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Several single-nucleotide polymorphism (SNP) genome-wide association studies (GWASs) have been completed in multiple sclerosis (MS). Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. In this study we report the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case Control Consortium non-synonymous SNP (nsSNP) screen. In total, the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in membrane metalloendopeptidase-like 1 (MMEL1; odds ratio=1.16, P=3.54 × 10⁻⁶) in MS susceptibility.

Abstract Background Classic Kaposi's Sarcoma (cKS) is a rare vascular tumor associated with Human Herpesvirus 8 (KSHV) infection, nevertheless not all KSHV-infected individuals have cKS. Objective We investigated whether particular KIR/HLA receptor/ligand genotypes would be preferentially present in KSHV-infected and uninfected individuals who have or have not developed cKS. Methods KIR/HLA genotypes were analyzed by molecular genotyping in 50 KSHV-infected individuals who did or did not have cKS and in 33 age-and sex-matched KSHV seronegative individuals. Results There was no association of individual KIR, HLA or receptor ligand combinations with KSHV infection. However, activating KIR and KIR/HLA genotypes were significantly more frequent in cKS cases, specifically KIR3DS1, KIR2DS1, and KIR2DS1 with its HLA-C2 ligand. Conclusion A nonspecific inflammatory response triggered by activation of NK cells upon KIR-HLA interaction could be associated with the pathogenesis of KS.

Compelling evidence has been presented in favor of herpes simplex virus type 1 (HSV1) being one of the causative agents of Alzheimer's disease (AD). The success of HSV1 as a pathogen relates to its sophisticated strategies to evade host immunosurveillance. One strategy involves encoding a decoy Fcγ receptor (FcγR) that thwarts the Fcγ-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), a potent host immunosurveillance mechanism against virally infected cells. The decoy FcγR binds to antibodies of all IgG subclasses, except IgG3; therefore, IgG3 would be expected to play an important role in viral clearance by neutralization and ADCC, and thus contribute to protection from HSV1-spurred diseases. Previous studies have shown significant association between anti-HSV1 IgG3 antibodies and cortical thinning of the areas of the brain typically altered in AD and also targeted by HSV1. The aim of the present investigation was to determine whether GM (γ marker) 5 and GM 21 allotypes, hereditary allelic determinants expressed on IgG3, together with brain biomarkers of neural integrity, contributed to neurodegeneration-as measured by mini-mental state examination (MMSE) score-in patients with AD. Multiple regression analyses showed that the homozygous GM 5/5 genotype, preserved right hippocampus, and right insula thickness were associated with higher MMSE scores (p < 0.001), whereas the opposite pattern and GM 5/21 genotype were associated with worse clinical profiles. Influence of GM 5/21-expressing IgG3 antibodies on the ADCC of HSV1-infected neurons could, at least partially, explain these results., Competing Interests: Declaration of competing interest The authors report no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Autism Spectrum disorders (ASD) are diagnosed more often in males than in females, by a ratio of about 3:1; this is likely to be due to a difference in risk burden between the sexes and/or to "compensatory skills" in females, that may delay the diagnosis of ASD. Identifying specific risk factors for ASD in females may be important in facilitating early diagnosis. We investigated whether HLA- class I: -A, -B, -C and class II -DRB1 alleles, which have been suggested to play a role in the development of ASD, can be considered as sex-related risk/protective markers towards the ASD. We performed HLA allele genotyping in 178 Italian children with ASD, 94 healthy siblings, and their parents. HLA allele distribution was compared between children with ASD, sex-matched healthy siblings, and a cohort of healthy controls (HC) enrolled in the Italian bone marrow donor registry. Allele transmission from parents to children with ASD and their siblings was also assessed. Our findings suggest that HLA-A*02 , B*38 , and C*12 alleles are more frequently carried by females with ASD compared to both HC and healthy female siblings, indicating these alleles as potential risk factors for ASD in females. Conversely, the HLA-A*03 allele was more commonly transmitted to healthy female siblings, suggesting it might have a protective effect. Additionally, the HLA-B*44 allele was found to be more prevalent in boys with ASD, indicating it is a potential risk factor for male patients. This is the first Italian study of sex-related HLA association with ASD. If confirmed, these results could facilitate early ASD diagnosis in female patients, allowing earlier interventions, which are crucial in the management of neurodevelopmental disorders.

Activation of the purinergic receptor P2X7 (P2X7R) is believed to be deleterious in autoimmune diseases and it was hypothesized to play a role in the pathogenesis of MS. P2X7R is an ATP-gated non-selective cationic channel; its activation can be driven by high concentrations of ATP and leads to the generation of large, cytolytic conductance pores. P2X7R activation can also result in apoptosis as a consequence of the activation of the caspase cascade via P2X7R-dependent stimulation of the NLRP3 inflammasome. We measured P2X7R in oligodendrocyte derived extracellular vesicles (ODEVs) in MS patients and in healthy subjects. Sixty-eight MS patients (50 relapsing-remitting, RR-MS, 18 primary progressive, PP-MS) and 57 healthy controls (HC) were enrolled. ODEVs were enriched from serum by a double step immunoaffinity method using an anti OMGp (oligodendrocyte myelin glycoprotein) antibody. P2X7R concentration was measured in ODEVs lysates by ELISA. One-way Anova test showed that P2X7R in ODEVs is significantly higher in PP-MS (mean: 1742.89 pg/mL) compared both to RR-MS (mean: 1277.33 pg/mL) (p < 0.001) and HC (mean: 879.79 pg/mL) (p < 0.001). Comparison between RR-MS and HC was also statistically significant (p < 0.001). Pearson's correlations showed that P2RX7 in ODEVs was positively correlated with EDSS (p = 0.002, r = 0.38, 0.15-0.57 95% CI) and MSSS (p = 0.004, r = 0.34, 0.12-0.54 95% CI) scores, considering MS patients together (PP-MS + RR-MS) and with disease duration in PP-MS group (p = 0.02, r = 0.53, 0.09-0.80 95% CI). Results suggest that ODEVs-associated P2X7R levels could be a biomarker for MS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Age represents a significant risk factor for the development of Alzheimer's disease (AD); however, recent research has documented an influencing role of sex in several features of AD. Understanding the impact of sex on specific molecular mechanisms associated with AD remains a critical challenge to creating tailored therapeutic interventions., Methods: The exploration of the sex-based differential impact on disease (SDID) in AD used a systematic review to first select transcriptomic studies of AD with data regarding sex in the period covering 2002 to 2021 with a focus on the primary brain regions affected by AD - the cortex (CT) and the hippocampus (HP). A differential expression analysis for each study and two tissue-specific meta-analyses were then performed. Focusing on the CT due to the presence of significant SDID-related alterations, a comprehensive functional characterization was conducted: protein-protein network interaction and over-representation analyses to explore biological processes and pathways and a VIPER analysis to estimate transcription factor activity., Results: We selected 8 CT and 5 HP studies from the Gene Expression Omnibus (GEO) repository for tissue-specific meta-analyses. We detected 389 significantly altered genes in the SDID comparison in the CT. Generally, female AD patients displayed more affected genes than males; we grouped said genes into six subsets according to their expression profile in female and male AD patients. Only subset I (repressed genes in female AD patients) displayed significant results during functional profiling. Female AD patients demonstrated more significant impairments in biological processes related to the regulation and organization of synapsis and pathways linked to neurotransmitters (glutamate and GABA) and protein folding, Aβ aggregation, and accumulation compared to male AD patients. These findings could partly explain why we observe more pronounced cognitive decline in female AD patients. Finally, we detected 23 transcription factors with different activation patterns according to sex, with some associated with AD for the first time. All results generated during this study are readily available through an open web resource Metafun-AD (https://bioinfo.cipf.es/metafun-ad/)., Conclusion: Our meta-analyses indicate the existence of differences in AD-related mechanisms in female and male patients. These sex-based differences will represent the basis for new hypotheses and could significantly impact precision medicine and improve diagnosis and clinical outcomes in AD patients., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Background: The incidence of Alzheimer's disease (AD)-the most frequent cause of dementia-is expected to increase as life expectancies rise across the globe. While sex-based differences in AD have previously been described, there remain uncertainties regarding any association between sex and disease-associated molecular mechanisms. Studying sex-specific expression profiles of regulatory factors such as microRNAs (miRNAs) could contribute to more accurate disease diagnosis and treatment., Methods: A systematic review identified six studies of microRNA expression in AD patients that incorporated information regarding the biological sex of samples in the Gene Expression Omnibus repository. A differential microRNA expression analysis was performed, considering disease status and patient sex. Subsequently, results were integrated within a meta-analysis methodology, with a functional enrichment of meta-analysis results establishing an association between altered miRNA expression and relevant Gene Ontology terms., Results: Meta-analyses of miRNA expression profiles in blood samples revealed the alteration of sixteen miRNAs in female and 22 miRNAs in male AD patients. We discovered nine miRNAs commonly overexpressed in both sexes, suggesting a shared miRNA dysregulation profile. Functional enrichment results based on miRNA profiles revealed sex-based differences in biological processes; most affected processes related to ubiquitination, regulation of different kinase activities, and apoptotic processes in males, but RNA splicing and translation in females. Meta-analyses of miRNA expression profiles in brain samples revealed the alteration of six miRNAs in female and four miRNAs in male AD patients. We observed a single underexpressed miRNA in female and male AD patients (hsa-miR-767-5p); however, the functional enrichment analysis for brain samples did not reveal any specifically affected biological process., Conclusions: Sex-specific meta-analyses supported the detection of differentially expressed miRNAs in female and male AD patients, highlighting the relevance of sex-based information in biomedical data. Further studies on miRNA regulation in AD patients should meet the criteria for comparability and standardization of information., (© 2024. The Author(s).)

Parkinson's disease (PD) is a neurodegenerative disorder affecting about 10 million people worldwide with a prevalence of about 2% in the over-80 population. The disease brings in also a huge annual economic burden, recently estimated by the Michael J Fox Foundation for Parkinson's Research to be USD 52 billion in the United States alone. Currently, no effective cure exists, but available PD medical treatments are based on symptomatic prescriptions that include drugs, surgical approaches and rehabilitation treatment. Due to the complex biology of a PD brain, the design of clinical trials and the personalization of treatment strategies require the identification of accessible and measurable biomarkers to monitor the events induced by treatment and disease progression and to predict patients' responsiveness. In the present review, we strive to briefly summarize current knowledge about PD biomarkers, focusing on the role of extracellular vesicles as active or involuntary carriers of disease-associated proteins, with particular attention to those research works that envision possible clinical applications.

While there is substantial agreement on the diagnostic criteria for autism spectrum disorder, it is also acknowledged that it has a broad range of clinical presentations. This can complicate the diagnostic process and aggravate the choice of the most suitable rehabilitative strategy for each child. Attentional difficulties are among the most frequently reported comorbidities in autism spectrum disorder. We investigated the role of SNAP-25 polymorphisms. Synaptosome-associated protein 25 (SNAP25) is a presynaptic membrane-binding protein; it plays a crucial role in neurotransmission and has already been studied in numerous psychiatric disorders. It was also seen to be associated with hyperactivity in children with autism spectrum disorder. We collected clinical, behavioral and neuropsychological data on 41 children with a diagnosis of autism spectrum disorder, and then genotyped them for five single-nucleotide polymorphisms of SNAP-25. Participants were divided into two groups according to the Autism Diagnostic Observation Schedule (ADOS-2) Severity Score. In the group with the highest severity score, we found significant associations of clinical data with polymorphism rs363050 (A/G): children with the GG genotype had lower total IQ, more severe autistic functioning and more attentional difficulties. Our research could be the starting point for outlining a possible endophenotype among patients with autism spectrum disorder who are clinically characterized by severe autistic functioning and significant attentional difficulties.

Background: Literature states that parents of individuals affected by autism spectrum disorder (ASD) can present social and cognitive deficits, restricted behavior patterns and psychiatric difficulties, without meeting standard diagnostic criteria for ASD ("broader autism phenotype"). We explored the relationship between parenting of children affected by ASD and levels of empathy and lack of emotion understanding (alexithymia)., Methods: We enlisted 58 families in which a child was affected by ASD. Parents' empathy and alexithymia were respectively assessed by means of Empathy Quotient (EQ) and Toronto Alexithymia Scale (TAS-20). Additionally, we included the assessment of the perception of children's behavior through the Child Behavior Checklist (CBCL)., Results: Our findings suggest that most parents have normal empathy and do not show significant alexithymia. We found lower EQ and higher TAS-20 scores being more frequent in fathers. Moreover, each parent's empathy degree negatively relates to his/her alexithymia and vice versa, showing that these two features are inversely correlated. Our study unveiled a strong correlation between maternal empathy and alexithymia and child's externalizing problems, as reported by mothers., Conclusions: Our data reveal differences in mothers and fathers' empathy and alexithymia profiles and confirm the importance of considering both parents' points of view either in the diagnostic and the therapeutic interventions. Parental empathy and alexithymia levels not only play a fundamental role in the evaluation of child's difficulties but can also influence the development of a good relationship with the child for what concerns affective resonance.

Autism spectrum disorders (ASD) can present with different onset and timing of symptom development; children may manifest symptoms early in their first year of life, i.e., early onset (EO-ASD), or may lose already achieved skills during their second year of life, thus showing a regressive-type onset (RO-ASD). It is still controversial whether regression represents a neurobiological subtype of ASD, resulting from distinct genetic and environmental causes. We focused this study on the 25 kD synaptosomal-associated protein ( SNAP-25 ) gene involved in both post-synaptic formation and adhesion and considered a key player in the pathogenesis of ASD. To this end, four single nucleotide polymorphisms (SNPs) of the SNAP-25 gene, rs363050, rs363039, rs363043, and rs1051312, already known to be involved in neurodevelopmental and psychiatric disorders, were analyzed in a cohort of 69 children with EO-ASD and 58 children with RO-ASD. Both the rs363039 G allele and GG genotype were significantly more frequently carried by patients with EO-ASD than those with RO-ASD and healthy controls (HC). On the contrary, the rs1051312 T allele and TT genotype were more frequent in individuals with RO-ASD than those with EO-ASD and HC. Thus, two different SNAP-25 alleles/genotypes seem to discriminate between EO-ASD and RO-ASD. Notably, rs1051312 is located in the 3' untranslated region (UTR) of the gene and is the target of microRNA (miRNA) regulation, suggesting a possible epigenetic role in the onset of regressive autism. These SNPs, by discriminating two different onset patterns, may represent diagnostic biomarkers of ASD and may provide insight into the different biological mechanisms towards the development of better tailored therapeutic and rehabilitative approaches.

Better knowledge about the possible role of genetic factors in modulating the response to multiple sclerosis (MS) treatment, including rehabilitation, known to promote neural plasticity, could improve the standard of care for this disease. Vitamin D receptor ( VDR ) gene polymorphisms are associated with MS risk, probably because of the role played by vitamin D in regulating inflammatory and reparative processes. The aim of this study was to evaluate the association of the most important functional VDR SNPs ( TaqI ( T / C ), ApaI ( A / C ), and FokI ( C / T )) with functional outcome in MS patients undergoing multidisciplinary inpatient rehabilitation (MDR) treatment, in order to determine whether genetic profiling might be useful to identify subjects with a higher chance of recovery. To this end, 249 MS inpatients with a diagnosis of either progressive (pMS; n = 155) or relapsing remitting (RRMS; n = 94) disease who underwent MDR treatment (average duration = 5.1 weeks) were genotyped for VDR SNPs by real-time allelic discrimination. The rehabilitation outcome was assessed using the modified Barthel Index (mBI), Expanded Disability Status Scale (EDSS), and pain numerical rating scores (NRS) at the beginning and the end of MDR treatment. A positive correlation was observed in RRMS patients between the VDR TaqI major allele (TT) and mBI increase (i.e., better functional recovery), as assessed by the linear and logistic regression analysis adjusted for gender, age, disease duration, time of hospitalization, HLA-DRB1*15.01 positivity, and number of rehabilitative interventions (Beta = 6.35; p = 0.0002). The VDR-1 TaqI , ApaI , FokI : TCC haplotype was also associated with mBI increase in RRMS patients (Beta = 3.24; p = 0.007), whereas the VDR-2 : CAC haplotype was correlated with a lower mBI increase (Beta = -2.18 p = 0.04) compared with the other haplotypes. VDR TaqI major allele (TT), as well as the VDR-1 TaqI , ApaI , FokI : TCC haplotype could be associated with a better rehabilitation outcome in RRMS patients.

Background: Many studies have already shown that individuals suffering from autism spectrum disorders (ASD) present low levels of empathy: in fact, reduced emotional reciprocity is considered a clinically significant indicator of autistic functioning. We decided to investigate the role of empathy in determining pathological behaviors in children affected by ASD considering parents' point of view; and to evaluate the presence of differences between mothers and fathers' perception of their child's empathy and behaviors., Methods: We compared empathy levels in a sample of 58 patients with ASD as reported by a parent-filled questionnaire with the results of a global evaluation conducted by means of play observations, clinician-rated scales, a semistructured interview with both caregivers and parent-filled questionnaires., Results: The majority of ASD patients have low levels of empathy according to both parents' points of view; noteworthy, mothers and fathers are highly concordant in this respect. Children's levels of empathy negatively correlate with many behavioral problems, both internalizing and externalizing. Furthermore, we found that mothers tend to perceive more internalizing problems, while fathers are more willing to notice externalizing ones., Conclusions: Involving both caregivers in children's diagnostic assessment could deepen patient's evaluation and finally the therapeutic results. Mothers and fathers seem to be highly consistent in describing the psychological characteristics of their child, but not in respect to symptoms.

Introduction: Autism spectrum disorder (ASD) is accompanied by complex immune alterations and inflammation, and the possible role played by Natural Killer (NK) in such alterations is only barely understood., Methods: To address this question we analysed activating and inhibitory NK receptors, as well as NK cells phenotype and function in a group of mothers of children who developed ASD (ASD-MO; N=24) comparing results to those obtained in mothers of healthy children who did not develop (HC-MO; N=25)., Results: Results showed that in ASD-MO compared to HC-MO: 1) NK cells expressing the inhibitory receptor ILT2 are significantly decreased; 2) the activating HLA-G14bp+ polymorphism is more frequently observed and is correlated with the decrease of ILT2-expressing cells; 3) the CD56 bright and CD56 dim NK subsets are increased; 4) IFNγ and TNF production is reduced; and 5) perforin- and granzymes-releasing NK cells are increased even in unstimulated conditions and could not be upregulated by mitogenic stimulation., Discussion: Results herein reinforce the hypothesis that ASD relatives present traits similar to, but not as severe as the defining features of ASD (Autism endophenotype) and identify a role for NK cells impairment in generating the inflammatory milieu that is observed in ASD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Saresella, Marventano, Piancone, Bolognesi, Hernis, Zanzottera, La Rosa, Agliardi, Giraldo, Chiappedi, Guerini and Clerici.)

The vitamin D/Vitamin D receptor (VDR) axis is crucial for human health as it regulates the expression of genes involved in different functions, including calcium homeostasis, energy metabolism, cell growth and differentiation, and immune responses. In particular, the vitamin D/VDR complex regulates genes of both innate and adaptive immunity. Autoimmune diseases are believed to arise from a genetic predisposition and the presence of triggers such as hormones and environmental factors. Among these, a role for Vitamin D and molecules correlated to its functions has been repeatedly suggested. Four single nucleotide polymorphisms (SNPs) of the VDR gene, ApaI , BsmI , TaqI , and FokI , in particular, have been associated with autoimmune disorders. The presence of particular VDR SNP alleles and genotypes, thus, was observed to modulate the likelihood of developing diverse autoimmune conditions, either increasing or reducing it. In this work, we will review the scientific literature suggesting a role for these different factors in the pathogenesis of autoimmune conditions and summarize evidence indicating a possible VDR SNP involvement in the onset of these diseases. A better understanding of the role of the molecular mechanisms linking Vitamin D/VDR and autoimmunity might be extremely useful in designing novel therapeutic avenues for these disorders.

REM sleep behavior disorder (RBD) has a tighter link with synucleinopathies than other neurodegenerative disorders. Parkinson's Disease (PD) patients with RBD have a more severe motor and cognitive impairment; biomarkers for RBD are currently unavailable. Synaptic accumulation of α-Syn oligomers and their interaction with SNARE proteins is responsible for synaptic dysfunction in PD. We verified whether oligomeric α-Syn and SNARE components in neural-derived extracellular vesicles (NDEVs) in serum could be biomarkers for RBD. Forty-seven PD patients were enrolled, and the RBD Screening Questionnaire (RBDSQ) was compiled. A cut-off score > 6 to define probable RBD (p-RBD) and probable non-RBD (p non-RBD) was used. NDEVs were isolated from serum by immunocapture, and oligomeric α-Syn and SNARE complex components VAMP-2 and STX-1 were measured by ELISA. NDEVs' STX-1A resulted in being decreased in p-RBD compared to p non-RBD PD patients. A positive correlation between NDEVs' oligomeric α-Syn and RBDSQ total score was found ( p = 0.032). Regression analysis confirmed a significant association between NDEVs' oligomeric α-Syn concentration and RBD symptoms ( p = 0.033) independent from age, disease duration, and motor impairment severity. Our findings suggest that synuclein-mediated neurodegeneration in PD-RBD is more diffuse. NDEVs' oligomeric α-Syn and SNARE complex components' serum concentrations could be regarded as reliable biomarkers for the RBD-specific PD endophenotype.

SNAP-25 protein is a key protein of the SNARE complex that is involved in synaptic vesicles fusion with plasma membranes and neurotransmitter release, playing a fundamental role in neural plasticity. Recently the concentration of three specific miRNAs-miR-27b-3p, miR-181a-5p and miR-23a-3p -was found to be associated with a specific SNAP-25 polymorphism (rs363050). in silico analysis showed that all the three miRNAs target SNAP-25, but the effect of the interaction between these miRNAs and the 3'UTR of SNAP-25 mRNA is currently unknown. For this reason, we verified in vitro whether miR-27b-3p, miR-181a-5p and miR-23a-3p modulate SNAP-25 gene and protein expression. Initial experiments using miRNAs-co-transfected Vero cells and SNAP-25 3'UTR luciferase reporter plasmids showed that miR-181a-5p (p≤0.01) and miR-23a-3p (p<0.05), but not miR-27b-3p, modulate the luciferase signal, indicating that these two miRNAs bind the SNAP-25 3'UTR. Results obtained using human oligodendroglial cell line (MO3.13) transfected with miR-181a-5p or miR-27b-3p confirmed that miR-181a-5p and miR-23a-3p regulate SNAP-25 gene and protein expression. Interestingly, the two miRNAs modulate in an opposite way SNAP-25, as miR-181a-5p significantly increases (p<0.0005), whereas miR-23a-3p decreases (p<0.0005) its expression. These results for the first time describe the ability of miR-181a-5p and miR-23a-3p to modulate SNAP-25 expression, suggesting their possible use as biomarkers or as therapeutical targets for diseases in which SNAP-25 expression is altered., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Agostini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Approximately 15% of multiple sclerosis (MS) patients develop a progressive form of disease from onset; this condition (primary progressive-PP) MS is difficult to diagnose and treat, and is associated with a poor prognosis. Extracellular vesicles (EVs) of brain origin isolated from blood and their protein cargoes could function as a biomarker of pathological conditions. We verified whether MBP and MOG content in oligodendrocytes-derived EVs (ODEVs) could be biomarkers of MS and could help in the differential diagnosis of clinical MS phenotypes. A total of 136 individuals (7 clinically isolated syndrome (CIS), 18 PPMS, 49 relapsing remitting (RRMS)) and 70 matched healthy controls (HC) were enrolled. ODEVs were enriched from serum by immune-capture with anti-MOG antibody; MBP and MOG protein cargoes were measured by ELISA. MBP concentration in ODEVs was significantly increased in CIS (p < 0.001), RRMS (p < 0.001) and PPMS (p < 0.001) compared to HC and was correlated with disease severity measured by EDSS and MSSS. Notably, MBP concentration in ODEVs was also significantly augmented in PPMS compared to RRMS (p = 0.004) and CIS (p = 0.03). Logistic regression and ROC analyses confirmed these results. A minimally invasive blood test measuring the concentration of MBP in ODEVs is a promising tool that could facilitate MS diagnosis.

The early differential diagnosis of Parkinson's disease (PD) and atypical Parkinsonian syndromes (APS), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), is challenging because of an overlap of clinical features and the lack of reliable biomarkers. Neural-derived extracellular vesicles (NDEVs) isolated from blood provide a window into the brain's biochemistry and may assist in distinguishing between PD and APS. We verified in a case-control study whether oligomeric α-Synuclein and Tau aggregates isolated from NDEVs could allow the differential diagnosis of these conditions. Blood sampling and clinical data, including disease duration, motor severity, global cognition, and levodopa equivalent daily dose (LEDD), were collected from patients with a diagnosis of either PD (n = 70), PSP (n = 21), or CBD (n = 19). NDEVs were isolated from serum by immunocapture using an antibody against the neuronal surface marker L1CAM; oligomeric α-Synuclein and aggregated Tau were measured by ELISA. NDEVs analyses showed that oligomeric α-Synuclein is significantly augmented in PD compared to APS, whereas Tau aggregates are significantly increased in APS compared to PD (p < 0.0001). ROC analyses showed that these two biomarkers have a "good" power of classification (p < 0.0001 for both proteins), with high sensitivity and specificity, with NDEVs concentration of Tau aggregates and oligomeric α-Synuclein being respectively the best biomarker for PD/PSP and PD/CBD diagnostic differentiation. Logistic and multiple regression analysis confirmed that NDEVs-derived oligomeric α-Synuclein and Tau aggregates differentiate PD from CBD and PSP (p < 0.001). Notably, a positive correlation between NDEVs oligomeric α-Synuclein and disease severity (disease duration, p = 0.023; Modified H&Y, p = 0.015; UPDRS motor scores, p = 0.004) was found in PD patients and, in these same patients, NDEVs Tau aggregates concentration inversely correlated with global cognitive scores (p = 0.043). A minimally invasive blood test measuring the concentration of α-synuclein and Tau aggregates in NDEVs can represent a promising tool to distinguish with high sensitivity and specificity PD from CBD or PSP patients. Optimization and validation of these data will be needed to confirm the diagnostic value of these biomarkers in distinguishing synucleinopathies from taupathies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) that leads to progressive physical disability. Recent evidence has suggested that P2X7 receptor (P2X7R)-mediated purinergic signalling pathways play a role in MS-associated neuroinflammation, possibly contributing to disease pathogenesis. To evaluate possible associations between P2X7R polymorphisms and MS disease severity, we performed an association study of five non-synonymous SNPs coding variants of the P2X7R gene: rs1718119 Ala348Thr, rs2230911 Thr357Ser, rs2230912 Gln460Arg, rs3751143 Glu496Ala, and rs28360457 Arg307Gln, modulating P2X7R expression in 128 MS patients (relapsing remitting MS, RRMS: n = 94; secondary progressive, SPMS: n = 34). All patients were genotyped, and multiple sclerosis severity score (MSSS) was evaluated in every case; 189 healthy subjects were enrolled as well as controls. Results showed that P2X7R rs1718119(A) 348Thr and rs22390912(G) 464Arg, two SNPs of minor allele frequency (MAF) known to confer gain of function to the P2X7R protein, were associated with significantly higher MSSS in RRMS patients alone (SMRR (p < 0.001, p = 0.01, respectively)). Interestingly, two whole haplotypes resulted in having significant association with MSSS in these same patients. Thus: (1) the P2X7R-4 “ACGAG” haplotype, characterized by the co-presence of the rs1718119-rs2230912 AG MAF alleles, was associated with higher MSSS (Beta: 1.11 p = 0.04), and (2) the P2X7R-1 “GCAAG” complementary haplotype, which contains the rs1718119 and rs2230912 GA wild-type alleles, was more frequently carried by patients with lower MSSS and less severe disease (Beta: −1.54 p < 0.001). Although being preliminary and needing confirmation in an ampler cohort, these results suggest that 348Thr and 464Arg variants have a role as modulators of disease severity in RRMS patients.

Autism spectrum disorders (ASD) are characterized by a wide spectrum of clinical, behavioral, and cognitive manifestations. It is, therefore, crucial to investigate possible biomarkers associated with specific ASD phenotypes. Ample literature suggests a possible role for vitamin D (VD) in influencing ASD clinical phenotypes. We analyzed three vitamin D binding protein gene (DBP) functional polymorphisms (rs2282679, rs7041, and rs4588), which are involved in the modulation of vitamin D serum concentration in 309 ASD children and 831 healthy controls. Frequency comparisons of single nucleotide polymorphisms (SNPs) alleles, genotypes, and GC isoforms (GC1f, G1s, and GC2)—generated by the combination of rs7041 and rs4588 alleles—were correlated with ASD diagnostic, behavioral, and functioning scales. The GC1f isoform was significantly more frequent in ASD compared with controls (18.6% vs. 14.5% pc = 0.02). Significantly higher scores for item 15 of the Childhood Autism Rating Scale (CARS) and lower ones for the Children’s Global Assessment Scale (CGAS) functioning scales were seen in ASD carrying the GC1f isoform. In GC phenotype analysis, a gradient of severity for overall CARS scores and CARS item 15 was observed, with scores decreasing according to the presence of GC1f-GC1f > GC1f-GC1s > GC1s-GC1s > GC1f-GC2 > GC2-GC2 isoforms. Similarly, lower CGAS scores were seen in carriers of the GC1f-GC1f isoform, whereas higher scores were present in those carrying GC2-GC2 (p = 0.028). This is the first study to evaluate possible relationships between GC variants and the different aspects of ASD in Italian ASD children. Results, although needing to be validated in ampler cohorts, suggest that the GC1f isoform could be a marker of severity in ASD that may be useful in establishing the intensity of therapeutic and rehabilitative protocols.

The aim of this investigation was to determine if particular immunoglobulin GM (γ marker) alleles and genotypes were associated with Parkinson's disease (PD) and whether they contributed to the interindividual differences in the level of antibodies to herpes simplex virus type 1 (HSV1), which has been implicated in PD pathology. Using a case-control study design, 94 PD patients and 157 controls were characterized for anti-HSV1 IgG antibodies and genotyped for GM alleles expressed on IgG1 (3,17) and IgG2 (23 +, 23-). The homozygosity for the GM 3 and GM 23 alleles was significantly associated with susceptibility to PD (p = 0.004, 0.018, respectively). Also, GM 23 genotypes were significantly associated with anti-HSV1 IgG antibody levels in patients (p = 0.0021), but not in controls. These results suggest that GM genes may act as effect modifiers of the reported HSV1-PD association., Competing Interests: Declaration of Competing Interest The authors have no competing financial interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Competing Interests: None

HLA allelic distribution was analysed in a cohort of 96 Northern Italian subjects (53M/43F) (mean age 59.9 ± 13.3 years) from Lombardy who developed COVID-19 during the first two pandemic waves to investigate possible correlations between HLA molecules and disease severity. An important role of HLA- B and HLA-C loci in modulating the clinical severity of COVID-19 disease was identified. In particular, the HLA-B07 supertype was observed to be associated with a significant risk for severe disease; conversely, the HLA-B27 supertype and C*12:02 allele played a protective role as they were associated with milder disease. These associations were confirmed after applying a multinomial regression analysis to adjust the correlation for age, gender and comorbidities with COVID-19 severity. Though the power of results is limited by the small sample size, data herein contribute to shedding light on the role played by genetic background in COVID-19 infection.

Vascular alterations often overlap with neurodegeneration, resulting in mixed forms of dementia (MD) that are hard to differentiate from Alzheimer's Disease (AD). The 26 bp intergenic polymorphism of VAMP2 , a key component of SNARE complex, as well as its mRNA and protein levels are associated with neurological diseases. We evaluated ApoE4 and VAMP2 26 bp Ins/Del genotype distribution in 177 AD, 132 MD, 115 Mild Cognitive Impairment (MCI) and 250 individuals without cognitive decline (CT), as well as VAMP2 gene expression in a subset of 73 AD, 122 MD, 103 MCI and 140 CT. Forty-two MCI evolved to AD (22 MCI-AD) or MD (20 MCI-MD) over time. VAMP2 mRNA was higher in MD compared to AD ( p = 0.0013) and CT ( p = 0.0017), and in MCI-MD compared to MCI-AD ( p < 0.001) after correcting for age, gender, MMSE and ApoE4 +/- covariates ( p c = 0.004). A higher VAMP2 expression was observed in subjects carrying the minor allele Del compared to those carrying the Ins/Ins genotype ( p = 0.012). Finally, Del/Del genotype was more frequently carried by MD/MCI-MD compared to CT ( p c = 0.036). These results suggest that VAMP2 mRNA expression can discriminate mixed form of dementia from AD, possibly being a biomarker of AD evolution in MCI patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Costa, Ferri, Guerini, Rossi, Arosio and Clerici.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Genome-wide association studies identified over 200 risk loci for multiple sclerosis (MS) focusing on common variants, which account for about 50% of disease heritability. The goal of this study was to investigate whether low-frequency and rare functional variants, located in MS-established associated loci, may contribute to disease risk in a relatively homogeneous population, testing their cumulative effect (burden) with gene-wise tests. We sequenced 98 genes in 588 Italian patients with MS and 408 matched healthy controls (HCs). Variants were selected using different filtering criteria based on allelic frequency and in silico functional impacts. Genes showing a significant burden (n = 17) were sequenced in an independent cohort of 504 MS and 504 HC. The highest signal in both cohorts was observed for the disruptive variants (stop-gain, stop-loss, or splicing variants) located in EFCAB13 , a gene coding for a protein of an unknown function ( p < 10 -4 ). Among these variants, the minor allele of a stop-gain variant showed a significantly higher frequency in MS versus HC in both sequenced cohorts ( p = 0.0093 and p = 0.025), confirmed by a meta-analysis on a third independent cohort of 1298 MS and 1430 HC ( p = 0.001) assayed with an SNP array. Real-time PCR on 14 heterozygous individuals for this variant did not evidence the presence of the stop-gain allele, suggesting a transcript degradation by non-sense mediated decay, supported by the evidence that the carriers of the stop-gain variant had a lower expression of this gene ( p = 0.0184). In conclusion, we identified a novel low-frequency functional variant associated with MS susceptibility, suggesting the possible role of rare/low-frequency variants in MS as reported for other complex diseases., Competing Interests: MF is Editor-in-Chief of the Journal of Neurology, Associate Editor of Human Brain Mapping, Associate Editor of Radiology, and Associate Editor of Neurological Sciences; received compensation for consulting services and/or speaking activities from Alexion, Almirall, Bayer, Biogen, Celgene, Eli Lilly, Genzyme, Merck-Serono, Novartis, Roche, Sanofi, Takeda, and Teva Pharmaceutical Industries; and receives research support from Biogen Idec, Merck-Serono, Novartis, Roche, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and ARiSLA (Fondazione Italiana di Ricerca per la SLA). VM received compensation for speaking and/or for consultancy and support for travel expenses and participation in Congresses from Biogen, Merck-Serono, Novartis, Roche, Genzyme and Teva Pharmaceutical Industries. FMB has received compensation for consulting services and/or speaking activities from Teva Pharmaceutical Industries, Sanofi Genzyme, Merck-Serono, Biogen Idec, Roche, Medday, Excemed, and received research support from Merck, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla and Fondazione Cariplo. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clarelli, Barizzone, Mangano, Zuccalà, Basagni, Anand, Sorosina, Mascia, Santoro, Guerini, Virgilio, Gallo, Pizzino, Comi, Martinelli, Comi, De Bellis, Leone, Filippi, Esposito, Bordoni, Martinelli Boneschi and D'Alfonso.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic of coronavirus disease (COVID-19). Whereas in most cases COVID-19 is asymptomatic or pauci-symptomatic, extremely severe clinical forms are observed. In this case, complex immune dysregulations and an excessive inflammatory response are reported and are the main cause of morbidity and mortality. Natural killer cells are key players in the control of viral infection, and their activity is regulated by a tight balance between activating and inhibitory receptors; an alteration of NK activity was suggested to be associated with the development of severe forms of COVID-19. In this study, we analyzed peripheral NK cell subpopulations and the expression of activating and inhibitory receptors in 30 patients suffering from neurological conditions who recovered from mild, moderate, or severe SARS-CoV-2 infection, comparing the results to those of 10 SARS-CoV-2-uninfected patients. Results showed that an expansion of NK subset with lower cytolytic activity and an augmented expression of the 2DL1 inhibitory receptor, particularly when in association with the C2 ligand (KIR2DL1-C2), characterized the immunological scenario of severe COVID-19 infection. An increase of NK expressing the ILT2 inhibitory receptor was instead seen in patients recovering from mild or moderate infection compared to controls. Results herein suggest that the KIR2DL1-C2 NK inhibitory complex is a risk factor toward the development of severe form of COVID-19. Our results confirm that a complex alteration of NK activity is present in COVID-19 infection and offer a molecular explanation for this observation., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vitamin D modulates immune responses and its deficiency has been observed in more than 60% of bronchiectasis patients. Vitamin D binding protein (DBP) is coded by the GC gene, is involved in the transport of vitamin D, and includes a number of isoforms based on single nucleotide polymorphisms (SNPs) in the coding region at rs7041 and rs4855. We evaluated the possible clinical impact of DBP polymorphisms and isoforms in an observational, cross-sectional study conducted in 116 bronchiectasis patients, who were genetically characterized for rs4588 and rs7041 SNPs. Results showed that the GC1f isoform (rs7041/rs4588 A/G) correlated with a more severe disease (18.9% vs. 6.3%, p = 0.038), a higher incidence of chronic infections (63.6% vs. 42%, p = 0.041), and a lower BACI score (0.0 (0.0, 2.5) vs. 3.0 (0.0, 3.0), p = 0.035). Moreover, blood concentration of vitamin D was higher in patients carrying GC1s (median (IQR): 20.5 (14.3, 29.7 vs. 15.8 (7.6, 22.4), p = 0.037)). Patients carrying GC1f isoform have a more severe disease, more chronic infections and lower asthmatic comorbidity in comparison to those without the GC1f isoform. Presence of the GC1s isoform (rs7041/rs4588 C/G) seems to be associated to a milder clinical phenotype with increased vitamin D levels and lower comorbidities score.

Known multiple sclerosis (MS) susceptibility variants can only explain half of the disease's estimated heritability, whereas low-frequency and rare variants may partly account for the missing heritability. Thus, here we sought to determine the occurrence of rare functional variants in a large Italian MS multiplex family with five affected members. For this purpose, we combined linkage analysis and next-generation sequencing (NGS)-based whole exome and whole genome sequencing (WES and WGS, respectively). The genetic burden attributable to known common MS variants was also assessed by weighted genetic risk score (wGRS). We found a significantly higher burden of common variants in the affected family members compared to that observed among sporadic MS patients and healthy controls (HCs). We also identified 34 genes containing at least one low-frequency functional variant shared among all affected family members, showing a significant enrichment in genes involved in specific biological processes-particularly mRNA transport-or neurodegenerative diseases. Altogether, our findings point to a possible pathogenic role of different low-frequency functional MS variants belonging to shared pathways. We propose that these rare variants, together with other known common MS variants, may account for the high number of affected family members within this MS multiplex family.

The etiology of Parkinson's disease (PD) is presumably multifactorial and likely involves interactions between genetic and environmental factors, as well as mitochondrial dysfunction, oxidative stress and inflammation. Among environmental factors, Vitamin D was reported to associate with the risk of PD. Vitamin D activity is mediated by its binding to the vitamin D Receptor (VDR), a transcriptional factor for almost 3% of human genes. We genotyped for ApaI, BsmI, TaqI, FokI and rs1989969 VDR single nucleotide polymorphisms (SNPs) a cohort of 406 PD and 800 healthy controls (HC) and found a strong association between the FokI (rs2228570) VDR SNP and PD. Thus, the TT genotype and the T allele resulted associated with PD in the overall analyzed PD population. Gender-based stratification of data indicated that results were maintained for FokI TT genotype and T allele in male PD patients, whereas the FokI T allele alone was confirmed as a risk factor for PD in females. Co-segregation analyses indicated the TaqI ApaI FokI rs1989969 GCTG as a "risk" haplotype for PD. In a subgroup of patients and controls neural Vitamin D and VDR concentration was analyzed in extravesicles (NDEVs) isolated from peripheral blood: no differences emerged between PD and HC. NDEVs results will need to be validated in ampler cohort but we can speculate that, if at neuronal level the amounts of Vitamin D and of VDR are comparable, than the bioavailability of vitamin D and the efficacy of the vitamin D/VDR axis is differentially modulated in PD by VDR SNPs., (Copyright © 2021 Elsevier B.V. All rights reserved.)