Hong Quach

Considerations for normalization of DNA methylation data by Illumina 450K BeadChip assay in population studies.

Analysis of epigenetic mechanisms, particularly DNA methylation, is of increasing interest for epidemiologic studies examining disease etiology and impacts of environmental exposures. The Infinium HumanMethylation450 BeadChip® (450K), which interrogates over 480 000 CpG sites and is relatively cost effective, has become a popular tool to characterize the DNA methylome. For large-scale studies, minimizing technical variability and potential bias is paramount. The goal of this paper was to evaluate the performance of several existing and novel color channel normalizations designed to reduce technical variability and batch effects in 450K analysis from a large population study. Comparative assessment of 10 normalization procedures included the GenomeStudio® Illumina procedure, the lumi smooth quantile approach, and the newly proposed All Sample Mean Normalization (ASMN). We also examined the performance of normalizations in combination with correction for the two types of Infinium chemistry utilized on the 450K array. We observed that the performance of the GenomeStudio® normalization procedure was highly variable and dependent on the quality of the first sample analyzed in an experiment, which is used as a reference in this procedure. While the lumi normalization was able to decrease batch variability, it increased variation among technical replicates, potentially reducing biologically meaningful findings. The proposed ASMN procedure performed consistently well, both at reducing batch effects and improving replicate comparability. In summary, the ASMN procedure can improve existing color channel normalization, especially for large epidemiologic studies, and can be successfully implemented to enhance a 450K DNA methylation data pipeline.

Obesity during childhood and adolescence increases susceptibility to multiple sclerosis after accounting for established genetic and environmental risk factors

OBJECTIVE To investigate the association between obesity and multiple sclerosis (MS) while accounting for established genetic and environmental risk factors. METHODS Participants included members of Kaiser Permanente Medical Care Plan, Northern California Region (KPNC) (1235 MS cases and 697 controls). Logistic regression models were used to estimate odds ratios (ORs) with 95% confidence intervals (95% CI). Body mass index (BMI) or body size was the primary predictor of each model. Both incident and prevalent MS cases were studied. RESULTS In analyses stratified by gender, being overweight at ages 10 and 20 were associated with MS in females (p<0.01). Estimates trended in the same direction for males, but were not significant. BMI in 20s demonstrated a linear relationship with MS (p-trend=9.60×10(-4)), and a twofold risk of MS for females with a BMI≥30kg/m(2) was observed (OR=2.15, 95% CI 1.18, 3.92). Significant associations between BMI in 20s and MS in males were not observed. Multivariate modelling demonstrated that significant associations between BMI or body size with MS in females persisted after adjusting for history of infectious mononucleosis and genetic risk factors, including HLA-DRB1*15:01 and established non-HLA risk alleles. INTERPRETATION Results show that childhood and adolescence obesity confer increased risk of MS in females beyond established heritable and environmental risk factors. Strong evidence for a dose-effect of BMI in 20s and MS was observed. The magnitude of BMI association with MS is as large as other known MS risk factors.

Mendelian randomization shows a causal effect of low vitamin D on multiple sclerosis risk.

Objective: We sought to estimate the causal effect of low serum 25(OH)D on multiple sclerosis (MS) susceptibility that is not confounded by environmental or lifestyle factors or subject to reverse causality. Methods: We conducted mendelian randomization (MR) analyses using an instrumental variable (IV) comprising 3 single nucleotide polymorphisms found to be associated with serum 25(OH)D levels at genome-wide significance. We analyzed the effect of the IV on MS risk and both age at onset and disease severity in 2 separate populations using logistic regression models that controlled for sex, year of birth, smoking, education, genetic ancestry, body mass index at age 18–20 years or in 20s, a weighted genetic risk score for 110 known MS-associated variants, and the presence of one or more HLA-DRB1*15:01 alleles. Results: Findings from MR analyses using the IV showed increasing levels of 25(OH)D are associated with a decreased risk of MS in both populations. In white, non-Hispanic members of Kaiser Permanente Northern California (1,056 MS cases and 9,015 controls), the odds ratio (OR) was 0.79 (p = 0.04, 95% confidence interval (CI): 0.64–0.99). In members of a Swedish population from the Epidemiological Investigation of Multiple Sclerosis and Genes and Environment in Multiple Sclerosis MS case-control studies (6,335 cases and 5,762 controls), the OR was 0.86 (p = 0.03, 95% CI: 0.76–0.98). A meta-analysis of the 2 populations gave a combined OR of 0.85 (p = 0.003, 95% CI: 0.76–0.94). No association was observed for age at onset or disease severity. Conclusions: These results provide strong evidence that low serum 25(OH)D concentration is a cause of MS, independent of established risk factors.

Genome-Wide DNA Methylation Profiles Indicate CD8+ T Cell Hypermethylation in Multiple Sclerosis

Objective Determine whether MS-specific DNA methylation profiles can be identified in whole blood or purified immune cells from untreated MS patients. Methods Whole blood, CD4+ and CD8+ T cell DNA from 16 female, treatment naïve MS patients and 14 matched controls was profiled using the HumanMethylation450K BeadChip. Genotype data were used to assess genetic homogeneity of our sample and to exclude potential SNP-induced DNA methylation measurement errors. Results As expected, significant differences between CD4+ T cells, CD8+ T cells and whole blood DNA methylation profiles were observed, regardless of disease status. Strong evidence for hypermethylation of CD8+ T cell, but not CD4+ T cell or whole blood DNA in MS patients compared to controls was observed. Genome-wide significant individual CpG-site DNA methylation differences were not identified. Furthermore, significant differences in gene DNA methylation of 148 established MS-associated risk genes were not observed. Conclusion While genome-wide significant DNA methylation differences were not detected for individual CpG-sites, strong evidence for DNA hypermethylation of CD8+ T cells for MS patients was observed, indicating a role for DNA methylation in MS. Further, our results suggest that large DNA methylation differences for CpG-sites tested here do not contribute to MS susceptibility. In particular, large DNA methylation differences for CpG-sites within 148 established MS candidate genes tested in our study cannot explain missing heritability. Larger studies of homogenous MS patients and matched controls are warranted to further elucidate the impact of CD8+ T cell and more subtle DNA methylation changes in MS development and pathogenesis.

Smoking and risk of multiple sclerosis: evidence of modification by NAT1 variants.

Background: Tobacco smoke is an established risk factor for multiple sclerosis (MS). We hypothesized that variation in genes involved in metabolism of tobacco smoke constituents may modify MS risk in smokers. Methods: A three-stage gene-environment investigation was conducted for NAT1, NAT2, and GSTP1 variants. The discovery analysis was conducted among 1588 white MS cases and controls from the Kaiser Permanente Northern California Region HealthPlan (Kaiser). The replication analysis was carried out in 988 white MS cases and controls from Sweden. Results: Tobacco smoke exposure at the age of 20 years was associated with greater MS risk in both data sets (in Kaiser, odds ratio [OR] = 1.51 [95% confidence interval (CI) = 1.17–1.93]; in Sweden, OR = 1.35 [1.04–1.74]). A total of 42 NAT1 variants showed evidence for interaction with tobacco smoke exposure (Pcorrected < 0.05). Genotypes for 41 NAT1 single nucleotide polymorphisms (SNPs) were studied in the replication data set. A variant (rs7388368C>A) within a dense transcription factor-binding region showed evidence for interaction (Kaiser, OR for interaction = 1.75 [95% CI = 1.19–2.56]; Sweden, OR = 1.62 [1.05–2.49]). Tobacco smoke exposure was associated with MS risk among rs7388368A carriers only; homozygote individuals had the highest risk (A/A, OR = 5.17 [95% CI = 2.17–12.33]). Conclusions: We conducted a three-stage analysis using two population-based case-control datasets that consisted of a discovery population, a replication population, and a pooled analysis. NAT1 emerged as a genetic effect modifier of tobacco smoke exposure in MS susceptibility.

Estimation of Blood Cellular Heterogeneity in Newborns and Children for Epigenome-Wide Association Studies

Confounding by cellular heterogeneity has become a major concern for epigenome‐wide association studies (EWAS) in peripheral blood samples from population and clinical studies. Adjusting for white blood cell percentage estimates produced by the minfi implementation of the Houseman algorithm (minfi) during statistical analysis is now an established method to account for this bias in adults. However, minfi has not been benchmarked against white blood cell counts in children that may differ substantially from the reference dataset used in its estimation. We compared estimates of white blood cell type percentages produced by two methods, minfi and differential cell count (DCC), in a birth cohort at two time points (birth and 12 years of age). We found that both minfi and DCC had similar trends as children aged, and neither count method differed by sex among newborns (P > 0.10). However, minfi estimates did not correlate well with DCC in samples from newborns (ρ = −0.05 for granulocytes; ρ = −0.03 for lymphocytes). In older children, correlation improved substantially (ρ = 0.77 for granulocytes; ρ = 0.75 for lymphocytes), likely due to increasing similarity with minfis adult reference data as children aged. Our findings suggest that the minfi method may provide suitable estimates of white blood cell composition for samples from adults and older children, but may not currently be appropriate for EWAS involving newborns or young children. Environ. Mol. Mutagen. 56:751–758, 2015.

Seroprevalence of aquaporin-4-IgG in a northern California population representative cohort of multiple sclerosis.

IMPORTANCE Using an aquaporin-4 (AQP4) M1-isoform-specific enzyme-linked immunosorbent assay (ELISA) and a fixed transfected cell-based assay (CBA), we tested AQP4-IgG in a northern California population representative cohort of 3293 potential cases with multiple sclerosis (MS). Seropositive cases were tested additionally by fluorescence-activated cell sorting, a live transfected cell-based assay. OBSERVATIONS Sera samples were available in 1040 cases; 7 yielded positive results, 4 by ELISA alone and 3 by both ELISA and CBA. Clinical data (episodes of optic neuritis and longitudinally extensive transverse myelitis [reported on at least 1 magnetic resonance imaging spine]) supported the alternative diagnosis of neuromyelitis optica for 2 patients as seropositive by both ELISA and CBA. These 2 patients alone tested positive by a fluorescence-activated cell-sorting assay. The diagnosis of MS was considered correct in the other 5 patients. Thus, 5 ELISA results and 1 fixed CBA result were false positive. CONCLUSIONS AND RELEVANCE Sensitive serological evaluation for AQP4-IgG in this large population-representative cohort of predominantly white non-Hispanic patients with MS reveals that neuromyelitis optica spectrum disorder is rarely misdiagnosed as MS in contemporary US neurological practice (0.2%). The frequency of a false-positive result for ELISA and CBA in this MS cohort were 0.5% and 0.1%, respectively. This finding reflects the superior specificity of CBA and justifies caution in interpreting AQP4-IgG results obtained by ELISA.

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%-5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%-16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.

Evidence for a causal relationship between low vitamin D, high BMI, and pediatric-onset MS

Objective: To utilize Mendelian randomization to estimate the causal association between low serum vitamin D concentrations, increased body mass index (BMI), and pediatric-onset multiple sclerosis (MS) using genetic risk scores (GRS). Methods: We constructed an instrumental variable for vitamin D (vitD GRS) by computing a GRS for 3 genetic variants associated with levels of 25(OH)D in serum using the estimated effect of each risk variant. A BMI GRS was also created that incorporates the cumulative effect of 97 variants associated with BMI. Participants included non-Hispanic white individuals recruited from over 15 sites across the United States (n = 394 cases, 10,875 controls) and Sweden (n = 175 cases, 5,376 controls; total n = 16,820). Results: Meta-analysis findings demonstrated that a vitD GRS associated with increasing levels of 25(OH)D in serum decreased the odds of pediatric-onset MS (odds ratio [OR] 0.72, 95% confidence interval [CI] 0.55, 0.94; p = 0.02) after controlling for sex, genetic ancestry, HLA-DRB1*15:01, and over 100 non–human leukocyte antigen MS risk variants. A significant association between BMI GRS and pediatric disease onset was also demonstrated (OR 1.17, 95% CI 1.05, 1.30; p = 0.01) after adjusting for covariates. Estimates for each GRS were unchanged when considered together in a multivariable model. Conclusions: We provide evidence supporting independent and causal effects of decreased vitamin D levels and increased BMI on susceptibility to pediatric-onset MS.

Adverse socioeconomic position during the life course is associated with multiple sclerosis

Background Adverse socioeconomic position (SEP) in childhood and adulthood is associated with a proinflammatory phenotype, and therefore an important exposure to consider for multiple sclerosis (MS), a complex neuroinflammatory autoimmune disease. The objective was to determine whether SEP over the life course confers increased susceptibility to MS. Methods 1643 white, non-Hispanic MS case and control members recruited from the Kaiser Permanente Medical Care Plan, Northern California Region, for which comprehensive genetic, clinical and environmental exposure data have been collected were studied. Logistic regression models investigated measures of childhood and adulthood SEP, and accounted for effects due to established MS risk factors, including HLA-DRB1*15:01 allele carrier status, smoking history, history of infectious mononucleosis, family history of MS and body size. Results Multiple measures of childhood and adulthood SEP were significantly associated with risk of MS, including parents renting versus owning a home at age 10: OR=1.48, 95% CI 1.09 to 2.02, p=0.013; less than a college education versus at least a college education based on parental household: OR=1.28, 95% CI 1.01 to 1.63, p=0.041; low versus high life course SEP: OR=1.50, 95% CI 1.09 to 2.05, p=0.012; and low versus high social mobility: OR=1.74, 95% CI 1.27 to 2.39, p=5.7×10−4. Conclusions Results derived from a population-representative case–control study provide support for the role of adverse SEP in MS susceptibility and add to the growing evidence linking lower SEP to poorer health outcomes. Both genetic and environmental contributions to chronic conditions are important and must be characterised to fully understand MS aetiology.