Effects of myeloperoxidase (MPO) polymorphism and tobacco smoke on asthma and wheezing in Southern California children

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Content EFFECTS OF MYELOPEROXIDASE (MPO) POLYMORPHISM AND
TOBACCO SMOKE ON ASTHMA AND WHEEZING IN SOUTHERN
CALIFORNIA CHILDREN
Copyright 2004
by
Wei-wei Tsai
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(BIOSTATISTICS)
December 2004
Wei-wei Tsai
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ACKNOWLEGEMENTS
I would like to express my greatest thanks to my thesis committee chair,
Dr. Frank Gilliland, for his guidance, support, inspiration and commitment of time. I
would also like to thank my thesis committee members, Dr. James Gauderman and
Dr. Louis Dubeau for their expert advice. A special thanks to Dr. Yu-fen Li for her
help and support which motivate the completion of the write up. Thanks to my lab
partners Kaylene Lin and Yo-hsuang Tsao for their brilliant ideas to solve all the
experimental problems. Last but not least, thanks to the teammates of Children’s
Health Study, it has been a pleasure to work with this outstanding group.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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LIST OF TABLES
iv
Table 1. Selected characteristics of CHS participants 14
Table 2. Distribution of MPO genotypes by ethnicity in cases and controls 15
Table 3. Distribution of MPO genotypes by ethnicity in asthmatic and 15
non-asthmatic children
Table 4. Crude risk estimates for MPO genotypes, exposures and 17
asthma and wheezing outcomes
Table 5. Adjusted risk estimates for MPO genotypes, exposures and 18
asthma and wheezing outcomes
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ABSTRACT
v
This study examined the effects of myeloperoxidase (MPO) -463 G to A
polymorphism and tobacco smoke on asthma and wheezing in Southern California
children while controlling for towns of residence, age, grade, race, gender, family
history of asthma and atopy, and gestational age. The results showed that in utero
exposure to maternal smoking was statistically significantly associated with all
spectrum of wheezing and early onset of asthma of the participants while current
ETS did not have any association to the outcomes. There was no association found
between MPO genotypes and asthma and wheezing outcomes. The interactions
between MPO genotypes and tobacco smoke exposures were not statistically
significant.
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1
Introduction
Asthma affects more than 5% of the population of the US, including children.
The burden from asthma in the US has increased over the last two decades according
to CDC reports. The increased prevalence of lifetime asthma diagnosis, current
asthma, asthma attack, asthma outpatient visits, asthma emergency department visits
and asthma hospitalization suggest that exposures to environmental risk factors have
also increased during this time. Many epidemiological studies have demonstrated not
only the effects of environmental factors but also the effects of genetics on the
occurrence of asthma. Environmental tobacco smoke (ETS) is one of the most
common indoor air pollutants and has been associated in epidemiologic studies with
asthma, decreased lung function and respiratory illnesses in exposed individuals.
However, symptoms occur in only some individuals, suggesting that individual
genotypes determine sensitivity to environmental tobacco smoke exposure.
Asthma is a common complex disease, the pathogenesis of which is affected
by exposure to exogenous agents and modified by a series of genetic determinants
that regulate key elements in bronchopulmonary function. Risk or trigger factors of
asthma include several allergens and nutrients, some infections, neonatal factors,
pollution, and smoking. Tobacco smoke is thought to encourage inflammation of the
airways by activating the inflammatory cells, altering cell functions and subtypes,
and encouraging proinflammatory mediator release, neurogenic inflammation, and
oxidative stress (Romero Palacios 2004). As inflammation is often associated with an
increased generation of reactive oxygen species, and the biochemical environment in
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2
the asthmatic airways is favorable for free radical mediated reactions, it is rational to
say that oxidative stress could be mechanistically important in asthma. The
inflammatory cells recruited to the asthmatic airways have an exceptional capability
for producing reactive oxygen species and toxic oxidative products that directly
damage cellular macromolecules and tissues and causes airway inflammatory
responses that enhance asthma (Halliwell and Cross 1994). Activated eosinophils,
neutrophils, monocytes, and macrophages can generate superoxide (O2’) via the
membrane associated NADPH-dependent complex (Dworski 2000). Subsequently,
dismutation of O2' gives hydrogen peroxide (H2O2) and molecular O2. 0 2 ' and H2O2
are moderate oxidants; however, both species are critical for the formation of potent
cytotoxic radicals in biological systems through their interaction with other
molecules. The inflammatory response enhances oxidative stress and may contribute
to persistent inflammation, which is a pathologic hallmark of asthma. Thus, oxidant
defenses play a key role in development o f asthma and its subsequent severity.
Children with decreased oxidant defenses may have deficits in lung function,
increased risk of asthma and other respiratory illnesses from exposure to air
pollutants and ETS. Since oxidative stress is important to the pathogenesis o f these
outcomes, children who are exposed to high amounts of oxidative stress may be at
greater risk.
Environmental pollution has been linked to an increase in the prevalence of
bronchial hyperresponsiveness, allergic sensitization, and respiratory diseases in
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3
general (Heraud and Herbelin-Wagner 2002). There are studies on adults that show a
direct relation between incidence of chronic bronchitic processes and asthma and
exposure to ETS at work and traveling to and from work (Eisner 2002). High
concentrations of oxidants and pro-oxidants are contained in tobacco smoke.
Exposure to tobacco smoke produces oxidative stress in the lung and increases
occurrence o f asthma and wheezing that due to airway inflammation (Segala 1999).
The effects o f oxidants exposure on inflammation appear to be larger among
individuals with asthma (Schunemann, Muti et al. 1997). Exposure o f children to
ETS basically occurs at home and in play environments. Maternal smoking is the
main source o f in utero and early childhood exposure to tobacco smoke. As children
grow up, exposure to maternal smoking decreases and the influence of smoke from
other sources, such as ETS in public places, increases. However, exposure to tobacco
smoke particularly from maternal smoking is undoubtedly one o f the factors that
directly influence the development of asthma in children. Although there appears to
be evidence o f a relation between passive smoking and asthma, the relation between
active smoking and asthma is not so well defined. There are contradictory findings,
some o f which show a relation between smoking and asthma and others that do not.
A strong association was found between household ETS and the incidence of
wheezing and asthma in children of up to 6 years of age, but after that the association
was weaker, possibly attributable to the degree of exposure to ETS during growth
(Strachan and Cook 1998). The long-term prognosis for children with asthma was
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poorer if their parents smoked. No consistent relation was found between parental
smoking before or after birth and risk of allergic sensitization (positive skin test,
immunoglobulin-E concentrations, hay fever, or eczema, excluding asthma) in
children, although there was an increased bronchial hyperresponsiveness in children
o f mothers who smoked (Strachan and Cook 1998).
In a prospective study carried out in Australia, no relation was found between
asthma and ETS exposure in children, suggesting that there are other factors that
influence the onset o f asthma and that genetic characteristics moderate susceptibility
to environmental factors. It has been known that atopic disease runs in families and a
family history is the strongest risk factor for the development of allergies and asthma.
Molecular studies also have identified some specific atopic entities that are
determined genetically, such as eosinophils, interleukin-5 (Kaiser 2004). In Maputo,
childhood asthma was strongly associated with a family history of asthma and
rhinitis, the place o f residence, having smokers as parents and early weaning from
maternal breast milk (Mavale-Manuel, Alexandre et al. 2004). Similar in Japanese
adolescents and western populations, positive maternal allergic history was more
evidently associated with an increased prevalence of wheeze and rhinoconjunctivitis,
but not atopic asthma, than a positive paternal allergic history (Miyake, Yura et al.
2004). The study that evaluated exposure of dust mite allergen and risk for outcomes
related to allergy and asthma indicated that parental history is an important
independent variable in the relationship between early dust mite exposure and atopic
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outcomes. Increased exposure during infancy is associated with a higher risk for
sensitization in the presence of a positive parental history, but is protective among
children o f parents without a history of atopic disease (Cole Johnson, Ownby et al.
2004).
Genes involved in oxidant production and antioxidant defenses play a critical
role in the effects of air pollution and tobacco smoke. Glutathione S-transferases
(GST), xenobiotic-metabolising enzymes, are involved in the metabolic
detoxification of various environmental carcinogens. Particular genetic
polymorphisms o f these enzymes have been shown to influence individual
susceptibility against various pathologies including cancer, cardiovascular and
respiratory diseases. GSTM1 may play a role in asthma and wheezing occurrence
among those exposed to tobacco smoke, as it functions in pathways involved in
asthma pathogenesis and antioxidant defenses. Our group reported that the effects of
in utero exposure to maternal smoking on asthma and wheezing occurrence were
largely restricted to children with GSTM1 null genotype. Our findings indicate that
there are important long-term effects of in utero exposure in a genetically susceptible
group o f children (Gilliland, Li et al. 2002). The results from a meta-analysis
indicated that GSTM1 null genotype was associated with enhanced risk for lung,
bladder, and larynx cancer. GSTT1 null genotype was associated with increased
astrocytomas and meningiomas cancer risk. GSTP1 allelic polymorphism influences
the development of bladder cancer in smokers and occupational asthma. These
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epidemiological data suggest that genetic GST polymorphisms influence the
individual susceptibility to these diseases. However, no evidence of interaction
between GST genotype and smoking status was found in lung cancer (Habdous, Siest
et al. 2004). The other study of German schoolchildren found that in children lacking
the GSTM1 allele who were exposed to current ETS the risk for current asthma and
asthma symptoms was higher than in GSTM1 positive individuals without ETS
exposure. Hints o f an interaction between ETS exposure and GSTM1 deficiency
were identified. In utero smoke exposure in GSTT1 deficient children was associated
with significant decrements in lung function compared with GSTT1 positive children
not exposed to ETS. They concluded that GSTM1 and GSTT1 deficiency may
increase the adverse health effects of in utero and current smoke exposure (Kabesch,
Hoefler et al. 2004).
Tumor-necrosis factor (TNF)-alpha and Myeloperoxidase (MPO) are genes
involved in inflammatory oxidant production. TNF-alpha is a multifunctional
proinflammatory cytokine that provides a rapid form of host defense against
infection but is fatal in excess. It is known that TNF-alpha is released in allergic
responses from both mast cells and macrophages via IgE-dependent mechanisms,
and elevated levels have been demonstrated in the bronchoalveolar fluid (BALF) of
asthmatic subjects undergoing allergen challenge. Inhaled TNF-alpha increases
airway responsiveness to methacholine in normal and asthmatic subjects associated
with a sputum neutrophilia. Additional data indicate that TNF-alpha can upregulate
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adhesion o f molecules, facilitate the immigration o f inflammatory cells into the
airway wall and activate pro-fibrotic mechanisms in the subepithelium (Thomas
2001). These data suggest that TNF-alpha plays a role in the initiation o f allergic
asthmatic airway inflammation and the generation of airway hyper-reactivity.
Among a number of TNF-alpha polymorphisms which have been identified, the G to
A single nucleotide polymorphism at the promoter region of position -308 is well
studied and likely to be functional. This polymorphism is associated with the
increased level o f TNF-alpha and the A allele was significantly associated with self-
reported childhood asthma in the UK and Irish population but not in the South Asian
population (Winchester, Millwood et al. 2000).
MPO is a lysosomal haemoprotein located in the azurophilic granules of
polymorphonuclear (PMN) leukocytes and monocytes. It is part of the host defense
system o f human polymorphonuclear leukocytes, responsible for microbicidal
activity against a wide range o f organisms. In the stimulated PMN, MPO catalyzes
the production o f hypohalous acids, primarily hypochlorous acid in physiologic
situations, and other toxic intermediates that greatly enhance PMN microbicidal
activity (Klebanoff 1999). MPO is located in the nucleus as well as in the cytoplasm.
Intranuclear MPO may help to protect DNA against damage resulting from oxygen
radicals produced during myeloid cell maturation and function. Serum MPO level
was found to be increased during 0 3 -induced inflammation, as well as respiratory
infections. Additionally, MPO metabolizes tobacco smoke procarcinogens such as
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benzo(a)pyrene and aromatic amines into highly reactive intermediates (Piedrafita,
Molander et al. 1996). Because neutrophils are recruited in large numbers to the
lung in response to these pulmonary insults, such as O 3, respiratory infection, and
tobacco smoke, which are risk factors for asthma, MPO is suspected to play a role in
pathogenesis in asthma. There is a polymorphic site near MPO that may modify the
level of carcinogen metabolism. The G to A polymorphism, rs2333227, is located at
the promoter region 463-bp upstream of the MPO with minor allele (A) frequency of
0.25. The A allele is associated with a decreased transcriptional activity attributable
to the disruption of a SP1-binding site. We therefore examined whether carriers of
the A allele may be at reduced risk of asthma.
The Children’s Health Study (CHS) which began in 1993 have been recruited
children who attended public schools as 4th, 7th, and 10th graders in 12 communities
in Southern California (Peters, Avol et al. 1999). Buccal cell specimens were
collected from these participants and DNA were extracted for genotyping. We are
allowed to further investigate the association of MPO polymorphism and tobacco
smoke exposure to asthma and wheezing.
Methods
Participants. 6259 school-aged children with known asthma status from 12
communities in southern California who were recruited by CHS. Only 3681 buccal
cell specimens were collected among these children and were included in this
analysis. Details on the study design, community selection, subject recruitment, and
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9
assessment of health effects have been described elsewhere (Peters, Avol et al. 1999;
Peters, Avol et al. 1999). Baseline information was obtained from a written
questionnaire, including demographics, medical and family health history, indoor air
exposures, and household characteristics that filled out by parents or guardians at
study entry. Buccal cell specimens of children were collected at school visits during
Years 6-10 o f the study. Buccal cell specimens of older children who were graduated
from high school or moved were collected by mail if available. In our analysis, cases
were defined as either ever have asthma or wheezing, whereas controls were defined
as neither had asthma nor wheezing.
Procedures. Asthma and wheezing. Asthma and wheezing history for
children were categorized according to CHS questionnaires responded by
participants’ parents or guardians. A child with asthma was classified using the
answer o f “has a doctor ever diagnosed this child as having asthma?” in the
questionnaire. Early onset of asthma was defined as asthma diagnosed at age 5 or
younger. A child with persistent asthma was defined as any child was diagnosed with
asthma and had wheezing or had medication for asthma in one year before the study
entry. A child with active asthma was defined as ever ill with asthma in the past year
before the questionnaire was answered. A child with wheezing occurrence was
classified using the answer of “has your child’s chest ever sounded wheezy or
whistling including times when he or she had a cold?” in the questionnaire. Other
wheezing related outcomes in the past year before the questionnaire was answered
were classified as follow: any current wheezing for a child was defined as who ever
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wheezed with colds or ever wheezed without colds; persistent wheezing was defined
as a child ever wheezed for 3 or more days out of the week for a month or longer;
shortness with wheezing was defined as ever had episodes of shortness of breath
with wheezing; awakened at night by wheezing was defined as ever been awakened
at night by wheezing; wheezing with exercise was defined as ever wheezing after he
or she has been playing hard or exercising. More questions were assessed for the past
year before the interview, such as medication for asthma, treatment for wheezing,
and lifetime occurrence for each of the outcomes.
Tobacco smoke exposure. Exposure to household environmental tobacco
smoke and exposure to maternal smoking in utero were categorized according to
CHS questionnaires responded by participants’ parents or guardians. ETS was
defined as daily smoking inside the house by anyone in the household. Current and
past ETS statuses of a child were collected by looking at participant’s parents, other
members in the household and regular household visitors’ smoking statuses.
Maternal smoking in utero was defined as a child’s biological mother smoke while
she was pregnant with the child, include time when she was pregnant but did not yet
know that she was. On average, numbers of cigarettes are smoked inside the child’s
home each day was defined as 3 levels: none, 1-29 cigarettes/day, and 30 or more
cigarettes/day whereas 2 0 cigarettes are in one pack.
Covariates. Ethnicity was categorized to 5 groups: non-Hispanic white
(reference group), Hispanic, African American, Asian/Pacific Islanders, and others.
Gestational age was categorized to 3 groups: full term (reference group), 1 to less
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than 4 weeks early and 4 or more weeks early. Family history of asthma was defined
as any o f a child’s biological parents have ever been diagnosed with asthma. Family
history of atopy was defined as any of a child’s biological parents have ever been
diagnosed with hay fever or allergy.
Laboratory methods. Genomic DNA was isolated from buccal mucosal cells
using PURGENE™ DNA isolation Kit (Gentra Systems, Minneapolis, MN). The G
to A transition polymorphism at position -463 for human MPO was identified by
PCR and allelic discrimination assay performed on TaqMan, an ABI PRISM™ 7700
Sequence Detector (Applied Biosystems, Foster city, CA). Standard setup and
operation for allelic discrimination assay were used as detailed by ABI PRISM™
7700 Sequence Detection System user’s manual. The QuantiTect™ Probe PCR Kit
(Qiagen, Valencia, CA) was used for PCR reactions. Primers and fluorescent probes
were designed by using Primer Express™ applications-based primer design software
(Applied Biosystems, Foster city, CA). The forward primer for human MPO was 5’-
CTT GGG CTG GTA GTG CTA AAT TC-3’, and the reverse primer was 5’- GTA
ATT TTT GTA TTT TTC CTT AGG CAA GAA GC-3’. The MGB VIC probe for G
allele was 5’-TCC ACC CGC CTC AG-3’, and the MGB FAM probe for A allele
was 5’- TCC ACC TGC CTC AG-3’(Applied Biosystems, Foster city, CA). 10%
randomly selected samples were used as quality controls. 8 samples o f each genotype
for MPO were validated by using PCR/RFLP methods. Hardy-Weinberg Equilibrium
was checked for gene data validation.
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Statistical Analysis. Logistic regression was used to examine the
relationship between asthma and wheezing, MPO genotypes, ETS, and exposure to
maternal smoke in utero. Odds ratios were adjusted for variables such as town of
residence, age, grade, ethnicity, gender, family history if asthma, family history of
atopy, and gestational age in the regression model. Income and education, health
insurance status, and housing characteristics were considered as potential
confounders. Effect modification was assessed by fitting models with possible
interaction terms and testing its significance by using the likelihood ratio tests
comparing the full models and reduced models. If significant interaction term was
observed, stratified analysis was performed. Logistic regression models were tested
for 3 possible genetic inheritance modes: codominant, dominant, and recessive
inheritance. All analyses were conducted using SAS 9.1.3(SAS Institute, Cary, NC)
at significant level of 0.05.
Results
The frequencies of ethnicity in our population were 59.09% non-Hispanic
white, 26.65% Hispanic, 4.16% African American, 4.29% Asian/Pacific Islanders,
and 5.81% others. The frequencies of boys and girls were 46.37% and 53.63%,
respectively. The average age for these children was 10.89-year-old at study entry.
The family history o f atopy and asthma of the participants were 49.67% and 19.54%,
respectively. Among the participants, exposure to ETS was 34.46%; currently
exposure to ETS was 18.03%; exposure to maternal smoking in utero was 16.81%;
exposure to ETS in utero was 37.48%. On average, the frequency for non-smoking
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homes each day was 80.48%, 17.93% had 1-29 cigarettes smoked, and only 1.6%
had cigarettes smoked inside the child’s home each day. 15.36% of children had
physician diagnosed asthma, and only 9.16% of children had active asthma. 34.87%
of children had ever wheezed in their life; 23.75% of children currently had
wheezing occurrences. The outcome, asthma, was different by ethnicity, African-
Americans had a highest rate of 23.49% and Asian/Pacific Islanders had a lowest
rate o f 9.21% (Table 1).
The genotype frequencies of MPO were 64.35% of genotype GG, 31.20% of
genotype GA, and 4.44% of genotype AA. For Caucasians, the allele frequency for
A allele was 21.5%, similar to the reported frequencies in other studies: 23%
(Kantarci, Lesnick et al. 2002), 23.4% for (London, Lehman et al. 1997), 25.7% (Le
Marchand, Seifried et al. 2000), 21.2% (Cascorbi, Henning et al. 2000), 29.8%
(Schabath, Spitz et al. 2002), and 30.6% (Misra, Tangrea et al. 2001). The
distribution of genotypes was in Hardy-Weinberg Equilibrium. Among controls,
6.58% of African-Americans were AA genotype for MPO; but only 1.04% of
Asian/Pacific Islanders were AA genotype for MPO. AA genotype was more
common in African-Americans and Asian/Pacific Islander among cases; the
frequencies were 12.24% and 16.67%, respectively. The rest of the ethnic groups,
Non-Hispanic Whites, Hispanics and Others, the frequencies for MPO genotypes
were similar comparing cases and controls (Table 2). To compare the asthmatic and
non-asthmatic subjects, Hispanics and Asian/Pacific Islanders had different
distribution for AA genotypes which were much higher among asthmatic children
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TABLE 1. SLELECTED CHARACTERISTICS OF CHS PARTICIPANTS
Subset CHS
n ("/o)1 n
( % y
Gender
Male 1707 53.63 3014 48.15
Female 1974 46.37 3245 51.85
Age at study entry, year
8-9 1944 52.88 2905 46.63
10-11 787 21.41 1348 21.64
12-13 554 15.07 1001 16.07
>14 391 10.64 976 15.67
Ethnicity
Non-Hispanic Whites 2175 59.09 3425 54.72
Hispanics 981 26.65 1773 28.33
African-Americans 153 4.16 334 5.34
Asian/Pacific Islanders 158 4.29 289 4.62
Others 214 5.81 438 7.00
Family history of asthma 666 19.54 1105 19.55
Family history o f atopy 1674 49.67 2657 47.51
Gestational age
Full term 3178 88.67 5377 89.41
< 4 week early 249 6.95 398 6.62
> 4 week early 157 4.38 239 3.97
Ever ETS exposure 1218 34.36 2364 39.83
Current ETS exposure 646 18.03 1366 22.62
Exposure to maternal smoking in utero 598 16.81 1130 18.95
Exposure to ETS in utero 1296 37.48 2471 42.80
# if cigarettes/day smoked inside home
None 2770 80.48 4372 75.46
1-29 617 17.93 1307 22.56
>30 55 1.60 115 1.98
Asthma
Ever asthma 553 15.36 873 14.41
Active asthma* 307 9.16 495 8.72
Medication for asthma* 361 10.61 569 9.90
Early onset asthma* 269 8.11 392 7.03
Persistent asthma* 207 5.88 303 5.02
Wheezing
Ever wheezing 1218 34.87 1960 33.41
Current wheezing* 707 23.75 1133 22.52
Wheezed with cold* 629 21.69 1019 20.72
Wheezed without cold* 383 14.41 611 13.53
Persistent wheezing* 225 9.00 345 8.12
Shortness o f breath with wheezing* 321 12.37 542 12.19
Awakened at night by wheezing* 264 10.41 436 10.05
Wheezing after exercise* 377 14.23 623 13.77
Medication for wheezing* 402 15.02 639 14.06
ER for wheezing* 109 4.58 188 4.59
Total 3681 6259
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then non-asthmatic children, 7.26% versus 3.75% and 27.27% versus 1.63%(Table
3). 396 subjects had missing genotype data due to decline to provide a sample,
moved or graduated and were no longer under active follow-up in school and not
available for sample collection.
TABLE 2. DISTRIBUTION OF MPO GENOTYPES BY ETHNICITY IN
CASES AND CONTROLS*
Ethnicity
Non-Hi spanic
Whites
n(%)
Hispanics
n (%)
African-Americans
n (%)
Asian/Pacific
Islanders
n (%)
Others
n (%)
Cases
GG 456 (63.1) 187 (70.3) 22(44.9) 18(75.0) 44 (65.7)
GA 235 (32.5) 67 (25.2) 21 (42.9) 2(8.3) 22 (32.8)
AA 32 (4.4) 12(4.5) 6 (12.2) 4(16.7) 1(1.5)
Controls
GG 697 (61.8) 404(70.1) 42(55.3) 71 (74.0) 70 (69.3)
GA 375 (33.2) 149 (25.9) 29 (38.2) 24 (25.0) 30 (29.7)
AA 56 (5.0) 23 (4.0) 5 (6.6)
. Hi-0) .........
1 (1.0)
* Cases are defined as ever asthma or wheezing while controls have neither.
TABLE 3. DISTRIBUTION OF MPO GENOTYPES BY ETHNICITY IN
ASTHMATIC AND NON-ASTHMATIC CHILDREN
Ethnicity
Non-Hi spanic
Whites
n(%)
Hispanics
n(%)
African-Americans
n (%)
Asian/Pacific
Islanders
n(% )
Others
n(% )
Asthmatic
GG 200 (65.4) 87 (70.2) 17(50.0) 8 (72.7) 12(46.1)
GA 94 (30.7) 28 (22.6) 14(41.2) 0 (0.00) 14(53.9)
AA 12(3.9) 9 (7.3) 3 (8.8) 3 (27.3) 0 (0.00)
Non
asthmatic
GG 978 (61.1) 523 (70.0) 49 (52.1) 92 (74.8) 103 (69.6)
GA 545 (34.1) 196 (26.2) 37 (39.4) 29 (23.6) 43 (29.1)
AA 77 (4.8) 28 (3.8) 8 (8.5) 2(1.6) 2(1.3)
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Performing univariate analysis, we found that in utero exposure to maternal
smoking was statistically significantly associated with all spectrum of wheezing,
history of ever have asthma, early onset of asthma, and persistent asthma
of the participants; Current ETS was also associated with history of ever wheezing,
wheezed with cold and wheezed with cold of the participants; MPO genotypes was
associated with shortness o f breath by wheezing using the codominant and recessive
inheritance modes (Table 4). The estimated effects were changed slightly when the
model was adjusted by towns, age, grade, race, gender, gestational age, family
history of asthma, and family history of atopy. Although in utero exposure to
maternal smoking was still statistically significantly associated with a broad
spectrum of wheezing outcomes and early onset of asthma, MPO genotypes was no
longer shown the association. There was no association found between current ETS,
MPO genotypes and all asthma and wheezing outcomes (Table 5).
We assessed whether an interaction existed between MPO genotypes,
smoking exposures and family history of asthma and atopy. For history o f ever
asthma, the interactions were all non-significant between MPO genotypes and
lifetime ETS exposure, between MPO genotypes and in utero exposure of maternal
smoking and between MPO genotypes and family history of asthma and atopy. The
only significant interaction was between MPO genotypes and family history of
asthma and atopy using the dominant inheritance model for the outcome of asthma
persistence (P=0.02). Children who have family history of asthma and atopy with
MPO AA genotype were in a reduced risk to have persistent asthma comparing to
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TABLE 4. CRUDE RISK ESTIMATES FOR MPO GENOTYPES, EXPOSURES AND ASTHMA AND WHEEZING
OUTCOMES
Outcomes
MPO
In utero Exposure Current ETS
Codominant Dominant Recessive
GA
OR(95% Cl)
AA
OR(95% Cl)
GA/AA
OR(95% Cl)
AA
OR(95% Cl) OR(95% Cl) OR(95% Cl)
Asthma
Ever asthma 0.95 (0.77,1.17) 1.24 (0.81,1.92) 0.99 (0.81,1.20) 1.26 (0.82,1.94) 1.27(1.01,1.60)* 1.17(0.93,1.47)
Active asthma 0.89 (0.67,1.17) 1.24 (0.71,2.16) 0.93 (0.71,1.21) 1.29(0.74,1.23) 1.15(0.85,1.57) 0.94(0.69,1.29)
Medication for 0.91(0.71,1.18) 1.18(0.70,2.01) 0.95(0.74,1.20) 1.22 (0.72,2.05) 1.13(0.85,1.51) 0.97 (0.72,1.30)
asthma
Early onset asthma 1.01 (0.76,1.35) 1.36 (0.76,2.42) 1.06 (0.80,1.38) 1.35(0.76,2.39) 1.51 (1.11,2.04)* 1.04 (0.75,1.43)
Persistent asthma 0.99 (0.71,1.37) 1.12(0.55,2.25) 1.01 (0.74,1.37) 1.12(0.56,2.24) 1.41 (1.00,2.00)* 0.91 (0.62,1.33)
Wheezing
Ever wheezing 0.98 (0.83,1.15) 1.13(0.79,1.61) 0.99(0.85,1.16) 1.14(0.80,1.62) 1.85(1.55,2.22)* 1.29(1.08,1.54)*
Current wheezing 0.97(0.79,1.18) 1.39(0.93,2.06) 1.02 (0.85,1.22) 1.40 (0.95,2.07) 1.80(1.45,2.23)* 1.27(1.02,1.57)*
Wheezed with cold 1.00 (0.82,1.23) 1.29 (0.84,1.97) 1.04(0.86,1.26) 1.29(0.84,1.96) 1.76(1.40,2.21)* 1.29(1.03,1.62)*
Wheezed without
o a IH
0.95 (0.74,1.23) 1.55(0.96,2.50) 1.03 (0.81,1.30) 1.57(0.98,2.52) 1.62(1.23,2.14)* 1.11 (0.84,1.48)
CU 1U
Persistent wheezing 1.03 (0.75,1.42) 1.19(0.60,2.35) 1.05 (0.78,1.42) 1.18(0.60,2.31) 1.84(1.32,2.58)* 1.11 (0.77,1.59)
Shortness o f breath 1.00 (0.76,1.30) 1.79(1.09,2.92)* 1.09 (0.85,1.41) 1.79(1.10,2.90)* 1.86(1.39,2.48)* 1.24(0.92,1.67)
Awakened at night 0.91 (0.67,1.23) 1.45(0.82,2.57) 0.97 (0.73,1.29) 1.50 (0.85,2.63) 1.63(1.18,2.26)* 1.21 (0.87,1.67)
Wheezing after 0.99(0.77,1.27) 1.45 (0.88,2.38) 1.04 (0.82,1.32) 1.45 (0.89,2.37) 1.71 (1.30,2.25)* 1.30 (0.99,1.71)
exercise
Medication 1.01 (0.79,1.29) 1.46 (0.89,2.37) 1.07(0.85,1.35) 1.45 (0.90,2.35) 1.56 (1.19,2.06)* 1.19(0.91,1.56)
ER 1.28(0.84,1.97) 1.81(0.80,4.09) 1.35(0.90,2.02) 1.66(0.75,3.68) 2.15(1.37,3.38)* 1.53 (0.97,2.43)
* The odds ratio is statistically significant between the groups for the specific outcome (p< 0.05).
< 1
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TABLE 5. ADJUSTED1 RISK ESTIMATES FOR MPO GENOTYPES, EXPOSURES AND ASTHMA AND
WHEEZING OUTCOMES
Outcomes
MPO
In utero Exposure Current ETS
Codominant Dominant Recessive
GA
OR(95% Cl)
AA
OR(95% Cl)
GAJAA
OR(95% Cl)
AA
OR(95% Cl) OR(95% Cl) OR(95% Cl)
Asthma
Ever asthma 0.92(0.72,1.18) 1.21(0.74,1.98) 0.96 (0.76,1.21) 1.24(0.77,2.02) 1.26 (0.95,1.68) 1.16(0.87,1.564)
Active asthma 0.90 (0.65,1.24) 1.06 (0.56,2.03) 0.92 (0.68,1.25) 1.10(0.58,2.08) 1.19(0.82,1.74) 0.95 (0.64,1.41)
Medication for 0.93 (0.69,1.26) 1.06 (0.58,1.93) 0.95(0.72,1.26) 1.08 (0.60,1.96) 1.21 (0.86,1.71) 1.09 (0.77,1.55)
asthma
Early onset asthma 0.93 (0.66,1.31) 1.38(0.72,1.36) 0.99 (0.72,1.36) 1.41 (0.74,2.69) 1.68(1.16,2.43)* 1.00(0.76,1.51)
Persistent asthma 0.84(0.57,1.25) 0.96 (0.43,2.13) 0.86 (0.59,1.24) 1.01 (0.46,2.23) 1.45(0.94,2.23) 0.74(0.45,1.23)
Wheezing
Ever wheezing 0.92 (0.76,1.11) 1.13(0.76,1.68) 0.95 (0.79,1.13) 1.16(0.78,1.72) 1.79(1.44,2.23)* 1.22(0.98,1.51)
Current wheezing 0.89(0.70,1.12) 1.30 (0.82,2.04) 0.94 (0.76,1.17) 1.35(0.86,2.11) 1.75(1.34,2.28)* 1.19(0.91,1.55)
Wheezed with cold 0.95 (0.75,1.21) 1.18(0.73,1.92) 0.98(0.78,1.23) 1.20 (0.74,1.94) 1.67(1.27,2.21)* 1.21 (0.92,1.59)
Wheezed without
p rtl H
0.86 (0.64,1.16) 1.45 (0.84,2.50) 0.94 (0.71,1.24) 1.52 (0.89,2.61) 1.54(1.10,2.17)* 1.10(0.78,1.56)
tutu
Persistent wheezing 1.05 (0.73,1.52) 1.20 (0.56,2.57) 1.07 (0.75,1.52) 1.18 (0.56„2.49) 1.75(1.16,2.66)* 1.08 (0.70,1.67)
Shortness of breath 0.93 (0.68,1.28) 1.64(0.93,2.88) 1.02 (0.76,1.38) 1.68 (0.96,2.93) 2.02(1.42,2.85)* 1.33(0.93,1.90)
Awakened at night 0.90 (0.64,1.28) 1.28(0.66,2.47) 0.95 (0.69,1.32) 1.32 (0.69,2.53) 1.74(1.18,2.56)* 1.25 (0.86,1.84)
Wheezing after 0.96 (0.71,1.30) 1.29(0.73,2.30) 1.01(0.76,1.34) 1.31 (0.74,2.31) 1.82(1.29,2.57)* 1.38(0.98,1.95)
exercise
Medication 0.93 (0.69,1.24) 1.21(0.69,2.13) 0.97(0.73,1.27) 1.24(0.71,2.17) 1.59(1.14,2.21)* 1.22(0.88,1.70)
ER 1.25 (0.76,2.07) 1.64(0.66,4.07) 1.31 (0.82,2.10) 1.51 (0.62,3.66) 2.03(1.17,3.51)* 1.41 (0.81,2.46)
* The odds ratio is statistically significant between the groups for the specific outcome (p< 0.05).
1 Models are adjusted by towns, age, grade, race, gender, family history of asthma and atopy, and gestational age.
00
19
children who have family history of asthma and atopy with MPO GA and GG
genotypes (OR=0.17, 95% CI=(0.04, 0.74)). There was no significant gene-gene
interaction between MPO and GSTs.
Discussion
The genetic polymorphisms and enzyme activity of MPO have previously
been reported in numerous diseases and biological processes. The variant A allele
was found as a protective factor for lung cancer, especially in men, younger
individuals and current smoker (Schabath, Spitz et al. 2000). The protective effect
was also shown in larynx cancer (Cascorbi, Henning et al. 2000). Individuals with
the wild-type GG genotype were at a greater risk for acute promyelocytic leukemia,
suggesting that higher levels of MPO were associated with an increased risk for
leukemia (Reynolds, Rhees et al. 1999). Additionally, the wild-type GG genotype
was over-represented in patients with early onset of multiple sclerosis in women,
suggesting that higher levels of MPO in macrophages accelerate neural damage
(Nagra, Becher et al. 1997). During MPO-dependent biotransformation process,
MPO converts the tobacco smoke intermediate to the high reactive and carcinogenic
products. Since the variant A allele may be associated with weaker transcriptional
activity, less enzyme would be available for activation of the tobacco smoke
intermediate (Mallet, Mosebrook et al. 1991). Thus, the variant allele reduces
metabolic activity resulting in a protective effect for those diseases.
While many studies have shown that there is a protective effect from the
MPO variant allele for certain outcomes, there were also some studies have shown
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20
that MPO genotypes do not associate those outcomes directly or solely. Only MPO
genotype variants itself were not significantly associated with lung cancer risk.
However, the MPO GA genotype interacted with the presence of glutathione S-
transferase mu and theta (GSTM1 and GSTT1) genotypes to significantly reduce the
risk (Cajas-Salazar, Sierra-Torres et al. 2003). For Caucasian smokers, MPO variant
only showed slightly reduced in lung cancer risk, that was not statistically significant
(Feyler, Voho et al. 2002). For Caucasians, Japanese, and native Hawaiian ancestry
in Hawaii, MPO AA genotype was shown 50% decreased in lung cancer risk, that
was also not statistically significant (Le Marchand, Seifried et al. 2000). However,
our data did not show any evidence of protective effect of MPO variant allele for our
outcomes asthma and wheezing. The joint effects of MPO genotypes and other
environmental or genetic factors will be further investigated.
We found that there was a technical problem that due to a few failures of
TaqMan MPO genotyping assays at the stage of primers and probes design for
TaqMan assay, we found that there are several sequences, on chromosome 5, 7, 8,
and 11, have more than 95% identities with the MPO target sequence, on
chromosome 17, nearby the G to A single nucleotide polymorphism (SNP). Similar
sequence nearby the SNP site causes the interferences of probe binding. A larger
fragment of similar sequence causes the amplification of non-specific PCR products
that gave us invalid genotyping results. Our TaqMan assay was designed to avoid the
similar fragment on chromosome 5, 7, 8 and 11; so that our PCR products were
specific and two probes would bind to specific sequences. BLAST the target
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21
sequences is the most important step when designing genotyping assays. It might be
a reason for Hardy-Weinberg disequilibrium. It might also be a reason why other
reported minor allele frequencies were slightly higher than ours. Although there are
some exist assays have been designed, tested, and analyzed, there is always
possibility to get the wrong genotyping results due to careful less with the sequences.
This useful tool is provided at: http://www.ncbi.nlm.nih.gov/BLAST.
Our findings are consistent with a large literature links prenatal maternal
smoking to decreased lung growth and increased rates of respiratory tract infections,
otitis media, and childhood asthma and wheezing, with the severity of these
problems increasing with increased exposure (DiFranza, Aligne et al. 2004). Prenatal
exposure to cigarette smoke affects airway hyperresponsiveness by modulating the
lung cyclic adenosine monophosphate (cAMP) levels through changes in
phosphodiesterase-4D activity, and these effects are independent of significant
mucous production or leukocyte recruitment into the lung (Singh, Barrett et al.
2003). Maternal cigarette smoking can modify aspects of fetal immune function by
increasing the levels of neonatal T helper type 2 (EL-13 protein) responses. In
appropriate persistence of a T helper type 2 response pattern appears to increase
likelihood of allergic sensitization upon sufficient exposure to a variety of common
antigens (Holt, Macaubas et al. 1999). Additionally, maternal smoking in pregnancy
increases the risk of asthma during the first 7 years of life, and only a small fraction
of the effect seems to be mediated through fetal growth (DiFranza, Aligne et al.
2004). While maternal smoking during pregnancy remains the most significant
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22
source of such exposure and is likely to be responsible for diminished airway
function in early life, continuing postnatal tobacco smoke exposure will increase the
risk of respiratory infections, the combination of both being responsible for the two-
to four fold increased risk of wheezing illnesses observed during the first year of life
in infants whose parents smoke (Stocks and Dezateux 2003). Based on these
findings, it is possible that toxins from in utero exposure to maternal smoking
influence sensitization to common antigens, inflammation, decreased lung function,
and increased airway hyperresponsiveness with variable obstruction to increase the
occurrence of childhood asthma and wheezing.
There were some limitations that influence the interpretation of our results.
As in all cross-sectional studies, sample selection and self-reported information
might be problems for validity (Gilliland, Li et al. 2002). However, the selection bias
was not likely related to genotyping results. Further investigation will be needed to
examine the joint associations of MPO, other candidate genes and the exposure to
tobacco smoke.
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23
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Asset Metadata

Creator Tsai, Wei-wei (author)

Core Title Effects of myeloperoxidase (MPO) polymorphism and tobacco smoke on asthma and wheezing in Southern California children

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Master of Science

Degree Program Biostatistics

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, genetics,health sciences, public health,OAI-PMH Harvest

Language English

Advisor Gilliland, Frank (committee chair), Dubeau, Louis (committee member), Gauderman, William James (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-324037

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