Liquid Biopsy in Neurological Diseases

Liquid biopsy; MicroRNA; Neurological diseasesBiòpsia líquida; MicroARN; Malalties neurològiquesBiopsia líquida; MicroARN; Enfermedades neurológicasThe most recent and non-invasive approach for studying early-stage biomarkers is liquid biopsy. This implies the extraction and analysis of non-solid biological tissues (serum, plasma, saliva, urine, and cerebrospinal fluid) without undergoing invasive procedures to determine disease prognosis. Liquid biopsy can be used for the screening of several components, such as extracellular vesicles, microRNAs, cell-free DNA, cell-free mitochondrial and nuclear DNA, circulating tumour cells, circulating tumour DNA, transfer RNA, and circular DNA or RNA derived from body fluids. Its application includes early disease diagnosis, the surveillance of disease activity, and treatment response monitoring, with growing evidence for validating this methodology in cancer, liver disease, and central nervous system (CNS) disorders. This review will provide an overview of mentioned liquid biopsy components, which could serve as valuable biomarkers for the evaluation of complex neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, traumatic brain injury, CNS tumours, and neuroinfectious diseases. Furthermore, this review highlights the future directions and potential limitations associated with liquid biopsy.S.M. was supported by the grant from Instituto Salud Carlos III (PI20/01697)

Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure

Inflammasome; Pyroptosis; Multiple sclerosisInflamasoma; Piroptosis; Esclerosis múltipleInflamasoma; Piroptosi; Esclerosi múltipleBackground and Objectives Inflammasomes are involved in the pathogenesis of different neuroimmune and neurodegenerative diseases, including multiple sclerosis (MS). In a previous study by our group, the nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain–containing 3 (NLRP3) inflammasome was reported to be associated with the response to interferon-beta in MS. Based on recent data showing the potential for the oral therapy fingolimod to inhibit NLRP3 inflammasome activation, here we investigated whether fingolimod could also be implicated in the response to this therapy in patients with MS. Methods NLRP3 gene expression levels were measured by real-time PCR in peripheral blood mononuclear cells at baseline and after 3, 6, and 12 months in a cohort of patients with MS treated with fingolimod (N = 23), dimethyl fumarate (N = 21), and teriflunomide (N = 21) and classified into responders and nonresponders to the treatment according to clinical and radiologic criteria. In a subgroup of fingolimod responders and nonresponders, the percentage of monocytes with an oligomer of ASC was determined by flow cytometry, and the levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor (TNF)α, and galectin-3 were quantified by ELISA. Results NLPR3 expression levels were significantly increased in fingolimod nonresponders after 3 (p = 0.03) and 6 months (p = 0.008) of treatment compared with the baseline but remained similar in responders at all time points. These changes were not observed in nonresponders to the other oral therapies tested. The formation of an oligomer of ASC in monocytes after lipopolysaccharide and adenosine 5′-triphosphate stimulation was significantly decreased in responders (p = 0.006) but increased in nonresponders (p = 0.0003) after 6 months of fingolimod treatment compared with the baseline. Proinflammatory cytokine release from stimulated peripheral blood mononuclear cells was comparable between responders and nonresponders, but galectin-3 levels on cell supernatants, as a marker of cell damage, were significantly increased in fingolimod nonresponders (p = 0.02). Discussion The differential effect of fingolimod on the formation of an inflammasome-triggered ASC oligomer in monocytes between responders and nonresponders could be used as a response biomarker after 6 months of fingolimod treatment and suggests that fingolimod may exert their beneficial effects by reducing inflammasome signaling in a subset of patients with MS.MCIN/AEI/10.13039/501100011033 (grant PID2020-116709RB-I00)—Fundación Séneca (grant 20859/PI/18) and the Instituto Salud Carlos III (grant DTS21/00080). L. Hurtado-Navarro was supported by the fellowship 21214/FPI/19 (Fundación Séneca, Región de Murcia, Spain)

Serum neurofilament light chain levels predict long-term disability progression in patients with progressive multiple sclerosis

© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.Objective : There is a lack of sensitive and specific biomarkers for use in progressive multiple sclerosis (MS). The study aimed to assess the potential of serum neurofilament light chain (sNfL) levels as biomarker of disability progression in patients with progressive MS. Methods: We performed a prospective observational cohort study in 51 patients with progressive MS who participated in a 2-year phase II single-centre, randomised, double-blind, placebo-controlled trial of interferon-beta. Mean (SD) follow-up duration was 13.9 (6.2) years. Levels of sNfL were measured using a single molecule array immunoassay at baseline, 1, 2 and 6 years. Univariable and multivariable analyses were carried out to evaluate associations between sNfL levels and disability progression at short term (2 years), medium term (6 years) and long term (at the time of the last follow-up). Results: A sNfL cut-off value of 10.2¿pg/mL at baseline discriminated between long-term progressors and non-progressors with a 75% sensitivity and 67% specificity (adjusted OR 7.8; 95%¿CI 1.8 to 46.4; p=0.01). Similar performance to discriminate between long-term progressors and non-progressors was observed using age/body mass index-adjusted sNfL Z-scores derived from a normative database of healthy controls. A cut-off increase of 5.1¿pg/mL in sNfL levels between baseline and 6 years also discriminated between long-term progressors and non-progressors with a 71% sensitivity and 86% specificity (adjusted OR 49.4; 95%¿CI 4.4 to 2×103; p=0.008). Conclusions: sNfL can be considered a prognostic biomarker of future long-term disability progression in patients with progressive MS. These data expand the little knowledge existing on the role of sNfL as long-term prognostic biomarker in patients with progressive MS.Postprint (author's final draft

Circulating EZH2-positive T cells are decreased in multiple sclerosis patients

BACKGROUND: Recent studies in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), suggest an involvement of the histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) in important processes such as cell adhesion and migration. METHODS: Here, we aimed to expand these initial observations by investigating the role of EZH2 in MS. mRNA expression levels for EZH2 were measured by real-time PCR in peripheral blood mononuclear cells (PBMC) from 121 MS patients (62 untreated and 59 receiving treatment) and 24 healthy controls. RESULTS: EZH2 expression levels were decreased in PBMC from untreated patients compared to that from controls, and treatment significantly upregulated EZH2 expression. Expression of miR-124 was increased in MS patients compared to controls. Blood immunophenotyping revealed EZH2 expression mostly restricted to CD4+ and CD8+ T cells, and circulating EZH2+ CD4+ and CD8+ T cells were decreased in untreated MS patients compared to controls. CD8+ T cells expressing EZH2 exhibited a predominant central memory phenotype, whereas EZH2+ CD4+ T cells were of effector memory nature, and both T cell subsets produced TNF-α. EZH2+ T cells were enriched in the cerebrospinal fluid compartment compared to blood and were found in chronic active lesions from MS patients. EZH2 inhibition and microarray analysis in PBMC was associated with significant downregulation of key T cell adhesion molecules. CONCLUSION: These findings suggest a role of EZH2 in the migration of T cells in MS patients. The observation of TNF-α expression by CD4+ and CD8+ T cells expressing EZH2 warrants additional studies to explore more in depth the pathogenic potential of EZH2+-positive cells in MS

Influence of the LILRA3 Deletion on Multiple Sclerosis Risk : Original Data and Meta-Analysis

Altres ajuts: Junta de Andalucía (JA)- Fondos Europeos de Desarrollo Regional (FEDER) (grant number CTS2704 to FM).Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system. Genome-wide association studies (GWAS) have identified over hundred polymorphisms with modest individual effects in MS susceptibility and they have confirmed the main individual effect of the Major Histocompatibility Complex. Additional risk loci with immunologically relevant genes were found significantly overrepresented. Nonetheless, it is accepted that most of the genetic architecture underlying susceptibility to the disease remains to be defined. Candidate association studies of the leukocyte immunoglobulin-like receptor LILRA3 gene in MS have been repeatedly reported with inconsistent results. In an attempt to shed some light on these controversial findings, a combined analysis was performed including the previously published datasets and three newly genotyped cohorts. Both wild-type and deleted LILRA3 alleles were discriminated in a single-tube PCR amplification and the resulting products were visualized by their different electrophoretic mobilities. Overall, this meta-analysis involved 3200 MS patients and 3069 matched healthy controls and it did not evidence significant association of the LILRA3 deletion [carriers of LILRA3 deletion: p = 0.25, OR (95% CI) = 1.07 (0.95-1.19)], even after stratification by gender and the HLA-DRB1*15 : 01 risk allele

Natalizumab-related anaphylactoid reactions in MS patients are associated with HLA class II alleles

Altres ajuts: Ajuts per donar Suport als Grups de Recerca de Catalunya," sponsored by the "Agència de Gestió d'Ajuts Universitaris i de Recerca" (AGAUR), Generalitat de CatalunyaWe aimed to investigate potential associations between human leukocyte antigen (HLA) class I and class II alleles and the development of anaphylactic/anaphylactoid reactions in patients with multiple sclerosis (MS) treated with natalizumab. HLA class I and II genotyping was performed in patients with MS who experienced anaphylactic/anaphylactoid reactions and in patients who did not develop infusion-related allergic reactions following natalizumab administration. A total of 119 patients with MS from 3 different cohorts were included in the study: 54 with natalizumab-related anaphylactic/anaphylactoid reactions and 65 without allergic reactions. HLA-DRB1*13 and HLA-DRB1*14 alleles were significantly increased in patients who developed anaphylactic/anaphylactoid reactions (p = 3 × 10 −7 ; odds ratio [OR] = 8.96, 95% confidence interval [CI] = 3.40-23.64), with a positive predictive value (PPV) of 82%. In contrast, the HLA-DRB1*15 allele was significantly more represented in patients who did not develop anaphylactic/anaphylactoid reactions to natalizumab (p = 6 × 10 −4 ; OR = 0.2, 95% CI = 0.08-0.50), with a PPV of 81%. HLA-DRB1 genotyping before natalizumab treatment may help neurologists to identify patients with MS at risk for developing serious systemic hypersensitivity reactions associated with natalizumab administration

Exome sequencing study in patients with multiple sclerosis reveals variants associated with disease course

BACKGROUND: It remains unclear whether disease course in multiple sclerosis (MS) is influenced by genetic polymorphisms. Here, we aimed to identify genetic variants associated with benign and aggressive disease courses in MS patients. METHODS: MS patients were classified into benign and aggressive phenotypes according to clinical criteria. We performed exome sequencing in a discovery cohort, which included 20 MS patients, 10 with benign and 10 with aggressive disease course, and genotyping in 2 independent validation cohorts. The first validation cohort encompassed 194 MS patients, 107 with benign and 87 with aggressive phenotypes. The second validation cohort comprised 257 patients, of whom 224 patients had benign phenotypes and 33 aggressive disease courses. Brain immunohistochemistries were performed using disease course associated genes antibodies. RESULTS: By means of single-nucleotide polymorphism (SNP) detection and comparison of allele frequencies between patients with benign and aggressive phenotypes, a total of 16 SNPs were selected for validation from the exome sequencing data in the discovery cohort. Meta-analysis of genotyping results in two validation cohorts revealed two polymorphisms, rs28469012 and rs10894768, significantly associated with disease course. SNP rs28469012 is located in CPXM2 (carboxypeptidase X, M14 family, member 2) and was associated with aggressive disease course (uncorrected p value < 0.05). SNP rs10894768, which is positioned in IGSF9B (immunoglobulin superfamily member 9B) was associated with benign phenotype (uncorrected p value < 0.05). In addition, a trend for association with benign phenotype was observed for a third SNP, rs10423927, in NLRP9 (NLR family pyrin domain containing 9). Brain immunohistochemistries in chronic active lesions from MS patients revealed expression of IGSF9B in astrocytes and macrophages/microglial cells, and expression of CPXM2 and NLRP9 restricted to brain macrophages/microglia. CONCLUSIONS: Genetic variants located in CPXM2, IGSF9B, and NLRP9 have the potential to modulate disease course in MS patients and may be used as disease activity biomarkers to identify patients with divergent disease courses. Altogether, the reported results from this study support the influence of genetic factors in MS disease course and may help to better understand the complex molecular mechanisms underlying disease pathogenesis

The Rare IL22RA2 Signal Peptide Coding Variant rs28385692 Decreases Secretion of IL-22BP Isoform-1, -2 and -3 and Is Associated with Risk for Multiple Sclerosis.

The IL22RA2 locus is associated with risk for multiple sclerosis (MS) but causative variants are yet to be determined. In a single nucleotide polymorphism (SNP) screen of this locus in a Basque population, rs28385692, a rare coding variant substituting Leu for Pro at position 16 emerged significantly (p = 0.02). This variant is located in the signal peptide (SP) shared by the three secreted protein isoforms produced by IL22RA2 (IL-22 binding protein-1(IL-22BPi1), IL-22BPi2 and IL-22BPi3). Genotyping was extended to a Europe-wide case-control dataset and yielded high significance in the full dataset (p = 3.17 × 10-4). Importantly, logistic regression analyses conditioning on the main known MS-associated SNP at this locus, rs17066096, revealed that this association was independent from the primary association signal in the full case-control dataset. In silico analysis predicted both disruption of the alpha helix of the H-region of the SP and decreased hydrophobicity of this region, ultimately affecting the SP cleavage site. We tested the effect of the p.Leu16Pro variant on the secretion of IL-22BPi1, IL-22BPi2 and IL-22BPi3 and observed that the Pro16 risk allele significantly lowers secretion levels of each of the isoforms to around 50%-60% in comparison to the Leu16 reference allele. Thus, our study suggests that genetically coded decreased levels of IL-22BP isoforms are associated with augmented risk for MS

Effect of Specific Mutations in Cd300 Complexes Formation; Potential Implication of Cd300f in Multiple Sclerosis.

Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals