Cholesterol and matrisome pathways dysregulated in astrocytes and microglia.

The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.

Exome sequencing of individuals with Huntington's disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset.

The age at onset of motor symptoms in Huntington's disease (HD) is driven by HTT CAG repeat length but modified by other genes. In this study, we used exome sequencing of 683 patients with HD with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We discovered damaging coding variants in candidate modifier genes identified in previous genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier-onset HD. Nuclease activities of purified variants in vitro correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion similar to those observed with complete FAN1 knockout. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

Multi-omics integration analysis identifies novel genes for alcoholism with potential overlap with neurodegenerative diseases.

Identification of causal variants and genes underlying genome-wide association study (GWAS) loci is essential to understand the biology of alcohol use disorder (AUD) and drinks per week (DPW). Multi-omics integration approaches have shown potential for fine mapping complex loci to obtain biological insights to disease mechanisms. In this study, we use multi-omics approaches, to fine-map AUD and DPW associations at single SNP resolution to demonstrate that rs56030824 on chromosome 11 significantly reduces SPI1 mRNA expression in myeloid cells and lowers risk for AUD and DPW. Our analysis also identifies MAPT as a candidate causal gene specifically associated with DPW. Genes prioritized in this study show overlap with causal genes associated with neurodegenerative disorders. Multi-omics integration analyses highlight, genetic similarities and differences between alcohol intake and disordered drinking, suggesting molecular heterogeneity that might inform future targeted functional and cross-species studies.

Association Analysis of Chromosome X to Identify Genetic Modifiers of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is caused by an expanded (>35) CAG trinucleotide repeat in huntingtin (HTT). Age-at-onset of motor symptoms is inversely correlated with the size of the inherited CAG repeat, which expands further in brain regions due to somatic repeat instability. Our recent genetic investigation focusing on autosomal SNPs revealed that age-at-onset is also influenced by genetic variation at many loci, the majority of which encode genes involved in DNA maintenance/repair processes and repeat instability. OBJECTIVE: We performed a complementary association analysis to determine whether variants in the X chromosome modify HD. METHODS: We imputed SNPs on chromosome X for ∼9,000 HD subjects of European ancestry and performed an X chromosome-wide association study (XWAS) to test for association with age-at-onset corrected for inherited CAG repeat length. RESULTS: In a mixed effects model XWAS analysis of all subjects (males and females), assuming random X-inactivation in females, no genome-wide significant onset modification signal was found. However, suggestive significant association signals were detected at Xq12 (top SNP, rs59098970; p-value, 1.4E-6), near moesin (MSN), in a region devoid of DNA maintenance genes. Additional suggestive signals not involving DNA repair genes were observed in male- and female-only analyses at other locations. CONCLUSION: Although not genome-wide significant, potentially due to small effect size compared to the power of the current study, our data leave open the possibility of modification of HD by a non-DNA repair process. Our XWAS results are publicly available at the updated GEM EURO 9K website hosted at https://www.hdinhd.org/ for browsing, pathway analysis, and data download.

Polygenic contributions to alcohol use and alcohol use disorders across population-based and clinically ascertained samples.

BACKGROUND: Studies suggest that alcohol consumption and alcohol use disorders have distinct genetic backgrounds. METHODS: We examined whether polygenic risk scores (PRS) for consumption and problem subscales of the Alcohol Use Disorders Identification Test (AUDIT-C, AUDIT-P) in the UK Biobank (UKB; N = 121 630) correlate with alcohol outcomes in four independent samples: an ascertained cohort, the Collaborative Study on the Genetics of Alcoholism (COGA; N = 6850), and population-based cohorts: Avon Longitudinal Study of Parents and Children (ALSPAC; N = 5911), Generation Scotland (GS; N = 17 461), and an independent subset of UKB (N = 245 947). Regression models and survival analyses tested whether the PRS were associated with the alcohol-related outcomes. RESULTS: In COGA, AUDIT-P PRS was associated with alcohol dependence, AUD symptom count, maximum drinks (R2 = 0.47-0.68%, p = 2.0 × 10-8-1.0 × 10-10), and increased likelihood of onset of alcohol dependence (hazard ratio = 1.15, p = 4.7 × 10-8); AUDIT-C PRS was not an independent predictor of any phenotype. In ALSPAC, the AUDIT-C PRS was associated with alcohol dependence (R2 = 0.96%, p = 4.8 × 10-6). In GS, AUDIT-C PRS was a better predictor of weekly alcohol use (R2 = 0.27%, p = 5.5 × 10-11), while AUDIT-P PRS was more associated with problem drinking (R2 = 0.40%, p = 9.0 × 10-7). Lastly, AUDIT-P PRS was associated with ICD-based alcohol-related disorders in the UKB subset (R2 = 0.18%, p < 2.0 × 10-16). CONCLUSIONS: AUDIT-P PRS was associated with a range of alcohol-related phenotypes across population-based and ascertained cohorts, while AUDIT-C PRS showed less utility in the ascertained cohort. We show that AUDIT-P is genetically correlated with both use and misuse and demonstrate the influence of ascertainment schemes on PRS analyses.

Dysregulation of mitochondrial and proteolysosomal genes in Parkinson's disease myeloid cells.

An increasing number of identified Parkinson's disease (PD) risk loci contain genes highly expressed in innate immune cells, yet their role in pathology is not understood. We hypothesize that PD susceptibility genes modulate disease risk by influencing gene expression within immune cells. To address this, we have generated transcriptomic profiles of monocytes from 230 individuals with sporadic PD and healthy subjects. We observed a dysregulation of mitochondrial and proteasomal pathways. We also generated transcriptomic profiles of primary microglia from brains of 55 subjects and observed discordant transcriptomic signatures of mitochondrial genes in PD monocytes and microglia. We further identified 17 PD susceptibility genes whose expression, relative to each risk allele, is altered in monocytes. These findings reveal widespread transcriptomic alterations in PD monocytes, with some being distinct from microglia, and facilitate efforts to understand the roles of myeloid cells in PD as well as the development of biomarkers.

Genetic and Functional Analyses Point to FAN1 as the Source of Multiple Huntington Disease Modifier Effects.

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.

Genetic Risk Underlying Psychiatric and Cognitive Symptoms in Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene. It is diagnosed following a standardized examination of motor control and often presents with cognitive decline and psychiatric symptoms. Recent studies have detected genetic loci modifying the age at onset of motor symptoms in HD, but genetic factors influencing cognitive and psychiatric presentations are unknown. METHODS: We tested the hypothesis that psychiatric and cognitive symptoms in HD are influenced by the same common genetic variation as in the general population by 1) constructing polygenic risk scores from large genome-wide association studies of psychiatric and neurodegenerative disorders and of intelligence and 2) testing for correlation with the presence of psychiatric and cognitive symptoms in a large sample (n = 5160) of patients with HD. RESULTS: Polygenic risk score for major depression was associated specifically with increased risk of depression in HD, as was schizophrenia risk score with psychosis and irritability. Cognitive impairment and apathy were associated with reduced polygenic risk score for intelligence. CONCLUSIONS: Polygenic risk scores for psychiatric disorders, particularly depression and schizophrenia, are associated with increased risk of the corresponding psychiatric symptoms in HD, suggesting a common genetic liability. However, the genetic liability to cognitive impairment and apathy appears to be distinct from other psychiatric symptoms in HD. No associations were observed between HD symptoms and risk scores for other neurodegenerative disorders. These data provide a rationale for treatments effective in depression and schizophrenia to be used to treat depression and psychotic symptoms in HD.

Sex differences in the genetic predictors of Alzheimer's pathology.

Autopsy measures of Alzheimer's disease neuropathology have been leveraged as endophenotypes in previous genome-wide association studies (GWAS). However, despite evidence of sex differences in Alzheimer's disease risk, sex-stratified models have not been incorporated into previous GWAS analyses. We looked for sex-specific genetic associations with Alzheimer's disease endophenotypes from six brain bank data repositories. The pooled dataset included 2701 males and 3275 females, the majority of whom were diagnosed with Alzheimer's disease at autopsy (70%). Sex-stratified GWAS were performed within each dataset and then meta-analysed. Loci that reached genome-wide significance (P < 5 × 10-8) in stratified models were further assessed for sex interactions. Additional analyses were performed in independent datasets leveraging cognitive, neuroimaging and CSF endophenotypes, along with age-at-onset data. Outside of the APOE region, one locus on chromosome 7 (rs34331204) showed a sex-specific association with neurofibrillary tangles among males (P = 2.5 × 10-8) but not females (P = 0.85, sex-interaction P = 2.9 × 10-4). In follow-up analyses, rs34331204 was also associated with hippocampal volume, executive function, and age-at-onset only among males. These results implicate a novel locus that confers male-specific protection from tau pathology and highlight the value of assessing genetic associations in a sex-specific manner.

Genetic Modification of Huntington Disease Acts Early in the Prediagnosis Phase.

Age at onset of Huntington disease, an inherited neurodegenerative disorder, is influenced by the size of the disease-causing CAG trinucleotide repeat expansion in HTT and by genetic modifier loci on chromosomes 8 and 15. Stratifying by modifier genotype, we have examined putamen volume, total motor score (TMS), and symbol digit modalities test (SDMT) scores, both at study entry and longitudinally, in normal controls and CAG-expansion carriers who were enrolled prior to the emergence of manifest HD in the PREDICT-HD study. The modifiers, which included onset-hastening and onset-delaying alleles on chromosome 15 and an onset-hastening allele on chromosome 8, revealed no major effect in controls but distinct patterns of modification in prediagnosis HD subjects. Putamen volume at study entry showed evidence of reciprocal modification by the chromosome 15 alleles, but the rate of loss of putamen volume was modified only by the deleterious chromosome 15 allele. By contrast, both alleles modified the rate of change of the SDMT score, but neither had an effect on the TMS. The influence of the chromosome 8 modifier was evident only in the rate of TMS increase. The data indicate that (1) modification of pathogenesis can occur early in the prediagnosis phase, (2) the modifier loci act in genetic interaction with the HD mutation rather than through independent additive effects, and (3) HD subclinical phenotypes are differentially influenced by each modifier, implying distinct effects in different cells or tissues. Together, these findings indicate the potential benefit of using genetic modifier strategies for dissecting the prediagnosis pathogenic process in HD.

Sex-specific genetic predictors of Alzheimer's disease biomarkers.

Cerebrospinal fluid (CSF) levels of amyloid-ß 42 (Aß42) and tau have been evaluated as endophenotypes in Alzheimer's disease (AD) genetic studies. Although there are sex differences in AD risk, sex differences have not been evaluated in genetic studies of AD endophenotypes. We performed sex-stratified and sex interaction genetic analyses of CSF biomarkers to identify sex-specific associations. Data came from a previous genome-wide association study (GWAS) of CSF Aß42 and tau (1527 males, 1509 females). We evaluated sex interactions at previous loci, performed sex-stratified GWAS to identify sex-specific associations, and evaluated sex interactions at sex-specific GWAS loci. We then evaluated sex-specific associations between prefrontal cortex (PFC) gene expression at relevant loci and autopsy measures of plaques and tangles using data from the Religious Orders Study and Rush Memory and Aging Project. In Aß42, we observed sex interactions at one previous and one novel locus: rs316341 within SERPINB1 (p = 0.04) and rs13115400 near LINC00290 (p = 0.002). These loci showed stronger associations among females (ß = - 0.03, p = 4.25 × 10-8; ß = 0.03, p = 3.97 × 10-8) than males (ß = - 0.02, p = 0.009; ß = 0.01, p = 0.20). Higher levels of expression of SERPINB1, SERPINB6, and SERPINB9 in PFC was associated with higher levels of amyloidosis among females (corrected p values < 0.02) but not males (p > 0.38). In total tau, we observed a sex interaction at a previous locus, rs1393060 proximal to GMNC (p = 0.004), driven by a stronger association among females (ß = 0.05, p = 4.57 × 10-10) compared to males (ß = 0.02, p = 0.03). There was also a sex-specific association between rs1393060 and tangle density at autopsy (pfemale = 0.047; pmale = 0.96), and higher levels of expression of two genes within this locus were associated with lower tangle density among females (OSTN p = 0.006; CLDN16 p = 0.002) but not males (p ≥ 0.32). Results suggest a female-specific role for SERPINB1 in amyloidosis and for OSTN and CLDN16 in tau pathology. Sex-specific genetic analyses may improve understanding of AD's genetic architecture.

Population-specific genetic modification of Huntington's disease in Venezuela.

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2-21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies.

A modifier of Huntington's disease onset at the MLH1 locus.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in HTT. Many clinical characteristics of HD such as age at motor onset are determined largely by the size of HTT CAG repeat. However, emerging evidence strongly supports a role for other genetic factors in modifying the disease pathogenesis driven by mutant huntingtin. A recent genome-wide association analysis to discover genetic modifiers of HD onset age provided initial evidence for modifier loci on chromosomes 8 and 15 and suggestive evidence for a locus on chromosome 3. Here, genotyping of candidate single nucleotide polymorphisms in a cohort of 3,314 additional HD subjects yields independent confirmation of the former two loci and moves the third to genome-wide significance at MLH1, a locus whose mouse orthologue modifies CAG length-dependent phenotypes in a Htt-knock-in mouse model of HD. Both quantitative and dichotomous association analyses implicate a functional variant on ∼32% of chromosomes with the beneficial modifier effect that delays HD motor onset by 0.7 years/allele. Genomic DNA capture and sequencing of a modifier haplotype localize the functional variation to a 78 kb region spanning the 3'end of MLH1 and the 5'end of the neighboring LRRFIP2, and marked by an isoleucine-valine missense variant in MLH1. Analysis of expression Quantitative Trait Loci (eQTLs) provides modest support for altered regulation of MLH1 and LRRFIP2, raising the possibility that the modifier affects regulation of both genes. Finally, polygenic modification score and heritability analyses suggest the existence of additional genetic modifiers, supporting expanded, comprehensive genetic analysis of larger HD datasets.

Haplotype-based stratification of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat in HTT, resulting in an extended polyglutamine tract in huntingtin. We and others have previously determined that the HD-causing expansion occurs on multiple different haplotype backbones, reflecting more than one ancestral origin of the same type of mutation. In view of the therapeutic potential of mutant allele-specific gene silencing, we have compared and integrated two major systems of HTT haplotype definition, combining data from 74 sequence variants to identify the most frequent disease-associated and control chromosome backbones and revealing that there is potential for additional resolution of HD haplotypes. We have used the large collection of 4078 heterozygous HD subjects analyzed in our recent genome-wide association study of HD age at onset to estimate the frequency of these haplotypes in European subjects, finding that common genetic variation at HTT can distinguish the normal and CAG-expanded chromosomes for more than 95% of European HD individuals. As a resource for the HD research community, we have also determined the haplotypes present in a series of publicly available HD subject-derived fibroblasts, induced pluripotent cells, and embryonic stem cells in order to facilitate efforts to develop inclusive methods of allele-specific HTT silencing applicable to most HD patients. Our data providing genetic guidance for therapeutic gene-based targeting will significantly contribute to the developments of rational treatments and implementation of precision medicine in HD.

Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9.

A comprehensive genetics-based precision medicine strategy to selectively and permanently inactivate only mutant, not normal allele, could benefit many dominantly inherited disorders. Here, we demonstrate the power of our novel strategy of inactivating the mutant allele using haplotype-specific CRISPR/Cas9 target sites in Huntington's disease (HD), a late-onset neurodegenerative disorder due to a toxic dominant gain-of-function CAG expansion mutation. Focusing on improving allele specificity, we combined extensive knowledge of huntingtin (HTT) gene haplotype structure with a novel personalized allele-selective CRISPR/Cas9 strategy based on Protospacer Adjacent Motif (PAM)-altering SNPs to target patient-specific CRISPR/Cas9 sites, aiming at the mutant HTT allele-specific inactivation for a given diplotype. As proof-of-principle, simultaneously using two CRISPR/Cas9 guide RNAs (gRNAs) that depend on PAM sites generated by SNP alleles on the mutant chromosome, we selectively excised ∼44 kb DNA spanning promoter region, transcription start site, and the CAG expansion mutation of the mutant HTT gene, resulting in complete inactivation of the mutant allele without impacting the normal allele. This excision on the disease chromosome completely prevented the generation of mutant HTT mRNA and protein, unequivocally indicating permanent mutant allele-specific inactivation of the HD mutant allele. The perfect allele selectivity with broad applicability of our strategy in disorders with diverse disease haplotypes should also support precision medicine through inactivation of many other gain-of-function mutations.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Exome sequencing identifies a novel multiple sclerosis susceptibility variant in the TYK2 gene.

OBJECTIVE: To identify rare variants contributing to multiple sclerosis (MS) susceptibility in a family we have previously reported with up to 15 individuals affected across 4 generations. METHODS: We performed exome sequencing in a subset of affected individuals to identify novel variants contributing to MS risk within this unique family. The candidate variant was genotyped in a validation cohort of 2,104 MS trio families. RESULTS: Four family members with MS were sequenced and 21,583 variants were found to be shared among these individuals. Refining the variants to those with 1) a predicted loss of function and 2) present within regions of modest haplotype sharing identified 1 novel mutation (rs55762744) in the tyrosine kinase 2 (TYK2) gene. A different polymorphism within this gene has been shown to be protective in genome-wide association studies. In contrast, the TYK2 variant identified here is a novel, missense mutation and was found to be present in 10/14 (72%) cases and 28/60 (47%) of the unaffected family members. Genotyping additional 2,104 trio families showed the variant to be transmitted preferentially from heterozygous parents (transmitted 16: not transmitted 5; χ(2) = 5.76, p = 0.016). CONCLUSIONS: Rs55762744 is a rare variant of modest effect on MS risk affecting a subset of patients (0.8%). Within this pedigree, rs55762744 is common and appears to be a modifier of modest risk effect. Exome sequencing is a quick and cost-effective method and we show here the utility of sequencing a few cases from a single, unique family to identify a novel variant. The sequencing of additional family members or other families may help identify other variants important in MS.

Vitamin D metabolic pathway genes and risk of multiple sclerosis in Canadians.

BACKGROUND: Multiple sclerosis (MS) is determined by interactions between genes and environment and the influence of vitamin D adequacy has been proposed. Previous studies have shown that serum 25-hydroxyvitamin D (25(OH)D) levels are genetically influenced. Polymorphisms in vitamin D pathway genes are candidates for association with MS susceptibility. METHODS: MS patients (n=1364) and their unaffected first-degree relatives (n=1661) were ascertained through the Canadian Collaborative study. Seventy-one SNPs, across four genes [vitamin D receptor (VDR), 1-alpha-hydroxylase (CYP27B1) enzyme, vitamin D binding protein (DBP), 24-hydroxylase (CYP24)], were genotyped and tested for association with MS susceptibility using TDT in PLINK. Secondary analyses included stratification for HLA-DRB1*15 and parent of origin transmission effects. RESULTS: We found no significant association of vitamin D pathway genes with MS susceptibility after correction for multiple comparisons. However, the VDR Fok1 variant (rs2228570), selected for previously positive associations with MS susceptibility and 25(OH)D levels in MS patients showed marginally distorted transmission in DRB15-negative patients (p=0.03). There was no evidence for differential maternal versus paternal allele transmission. CONCLUSIONS: The findings fail to directly connect vitamin D metabolism genes to MS susceptibility, despite a large sample size and comprehensive gene coverage.

Parent-of-origin effects at the major histocompatibility complex in multiple sclerosis.

Multiple sclerosis (MS) susceptibility is characterized by maternal parent-of-origin effects and increased female penetrance. In 7796 individuals from 1797 MS families (affected individuals n = 2954), we further implicate epigenetic modifications within major histocompatibility complex (MHC) class II haplotypes as mediating these phenomena. Affected individuals with the main MS-associated allele HLA-DRB1*15 had a higher female-to-male ratio versus those lacking it (P = 0.00023). Distorted transmission of MHC haplotypes by both parent-of-origin and gender-of-affected-offspring was most evident in the maternal HLA-DRB1*15 transmission to affected female offspring (OR = 3.31, 95% CI = 2.59-4.24) contrasting with similarity among maternal transmission to affected male offspring (OR = 2.13, 95% CI = 1.44-3.14), paternal transmissions to affected female (OR = 2.14, 95% CI = 1.64-2.78) and male (OR = 2.16, 95% CI = 1.37-3.39) offspring. Significant parent-of-origin effects were observed in affected females (maternal: P = 9.33 x 10(-42); paternal: P = 1.12 x 10(-15); comparison: P = 0.0014), but not in affected males (maternal: P = 6.70 x 10(-8); paternal: P = 2.54 x 10(-6); comparison: P = 0.95). Conditional logistic regression analysis revealed further differential risk of HLA diplotypes. Risks for HLA-DRB1*15 and likely for other HLA-DRB1 haplotypes were restricted by (i) parent-of-origin, (ii) gender-of-offspring and (iii) trans epistasis in offspring. These findings may illuminate the gender bias characterizing autoimmunity overall. They raise questions about the concept of restricted antigen presentation in autoimmunity and suggest that gender-specific epigenetic interactions may be the driving forces behind the MHC haplotypic associations. Haplotype-specific epigenetic modifications at MHC class II and their decay appear to be at the heart of MS pathogenesis and inheritance of risk, providing the focus for gene-environment interactions that determine susceptibility and resistance.

Epistasis among HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci determines multiple sclerosis susceptibility.

Multiple sclerosis (MS), a common central nervous system inflammatory disease, has a major heritable component. Susceptibility is associated with the MHC class II region, especially HLA-DRB5*0101-HLA-DRB1*1501-HLA-DQA1*0102-HLA-DQB1*0602 haplotypes(hereafter DR2), which dominate genetic contribution to MS risk. Marked linkage disequilibrium (LD) among these loci makes identification of a specific locus difficult. The once-leading candidate, HLA-DRB1*15, localizes to risk, neutral, and protective haplotypes. HLA-DRB1*15 and HLA-DQB1*0602, nearly always located together on a small ancestral chromosome segment, are strongly MS-associated. One intervening allele on this haplotype, viz. HLA-DQA1*0102, shows no primary MS association. Two Canadian cohorts (n = 830 and n = 438 trios) genotyped for HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles were tested for association using TDT. To evaluate epistasis involving HLA-DRB1*15, transmissions from HLA-DRB1*15-negative parents were stratified by the presence/absence of HLA-DRB1*15 in affected offspring. All 3 alleles contribute to MS susceptibility through novel epistatic interactions. HLA-DQA1*0102 increased disease risk when combined with HLA-DRB1*1501 in trans, thereby unambiguously implicating HLA-DQ in MS susceptibility. Three-locus haplotypes demonstrated that HLA-DRB1*1501 and HLA-DQB1*0602 each influence risk. Transmissions of rare morcellated DR2 haplotypes showed no interaction with HLA-DQA1*0102. Incomplete haplotypes bearing only HLA-DRB1*1501 or HLA-DQB1*0602 did not predispose to MS. Balanced reciprocal transmission distortion can mask epistatic allelic association. These findings implicate epistasis among HLA class II alleles in human immune responses generally, provide partial explanation for intense linkage disequilibrium in the MHC, have relevance to animal models, and demonstrate key roles for DR2-specific interactions in MS susceptibility. MHC disease associations may be more generally haplotypic or diplotypic.