Running Head: Interleukin-4 promoter and Molds and asthma
Bing-Fang Hwang, Ph.D., I-Ping Liu, M.Sc., Tzu-Pi Huang, Ph.D.
Bing-Fang Hwang, Ph.D.
Department of Occupational Safety and Heath College of Public Health
China Medical University Taichung, Taiwan
I-Ping Liu, M.Sc.
Department of Occupational Safety and Heath College of Public Health
China Medical University Taichung, Taiwan
2Tzu-Pi Huang, Ph.D
Department of Plant Pathology National Chung Hsing University Taichung, Taiwan
Tzu-Pi Huang
Correspondence to:
Dr. Bing-Fang Hwang,
Department of Occupational Safety and Health, College of Public Health, China Medical University No 91 Hsueh-Shih Rd, Taichung, Taiwan, 40402 R.O.C.
E-mail: [email protected] Telephone: +886-4-22053366 ext 6208 Telefax: +886-4-22071861
ABSTRACT
PURPOSE: To assess the role of gene-environment interaction between interleukin-4 (IL-4) promoter and mold exposure on the development of asthma.
METHODS: We conducted a cohort based, incident case-control study. The case group consisted of 188 children with new asthma and the control group (n=376) was matched for age and sex. The outcome of interest was the development of asthma during the 6-year study period. The studied determinants were three polymorphisms of IL-4 promoter (TT; CT and CC) and three indicators of exposure including histories of water damage, presence of visible molds, and perceived mold odor in the home.
RESULTS: Apparent joint effects between IL-4 promoter and mold exposure were observed on both additive and multiplicative scales. Specially, the risk of asthma was significantly associated with children carrying CT genotype and visible mold exposure comparing with those carrying TT genotype without any exposure indicator (adjusted OR 2.14, 95% CI
1.05-4.34) (modified Rothman synergy index for directly use of odds and odds ratios (s) =1.41;
p for interaction=0.03). A similar tendency was found (s=1.30; p for interaction=0.04) for children who were exposed to mold odor and carried CT genotype (adjusted OR 1.99, 95% CI 1.03-4.41)
CONCLUSIONS: The results of this study suggest gene-environment interaction between IL-4 promoter and indoor mold problem may play an important role in childhood asthma.
Key words: Interleukin-4 promoter, molds, damp housing, asthma, interaction, effect modification
INTRODUCTION
Asthma is the most common chronic childhood disease (1). It is characterized by recurrent respiratory symptoms, reversible airway obstruction, airway inflammation and increase airway responsiveness (2). One of the most important pathways on development of asthma involves IgE antibody responses to ubiquitous allergens. Interleukin-4 (IL-4) plays an important role in IgE synthesis by activating the pre-T helper cells that trigger isotype switching from IgM/IgG to IgE in B cells by promoting Th2 cell development (3). It is reasonable to expect that increase expression of IL-4 might lead to the development of asthma and therefore the IL-4 promoter region (chromosome 5q31) seems to be the most likely site for polymorphisms. One of the IL-4 promoter polymorphisms -589C/T (also referred to rs number 2243250) is close to the
glucocorticoid response element that positively stimulates IL-4 expression (4). Few studies of genetic epidemiology have assessed the relation between IL-4 promoter and childhood asthma in different populations, but these studies provided inconsistent results (5-8).
In our systematic Medline search, we identified only two previous longitudinal studies (9-10) and a population-based incident case-control study (11) which investigated exposure prior to the onset of asthma in children. All the three studies report that children exposure to dampness problems and molds increase the incidence of asthma (9-11). However, potential modification of the association between exposure to molds and the development of asthma by IL-4 promoter (i.e. gene-environment interaction) has not yet investigated.
We conducted a cohort based, incident case-control study to assess the independent and joint effect of IL-4 promoter and molds exposure on the risk of childhood asthma.
METHODS
Data Collection and Study Design
The source population included 44,000 children born in Taoyuan City between January 1, 1995 and December 31, 2002. Taoyuan is an urban-suburban municipality located across the north-western boarder of Taiwan. In the October 2002 baseline survey, a modified Chinese version of The International Study of Asthma and Allergies in Childhood (ISAAC-C)
questionnaire was used to collect information on children‟s health, environmental exposures, and other relevant factors. The parents or other guardians were asked to provide information on children's personal characteristics, health, details of the environment, and other relevant factors (Table 1). We approached a random sample of 2,253 children aged 1-7 from the source population. The response rate was 85.3%. We excluded 331 children with asthma (n=150), without asthma but with at least one asthmatic symptoms (n=160), or incomplete questionnaire (n=21). The study population included 1,922 children free of asthma and non-asthmatic symptoms at the cohort entry (baseline). The study design has been described in detail previously (12).
In October 2008, we conducted a cohort-based incidence case-control study. We identified 188 new cases who developed asthma during the study period between October 15, 2002 and October 31, 2008. The definition of new asthma subjects was determined by a positive response to the question “Has a physician ever diagnosed your child as having asthma during 2002-2008?” at the follow-up survey. Five questions related to current asthmatic symptoms were also asked at both baseline and follow-up surveys:
1. In the past 12 months, has your child dyspnoea with wheezing in the chest? (wheezing) 2. In the past 12 months, has your child‟s sleep been disturbed because of wheezing?
(night wheezing)
3. In the past 12 months, has whizzing ever been severe enough to limit your child‟s
speech to only one or two words at a time between breaths? (dyspnoea at rest) 4. In the past 12 months, has your child‟s sounded wheezy during or after exercise?
(exercise wheeze)
5. In the past 12 months, has your child had a dry cough at night, apart from a cough associated with a cold or chest infection? (night cough)
Control subjects were selected without asthma or asthmatic symptoms applying
one-to-two matching for age and sex from 2002 baseline survey of 1,922 children. The criteria of control subjects was (1) no physician diagnosed asthma or dyspnoea with wheezing in the past; (2) no positive response to any of the five questions concerning current asthmatic
symptoms. The final study population constituted 188 case and 376 control subjects. The study protocol was approved by the Institutional Review Board of China Medical University, and it complied with the principles outlined in the Helsinki Declaration.
IL-4 promoter Genotyping
Cotton swabs containing oral mucosa were collected and were immediately maintained at
−80°C throughout the transfer and storage. Genomic DNA will be isolated using phenol/chloroform extraction method.
The cotton swabs directly immersed in 300 μL cell lyses buffer (50 mm Tris-HCl, 1 mm EDTA, 0.1 m NaCl, pH 8.0) containing 2% SDS and 20 μg/mL proteinase K in a 1.5 mL micro centrifuge tube. After incubation overnight at 55°C, the swabs were discarded and the DNA in supernatants were purified by phenol/chloroform extraction and then precipitated with ethanol.
DNA fragments including the -589C/T variant were amplified a 198 base pair (bp) long by allele-specific polymerase chain reaction (PCR). The forward primer sequence from -683 to -633 nucleotides is 5' TGG GTA AGG ACC TTA TGG ACC 3' whereas the reverse primer sequence from -486 to -505 is 5' GGT GGC ATC TTG GAA ACT GT 3'. The reverse primer
was designed to contain a base substitution C→T at the fourth last nucleotide from its 3'end.
The individual PCR reaction vial contains a final volume of 20 μl solution. Two-hundred nanograms of DNA samples was added to 8 μl of PCR master mix consisting of 2.0 μl of 10X Mg Free PCR Buffer, 2.0 μl of 10 μM forward primer, 2.0 μl of 10 μM reverse primer and 2.0 ml of 10 mM dNTP. An appropriate amount of sterile ultrapure water (which totals up to 20 μl) was added to each of the microfuge tube. One micro liter of 5 units/μl Taq DNA polymerase was added to the reaction vial only after 5 minutes of pre-denaturation process prior to performing 'hot start' PCR. The PCR was performed using the Thermal Cycler (Applied Biosystems 9800®) for 35 cycles. The temperature for the initial denaturation of DNA was 94
°C for 1 minute, annealing at 60 °C for 1 minute and extension 72°C for 1 minute and a final extension at 72 °C for 5 minutes following the last cycle. The PCR product was subjected to BsmFI digestion for 2 hours at 37°C and electrophoresed on a 2.5% agarose gel with ethidium
bromide staining. The IL-4 promoter -589 C/T polymorphisms was visualized as 198 bp fragments for TT genotype, 120 pb and 78 pb fragments for CC genotype and 198 bp, 120 bp, 78 pb fragments for CT genotype. All assays were performed by a laboratory worker unaware of the clinical status of individual subjects, and genotype assignments were based on two consistent experimental results. About 15% of randomly selected samples were directly sequenced, and all of them were concordant with the initial genotyping results.
Environmental Determinants of Interest
We used three indicators of exposure defined from the answers to following structured questions at baseline:
Mold odor. "Have you perceived mold odor in your dwelling during the past 12 months?" (No;
Yes, almost daily; Yes, 1-3 days a week; Yes, 1-3 days a month)
Visible mold. "Have you ever had visible mold in your dwelling?"(No; Yes, during the past 12
months; Yes, only earlier.)
Water damage. "Have you ever had water damage in your dwelling?" (No; Yes, during the past 12 months; Yes, only earlier.)
Any exposure indicator. Presence of any of the three exposure indicators.
We decided to focus on exposures documented prior to the study period to ensure a plausible temporal sequence between exposure and the studied outcome for the causal inference.
Covariates
Information on potential confounders was obtained from the baseline questionnaire. The covariates in the present analyses included parental education, duration of breastfeeding, daily activity, furry/feathery pets and environmental tobacco smoke (ETS) (Table 1). The duration of breastfeeding was categorized into i) less than 1 months, ii) 1 to 5 months and iii) 6 months or longer. Daily activity was counted hours per day spent at activity levels corresponding to high (>4 hour/day), medium (2-4 hours/day) and low (<2 hours/day) as the reference category.
Parents education was categorized into i) < 9 years, ii) 10-12 years, iii) 13-16 years and iv) >
17 years, and four indicators variables were formed with i) as a reference category. ETS exposures were defined as paternal smoke only, maternal smoke only, or both paternal and maternal smoke exposure (yes) and none (no). Other covariates, such ascockroaches noted monthly, furry/feathery pets were dichotomous. Parental atopy was defined as the father or mother of the index child ever having been diagnosed as having asthma, or allergic rhinitis, or atopic eczema.
Statistical Methods
First, we estimated the incidence rate of asthma during the 6-year study period according to polymorphisms of IL-4 promoter and indicators of exposure to dampness and molds. In the crude analysis, matched odds ratio of the relations between exposure and outcome relations were estimated. We estimated adjusted odds ratios applying conditional logistic regression analysis. The matched odds ratios were adjusted for the aforementioned covariates.
Second, we studied the additive joint effects of IL-4 promoter and the two most relevant exposure indicators, namely „mold odor‟ and „visible mold‟ on the risk of asthma.
Because of small number of CC genotype, we compared the odds ratios (OR) of asthma in four exposure categories: 1) TT genotype and no exposure (OR00, reference category); 2) CT genotype and no exposure (OR10); 3) TT genotype and exposure (OR01); and 4) CT genotype and exposure (OR11). Then their odds (O) in four exposure categories (O00, O01, O10 and O11) were derived from the same conditional logistic regression model adjusting for the covariates.
On an additive scale, the attributable proportion due to interaction calculated by substituting odds ratios for risk ratios (AP) was suggested by Kalilani and Atashili (13):
Finally, the Rothman synergy index calculated by substituting odds ratios for risk ratios and its 95% CI were used to assess the joint effect of the two factors (14). The synergy index (S) modified for direct use of odds and odds ratios was employed for more accurate assessment of additive interaction (13). The synergy index was calculated using the following formula:
.
An observed synergy index value that departs substantially from the expected additive null, i.e., synergy index not equal to 1, suggests an additive interaction effect. The IR values and their variance covariance matrix were then used to calculate values for synergy index and 95% CIs (15). SAS version 9.2 was used for all statistical analyses. Furthermore, we studied the
multiplicative joint effect of IL-4 promoter and exposure by introducing interaction terms in the model. All tests assumed a two-sided alternative hypothesis and a 0.05 significance level.
RESULTS
Characteristics of Case and Control Subjects
Table 1 compares the demographic and environmental characteristics between the case and control subjects at baseline. The case subjects had lower duration of breastfeeding, more time spent outdoors and higher proportion of parental atopy compared with the control subjects, and were more commonly exposed to cockroaches (94.7 vs. 90.2%) and furry or feathery pets (30.3 vs. 27.1%) in the home.
Independent Effects of IL-4 Promoter and Exposure to Dampness and Mold problems
Hardy-Weinberg equilibrium tests showed non-significance (p>0.05) in both case and control groups. IL-4 promoter (CT vs. TT genotype) was not significantly associated with the risk of asthma with an adjusted odds ratio of 1.17 (95% CI 0.76-1.79) as shown in the Table 2.
There was a positive association between visible mold and mold odor (r=0.30). Water damage was not associated with visible mold, but positively associated with mold odor (r=0.22) (Table 3).Table 4 presents the matched odds ratios for asthma according to the three exposure indicators at baseline, as well as odds ratios contrasted to the reference category of no exposure. The risk of asthma was related to any indicator of exposure (adjusted OR 1.43, 95%
CI 1.01-2.13), presence of mold odor (adjusted OR 1.61, 95% CI 1.02-2.68), and visible mold (adjusted OR 1.50, 95% CI 1.01-2.31).
Joint Effect of IL-4 Promoter and Visible Mold
Table 4 shows the incidence rates of asthma in four categories representing the reference, independent effects of IL-4 promoter and any exposure indicator, and their additive joint effect.
Children carrying CT genotype without visible mold didn‟t have a significantly increased risk of asthma with an adjusted odds ratio (OR10) of 0.97 (95% CI 0.45 – 2.07)(Table 5). The effect of visible mold exposure on children with TT genotype increased with an OR01 of 1.35 (95%
CI 0.82-2.22). In children with both CT genotype and visible mold, the adjusted OR11 of asthma was 2.14 (95%CI 1.05-4.34), compared with children of the reference category. Thus the attributable proportion due to interaction of CT genotype and visible mold (AP) was 11.4%.
Additionally, the Rothman synergy index (s) calculated by substituting odds ratios for risk ratios was 1.41 (95% CI 1.01-3.47) greater than 1. It suggests additive and multiplicative interactions (p for interaction =0.03) between IL-4 promoter and visible mold exposure (Table 5).
Joint Effect of IL-4 Promoter and Exposure to Mold Odor
One the basis of the table 5, the joint effect for CT genotype and exposure to mold odor (adjusted OR11 1.99 95% CI 1.03-4.41), corresponded with the independent effects for CT genotype (adjusted OR01 0.99 95% CI 0.45-2.22) and exposure to mold odor (adjusted OR10 1.46 95% CI 0.83-2.59). Thus there was an apparent additive interaction (AP=8.9%) between IL-4 promoter and mold odor exposure (s=1.30 95% CI 1.03-2.35). In addition, a multiplicative interaction between IL-4 promoter and mold odor exposure (p for interaction =0.04) was also found (Table 6).
.
DISCUSSION
The results of present study, we found approximately 43%, 61%, and 50% increased the risk of development of asthma for children living in homes with any mold problem, mold odor, or visible mold respectively. Although the IL-4 promoter did not predict asthma, the results indicate that the joint effect of IL-4 promoter, representing genetic constitution, and exposure to visible mold and mold odor was stronger than expected on the basis of their independent effects in additive and multiplicative scales.
Validity of Results
A cohort-based incident case-control study offers an appropriate approach to assess the role of mold problems on the development of asthma. The prospective study design minimizes selection bias and information bias. A selection bias will be eliminated if the parents of children with asthma are more likely to change housing conditions after the first symptoms and signs of asthma compared with parents of healthy children. Information bias will not introduce if the parents of the symptomatic children report or recall similar exposure indicators
differently from the parents of healthy children.
The exposure assessment was based parental reporting at baseline rather than objective measurements, which is a limitation of the present study. Objective measurements were not yet used in any of the epidemiologic studies conducted at the time of the data collection. Visual observation by a trained person would also have improved the exposure assessment (11, 16).
However, our exposure information was collected before the onset of the asthma and therefore any bias due to awareness of the disease or exposure to molds was avoided. Another limitation was that it‟s not possible to address the changes in children‟s living environment during the 6-year period based on two surveys (baseline and follow-up).
We were able to take into account most of the known potential confounders related to individual characteristics, other environmental exposure in the condition logistic regression
analysis. However, dampness problems may also be related to other indoor environmental factors, such as dust mites. Dampness problems may also imply low ventilation rate and consequently increase the levels of indoor pollutants. We cannot rule out these indoor environmental factors will influence our results.
Synthesis with Previous Knowledge
We identified only three previous prospective cohort or incident case-control studies with incident asthma in children as the outcome of interest (9-11). Wickman and colleagues conducted a population-based birth cohort study of 4089 children in Stockholm, where they reported an increased risk of asthma among children in damp home environment during the first two years of life compared with unexposed with an adjusted odds ratio of 1.75 (95% CI 1.26-2.43). The exposure was defined as smell and visible signs of mold, water damage inside construction, and persistent windowpane condensation in dwellings with double-glazing (9).
Jaakkola and colleagues conducted a 6-year cohort study of 1916 children in Finland using self-reported exposure indicated an association between the risk of asthma and mold odor with an adjusted incidence rate ratio of 2.44 (95% CI 1.07-5.60), but not visible mold, and water damage (10). Pekkane and colleagues conducted an incident case-control study in Finland and found the presence of visible mold and moisture damage in main living parts increase the risk of asthma (11). The present study strengths the evidence that home dampness due to any problem of mold, mold odor, or visible mold increase the risk of development of asthma in childhood.
An important rationale for presenting the interaction on the additive scale is that it fits with the sufficient-component concept of causality (17). It has been suggested that the additive scale is more appropriate to assess “biologic interaction” which is implied by terms such as synergism or antagonism (18). We further performed test interaction on multiplicative scale as used in most genetic epidemiology literature. Our study shows apparent joint effects between IL-4 promoter and molds exposure on both additive and multiplicative scales.
The specific causal agents of asthma related to indoor dampness and mold problems are not well understood, and several potential causes have been suggested including molds, bacteria, house dust mites, and enhanced emission of chemicals from surface materials. Our results suggest that mold odor, and visible mold are important indicators of relevant exposure.
Several biological mechanisms by which indoor molds, particular concerning Penicillium, Aspergillus, Cladosporium and Alternaria could induce asthma have been suggested including immunoglobulin E (IgE) or immunoglobulin G (IgG)-mediated hypersensitivity reactions, toxic reactions caused by mycotoxins, and nonspecific inflammatory reactions caused by irrigative volatile organic compounds produced by microbes (MVOCs) or cell wall components, such as 1, 3-ß-D-glucan and ergosterol (20-26).
We found IL-4 promoter might not be a determinant of developing asthma in childhood, which was inconsistent with a recent meta-analysis of 14 studies, representing a pooled total of 2,476 asthmatic cases and 2,339 controls (8). The differences in direction of effects with IL-4 promoter genotypes may be due to chance, insufficient power, different populations or ethnic
We found IL-4 promoter might not be a determinant of developing asthma in childhood, which was inconsistent with a recent meta-analysis of 14 studies, representing a pooled total of 2,476 asthmatic cases and 2,339 controls (8). The differences in direction of effects with IL-4 promoter genotypes may be due to chance, insufficient power, different populations or ethnic