, MSBase. We assembled a cohort of deeply phenotyped individuals of European ancestry with relapse-onset multiple sclerosis. We used unbiased genome-wide association study and machine learning approaches to assess the genetic contribution to longitudinally defined multiple sclerosis severity phenotypes in 1813 individuals. Our primary analyses did not identify any genetic variants of moderate to large effect sizes that met genome-wide significance thresholds. The strongest signal was associated with rs7289446 (ß = -0.4882, P = 2.73 × 10-7), intronic to SEZ6L on chromosome 22. However, we demonstrate that clinical outcomes in relapse-onset multiple sclerosis are associated with multiple genetic loci of small effect sizes. Using a machine learning approach incorporating over 62 000 variants together with clinical and demographic variables available at multiple sclerosis disease onset, we could predict severity with an area under the receiver operator curve of 0.84 (95% CI 0.79-0.88). Our machine learning algorithm achieved positive predictive value for outcome assignation of 80% and negative predictive value of 88%. This outperformed our machine learning algorithm that contained clinical and demographic variables alone (area under the receiver operator curve 0.54, 95% CI 0.48-0.60). Secondary, sex-stratified analyses identified two genetic loci that met genome-wide significance thresholds. One in females (rs10967273; ßfemale = 0.8289, P = 3.52 × 10-8), the other in males (rs698805; ßmale = -1.5395, P = 4.35 × 10-8), providing some evidence for sex dimorphism in multiple sclerosis severity. Tissue enrichment and pathway analyses identified an overrepresentation of genes expressed in CNS compartments generally, and specifically in the cerebellum (P = 0.023). These involved mitochondrial function, synaptic plasticity, oligodendroglial biology, cellular senescence, calcium and G-protein receptor signalling pathways. We further identified six variants with strong evidence for regulating clinical outcomes, the strongest signal again intronic to SEZ6L (adjusted hazard ratio 0.72, P = 4.85 × 10-4). Here we report a milestone in our progress towards understanding the clinical heterogeneity of multiple sclerosis outcomes, implicating functionally distinct mechanisms to multiple sclerosis risk. Importantly, we demonstrate that machine learning using common single nucleotide variant clusters, together with clinical variables readily available at diagnosis can improve prognostic capabilities at diagnosis, and with further validation has the potential to translate to meaningful clinical practice change.

Exome sequencing in multiple sclerosis families identifies 12 candidate genes and nominates biological pathways for the genesis of disease.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.

Targeted resequencing reveals rare variants enrichment in multiple sclerosis susceptibility genes.

Although genome-wide association studies have identified a number of common variants associated with multiple sclerosis (MS) susceptibility, little is known about the relevance of rare variants. Here, we aimed to explore the role of rare variants in 14 MS risk genes (FCRL1, RGS1, TIMMDC1, HHEX, CXCR5, LTBR, TSFM, GALC, TRAF3, STAT3, TNFSF14, IFI30, CD40, and CYP24A1) by targeted resequencing in an Iberian population of 524 MS cases and 546 healthy controls. Four rare variants-enriched regions within CYP24A1, FCRL1, RGS1, and TRAF3 were identified as significantly associated with MS. Functional studies revealed significantly decreased regulator of G protein signaling 1 (RGS1) gene expression levels in peripheral blood mononuclear cells from MS patients with RGS1 rare variants compared to noncarriers, whereas no significant differences in gene expression were observed for CYP24A1, FCRL1, and TRAF3 between rare variants carriers and noncarriers. Immunophenotyping showed significant decrease in RGS1 expression in peripheral blood B lymphocytes from MS patients with RGS1 rare variants relative to noncarriers. Lastly, peripheral blood mononuclear cell from MS patients carrying RGS1 rare variants showed significantly lower induction of RGS1 gene expression by interferon-ß compared to MS patients lacking RGS1 variants. The presence of rare variants in RGS1 reinforce the ideas of high genetic heterogeneity and a role of rare variants in MS pathogenesis.

SP140 regulates the expression of immune-related genes associated with multiple sclerosis and other autoimmune diseases by NF-κB inhibition.

SP140 locus has been associated with multiple sclerosis (MS) as well as other autoimmune diseases by genome-wide association studies (GWAS). The causal variant of these associations (rs28445040-T) alters the splicing of the SP140 gene transcripts reducing the protein expression. We aimed to understand why the reduction of SP140 expression produced by the risk variant can increase the susceptibility to MS. To this end, we determined by RNA sequencing (RNA-seq) analysis the differentially expressed genes after SP140 silencing in lymphoblastoid cell lines (LCLs). We analyzed these genes by gene ontology (GO), comparative transcriptome profiles, enrichment of transcription factors (TFs) in the promoters of these genes and colocalization with GWAS risk variants. We also monitored the activity of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in SP140-silenced cells by luciferase reporter system. We identified 100 genes that were up-regulated and 22 genes down-regulated in SP140-silenced LCLs. GO analysis revealed that genes affected by SP140 were involved in regulation of cytokine production, inflammatory response and cell-cell adhesion. We observed enrichment of NF-κB TF in the promoter of up-regulated genes and NF-κB-increased activity in SP140-silenced cell lines. We showed enrichment of genes regulated by SP140 in GWAS-detected risk loci for MS (14.63 folds), Crohn's disease (4.82 folds) and inflammatory bowel disease (4.47 folds), not observed in other unrelated immune diseases. Our findings showed that SP140 is an important repressor of genes implicated in inflammation, suggesting that decreased expression of SP140, promoted by the rs28445040-T risk variant, may lead to up-regulation of these genes by means of NF-κB inhibition in B cells.

A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis.

Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.

The multiple sclerosis-associated regulatory variant rs10877013 affects expression of CYP27B1 and VDR under inflammatory or vitamin D stimuli.

BACKGROUND: Vitamin D deficit is considered an important risk factor for many inflammatory and autoimmune diseases. OBJECTIVE: To investigate the influence of the multiple sclerosis (MS)-associated regulatory variant rs10877013 on the expression of genes involved in vitamin D activation (CYP27B1), vitamin D receptor (VDR), and vitamin D degradation (CYP24A1) under inflammatory environment or vitamin D. METHODS: We used lipopolysaccharide and interferon-gamma (LPS+IFNγ) activated monocytes from 119 individuals and vitamin D-stimulated lymphoblastoid cell lines (LCLs, n = 109) of 1000 genomes to quantify the mRNA expression of vitamin D genes by quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: We found that CYP27B1 mRNA expression level was associated with the rs10877013 genotypes (p = 5.0E-6) in LPS+IFNγ treated monocytes, but not in vitamin D-stimulated LCLs. Inversely, rs10877013 genotypes were associated with VDR expression in LCLs (p = 6.0E-4) but not in monocytes. Finally, CYP24A1 was highly induced by the active form of vitamin D and its expression correlated with the expression of VDR in LCLs but neither the MS-associated variant in the region (rs2248359) nor any other variant located in 1 Mb around CYP24A1 was associated with its expression. CONCLUSIONS: The MS-associated variant rs10877013 is a genetic determinant that affects the functioning of the vitamin D system linking environmental and genetic factors.

Genome-wide significant association with seven novel multiple sclerosis risk loci.

OBJECTIVE: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. METHODS: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. RESULTS: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10(-8)) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10(-12)), CD28 (rs6435203, p=1.35×10(-9)), LPP (rs4686953, p=3.35×10(-8)), ETS1 (rs3809006, p=7.74×10(-9)), DLEU1 (rs806349, p=8.14×10(-12)), LPIN3 (rs6072343, p=7.16×10(-12)) and IFNGR2 (rs9808753, p=4.40×10(-10)). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. CONCLUSIONS: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases.

A new risk variant for multiple sclerosis at the immunoglobulin heavy chain locus associates with intrathecal IgG, IgM index and oligoclonal bands.

BACKGROUND: Recent findings have shown a correlation between the intrathecal IgG index and variants at the immunoglobulin heavy chain (IGHC) locus in patients with multiple sclerosis (MS). OBJECTIVES: The objective of this paper is to analyse the association of the locus with MS susceptibility and its relationship with intrathecal immunoglobulin (Ig) parameters. METHODS: We genotyped the rs11621145 variant, located at the IGHC locus, in 2726 patients with MS and 2133 healthy controls. Associations of intrathecal IgG and IgM indexes with rs11621145 were analysed by linear regression analysis in 538 MS patients. RESULTS: We found that rs11621145 showed statistically significant evidence for association with susceptibility to MS (odds ratio = 0.69, p = 1.053E-09), though validation of this result in additional cohorts would be desirable. We confirmed the association between the IgG index and the rs11621145 (p = 6.85E-07, Beta = 0.207). Furthermore, rs11621145 was inversely correlated with IgM index (p = 7.24E-04, Beta = -0.277), and therefore marks a decreased likelihood of presenting IgM oligoclonal bands (odds ratio = 0.38, p = 2.35E-06). CONCLUSIONS: Our results suggest that the polymorphism of the IGHC locus could be altering the switching of the Ig isotype in B cells and it may be interfering with T-dependent and T-independent antibody responses.

HERV-W polymorphism in chromosome X is associated with multiple sclerosis risk and with differential expression of MSRV.

BACKGROUND: Multiple Sclerosis (MS) is an autoimmune demyelinating disease that occurs more frequently in women than in men. Multiple Sclerosis Associated Retrovirus (MSRV) is a member of HERV-W, a multicopy human endogenous retroviral family repeatedly implicated in MS pathogenesis. MSRV envelope protein is elevated in the serum of MS patients and induces inflammation and demyelination but, in spite of this pathogenic potential, its exact genomic origin and mechanism of generation are unknown. A possible link between the HERV-W copy on chromosome Xq22.3, that contains an almost complete open reading frame, and the gender differential prevalence in MS has been suggested. RESULTS: MSRV transcription levels were higher in MS patients than in controls (U-Mann-Whitney; p = 0.004). Also, they were associated with the clinical forms (Spearman; p = 0.0003) and with the Multiple Sclerosis Severity Score (MSSS) (Spearman; p = 0.016). By mapping a 3 kb region in Xq22.3, including the HERV-W locus, we identified three polymorphisms: rs6622139 (T/C), rs6622140 (G/A) and rs1290413 (G/A). After genotyping 3127 individuals (1669 patients and 1458 controls) from two different Spanish cohorts, we found that in women rs6622139 T/C was associated with MS susceptibility: [χ2; p = 0.004; OR (95% CI) = 0.50 (0.31-0.81)] and severity, since CC women presented lower MSSS scores than CT (U-Mann-Whitney; p = 0.039) or TT patients (U-Mann-Whitney; p = 0.031). Concordantly with the susceptibility conferred in women, rs6622139*T was associated with higher MSRV expression (U-Mann-Whitney; p = 0.003). CONCLUSIONS: Our present work supports the hypothesis of a direct involvement of HERV-W/MSRV in MS pathogenesis, identifying a genetic marker on chromosome X that could be one of the causes underlying the gender differences in MS.

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis.

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.

Identification of a functional variant in the KIF5A-CYP27B1-METTL1-FAM119B locus associated with multiple sclerosis.

BACKGROUND AND AIM: Several studies have highlighted the association of the 12q13.3-12q14.1 region with coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis (MS); however, the causal variants underlying diseases are still unclear. The authors sought to identify the functional variant of this region associated with MS. METHODS: Tag-single nucleotide polymorphism (SNP) analysis of the associated region encoding 15 genes was performed in 2876 MS patients and 2910 healthy Caucasian controls together with expression regulation analyses. RESULTS: rs6581155, which tagged 18 variants within a region where 9 genes map, was sufficient to model the association. This SNP was in total linkage disequilibrium (LD) with other polymorphisms that associated with the expression levels of FAM119B, AVIL, TSFM, TSPAN31 and CYP27B1 genes in different expression quantitative trait loci studies. Functional annotations from Encyclopedia of DNA Elements (ENCODE) showed that six out of these rs6581155-tagged-SNPs were located in regions with regulatory potential and only one of them, rs10877013, exhibited allele-dependent (ratio A/G=9.5-fold) and orientation-dependent (forward/reverse=2.7-fold) enhancer activity as determined by luciferase reporter assays. This enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-enhancer of several genes has been described, possibly affecting their expression simultaneously. CONCLUSIONS: This study determines a functional variant which alters the enhancer activity of a regulatory element in the locus affecting the expression of several genes and explains the association of the 12q13.3-12q14.1 region with MS.

Closing the case of APOE in multiple sclerosis: no association with disease risk in over 29 000 subjects.

BACKGROUND: Single nucleotide polymorphisms (SNPs) rs429358 (ε4) and rs7412 (ε2), both invoking changes in the amino-acid sequence of the apolipoprotein E (APOE) gene, have previously been tested for association with multiple sclerosis (MS) risk. However, none of these studies was sufficiently powered to detect modest effect sizes at acceptable type-I error rates. As both SNPs are only imperfectly captured on commonly used microarray genotyping platforms, their evaluation in the context of genome-wide association studies has been hindered until recently. METHODS: We genotyped 12 740 subjects hitherto not studied for their APOE status, imputed raw genotype data from 8739 subjects from five independent genome-wide association studies datasets using the most recent high-resolution reference panels, and extracted genotype data for 8265 subjects from previous candidate gene assessments. RESULTS: Despite sufficient power to detect associations at genome-wide significance thresholds across a range of ORs, our analyses did not support a role of rs429358 or rs7412 on MS susceptibility. This included meta-analyses of the combined data across 13 913 MS cases and 15 831 controls (OR=0.95, p=0.259, and OR 1.07, p=0.0569, for rs429358 and rs7412, respectively). CONCLUSION: Given the large sample size of our analyses, it is unlikely that the two APOE missense SNPs studied here exert any relevant effects on MS susceptibility.

Role of the small GTPase Rab27a during herpes simplex virus infection of oligodendrocytic cells.

BACKGROUND: The morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection. RESULTS: Herein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells. CONCLUSIONS: Altogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

Replication study of 10 genes showing evidence for association with multiple sclerosis: validation of TMEM39A, IL12B and CBLB [correction of CLBL] genes.

BACKGROUND AND OBJECTIVES: Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. METHODS: Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. RESULTS: Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). CONCLUSIONS: Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p(M-H)=0.001; OR(M-H) (95% CI)= 0.84 (0.75-0.93)], IL12B [rs6887695: p(M-H)=0.03; OR(M-H) (95% CI)= 1.09 (1.01-1.17)] and CBLB [rs9657904: p(M-H)=0.01; OR(M-H) (95% CI)= 0.89 (0.81-0.97)].

Characterization of the MAL2-positive compartment in oligodendrocytes.

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.

A New Risk Variant for Multiple Sclerosis at 11q23.3 Locus Is Associated with Expansion of CXCR5+ Circulating Regulatory T Cells.

Genome-wide association studies and meta-analysis have contributed to the identification of more than 200 loci associated with multiple sclerosis (MS). However, a proportion of MS heritability remains unknown. We aimed to uncover new genetic variants associated with MS and determine their functional effects. For this, we resequenced the exons and regulatory sequences of 14 MS risk genes in a cohort of MS patients and healthy individuals (n = 1,070) and attempted to validate a selection of signals through genotyping in an independent cohort (n = 5,138). We identified three new MS-associated variants at C-X-C motif chemokine receptor 5 (CXCR5), Ts translation elongation factor, mitochondrial (TSFM) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Rs10892307 resulted in a new signal at the CXCR5 region that explains one of the associations with MS within the locus. This polymorphism and three others in high linkage disequilibrium mapped within regulatory regions. Of them, rs11602393 showed allele-dependent enhancer activity in the forward orientation as determined by luciferase reporter assays. Immunophenotyping using peripheral blood mononuclear cells from MS patients associated the minor allele of rs10892307 with increased percentage of regulatory T cells expressing CXCR5. This work reports a new signal for the CXCR5 MS risk locus and points to rs11602393 as the causal variant. The expansion of CXCR5+ circulating regulatory T cells induced by this variant could cause its MS association.

Splice-site variant in ACSL5: a marker promoting opposing effect on cell viability and protein expression.

Long-chain Acyl-CoA synthetases (ACSLs) activate fatty acids (FAs) by thioesterification with Coenzyme A (CoA), generating FA-CoAs. These products are essential for lipid metabolism and carcinogenesis. In previous study, we identified an intronic variant rs2256368:A>G, whose G allele promotes exon 20 skipping in up to 43% of ACSL5 transcripts but its functional relevance is unclear. Here, we compared the expression of splice (Spl) and nonsplice (NSpl) ACSL5 variants and the effect on cell viability under culture conditions that force cells to metabolize fatty acids. We found that lymphoblastoid cell lines from 1000 Genomes Project, bearing Spl genotypes, showed a reduced expression of total ACSL5 protein due to an inefficient translation of the Spl RNA. These cells impaired growth in cultures with phorbol myristate acetate-ionomycin (PMA-Io) or medium deprived of glucose, while production of reactive oxygen species increased in PMA-Io. Specific ACSL5-isoform transfection in HEK239T (kidney), U87 (astroglioma), and HOG (oligodendrocyte) cells showed the Spl protein to be the causal factor of cell-growth inhibition, despite its reduced protein expression. Our findings indicate that the variant rs2256368:A>G can predict a growth inhibitory activity, caused by the Spl isoform of ACSL5 protein, opposed to the activity of the NSpl. Deep understanding of its functioning might have application in metabolic diseases and cancer.