Bettina Kulle Andreassen

Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns

Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illuminas HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring.

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.

Oligoclonal band status in Scandinavian multiple sclerosis patients is associated with specific genetic risk alleles.

The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in multiple sclerosis (MS). We applied data from Norwegian, Swedish and Danish (i.e. Scandinavian) MS patients from a genome-wide association study (GWAS) to search for genetic differences in MS relating to OCB status. GWAS data was compared in 1367 OCB positive and 161 OCB negative Scandinavian MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. HLA-DRB1 genotypes were analyzed in a subset of the OCB positive (n = 2781) and OCB negative (n = 292) MS patients and compared to 890 healthy controls. Results from the genome-wide analyses showed that single nucleotide polymorphisms (SNPs) from the HLA complex and six other loci were associated to OCB status. In SNPs selected for replication, combined analyses showed genome-wide significant association for two SNPs in the HLA complex; rs3129871 (p = 5.7×10−15) and rs3817963 (p = 5.7×10−10) correlating with the HLA-DRB1*15 and the HLA-DRB1*04 alleles, respectively. We also found suggestive association to one SNP in the Calsyntenin-2 gene (p = 8.83×10−7). In HLA-DRB1 analyses HLA-DRB1*15∶01 was a stronger risk factor for OCB positive than OCB negative MS, whereas HLA-DRB1*04∶04 was associated with increased risk of OCB negative MS and reduced risk of OCB positive MS. Protective effects of HLA-DRB1*01∶01 and HLA-DRB1*07∶01 were detected in both groups. The groups were different with regard to age at onset (AAO), MS outcome measures and gender. This study confirms both shared and distinct genetic risk for MS subtypes in the Scandinavian population defined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS with implications for patient management, which need to be further studied.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

ABSTRACT Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Association of genetic markers with CSF oligoclonal bands in multiple sclerosis patients.

Objective to explore the association between genetic markers and Oligoclonal Bands (OCB) in the Cerebro Spinal Fluid (CSF) of Italian Multiple Sclerosis patients. Methods We genotyped 1115 Italian patients for HLA-DRB1*15 and HLA-A*02. In a subset of 925 patients we tested association with 52 non-HLA SNPs associated with MS susceptibility and we calculated a weighted Genetic Risk Score. Finally, we performed a Genome Wide Association Study (GWAS) with OCB status on a subset of 562 patients. The best associated SNPs of the Italian GWAS were replicated in silico in Scandinavian and Belgian populations, and meta-analyzed. Results HLA-DRB1*15 is associated with OCB+: p = 0.03, Odds Ratio (OR) = 1.6, 95% Confidence Limits (CL) = 1.1–2.4. None of the 52 non-HLA MS susceptibility loci was associated with OCB, except one SNP (rs2546890) near IL12B gene (OR: 1.45; 1.09–1.92). The weighted Genetic Risk Score mean was significantly (p = 0.0008) higher in OCB+ (7.668) than in OCB− (7.412) patients. After meta-analysis on the three datasets (Italian, Scandinavian and Belgian) for the best associated signals resulted from the Italian GWAS, the strongest signal was a SNP (rs9320598) on chromosome 6q (p = 9.4×10−7) outside the HLA region (65 Mb). Discussion genetic factors predispose to the development of OCB.

Association Analysis of PALB2 and BRCA2 in Bipolar Disorder and Schizophrenia in a Scandinavian Case-Control Sample

A recent genome‐wide association study (GWAS) found significant association between the PALB2 SNP rs420259 and bipolar disorder (BD). The intracellular functions of the expressed proteins from the breast cancer risk genes PALB2 and BRCA2 are closely related. Therefore, we investigated the relation between genetic variants in PALB2 and BRCA2 and BD. Due to increasing evidence of genetic overlap between BD and schizophrenia (SCZ), we also investigated association with SCZ. In a Scandinavian case–control sample (n = 686/2,538) we found the BRCA2 SNP rs9567552 to be significantly associated with BD (Nominal P = 0.00043). Additionally, we replicated the association between PALB2 SNP rs420259 and BD (Nominal P = 0.025). We then combined our sample with another Nordic case–control sample (n = 435/11,491) from Iceland, and added results from the Wellcome Trust Case Control Consortium (WTCCC) (n = 1,868/2,938) and the STEP‐UCL/ED‐DUB‐STEP2 study (n = 2,558/3,274) in a meta‐analysis which revealed a P‐value of 1.2 × 10−5 for association between PALB2 SNP rs420259 and BD (n = 5,547/20,241). Neither the PALB2 SNP rs420259 nor the BRCA2 SNP rs9567552 were nominally significantly associated with the SCZ phenotype in our Scandinavian sample (n = 781/2,839). Our findings support PALB2 and BRCA2 as risk genes specifically for BD, and suggest that altered DNA repair related to neurogenesis may be involved in BD pathophysiology.

Two new loci and gene sets related to sex determination and cancer progression are associated with susceptibility to testicular germ cell tumor

Genome-wide association (GWA) studies have reported 19 distinct susceptibility loci for testicular germ cell tumor (TGCT). A GWA study for TGCT was performed by genotyping 610 240 single-nucleotide polymorphisms (SNPs) in 1326 cases and 6687 controls from Sweden and Norway. No novel genome-wide significant associations were observed in this discovery stage. We put forward 27 SNPs from 15 novel regions and 12 SNPs previously reported, for replication in 710 case-parent triads and 289 cases and 290 controls. Predefined biological pathways and processes, in addition to a custom-built sex-determination gene set, were subject to enrichment analyses using Meta-Analysis Gene Set Enrichment of Variant Associations (M) and Improved Gene Set Enrichment Analysis for Genome-wide Association Study (I). In the combined meta-analysis, we observed genome-wide significant association for rs7501939 on chromosome 17q12 (OR = 0.78, 95% CI = 0.72-0.84, P = 1.1 × 10(-9)) and rs2195987 on chromosome 19p12 (OR = 0.76, 95% CI: 0.69-0.84, P = 3.2 × 10(-8)). The marker rs7501939 on chromosome 17q12 is located in an intron of the HNF1B gene, encoding a member of the homeodomain-containing superfamily of transcription factors. The sex-determination gene set (false discovery rate, FDRM < 0.001, FDRI < 0.001) and pathways related to NF-κB, glycerophospholipid and ether lipid metabolism, as well as cancer and apoptosis, was associated with TGCT (FDR < 0.1). In addition to revealing two new TGCT susceptibility loci, our results continue to support the notion that genes governing normal germ cell development in utero are implicated in the development of TGCT.

No association between dgkh and bipolar disorder in a Scandinavian case–control sample

Single nucleotide polymorphisms (SNPs) in diacylglycerol kinase eta (DGKH) have recently been shown to be associated with bipolar disorder (BD). To replicate this finding, we carried out a gene-wide genotyping of 36 tagSNPs in DGKH and performed a population-based association study on two Scandinavian samples, with successful genotyping of 594 BD cases and 1421 healthy controls. We found no significant association after multiple-testing correction between any of these SNPs and BD in our sample. Thus, it is unlikely that these genetic variations confer susceptibility to BD in this large Scandinavian sample.

Identifying Novel Gene Variants in Coronary Artery Disease and Shared Genes With Several Cardiovascular Risk Factors

RATIONALE Coronary artery disease (CAD) is a critical determinant of morbidity and mortality. Previous studies have identified several cardiovascular disease risk factors, which may partly arise from a shared genetic basis with CAD, and thus be useful for discovery of CAD genes. OBJECTIVE We aimed to improve discovery of CAD genes and inform the pathogenic relationship between CAD and several cardiovascular disease risk factors using a shared polygenic signal-informed statistical framework. METHODS AND RESULTS Using genome-wide association studies summary statistics and shared polygenic pleiotropy-informed conditional and conjunctional false discovery rate methodology, we systematically investigated genetic overlap between CAD and 8 traits related to cardiovascular disease risk factors: low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, type 2 diabetes mellitus, C-reactive protein, body mass index, systolic blood pressure, and type 1 diabetes mellitus. We found significant enrichment of single-nucleotide polymorphisms associated with CAD as a function of their association with low-density lipoprotein, high-density lipoprotein, triglycerides, type 2 diabetes mellitus, C-reactive protein, body mass index, systolic blood pressure, and type 1 diabetes mellitus. Applying the conditional false discovery rate method to the enriched phenotypes, we identified 67 novel loci associated with CAD (overall conditional false discovery rate <0.01). Furthermore, we identified 53 loci with significant effects in both CAD and at least 1 of low-density lipoprotein, high-density lipoprotein, triglycerides, type 2 diabetes mellitus, C-reactive protein, systolic blood pressure, and type 1 diabetes mellitus. CONCLUSIONS The observed polygenic overlap between CAD and cardiometabolic risk factors indicates a pathogenic relation that warrants further investigation. The new gene loci identified implicate novel genetic mechanisms related to CAD.

Altered DNA methylation profile in Norwegian patients with Autoimmune Addison’s Disease

Autoimmune Addisons Disease (AAD) is an endocrine and immunological disease of uncertain pathogenesis resulting from the immune systems destruction of the hormone producing cells of the adrenal cortex. The underlying molecular mechanisms are largely unknown, but it is commonly accepted that a combination of genetic susceptibility and environmental impact is critical. In the present study, we identified multiple hypomethylated gene promoter regions in patients with isolated AAD using DNA isolated from CD4+ T cells. The identified differentially methylated regions were distributed evenly across the 10.5-kb-promoter regions covered by the array, and a substantial number localized to promoters of genes involved in immune regulation and autoimmunity. This study reveals a hypomethylated status in CD4+ T cells from AAD patients and indicates differential methylation of promoters of key genes involved in immune responses.