Next-Generation Sequencing Identifies Extended HLA Class I and II Haplotypes Associated With Early-Onset and Late-Onset Myasthenia Gravis in Italian, Norwegian, and Swedish Populations.

Genetic susceptibility to myasthenia gravis (MG) associates with specific HLA alleles and haplotypes at the class I and II regions in various populations. Previous studies have only examined alleles at a limited number of HLA loci that defined only broad serotypes or alleles defined at the protein sequence level. Consequently, genetic variants in noncoding and untranslated HLA gene segments have not been fully explored but could also be important determinants for MG. To gain further insight into the role of HLA in MG, we applied next-generation sequencing to analyze sequence variation at eleven HLA genes in early-onset (EO) and late-onset (LO) non-thymomatous MG patients positive for the acetylcholine receptor (AChR) antibodies and ethnically matched controls from Italy, Norway, and Sweden. For all three populations, alleles and haplotype blocks present on the ancestral haplotype AH8.1 were associated with risk in AChR-EOMG patients. HLA-B*08:01:01:01 was the dominant risk allele in Italians (OR = 3.28, P = 1.83E-05), Norwegians (OR = 3.52, P = 4.41E-16), and in Swedes HLA-B*08:01 was the primary risk allele (OR = 4.24, P <2.2E-16). Protective alleles and haplotype blocks were identified on the HLA-DRB7, and HLA-DRB13.1 class II haplotypes in Italians and Norwegians, whereas in Swedes HLA-DRB7 exhibited the main protective effect. For AChR-LOMG patients, the HLA-DRB15.1 haplotype and associated alleles were significantly associated with susceptibility in all groups. The HLA-DR13-HLA-DR-HLA-DQ haplotype was associated with protection in all AChR-LOMG groups. This study has confirmed and extended previous findings that the immunogenetic predisposition profiles for EOMG and LOMG are distinct. In addition, the results are consistent with a role for non-coding HLA genetic variants in the pathogenesis of MG.

A splice acceptor variant in HLA-DRA affects the conformation and cellular localization of the class II DR alpha-chain.

Class II human leucocyte antigen (HLA) proteins are involved in the immune response by presenting pathogen-derived peptides to CD4+ T lymphocytes. At the molecular level, they are constituted by α/ß-heterodimers on the surface of professional antigen-presenting cells. Here, we report that the acceptor variant (rs8084) in the HLA-DRA gene mediates the transcription of an alternative version of the α-chain lacking 25 amino acids in its extracellular domain. Molecular dynamics simulations suggest this isoform undergoes structural refolding which in turn affects its stability and cellular trafficking. The short HLA-DRA isoform cannot reach the cell surface, although it is still able to bind the corresponding ß-chain. Conversely, it remains entrapped within the endoplasmic reticulum where it is targeted for degradation. Furthermore, we demonstrate that the short isoform can be transported to the cell membrane via interactions with the peptide-binding site of canonical HLA heterodimers. Altogether, our findings indicate that short HLA-DRA functions as a novel intact antigen for class II HLA molecules.

Ataxin-1 regulates B cell function and the severity of autoimmune experimental encephalomyelitis.

Ataxin-1 (ATXN1) is a ubiquitous polyglutamine protein expressed primarily in the nucleus where it binds chromatin and functions as a transcriptional repressor. Mutant forms of ataxin-1 containing expanded glutamine stretches cause the movement disorder spinocerebellar ataxia type 1 (SCA1) through a toxic gain-of-function mechanism in the cerebellum. Conversely, ATXN1 loss-of-function is implicated in cancer development and Alzheimer's disease (AD) pathogenesis. ATXN1 was recently nominated as a susceptibility locus for multiple sclerosis (MS). Here, we show that Atxn1-null mice develop a more severe experimental autoimmune encephalomyelitis (EAE) course compared to wildtype mice. The aggravated phenotype is mediated by increased T helper type 1 (Th1) cell polarization, which in turn results from the dysregulation of B cell activity. Ataxin-1 ablation in B cells leads to aberrant expression of key costimulatory molecules involved in proinflammatory T cell differentiation, including cluster of differentiation (CD)44 and CD80. In addition, comprehensive phosphoflow cytometry and transcriptional profiling link the exaggerated proliferation of ataxin-1 deficient B cells to the activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) pathways. Lastly, selective deletion of the physiological binding partner capicua (CIC) demonstrates the importance of ATXN1 native interactions for correct B cell functioning. Altogether, we report a immunomodulatory role for ataxin-1 and provide a functional description of the ATXN1 locus genetic association with MS risk.

miRNA contributions to pediatric-onset multiple sclerosis inferred from GWAS.

OBJECTIVE: Onset of multiple sclerosis (MS) occurs in childhood for approximately 5% of cases (pediatric MS, or ped-MS). Epigenetic influences are strongly implicated in MS pathogenesis in adults, including the contribution from microRNAs (miRNAs), small noncoding RNAs that affect gene expression by binding target gene mRNAs. Few studies have specifically examined miRNAs in ped-MS, but individuals developing MS at an early age may carry a relatively high burden of genetic risk factors, and miRNA dysregulation may therefore play a larger role in the development of ped-MS than in adult-onset MS. This study aimed to look for evidence of miRNA involvement in ped-MS pathogenesis. METHODS: GWAS results from 486 ped-MS cases and 1362 controls from the U.S. Pediatric MS Network and Kaiser Permanente Northern California membership were investigated for miRNA-specific signals. First, enrichment of miRNA-target gene network signals was evaluated using MIGWAS software. Second, SNPs in miRNA genes and in target gene binding sites (miR-SNPs) were tested for association with ped-MS, and pathway analysis was performed on associated target genes. RESULTS: MIGWAS analysis showed that miRNA-target gene signals were enriched in GWAS (P = 0.038) and identified 39 candidate biomarker miRNA-target gene pairs, including immune and neuronal signaling genes. The miR-SNP analysis implicated dysregulation of miRNA binding to target genes in five pathways, mainly involved in immune signaling. INTERPRETATION: Evidence from GWAS suggests that miRNAs play a role in ped-MS pathogenesis by affecting immune signaling and other pathways. Candidate biomarker miRNA-target gene pairs should be further studied for diagnostic, prognostic, and/or therapeutic utility.

A specific amino acid motif of HLA-DRB1 mediates risk and interacts with smoking history in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease in which genetic risk has been mapped to HLA, but precise allelic associations have been difficult to infer due to limitations in genotyping methodology. Mapping PD risk at highest possible resolution, we performed sequencing of 11 HLA genes in 1,597 PD cases and 1,606 controls. We found that susceptibility to PD can be explained by a specific combination of amino acids at positions 70-74 on the HLA-DRB1 molecule. Previously identified as the primary risk factor in rheumatoid arthritis and referred to as the "shared epitope" (SE), the residues Q/R-K/R-R-A-A at positions 70-74 in combination with valine at position 11 (11-V) is highly protective in PD, while risk is attributable to the identical epitope in the absence of 11-V. Notably, these effects are modified by history of cigarette smoking, with a strong protective effect mediated by a positive history of smoking in combination with the SE and 11-V (P = 10-4; odds ratio, 0.51; 95% confidence interval, 0.36-0.72) and risk attributable to never smoking in combination with the SE without 11-V (P = 0.01; odds ratio, 1.51; 95% confidence interval, 1.08-2.12). The association of specific combinations of amino acids that participate in critical peptide-binding pockets of the HLA class II molecule implicates antigen presentation in PD pathogenesis and provides further support for genetic control of neuroinflammation in disease. The interaction of HLA-DRB1 with smoking history in disease predisposition, along with predicted patterns of peptide binding to HLA, provide a molecular model that explains the unique epidemiology of smoking in PD.

Genome sequencing uncovers phenocopies in primary progressive multiple sclerosis.

OBJECTIVE: Primary progressive multiple sclerosis (PPMS) causes accumulation of neurological disability from disease onset without clinical attacks typical of relapsing multiple sclerosis (RMS). However, whether genetic variation influences the disease course remains unclear. We aimed to determine whether mutations causative of neurological disorders that share features with multiple sclerosis (MS) contribute to risk for developing PPMS. METHODS: We examined whole-genome sequencing (WGS) data from 38 PPMS and 81 healthy subjects of European ancestry. We selected pathogenic variants exclusively found in PPMS patients that cause monogenic neurological disorders and performed two rounds of replication genotyping in 746 PPMS, 3,049 RMS, and 1,000 healthy subjects. To refine our findings, we examined the burden of rare, potentially pathogenic mutations in 41 genes that cause hereditary spastic paraplegias (HSPs) in PPMS (n = 314), secondary progressive multiple sclerosis (SPMS; n = 587), RMS (n = 2,248), and healthy subjects (n = 987) genotyped using the MS replication chip. RESULTS: WGS and replication studies identified three pathogenic variants in PPMS patients that cause neurological disorders sharing features with MS: KIF5A p.Ala361Val in spastic paraplegia 10; MLC1 p.Pro92Ser in megalencephalic leukodystrophy with subcortical cysts, and REEP1 c.606 + 43G>T in Spastic Paraplegia 31. Moreover, we detected a significant enrichment of HSP-related mutations in PPMS patients compared to controls (risk ratio [RR] = 1.95; 95% confidence interval [CI], 1.27-2.98; p = 0.002), as well as in SPMS patients compared to controls (RR = 1.57; 95% CI, 1.18-2.10; p = 0.002). Importantly, this enrichment was not detected in RMS. INTERPRETATION: This study provides evidence to support the hypothesis that rare Mendelian genetic variants contribute to the risk for developing progressive forms of MS. Ann Neurol 2018;83:51-63.

Prognostic biomarkers of IFNb therapy in multiple sclerosis patients.

BACKGROUND: Interferon beta (IFNb) reduces relapse frequency and disability progression in patients with multiple sclerosis (MS). OBJECTIVES: Early identification of prognostic biomarkers of IFNb-treated patients will allow more effective management of MS. METHODS: The IMPROVE study evaluated subcutaneous IFNb versus placebo in 180 patients with relapsing-remitting MS. Magnetic resonance imaging scans, clinical assessments, and blood samples were obtained at baseline and every 4 weeks from every participant. Thirty-nine biomarkers (32 transcripts; seven proteins) were studied in 155 patients from IMPROVE. Therapeutic response was defined by absence of new combined unique lesions, relapses, and sustained increase in Expanded Disability Status Scale over 1 year. A machine learning approach was used to examine the association between biomarker expression and treatment response. RESULTS: While baseline levels of individual genes were relatively poor predictors, combinations of three genes were able to identify subjects with sub-optimal therapeutic responses. The triplet CASP2/IRF4/IRF6, previously identified in an independent dataset, was tested among other combinations. This triplet showed acceptable predictive accuracy (0.68) and specificity (0.88), but had relatively low sensitivity (0.22) resulting in an area under the curve (AUC) of 0.63. Other combinations of biomarkers resulted in AUC of up to 0.80 (e.g. CASP2/IL10/IL12Rb1). CONCLUSIONS: Baseline expression, or induction ratios, of specific gene combinations correlate with future therapeutic response to IFNb, and have the potential to be prognostically useful.

Evaluating the genomic and sequence integrity of human ES cell lines; comparison to normal genomes.

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count, and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation, using both microarray and sequence-based analyses, provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines, the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly, genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes, such as tumor suppressors and genetic disease genes, do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens, such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents.

Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis.

Monozygotic or 'identical' twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), an autoimmune demyelinating disease and common cause of neurodegeneration and disability in young adults, disease discordance in monozygotic twins has been interpreted to indicate environmental importance in its pathogenesis. However, genetic and epigenetic differences between monozygotic twins have been described, challenging the accepted experimental model in disambiguating the effects of nature and nurture. Here we report the genome sequences of one MS-discordant monozygotic twin pair, and messenger RNA transcriptome and epigenome sequences of CD4(+) lymphocytes from three MS-discordant, monozygotic twin pairs. No reproducible differences were detected between co-twins among approximately 3.6 million single nucleotide polymorphisms (SNPs) or approximately 0.2 million insertion-deletion polymorphisms. Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and insertion-deletion genotypes, or the expression of approximately 19,000 genes in CD4(+) T cells. Only 2 to 176 differences in the methylation of approximately 2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to approximately 800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or between normal and cancerous tissues. In the first systematic effort to estimate sequence variation among monozygotic co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first, to our knowledge, female, twin and autoimmune disease individual genome sequences reported.

Abrogation of T cell quiescence characterizes patients at high risk for multiple sclerosis after the initial neurological event.

Clinically isolated syndrome (CIS) refers to the earliest clinical manifestation of multiple sclerosis (MS). Currently there are no prognostic biological markers that accurately predict conversion of CIS to clinically definite MS (CDMS). Furthermore, the earliest molecular events in MS are still unknown. We used microarrays to study gene expression in naïve CD4(+) T cells from 37 CIS patients at time of diagnosis and after 1 year. Supervised machine-learning methods were used to build predictive models of disease conversion. We identified 975 genes whose expression segregated CIS patients into four distinct subgroups. A subset of 108 genes further discriminated patients in one of these (group 1) from other CIS patients. Remarkably, 92% of patients in group 1 converted to CDMS within 9 months. Consistent down-regulation of TOB1, a critical regulator of cell proliferation, was characteristic of group 1 patients. Decreased TOB1 expression at the RNA and protein levels also was confirmed in experimental autoimmune encephalomyelitis. Finally, a genetic association was observed between TOB1 variation and MS progression in an independent cohort. These results indicate that CIS patients at high risk of conversion have impaired regulation of T cell quiescence, possibly resulting in earlier activation of pathogenic CD4(+) cells.

Interleukin 7 receptor alpha chain (IL7R) shows allelic and functional association with multiple sclerosis.

Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7R) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 x 10(-7)). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

Clustering of autoimmune diseases in families with a high-risk for multiple sclerosis: a descriptive study.

BACKGROUND: Autoimmune mechanisms are thought to have a major role in the pathogenesis of multiple sclerosis. We aimed to identify coexisting autoimmune phenotypes in patients with multiple sclerosis from families with several members with the disease and in their first-degree relatives. METHODS: A total of 176 families (386 individuals and 1107 first-degree relatives) were characterised for a history of other autoimmune disorders. Family-based or case-control analyses were done to assess the association of cytotoxic T-lymphocyte-antigen 4 (CTLA4) and protein tyrosine phosphatase (PTPN22) variants with susceptibility to multiple sclerosis. FINDINGS: 46 (26%) index cases reported at least one coexisting autoimmune disorder. The most common were Hashimoto thyroiditis (10%), psoriasis (6%), inflammatory bowel disease (3%), and rheumatoid arthritis (2%). 112 (64%) families with a history of multiple sclerosis reported autoimmune disorders (excluding multiple sclerosis) in one or more first-degree relatives, whereas 64 (36%) families reported no history of autoimmunity. Similar to index cases, Hashimoto thyroiditis, psoriasis, and inflammatory bowel disease were also the most common disorders occurring in family members. A common variant within CTLA4 was strongly associated with multiple sclerosis in families who had other autoimmune diseases (p=0.009) but not in families without a history of other autoimmune disorders (p=0.90). INTERPRETATION: The presence of various immune disorders in families with several members with multiple sclerosis suggests that the disease might arise on a background of a generalised susceptibility to autoimmunity. This distinct multiple-sclerosis phenotype, defined by its association with other autoimmune diseases, segregates with specific genotypes that could underlie the common susceptibility.