Asociación entre metilación del ADN y tabaquismo en pacientes con isquemia crónica amenazante de extremidades inferiores / Association between DNA methylation and smoking in patients with chronic limb-threatening ischemia

Introducción y objetivo: estudiar la asociación entre tabaquismo y el nivel de metilación de dos regiones genómicas en pacientes con enfermedad arterial periférica (EAP). Método: estudio transversal de 297 pacientes (edad media: 69,6 años; varones: 78,5 %) diagnosticados de isquemia crónica de extremidades inferiores en diferentes estadios clínicos entre marzo de 2016 y diciembre de 2019en el servicio de cirugía vascular del Hospital del mar (Barcelona). Se analizó la metilación de Cg02156642 y deCg03636183 asociados en otros estudios al tabaquismo. Se realizó una regresión lineal múltiple para discriminar lasvariables asociadas al nivel de metilación. Se calculó el área bajo la curva ROC para discriminar el nivel de metilaciónentre fumadores y no fumadores. Resultados: de la muestra, 46 pacientes (15,5 %) eran no fumadores; 132 (44,4 %), exfumadores y 119 (40,1 %),fumadores. No se observó una asociación entre la exposición al tabaco y el nivel de metilación del Cg02156642,pero sí con el de Cg03636183: los fumadores presentaban menor nivel de metilación y, además, a más carga detabaco menos metilación (Rho de Spearman: -0,324; p < 0,001).Un nivel de metilación en este CpG del 80 % tiene una sensibilidad (S) del 90,0 % y una especificidad (E) del83,5 % para discriminar entre fumadores y nunca fumadores. Para discriminar entre fumadores y exfumadores,un nivel de metilación del 75 % tiene una S del 69 % y una E del 56,9 %.Al ajustar por todas las variables relacionadas con la metilación, la magnitud de esta asociación entre Cg03636183y tabaquismo se mantenía signifi cativa entre los nunca fumadores y los fumadores. Conclusiones: la metilación del cpg cg03636183 se asocia a tabaquismo en pacientes con eap y está directamenterelacionada con la carga de tabaco. Este biomarcador podría utilizarse en la práctica clínica para valorar el consumode tabaco de nuestros pacientes.(AU)

Introduction and objective: to study the association between smoking and the methylation level of 2 genomicregions in patients with peripheral artery disease (PAD). Method: cross-sectional study of 297 patients (mean age, 69.6 years; males, 78.5%) diagnosed with chronic lowerextremity ischemia at various clinical stages from march 2016 through December 2019 at the Vascular Surgery Unitof Hospital del mar, Barcelona, Catalonia, Spain. methylation analysis of Cg02156642 and Cg03636183, previouslyassociated with smoking in former studies was performed. multiple linear regression was conducted to identifyvariables associated with methylation levels. The area under the ROC curve was estimated to discriminate meth-ylation levels between smokers and non-smokers. Results: among the sample, 46 patients (15.5%) were non-smokers, 132 (44.4%) were former smokers, and 119(40.1%) were current smokers. No association was seen between tobacco exposure and methylation levels ofCg02156642. However, an association was found with Cg03636183: smokers had lower methylation levels, anda higher smoking load was associated with lower methylation (Spearman's Rho, -0.324; p < .001). A methylationlevel of 80% in this region showed a 90.0% sensitivity and an 83.5% specificity to discriminate between smokersand never smokers. To discriminate between smokers and former smokers, a methylation level of 75% had an 69%sensitivity and an 56.9% specificity. After adjusting for all variables associated with methylation, the associationbetween Cg03636183 and smoking remained significant among never smokers and smokers. Conclusions: methylation of the Cg03636183 region is associated with smoking in patients with PAD and is directlyassociated with the smoking load. This biomarker could be used in the routine clinical practice to assess tobaccouse in our patients.(AU)

Predictive factors and early biomarkers of response in multiple sclerosis patients treated with natalizumab.

There are an increasing number of treatments available for multiple sclerosis (MS). The early identification of optimal responders to individual treatments is important to achieve individualized therapy. With this aim, we performed a multicenter retrospective longitudinal study including 186 MS patients treated with natalizumab who were followed for 2 years. We analyzed the following variables at recruitment: sex, current age, age at disease onset, disease duration, EDSS, number of T2 and Gd + lesions, IgG and IgM oligoclonal bands, HLA class II (DR, DRB, DQA, DQB, and DRB1*15:01), IgG and IgM antibody titers against human herpesvirus 6 (HHV-6) and the antibody response to Epstein-Barr virus (EBV) through the measurement of the anti-EBNA-1 and anti-VCA IgG titers, in relation to clinical response (no relapses or disability progression), and to NEDA-3 (no evidence of disease activity in terms of clinical response and no changes in MRI scans either) after 2-years follow-up. Baseline EDSS score, baseline EBNA-1 IgG titers and percentage change of HHV6 IgG titers between baseline and 6 month visits were significantly different in clinical responders and in NEDA-3 status (all of them remained significant in the multivariate analysis). We identified three variables for the early identification of natalizumab optimal responders in a rapid and cost-effective approach.

Skin Phototype Could Be a Risk Factor for Multiple Sclerosis.

Environmental and genetic factors are assumed to be necessary for the development of multiple sclerosis (MS), however its interactions are still unclear. For this reason here, we have not only analyzed the impact on increased risk of MS of the best known factors (HLA-DRB1*15:01 allele, sun exposure, vitamin D levels, smoking habit), but we have included another factor (skin phototype) that has not been analyzed in depth until now. This study included 149 MS patients and 147 controls. A multivariate logistic regression (LR) model was carried out to determine the impact of each of the factors on the increased risk of MS. Receiver Operating Characteristics (ROC) analysis was performed to evaluate predictive value of the models. Our multifactorial LR model of susceptibility showed that females with light brown skin (LBS), smokers and who had HLA-DRB1*15:01 allele had a higher MS risk (LBS: OR = 5.90, IC95% = 2.39-15.45; smoker: OR = 4.52, IC95% = 2.69-7.72; presence of HLA-DRB1*15:01: OR = 2.39, IC95% = 1.30-4.50; female: OR = 1.88, IC95% = 1.08-3.30). This model had an acceptable discriminant value with an Area Under a Curve AUC of 0.76 (0.69-0.82). Our study indicates that MS risk is determined by complex interactions between sex, environmental factors, and genotype where the milieu could provide the enabling proinflammatory environment that drives an autoimmune attack against myelin by self-reactive lymphocytes.

Multi-centre validation of a flow cytometry method to identify optimal responders to interferon-beta in multiple sclerosis.

BACKGROUND AND OBJECTIVES: Percentages of blood CD19+CD5+ B cells and CD8+perforin+ T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. METHODS: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. RESULTS: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19+CD5+ B cell and 0.89 (CI: 0.85-0.93) for CD8+ perforin+ T cell quantification; Spearman r was 0.92 (0.89-0.95; p <0.0001) and 0.92 (0.88-0.95; p <0.0001) respectively. All centres obtained z-scores≤0.5 for both biomarkers. CONCLUSION: Homogenous percentages of CD19+CD5+ B cells and CD8 perforin+ T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.

Cross-reactivity of antibodies against interferon beta in multiple sclerosis patients and interference of the JAK-STAT signaling pathway.

Interferon beta (IFNß) therapy has immunogenic properties and induces the development of neutralizing antibodies (NAbs). From the extensive literature focused in the development of NAbs in multiple sclerosis (MS) patients, their ability to cross-react has been deficiently evaluated, despite having important consequences in the clinical practice. Here, the relation between the cross-reactivity and the NAbs titers has been evaluated in MS patients, by inhibition of the antiviral activity of IFNß by bioassay and through the interference with the activation of the IFNß pathway (JAK-STAT), by phosphoflow. Thus, patients with intermediate-high titers of NAbs, determined by bioassay, had a 79-fold increased risk of cross-reactivity compared to patients with low titers. The cross-reactivity is also demonstrated because NAbs positive sera were able to decrease significantly the activation of pSTAT1 achieved by other different IFNß molecules in the cells patients. Besides, a linear relationship between the STAT1 phosphorylation and NAbs titers was found. The study demonstrates that cross-reactivity increases with the titer of antibodies, which has important implications in clinical practice when switching the treatment. The direct relationship between the NAbs titer and the activation of STAT1 suggest that its determination could be an indirect method to identify the presence of NAbs.

Global methylation correlates with clinical status in multiple sclerosis patients in the first year of IFNbeta treatment.

The alteration of DNA methylation patterns are a key component of disease onset and/or progression. Our objective was to evaluate the differences in Long Interspersed Nuclear Element-1 (LINE-1) methylation levels, as a surrogate marker of global DNA methylation, between multiple sclerosis (MS) patients and healthy controls. In addition, we assessed the association of LINE-1 methylation with clinical disease activity in patients treated with IFNbeta (IFNß). We found that individuals with high levels of LINE-1 methylation showed 6-fold increased risk of suffering MS. Additionally, treated MS patients who bear high LINE-1 methylation levels had an 11-fold increased risk of clinical activity. Moreover, a negative correlation between treatment duration and percentage of LINE-1 methylation, that was statistically significant exclusively in the group of patients without clinical activity, was observed. Our data suggest that in MS patients, a slight global DNA hypermethylation occurs that may be related to the pathophysiology of the disease. In addition, global DNA methylation levels could play a role as a biomarker for the differential clinical response to IFNß.

Activation of the JAK-STAT Signaling Pathway after In Vitro Stimulation with IFNß in Multiple Sclerosis Patients According to the Therapeutic Response to IFNß.

Interferon beta (IFNß) is a common treatment used for multiple sclerosis (MS) which acts through the activation of the JAK-STAT pathway. However, this therapy is not always effective and currently there are no reliable biomarkers to predict therapeutic response. We postulate that the heterogeneity in the response to IFNß therapy could be related to differential activation patterns of the JAK-STAT signaling pathway. Our aim was to evaluate the basal levels and the short term activation of this pathway after IFNß stimulation in untreated and IFNß treated patients, as well as according to therapeutic response. Therefore, cell surface levels of IFNAR subunits (IFNAR1 and IFNAR2) and the activated forms of STAT1 and STAT2 were assessed in peripheral blood mononuclear cells from MS patients by flow cytometry. Basal levels of each of the markers strongly correlated with the expression of the others in untreated patients, but many of these correlations lost significance in treated patients and after short term activation with IFNß. Patients who had undergone IFNß treatment showed higher basal levels of IFNAR1 and pSTAT1, but a reduced response to in vitro exposure to IFNß. Conversely, untreated patients, with lower basal levels, showed a greater ability of short term activation of this pathway. Monocytes from responder patients had lower IFNAR1 levels (p = 0.039) and higher IFNAR2 levels (p = 0.035) than non-responders just after IFNß stimulation. A cluster analysis showed that levels of IFNAR1, IFNAR2 and pSTAT1-2 in monocytes grouped 13 out of 19 responder patients with a similar expression pattern, showing an association of this pattern with the phenotype of good response to IFNß (p = 0.013). Our findings suggest that an activation pattern of the IFNß signaling pathway in monocytes could be associated with a clinical phenotype of good response to IFNß treatment and that a differential modulation of the IFNAR subunits in monocytes could be related with treatment effectiveness.

TRAIL and TRAIL receptors splice variants during long-term interferon ß treatment of patients with multiple sclerosis: evaluation as biomarkers for therapeutic response.

OBJECTIVE: We aimed to assess the effects of interferon ß (IFNß) treatment on the expression of the splice variants of the Tumour necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) and its receptors in different cell subpopulations (CD14+, CD4+ and CD8+) from patients with multiple sclerosis (MS), and to determine whether this expression discriminated responders from non-responders to IFNß therapy. METHODS: We examined mRNA expression of the TRAIL and TRAIL receptors variants in patients with MS, at baseline and after one year of IFNß therapy, according to responsiveness to this drug. RESULTS: Long-term therapy with IFNß increased the expression of TRAIL-α in T cell subsets exclusively from responders and decreased the expression of the isoform 2 of TRAILR-2 in monocytes from responders as well as non-responders. Lower expression of TRAIL-α, and higher expression of TRAIL-ß in monocytes and T cells, was found before the onset of IFNß therapy in patients who will subsequently become responders. Baseline expression of TRAILR-1 was also significantly higher in monocytes and CD4+ T cells from responders. CONCLUSIONS: The present study shows that long-term IFNß treatment has a direct influence on TRAIL-α and TRAILR-2 isoform 2 expression. Besides, receiver operating characteristic analysis revealed that the baseline expression of TRAIL-α in monocytes and T cells, and that of TRAILR-1 in monocytes and CD4+ T cells, showed a predictive value of the clinical response to IFNß therapy, pointing to a role of TRAIL system in the mechanism of action of IFNß in MS that will need further investigation.

Cell-specific effects in different immune subsets associated with SOCS1 genotypes in multiple sclerosis.

BACKGROUND: Single nucleotide polymorphisms (SNPs) near SOCS1 are associated with multiple sclerosis (MS), but the most important SNPs in the area and mechanisms by which they influence the disease are unknown. METHODS: A haplotype-tagging association study was performed covering 60.5kbp around SOCS1, and the index SNP was validated in a total of 2292 individuals. mRNA expression of SOCS1 and nearby genes was measured in MS patients with different disease courses and healthy controls. SOCS1 protein expression was studied by flow cytometry in a separate cohort of patients and controls. Differentiation and maturation of monocyte-derived dendritic cells (moDCs) were also studied. RESULTS: One SNP, rs423674, reached genome-wide significance. No genotype-specific mRNA expression differences were seen, but, by flow cytometry, significant interactions were observed between genotypes for rs423674 and disease activity (relapse or remission) in B cells and regulatory T cells. Furthermore, homozygotes for the risk allele (GG) showed higher levels of CD1a and CD86 than carriers of the protective allele (GT) in immature moDCs and a greater increase of HLA-DR+ cell percentage than GT heterozygotes upon maturation. CONCLUSIONS: rs423674, or its genetic proxies, may influence MS risk by modulating SOCS1 expression in a cell-specific manner and by influencing dendritic cell function.

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.

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4), pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

The CD4+ T-cell subset lacking expression of the CD28 costimulatory molecule is expanded and shows a higher activation state in multiple sclerosis.

Multiple sclerosis (MS) is a chronic debilitating disease, in which T-cells are considered to play a pivotal role. CD28 is the quintessential costimulatory molecule on T-cells and its expression declines progressively with repeated stimulations, leading to the generation of CD28(-) T-cells. Our aim was to examine whether CD4(+)CD28(-) T-cells were enriched in MS patients, and characterize the phenotype of this subset in MS patients and healthy controls (HC). All these changes could provide these CD4(+)CD28(-) T-cell characteristics that might be involved in the pathogenesis of MS, turning this T-cell subset into a potential target for future therapeutic strategies.

TRAIL/TRAIL receptor system and susceptibility to multiple sclerosis.

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10(-4), OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10(-5), OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.

Kinetics and incidence of anti-natalizumab antibodies in multiple sclerosis patients on treatment for 18 months.

Natalizumab is a monoclonal antibody shown to be highly effective in the treatment of relapsing-remitting multiple sclerosis (RRMS). Patients treated with natalizumab can develop antibodies directed against this agent that may affect the efficacy and safety of the drug. In this observational study, the kinetics of the appearance and the incidence of anti-natalizumab antibodies were followed prospectively for 18 months in a cohort of 64 consecutive patients treated with natalizumab for relapsing MS. Blood samples were drawn immediately before starting natalizumab therapy and each month afterwards. The presence of antibodies against natalizumab was assessed by enzyme-linked immunosorbent assay (ELISA) in all patients. Anti-natalizumab antibodies were detected in nine (14.1%) natalizumab-treated patients, three (4.68%) of whom were transiently positive while six (9.37%) were persistently positive (these patients discontinued natalizumab). All positive titres were observed during the first 4 months of treatment. One patient with a hypersensitivity reaction also had persistent antibodies. We conclude that antibodies against natalizumab develop early, within the first 6 months of therapy with natalizumab. Although no antibodies were detected after 4 months of therapy in this particular study, this does not rule out their development later on in exceptional cases.

HLA class II alleles in patients with multiple sclerosis in the Biscay province (Basque Country, Spain).

Genetic susceptibility to multiple sclerosis (MS) is associated with genes of the major histocompatibility complex, particularly with the HLA DRB1*1501-DQA1*0102-DQB1*0602 haplotype in Caucasians. To investigate the association of DRB1, DQA1 and DQB1 alleles and haplotypes with MS in Biscay, Basque Country, northern Spain, we examined 197 patients and 200 regionally matched controls. High resolution HLA class II typing was performed by polymerase chain reaction followed by sequence-specific oligonucleotide probe hybridization. Several alleles were overrepresented in MS patients compared with those of controls: DRB1*0402, DRB1*1303, DRB1*1501, DQA1*0102, DQB1*0301, and DQB1*0602. DQB1*0602 was the only potentially predisposing allele for MS that withstood Bonferroni correction and maintained the association in a logistic regression model. On the other hand, several alleles showed lower frequencies in the MS group: DRB1*0101, DQA1*0101, DQB1*0303, and DQB1*0501, but only DRB1*0101 and DQB1*0303 maintained a negative association with the disease in the regression analysis. Three haplotypes were identified as potentially predisposing for MS in our population: DRB1*1501-DQA1*0102-DQB1*0602, DRB1*0402-DQA1*0301-DQB1*0302, and HLA-DRB1*013-DQA1*05-DQB1*0301. Additionally, three haplotypes associated with a lower risk for MS were identified, exhibiting DRB1*0101-DQA1*0101-DQB1*0501 the strongest negative association with MS [12% in controls vs. 3.8% in MS, Pc = 0.00047, OR = 0.290 (95% CI = 0.160­0.528)], and suggesting, therefore, a putative protective role for this haplotype in the population under study.

IFNAR1 and IFNAR2 polymorphisms confer susceptibility to multiple sclerosis but not to interferon-beta treatment response.

We investigated the role of three polymorphisms in the IFNAR1 (SNPs 18417 and -408) and IFNAR2 (SNP 11876) genes in multiple sclerosis (MS) susceptibility and in the IFNbeta treatment response in a group of 147 patients and 210 controls undergoing interferon therapy during the last 2 years. Only the 18417 and the 11876 SNPs showed an association with disease susceptibility (p=0.001 and 0.035, respectively) although no differential genotype distribution were observed between interferon responders and non-responder MS patients. No alteration of the expression level of IFNAR-1 was observed with respect to the -408 genotypes or to interferon treatment response. These data suggest a role for the IFNAR pathway in susceptibility to MS.