Cilla Söderhäll

Differential DNA Methylation in Purified Human Blood Cells: Implications for Cell Lineage and Studies on Disease Susceptibility

Methylation of cytosines at CpG sites is a common epigenetic DNA modification that can be measured by a large number of methods, now even in a genome-wide manner for hundreds of thousands of sites. The application of DNA methylation analysis is becoming widely popular in complex disorders, for example, to understand part of the “missing heritability”. The DNA samples most readily available for methylation studies are derived from whole blood. However, blood consists of many functionally and developmentally distinct cell populations in varying proportions. We studied whether such variation might affect the interpretation of methylation studies based on whole blood DNA. We found in healthy male blood donors there is important variation in the methylation profiles of whole blood, mononuclear cells, granulocytes, and cells from seven selected purified lineages. CpG methylation between mononuclear cells and granulocytes differed for 22% of the 8252 probes covering the selected 343 genes implicated in immune-related disorders by genome-wide association studies, and at least one probe was differentially methylated for 85% of the genes, indicating that whole blood methylation results might be unintelligible. For individual genes, even if the overall methylation patterns might appear similar, a few CpG sites in the regulatory regions may have opposite methylation patterns (i.e., hypo/hyper) in the main blood cell types. We conclude that interpretation of whole blood methylation profiles should be performed with great caution and for any differences implicated in a disorder, the differences resulting from varying proportions of white blood cell types should be considered.

MMP12, lung function, and COPD in high-risk populations.

BACKGROUND Genetic variants influencing lung function in children and adults may ultimately lead to the development of chronic obstructive pulmonary disease (COPD), particularly in high-risk groups. METHODS We tested for an association between single-nucleotide polymorphisms (SNPs) in the gene encoding matrix metalloproteinase 12 (MMP12) and a measure of lung function (prebronchodilator forced expiratory volume in 1 second [FEV(1)]) in more than 8300 subjects in seven cohorts that included children and adults. Within the Normative Aging Study (NAS), a cohort of initially healthy adult men, we tested for an association between SNPs that were associated with FEV(1) and the time to the onset of COPD. We then examined the relationship between MMP12 SNPs and COPD in two cohorts of adults with COPD or at risk for COPD. RESULTS The minor allele (G) of a functional variant in the promoter region of MMP12 (rs2276109 [-82A-->G]) was positively associated with FEV(1) in a combined analysis of children with asthma and adult former and current smokers in all cohorts (P=2x10(-6)). This allele was also associated with a reduced risk of the onset of COPD in the NAS cohort (hazard ratio, 0.65; 95% confidence interval [CI], 0.46 to 0.92; P=0.02) and with a reduced risk of COPD in a cohort of smokers (odds ratio, 0.63; 95% CI, 0.45 to 0.88; P=0.005) and among participants in a family-based study of early-onset COPD (P=0.006). CONCLUSIONS The minor allele of a SNP in MMP12 (rs2276109) is associated with a positive effect on lung function in children with asthma and in adults who smoke. This allele is also associated with a reduced risk of COPD in adult smokers.

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.

Variants in a Novel Epidermal Collagen Gene (COL29A1) Are Associated with Atopic Dermatitis

Atopic dermatitis (AD) is a common chronic inflammatory skin disorder and a major manifestation of allergic disease. AD typically presents in early childhood often preceding the onset of an allergic airway disease, such as asthma or hay fever. We previously mapped a susceptibility locus for AD on Chromosome 3q21. To identify the underlying disease gene, we used a dense map of microsatellite markers and single nucleotide polymorphisms, and we detected association with AD. In concordance with the linkage results, we found a maternal transmission pattern. Furthermore, we demonstrated that the same families contribute to linkage and association. We replicated the association and the maternal effect in a large independent family cohort. A common haplotype showed strong association with AD (p = 0.000059). The associated region contained a single gene, COL29A1, which encodes a novel epidermal collagen. COL29A1 shows a specific gene expression pattern with the highest transcript levels in skin, lung, and the gastrointestinal tract, which are the major sites of allergic disease manifestation. Lack of COL29A1 expression in the outer epidermis of AD patients points to a role of collagen XXIX in epidermal integrity and function, the breakdown of which is a clinical hallmark of AD.

A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis

Atopic dermatitis (AD) is the most common dermatological disease of childhood. Many children with AD have asthma and AD shares regions of genetic linkage with psoriasis, another chronic inflammatory skin disease. We present here a genome-wide association study (GWAS) of childhood-onset AD in 1563 European cases with known asthma status and 4054 European controls. Using Illumina genotyping followed by imputation, we generated 268 034 consensus genotypes and in excess of 2 million single nucleotide polymorphisms (SNPs) for analysis. Association signals were assessed for replication in a second panel of 2286 European cases and 3160 European controls. Four loci achieved genome-wide significance for AD and replicated consistently across all cohorts. These included the epidermal differentiation complex (EDC) on chromosome 1, the genomic region proximal to LRRC32 on chromosome 11, the RAD50/IL13 locus on chromosome 5 and the major histocompatibility complex (MHC) on chromosome 6; reflecting action of classical HLA alleles. We observed variation in the contribution towards co-morbid asthma for these regions of association. We further explored the genetic relationship between AD, asthma and psoriasis by examining previously identified susceptibility SNPs for these diseases. We found considerable overlap between AD and psoriasis together with variable coincidence between allergic rhinitis (AR) and asthma. Our results indicate that the pathogenesis of AD incorporates immune and epidermal barrier defects with combinations of specific and overlapping effects at individual loci.

The chitinase-like protein YKL-40: a possible biomarker of inflammation and airway remodeling in severe pediatric asthma

BACKGROUND Problematic severe childhood asthma includes a subgroup of patients who are resistant to therapy. The specific mechanisms involved are unknown, and novel biomarkers are required to facilitate treatment and diagnosis of therapy-resistant asthma. The chitinase-like protein YKL-40 has been related to asthma and airway remodeling. OBJECTIVES To compare serum YKL-40 levels in children with severe, therapy-resistant asthma (n = 34), children with controlled persistent asthma (n = 39), and healthy controls (n = 27), and to investigate correlations with biomarkers of inflammation and airway remodeling. METHODS The study protocol included questionnaires, measurement of exhaled nitric oxide in exhaled air, blood sampling for inflammatory biomarkers, and high-resolution computed tomography of the lungs to identify bronchial wall thickening (therapy-resistant only). Serum YKL-40 levels were measured by ELISA, and all asthmatic children were genotyped for a CHI3L1 promoter single nucleotide polymorphism (rs4950928). RESULTS Serum YKL-40 levels were significantly higher in children with therapy-resistant asthma than in healthy children (19.2 ng/mL vs 13.8 ng/mL, P = .03). Among children with severe, therapy-resistant asthma, YKL-40 levels correlated with fraction of exhaled nitric oxide in exhaled air (r = 0.48, P = .004), blood neutrophils (r = 0.63, P < .001), and bronchial wall thickening on high-resolution computed tomography (r = 0.45, P = .01). Following adjustment for CHI3L1 genotype, significantly greater levels of YKL-40 were found in children with therapy-resistant asthma than in children with controlled asthma. CONCLUSIONS YKL-40 levels are increased in children with severe, therapy-resistant asthma compared to healthy children, and also compared to children with controlled asthma following correction for genotype.

Transcriptome analysis reveals upregulation of bitter taste receptors in severe asthmatics

The causes of severe childhood asthma are poorly understood. Our aim was to define global patterns of gene expression in children with severe therapy-resistant and controlled asthma. White blood cells were isolated and the global transcriptome profile was characterised using the Affymetrix Human Gene ST 1.0 chip in children with severe, therapy-resistant asthma (n=17), controlled asthma (n=19) and healthy controls (n=18). Receptor expression was studied in separated leukocyte fractions from asthmatic adults (n=12). Overall, 1378 genes were differentially expressed between children with severe/controlled asthma and controls. Three significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways were represented: natural killer cell-mediated cytotoxicity (upregulated in controlled asthma); N-glycan biosynthesis (downregulated in severe asthma); and bitter taste transduction receptors (TAS2Rs) (upregulated in severe asthma). Quantitative PCR experiments confirmed upregulation of TAS2Rs in severe asthmatics. TAS2R expression was replicated in leukocytes from adult asthmatics, in which TAS2R agonists also inhibited LPS-induced cytokine release. Significant correlations between expression of TAS2Rs and clinical markers of asthma severity were found in both adults and children. In conclusion, specific gene expression patterns were observed in children with severe, therapy-resistant asthma. The increased expression of bronchodilatory TAS2Rs suggests a new target for the treatment of asthma.

Assessment of the neuropeptide S system in anxiety disorders

BACKGROUND The G protein-coupled receptor neuropeptide S receptor 1 (NPSR1) and its ligand neuropeptide S (NPS) form a signaling system mainly implicated in susceptibility to asthma and inflammatory disorders in humans and regulation of anxiety and arousal in rodents. We addressed here the role of NPS and NPSR1 as susceptibility genes for human anxiety disorders. METHODS We performed comprehensive association analysis of genetic variants in NPS and NPSR1 in three independent study samples. We first studied a population-based sample (Health 2000, Finland) of 321 anxiety disorder patients and 1317 control subjects and subsequently a Spanish clinical panic disorder sample consisting of 188 cases and 315 control subjects. In addition, we examined a birth cohort of 2020 children (Barn Allergi Miljö Stockholm Epidemiologi [BAMSE], Sweden). We then tested whether alleles of the most significantly associated single nucleotide polymorphisms alter DNA-protein complex formation in electrophoretic mobility shift assays. Finally, we compared acute stress responses on the gene expression level in wild-type and Npsr1(-/-) mice. RESULTS We confirmed previously observed epidemiological association between anxiety and asthma in two population-based cohorts. Single nucleotide polymorphisms within NPS and NPSR1 associated with panic disorder diagnosis in the Finnish and Spanish samples and with parent-reported anxiety/depression in the BAMSE sample. Moreover, some of the implicated single nucleotide polymorphisms potentially affect transcription factor binding. Expression of neurotrophin-3, a neurotrophic factor connected to stress and panic reaction, was significantly downregulated in brain regions of stressed Npsr1(-/-) mice, whereas interleukin-1 beta, an active stress-related immunotransmitter, was upregulated. CONCLUSIONS Our results suggest that NPS-NPSR1 signaling is likely involved in anxiety.

Epigenome-Wide Meta-Analysis of Methylation in Children Related to Prenatal NO2 Air Pollution Exposure

Background: Prenatal exposure to air pollution is considered to be associated with adverse effects on child health. This may partly be mediated by mechanisms related to DNA methylation. Objectives: We investigated associations between exposure to air pollution, using nitrogen dioxide (NO2) as marker, and epigenome-wide cord blood DNA methylation. Methods: We meta-analyzed the associations between NO2 exposure at residential addresses during pregnancy and cord blood DNA methylation (Illumina 450K) in four European and North American studies (n = 1,508) with subsequent look-up analyses in children ages 4 (n = 733) and 8 (n = 786) years. Additionally, we applied a literature-based candidate approach for antioxidant and anti-inflammatory genes. To assess influence of exposure at the transcriptomics level, we related mRNA expression in blood cells to NO2 exposure in 4- (n = 111) and 16-year-olds (n = 239). Results: We found epigenome-wide significant associations [false discovery rate (FDR) p < 0.05] between maternal NO2 exposure during pregnancy and DNA methylation in newborns for 3 CpG sites in mitochondria-related genes: cg12283362 (LONP1), cg24172570 (3.8 kbp upstream of HIBADH), and cg08973675 (SLC25A28). The associations with cg08973675 methylation were also significant in the older children. Further analysis of antioxidant and anti-inflammatory genes revealed differentially methylated CpGs in CAT and TPO in newborns (FDR p < 0.05). NO2 exposure at the time of biosampling in childhood had a significant impact on CAT and TPO expression. Conclusions: NO2 exposure during pregnancy was associated with differential offspring DNA methylation in mitochondria-related genes. Exposure to NO2 was also linked to differential methylation as well as expression of genes involved in antioxidant defense pathways. Citation: Gruzieva O, Xu CJ, Breton CV, Annesi-Maesano I, Antó JM, Auffray C, Ballereau S, Bellander T, Bousquet J, Bustamante M, Charles MA, de Kluizenaar Y, den Dekker HT, Duijts L, Felix JF, Gehring U, Guxens M, Jaddoe VV, Jankipersadsing SA, Merid SK, Kere J, Kumar A, Lemonnier N, Lepeule J, Nystad W, Page CM, Panasevich S, Postma D, Slama R, Sunyer J, Söderhäll C, Yao J, London SJ, Pershagen G, Koppelman GH, Melén E. 2017. Epigenome-wide meta-analysis of methylation in children related to prenatal NO2 air pollution exposure. Environ Health Perspect 125:104–110; http://dx.doi.org/10.1289/EHP36

Genome-wide association study of body mass index in 23 000 individuals with and without asthma

Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co‐morbidity between these conditions.