Malin Almgren

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.

Adenovirus-36 Is Associated with Obesity in Children and Adults in Sweden as Determined by Rapid ELISA

Background Experimental and natural human adenovirus-36 (Adv36) infection of multiple animal species results in obesity through increasing adipogenesis and lipid accumulation in adipocytes. Presence of Adv36 antibodies detected by serum neutralization assay has previously been associated with obesity in children and adults living in the USA, South Korea and Italy, whereas no association with adult obesity was detected in Belgium/the Netherlands nor among USA military personnel. Adv36 infection has also been shown to reduce blood lipid levels, increase glucose uptake by adipose tissue and skeletal muscle biopsies, and to associate with improved glycemic control in non-diabetic individuals. Principal Findings Using a novel ELISA, 1946 clinically well-characterized individuals including 424 children and 1522 non-diabetic adults, and 89 anonymous blood donors, residing in central Sweden representing the population in Stockholm area, were studied for the presence of antibodies against Adv36 in serum. The prevalence of Adv36 positivity in lean individuals increased from ∼7% in 1992–1998 to 15–20% in 2002–2009, which paralleled the increase in obesity prevalence. We found that Adv36-positive serology was associated with pediatric obesity and with severe obesity in females compared to lean and overweight/mildly obese individuals, with a 1.5 to 2-fold Adv36 positivity increase in cases. Moreover, Adv36 positivity was less common among females and males on antilipid pharmacological treatment or with high blood triglyceride level. Insulin sensitivity, measured as lower HOMA-IR, showed a higher point estimate in Adv36-positive obese females and males, although it was not statistically significant (p = 0.08). Conclusion Using a novel ELISA we show that Adv36 infection is associated with pediatric obesity, severe obesity in adult females and lower risk of high blood lipid levels in non-diabetic Swedish individuals.

Cesarean delivery and hematopoietic stem cell epigenetics in the newborn infant: implications for future health?

OBJECTIVE Cesarean section (CS) has been associated with a greater risk for asthma, diabetes, and cancer later in life. Although elective CS continues to rise, it is unclear whether and how it may contribute to compromised future health. Our aim was to investigate the influence of mode of delivery on the epigenetic state in neonatal hematopoietic stem cells. STUDY DESIGN This was an observational study of 64 healthy, singleton, newborn infants (33 boys) born at term. Cord blood was sampled after elective CS (n = 27) and vaginal delivery. Global deoxyribonucleic acid (DNA) methylation in hematopoietic stem cells (CD34+) was determined by luminometric methylation assay, and genome-wide, locus-specific DNA methylation analysis was performed by Illumina Infinium 450K (Illumina, San Diego, CA), validated by bisulfite-pyrosequencing. RESULTS CD34+ cells from infants delivered by CS were globally more DNA methylated (+2%) than DNA from infants delivered vaginally (P = .02). In relation to mode of delivery, a locus-specific analysis identified 343 loci with a difference in DNA methylation of 10% or greater (P < .01). A majority of the differentially methylated loci in neonatal CD34+ cells (76%) were found to be hypermethylated after vaginal delivery. In these infants, the degree of DNA methylation in 3 loci correlated to the duration of labor. The functional relevance of differentially methylated loci involved processes such as immunoglobulin biosynthetic process, regulation of glycolysis and ketone metabolism, and regulation of the response to food. CONCLUSION A possible interpretation is that mode of delivery affects the epigenetic state of neonatal hematopoietic stem cells. Given the functional relevance indicated, our findings may have important implications for health and disease in later life.

Population-based study of antiepileptic drug exposure in utero-Influence on head circumference in newborns

PURPOSE To study the effect of AED exposure on head circumference in the newborn. METHODS Data on all Swedish singletons births between 1995 and 2005, over 900,000 births, were obtained from the Swedish Medical Birth Registry. The effects of AEDs on birth-weight-adjusted mean head circumference (bw-adj-HC) were estimated by comparison with data from all births in an analysis which was adjusted for year of birth, maternal age, parity, maternal smoking, and maternal body mass index. RESULTS A significant reduction of mean bw-adj-HC was seen after both carbamazepine (CBZ) (standard deviation scores (SDS)=0.15, p<0.001) and valproic acid (VPA) (SDS=0.10, p=0.04) in monotherapy. No effect on mean bw-adj-HC was seen for phenytoin, clonazepam, lamotrigine and gabapentin. There was a significant increase in the occurrence of microcephaly (bw-adj-HC smaller than 2 SD below the mean) after any AED polytherapy (OR=2.85, 95% CI: 1.74-4.78) but not after AED monotherapy or monotherapy with CBZ or VPA. CBZ or VPA was taken by 71% of the pregnant mothers on AED, and the usage increased over time. CONCLUSIONS CBZ and VPA in monotherapy during pregnancy reduce mean bw-adj-HC. AED polytherapy increases the rate of microcephaly but no significant effect is seen of AED monotherapy. The possible significance for the further development of the child is uncertain but should be explored.

A truncated Kv1.1 protein in the brain of the megencephaly mouse: expression and interaction

BackgroundThe megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice.ResultsWe found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons.ConclusionThe megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.

Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis

Significance Vitamin D has been suggested to be associated with beneficial immunomodulation in autoimmune diseases. We demonstrate that the protective effect of vitamin D in an animal model of multiple sclerosis (MS) is linked to multiple signaling and metabolic pathways critical for T-cell activation and differentiation into pathogenic T helper (Th) 1 and Th17 subsets in vivo. This effect is mediated by epigenetic mechanisms as reflected by genome-wide reduction of DNA methylation and upregulation of microRNAs, with concomitant downregulation of their protein-coding target genes. Our data support the role of vitamin D in modulating risk for human disease, because orthologues of nearly 50% of MS candidate risk genes changed their expression in vivo in CD4+ T cells upon vitamin D supplementation. Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration

ABSTRACT Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro. Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro. Remarkably, 86% (351/406) of these CpGs were located <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible. IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

Carbamazepine protects against neuronal hyperplasia and abnormal gene expression in the megencephaly mouse

Carbamazepine (CBZ) is an anticonvulsant drug used to treat epilepsy and mood disorders. However, it can cause birth defects like reduced head circumference. It was recently shown to protect against brain overgrowth and seizure-induced abnormal plasticity in the megalencephalic mice Kv1.1(mceph/mceph), (mceph/mceph) despite remaining seizures. The mceph/mceph mouse displays two-fold enlarged hippocampus due to more neurons and astrocytes. Using stereology, we found that CBZ normalized the number of neurons and astrocytes in mceph/mceph hippocampus. To characterize CBZs protective ability on brain growth we studied the gene expression profile of mceph/mceph and wild type hippocampus, with and without CBZ treatment. Microarray analysis revealed transcripts involved in proliferation, differentiation and apoptosis including; NPY, Penk, Vgf, Mlc1, Sstr4, ApoD, Ndn, Aatk, Rgs2 and Gabra5, where Vgf may be of particular interest. The results also support CBZs effect on synaptic transmission through GABA A receptors, which could promote apoptotic neurodegeneration, affecting cell number.

Benefits of skin-to-skin contact during the neonatal period: Governed by epigenetic mechanisms?

The perinatal period experiences are important for later life physiology. Prematurely born babies have been shown to benefit from close contact with their mothers, and evidence suggests that epigenetic mechanisms are involved in these early imprints. This mini review is summarizing current praxis and discusses the need for more and larger studies.

Epigenetic influences on aging: a longitudinal genome-wide methylation study in old Swedish twins

ABSTRACT Age-related changes in DNA methylation were observed in cross-sectional studies, but longitudinal evidence is still limited. Here, we aimed to characterize longitudinal age-related methylation patterns using 1011 blood samples collected from 385 Swedish twins (age at entry: mean 69 and standard deviation 9.7, 73 monozygotic and 96 dizygotic pairs) up to five times (mean 2.6) over 20 years (mean 8.7). We identified 1316 age-associated methylation sites (P<1.3×10−7) using a longitudinal epigenome-wide association study design. We measured how estimated cellular compositions changed with age and how much they confounded the age effect. We validated the results in two independent longitudinal cohorts, where 118 CpGs were replicated in Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS, 390 samples) (P<3.9×10−5), 594 in Lothian Birth Cohort (LBC, 3018 samples) (P<5.1×10−5) and 63 in both. Functional annotation of age-associated CpGs showed enrichment in CCCTC-binding factor (CTCF) and other transcription factor binding sites. We further investigated genetic influences on methylation and found no interaction between age and genetic effects in the 1316 age-associated CpGs. Moreover, in the same CpGs, methylation differences within twin pairs increased with 6.4% over 10 years, where monozygotic twins had smaller intra-pair differences than dizygotic twins. In conclusion, we show that age-related methylation changes persist in a longitudinal perspective, and are fairly stable across cohorts. The changes are under genetic influence, although this effect is independent of age. Moreover, methylation variability increase over time, especially in age-associated CpGs, indicating the increase of environmental contributions on DNA methylation with age.