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Despite improvement of current treatment strategies and novel targeted drugs, relapse and treatment resistance largely determine the outcome for acute myeloid leukemia (AML) patients. To identify the underlying molecular characteristics, numerous studies have been aimed to decipher the genomic- and transcriptomic landscape of AML. Nevertheless, further molecular changes allowing malignant cells to escape treatment remain to be elucidated. Mass spectrometry is a powerful tool enabling detailed insights into proteomic changes that could explain AML relapse and resistance. Here, we investigated AML samples from 47 adult and 22 pediatric patients at serial time-points during disease progression using mass spectrometry-based in-depth proteomics. We show that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse. Further, higher levels of granzymes and lower levels of the anti-inflammatory protein CR1/CD35 suggest an inflammatory signature promoting disease progression. Finally, through a proteogenomic approach, we detected novel peptides, which present a promising repertoire in the search for biomarkers and tumor-specific druggable targets. Altogether, this study highlights the importance of proteomic studies in holistic approaches to improve treatment and survival of AML patients. Title in the list of papers of Svea Stratmann thesis: Proteogenomic analysis of relapsed acute myeloid leukemia in adults and childrenAuthors in the list of papers of Svea Stramann: S. Stratmann, M. Vesterlund, H.M. Umer, A. Skaftason, M. Krogh Herlin, C. Sundström, A. Eriksson, M. Höglund, J. Palle, J. Abrahamson, K. Jahnukainen, M. Cheng Munthe-Kaas, B. Zeller, K. Pokrovskaja Tamm, L. Cavelier, J. Lehtiö, L. Holmfeld

Background/aim Previous reports have associated the KMT2A-ELL fusion gene, generated by t(11;19)(q23;p13.1), with acute myeloid leukemia (AML). We herein report a KMT2A-ELL and a novel ZNF56-KMT2A fusion genes in a pediatric T-lineage acute lymphoblastic leukemia (T-ALL). Materials and methods Genetic investigations were performed on bone marrow of a 13-year-old boy diagnosed with T-ALL. Results A KMT2A-ELL and a novel ZNF56-KMT2A fusion genes were generated on der(11)t(11;19)(q23;p13.1) and der(19)t(11;19)(q23;p13.1), respectively. Exon 20 of KMT2A fused to exon 2 of ELL in KMT2A-ELL chimeric transcript whereas exon 1 of ZNF56 fused to exon 21 of KMT2A in ZNF56-KMT2A transcript. A literature search revealed four more T-ALL patients carrying a KMT2A-ELL fusion. All of them were males aged 11, 11, 17, and 20 years. Conclusion KMT2A-ELL fusion is a rare recurrent genetic event in T-ALL with uncertain prognostic implications. The frequency and impact of ZNF56-KMT2A in T-ALL are unknown.

Purpose of the study: This study was aimed at identifying epigenome signature associated with risk of pediatric leukemia and uncovering molecular precursors of leukemia at birth in the blood of children before they develop the disease. Pediatric cancer is the leading cause of disease-related mortality in children and adolescents, with increasing incidence worldwide and lifelong sequelae in survivors. The most common form is leukemia, the causes of which are largely unknown. Growing evidence points to an origin in utero, when global redistribution of the epigenome modifications occurs driving tissue differentiation. Here, we sought to identify genome-wide differentially methylated genes at birth in newborns who later developed pediatric precursor B-cell ALL (pre-B ALL), compared with those who did not. Experimental procedures: Epigenome-wide DNA methylation was profiled in neonatal blood, with follow-up to pediatric pre-B ALL, using double-blinded analyses between prospective cohorts extending from birth to diagnosis and retrospective studies backtracking from clinical disease to birth. Validation was done using an independent technology and population (totaling 317 cases and 483 control) and complemented with pan-tissue methylation-stability (n=5,023 tissues; 30 types) and methylation-expression (n=2,294 tissues; 26 types) analyses. At diagnosis, methylation analysis was performed in leukemia tissues from pre-B ALL patients (n=644) with at least ten-year follow-up. Results: We found a limited number of loci (among which an imprinted tumor suppressor gene) as being significantly hypermethylated at birth in nested cases relative to controls in all tested populations, including European and Hispanic ancestries. Some DMRs were found to be stable over follow-up years after birth and across surrogate blood and target bone marrow tissues. Differential methylation was found to be associated with a change in gene expression and with worse pre-B ALL patient survival, supporting a functional and translational role for differential methylation. Conclusions: Our results provide proof-of-concept to detect at birth epigenetic alterations predisposing to childhood leukemia, reproducible in three continents and two ethnicities. DNA methylation alterations evident before diagnosis could be precursors of pediatric pre-B ALL development and actionable targets for risk assessment and prognosis. Citation Format: Akram Ghantous, Semira Gonseth Nusslé, Farah Nassar, Natalia Spitz, Alexei Novoloaca, Olga Krali, Ritu Roy, Shaobo Li, Maxime Caron, Lilys Lam, Peter Daniel Fransquet, John Casement, John Strathdee, Mark S. Pearce, Helen M. Hansen, Adam J. De Smith, Daniel Sinnett, Siri Eldevik Håberg, Jill McKay, Jessica Nordlund, Per Magnus, Terence Dwyer, Richard Saffery, Joseph Leo Wiemels, Monica Cheng Munthe-Kaas, Zdenko Herceg. Epigenome-wide DNA methylation alterations precede diagnosis since birth and affect prognosis of pediatric B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB362.

Background/aim Fusion of histone-lysine N-methyltransferase 2A gene (KMT2A) with the Rho guanine nucleotide exchange factor 12 gene (ARHGEF12), both located in 11q23, was reported in some leukemic patients. We report a KMT2A-ARHGEF12 fusion occurring during treatment of a pediatric acute myeloid leukemia (AML) with topoisomerase II inhibitors leading to a secondary acute lymphoblastic leukemia (ALL). Materials and methods Multiple genetic analyses were performed on bone marrow cells of a girl initially diagnosed with AML. Results At the time of diagnosis with AML, the t(9;11)(p21;q23)/KMT2A-MLLT3 genetic abnormality was found. After chemotherapy resulting in AML clinical remission, a 2 Mb deletion in 11q23 was found generating a KMT2A-ARHGEF12 fusion gene. When the patient later developed B lineage ALL, a t(14;19)(q32;q13), loss of one chromosome 9, and KMT2A-ARHGEF12 were detected. Conclusion The patient sequentially developed AML and ALL with three leukemia-specific genomic abnormalities in her bone marrow cells, two of which were KMT2A-rearrangements.

Background - The rapidly expanding era of “omics” research is highly dependent on the availability of quality-proven biological material, especially for rare conditions such as pediatric malignancies. Professional biobanks provide such material, focusing on standardized collection and handling procedures, distinctive quality measurements, traceability of storage conditions, and accessibility. For pediatric malignancies, traditional tumor biobanking is challenging due to the rareness and limited amount of tissue and blood samples. The higher molecular heterogeneity, lower mutation rates, and unique genomic landscapes, however, renders biobanking of this tissue even more crucial. Aim - The aim of this study was to test and establish methods for a prospective and centralized biobank for infants, children, and adolescents up to 18 years of age diagnosed with cancer in Norway. Methods - Obtain judicial and ethical approvals and administration through a consortium, steering committee, and advisory board. Develop pipelines including SOPs for all aspects in the biobank process, including collection, processing and storing of samples and data, as well of quality controlling, safeguarding, distributing, and transport. Results - The childhood cancer biobanking started at Oslo University Hospital in March 2017 and was from 2019 run as a national Norwegian Childhood Cancer Biobank. Informed consent and biological samples are collected regionally and stored centrally. Approximately 12 000 samples from 510 patients and have been included by January 1, 2021, representing a 96% consent and participation rate among our newly diagnosed patients. Conclusion - A well-functioning nationwide collection and centralized biobank with standardized procedures and national storage for pediatric malignancies has been established with a high acceptance among families.

Background/Aim: Previous reports have associated the KMT2A-ELL fusion gene, generated by t(11;19)(q23;p13.1), with acute myeloid leukemia (AML). We herein report a KMT2A-ELL and a novel ZNF56-KMT2A fusion genes in a pediatric T-lineage acute lymphoblastic leukemia (T-ALL). Materials and Methods: Genetic investigations were performed on bone marrow of a 13-year-old boy diagnosed with T-ALL. Results: A KMT2A-ELL and a novel ZNF56-KMT2A fusion genes were generated on der(11)t(11;19)(q23;p13.1) and der(19)t(11;19)(q23;p13.1), respectively. Exon 20 of KMT2A fused to exon 2 of ELL in KMT2A-ELL chimeric transcript whereas exon 1 of ZNF56 fused to exon 21 of KMT2A in ZNF56-KMT2A transcript. A literature search revealed four more T-ALL patients carrying a KMT2A-ELL fusion. All of them were males aged 11, 11, 17, and 20 years. Conclusion: KMT2A-ELL fusion is a rare recurrent genetic event in T-ALL with uncertain prognostic implications. The frequency and impact of ZNF56-KMT2A in T-ALL are unknown.

Background/Aim: Fusion of histone-lysine N-methyltransferase 2A gene (KMT2A) with the Rho guanine nucleotide exchange factor 12 gene (ARHGEF12), both located in 11q23, was reported in some leukemic patients. We report a KMT2A-ARHGEF12 fusion occurring during treatment of a pediatric acute myeloid leukemia (AML) with topoisomerase II inhibitors leading to a secondary acute lymphoblastic leukemia (ALL). Materials and Methods: Multiple genetic analyses were performed on bone marrow cells of a girl initially diagnosed with AML. Results: At the time of diagnosis with AML, the t(9;11)(p21;q23)/KMT2A-MLLT3 genetic abnormality was found. After chemotherapy resulting in AML clinical remission, a 2 Mb deletion in 11q23 was found generating a KMT2A-ARHGEF12 fusion gene. When the patient later developed B lineage ALL, a t(14;19)(q32;q13), loss of one chromosome 9, and KMT2A-ARHGEF12 were detected. Conclusion: The patient sequentially developed AML and ALL with three leukemia-specific genomic abnormalities in her bone marrow cells, two of which were KMT2A-rearrangements.

Dysregulated tyrosine kinases in myeloid/lymphoid neoplasms with eosinophilia are rare, but do occur in children. To increase awareness of this diagnosis, we present a child who was diagnosed after a 3-year disease history. The patient was initially treated according to a T-cell lymphoblastic lymphoma protocol, but genetic analyses at recurrence revealed microdeletions resulting in an in-frame fusion of ZMYM2 and FLT3. Treatment with sorafenib, an FLT3 tyrosine kinase inhibitor, rapidly resulted in significant reduction of lymphadenopathy and normalization of white blood cell and eosinophil counts. At 17 months of treatment, he remains in complete hematologic, but not molecular remission.

BackgroundAccumulating evidence links paternal adiposity in the peri-conceptional period to offspring health outcomes. DNA methylation has been proposed as a mediating mechanism, but very few studies have explored this possibility in humans.Methods and findingsIn the Pregnancy And Childhood Epigenetics (PACE) consortium, we conducted a meta-analysis of co-ordinated epigenome-wide association studies (EWAS) of paternal prenatal Body Mass Index (BMI) (with and without adjustment for maternal BMI) in relation to DNA methylation in offspring blood at birth (13 datasets; total n= 4,894) and in childhood (six datasets; total n = 1,982). We found little evidence of association at either time point: for all CpGs, the False Discovery Rate-adjusted P-values were >0.05. In sex-stratified analyses, we found just four CpGs where there was robust evidence of association in female offspring. To compare our findings to those of other studies, we conducted a systematic review, which identified seven studies, including five candidate gene studies showing associations between paternal BMI/obesity and offspring or sperm DNA methylation at imprinted regions. However, in our own study, we found very little evidence of enrichment for imprinted genes.ConclusionOur findings do not support the hypothesis that paternal BMI around the time of pregnancy is associated with offspring blood DNA methylation, even at imprinted regions.Author SummaryPrevious small, mostly candidate gene studies have shown associations between paternal pre-pregnancy BMI and offspring blood DNA methylation. However, in our large meta-analysis of co-ordinated EWAS results from a total of 19 datasets across two timepoints, we found little evidence to support these findings, even at imprinted regions. This does not rule out the possibility of a paternal epigenetic effect in different tissues, at regions not covered by the 450k array, via different mechanisms, or in populations with greater extremes of paternal BMI. More research is warranted to help understand the size and nature of contributions of paternal adiposity to offspring epigenetics and health outcomes.

Background Preterm birth and shorter duration of pregnancy are associated with increased morbidity in neonatal and later life. As the epigenome is known to have an important role during fetal development, we investigated associations between gestational age and blood DNA methylation in children. Methods We performed meta-analysis of Illumina’s HumanMethylation450-array associations between gestational age and cord blood DNA methylation in 3648 newborns from 17 cohorts without common pregnancy complications, induced delivery or caesarean section. We also explored associations of gestational age with DNA methylation measured at 4–18 years in additional pediatric cohorts. Follow-up analyses of DNA methylation and gene expression correlations were performed in cord blood. DNA methylation profiles were also explored in tissues relevant for gestational age health effects: fetal brain and lung. Results We identified 8899 CpGs in cord blood that were associated with gestational age (range 27–42 weeks), at Bonferroni significance, P − 7, of which 3343 were novel. These were annotated to 4966 genes. After restricting findings to at least three significant adjacent CpGs, we identified 1276 CpGs annotated to 325 genes. Results were generally consistent when analyses were restricted to term births. Cord blood findings tended not to persist into childhood and adolescence. Pathway analyses identified enrichment for biological processes critical to embryonic development. Follow-up of identified genes showed correlations between gestational age and DNA methylation levels in fetal brain and lung tissue, as well as correlation with expression levels. Conclusions We identified numerous CpGs differentially methylated in relation to gestational age at birth that appear to reflect fetal developmental processes across tissues. These findings may contribute to understanding mechanisms linking gestational age to health effects.

Aim: Cigarette smoking influences DNA methylation genome wide, in newborns from pregnancy exposure and in adults from personal smoking. Whether a unique methylation signature exists for in utero exposure in newborns is unknown. Materials & methods: We separately meta-analyzed newborn blood DNA methylation (assessed using Illumina450k Beadchip), in relation to sustained maternal smoking during pregnancy (9 cohorts, 5648 newborns, 897 exposed) and adult blood methylation and personal smoking (16 cohorts, 15907 participants, 2433 current smokers). Results & conclusion: Comparing meta-analyses, we identified numerous signatures specific to newborns along with many shared between newborns and adults. Unique smoking-associated genes in newborns were enriched in xenobiotic metabolism pathways. Our findings may provide insights into specific health impacts of prenatal exposure on offspring.

Epigenetic modifications, including DNA methylation, represent a potential mechanism for environmental impacts on human disease. Maternal smoking in pregnancy remains an important public health problem that impacts child health in a myriad of ways and has potential lifelong consequences. The mechanisms are largely unknown, but epigenetics most likely plays a role. We formed the Pregnancy And Childhood Epigenetics (PACE) consortium and meta-analyzed, across 13 cohorts (n = 6,685), the association between maternal smoking in pregnancy and newborn blood DNA methylation at over 450,000 CpG sites (CpGs) by using the Illumina 450K BeadChip. Over 6,000 CpGs were differentially methylated in relation to maternal smoking at genome-wide statistical significance (false discovery rate, 5%), including 2,965 CpGs corresponding to 2,017 genes not previously related to smoking and methylation in either newborns or adults. Several genes are relevant to diseases that can be caused by maternal smoking (e.g., orofacial clefts and asthma) or adult smoking (e.g., certain cancers). A number of differentially methylated CpGs were associated with gene expression. We observed enrichment in pathways and processes critical to development. In older children (5 cohorts, n = 3,187), 100% of CpGs gave at least nominal levels of significance, far more than expected by chance (p value < 2.2 × 10−16). Results were robust to different normalization methods used across studies and cell type adjustment. In this large scale meta-analysis of methylation data, we identified numerous loci involved in response to maternal smoking in pregnancy with persistence into later childhood and provide insights into mechanisms underlying effects of this important exposure. The BAMSE cohort was supported by The Swedish Research Council, The Swedish Heart-Lung Foundation, Freemason Child House Foundation in Stockholm, MeDALL (Mechanisms of the Development of ALLergy), a collaborative project conducted within the European Union (grant agreement No. 261357), Centre for Allergy Research, Stockholm County Council (ALF), Swedish foundation for strategic research (SSF, RBc08-0027, EpiGene project), the Strategic Research Programme (SFO) in Epidemiology at Karolinska Institutet, The Swedish Research Council Formas and the Swedish Environment Protection Agency.

Birthweight is associated with health outcomes across the life course, DNA methylation may be an underlying mechanism. In this meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, we find that DNA methylation in neonatal blood is associated with birthweight at 914 sites, with a difference in birthweight ranging from −183 to 178 grams per 10% increase in methylation (PBonferroni, Birthweight has been found to associate with later-life health outcomes. Here the authors perform a meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, identifying differentially methylated CpGs in neonatal blood that associate with birthweight.

Pre-pregnancy maternal obesity is associated with adverse offspring outcomes at birth and later in life. Individual studies have shown that epigenetic modifications such as DNA methylation could contribute. Within the Pregnancy and Childhood Epigenetics (PACE) Consortium, we meta-analysed the association between pre-pregnancy maternal BMI and methylation at over 450,000 sites in newborn blood DNA, across 19 cohorts (9,340 mother-newborn pairs). We attempted to infer causality by comparing the effects of maternal versus paternal BMI and incorporating genetic variation. In four additional cohorts (1,817 mother-child pairs), we meta-analysed the association between maternal BMI at the start of pregnancy and blood methylation in adolescents. In newborns, maternal BMI was associated with small (

Background Several studies have reported age-associated changes in DNA methylation in the first few years of life and in adult populations, but the extent of such changes during childhood is less well studied. The goals of this study were to investigate to what degree intra-individual changes in DNA methylation are associated with aging during childhood and dissect the methylation changes directly associated with aging from the effect mediated through variation in cell-type composition (CTC). Results We performed reduced representation bisulfite sequencing (RRBS) in peripheral whole-blood samples collected at 2, 10, and 16 years of age. We identified age-associated longitudinal changes in DNA methylation at 346 CpGs in 178 genes. Analyses separating the effect mediated by CTC variability across age identified 26 CpGs located in 12 genes that associated directly with age. Hence, the CTC changes across age appear to act as a mediator of the observed DNA methylation associated with age. The results were replicated using EpiTYPER in a second sample set selected from the same cohort. Gene ontology analyses revealed enrichment of transcriptional regulation and developmental processes. Further, comparisons of the mean DNA methylation differences between the time points reveal greater differences between 2 to 10 years and 10 to 16 years, suggesting that the identified age-associated DNA methylation patterns manifests in early childhood. Conclusions This study reveals insights into the epigenetic dynamics associated with aging early in life. Such information could ultimately provide clues and point towards molecular pathways that are susceptible to aging-related disease-associated epigenetic dysregulation. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0277-3) contains supplementary material, which is available to authorized users.

MoBa and the International Childhood Cancer Cohort Consortium (I4C) Terence Dwyer, Gabriella Tikellis, Akram Ghantous, Stanley Lemeshow, Siri E. Haberg, Jorn Olsen, Ora Paltiel, Jean Golding, Martha S. Linet, Zdenko Herceg, Monica C. Munthe-Kaas and Camilla Stoltenberg 1) The George Institute for Global Health, Nuffield Department of Population Health, University of Oxford, Oxford, UK 2) The International Agency for Research on Cancer, Lyon, France 3) Murdoch Children’s Research Institute, Royal Children’s Hospital, University of Melbourne, Melbourne, Australia 4) The Ohio State University College of Public Health, Columbus, Ohio 5) Norwegian Institute of Public Health, Oslo, Norway 6) Department of Epidemiology, University of California, Los Angeles, CA, USA 7) School of Public Health and Department of Hematology, Hadassah-Hebrew University, Jerusalem, Israel 8) Centre for Child & Adolescent Health, School of Social & Community Medicine, University of Bristol, Bristol, UK 9) Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 10) Department of Pediatrics, Oslo University Hospital, Oslo, Norway

Background Several CD14 gene–environment interactions in relation to the development of allergic diseases have been reported, but the underlying biological mechanisms are unclear. We recently showed that CD14 methylation increased during childhood, parallelling a decreased impact of CD14 polymorphisms on soluble CD14 levels. Here, we aim to explore whether environmental stimuli during childhood affects CD14 methylation, thereby providing a biological mechanism through which environment may modulate genetic effect. Methods CD14 methylation levels were quantified in 157 children from the prospective Environment and Childhood Asthma birth cohort at ages 2 and 10. Associations between CD14 methylation levels and house dust levels of endotoxin, β(1,3)-glucans (at 2 yr only), allergens (dog, cat, and house dust mite), pet keeping and tobacco smoke exposure (TSE; questionnaire data) at 2 and 10 yr were explored. Results Children in homes without pets had larger increases in CD14 methylation through childhood (2–10 yr) compared with children with pets (2.1% increase (p = 0.003) vs. 0.4% decrease (n.s.), global p = 0.04). At 10 yr of age, lower CD14 methylation values were found in children with pets compared with children without pets at both 2 and 10 yr (5.4% vs. 7.5% [p = 0.02]). A similar trend was detected for TSE; children not exposed show larger increases in CD14 methylation, most pronounced in school-age girls exposed vs. not exposed to tobacco (5.5% vs. 7.5% methylation, p = 0.037). Conclusion Pet keeping and TSE appears to limit increase in CD14 methylation from 2 to 10 yr of age. This may partly explain the diverging CD14 allele associations with allergic diseases detected in different environments.

Background: The CHRNA 3 and 5 genes on chromosome 15 encode the alpha subunits of the nicotinic acetylcholine receptor, mediating airway cholinergic activity. Polymorphisms are associated with cigarette smoking, chronic obstructive pulmonary disease, and lung cancer. Aims: To determine possible associations between CHRNA 3/5 SNP rs8034191 and asthma or lung function in children in one local and one replicate multinational population, and assess if tobacco smoke modified the associations. Materials and methods: The rs8034191 SNP genotyped in 551 children from the environment and childhood asthma (ECA) birth cohort study in Oslo, Norway, and in 516 families from six European centers [the Genetics of Asthma International Network (GAIN) study] was tested for genotypic or allelic associations to current or history of asthma, allergic sensitization (= one positive skin prick tests), bronchial hyperresponsiveness (BHR), and lung function (FEV1% of predicted and FEV1/FVC ratio over/ below the 5th percentile). Results: Although the TT and CT genotypes at SNP rs 8034191 were overall significantly associated with BHR (OR = 3.9, 95% CI 1.510.0, p = 0.005), stratified analyses according to exposure to maternal smoking in-utero or indoor smoking at 10 yrs of age showed significant association (OR = 4.4, 95% CI 1.512.6, p = 0.006 and OR 5.6, 95% CI 1.718.5, p = 0.004, respectively) only in the non-exposed and not in exposed children. The SNPBHR association was replicated in the non-tobacco-smoke-exposed subjects in one of the GAIN centers (BHR associated with the T allele (p = 0.034)), but not in the collated GAIN populations. Asthma, allergic sensitization, and lung function were not associated with the rs8034191 alleles. Conclusion: An interaction between tobacco smoke exposure and a CHRNA3/5 polymorphism was found for BHR in children, but CHRNA3/5 was not associated with asthma or lung function.

Studies addressing the relationship between pet keeping and development of asthma and allergies may be influenced by pet avoidance in families with a history of allergic disease. Following a cohort of 1019 children in Oslo till 10 years of age, we studied the association of pet keeping with socio-economic factors and allergic disease in the family. A family history of asthma and rhinoconjunctivitis was not significantly associated with pet ownership at birth or with pet removal by 10 years. Acquiring cats and dogs was less likely if the child had allergic rhinoconjunctivitis, whereas no association was seen with asthma (in any family member). Single parenthood increased the likelihood of acquiring a cat, smoking parents more often had cats or dogs, and having older siblings was associated with keeping dogs and other furry pets. Among 319 families reporting pet avoidance, 70% never had pets, 8% had given up pets, and 22% avoided a particular type of pet only. Twenty-four per cent of the parents failed to retrospectively report pet keeping during the child's first year of life. Overall, allergic rhinitis, but not asthma was associated with actual pet avoidance, whereas the strongest predictors for keeping pets were found to be socio-economic factors. Practical implications Allergic disease in a child most often does not lead to the removal of the family's furry pet. Pet avoidance is associated with allergic symptoms, but not asthma. Socio-economic factors like parental education, single parenthood and smoking affects the families' decisions on pet keeping, including the type of pets the families will avoid or acquire. The large recall error demonstrated points to the need for prospective data regarding pet keeping.

Early intervention with inhaled corticosteroid (ICS) treatment for lung function development in childhood is debated. In view of lung function at birth, we aimed to assess if early use of ICS influenced lung function at 10 yrs of age. A 10-yr follow-up study of 614/802 children (mean age 10.9 +/- 0.9 yrs) with lung function measurements at birth in the Environment and Childhood Asthma study in Oslo included information on ICS treatment (124 with history of asthma) obtained at 2 and 10 yrs by parental interviews. Main outcomes at 10 yrs were the best values (% predicted and Z-scores) of forced expiratory volume in 1 s (FEV(1)) and mid-expiratory flow. The main explanatory factors were never, past or current use of ICS and Z-scores of the tidal flow-volume ratio t(PTEF)/t(E) [time to peak expiratory flow (t(PTEF)) and total expiratory time (t(E))] at birth. ICS treatment, reported by 11.9% of children in the population sample and 71.6% with current asthma, did not significantly influence lung function from birth to 10 yrs. The best values (and Z-scores) of FEV(1), and mid-expiratory flow were similar (p > 0.1) in subjects receiving ICS during and after 0-3 yrs of age, after 3 yrs only or currently compared with steroid naive children. Almost half of the change in lung function 0-10 yrs was explained by gender, a history of asthma and t(PTEF)/t(E) at birth. ICS treatment for asthma, reported in every eighth child by age 10 yrs, did not significantly improve lung function from birth to 10 yrs.

Background: Limited information is available regarding the prevalence of severe asthma in children. The present study aimed at investigating the prevalence of severe asthma in an urban child population; secondarily evaluating the applicability of the chosen definition by clinical characteristics. Methods: Children enrolled in the prospective birth cohort; the Environment and Childhood Asthma Study in Oslo; were reinvestigated at the age of 10 years (n ¼ 1019). A representative population based cohort of 616 children [mean age 10.9 (SD 0.9) years] with lung function measurements at birth was used for prevalence estimates, whereas all 1019 children (154 with current asthma) attending the 10-year follow-up were included for verification of the definition of severe asthma. Clinical investigations included spirometry, tests of bronchial hyperresponsiveness, skin prick tests and exhaled nitric oxide. Severe asthma was defined as poorly controlled asthma despite treatment with ‡ 800 lg budesonide or equivalent; assessed by a detailed structured interview. Results: The population point prevalence at age 10 years of current severe asthma was 0.5% (three of 616) and among children with current asthma 4.5% (three of 67). The 10/154 children identified as suffering from severe asthma more often had severe bronchial hyperresponsiveness (PD20 methacholine

Background: The HLA (human leukocyte antigen) class II genes DQB1 and DRB1 and the Tumor Necrosis Factor α gene (TNFA) within the HLA complex (chromosome 6p21) have been associated with asthma and allergy. Due to the strong linkage disequilibrium characterizing this complex and the multiple asthma/allergy expressions, we aimed to determine which of these genes were primarily involved in specific asthma/allergy traits. Methods: The DRB1–DQB1 alleles and TNFA-308 polymorphism were genotyped in 959 children from the Environment and Childhood Asthma study and analyzed for possible associations with allergic and non-allergic asthma (with/without at least one positive skin prick test for allergens) and specific allergic sensitization, as well as bronchial hyperresponsiveness and total IgE, using both allele and extended haplotype analyses. Results: Different genes within the HLA complex were associated with separate asthma and allergy traits. Nonallergic asthma was associated with both the TNFA-308A allele [Odds ratio (OR) 1.7 (1.3–2.3)] and DRB1*03 allele [OR 1.6(1–2.6)], but extended DRB1*03-TNFA-308 haplotype analysis suggested that the DRB1–DQB1 association was secondary to linkage disequilibrium with the TNFA-308 polymorphism. Allergies were associated with HLA class II alleles only; birch sensitization with DQB1*0603-DRB1*13 [OR 2.3 (1.4–4.0)] and mugwort sensitization with DQB1*0609-DRB1*13 [OR 7.1 (1.9–27.0)] and DQB1*0501-DRB1*01 [OR 2.0 (1.0–4.0)]. Conclusions: Our data suggests that asthma is not associated with DRB1 or DQB1 but rather TNFA or a gene(s) in linkage disequilibrium, while sensitization to specific allergens is associated with particular alleles at the DQ and/or DR loci. A novel association between DQB1*0603-DRB1*13 and birch allergy is identified.

Background: Eosinophil cationic protein (ECP) is a potent cytotoxic secretory protein with bactericidal and antiviral properties. ECP is released by activated eosinophils and regarded as a marker of eosinophilic inflammation. High levels of ECP have been reported in cases of active asthma and other allergic diseases. This study aimed to assess whether three single-nucleotide polymorphisms (SNPs) in the ECP gene (RNASE3) on chromosome 14 q24–q31 or their haplotypes are associated with asthma, allergy, or related phenotypes. Methods: The three SNPs −38CA, +371CG and +499CG in RNASE3 and their haplotypes were analyzed for associations with asthma, serum-ECP (s-ECP) levels, allergic sensitization (positive skin-prick test to common allergens), bronchial hyperresponsiveness (BHR) assessed by methacholine inhalation, and serum-IgE (s-IgE) levels in 177 families from Norway and the Netherlands identified through siblings with asthma. Results: Transmission disequilibrium test (TDT) demonstrated significant associations between the A-G-G haplotype and asthma as well as the specific phenotypes allergic asthma (but not non-allergic asthma), high s-ECP, high s-IgE and BHR, while the C-G-G haplotype was associated with reduced occurrence of these traits. In addition, the −38A allele was associated with high s-ECP levels and allergic asthma. Conclusion: The present study suggests that the A-G-G haplotype in the RNASE3 gene influences the development of asthma, in particular, an allergic form of asthma. Furthermore, as the −38CA SNP lies in close vicinity of known intron-regulatory sites, results of SNP analysis suggest that the detected association is possibly linked to a genetic transcriptional control of s-ECP levels.

Summary Background Several candidate genes have been implicated in the etiology of asthma, including the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene result in derangements of mucociliary clearance. Homozygotes for CFTR mutations develop cystic fibrosis (CF), a disorder characterized mainly by lung and pancreas disease. Objective To investigate whether there was an increased frequency of CFTR mutations in asthma patients. Methods Seven hundred and three subjects aged 10–11 years from the environment and childhood asthma (ECA) study were included in the present study. Possible associations between asthma, reduced lung function, bronchial hyperresponsiveness (BHR), and increased or decreased nitrogen oxide (NO) levels (based on structural parental interview, spirometry, PD 20 methacholine challenge test and exhaled NO measurements), and the five most common CFTR mutations in Norway (ΔF508, R117H, R117C, 4005+2T→C, 394delTT), the modulating polymorphisms IVS8(TG) m T n and the IVS8-5T were investigated. Results No association were found between asthma, reduced lung function, BHR or exhaled NO levels and CF heterozygosity. However, the IVS8(TG) 11 T 7 haplotype was associated with normal lung function. Conclusions Our results do not support the hypothesis that CFTR mutations or polymorphisms play a role in the pathogenesis of asthma in children. However, the distribution of Tn(TG)m haplotypes differed between individuals with reduced lung function and individuals with normal lung function.

Background Several genomic regions are reported to be associated with the development of asthma and allergy, including chromosome 2q33. This region harbors the candidate gene cytotoxic T-lymphocyte antigen 4 (CTLA-4) , an important regulator of T-cell activation and differentiation. Objective We sought to explore possible associations between CTLA-4 polymorphisms and allergy and asthma. Methods Seven single nucleotide polymorphisms (SNPs; MH30, −1147CT, +49AG, CT60, JO31, JO30, JO27_1) in CTLA-4 were analyzed for associations with total serum IgE, allergic sensitization (positive skin prick test to common allergens), bronchial hyperresponsiveness (BHR) to methacholine, asthma, and lung function (FEV 1 % of predicted) in 364 asthmatic families from 3 European countries. Results Transmission disequilibrium test analysis showed that several SNPs were significantly associated with serum IgE levels, allergy, asthma, and FEV 1 % predicted below 80%, but not with BHR, and CTLA-4 polymorphisms of potentially direct pathogenic significance in atopic disorders were identified. Conclusion We identified associations between 4 newly discovered SNPs in the CTLA-4 gene and serum IgE levels, allergy, asthma, and reduced lung function, but not BHR, suggesting an important role for CTLA-4 in atopy and reduced lung function in asthmatic subjects rather than asthma per se. The particular SNP alleles found positively associated with our phenotypes were recently shown to be associated negatively with autoimmune disorders. Although a skewing toward a T H 1 reactivity pattern is believed to characterize autoimmune diseases, atopic diseases are considered T H 2-mediated. Hence, our data suggest a role for CTLA-4 polymorphisms in determining the T H 1/T H 2 balance and identify CTLA-4 signaling as a potential therapeutic target in atopic disease.

Aim To determine associations between ADRB2 polymorphisms and lung function through childhood, and possible modification by gender, pet keeping or tobacco smoke. Methods Four ADRB2 single nucleotide polymorphisms (rs1042711, rs1042713, rs1042714 and rs1800888) were genotyped in 953 children from the prospective birth cohort ‘Environment and Childhood Asthma’ study and analysed for association with flow-volume parameters at birth (tidal breathing) and at 10 years of age (maximally forced), stratified by environmental exposures. Results The risk of reduced lung function was reduced in 10-year-old children carrying the most common ADRB2 haplotype (CGGC) (OR 0.45 (95% CI 0.25, 0.82)), whereas there was no association between lung function at birth and ADRB2 haplotypes. Tobacco smoke exposure, gender and pet keeping did not significantly interact with the haplotypes in influencing lung function. Conclusion This study demonstrates a possible protective effect by the ADRB2 haplotype I (CGGC) on reduced FEV1 in 10-year-old children, whereas no ADRB2 geno-/haplotypes were significantly associated with neonatal lung function. The ADRB2 gene thus appears to contribute to lung function development in childhood, independently of smoking, pets and gender.

Sachs-Olsen C, Lodrup Carlsen KC, Mowinckel P, Haland G, Devulapalli CS, Munthe-Kaas MC, Carlsen K-H. Diagnostic value of exhaled nitric oxide in childhood asthma and allergy. Pediatr Allergy Immunol 2010: 21: e213–e221. © 2010 John Wiley & Sons A/S Fractional exhaled nitric oxide (FENO) has been proposed as a diagnostic test of asthma. We aimed to investigate in a population based birth cohort of children the usefulness of FENO as a diagnostic tool. The 10-yr follow up of the Environment and Childhood Asthma Study in Oslo included 616 children representative of the prospective birth cohort. Both FENO (single breath technique) and skin prick test (SPT) were measured in 331, limited at the time by equipment availability. Structural parental interview, spirometry, methacholine challenge and exercise test were performed. FENO was significantly elevated in children with current asthma (geometric mean 9.6 (95% confidence interval (CI) (6.9, 13.4) p.p.b.) compared with healthy children (5.8 (5.4, 6.3) p.p.b.; p

Background CD14 is a pattern-recognition receptor for environmental LPS, and engagement of the CD14-LPS complex activates innate host defense mechanisms. Single nucleotide polymorphisms (SNPs) in the CD14 gene have been associated with soluble CD14 (sCD14) levels, but inconsistencies between studies suggest the presence of regulatory mechanisms hitherto not well understood. Objective We sought to investigate possible associations between CD14 SNPs and sCD14 levels at different time points in childhood (at birth [cord blood] and 2 and 10 years) and to explore whether these associations were related to CD14 gene methylation. Methods Four SNPs, rs2569191 (−1145GA), rs5744455 (−550CT or −651CT), rs2569190 (−159CT or −260CT), and rs4914 in CD14 were genotyped in 762 children from the Environmental and Childhood Asthma study. Genotype frequencies were analyzed for association with sCD14 levels in 660 babies, 346 children at age 2 years, and 360 children at age 10 years. In a subgroup of 157 children with DNA available at both 2 and 10 years of age, CD14 methylation patterns were determined and analyzed against detected CD14 gene–sCD14 associations. Results rs2569191, rs5744455, and rs2569190 were associated with sCD14 levels at birth and 2 years, but only rs5744455 was associated with sCD14 levels at 10 years. CD14 methylation increased significantly from age 2 to 10 years, and the level of methylation was inversely correlated with sCD14 levels at 10 years. Conclusion The reduced effect of CD14 polymorphisms on sCD14 levels from early to late childhood paralleled a small but significant increase in CD14 methylation during the same period.

The causal relationship between lower respiratory tract infections (LRIs) in early life and reduced lung function later in childhood is unsettled. Therefore, we assessed whether LRIs the first 2 yr of life influenced lung function development from birth to school age. In the prospective Oslo birth cohort, 'the Environment and Childhood Asthma (ECA) study' lung function was measured at birth in 802 infants by tidal flow volume loops and in 664 infants by passive respiratory mechanics and half yearly questionnaires, including LRI questions, were completed until 2 yr of age. The present study includes 607 children with information about LRIs the first 2 yr of life and successfully forced expiratory flow (FEF) volume measurements at the 10-yr follow-up assessment. At 10 yr of age, FEF at 50% of forced vital capacity (FEF(50)) (mean 95% confidence interval) was reduced in children with at least one bronchiolitis (85.0, 80.6-89.5, p = 0.020) or bronchitis (86.2, 82.6-89.8, p = 0.030) or > or =3 LRIs (83.4, 78.1-88.8, p = 0.017) when compared with no LRIs (90.6, 88.8-92.5) by 2 yr of life. The effects were significant in girls only when stratifying for gender. Among girls with later bronchiolitis compliance of the respiratory system (3.64, 3.17-4.10 vs. 4.18, 3.98-4.37, p = 0.031) and the ratio of time to peak tidal expiratory flow to total expiratory time (t(PTEF)/t(E)) measured at birth was significantly reduced (0. 26, 0.23-0.29 vs. 0.32, 0.30-0.33, p = 0.005) when compared with children with no LRIs. Change in lung function from birth (by t(PTEF)/t(E)) to 10 yr of age was not significantly associated with LRIs the first 2 yr of life, and LRIs by 2 yr of life were not significantly associated with lung function at 10 yr of age in regression analyses including lung function at birth and other possible predictors of lung function at 10 yr. In our study, LRIs during the first 2 yr of life did not impair lung function development from birth until 10 yr of age.

Type 1 diabetes (T1D) and allergic asthma are immune-mediated diseases. Pattern recognition receptors are proteins expressed by cells in the immune system to identify microbial pathogens and endogenous ligands. Toll-like receptors (TLRs) and CD14 are members of this family and could represent a molecular link between microbial infections and immune-mediated diseases. Diverging hypotheses regarding whether there exists a common or inverse genetic etiology behind these immune-mediated diseases have been presented. We aimed to test whether there exist common or inverse associations between polymorphisms in the pattern recognition receptors TLR2, TLR4 and CD14 and T1D and allergic asthma. Eighteen single nucleotide polymorphisms (SNPs) were genotyped in TLR2 (2), TLR4 (12) and CD14 (4) in 700 T1D children, 357 nuclear families with T1D children and 796 children from the 'Environment and Childhood Asthma' study. Allele and haplotype frequencies were analyzed in relation to diseases and in addition transmission disequilibrium test analyses were performed in the family material. Both T1D and allergic asthma were significantly associated with the TLR2 rs3804100 T allele and further associated with the haplotype including this SNP, possibly representing a susceptibility locus common for the two diseases. Neither TLR4 nor CD14 were associated with T1D or allergic asthma.

Background: Predicting school-age asthma from obstructive airways disease (OAD) in early life is difficult, even when parental and children’s atopic manifestations are taken into consideration. Objective: To assess if the severity of OAD in the first 2 years of life predicts asthma at 10 years of age. Methods: From a nested case control study within the Environment and Childhood Asthma study, 233 2-year-old subjects with recurrent (⩾2 episodes) bronchial obstruction (rBO+) and 216 subjects without bronchial obstruction (rBO−) underwent clinical examination, parental interview, treadmill test and metacholine bronchial hyperresponsiveness (BHR) measurement at 10 years. A severity score at 2 years was calculated by frequency, persistence of bronchial obstruction and hospital admissions because of OAD. Main outcomes: Current asthma at 10 years (asthma with symptoms and/or asthma medication during the past year and/or positive treadmill test). Secondary outcome was metacholine BHR at 10 years. Results: Compared with rBO− subjects, adjusted odds ratio (95% CI) of current asthma among rBO+ was 7.9 (4.1, 15.3), and among rBO+ with a severity score of >5, 20.2 (9.9, 41.3). In receiver operated characteristic analysis, positive and negative predictive values demonstrated the applicability and value of the score, with an optimal cut-off at severity score 5. Children with severity score >5 had severe BHR more often (PD 20 metacholine Conclusion: Using a simple scoring system, a high severity score of OAD by 2 years of age is a strong risk factor for, and may predict, current asthma at 10 years of age.

Background: The western world's increase in childhood asthma is suggested to level off. We aimed to investigate asthma prevalence in 10-year-old children within the prospective birth cohort Environment and Childhood Asthma (ECA) Study in Oslo established in 1992/1993. Subjects and methods: Six hundred and sixteen (77%) of 803 children (mean age 10.9 ± 0.9 (SD) years) with lung function measurements at birth were reinvestigated at age 10 years. At birth they corresponded to the entire birth cohort (n = 3754) regarding gender, socio-demographic factors, parental allergic diseases, pet keeping and maternal smoking. Results from structured parental interview, spirometry, and skin prick test for inhalant and food allergens are presented. Asthma definition required minimum two positive criteria, (i) doctor's diagnosis of asthma, (ii) wheeze and/or chest tightness, (iii) use of anti-asthmatic treatment. Current asthma required asthma definition plus either (ii) and/or (iii) in the last 12 months, and/or ≥10% fall in forced expired volume in 1 s after treadmill running. Results: Lifetime prevalence of asthma was 20.2%; current asthma 11.1%, doctor diagnosis of asthma 16.1% and wheezes ever 30.3%. Allergic sensitization (29.3% overall) was more common among children with current (56.3%) compared to asymptomatic (last 12 months) (26.0%) or no asthma (27.6%) (P

SummaryBackgroundIt is debated whether early treatment with inhaled corticosteroids (ICS) can change the natural course of childhood asthma.AimTo assess if ICS treatment before 2 years of age in children with obstructive airways disease reduces current asthma at 10 years of age.MethodsChildren with (n = 233) and without (n = 219) recurrent (r) bronchial obstruction (BO) attending clinical examination at 2 years of age in the birth cohort Environment and Childhood Asthma study in Oslo, were reinvestigated at 10 years of age. Current asthma (CA) at 10 years was defined as asthma with either symptoms and/or asthma treatment during the last year, and/or ⩾10% fall in forced expired volume in 1s after standardized treadmill run. The risk of CA was assessed by logistic regression and propensity modelling (including gender, parental atopy and severity score at 2 years) in children with rBO who received ICS or not by 2 years.ResultsCA was found in 97 children, more often among rBO children with (56.9%) and without ICS treatment (30.8%) compared to no-BO children (5.5%) (p