Interactions between High Seroreactivity to Human Herpes Virus 6A and Epstein-Barr Virus in MS Development: A Presymptomatic Case-Control Study.

Synergistic interactions between human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) are hypothesized in the etiopathogenesis of multiple sclerosis (MS). This study investigated if HHV-6A and EBV seroreactivities interact regarding the risk of developing MS. Antibodies against viral antigens were analyzed in biobank samples from 670 individuals who later developed MS and matched controls. Additive interactions were analyzed. A significant interaction between HHV-6A and EBNA-1 seroreactivities was observed in study participants above the median age of 24.9 years (attributable proportion due to interaction = 0.45). This finding supports the hypothesis that HHV-6A and EBV infections interact in MS development. ANN NEUROL 2024.

HLA Associations of Intrathecal IgG Production against Specific Viruses in Multiple Sclerosis.

OBJECTIVE: Specific human leucocyte antigen (HLA) alleles are not only associated with higher risk to develop multiple sclerosis (MS) and other autoimmune diseases, but also with the severity of various viral and bacterial infections. Here, we analyzed the most specific biomarker for MS, that is, the polyspecific intrathecal IgG antibody production against measles, rubella, and varicella zoster virus (MRZ reaction), for possible HLA associations in MS. METHODS: We assessed MRZ reaction from 184 Swiss patients with MS and clinically isolated syndrome (CIS) and 89 Swiss non-MS/non-CIS control patients, and performed HLA sequence-based typing, to check for associations of positive MRZ reaction with the most prevalent HLA alleles. We used a cohort of 176 Swedish MS/CIS patients to replicate significant findings. RESULTS: Whereas positive MRZ reaction showed a prevalence of 38.0% in MS/CIS patients, it was highly specific (97.7%) for MS/CIS. We identified HLA-DRB1*15:01 and other tightly linked alleles of the HLA-DR15 haplotype as the strongest HLA-encoded risk factors for a positive MRZ reaction in Swiss MS/CIS (odds ratio [OR], 3.90, 95% confidence interval [CI] 2.05-7.46, padjusted = 0.0004) and replicated these findings in Swedish MS/CIS patients (OR 2.18, 95%-CI 1.16-4.02, padjusted = 0.028). In addition, female MS/CIS patients had a significantly higher probability for a positive MRZ reaction than male patients in both cohorts combined (padjusted <0.005). INTERPRETATION: HLA-DRB1*15:01, the strongest genetic risk factor for MS, and female sex, 1 of the most prominent demographic risk factors for developing MS, predispose in MS/CIS patients for a positive MRZ reaction, the most specific CSF biomarker for MS. ANN NEUROL 2024;95:1112-1126.

Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A. A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI). Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis). In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Genetics of immune response to Epstein-Barr virus: prospects for multiple sclerosis pathogenesis.

Epstein-Barr virus (EBV) infection has been advocated as a prerequisite for developing multiple sclerosis (MS) and possibly the propagation of the disease. However, the precise mechanisms for such influences are still unclear. A large-scale study investigating the host genetics of EBV serology and related clinical manifestations, such as infectious mononucleosis (IM), may help us better understand the role of EBV in MS pathogenesis. This study evaluates the host genetic factors that influence serological response against EBV and history of IM and cross-evaluates them with MS risk and genetic susceptibility in the Swedish population. Plasma IgG antibody levels against EBV nuclear antigen-1 (EBNA-1, truncated=aa[325-641], peptide=aa[385-420]) and viral capsid antigen p18 (VCAp18) were measured using bead-based multiplex serology for 8744 MS cases and 7229 population-matched controls. The MS risk association for high/low EBV antibody levels and history of IM was compared to relevant clinical measures along with sex, age at sampling, and associated HLA allele variants. Genome-wide and HLA allele association analyses were also performed to identify genetic risk factors for EBV antibody response and IM history. Higher antibody levels against VCAp18 (OR=1.74, 95% CI=1.60-1.88) and EBNA-1, particularly the peptide (OR=3.13, 95% CI=2.93-3.35), were associated with an increased risk for MS. The risk increased with higher anti-EBNA-1 IgG levels up to twelve times the reference risk. We also identified several independent HLA haplotypes associated with EBV serology overlapping with known MS risk alleles (e.g., DRB1*15:01). Although there were several candidates, no variants outside the HLA region reached genome-wide significance. Cumulative HLA risk for anti-EBNA-1 IgG levels, particularly the peptide fragment, was strongly associated with MS. In contrast, the genetic risk for high anti-VCAp18 IgG levels was not as strongly associated with MS risk. IM history was not associated with class II HLA genes but negatively associated with A*02:01, which is protective against MS. Our findings emphasize that the risk association between anti-EBNA-1 IgG levels and MS may be partly due to overlapping HLA associations. Additionally, the increasing MS risk with increasing anti-EBNA-1 levels would be consistent with a pathogenic role of the EBNA-1 immune response, perhaps through molecular mimicry. Given that high anti-EBNA-1 antibodies may reflect a poorly controlled T-cell defense against the virus, our findings would be consistent with DRB1*15:01 being a poor class II antigen in the immune defense against EBV. Lastly, the difference in genetic control of IM supports the independent roles of EBNA-1 and IM in MS susceptibility.

A Genetic Risk Variant for Multiple Sclerosis Severity is Associated with Brain Atrophy.

The minor allele of the genetic variant rs10191329 in the DYSF-ZNF638 locus is associated with unfavorable long-term clinical outcomes in multiple sclerosis patients. We investigated if rs10191329 is associated with brain atrophy measured by magnetic resonance imaging in a discovery cohort of 748 and a replication cohort of 360 people with relapsing multiple sclerosis. We observed an association with 28% more brain atrophy per rs10191329*A allele. Our results encourage stratification for rs10191329 in clinical trials. Unraveling the underlying mechanisms may enhance our understanding of pathophysiology and identify treatment targets. ANN NEUROL 2023;94:1080-1085.

Spasticity treatment patterns among people with multiple sclerosis: a Swedish cohort study.

BACKGROUND: Spasticity is common among people with multiple sclerosis (MS), but there are few studies of spasticity treatment patterns. We aim to describe associations with spasticity treatment measured primarily by oral baclofen use. METHODS: This cohort study using Swedish registers included 1826 and 3519 people with incident and prevalent MS (pwIMS, pwPMS) respectively, followed from 2005 to 2014. Cox regression assessed factors associated with new baclofen prescriptions and its discontinuation. RESULTS: A total of 10% of pwIMS and 19% of pwPMS received baclofen, a drug prescribed specifically for spasticity in Sweden, of which many patients had relapsing-remitting course. Prescriptions occurred soon after MS diagnosis: pwIMS received baclofen typically within 6 months of diagnosis, and pwPMS within 3 years. Younger patients compared with older patients were three times more likely to receive baclofen with similar disability level measured using Expanded Disability Severity Scores (EDSS). Patients aged 18-44 years with EDSS 3.0-5.0 have an HR for baclofen use of 5.62 (95% CI 2.91 to 10.85) and EDSS 6+ have an HR of 15.41 (95% CI 7.07 to 33.58) compared with individuals with EDSS 0-2.5. In comparison, patients aged 45+ years with EDSS 3.0-5.0 have an HR of 2.05 (95% CI 1.10 to 3.82) and EDSS 6+ an HR of 4.26 (95% CI 1.96 to 9.17). Baclofen discontinuation was high: 49% (95% CI 0.42 to 0.57) of pwIMS discontinued within 150 days of dispensation, 90% discontinued within 2 years including patients with progressive course or higher EDSS. Associations among pwPMS and sensitivity analyses including additional treatments were similar. CONCLUSIONS: Younger patients with MS are more likely to receive baclofen compared with older patients with MS. High rates of baclofen discontinuation highlight the need for more tolerable and efficacious spasticity treatments and monitoring of spasticity among people with MS.

Free vitamin D3 index and vitamin D-binding protein in multiple sclerosis: A presymptomatic case-control study.

BACKGROUND AND PURPOSE: High levels of 25-hydroxyvitamin D3 (25[OH]D3 ) are associated with a lower risk for multiple sclerosis (MS). The bioavailability of 25(OH)D3 is regulated by its main plasma carrier, vitamin D-binding protein (DBP). Free 25(OH)D3 can be estimated by also measuring DBP concentration. In addition, DBP has immunomodulatory functions that may independently affect MS pathogenesis. No previous studies have assessed free 25(OH)D3 or DBP in presymptomatically collected samples. This study was undertaken to assess free 25(OH)D3 and DBP as risk factors for MS. METHODS: A nested case-control study was performed with presymptomatic serum samples identified through cross-linkage of MS registries and Swedish biobanks. Concentration of 25(OH)D3 was measured with liquid chromatography and DBP levels with sandwich immunoassay. Free 25(OH)D3 was approximated as free vitamin D3 index: (25[OH]D3 /DBP) × 103 . MS risk was analyzed by conditional logistic regression, calculating odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: Serum samples from 660 pairs of matched cases and controls were included. At <20 years of age, high levels of free vitamin D3 index were associated with a lower risk of MS (highest vs. lowest quintile: OR = 0.37, 95% CI = 0.15-0.91, p for trend across quintiles = 0.04). At age 30-39 years, high levels of DBP were associated with a lower MS risk (highest vs. lowest quintile: OR = 0.36, 95% CI = 0.15-0.85, p for trend = 0.02). CONCLUSIONS: These findings support the hypothesis that high levels of free 25(OH)D3 at a young age reduce the risk of MS later in life. They also implicate a role for DBP in MS etiology.

MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis.

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor's downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.

Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk.

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

Cytomegalovirus seropositivity is associated with reduced risk of multiple sclerosis-a presymptomatic case-control study.

BACKGROUND AND PURPOSE: Epstein-Barr virus (EBV) and human herpesvirus 6A (HHV-6A) are associated with increased risk of multiple sclerosis (MS). Conversely, infection with cytomegalovirus (CMV) has been suggested to reduce the risk of MS but supporting data from presymptomatic studies are lacking. Here, it was sought to increase the understanding of CMV in MS aetiology. METHODS: A nested case-control study was performed with presymptomatically collected blood samples identified through crosslinkage of MS registries and Swedish biobanks. Serological antibody response against CMV, EBV and HHV-6A was determined using a bead-based multiplex assay. Odds ratio (OR) with 95% confidence interval (CI) for CMV seropositivity as a risk factor for MS was calculated by conditional logistic regression and adjusted for EBV and HHV-6A seropositivity. Potential interactions on the additive scale were analysed by calculating the attributable proportion due to interaction (AP). RESULTS: Serum samples from 670 pairs of matched cases and controls were included. CMV seropositivity was associated with a reduced risk for MS (OR = 0.70, 95% CI 0.56-0.88, p = 0.003). Statistical interactions on the additive scale were observed between seronegativity for CMV and seropositivity against HHV-6A (AP 0.34, 95% CI 0.06-0.61) and EBV antigen EBNA-1 (amino acid 385-420) at age 20-39 years (AP 0.37, 95% CI 0.09-0.65). CONCLUSIONS: Cytomegalovirus seropositivity is associated with a decreased risk for MS. The protective role for CMV infection in MS aetiology is further supported by the interactions between CMV seronegativity and EBV and HHV-6A seropositivity.

The influence of human leukocyte antigen-DRB1*15:01 and its interaction with smoking in MS development is dependent on DQA1*01:01 status.

BACKGROUND: HLA-DRB1*15:01, absence of HLA-A*02:01, and smoking interact to increase multiple sclerosis (MS) risk. OBJECTIVE: To analyze whether MS-associated human leukocyte antigen (HLA) alleles, apart from DRB1*15:01 and absence of A*02:01, interact with smoking in MS development, and to explore whether the established HLA-smoking interaction is affected by the DQA1*01:01 allele, which confers a protective effect only in the presence of DRB1*15:01. METHODS: In two Swedish population-based case-control studies (5838 cases, 5412 controls), subjects with different genotypes and smoking habits were compared regarding MS risk, by calculating odds ratios with 95% confidence intervals employing logistic regression. Interaction on the additive scale between different genotypes and smoking was evaluated. RESULTS: The DRB1*08:01 allele interacted with smoking to increase MS risk. The interaction between DRB1*15:01 and both the absence of A*02:01 and smoking was confined to DQA1*01:01 negative subjects, whereas no interactions occurred among DQA1*01:01 positive subjects. CONCLUSION: Multifaceted interactions take place between different class II alleles and smoking in MS development. The influence of DRB1*15:01 and its interaction with the absence of A*02:01 and smoking is dependent on DQA1*01:01 status which may be due to differences in the responding T-cell repertoires.

Factors associated with and long-term outcome of benign multiple sclerosis: a nationwide cohort study.

OBJECTIVE: Benign multiple sclerosis (BMS) is often defined by the Expanded Disability Status Scale (EDSS) score of ≤3.0 after ≥15 years of disease duration. This classification's clinical relevance remains unclear as benign patients may suffer other impairments and advance towards a progressive course, prompting our objective to holistically investigate factors associated with BMS and its long-term prognosis. METHODS: Benign cases were identified in the Swedish Multiple Sclerosis registry. Baseline clinical data, demographic features and influence of multiple sclerosis (MS) major risk alleles on likelihood of benign course were investigated. Physical disability (EDSS), cognitive function (Symbol Digit Modalities Test; SDMT) and self-reported and socioeconomic differences between benign and non-benign patients were evaluated using generalised estimation equations models. RESULTS: 11222 patients (2420 benign/8802 non-benign) were included. Benign patients were more likely to be female and younger at MS onset, have fewer relapses within the first two and 5 years from onset and fully recover from the first relapse (p<0.001). No association between human leucocyte antigen (HLA) DRB1*15:01 carriership (OR: 0.97, 95% CI: 0.86 to 1.09) or HLA-A*02:01 lacking (OR: 0.99, 95% CI: 0.87 to 1.11) and benign/non-benign was found. Non-benign patients accumulated an extra 0.04 (95% CI 0.03 to 0.04, p<0.001) EDSS score/year, lost an extra 0.3 (95% CI - 0.39 to - 0.18, p<0.001) SDMT score/year and deteriorated faster in self-reported impact and socioeconomic measures (p<0.001). CONCLUSION: Patients with BMS have a better disease course as they progress more slowly at the group level in all respects. Lack of an association with major genetic risk factors indicates that MS course is most likely influenced by either environmental factor(s) or genetic factors outside the HLA region.

The immune response of the human brain to abdominal surgery.

OBJECTIVE: Surgery launches a systemic inflammatory reaction that reaches the brain and associates with immune activation and cognitive decline. Although preclinical studies have in part described this systemic-to-brain signaling pathway, we lack information on how these changes appear in humans. This study examines the short- and long-term impact of abdominal surgery on the human brain immune system by positron emission tomography (PET) in relation to blood immune reactivity, plasma inflammatory biomarkers, and cognitive function. METHODS: Eight males undergoing prostatectomy under general anesthesia were included. Prior to surgery (baseline), at postoperative days 3 to 4, and after 3 months, patients were examined using [11 C]PBR28 brain PET imaging to assess brain immune cell activation. Concurrently, systemic inflammatory biomarkers, ex vivo blood tests on immunoreactivity to lipopolysaccharide (LPS) stimulation, and cognitive function were assessed. RESULTS: Patients showed a global downregulation of gray matter [11 C]PBR28 binding of 26 ± 26% (mean ± standard deviation) at 3 to 4 days postoperatively compared to baseline (p = 0.023), recovering or even increasing after 3 months. LPS-induced release of the proinflammatory marker tumor necrosis factor-α in blood displayed a reduction (41 ± 39%) on the 3rd to 4th postoperative day, corresponding to changes in [11 C]PBR28 distribution volume. Change in Stroop Color-Word Test performance between postoperative days 3 to 4 and 3 months correlated to change in [11 C]PBR28 binding (p = 0.027). INTERPRETATION: This study translates preclinical data on changes in the brain immune system after surgery to humans, and suggests an interplay between the human brain and the inflammatory response of the peripheral innate immune system. These findings may be related to postsurgical impairments of cognitive function. Ann Neurol 2017;81:572-582.

Genetic risk factors for pediatric-onset multiple sclerosis.

BACKGROUND: Strong evidence supports the role of both genetic and environmental factors in pediatric-onset multiple sclerosis (POMS) etiology. OBJECTIVE: We comprehensively investigated the association between established major histocompatibility complex (MHC) and non-MHC adult multiple sclerosis (MS)-associated variants and susceptibility to POMS. METHODS: Cases with onset <18 years (n = 569) and controls (n = 16,251) were included from the United States and Sweden. Adjusted logistic regression and meta-analyses were performed for individual risk variants and a weighted genetic risk score (wGRS) for non-MHC variants. Results were compared to adult MS cases (n = 7588). RESULTS: HLA-DRB1*15:01 was strongly associated with POMS (odds ratio (OR)meta = 2.95, p < 2.0 × 10-16). Furthermore, 28 of 104 non-MHC variants studied (23%) were associated (p < 0.05); POMS cases carried, on average, a higher burden of these 28 variants compared to adults (ORavg = 1.24 vs 1.13, respectively), though the difference was not significant. The wGRS was strongly associated with POMS (ORmeta = 2.77, 95% confidence interval: 2.33, 3.32, p < 2.0 × 10-16) and higher, on average, when compared to adult cases. Additional class III risk variants in the MHC region associated with POMS were revealed after accounting for HLA-DRB1*15:01 and HLA-A*02. CONCLUSION: Pediatric and adult MS share many genetic variants suggesting similar biological processes are present. MHC variants beyond HLA-DRB1*15:01 and HLA-A*02 are also associated with POMS.

Parent-of-origin effects implicate epigenetic regulation of experimental autoimmune encephalomyelitis and identify imprinted Dlk1 as a novel risk gene.

Parent-of-origin effects comprise a range of genetic and epigenetic mechanisms of inheritance. Recently, detection of such effects implicated epigenetic mechanisms in the etiology of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. We here sought to dissect the magnitude and the type of parent-of-origin effects in the pathogenesis of experimental neuroinflammation under controlled environmental conditions. We investigated inheritance of an MS-like disease in rat, experimental autoimmune encephalomyelitis (EAE), using a backcross strategy designed to identify the parental origin of disease-predisposing alleles. A striking 37-54% of all detected disease-predisposing loci depended on parental transmission. Additionally, the Y chromosome from the susceptible strain contributed to disease susceptibility. Accounting for parent-of-origin enabled more powerful and precise identification of novel risk factors and increased the disease variance explained by the identified factors by 2-4-fold. The majority of loci displayed an imprinting-like pattern whereby a gene expressed only from the maternal or paternal copy exerts an effect. In particular, a locus on chromosome 6 comprises a well-known cluster of imprinted genes including the paternally expressed Dlk1, an atypical Notch ligand. Disease-predisposing alleles at the locus conferred lower Dlk1 expression in rats and, together with data from transgenic overexpressing Dlk1 mice, demonstrate that reduced Dlk1 drives more severe disease and modulates adaptive immune reactions in EAE. Our findings suggest a significant epigenetic contribution to the etiology of EAE. Incorporating these effects enables more powerful and precise identification of novel risk factors with diagnostic and prognostic implications for complex disease.

Natural polymorphisms in Tap2 influence negative selection and CD4∶CD8 lineage commitment in the rat.

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.

Combining genetic mapping with genome-wide expression in experimental autoimmune encephalomyelitis highlights a gene network enriched for T cell functions and candidate genes regulating autoimmunity.

The experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system commonly used to study multiple sclerosis (MS). We combined clinical EAE phenotypes with genome-wide expression profiling in spleens from 150 backcross rats between susceptible DA and resistant PVG rat strains during the chronic EAE phase. This enabled correlation of transcripts with genotypes, other transcripts and clinical EAE phenotypes and implicated potential genetic causes and pathways in EAE. We detected 2285 expression quantitative trait loci (eQTLs). Sixty out of 599 cis-eQTLs overlapped well-known EAE QTLs and constitute positional candidate genes, including Ifit1 (Eae7), Atg7 (Eae20-22), Klrc3 (eEae22) and Mfsd4 (Eae17). A trans-eQTL that overlaps Eae23a regulated a large number of small RNAs and implicates a master regulator of transcription. We defined several disease-correlated networks enriched for pathways involved in cell-mediated immunity. They include C-type lectins, G protein coupled receptors, mitogen-activated protein kinases, transmembrane proteins, suppressors of transcription (Jundp2 and Nr1d1) and STAT transcription factors (Stat4) involved in interferon signaling. The most significant network was enriched for T cell functions, similar to genetic findings in MS, and revealed both established and novel gene interactions. Transcripts in the network have been associated with T cell proliferation and differentiation, the TCR signaling and regulation of regulatory T cells. A number of network genes and their family members have been associated with MS and/or other autoimmune diseases. Combining disease and genome-wide expression phenotypes provides a link between disease risk genes and distinct molecular pathways that are dysregulated during chronic autoimmune inflammation.

MOG-induced experimental autoimmune encephalomyelitis in the rat species triggers anti-neurofascin antibody response that is genetically regulated.

BACKGROUND: Ιn multiple sclerosis (MS), axonal damage leads to permanent neurological disabilities and the spreading of the autoimmune response to axonal antigens is implicated in disease progression. Experimental autoimmune encephalomyelitis (EAE) provides an animal model that mimics MS. Using different EAE models, we investigated the pathophysiological basis of epitope spreading to neurofascin, a protein localized at the node of Ranvier and its regulation by non-MHC genes. METHODS: We used two different EAE models in DA rat; one which is induced with myelin oligodendrocyte glycoprotein (MOG) which leads to disease characterized by profound demyelination, and the second which is induced with myelin basic protein (MBP) peptide 63-88 which results in severe central nervous system (CNS) inflammation but little or no demyelination. We determined anti-neurofascin antibody levels during the course of disease. Furthermore, the anti-neurofascin IgG response was correlated with clinical parameters in 333 (DAxPVG.1AV1) x DA rats on which we performed linkage analysis to determine if epitope spreading to neurofascin was affected by non-MHC genes. RESULTS: Spreading of the antibody response to neurofascin occurred in demyelinating MOG-induced EAE but not in EAE induced with MBP peptide 63-88. Anti-neurofascin IgG levels correlated with disease severity in (DAxPVG.1AV1) x DA rats, and a genomic region on chromosome 3 was found to influence this response. CONCLUSIONS: Inter-molecular epitope spreading to neurofascin correlates with disease severity in MOG-EAE is dependent on extensive demyelination and is influenced by non-MHC genes. The findings presented here may shed light on factors involved in the severity of MS and its genetics.

Acute treatment with valproic acid and l-thyroxine ameliorates clinical signs of experimental autoimmune encephalomyelitis and prevents brain pathology in DA rats.

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS) in young adults. Chronic treatments with histone deacetylase inhibitors (HDACis) have been reported to ameliorate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by targeting immune responses. We have recently shown that the HDAC inhibition/knockdown in the presence of thyroid hormone (T3) can also promote oligodendrocyte (OL) differentiation and expression of myelin genes in neural stem cells (NSCs) and oligodendrocyte precursors (OPCs). In this study, we found that treatment with an HDACi, valproic acid (VPA), and T3, alone or in combination, directly affects encephalitogenic CD4+ T cells. VPA, but not T3, compromised their proliferation, while both molecules reduced the frequency of IL-17-producing cells. Transfer of T3, VPA and VPA/T3 treated encephalitogenic CD4+ T cells into naïve rats induced less severe EAE, indicating that the effects of these molecules are persistent and do not require their maintenance after the initial stimuli. Thus, we investigated the effect of acute treatment with VPA and l-thyroxine (T4), a precursor of T3, on myelin oligodendrocyte glycoprotein-induced EAE in Dark Agouti rats, a close mimic of MS. We found that a brief treatment after disease onset led to sustained amelioration of EAE and prevention of inflammatory demyelination in the CNS accompanied with a higher expression of myelin-related genes in the brain. Furthermore, the treatment modulated immune responses, reduced the number of CD4+ T cells and affected the Th1 differentiation program in the brain. Our data indicate that an acute treatment with VPA and T4 after the onset of EAE can produce persistent clinically relevant therapeutic effects by limiting the pathogenic immune reactions while promoting myelin gene expression.

Recovering Misidentified Samples Through Genetic Discordance Clustering.

The many logistical and technical challenges associated with sample and data handling in largescale genotyping studies can increase the risk of sample misidentification, which may compromise subsequent analyses. However, the standard quality assurance methods typical for large genotyping arrays can often be further utilized to identify and recover problematic samples. This article emphasizes the importance of identifying and correcting underlying sample misidentification rather than simply excluding known discrepancies, which may potentially include undetected issues. Lastly, we provide a screening protocol to complement standard quality assessments as a guideline for identifying mismatched samples and a tool for assessing the most common causes of sample misidentification. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.