For Review Only. Sex-specific development of asthma differs between farm and non-farm children - a. cohort study. Jon Genuneit. Online Data Supplement

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1 Page 12 of Sex-specific development of asthma differs between farm and non-farm children - a cohort study. 3 Jon Genuneit 4 5 Online Data Supplement

2 Page 13 of Methods A prospective, population-based cohort study was conducted in rural areas around Ulm (Baden-Württemberg), southern Germany, with baseline assessment among 6-10 year old school children in 2006 as part of the GABRIELA study. Details of the study are published elsewhere.(e1) In brief, from n=23,040 participants in Baden-Württemberg, n=11,169 agreed to participate in a second phase of baseline including an in-depth assessment of environmental exposure and potential follow-ups. Disproportionate random samples stratified for categories of exposure to farming environments were drawn (in total n=2,497). Of these, parental questionnaires for 2,248 children were received. Figure E1 depicts the participation at each follow-up. The ethics committee of Ulm University approved the study. Accounting for the complex survey design, the data were analyzed with stratified weighted cox and logistic regression models using the Taylor series method to estimate variances. The sampling weights were calculated as fixed weights scaling the 2,248 baseline participants up to the 11,169 eligible participants. The baseline assessment was conducted in elementary schools which in the German school system have four grades spanning mostly from age six to ten years. Thus, at baseline in 2006, children from four consecutive years of birth (roughly ) were recruited. In the follow-up in 2013, most of the children were therefore years old, the age at which they were censored. Because they are approximately equally distributed across the four consecutive years of birth, about 25% are censored with each year from age 13 years onwards. The children s body weight and height was measured by trained fieldworkers at baseline and was parent-reported at the follow-ups in The parents were encouraged to measure weight and height and to indicate whether they provide measured or estimated figures. At the follow-ups in 2011, 2012, and 2013, the proportion of parents who indicated measured figures for both, weight and height, were 84%, 79%, and 78%, respectively. In addition, in 2013, the parents were asked to copy the child s body weight and

3 Page 14 of height from routine health examinations which are performed and documented by the caring pediatricians around the ages of 3 days, 4 weeks, 3 months, 6 months, 1 year, 2, 3, 4, 5, 7, and 9 years. The child s body mass index (BMI) was calculated as body weight divided by body height squared at each age at which both figures were available and standardized on German reference data.(e2) For the present analyses, BMI from age four years onwards was included. This was deemed sufficient because asthma diagnosis was only analyzed from age six years onwards. Children below the 10 th percentile were classified as underweight, children above the 90 th percentile were classified as overweight, and children in between were classified as normal (reference). The median number of available BMI measurements was five. Since BMI can change over time, BMI was fitted as a time-dependent explanatory variable in the stratified weighted cox regression models. At ages at which no BMI measurement was available, the BMI was imputed to be the previous measured BMI. In the logistic regression models, BMI in the year of life preceding asthma diagnosis or end of observation was fitted as a covariate. Physical activity was not included as a covariate because it was shown in the Swiss GABRIELA cohort that physical activity did not mediate or confound the protective farmeffect on asthma.(e3) Furthermore, in the Ulm cohort, data on physical activity was only ascertained at baseline at which the children were roughly six to ten years old. Supposedly, physical activity is age-dependent but this study lacks data on physical activity at various ages including the year of life preceding asthma diagnosis for most of the asthma cases and the year of life preceding the end of observation for most of the other participants. Thus, unlike BMI, physical activity could not be modelled as a time-dependent exposure or in relation to the age at asthma diagnosis which was a further reason why physical activity was not considered as a covariate.

4 Page 15 of References E1. Genuneit J, Büchele G, Waser M, Kovacs K, Debinska A, Boznanski A, Strunz-Lehner C, Horak E, Cullinan P, Heederik D, Braun-Fahrländer C, von Mutius E. The GABRIEL Advanced Surveys: study design, participation and evaluation of bias. Paediatr Perinat Epidemiol 2011;25: E2. Kromeyer-Hauschild K, Wabitsch M, Kunze D, Geller F, Geiß HC, Hesse V, von Hippel A, Jaeger U, Johnsen D, Korte W, Menner K, Müller G, Müller JM, Niemann-Pilatus A, Remer T, Schaefer F, Wittchen H-U, Zabransky S, Zellner K, Ziegler A, Hebebrand J. Perzentile für den Body-mass-Index für das Kindes- und Jugendalter unter Heranziehung verschiedener deutscher Stichproben. Monatsschrift Kinderheilkunde 2001;149: E3. Bringolf-Isler B, Graf E, Waser M, Genuneit J, von Mutius E, Loss G, Kriemler S, Braun- Fahrländer C. Association of physical activity, asthma, and allergies: a cohort of farming and nonfarming children. J Allergy Clin Immunol 2013;132: e4. 69

5 Page 16 of 18 Baseline (2006/07) n=2248 for details see Ref. E15 1 st Follow-up (2010) n=1928 (87%) drop out n=27 2 nd Follow-up (2011) n=1804 (81%) drop out n=75 3 rd Follow-up (2012) n=1770 (82%) drop out n=62 4 th Follow-up (2013) n=1547 (74%) Net study population with at least one follow-up n=2064 (92%) follow-up: 52 (3%) 2 follow-ups: 171 (8%) 3 follow-ups: 461 (22%) 4 follow-ups: 1380 (67%) Figure E1: Flow chart of follow-up including the year of assessment and participation in n (%) after correction for drop-out

6 Page 17 of Table E1: Characteristics of the study population (in unweighted n (weighted %)) all farm non-farm girls boys 2, (11.5) 1,181 (88.5) 1,037 (50.4) 1,027 (49.6) 74 number of siblings (7.8) 34 (3.7) 91 (8.2) 65 (7.7) 63 (8.3) (45.5) 280 (33.4) 535 (47.2) 405 (46.3) 415 (44.7) (30.8) 334 (37.0) 370 (30.0) 352 (30.5) 354 (31.1) (15.7) 223 (25.8) 178 (14.5) 210 (15.5) 192 (15.9) familial allergies 997 (55.1) 352 (41.7) 640 (56.9) 512 (57.0) 485 (53.1) parental smoking 1069 (58.6) 370 (46.3) 691 (60.1) 546 (59.2) 523 (58.1) parental high education 538 (31.0) 150 (17.4) 386 (33.0) 266 (30.9) 272 (31.2) body mass index (BMI) 1 underweight 99 (6.0) 34 (4.3) 65 (6.3) 47 (6.4) 52 (5.6) normal 1830 (90.1) 777 (90.1) 1043 (90.0) 919 (88.8) 911 (91.4) overweight 87 (3.9) 46 (5.6) 41 (3.7) 51 (4.8) 36 (3.0) asthma diagnosis 191 (10.7) 59 (6.8) 132 (11.4) 86 (10.3) 105 (11.2) 75 1 in the year of life preceding asthma diagnosis or end of observation

7 Page 18 of Table E2: Characteristics of the study population stratified by living on a farm at baseline and sex (in unweighted n (weighted %)) farm non-farm girls boys girls boys 446 (5.8) 426 (5.7) 588 (44.0) 593 (44.5) number of siblings 0 16 (3.1) 18 (4.4) 49 (8.3) 42 (8.1) (30.8) 149 (36.0) 273 (48.4) 262 (46.1) (39.0) 158 (35.1) 175 (29.3) 195 (30.8) (27.1) 101 (24.5) 87 (14.0) 91 (15.0) familial allergies 184 (41.7) 168 (41.6) 327 (59.3) 313 (54.7) parental smoking 197 (45.9) 173 (46.7) 347 (61.0) 344 (59.3) parental high education 75 (16.3) 75 (18.5) 190 (32.9) 196 (33.1) body mass index (BMI) 1 underweight 13 (3.0) 21 (5.6) 34 (6.9) 31 (5.7) normal 403 (91.8) 374 (88.5) 514 (88.4) 529 (91.6) overweight 25 (5.2) 21 (5.9) 26 (4.7) 15 (2.7) asthma diagnosis 25 (5.7) 34 (7.9) 61 (11.0) 71 (11.7) 1 in the year of life preceding asthma diagnosis or end of observation

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