Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Genetic variation in inducible nitric oxide synthase promoter, residential traffic related air pollution and exhaled nitric oxide in children
(USC Thesis Other)
Genetic variation in inducible nitric oxide synthase promoter, residential traffic related air pollution and exhaled nitric oxide in children
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
GENETIC VARIATION IN INDUCIBLE NITRIC OXIDE SYNTHASE PROMOTER,
RESIDENTIAL TRAFFIC RELATED AIR POLLUTION AND EXHALED NITRIC
OXIDE IN CHILDREN
by
Pi-chu Kaylene Lin
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Applied Biostatistics and Epidemiology)
AUGUST 2013
Copyright 2013 Pi-Chu Kaylene Lin
ii
ACKNOWLEDGEMENTS
I am greatly indebted to my thesis committee chair and advisor, Dr. Frank D.
Gilliland for long-term support and encouragement to all aspects of my work on this
thesis as well as my life at USC. I would like to express my deepest gratitude to Dr. Md.
Towhid Salam. This manuscript would not have been possible without his vision and
guidance. I would like to express my upmost appreciation to Dr. W. James Gauderman
for reviewing the thesis. I would also like to thank Dr. Sandy Eckel for her insightful
comments and suggestions and Xia Iona Li for support with database management. It has
been my honor to work with and learn from such dedicated mentors and researchers.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES iv
LIST OF FIGURES v
ABBREVIATIONS vi
ABSTRACT vii
INTRODUCTION 1
METHODS AND PROCEDURES 3
Study Design and Subjects 3
FeNO measurement 3
Traffic-related exposure 4
Buccal sample collection and processing 4
Selection of NOS2 promoter haplotypes 4
Assessment of covariates 5
Statistical analysis 5
RESULTS 7
Characteristics of the study population and association with FeNO 7
Distribution and correlation of exposure and NOS2 promoter haplotypes 7
Influence of NOS2 promoter haplotypes on road length measures and FeNO
association
9
Influence of Asthma on the associations of NOS2 haplotype and road length
on FeNO
11
DISCUSSION 14
CONCLUSION 17
REFERENCES 18
iv
LIST OF TABLES
Table 1. Selected characteristics of the study population and their bivariate
relationships with FeNO
8
Table 2. Distributions and correlations of local road lengths 9
Table 3. Pairwise Spearman correlation and Haplotype frequency of the NOS2
promoter haplotypes
9
Table 4. Influence of NOS2 promoter haplotypes on the association between local
road lengths measures and FeNO
10
Table 5. Joint effects of NOS2 H1 promoter haplotype and length of local road
within 100m and 200m buffers on FeNO
11
Table 6. Joint effects of asthma, NOS2 promoter haplotype H1 and road length on
FeNO
13
v
LIST OF FIGURES
Figure 1. Influence of NOS2 H1 haplotype copies on the association of length of
local roads around home and FeNO
12
vi
ABBREVIATIONS
CHS: Children’s Health Study
FeNO: Fractional concentration of exhaled nitric oxide
htSNP: Haplotype-tagging single nucleotide polymorphism
iNOS: Inducible nitric oxide synthase
NO: Nitric oxide
NO2: Nitrogen dioxide
NOS1: Nitric oxide synthase isoform 1
NOS2: Nitric oxide synthase isoform 2
NOS3: Nitric oxide synthase isoform 3
ROS: Reactive oxygen species
SNP: Single nucleotide polymorphism
TRP: Traffic related pollution
vii
ABSTRACT
Background: Fractional concentration of nitric oxide in exhaled air (FeNO) is a
biomarker of airway inflammation. Nitric oxide synthase 2 (NOS2) in airway epithelium
has been recognized as the major source of NO in exhaled breath. Earlier work has shown
that common promoter haplotypes in NOS2 and total length of local roads around homes,
a metric of residential traffic related pollution, affect FeNO level in children.
Aims: The aims of this study were to examine the joint associations of NOS2 promoter
haplotypes and length of local roads around homes and FeNO and to assess the influence
of asthma on these associations in children.
Methods: The study included 2,457 (7 to 11 year-old) children of the Southern California
Children’s Health Study. FeNO was measured at school during 2005-2006. Lengths of
local roads within circular buffers (50m, 100m and 200m) around the participant
residence were estimated using GIS methods. Two common promoter haplotypes in
NOS2 that have been associated with FeNO, asthma and lung function growth in children
were selected. Linear regression was utilized to examine the independent and joint
associations of NOS2 promoter haplotypes and road length measures on FeNO.
Results: We observed joint effects of length of local roads within 100m and 200m buffer
and the most common haplotype for FeNO (P-values for interaction ≤0.03). In children
who had ≤250m of road within100m buffer around home, those with two copies of the
haplotype had significantly lower FeNO (adjusted mean FeNO=10.71ppb; 95%
confidence intervals (CI): 9.10-12.61) than those with no copies (adjusted mean FeNO
14.18ppb; 95% CI: 13.09-15.36). This protective effect of the haplotype was not
observed in children who had >250m road lengths within 100m buffer. Similar joint
viii
effects of this haplotype and road lengths within 200m buffer on FeNO was observed.
These joint effects were not influenced by asthma.
Conclusion: Our results indicate that the protective effect of a common NOS2 promoter
haplotype on NO synthesis in the airway is not evident in children who live near higher
levels of local traffic.
1
INTRODUCTION
The fractional concentration of nitric oxide (NO) in exhaled air (FeNO) is a
noninvasive biomarker of airway inflammation (1-3) and predicts asthma risk in children
(4, 5). Nitric oxide (NO) plays an important role in regulating airway smooth muscle tone
and is involved in a variety of physiological and pathological processes (6). NO is
generated endogenously from L-arginine by NO synthase (NOS). Two constitutive
(encoded by NOS1 and NOS3) and one inducible isoforms of NOS (iNOS, encoded by
NOS2) are the sources of airway epithelium-derived NO (7); nevertheless, FeNO appears
to be predominantly determined by NOS2 (8).
Atopic conditions (asthma and allergy), genetic, and environmental exposures are
some of the major determinants of FeNO level. We and others have found that children
with asthma and allergic rhinitis have significantly higher FeNO than children without
these conditions (9, 10). Using data from the Southern California Children's Health Study
(CHS), we found that the two most common promoter haplotypes in NOS2 significantly
affected FeNO levels in children (11, 12) and were also associated with asthma risk and
lung function growth in children (13).
Traffic exhaust is considered to be the one of the main sources of environmental
pollution in urban areas. Higher FeNO has been attributed to urban pollution (14, 15).
Reactive oxygen species (ROS) and free radicals generated by traffic exhaust have
adverse health effect on the respiratory system in children (16). We and others have
found that length of roads around the home was associated with higher FeNO in children
(17-19).
2
Based on the aforementioned evidence, we hypothesized that 1) common NOS2
promoter haplotypes influence the relationship between road length around homes and
FeNO and 2) the joint effects of NOS2 promoter haplotypes and length of road traffic
exposure on FeNO vary by asthma status in children.
3
METHODS AND PROCEDURES
Study Design and Subjects
The subjects were kindergarten and first grade school children (7-11 years old)
who were recruited in a new cohort of the Children’s Health Study in 2003 from 12
communities of southern California. The detailed study design and subject recruitment
have been described earlier (11, 20). Briefly, parents or guardians of study participants
completed the written informed consent and questionnaires and the University of
Southern California Institutional Review Board approved the protocol. In 2005-2006,
FeNO data were acquired from 2,948 children, of whom 2605 children were non-
Hispanic and Hispanic white. Due to missing genotyping data on 90 children, there were
2515 Hispanic and non-Hispanic children who had both genotype and FeNO data.
Among these children, local length of road measures was not available on 58 children.
Therefore, a total of 2457 children were accordingly included in the final analysis.
FeNO measurement
Details of FeNO collection, quality control and data transformation have been
described earlier (21, 22). FeNO was collected from children at school during October to
June in 2005-2006 using an offline (100 ml/sec flow) technique based on American
Thoracic Society (ATS) guidelines (23). Collection was mostly processed in the
midmorning to noon to avoid potential effects of traffic-related peaks in ambient nitrogen
oxide (NO) and recent food intake. Children with acute respiratory infection within the
prior 3 days were excluded or scheduled revisiting. The offline measurements were
validated by an online (50 ml/sec flow) technique which was performed in a subset group
4
(N=361) of participants. We were able to reliably predict online FeNO from offline
FeNO (adjusted R2 = 0.94) by incorporating ambient NO, and lag time between time of
collection and FeNO measurement in a regression model. Offline FeNO data were
transformed to predicted online FeNO data for these analyses, as in our previous work.
Traffic-related exposure
Because our earlier work showed that total local road lengths within 50m, 100m
and 200m buffers around subjects' home were associated with higher FeNO, we
evaluated the joint effects of the two most common NOS2 promoter haplotypes and these
road length measures on FeNO in this study. The road length within these circular buffers
around each participant’s residence was calculated based on TeleAtlas Multi-Net road
class data (TeleAtlas 2002). Data of length of local roads obtained from major or minor
collectors were equivalent to functional road class (FRC) 5 or FRC6, respectively (19).
Buccal sample collection and processing
Buccal cell specimens were collected from children at school under the
supervision of study staff. Genomic DNA was extracted from buccal cell pellets using
PUREGENE DNA isolation kit (cat #D-5000; GENTRA, Minneapolis, MN). The
extracted DNA samples were resuspended in the hydration solution (GENTRA) and
stored at -80°C (11).
Selection of NOS2 promoter haplotypes
The criteria for haplotype-tagged single nucleotide polymorphism (htSNPs)
selection and genotyping methods have been described in detail previously (11). Seven
SNPs were included to estimate the haplotype in NOS2 promoter region. These htSNPs
5
had minor allele frequency ≥0.05 and were able to explained >90% of the haplotype
diversity of the NOS2 promoter region. Haplotypes for each racial/ethnic group were
estimated using the TagSNPs program (http://www-rcf.usc.edu/~stram/tagSNPs.html).
We used 233 ancestry informative markers (AIMs) to determine genetic ancestry and
account for population stratification. Genotyping was done using the Illumina BeadArray
platform. All SNPs had call rates greater than 99%. We used the STRUCTURE program
(available at http://pritch.bsd.uchicago.edu/structure.html) to differentiate the four major
ancestral populations. More information on the basic algorithm of the program and
methods utilized in similar multiethnic populations has been published elsewhere (11, 24-
26).
Assessment of covariates
Information of race/ethnicity, physician-diagnosed asthma, history of respiratory
allergy (allergic rhinitis and/or hay fever), asthma medication use during the past 12
months, annual family income, parental education and exposure to secondhand tobacco
smoke were based on parental reports. Height and weight were measured on the day of
FeNO testing. Age- and sex-specific body mass index (BMI) percentiles was computed
using the Centers for Disease Control and Prevention body mass index growth charts
(http://www.cdc.gov/NCCDPHP/dnpa/growthcharts/resources/sas.htm) and categories of
BMI (e.g., underweight, normal, overweight and obese) were defined.
Statistical analysis
Predicted online FeNO was natural-log-transformed because the FeNO
distribution was right-skewed. The road length measures were centered at their respective
6
means and were scaled to 100m, 300m, and 1000m for total length of roads in 50m,
100m, and 200m buffers, respectively. Descriptive analyses were performed to describe
characteristics of the study population. Spearman correlations were used for pairwise
correlations of NOS2 promoter haplotypes and of road length measures. Linear regression
models with appropriate interaction terms were used to examine the joint effects of NOS2
promoter haplotypes and road length measures on FeNO level using likelihood ratio tests
(LRTs). Pairwise comparisons were done to identify statistically significant difference in
adjusted geometric mean FeNO level between categories of children who had different
profiles based on haplotype copy number and dichotomized road length measures. All the
models were adjusted for race/ethnicity, genetic ancestry and community of residence.
We did not adjust for further covariates (i.e., child's asthma status, history of respiratory
allergy, age and SES) because none of these factors changed the effect estimate by more
than 5%. All tests were two-sided at a 5% significance level. All analyses were
performed using the Statistical Analysis System software (SAS version 9.3; SAS Institute
Inc., Cary, NC).
7
RESULTS
Characteristics of the study population and association with FeNO
The average age of children was 9 years with nearly equal proportion of boys and
girls (Table 1). The majority of subjects were Hispanic white (61.3%). More than half of
the children (56%) had history of respiratory allergy and 12.6% of children had
physician-diagnosed asthma. Age, asthma and respiratory allergy were positively
associated with elevated FeNO (all P <0.0001); however, sex, race/ethnicity,
socioeconomic status (reflected by parental education and annual family income) and
BMI were not significantly associated with FeNO.
Distribution and correlation of exposure and NOS2 promoter haplotypes
The distributions and correlations of local road lengths around homes are
presented in Table 2. Within 50m, 100m, and 200m circular buffers around subjects'
home, the average road lengths were 116m, 365m, and 1344m, respectively. These traffic
exposure metrics were statistically significantly correlated (P <0.0001), with stronger
correlation between road lengths within 100m and 200m buffers. The two most common
NOS2 promoter haplotypes, H1 and H2, represented more than 60% of the haplotype
diversity in the study population (Table 3). These haplotypes have modest negative
correlation (Spearman correlation coefficient = -0.39, P <0.0001).
8
Table 1. Selected characteristics of the study population and their bivariate relationships
with FeNO
*
Characteristics N
†
%
Geometric Mean FeNO
(ppb) (95%CI)]
P-value
‡
Age (years) [mean range]
**
2457 9.3 (7.3-11.5) 14.0% (9.3 to 18.9) <0.0001
Sex
Girls 1248 50.8 15.01 (13.61 to 16.56) 0.33
Boys 1209 49.2 14.60 (13.24 to 16.10)
Race/Ethnicity
Non-Hispanic white 951 38.7 14.56 (13.67 to 16.58) 0.33
Hispanic white 1506 61.3 15.05 (13.15 to 16.13)
Asthma
No 2148 87.4 13.12 (12.04 to 14.30) <0.0001
Yes 309 12.6 16.70 (14.85 to 18.79)
History of respiratory allergy
No 1079 44.0 13.80 (12.46 to 15.28) <0.0001
Yes 1376 56.0 15.88 (14.46 to 17.45)
Exposure to secondhand smoke
No 2122 95.0 14.78 (13.74 to 15.92) 0.97
Yes 113 5.0 14.82 (12.86 to 17.09)
Body mass index categories
Underweight (<5
th
percentile) 44 1.8 15.35 (12.21 to 19.31) 0.97
Normal (5
th
-85
th
percentile) 1505 61.5 14.60 (13.47 to 15.84)
Overweight (85
th
to 95
th
percentile) 381 15.6 14.57 (13.18 to 16.11)
Obese (≥95
th
percentile) 517 21.1 14.71 (13.39 to 16.15)
Parental education
<12
th
grade 500 21.3 15.67 (14.02 to 17.51) 0.13
12
th
grade 436 18.6 14.89 (13.34 to 16.62)
Some college 894 38.1 15.38 (13.92 to 17.00)
College 278 11.8 14.48 (12.81 to 16.36)
Some graduate 240 10.2 13.69 (12.06 to 15.54)
Annual family income ($)
<14,999 312 14.8 15.11 (13.40 to 17.04) 0.79
15,000-49,999 695 33.1 14.73 (13.28 to 16.33)
≥49,999 1096 52.1 14.59 (13.26 to 16.05)
*Study subjects included Hispanic and Non-Hispanic white children in the Children’s Health Study who had FeNO
measured in 2005-2006 and had genotypic and exposure (local road lengths around homes) data available.
†
Numbers do not always add up due to missing values.
‡
P-values testing overall association of the variable with FeNO level.
**
Age (continuous) is presented as mean and range; percent difference in FeNO per year increment in age is presented
with 95% CI.
Table 2. Distributions and correlations of local road lengths
Local road lengths within circular
buffers (m)
Mean SD Median
25
th
percentile
75
th
percentile
Local
road
length in
50m
buffer
Local
road
length in
100m
buffer
Local road length in 50m buffer (m) 115.6 48.5 100.0 98.2 145.1
Local road length in 100m buffer (m) 365.2 155.3 362.2 258.3 469.4 0.60*
Local road length in 200m buffer (m) 1344.3 514.6 1380.9 1014.6 1709.3 0.42* 0.77*
*P <0.0001 for Spearman correlation coefficients
SD, standard deviation
9
Table 3. Haplotype frequency of the NOS2 promoter haplotypes
Haplotype*
Haplotype frequency
Non-Hispanic white Hispanic white
h0111101 (H1) 0.35 0.31
h1000010 (H2) 0.27 0.37
*SNP order in NOS2A promoter haplotypes is rs4795080-rs2779253-rs1889022-rs10853181-rs2531866-rs1014025-
rs2531872 3. Within each haplotype (h), ‘0’ and ‘1’ represent the common and the variant alleles at the ordered SNP
position, respectively.
Influence of NOS2 promoter haplotypes on road length measures and FeNO
association
NOS2 H1 promoter haplotype modified the associations of length of local roads
around 100m and 200m buffers and FeNO (both p-values for interaction ≤0.03; Table 4).
However, we did not found any significant interactions between NOS2 H2 promoter
haplotype and any of the local road length measures around home. To further investigate
the joint effects of copies of H1 haplotypes (i.e., 0, 1 and 2) and local road lengths within
100m and 200m buffer on FeNO level, we dichotomized the road length measures by
choosing a cutpoint close to their 25th percentile values (i.e., 250m and 1000m for road
lengths around 100m and 200m buffers, respectively). We found that children with 2
copies of the H1 haplotype who had fewer roads within 100m and 200m buffer around
their homes had significantly lower FeNO compared to children who had similar
exposure but no copies of the H1 haplotype (Table 5 and Figure 1). Furthermore, this
protective effect of H1 haplotype on FeNO was abrogated in children who lived in homes
surrounded by more roads (i.e. had higher road lengths). Selecting different cut-points for
road lengths using quartiles provided similar results (data not shown).
10
Table 4. Influence of NOS2 promoter haplotypes on the association between local road lengths measures and FeNO
Factor*
Estimates
(95% CI, ppb)
†
Factor*
Estimates
(95% CI; ppb)
†
Joint effects of H1 haplotype and road lengths within 50m buffer Joint effects of H2 haplotype and road lengths within 50m buffer
H1 -0.047 (-0.086 to -0.009) H2 0.017 (-0.020 to 0.054)
Local road lengths within 50m buffer 0.051 (-0.001 to 0.102) Local road lengths within 50m buffer 0.053 (0.002 to 0.105)
H1 x Local road lengths within 50m buffer 0.067 (-0.010 to 0.144) H2 x Local road lengths within 50m buffer 0.001 (-0.072 to 0.073)
Interaction p-value 0.09 Interaction p-value 0.99
Joint effects of H1 haplotype and road lengths within 100m buffer Joint effects of H2 haplotype and road lengths within 100m buffer
H1 -0.047 (-0.085 to -0.008) H2 0.017 (-0.020 to 0.054)
Local road lengths within 100m buffer 0.055 (0.006 to 0.105) Local road lengths within 100m buffer 0.057 (0.008 to 0.107)
H1 x Local road lengths within 100m buffer 0.087 (0.014 to 0.160) H2 x Local road lengths within 100m buffer -0.001 (-0.070 to 0.068)
Interaction p-value 0.02 Interaction p-value 0.98
Joint effects of H1 haplotype and road lengths within 200m buffer Joint effects of H2 haplotype and road lengths within 200m buffer
H1 -0.046 (-0.084 to -0.007) H2 0.017 (-0.019 to 0.054)
Local road lengths within 200m buffer 0.063 (0.010 to 0.117) Local road lengths within 200m buffer 0.066 (0.013 to 0.120)
H1 x Local road lengths within 200m buffer 0.080 (0.007 to 0.154) H2 x Local road lengths within 200m buffer 0.014 (-0.057 to 0.085)
Interaction p-value 0.03 Interaction p-value 0.70
*Road length variables were centered at their respective mean values. The 'x' between factors represents interaction terms.
†Estimates (95% confidence intervals) represent natural log transformed FeNO obtain from multivariate liner regression models associated with each factor. All models
were adjusted for race/ethnicity, ancestry and community of residence. The estimates for road lengths were scaled to 100m, 300m, and 1000m for total length of roads in
50m, 100m, and 200m buffers, respectively. The estimate for each haplotype is for per-copy of the haplotype compared to those with no copies of the respective
haplotype.
‡P-values for interaction for total length of local roads within each buffer by NOS2 haplotypes were obtained from likelihood ratio tests from non-stratified models with
appropriate interaction terms and were based on 1 degree of freedom. Statistically significant interaction P-values are in bold.
11
Table 5. Joint effects of NOS2 H1 promoter haplotype and length of local road within
100m and 200m buffers on FeNO
Number of haplotype
H1copy
Length of local road within 100m buffer around home*
≤ 250m > 250m
Adjusted Geometric Mean FeNO
(95% CI; ppb)†
Adjusted Geometric Mean FeNO
(95% CI; ppb)
0 14.18 (13.09 to 15.36)
‡
14.17 (13.59 to 14.79)
‡
1 13.36 (12.43 to 14.36) 13.75 (13.19 to 14.33)
‡
2 10.71 (9.10 to 12.61) 13.51 (12.27 to 14.86)
Number of haplotype
H1copy
Length of local road within 200m buffer around home*
≤ 1000m > 1000m
Adjusted Geometric Mean FeNO
(95% CI; ppb)
Adjusted Geometric Mean FeNO
(95% CI; ppb)
0 14.65 (13.50 to 15.89)
‡
14.05 (13.46 to 14.66)
‡
1 13.00 (12.07 to 14.00) 13.88 (13.31 to 14.47)
‡
2 11.29 (9.71 to 13.12)
13.42 (12.15 to 14.82)
*Road length cutoffs were selected approximately at the 25th percentile distributions.
†All models adjusted for race/ethnicity, an index of genetic ancestry and community of residence.
‡
Adjusted geometric mean FeNO is significantly different compared to the adjusted geometric mean FeNO level in
children who had 2 copies of the H1 haplotype and had less than 250m and 1000m total local road lengths within 100m
and 200m buffers, respectively.
Influence of Asthma on the associations of NOS2 haplotype and road length on FeNO
We evaluated the joint effects of asthma, NOS2 H1 promoter haplotype and
length of local roads around home on FeNO in children (Table 6). While children with
asthma had significantly higher FeNO than those without asthma, there was no
statistically significant three-way interactive effects of asthma, NOS2 H1 haplotype and
road lengths on FeNO.
12
Figure 1. Influence of NOS2 H1 haplotype copies on the association of length of local
roads around home and FeNO. The x-axis shows H1 haplotype copies (0, 1, and 2) within
categories of local road-lengths within (A) 100m and (B) 200m circular buffers of home.
The circles represent adjusted geometric mean FeNO and the vertical bars represent 95%
confidence intervals. The 95%CIs for the adjusted geometric means that cross the dashed
horizontal line in each figure are not statistically significantly different from the adjusted
geometric mean FeNO in children who had 2 copies of the H1 haplotype and had less
than 250m and 1000m total local road lengths within 100m and 200m buffers,
respectively.
13
Table 6. Joint effects of asthma, NOS2 promoter haplotype H1 and road length on FeNO
Factors
*
Estimates (95% CI, ppb)
†
P value
‡
Length of local road within 100-m buffer around home
H1 -0.03 (-0.07 to 0.01) 0.10
Local road lengths within 100m buffer 0.04 (-0.01 to 0.09) 0.12
Asthma 0.31 (0.24 to 0.39) <.0001
H1x Local road lengths within 100m buffer 0.08 (0.00 to 0.16) 0.05
H1x Asthma -0.12 (-0.23 to -0.00) 0.05
Asthma x Local road lengths within 100m buffer 0.11 (-0.02 to 0.25) 0.10
Asthma x H1 x Local road lengths within 100m buffer -0.03 (-0.22 to 0.17) 0.79
Length of local road within 200-m buffer around home
H1 -0.03 (-0.07 to 0.01) 0.12
Local road lengths within 200m buffer 0.06 (0.01 to 0.12) 0.02
Asthma 0.31 (0.24 to 0.39) <.0001
H1x Local road lengths within 200m buffer 0.08 (0.00 to 0.16) 0.04
H1x Asthma -0.12 (-0.23 to 0.00) 0.05
Asthma x Local road lengths within 200m buffer -0.00 (-0.14 to 0.14) 0.96
Asthma x H1 x Local road lengths within 200m buffer -0.04 (-0.25 to 0.17) 0.69
*
Road length variables were centered at their respective mean values. The 'x' between factors represents interaction
terms.
†
Estimates (95% confidence intervals) represent natural log transformed FeNO associated with each factor. All models
were adjusted for race/ethnicity, ancestry and community of residence. The estimates for road lengths were scaled to
300m, and 1000m for total length of roads in 100m, and 200m buffers, respectively.
‡
P-values for the association of each of the main effects and interaction terms with FeNO.
14
DISCUSSION
We found that one of the most common NOS2 promoter haplotypes and length of
local road within 100m and 200m buffers jointly influence FeNO level in children. Our
results show that the protective effect of the promoter haplotype on FeNO was evident in
children who had fewer roads around their homes. However, in children who lived in
homes surrounded by more roads, the protective effect of the haplotype was lost. This
novel finding provides evidence that joint evaluation of DNA sequence variants in NOS2
and traffic-related exposure is needed to understand interindividual differences in FeNO
levels in children.
Using data from the CHS cohort, we previously reported that local road length
within 100m and 200m buffers were significantly associated with higher FeNO (19), and
per copy of NOS2 promoter haplotype H1 was significantly associated with 6.2% lower
FeNO (11). In this paper, we extend these findings by showing joint effects of genetic
and environmental exposures on FeNO. We speculate that in children carrying two copies
of the H1 haplotype, the protective effects of the haplotype on FeNO is lost due to higher
traffic-related exposure, as the latter may induce iNOS enzymatic activity. Although the
tagSNPs were able to characterize the haplotype diversity of the NOS2 promoter region,
more research is warranted to identify the causal variants that may mediate these effects
on FeNO. Furthermore, experimental studies are required to evaluate iNOS enzyme
activity in relation to the DNA sequence variations in NOS2 promoter and traffic-related
exposures.
To limit the number of hypothesis tests, we restricted our testing of gene-
environment interaction by investigating the joint effects of two of the most common
15
promoter haplotypes of NOS2 (the major determinant of NO in airway) and traffic
measures that have been previously found to be associated with FeNO (11, 17-19). Of the
six tests we conducted for joint effects (Table 4), two were statistically significant
suggesting that the observed results are less likely due to chance.
We used one of the surrogates for traffic-related exposure that has been
consistently associated with higher FeNO in earlier studies (17-19), while other traffic
metrics (e.g., modeled total oxides of nitrogen [NOx], traffic density, distance to freeway
and major roads) did not show significant associations with FeNO in our previous
analysis (19). It is currently unknown which specific source of traffic-related exposure
(fresh gasoline/diesel exhaust, brake wear metals, re-suspended road dust, etc) underlie
the observed relationships. Near-road traffic emissions can be complexly impacted by
many factors, such as temporal and spatial patterns of traffic activity and meteorology
(27). It could be also impacted by exposures related to acceleration and brake wear due to
traffic stop signs in residential areas (19). It is also interesting to note that traffic count
data in local roads are often unavailable from the state or city departments of
transportation while such data from freeways and major roads are often available, which
may have limited estimation of near-home traffic-related exposure modeling. Further
research is needed to better understand the local sources of residential-level traffic-related
exposures so that better exposure estimation modeling approach could be utilized to
capture short- and long-term traffic related exposures near homes.
The strengths of the study include relative large sample size from a well-
characterized population-based study. Children living in 12 Southern California
communities had a wide contrast in local road length measures. We also minimized
16
population stratification by adjusting for genetic ancestry in all our models. However,
interpretation of our results requires the consideration of some study limitations. We used
parental report to define the health outcomes, and concern has been raised that parental
report might not reflect physician diagnosis. Based on medical records review, we have
previously found strong evidence that parental report reflected physician diagnosis (28).
Although asthma prevalence rate in our study population are in line with national
prevalence estimates, the study was not adequately powered to detect significant three-
way interactive effects of asthma, NOS2 haplotype and road length measures. Genotyping
data were only available on Hispanic and non-Hispanic white subjects, so the study
findings may not be generalizable to other ethnic groups.
17
CONCLUSION
We found that the protective role of a common NOS2 promoter haplotype on
FeNO level in children was lost when children’s lived near busy traffic. Because FeNO is
a biomarker of airway inflammation, our findings suggest that at high level of traffic-
related exposure, children may not benefit from having a protective DNA sequence
variants in NOS2 promoter. Because the NOS2 promoter haplotype frequency that
showed joint effects with traffic exposure for FeNO is quite common, our findings may
have implications for public health and transportation planning in residential areas so that
traffic-related exposures could be reduced to protect children’s respiratory health.
18
REFERENCES
1. Barnes PJ and Kharitonov SA. Exhaled nitric oxide: a new lung function test. Thorax
1996;51: 233-237.
2. Saito J, Inoue K, Sugawara A, et al. Exhaled nitric oxide as a marker of airway
inflammation for an epidemiologic study in schoolchildren. J Allergy Clin Immunol
2004;114:512-516.
3. Pijnenburg MWH, de Jongste JC. Exhaled nitric oxide in childhood asthma: a review.
Clinical and Experimental Allergy 2008;38:246-259.
4. Olin AC, Rosengren A, Thelle DS, et al. Increased fraction of exhaled nitric oxide
predicts new-onset wheeze in a general population. Am J Respir Crit Care Med
2010;181:324-327.
5. Bastain TM, Islam T, Berhane KT, et al. Exhaled nitric oxide, susceptibility and new-
onset asthma in the Children's Health Study. Eur Respir J 2011;37:523-531.
6. Ghosh S and Erzurum SC. Nitric Oxide Metabolism in Asthma Pathophysiology.
Biochim Biophys Acta 2011;1810:1008-1016.
7. Sherman TS, Chen Z, Yuhanna IS, et al. Nitric oxide synthase isoform expression in
the developing lung epithelium. Am J Physiol 1999;276:383-390.
8. Lane C, Knight D, Burgess S, et al. Epithelial inducible nitric oxide synthase activity is
the major determinant of nitric oxide concentration in exhaled breath. Thorax
2004;59:757–760.
9. Langley SJ, Goldthorpe S, Custovic A, et al. Relationship among pulmonary function,
bronchial reactivity, and exhaled nitric oxide in a large group of asthmatic patients. Ann
Allergy Asthma Immunol 2003;91:398-404.
10. Yamamoto M, Tochino Y, Chibana K, et al. Nitric oxide and related enzymes in
asthma: relation to severity, enzyme function and inflammation. Clin Exp Allergy
2012;42:760-768.
11. Salam MT, Bastain TM, Rappaport EB, et al. Genetic variations in nitric oxide
synthase and arginase influence exhaled nitric oxide levels in children. Allergy
2011;66:412-419.
12. Salam MT, Byun HM, Lurmann F, et al. Genetic and epigenetic variations in
inducible nitric oxide synthase promoter, particulate pollution, and exhaled nitric oxide
levels in children. J Allergy Clin Immunol 2012;129:232-239.
19
13. Islam T, Breton C, Salam MT, et al. Role of inducible nitric oxide synthase in asthma
risk and lung function growth during adolescence. Thorax 2010;65:139-145.
14. Mar TF, Jansen K, Shepherd K, et al. Exhaled nitric oxide in children with asthma
and short-term PM2.5 exposure in Seattle. Environ Health Perspect 2005;113:1791-1794.
15. Koenig JQ, Mar TF, Allen RW, et al. Pulmonary effects of indoor- and outdoor-
generated particles in children with asthma. Environ Health Perspect 2005;113:499-503.
16. Kelly FJ. Oxidative stress: its role in air pollution and adverse health effects. Occup
Environ Med 2003;60:612-616.
17. Holguin F, Flores S, Ross Z, et al. Traffic-related exposures, airway function,
inflammation, and respiratory symptoms in children. Am J Respir Crit Care Med
2007;176:1236-1242.
18. Dales R, Wheeler A, Mahmud M, et al. The influence of living near roadways on
spirometry and exhaled nitric oxide in elementary schoolchildren. Environ Health
Perspect 2008;116:1423-1427.
19. Eckel SP, Berhane K, Salam MT, et al. Residential traffic-related pollution exposures
and exhaled nitric oxide in the children's health study. Environ Health Perspect
2011;119:1472-1477.
20. McConnell R, Berhane K, Yao L, et al. Traffic, susceptibility, and childhood asthma.
Environ Health Perspect 2006;114:766-772.
21. Linn WS, Berhane KT, Rappaport EB, et al. Relationships of online exhaled, offline
exhaled, and ambient nitric oxide in an epidemiologic survey of schoolchildren. J Expo
Sci Environ Epidemiol 2009;19:674-681.
22. Linn WS, Rappaport EB, Berhane KT, et al. Exhaled nitric oxide in a population-
based study of southern California schoolchildren. Respir Res 2009;10:28.
23. Recommendations for standardized procedures for the on-line and off-line
measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and
children-1999. This official statement of the American Thoracic Society was adopted by
the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 1999;160:2104-2117.
24. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using
multilocus genotype data. Genetics 2000;155: 945-959.
25. Falush D, Stephens M, Pritchard JK. Inference of population structure using
multilocus genotype data: dominant markers and null alleles. Mol Ecol Notes 2007;7:
574-578.
20
26. Shtir CJ, Marjoram P, Azen S, et al. Variation in genetic admixture and population
structure among Latinos: the Los Angeles Latino eye study (LALES). BMC Genet
2009;10:71.
27. Baldauf R, Watkins N, Heist D, et al. Near-road air quality monitoring: Factors
affecting network design and interpretation of data. Air Qual Atmos Health 2009;2:1-9.
28. Salam MT, Gauderman WJ, McConnell R, et al. Transforming growth factor- 1 C-
509T polymorphism, oxidant stress, and early-onset childhood asthma. Am J Respir Crit
Care Med 2007;176:1192-1199.
Abstract (if available)
Abstract
Background: Fractional concentration of nitric oxide in exhaled air (FeNO) is a biomarker of airway inflammation. Nitric oxide synthase 2 (NOS2) in airway epithelium has been recognized as the major source of NO in exhaled breath. Earlier work has shown that common promoter haplotypes in NOS2 and total length of local roads around homes, a metric of residential traffic related pollution, affect FeNO level in children. ❧ Aims: The aims of this study were to examine the joint associations of NOS2 promoter haplotypes and length of local roads around homes and FeNO and to assess the influence of asthma on these associations in children. ❧ Methods: The study included 2,457 (7 to 11 year-old) children of the Southern California Children’s Health Study. FeNO was measured at school during 2005-2006. Lengths of local roads within circular buffers (50m, 100m and 200m) around the participant residence were estimated using GIS methods. Two common promoter haplotypes in NOS2 that have been associated with FeNO, asthma and lung function growth in children were selected. Linear regression was utilized to examine the independent and joint associations of NOS2 promoter haplotypes and road length measures on FeNO. ❧ Results: We observed joint effects of length of local roads within 100m and 200m buffer and the most common haplotype for FeNO (P-values for interaction ≤0.03). In children who had ≤250m of road within 100m buffer around home, those with two copies of the haplotype had significantly lower FeNO (adjusted mean FeNO=10.71ppb
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Association of traffic-related air pollution and lens opacities in the Los Angeles Latino Eye Study
PDF
Airway inflammation and respiratory health in the Southern California children's health study
Asset Metadata
Creator
Lin, Pi-Chu Kaylene
(author)
Core Title
Genetic variation in inducible nitric oxide synthase promoter, residential traffic related air pollution and exhaled nitric oxide in children
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Applied Biostatistics and Epidemiology
Publication Date
07/29/2013
Defense Date
06/24/2013
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Children’s Health Study,CHS,FeNO,fractional concentration of exhaled nitric oxide,haplotype-tagging single nucleotide polymorphism,htSNP,inducible nitric oxide synthase,iNOS,nitric oxide,nitric oxide synthase isoform 2,nitrogen dioxide,NO,NO2,NOS2,OAI-PMH Harvest,reactive oxygen species,ROS,single nucleotide polymorphism,SNP,traffic related pollution,TRP
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Gilliland, Frank D. (
committee chair
), Gauderman, William James (
committee member
), Salam, Md. Towhid (
committee member
)
Creator Email
kaylenelin@hotmail.com,pichulin@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-303576
Unique identifier
UC11293771
Identifier
etd-LinPiChuKa-1868.pdf (filename),usctheses-c3-303576 (legacy record id)
Legacy Identifier
etd-LinPiChuKa-1868-0.pdf
Dmrecord
303576
Document Type
Thesis
Format
application/pdf (imt)
Rights
Lin, Pi-Chu Kaylene
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
Children’s Health Study
CHS
FeNO
fractional concentration of exhaled nitric oxide
haplotype-tagging single nucleotide polymorphism
htSNP
inducible nitric oxide synthase
iNOS
nitric oxide
nitric oxide synthase isoform 2
nitrogen dioxide
NO
NO2
NOS2
reactive oxygen species
ROS
single nucleotide polymorphism
SNP
traffic related pollution
TRP