The shared ancestry between the C9orf72 hexanucleotide repeat expansion and intermediate-length alleles using haplotype sharing trees and HAPTK.

The C9orf72 hexanucleotide repeat expansion (HRE) is a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The inheritance is autosomal dominant, but a high proportion of subjects with the mutation are simplex cases. One possible explanation is de novo expansions of unstable intermediate-length alleles (IAs). Using haplotype sharing trees (HSTs) with the haplotype analysis tool kit (HAPTK), we derived majority-based ancestral haplotypes of HRE samples and discovered that IAs containing ≥18-20 repeats share large haplotypes in common with the HRE. Using HSTs of HRE and IA samples, we demonstrate that the longer IA haplotypes are largely indistinguishable from HRE haplotypes and that several ≥18-20 IA haplotypes share over 5 Mb (>600 markers) haplotypes in common with the HRE haplotypes. These analysis tools allow physical understanding of the haplotype blocks shared with the majority-based ancestral haplotype. Our results demonstrate that the haplotypes with longer IAs belong to the same pool of haplotypes as the HRE and suggest that longer IAs represent potential premutation alleles.

Limbic-predominant age-related TDP-43 encephalopathy in the oldest old: a population-based study.

Population-based cohort studies are essential for understanding the pathological basis of dementia in older populations. Previous studies have shown that limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC) increases with age, but there have been only a few studies, which have investigated this entity in a population-based setting. Here we studied the frequency of LATE-NC and its associations with other brain pathologies and cognition in a population aged ≥ 85 years. The population-based Vantaa 85+ study cohort includes all 601 individuals aged ≥ 85 years who were living in Vantaa, Finland in 1991. A neuropathological examination was performed on 304 subjects (50.5%) and LATE-NC staging was possible in 295 of those. Dementia status and Mini-Mental State Examination (MMSE) scores were defined in the baseline study and 3 follow-ups (1994-99). The LATE-NC stages were determined based on TDP-43 immunohistochemistry, according to recently updated recommendations. Arteriolosclerosis was digitally assessed by calculating the average sclerotic index of five random small arterioles in amygdala and hippocampal regions, and frontal white matter. The association of LATE-NC with arteriolosclerosis and previously determined neuropathological variables including Alzheimer's disease neuropathological change (ADNC), Lewy-related pathology (LRP), hippocampal sclerosis (HS), and cerebral amyloid angiopathy (CAA), and cognitive variables were analysed by Fisher's exact test, linear and logistic regression (univariate and multivariate) models. LATE-NC was found in 189 of 295 subjects (64.1%). Stage 2 was the most common (28.5%) and stage 3 the second most common (12.9%), whereas stages 1a, 1b and 1c were less common (9.5%, 5.1% and 8.1%, respectively). Stages 1a (P < 0.01), 2 (P < 0.001) and 3 (P < 0.001) were significantly associated with dementia and lower MMSE scores. LATE-NC was associated with ADNC (P < 0.001), HS (P < 0.001), diffuse neocortical LRP (P < 0.002), and arteriolosclerosis in amygdala (P < 0.02). In most cases LATE-NC occurred in combination alongside other neuropathological changes. There were only six subjects with dementia who had LATE-NC without high levels of ADNC or LRP (2% of the cohort, 3% of the cases with dementia), and five of these had HS. In all multivariate models, LATE-NC was among the strongest independent predictors of dementia. When LATE-NC and ADNC were assessed in a multivariate model without other dementia-associated pathologies, the attributable risk was higher for LATE-NC than ADNC (24.2% vs. 18.6%). This population-based study provides evidence that LATE-NC is very common and one of the most significant determinants of dementia in the general late-life aged population.

Identification of genetic risk loci and prioritization of genes and pathways for myasthenia gravis: a genome-wide association study.

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

High Epstein-Barr virus capsid antigen IgG level associates with the carriership of CD8+ T cell somatic mutations in the STAT3 SH2 domain.

High carrier prevalence of STAT3 SH2 domain somatic mutations was recently discovered in CD8+ T cells. We found these low-allele-fraction clones in 26% of donors, without difference between multiple sclerosis (MS) patients and controls. Here we tested whether anti-viral antibodies associate with the carriership of these mutant clones. We compared antibody responses against common viruses in mutation carriers vs. non-carriers. Plasma samples of 152 donors (92 MS patients, 60 controls) were analyzed for antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6A and parvovirus B19. The mutation carrier status associated with EBV VCA IgG level (p = 0.005) and remained significant after logistic regression (p = 0.036). This association was contributed similarly by MS patients and controls. These results suggest that EBV contributes to the generation or growth of these clones. The pathogenic role of the STAT3 mutant clones in MS is presently unclear, but their detailed characterization warrants further study.

Cultured lymphocytes' mitochondrial genome integrity is not altered by cladribine.

Cladribine tablets are a treatment for multiple sclerosis with effects on lymphocytes, yet its mode of action has not been fully established. Here, we analyzed the effects of cladribine on mitochondrial DNA integrity in lymphocytes. We treated cultured human T-cell lines (CCRF-CEM and Jurkat) with varying concentrations of cladribine to mimic the slow cell depletion observed in treated patients. The CCRF-CEM was more susceptible to cladribine than Jurkat cells. In both cells, mitochondrial protein synthesis, mitochondrial DNA copy number, and mitochondrial cytochrome-c oxidase-I mRNA mutagenesis was not affected by cladribine, while caspase-3 cleavage was detected in Jurkat cells at 100 nM concentration. Cladribine treatment at concentrations up to 10 nM in CCRF-CEM and 100 nM in Jurkat cells did not induce significant increase in mitochondrial DNA mutations. Peripheral blood mononuclear cells from eight multiple sclerosis patients and four controls were cultured with or without an effective dose of cladribine (5 nM). However, we did not find any differences in mitochondrial DNA somatic mutations in lymphocyte subpopulations (CD4+, CD8+, and CD19+) between treated versus nontreated cells. The overall mutation rate was similar in patients and controls. When different lymphocyte subpopulations were compared, greater mitochondrial DNA mutation levels were detected in CD8+ (P = 0.014) and CD4+ (P = 0.038) as compared to CD19+ cells, these differences were independent of cladribine treatment. We conclude that T cells have more detectable mitochondrial DNA mutations than B cells, and cladribine has no detectable mutagenic effect on lymphocyte mitochondrial genome nor does it impair mitochondrial function in human T-cell lines.

Incidental demyelination in magnetic resonance imaging and 10-year risk of multiple sclerosis: A data lake cohort study.

BACKGROUND AND PURPOSE: There is an absence of data from large population-based cohort studies on the incidence of radiologically isolated syndrome (RIS). The incidence of RIS and the subsequent risk for multiple sclerosis (MS) were investigated. METHODS: A population-based, retrospective cohort study was conducted using a data-lake-based analysis of digitalized radiology reports. All brain and spinal cord magnetic resonance imaging (MRI) in people aged 16-70 during the years 2005-2010 (n = 102,224) were screened using optimized search terms to detect cases with RIS. The subjects with RIS were followed up until January 2022. RESULTS: The cumulative incidence of RIS was 0.03% when all MRI modalities were included and 0.06% when only brain MRI was included according to MAGNIMS 2018 recommendation criteria. With the Okuda 2009 criteria, the respective figures were 0.03% and 0.05% (86% concordance). The overall risk for MS after RIS was similar, 32% by using the MAGNIMS and 32% by using the Okuda definition of RIS. Individuals aged <35.5 years exhibited the most significant predisposition to MS (80%), whilst those >35.5 years had less than 10% risk of MS. MS diagnosed after RIS constituted 0.8% of the incident MS cases in the population during 2005-2010. CONCLUSIONS: A population-wide context was provided for the incidence of RIS and its relationship to MS. MAGNIMS recommendations were only slightly more sensitive to detect RIS compared to the Okuda criteria. RIS has a subtle effect on the overall incidence of MS, yet the risk for MS in individuals under the age of 35.5 years is substantial.

Primary age-related tauopathy in a Finnish population-based study of the oldest old (Vantaa 85+).

AIMS: Few studies have investigated primary age-related tauopathy (PART) in a population-based setting. Here, we assessed its prevalence, genetic background, comorbidities and features of cognitive decline in an unselected elderly population. METHODS: The population-based Vantaa 85+ study includes all 601 inhabitants of Vantaa aged ≥ 85 years in 1991. Neuropathological assessment was possible in 301. Dementia (DSM IIIR criteria) and Mini-Mental State Examination (MMSE) scores were assessed at the baseline of the study and follow-ups. PART subjects were identified according to the criteria by Crary et al and were compared with subjects with mild and severe Alzheimer's disease (AD) neuropathological changes. The effects of other neuropathologies were taken into account using multivariate and sensitivity assays. Genetic analyses included APOE genotypes and 29 polymorphisms of the MAPT 3' untranslated region (3'UTR region). RESULTS: The frequency of PART was 20% (n = 61/301, definite PART 5%). When PART subjects were compared with those with severe AD pathology, dementia was less common, its age at onset was higher and duration shorter. No such differences were seen when compared with those with milder AD pathology. However, both AD groups showed a steeper decline in MMSE scores in follow-ups compared with PART. APOE ε4 frequency was lower, and APOE ε2 frequency higher in the PART group compared with each AD group. The detected nominally significant associations between PART and two MAPT 3'UTR polymorphisms and haplotypes did not survive Bonferroni correction. CONCLUSIONS: PART is common among very elderly. PART subjects differ from individuals with AD-type changes in the pattern of cognitive decline, associated genetic and neuropathological features.

Investigation of Autosomal Genetic Sex Differences in Parkinson's Disease.

OBJECTIVE: Parkinson's disease (PD) is a complex neurodegenerative disorder. Men are on average ~ 1.5 times more likely to develop PD compared to women with European ancestry. Over the years, genomewide association studies (GWAS) have identified numerous genetic risk factors for PD, however, it is unclear whether genetics contribute to disease etiology in a sex-specific manner. METHODS: In an effort to study sex-specific genetic factors associated with PD, we explored 2 large genetic datasets from the International Parkinson's Disease Genomics Consortium and the UK Biobank consisting of 13,020 male PD cases, 7,936 paternal proxy cases, 89,660 male controls, 7,947 female PD cases, 5,473 maternal proxy cases, and 90,662 female controls. We performed GWAS meta-analyses to identify distinct patterns of genetic risk contributing to disease in male versus female PD cases. RESULTS: In total, 19 genomewide significant regions were identified and no sex-specific effects were observed. A high genetic correlation between the male and female PD GWAS were identified (rg = 0.877) and heritability estimates were identical between male and female PD cases (~ 20%). INTERPRETATION: We did not detect any significant genetic differences between male or female PD cases. Our study does not support the notion that common genetic variation on the autosomes could explain the difference in prevalence of PD between males and females cases at least when considering the current sample size under study. Further studies are warranted to investigate the genetic architecture of PD explained by X and Y chromosomes and further evaluate environmental effects that could potentially contribute to PD etiology in male versus female patients. ANN NEUROL 2021;90:41-48.

Genetic modifiers of risk and age at onset in GBA associated Parkinson's disease and Lewy body dementia.

Parkinson's disease is a genetically complex disorder. Multiple genes have been shown to contribute to the risk of Parkinson's disease, and currently 90 independent risk variants have been identified by genome-wide association studies. Thus far, a number of genes (including SNCA, LRRK2, and GBA) have been shown to contain variability across a spectrum of frequency and effect, from rare, highly penetrant variants to common risk alleles with small effect sizes. Variants in GBA, encoding the enzyme glucocerebrosidase, are associated with Lewy body diseases such as Parkinson's disease and Lewy body dementia. These variants, which reduce or abolish enzymatic activity, confer a spectrum of disease risk, from 1.4- to >10-fold. An outstanding question in the field is what other genetic factors that influence GBA-associated risk for disease, and whether these overlap with known Parkinson's disease risk variants. Using multiple, large case-control datasets, totalling 217 165 individuals (22 757 Parkinson's disease cases, 13 431 Parkinson's disease proxy cases, 622 Lewy body dementia cases and 180 355 controls), we identified 1691 Parkinson's disease cases, 81 Lewy body dementia cases, 711 proxy cases and 7624 controls with a GBA variant (p.E326K, p.T369M or p.N370S). We performed a genome-wide association study and analysed the most recent Parkinson's disease-associated genetic risk score to detect genetic influences on GBA risk and age at onset. We attempted to replicate our findings in two independent datasets, including the personal genetics company 23andMe, Inc. and whole-genome sequencing data. Our analysis showed that the overall Parkinson's disease genetic risk score modifies risk for disease and decreases age at onset in carriers of GBA variants. Notably, this effect was consistent across all tested GBA risk variants. Dissecting this signal demonstrated that variants in close proximity to SNCA and CTSB (encoding cathepsin B) are the most significant contributors. Risk variants in the CTSB locus were identified to decrease mRNA expression of CTSB. Additional analyses suggest a possible genetic interaction between GBA and CTSB and GBA p.N370S induced pluripotent cell-derived neurons were shown to have decreased cathepsin B expression compared to controls. These data provide a genetic basis for modification of GBA-associated Parkinson's disease risk and age at onset, although the total contribution of common genetics variants is not large. We further demonstrate that common variability at genes implicated in lysosomal function exerts the largest effect on GBA associated risk for disease. Further, these results have implications for selection of GBA carriers for therapeutic interventions.

Shared polygenic risk and causal inferences in amyotrophic lateral sclerosis.

OBJECTIVE: To identify shared polygenic risk and causal associations in amyotrophic lateral sclerosis (ALS). METHODS: Linkage disequilibrium score regression and Mendelian randomization were applied in a large-scale, data-driven manner to explore genetic correlations and causal relationships between >700 phenotypic traits and ALS. Exposures consisted of publicly available genome-wide association studies (GWASes) summary statistics from MR Base and LD-hub. The outcome data came from the recently published ALS GWAS involving 20,806 cases and 59,804 controls. Multivariate analyses, genetic risk profiling, and Bayesian colocalization analyses were also performed. RESULTS: We have shown, by linkage disequilibrium score regression, that ALS shares polygenic risk genetic factors with a number of traits and conditions, including positive correlations with smoking status and moderate levels of physical activity, and negative correlations with higher cognitive performance, higher educational attainment, and light levels of physical activity. Using Mendelian randomization, we found evidence that hyperlipidemia is a causal risk factor for ALS and localized putative functional signals within loci of interest. INTERPRETATION: Here, we have developed a public resource (https://lng-nia.shinyapps.io/mrshiny) which we hope will become a valuable tool for the ALS community, and that will be expanded and updated as new data become available. Shared polygenic risk exists between ALS and educational attainment, physical activity, smoking, and tenseness/restlessness. We also found evidence that elevated low-desnity lipoprotein cholesterol is a causal risk factor for ALS. Future randomized controlled trials should be considered as a proof of causality. Ann Neurol 2019;85:470-481.

Lewy-related pathology exhibits two anatomically and genetically distinct progression patterns: a population-based study of Finns aged 85.

According to a generally accepted concept Lewy-related pathology (LRP) follows hierarchical caudo-rostral progression. LRP is also frequently present concomitantly with Alzheimer's disease (AD), and it has been hypothesized that AD-associated LRP forms a distinct type of α-synucleinopathy, where LRP originates in the amygdala. The frequency of distinct forms of LRP progression types has not been studied in a population-based setting. We investigated the distribution and progression of LRP and its relation to AD pathology and apolipoprotein (APOE) ε4 in a population-based sample of Finns aged over 85 years (N = 304). Samples from spinal cord to neocortical areas representing 11 anatomical sites without any hierarchical selection were analyzed immunohistochemically (α-synuclein antibody clone 5G4). LRP was present in 124 individuals (41%) and according to DLB Consortium guidelines 19 of them were categorized as brainstem, 10 amygdala-predominant, 41 limbic, and 43 diffuse neocortical type, whereas 11 could not be classified. To determine the LRP progression patterns, a systematic anatomical scoring was carried out by taking into account the densities of the semiquantitative LRP scores in each anatomic site. With this scoring 123 (99%) subjects could be classified into two progression pattern types: 67% showed caudo-rostral and 32% amygdala-based progression. The unsupervised statistical K-means cluster analysis was used as a supplementary test and supported the presence of two progression patterns and had a 90% overall concordance with the systematic anatomical scoring method. Severe Braak NFT stage, high CERAD score and APOE ε4 were significantly (all p < 0.00001) associated with amygdala-based, but not with caudo-rostral progression type (all p > 0.2). This population-based study demonstrates two distinct common LRP progression patterns in the very elderly population. The amygdala-based pattern was associated with APOE ε4 and AD pathology. The results confirm the previous progression hypotheses but also widen the concept of the AD-associated LRP.

Multiple sclerosis in Finland 2018-Data from the national register.

OBJECTIVES: Finland is a high-risk multiple sclerosis (MS) region, but a national MS register has not existed until 2014. In this paper, we present the Finnish MS register variables and data collected by 31 December 2018. MATERIALS AND METHODS: Numbers and data counts of MS patients in the register (ICD-10 code G35) are presented. The disease types and proportion of patients receiving disease-modifying treatments (DMTs) were analysed in five hospital districts with most complete data sets. MS prevalence in Finland was estimated using administrative hospital discharge data as an additional resource. RESULTS: There were a total of 8722 MS patients in the Finnish MS register by 31 December 2018 (71.5% females). Mean age at MS diagnosis was 38.7 years and peak prevalence was at age 50-54 years. Disease course was relapsing remitting (RRMS) in 66.7%, secondary progressive (SPMS) in 13.5%, and primary progressive (PPMS) in 7.9% of the 5365 MS patients in the selected districts with most complete data. A total of 66.0% of RRMS patients, 19.6% of SPMS patients and 9.9% of PPMS patients were receiving DMTs. By combining MS register data with databases of those hospitals that had not joined the register, the nationwide prevalence estimate was between 10 and 11 thousand patients (corresponding to crude prevalence 180-200/100 000). CONCLUSIONS: The Finnish MS register is currently used in 15/21 Finnish hospital districts. By register integration into the electronic patient files, the coverage of the register has increased to approximately 80% of the estimated Finnish MS population.

A novel class of somatic mutations in blood detected preferentially in CD8+ cells.

Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4+, CD8+, CD19+ and CD4-/CD8-/CD19- cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune-related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%-4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8+ cells (85% of mutations). In follow-up after a median time of 2.3years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies.

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100ß, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

Cerebrospinal fluid biomarkers for Alzheimer's disease in patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis with the C9ORF72 repeat expansion.

BACKGROUND: The C9ORF72 expansion is one of the most common causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The C9ORF72 expansion is associated with TDP-43 and p62 neuropathology, and amyloid plaques and neurofibrillary tangles are not common in patients with the C9ORF72 expansion. Therefore, we hypothesized that cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease [AD; Aß1-42, total tau (T-tau) and phospho-tau] are normal in these patients. METHODS: The CSF Aß1-42, T-tau and phospho-tau levels were measured in 40 Finnish patients with the C9ORF72 expansion (29 FTLD, 10 ALS and 1 FTLD-ALS) using ELISA. RESULTS: A decreased Aß1-42 level was found in 25% of cases, while there were only single cases with changes in the t-Tau or phospho-tau level. The patients with abnormal biomarkers fulfilled the clinical criteria of the behavioral variant frontotemporal dementia and expressed no clinical signs of AD. CONCLUSIONS: In clinical diagnostics, a decreased CSF Aß1-42 level does not exclude the C9ORF72 expansion associated with FTLD.

A predominant role for the HLA class II region in the association of the MHC region with multiple sclerosis.

Genetic susceptibility to multiple sclerosis is associated with genes of the major histocompatibility complex (MHC), particularly HLA-DRB1 and HLA-DQB1 (ref. 1). Both locus and allelic heterogeneity have been reported in this genomic region. To clarify whether HLA-DRB1 itself, nearby genes in the region encoding the MHC or combinations of these loci underlie susceptibility to multiple sclerosis, we genotyped 1,185 Canadian and Finnish families with multiple sclerosis (n = 4,203 individuals) with a high-density SNP panel spanning the genes encoding the MHC and flanking genomic regions. Strong associations in Canadian and Finnish samples were observed with blocks in the HLA class II genomic region (P < 4.9 x 10(-13) and P < 2.0 x 10(-16), respectively), but the strongest association was with HLA-DRB1 (P < 4.4 x 10(-17)). Conditioning on either HLA-DRB1 or the most significant HLA class II haplotype block found no additional block or SNP association independent of the HLA class II genomic region. This study therefore indicates that MHC-associated susceptibility to multiple sclerosis is determined by HLA class II alleles, their interactions and closely neighboring variants.