Jorge R. Oksenberg

There is increasing evidence that human leukocyte antigen (HLA) allelic variants play a role in the genetic predisposition to Parkinson disease (PD). In this regard, we have recently reported that the HLA-DRB1*04:01 allele, typified by residues Q/R-K/R-R-A-A at positions 70-74 in the HLA-DRβ1 chain in combination with valine at position 11 (11-V), confers moderate protective effects. In this study, we tested whether HLA-DRB1*04:01 can modulate PD pathology in vivo. We induced the alpha-synuclein preformed fibrils seeding PD model in humanized mice expressing the HLA-DRB1*04:01 or HLA-DRB1*04:02 allele. We then performed a comprehensive histopathologic and molecular characterization of the main disease phenotypes by combining quantitative histopathology, RNA-sequencing, flow cytometry immunophenotyping, and cytokine profiling. Mice expressing HLA-DRB1*04:01 display limited alpha-synuclein aggregation and lower dopaminergic axonal injury compared with mice expressing HLA-DRB1*04:02, along with reduced systemic inflammation. We also show that the microglia compartment in HLA-DRB1*04:01 mice is in an activated steady state, possibly suggesting their mechanistic involvement in alpha-synuclein clearance. Altogether, our findings provide the first experimental evidence of the genetic association between the HLA locus and PD susceptibility and support a neuroprotective function for the HLA-DRB1*04:01 allele against alpha-synuclein pathology.

Neuromyelitis optica spectrum disorders (NMOSDs) comprise inflammatory processes of the CNS. Most patients with NMOSD have serum immunoglobulin (Ig) G autoantibodies directed against the astrocytic water channel aquaporin-4 (AQP4-IgG). In this study, we analyzed HLA allelic frequencies in a large cohort of patients with NMOSD, stratified by ethnicity and AQP4-IgG status, compared with healthy controls. Next-generation sequencing-based HLA class I and II genotyping was performed in 174 White, 45 Black, and 41 Hispanic AQP4-IgG-positive (AQP4-IgG+) patients with NMOSD; 49 White patients with AQP4-IgG-negative (AQP4-IgG-) NMOSD; and 2,427 White, 244 Black, and 155 Hispanic controls. Correction for multiple testing was performed using the Bonferroni method. In White AQP4-IgG+ patients with NMOSD, the most significantly associated alleles were HLA-DQA1*05:01:01 (30.1% vs 11.1%, odds ratio 3.43 [95% CI 2.65-4.42], corrected p = 8.95E-17), HLA-DQB1*02:01:01 (29.9% vs 11.3%, 3.34 [2.58-4.31], corrected p = 4.33E-16), and HLA-DRB1*03:01:01 (29.2% vs 11.6%, 3.15 [2.43-4.06], corrected p = 3.66E-14), followed by HLA-B*08:01:01 (26% vs 10.4%, 3.02 [2.3-3.94] corrected p = 8.79E-12), HLA-C*07:01:01 (27.8% vs 14%, 2.36 [1.81-3.04], corrected p = 2.44E-07), and HLA-DRB3*01:01:02 (28% vs 14.7%, 2.27 [1.73-2.95], corrected p = 2.19E-06). The frequency of HLA-DRB1*08:04:01 was higher in Black AQP4-IgG+ patients with NMOSD than in Black controls but did not achieve statistical significance (19.3% vs 5.7%, 3.92 [1.91-7.86], corrected p = 0.08). Nevertheless, when compared with a larger cohort of Black controls (n = 16,178), the frequency of HLA-DRB1*08:04 (19.3% vs 5.1%, 4.46 [2.45-7.66], corrected p = 1.88E-03) was significantly higher in Black AQP4-IgG+ patients with NMOSD. No significant HLA associations were detected in AQP4-IgG+ Hispanic patients or White AQP4-IgG- patients with NMOSD. This study confirms the previously recognized association of HLA-DRB1*03:01 with AQP4-IgG+ NMOSD in White patients and extends this association to the HLA-DRB1*03:01:01∼HLA-DQA1*05:01:01∼HLA-DQB1*02:01:01 haplotype. Furthermore, it identifies an association of HLA-DRB1*08:04 with AQP4-IgG+ NMOSD in Black patients. However, no HLA associations were detected in White AQP4-IgG- patients with NMOSD. The immunogenetic differences between AQP4-IgG+ and AQP4-IgG- NMOSD support pathophysiologic distinctions between these entities.

Multiple sclerosis (MS) is a chronic progressive, demyelinating autoimmune CNS disease. Autoantibodies to the motif P-(SA)-x-(SGA)-R-(SN)-(LRKH) are a class of predictive markers specific to MS that could add to emerging diagnostic criteria for MS. In this study, we describe the discovery of an MS patient-derived monoclonal antibody (mAb) specific to this motif from memory B cells, and we develop a proof-of-principle autoantibody test to report the prevalence of seropositivity, predict MS early in disease, and identify underlying tolerance-breaking antigens in Epstein-Barr virus (EBV) and the CNS. Peptide tetramers containing the motif were used to screen activated memory B cells, collected from a patient with MS, on the Beacon Optofluidic system. A mAb to the motif and serum samples from clinically diagnosed patients with MS and healthy controls were used to qualify a Luminex xMAP autoantibody serologic test. Antigen discovery methods included phage-immunoprecipitation sequencing (PhIP-Seq), biolayer interferometry, human protein microarrays, isoelectric focusing gels and blots, and immunofluorescence staining of mouse brain cell cultures. A mAb cloned from an MS patient's memory B cells binds diverse peptides containing the MS signature motif with affinities ranging from 2 to 25 nM. Consensus peptides defined by alanine scanning PhIP-Seq were used to develop an autoimmune IgG test with a sensitivity equivalent to 0.5 ng/mL mAb, a precision of <11%, and a positivity rate of 11% among a cohort of 179 patients with MS (n = 91 healthy and n = 49 unrelated neurologic disease serum samples were negative). Utility of the assay for prodromal MS was demonstrated using retrospective, longitudinal samples from cases in the Department of Defense Serum Repository. The mAb identified EBV tegument protein BRRF2 as a tolerance-breaking antigen with nanomolar affinity, containing the motif with reactivity to oligoclonal bands in CSF from MS signature-positive patients. Immunocytochemical staining of mixed mouse neuronal cells showed the dominant cross-reactive human antigen to be vimentin. We describe a test for MS signature autoantibodies that could be used to support MS diagnosis, prodromal research, and early interventions. The integration of this assay with other emerging biomarkers will advance progress toward a combined predictive risk score for MS.

Psoriasis is a common, debilitating immune-mediated skin disease. Genetic studies have identified biological mechanisms of psoriasis risk, including those targeted by effective therapies. However, the genetic liability to psoriasis is not fully explained by variation at robustly identified risk loci. To refine the genetic map of psoriasis susceptibility we meta-analysed 18 GWAS comprising 36,466 cases and 458,078 controls and identified 109 distinct psoriasis susceptibility loci, including 46 that have not been previously reported. These include susceptibility variants at loci in which the therapeutic targets IL17RA and AHR are encoded, and deleterious coding variants supporting potential new drug targets (including in STAP2, CPVL and POU2F3). We conducted a transcriptome-wide association study to identify regulatory effects of psoriasis susceptibility variants and cross-referenced these against single cell expression profiles in psoriasis-affected skin, highlighting roles for the transcriptional regulation of haematopoietic cell development and epigenetic modulation of interferon signalling in psoriasis pathobiology.

The impact of menopause on the brain is not well understood. Hormonal changes, including puberty and pregnancy, influence the onset and course of multiple sclerosis (MS). After menopause, a worsening of MS disease trajectory measured on the clinician-rated Expanded Disability Status Scale (EDSS) was reported in some, but not all, studies. Evaluating the association between menopause and more objective measures of CNS injury is warranted. This study sought to assess the trajectory of objective functional outcomes and disease biomarkers in women with MS before and after menopause in a longitudinal prospective observational cohort. Data were collected prospectively from a longitudinally followed MS cohort, including the performance-based Multiple Sclerosis Functional Composite (MSFC) as the primary functional outcome and the paraclinical marker of neuronal injury serum neurofilament light chain (sNfL) as the primary biomarker outcome. Outcomes were analyzed using segmented linear mixed model regressions adjusted for age, BMI, and tobacco use, with a change in slope at the time of menopause, as the a priori inflection point. One hundred and eighty-four postmenopausal women met inclusion criteria. Participants were followed for a median of 13 years (interquartile range [IQR] = 4, range: 1-17). The median MS duration was 24 years (IQR = 13, range: 3-64), and the median EDSS score was 2.5 (IQR = 2, range: 0-8). The median age at natural menopause was 50 years (IQR = 5, range: 33-60); 17% of participants used any systemic menopausal hormone therapy. Menopause reflected an inflection point in MSFC worsening (slope difference 0.08, 95% CI 0.01, 0.14, p = 0.0163) and increase in serum neurofilament light chain (slope difference -0.95, 95% CI -1.74 to -0.16, p = 0.0194) while the opposite was found for EDSS (slope difference 0.05, 95% CI 0.01-0.09, p = 0.0200). Findings remained significant after adjustment for multiple covariates. When using additional nonlinear regression modeling, similar inflection points were found (within 3 years of the final menstrual period) for sNfL and EDSS but not MSFC. The menopausal transition may represent an inflection in accumulation of neuronal injury and functional decline in MS.

Background: The HLA-DRB1 gene in the major histocompatibility complex (MHC) region in chromosome 6p21 is the strongest genetic factor identified as influencing multiple sclerosis (MS) susceptibility. DNA methylation changes associated with MS have been consistently detected at the MHC region. However, understanding the full scope of epigenetic regulations of the MHC remains incomplete, due in part to the limited coverage of this region by standard whole genome bisulfite sequencing or array-based methods.Methods: We developed and validated an MHC capture protocol coupled with bisulfite sequencing and conducted a comprehensive analysis of the MHC methylation landscape in blood samples from 147 treatment naïve MS study participants and 129 healthy controls.Results: We identified 132 differentially methylated region (DMRs) within MHC region associated with disease status. The DMRs overlapped with established MS risk loci. Integration of the MHC methylome with human leukocyte antigen (HLA) genetic data indicate that the methylation changes are significantly associated with HLA genotypes. Using DNA methylation quantitative trait loci (mQTL) mapping and the causal inference test (CIT), we identified 643 cis-mQTL-DMRs paired associations, including 71 DMRs possibly mediating causal relationships between 55 single nucleotide polymorphisms (SNPs) and MS risk.Results: The results describe MS-associated methylation changes in MHC region and highlight the association between HLA genotypes and methylation changes. Results from the mQTL and CIT analyses provide evidence linking MHC region variations, methylation changes, and disease risk for MS.

Genome-wide association studies (GWAS) successfully identified multiple sclerosis (MS) susceptibility variants. Despite this notable progress, understanding the biological context of these associations remains challenging, due in part to the complexity of linking GWAS results to causative genes and cell types. Here, we aimed to address this gap by integrating GWAS data with single-cell and bulk chromatin accessibility data and histone modification profiles from immune and nervous systems. MS-GWAS associations are significantly enriched in regulatory regions of microglia and peripheral immune cell subtypes, especially B cells and monocytes. Cell-specific polygenic risk scores were developed to examine the cumulative impact of the susceptibility genes on MS risk and clinical phenotypes, showing significant associations with risk and brain white matter volume. The findings reveal enrichment of GWAS signals in B cell and monocyte/microglial cell-types, consistent with the known pathology and presumed targets of effective MS therapeutics.

Polygenic inheritance plays a pivotal role in driving multiple sclerosis susceptibility, an inflammatory demyelinating disease of the CNS. We developed polygenic risk scores (PRS) of multiple sclerosis and assessed associations with both disease status and severity in cohorts of European descent. The largest genome-wide association dataset for multiple sclerosis to date (n = 41 505) was leveraged to generate PRS scores, serving as an informative susceptibility marker, tested in two independent datasets, UK Biobank [area under the curve (AUC) = 0.73, 95% confidence interval (CI): 0.72–0.74, P = 6.41 × 10⁻¹⁴⁶] and Kaiser Permanente in Northern California (KPNC, AUC = 0.8, 95% CI: 0.76–0.82, P = 1.5 × 10⁻⁵³). Individuals within the top 10% of PRS were at higher than 5-fold increased risk in UK Biobank (95% CI: 4.7–6, P = 2.8 × 10⁻⁴⁵) and 15-fold higher risk in KPNC (95% CI: 10.4–24, P = 3.7 × 10⁻¹¹), relative to the median decile. The cumulative absolute risk of developing multiple sclerosis from age 20 onwards was significantly higher in genetically predisposed individuals according to PRS. Furthermore, inclusion of PRS in clinical risk models increased the risk discrimination by 13% to 26% over models based only on conventional risk factors in UK Biobank and KPNC, respectively. Stratifying disease risk by gene sets representative of curated cellular signalling cascades, nominated promising genetic candidate programmes for functional characterization. These pathways include inflammatory signalling mediation, response to viral infection, oxidative damage, RNA polymerase transcription, and epigenetic regulation of gene expression to be among significant contributors to multiple sclerosis susceptibility. This study also indicates that PRS is a useful measure for estimating susceptibility within related individuals in multicase families. We show a significant association of genetic predisposition with thalamic atrophy within 10 years of disease progression in the UCSF-EPIC cohort (P < 0.001), consistent with a partial overlap between the genetics of susceptibility and end-organ tissue injury. Mendelian randomization analysis suggested an effect of multiple sclerosis susceptibility on thalamic volume, which was further indicated to be through horizontal pleiotropy rather than a causal effect. In summary, this study indicates important, replicable associations of PRS with enhanced risk assessment and radiographic outcomes of tissue injury, potentially informing targeted screening and prevention strategies.

Significance: Cellular and molecular mediators driving multiple sclerosis (MS) pathology have been discovered to a great extent. However, the early molecular events leading to aberrant immune responses remain largely unknown. In this study, we combined bisulfite sequencing with transcriptome profiling to characterize the epigenetic landscape of four peripheral immune cell populations isolated from newly diagnosed, untreated MS patients and healthy individuals. We demonstrate widespread hypomethylation in CD19 ⁺ B cells at clinical disease onset. Notably, this epigenetic signature is functionally linked with the overactivation of B cells. Altogether, our results pinpoint the role of aberrant DNA methylation in connecting defects in the periphery with central nervous system autoimmunity and corroborate the key role of B cells in the initial stages of MS.