Online Repository Material: Text

Online Repository Material: Text

Content:

• Methods
• Description of studies in discovery and replication study
• Methodological consideration related to genome-wide interaction studies
• In utero smoke exposure GWIS - Details on the 27 SNPs in the discovery sample with a P-value<10-4
• Childhood tobacco smoke exposure GWIS - details on the 31 SNPs in the discovery sample with a P-value<10-4
• References
• Acknowledgements

Methods

Subjects

Data from nine individual studies were included in two meta-analyses: a meta-analysis on the interaction between single nucleotide polymorphisms (SNPs) and in utero smoke exposure and a meta-analysis on the interaction between SNPs and childhood smoke exposure (Table E1, Online Repository). All cases and controls were of European descent and two studies had a family structure. We focused on childhood onset asthma, because this disease has a strong genetic background interacting with environmental exposures. Childhood onset asthma was defined as asthma diagnosed by a doctor before the age of 16 years, which is consistent with the definition in the GABRIEL consortiumE1. In uterotobacco smoke exposure was defined as ‘exposure to maternal tobacco smoking at any time during pregnancy’. Childhood tobacco smoke exposure was defined as ‘exposure to passive tobacco smoking at any time from birth until 16 years of age’. Details on the outcome and exposure definition for the individual studies are provided in the Tables E1 to E4 (Online Repository).

Genotyping and quality control

Genotyping was carried out using the Illumina Human610 quad array ( at CEA-Centre National de Génotypage, Evry, France. Details on the genotyping method have been described previously.E1 We restricted our meta-analyses to SNPs fulfilling the following quality control criteria in each study: genotype missing rate <3% in cases and controls, minor allele frequency >5% in controls and consistency with Hardy-Weinberg equilibrium in controls (P-value>10-4). Samples were considered with >95% genotyping success rate. We excluded putative non-European samples, identified using EIGENSTRAT2.0 software.

Statistical analyses

The effects of in utero tobacco smoke exposure and childhood tobacco smoke exposure were analyzed separately. All individual studies were analyzed using a logistic regression model containing the genetic effect, the effect of tobacco smoke exposure and an interaction term indicating the interaction between the genetic effect and tobacco smoke exposure. This way deviation from a multiplicative model is tested. Gender, age and informative principal components for within-Europe diversity were included as covariates. For the studies containing family data, a cluster variable indicating the family relations was included. An additive genetic model was fitted to the data. For the discovery meta-analysis, we meta-analyzed the estimate of the interaction term of the individual studies. We calculated pooled interaction odds ratios (ORint) and 95% confidence intervals (CI) assuming a fixed effect model and a random effect model. A test for heterogeneity between studies was based on the Cochran’s Q statistic. We selected SNPs with an interaction P-value <10-4 based on the fixed effect model, and without heterogeneity (P-value Q-statistic <0.05) for follow-up in a replication set. Genome-wide significance would be reached at a P-value <9.3*10-8 with the number of SNPs tested. Our threshold for further follow-up was an arbitrary chosen P-value of <10-4 based on the number of SNPs meeting that threshold to select SNPs for the replication study. The selected SNPs were tested in a replication study consisting of four independent studies (Description of studies see Tables E1 to E4, Online Repository) and we meta-analyzed these in the combined set of discovery and replication cohorts as well. The effect of the selected SNPs among exposed and unexposed separately was also calculated in a stratified analysis. Analyses were conducted using Plink 1.07E2 and RE3. For annotation and inspection of linkage disequilibrium (LD) patterns WGAviewerE4 was used. A network analyses was performed for the SNPs with the largest interaction effect using GeneMANIA algorithm ( to inspect a possible common pathway.

Description of studies in discovery and replication study

British 1958 birth cohort (B58C)

The 1958 British birth cohort is an ongoing follow-up of persons born in Britain during one week in 1958 ( At age 44-45, a full biomedical examination was performed from which DNA samples were prepared for use as a nationally representative reference series for genetic case-control studies.E5 About half of the cohort members with a history of asthma ascertained at any age up to 42 years, and a similar number of non-asthmatic controls, were included in the GABRIEL meta-analysisE1. For the purpose of this interaction analysis, childhood asthmatics were defined as persons reporting asthma ever at any follow-up whose parents also reported that they had asthma and/or wheezy bronchitis at any age up to and including 16 years. Maternal smoking after the fourth month of pregnancy was ascertained by interview with a midwife shortly after birth. Postnatal smoke exposure was ascertained from interview questions about maternal smoking and paternal smoking, administered at the 16-year follow-up.

ALSPAC

The Avon Longitudinal Study of Parents and Children (ALSPAC) is a population-based, birth cohort recruited during pregnancy. Pregnant women resident in Avon, United Kingdom with estimated dates of delivery between 1st April 1991 and 31st December 1992 were recruited through antenatal clinics.E6 From 14,451 women recruited, there were 14,072 live births and 13,988 children were alive at age one year. Children were followed from birth using self-completion questionnaires sent to their mothers at approximately annual intervals and, from 7 years of age, in yearly dedicated research clinics. Asthma is defined as an affirmative answer to a question at 91 months (approximately 7½ years), “Has a doctor ever told you that your child had asthma?” Data on passive smoke exposure in utero was collected from questionnaires sent to mothers during pregnancy at 18 weeks gestation. Postnatal exposure was derived from two questionnaires sent to mothers 5 or 6 times between 6 months and 78 months after birth. In utero smoke exposure was defined as “The mother smoked regularly in first 3 months of pregnancy (1 cig/day)”. Childhood smoke exposure was defined as a positive response at any time to “The mother smokes regularly (1 cigs/day currently smoked)” or “The child is exposed to a room or enclosed place where people are smoking during weekdays or weekends”. A total of 3,030 children had DNA available for genotyping and data on asthma at 91 months as well as other asthma related phenotypes. Of these, 625 (20.6%) had asthma and an equal number of controls was selected at random from the remainder. After ALSPAC-QC a total of 1,139 subjects were retained in the GWI study analysis on in utero smoke exposure and 1,025 subjects were retained in the GWI study analysis on childhood smoke exposure.

BAMSE

Between 1994 and 1996, 4,089 newborn infants were recruited in the BAMSE study, and questionnaire data on baseline study characteristics were obtained.E7The catchment area included central and north-western parts of Stockholm. At approximately one, two, four, and eight years of age, parents completed questionnaires on their children’s symptoms related to asthma and other allergic diseases. The response rates were 96%, 94%, 92% and 84%, respectively. At eight years of age, all children of the BAMSE study were invited to clinical testing where blood samples were obtained from 2,480 children. DNA was extracted from 2,033 samples after exclusion of samples with too little blood, lack of questionnaire data, or if parental consent to genetic analysis of the sample was not obtained. From these samples, all children with a doctor’s diagnosis of asthma (ever) were selected as cases and children with no history of asthma or other allergic diseases were selected as controls. Data on passive smoke exposure was collected from repeated questionnaires filled out by parents. In utero smoke exposure was defined as “The mother smoked at least one cigarette per day in any point of time during the pregnancy” as reported in the baseline questionnaire at approximately 2 months. Childhood smoke exposure was defined as “Any of the parents smoked at least one cigarette per day” at the time of any questionnaire, distributed at 2 months, 1, 2, 4 and 8 years. After genome-wide data QC and exclusion of subjects with missing values on smoke exposure a total of 484 subjects were retained in the GWI study analyses.

ECRHS

Sixteen centres (eight countries) in the European Community Respiratory Health Survey (ECRHS) have contributed samples to GABRIEL ( In each centre, a representative community-based sample of at least 3000 adults aged 20-44 years were invited to complete a brief postal questionnaire asking about respiratory symptoms (ECRHS I - Stage 1) between 1991-1993. A random sample of these (600 per centre) underwent intensive further investigation (ECRHS I - Stage 2 – random sample). Participants who had symptoms highly suggestive of asthma but who had not been selected at random to take part in Stage 2, were also invited to undergo intensive investigations (ECRHS I - Stage 2- enriched sample). About ten years later all adults who had taken part in Stage 2 were recontacted (ECRHS II) and again asked about respiratory symptoms. Samples suitable for DNA extraction were collected. For the GABRIEL initiative all cases of asthma were identified (participants from the random or enriched sample who said yes to the question „Have you ever had asthma? at either ECRHS I or ECRHS II). Controls were a random sample who answered “no to the same question in both surveys”. In utero smoke exposure was defined as any smoking by the mother during pregnancy. Childhood smoke exposure was defined as either mother or father smoked regularly during childhood (ECRHS1 main questionnaire).

EGEA

EGEA is a 12-year longitudinal survey which combines a case-control study and a family study ( The first survey (EGEA1) took place between 1992 and 1995.E10 The study population included 388 asthmatics recruited in chest clinics and their 1,244 family members plus 415 population-based controls (total of 2,047 subjects). The probands (asthmatics and controls) were between 7 and 70 years old at time of study. All probands and their two parents were of European ancestry and were born in France. The second survey (EGEA2) was conducted between 2003 and 2007 and included follow-up data in 1,543 subjects from the initial cohort and 58 new family members.E11 Data collected through face-to-face interviews and examination included extensive phenotypic characterization (detailed clinical data based on standardized questionnaire, skin prick tests, lung function tests, bronchial responsiveness, blood samples, white blood cell counts, total IgE), data on risk factors (environmental exposures, diet, physical activity, hormone-related events) and drug consumption. For the GABRIEL initiative, asthma was defined by a positive answer to the question "Have you ever had asthma attack "at EGEA1 (or at EGEA2 if not examined at EGEA1). In utero smoke exposure was defined in adults as an affirmative answer on the question “When your mother was pregnant, in particular with you, did she stop smoking during pregnancy?” Childhood smoke exposure was defined as an affirmative answer on either question “Did your mother ever smoke during your childhood (before 16 years old)?” or “Did your father ever smoke during your childhood?” (for adults) and “Did his/her mother ever smoke when he/she was younger than 2 years?” or “Did his/her father ever smoke when he/she was younger than 2 years?” or “Did his/her father or mother or someone smoke in the household at the time of the questionnaire in EGEA1?” (for children).

GABRIELA

The GABRIELA study was carried out as a population survey in five rural regions of Europe: Baden-Württemberg and Bavaria in Germany, North/Central Switzerland, Tyrol in Austria, and Lower Silesia in Poland. The Polish center was not included in the GWI study analysis. Children aged 5-13 years were recruited through primary schools. A recruitment questionnaire was sent out to 132,366 children, of whom 79,888 (60.4%) returned their completed questionnaires (phase 1), containing questions on asthma. Of these children, 34,491 were eligible for phase 2 as defined by a documented parental consent to dust sampling, blood sampling, and genetic analyses and nationality of their study country. To enrich informative observations a stratified random selection process was applied in two steps. In a first step, 9,668 children were selected for phase 2 within the three exposure strata per center. In the second step, 1,708 children were selected for genotyping within exposure and outcome strata per center. In utero smoke exposure was defined as any smoking by the mother during pregnancy. Childhood smoke exposure was defined as current smoking by the father or the mother. Details on the study design are described in: Genuneit J et al., The GABRIEL Advanced Surveys: study design, participation, and evaluation of bias.E12

PIAMA

The PIAMA study is a birth cohort study of children born between 1996-1997. Details of the study design have been published previously.E13 10,232 pregnant women completed a validated screening questionnaire at their prenatal health care clinic (n=52). Based on this screening, 7,862 women were invited to participate, of whom 4,146 women agreed and gave informed consent. Mothers reporting a history of asthma, current hay fever or allergy to pets or house dust mite were defined as allergic. Children were recruited during the first trimester of pregnancy. Follow-up of the children took place at 3 months of age and yearly from 1 to 8 years of age. At eight years of age, all children were invited to clinical testing including collection of blood samples for DNA extraction. The Medical Ethical Committees of the participating institutes approved the study, and all participants gave written informed consent. The response rates to the annual questionnaires ranged from 3764 at age 1 to 3269 at age 8 years. For the GWI study, DNA from childhood asthma cases (children who have ever had a doctor’s diagnosis of asthma at age 8 years) and a set of non-asthmatic controls (children who never had a doctor’s diagnosis of asthma and who have never wheezed at age 8 years) were provided. In utero smoke exposure was defined as any smoking by the mother during pregnancy from week 4 of pregnancy. Childhood smoke exposure was defined as any smoking by the father or the mother inside the house between the child’s birth and 8 years of age.

SAPALDIA

SAPALDIA contributed all self-reported asthma cases as well as a random sample of controls. These subjects were obtained from among 6,055 SAPALDIA cohort subjects that participated in both, the baseline (1991) and follow-up (2002) examinations and agreed to providing blood for genetic analysis. SAPALDIA is a population-based cohort that originally recruited subjects aged 18 to 60 from population registries in eight Swiss communities representing the three largest language groups (German, French, Italian) as well as different levels of air pollution, altitude and degrees of urbanization. At both baseline and follow-up examination subjects underwent spirometry as well as a detailed interview on respiratory health, smoking history and lifestyle factors. At follow-up, 8,047 of 9,651 baseline subjects re-participated in at least one part of the study and a formal biobank was established. SAPALDIA questions about smoking and asthma status were equivalent to those used by the ECRHS. Asthma status was defined by an affirmative answer to the question “Have you ever had asthma” at baseline and/or follow-up interview. Childhood smoke exposure was defined as positive answer to either of the questions “Did your father regularly smoke when you were a child?” or “Did your mother regularly smoke when you were a child?”. In utero smoke exposure was present when the person’s mother either continuously smoked, or reduced or stopped smoking during pregnancy. Exposure during pregnancy was asked upon affirmatively answering the question on regular smoking of the mother during the person’s childhood.

TOMSK

TOMSK is a population-based family study conducted by the Research Institute of Medical Genetics and SiberianStateMedicalUniversity (Tomsk, Russia) from 1998 onwards.E14 Both nuclear families and extended pedigrees were recruited through atopic bronchial asthmatic probands. All participants were Russians or of a mixed ethnic origin due to marriages between Russians and major East Slavonic populations (Ukrainians, Byelorussians). Altogether, 196 families were studied, out of which 150 families were recruited in TomskRegionChildrenHospital and TomskRegionHospital (Tomsk, Russia), and 46 families were recruited in the city of Irkutsk hospitals by the staff of the Irkutsk State Institute of Doctors Advanced Training (Irkutsk, Russia). Both probands and their relatives were clinically examined to establish diagnosis of asthma and atopy by the GINA criteria (Global Initiative for Asthma: Global Strategy for Asthma Management and Prevention. Besides the clinical examination, laboratory and functional testing were conducted to assess common IgE levels (solid-phase immune-enzyme assay), specific sensitization (skin-prick tests), lung volumes (spirometry), and airway responsiveness (bronchoprovocative tests with methacholine). For the GWI study analysis, only children were included. Childhood smoke exposure was defined as an affirmative answer to the question “Are you currently exposed to passive smoking?”.