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The ADRB3 TRP64ARG variant and obesity in African American breast cancer cases

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The ADRB3 TRP64ARG variant and obesity in African American breast cancer cases

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THE ADRB3 TRP64ARG VARIANT AND OBESITY IN AFRICAN AMERICAN
BREAST CANCER CASES

by

Xuejun Li

____________________________________________________________________

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOSTATISTICS)

December 2006

Copyright 2006 Xuejun Li

TABLE OF CONTENTS

List of Tables iii
Abstract iv
Introduction 1
Materials and Methods 3
Results 10
Discussion 15
Conclusion 22
References 23

ii

LIST OF TABLES

Table 1: Population Characteristics of HEAL Study Subjects. 10

Table 2: Age adjusted mean visceral and subcutaneous abdominal fat
by genotype 11

Table 3: Relative odds of being obese associated with ADRB3 genotype
overall and by menopausal status, physical activity level, and
chemotherapy 13

Table 4: Age adjusted mean BMI (kg/m
2
) for each genotype across
different levels of menopausal status, physical activity, and
chemotherapy 14

iii

ABSTRACT

Weight is an important determinant of disease-free survival in women
diagnosed with breast cancer. ADRB3 is a candidate obesity gene that plays an
active role in lipid metabolism in visceral fat tissue. A missense change at codon 64
of this gene has been associated with obesity-related phenotypes and may be relevant
to weight and obesity among breast cancer cases. The current analysis includes 219
women from the Los Angeles component of the HEAL Study – a population-based
cohort of African-American (AA) breast cancer patients. We found AA women who
were homozygous for the ADRB3 wild type allele had significantly higher mean
visceral fat levels than women who carried the variant (p=0.04) and were
significantly more likely to have a BMI >30 kg/m
2
(OR=2.1, 95% CI 1.1-4.2). The
association with BMI was most pronounced among women who were
premenopausal, who had taken chemotherapy, or who were not physically active.

iv

INTRODUCTION
Breast cancer is the second most common cancer in women as well as the
second leading cause of cancer deaths (American Cancer Society 2005). One
important predictor of breast cancer risk and prognosis for women is weight
(Chlebowski, Aiello et al. 2002). Several observational studies have found that
women who are overweight or obese at the time of breast cancer diagnosis, as well
as those who gain weight after diagnosis, are at greater risk for breast cancer
recurrence and death compared with non-overweight women (Demark-Wahnefried,
Rimer et al. 1997; Newman, Lees et al. 1997; Goodwin, Esplen et al. 1998; Daling,
Malone et al. 2001; Chlebowski, Aiello et al. 2002). The relationship between
obesity and breast cancer may be explained, at least in part, by women’s lifetime
exposure to estrogens. Premenopausal obesity has been associated with a cessation
of ovulation, assumed to have a protective role for breast cancer (Weiderpass,
Braaten et al. 2004), while postmenopausal obesity has been associated with an
increased risk of breast cancer, attributed to greater estrogen biosynthesis in
peripheral adipose tissue (Probst-Hensch, Pike et al. 2000). There is some evidence
that weight control measures such as physical activity may improve survival in
women diagnosed with breast cancer (Holmes, Chen et al. 2005) and therefore,
genetic determinants of weight or weight gain may also be important determinants of
survival in breast cancer cases.
Family studies suggest that human obesity has both environmental and
genetic determinants (Bouchard and Perusse 1993; Maes, Neale et al. 1997). Efforts
1

to identify candidate obesity genes have focused on genes active in adipose tissue
due to the crucial role of the adipose cells in regulating the storage and mobilization
of lipids. The ADRB3 gene has been targeted as a candidate obesity gene because of
its role in the regulation of lipolysis and thermogenesis (Emorine, Blin et al. 1994).
The ADRB3 gene is highly conserved between humans and rodents and it is believed
that its functional impairment may lead to obesity by altering metabolic processes of
fat tissue (Emorine, Blin et al. 1994; Oizumi, Daimon et al. 2001). The receptor is
expressed in visceral and subcutaneous fat tissue but is more abundant and active in
visceral tissue (Emorine, Blin et al. 1994). A missense change at codon 64 of this
gene (Trp64Arg) has been identified and has been associated with measures of
obesity (Widen, Lehto et al. 1995; Mitchell, Blangero et al. 1998; Oizumi, Daimon et
al. 2001), weight gain (Clement, Vaisse et al. 1995), insulin resistance (Widen, Lehto
et al. 1995), and type II diabetes (Oizumi, Daimon et al. 2001).
In the current study, we examined the association between the codon 64
ADRB3 missense change and measures of weight and obesity among African
American breast cancer cases. We assessed the relationship of the ADRB3 genotypes
with visceral and subcutaneous abdominal fat, body mass index (BMI), weight
change, and measures of central obesity (waist, hip, waist-to-hip-ratio (WHR))
approximately 30 months after diagnosis of breast cancer, when most women had
finished their primary cancer treatment. The analysis was completed to better
understand the determinants of weight in breast cancer cases, an important predictor
of disease-free and total survival.
2

MATERIALS AND METHODS
The analysis included 219 breast cancer cases (approximately 30 months
post-diagnosis) from the Los Angeles component of the Health, Eating, Activity, and
Lifestyle (HEAL) Study, a population-based prospective cohort study of women with
breast cancer. The HEAL study includes 1,223 pre- and post-menopausal breast
cancer patients from California, Washington, and New Mexico (McTiernan, Rajan et
al. 2003) and was designed to evaluate the roles of hormones, genetics, diet, and
physical activity on breast cancer prognosis and survival (Irwin, Crumley et al. 2003;
McTiernan, Rajan et al. 2003; Irwin, McTiernan et al. 2004). Women were recruited
into the HEAL Study through Surveillance, Epidemiology, End Results (SEER)
registries in New Mexico, Los Angeles County (CA), and Western Washington.
Details of the aims, study design, and recruitment procedures have been published
previously (McTiernan, Rajan et al. 2003).
Participants for the Los Angeles component of the HEAL study included 366
African American (AA) women with stage 0–IIIA breast cancer who had
participated in the Los Angeles portion of the Women’s Contraceptive and
Reproductive Experiences (CARE) Study, a case-control study of invasive breast
cancer, or who had participated in a parallel case-control study of in situ breast
cancer (INSITU). Eligible participants from these two studies were women who
were diagnosed with breast cancer between May 1995 and May 1998, who were
aged 35–64 years at diagnosis, and who were born in the United States and therefore
able to speak English (Irwin, McTiernan et al. 2004).
3

The analysis was restricted to 219 (of the original 366) AA women who had
provided a blood sample for genetic analysis and who had complete anthropometric
and questionnaire data. Cases excluded for missing information included: 105
women who did not complete a 24-month follow-up interview, 1 woman with
missing weight, 29 women with missing genotype data, 9 women with missing hip
and waist measurements, and 3 patients who were missing fat free mass and percent
body fat measurements.
The Los Angeles component of the study was performed after receiving
approval by the Institutional Review Board at the University of Southern California.

DATA COLLECTION AND DEFINITIONS.
Questionnaire variables. Questionnaire data was collected through in-person
interviews approximately 30 months after breast cancer diagnosis (24 months after
the baseline interview). In-person interviews were conducted with each participant to
collect information on demographics, reproductive and menstrual history (age at
menarche, regularity of periods when menstruating, age at menopause, type of
menopause), hysterectomy and oophorectomy status, history of oral contraceptive
and hormone replacement therapy use, medical history including history of
endocrine problems and other medical problems, history of tobacco, caffeine, and
alcohol use, maximal adult height, height at age 18 and 65, and previous weight
(ages 18, 35, 50, 65 years and 5 years prior to diagnosis). Anthropometric
measurements including weight, height, skinfold thickness (tricep, subscapular,
4

thigh, calf), and circumference (waist, hip, midarm, midthigh, calf) were made and
serum samples were collected at the 24-month follow-up interview. Treatment
information, including chemotherapy and tamoxifen history, was obtained from
baseline and follow-up interviews, examination of medication bottles, medical record
review, and summary abstracts from the SEER registries.
Anthropometrics & Bioelectrical Impedance. Anthropometric measurements were
taken by trained interviewers using standardized HEAL protocols at the 24-month
follow-up interview (McTiernan, Rajan et al. 2003). Weight was measured on a
fully clothed respondent using a portable scale that was calibrated on a monthly
basis. Height was self-reported by participants. Body mass index was computed as
weight in kilograms divided by self-reported height in meters squared. Patients were
classified as obese if their body mass index was greater than or equal to 30 kg/m
2
,
based on the World Health Organization (WHO) definition of obesity (Ogden, Flegal
et al. 2002).
Waist and hip measurements were taken at the time of each woman’s
magnetic resonance imaging (MRI) appointment. If the respondent refused the MRI,
the anthropometric measurements were done at the patient’s home. Waist
circumference was measured in centimeters just above the superior margin of the
iliac crest. Hip circumference was measured in centimeters at the maximal posterior
projection of the buttocks (McTiernan, Rajan et al. 2003). All measurements were
completed by one trained USC interviewer. MRI was completed on Los Angeles
HEAL participants at the Los Angeles County-USC Imaging Science Center; single
5

slice T1-weighted images with 1 cm thickness were acquired axially at the
umbilicus. Inter-abdominal fat and extra-abdominal fat was outlined and recorded in
square centimeters as determined by the study radiologist (Dr. Pat Colletti) and
image results were archived in electronic and film formats. Inter-abdominal and
extra-abdominal fat measurements were reviewed and confirmed by a second
radiologist at the University of New Mexico.
Physical activity assessment. Information on physical activity was collected
at baseline (on physical activity levels prior to diagnosis) and at the 24-month
follow-up interview. The questionnaire at the 24-month follow-up interview was
based on the Modifiable Activity Questionnaire developed by Kriska and colleagues
(Kriska 1997), which was designed to be easily modified for use with different
populations and has been shown to be reliable and valid. The type, duration, and
frequency of activities performed in the past year were assessed. The
sports/recreation and household activity section of the questionnaire addressed 29
popular activities, such as fast walking, moderate/slow walking, jogging, aerobic,
tennis, household cleaning, and yard work.
Hours of activity per week for each activity type were calculated by
multiplying frequency of the activity by duration. Two mutually exclusive groups
were created based on type of activity (sports/recreation or household/gardening).
Each activity was categorized as light- (< 3 METs), moderate- (3-6 METs), or
vigorous (>6 METs) intensity based on Ainsworth et al.’s Compendium of Physical
Activities (Ainsworth, Haskell et al. 2000) , where a MET is defined as the ratio of
6

the associated metabolic rate for a specific activity divided by the resting metabolic
rate (RMR). We also considered both moderate and vigorous intensity activities
combined.
Blood draw, DNA extraction, and polymorphism determination. Fasting
blood samples (35 ml) were obtained from each participant at the 24-month follow-
up interview. Blood was processed within 3 hours of collection; serum and buffy
coat were stored in 1.8-ml aliquot tubes at -70 to –80 °C. Genomic DNA was
extracted from peripheral blood leukocytes using a Qiagen kit. The ADRB3
Trp64Arg variant for all samples was determined by allelic discrimination in a
fluorogenic Taqman assay at Albany Molecular Research in Bothell, Washington,
with the ABI 7700 Sequence Detection System (Applied Biosystems, Foster City,
CA), which has been described (Livak 1999). The PCR primers were: (sense) 5’-
GGCCATCGCC3’; and (antisense) 5’- GGACTCCGAG -3’. The sense and
antisense PCR primers were used at a final concentration of 900nM in 25µl PCR
solution. Both fluorogenic wild-type and variant allele-specific probes were
complementary to their corresponding antisense strands and were labeled with the
TAMRA quencher at the 3’ end, with the 6-FAM reporter dye and the VIC reporter
dye at the 5’ end. The wild type and variant fluorogenic probes were used at a final
concentration of 100 nM and detected alleles from approximately 40 ng of genomic
DNA template. Temperature cycling was performed as followed: 50
○
C for 2 minutes
and 95
○
C for 10 minutes, followed by 40 cycles of 95
○
C for 15 s and 62
○
C for 1
minute. The data was collected and analyzed with ABI Prism 7700 Sequence
7

Detection System version 1.6.5. All Taqman genotyping calls were confirmed by
sequencing several samples of each allele.
Stage of disease and cancer treatment. Stage of disease was determined by
medical record review from summary data assembled by the Los Angeles County
Cancer Surveillance Program (CSP). The CSP has been a National Cancer Institute-
funded SEER registry since 1992 and is estimated to be more than 99% complete.
Participants were classified as having in situ, stage I (localized), or stage II–IIIA
(regional) breast cancer using the SEER stage of disease classification (SEER
Program (National Cancer Institute (U.S.)), Fritz et al. 1998).

STATISTICAL ANALYSES
Statistical analyses were performed on logarithmically transformed values for
weight and BMI, and geometric mean values with 95% confidence intervals (CI) are
presented. Analysis of covariance was used to test the hypotheses that mean visceral
abdominal fat, subcutaneous abdominal fat, BMI, BMI gain from 5 years prior to
diagnosis to the 24-month follow-up interview, and mean hip and waist
circumference vary by ADRB3 genotype. Odds ratios and 95% confidence intervals
were calculated using unconditional logistic regression to determine if the ADRB3
genotype was associated with obesity status (defined as having a BMI ≥30 kg/m
2
).
All models were adjusted for age at interview. We tested for effect modification by
the covariates using the Wald’s test. Stratified analysis by history of chemotherapy
(ever versus never), moderate/vigorous physical activity (above and below median
8

MET value), menopausal status at baseline and 24-month interviews
(premenopausal/post), smoking history (current, past, never), tamoxifen therapy
(yes/no) and radiation therapy (yes/no) separately were completed to evaluate
potential effect modification. Two-sided p-values comparing ADRB3 wildtype to the
variant receptor are presented. Calculations were performed using the PROC GLM
procedure in SAS Version 8.2 (SAS Institute, Cary, NC).

9

RESULTS
The mean age at the baseline interview (approximately 6 months after
diagnosis) of the 219 breast cancer survivors in the analysis was 51years, of whom
102 (46.6%) were classified as obese (BMI>30kg/m
2
) (Table 1). The mean visceral
and subcutaneous abdominal fat measurements were 96.6 cm
2
and 391.7 cm
2
,
respectively. At the 24-month interviews, 204 women (93.2%) reported they
participated regularly in moderate or vigorous physical activity (MET ≥3) and the
mean hours of moderate and vigorous physical activity combined was approximately
20 per week. Among the total participants, 170 (77.6%) were ADRB3 homozygous
wild type (Trp64Trp) and 49 (22.4%) had 1 or 2 copies of the variant (44 had
Trp64Arg and 5 had Arg64Arg). The 12.3% minor allele (A) frequency in our
African-American sample is similar to the frequency reported for other African-
American (12%) and Mexican-American populations (13%), but higher than the
frequency reported for Caucasians (8%), and lower than the frequency reported for
Pima Indians (31%) (Walston, Silver et al. 1995). The genotypes were in Hardy-
Weinberg equilibrium.
Table 1. Population Characteristics of HEAL Study Subjects (N=219).
Continuous
variables
Mean (SD) Continuous
variables
Mean (SD)
Age at interview 51.0 (7.8) Hip (cm) 114.2 (16.1)
Weight (Kg) 83.5 (21.5) Calculated Fat Free Mass 47.4 (7.8)
Height at 18 (cm) 165.3 (7.2) Visceral abdominal fat (cm
2
)

* 96.6 (49.9)
BMI (kg/m
2
) 30.5 (7.3) Subcutaneous abdominal fat
(cm
2
)

*
391.7(188.5
)
Waist (cm) 97.1 (15.6) MET HR/WK of moderate or
vigorous physical activity
20.3 (21.9)
10

Table 1. Continued.
*79 women were not tested for internal/external abdominal fat
Categorical variables N (%) Categorical variables N (%)
ADR β3 gene Age Group
Trp64Trp 170 (77.6) 36 – 45 60 (27.4)
Trp64Arg, Arg64Arg 49 (22.4) 46 – 50 44 (20.1)
51 – 55 48 (21.9)
BMI 56 – 65 67 (30.6)
BMI>30kg/m
2
102 (46.6)
25 < BMI ≤30kg/m
2
65 (29.7) Smoking Status
BMI ≤25kg/m
2
52 (23.7) Never 108 (49.3)
Past 73 (33.3)
Diagnostic Stage Current 38 (17.4)
0 = In situ 43 (19.6)
1 = Localized 94 (42.9) History of Tamoxifen Use 86 (39.3)
2 = Regional 82 (37.5)
History of Radiation
Therapy
82 (37.4)
Menopausal Statusº
Pre 89 (40.6) History of Chemotherapy 92 (42.0)
Post 103 (47.1)
Unknown 27 (12.3)
ºmenopausal status at baseline interview
In our African-American sample, women who carried the ADRB3
homozygous wild type allele had significantly higher mean levels of visceral
abdominal fat compared to women who carried the variant allele (p = 0.04); the
association between ADRB3 genotype and subcutaneous abdominal fat was not
significant (p = 0.50) (see Table 2). Differences in hip circumference, waist
circumference, and waist to hip ratio did not differ significantly by ADRB3 genotype.
Table 2. Age adjusted mean visceral and subcutaneous abdominal fat by
genotype (N=140)*.
Trp64Trp
Mean (95% CI)
Trp64Arg, Arg64Arg
Mean (95% CI)
P

Visceral abdominal
fat (cm
2
)
101.0 (92.0 – 110.0) 80.4 (63.1 – 97.7) 0.04
Subcutaneous
abdominal fat (cm
2
)
398.3 (362.7 – 433.9) 367.5 (299.3 – 435.7) 0.50
*79 women were not tested for abdominal fat
11

We examined the association between ADRB3 genotype and BMI,
considering BMI as both a categorical (obese: BMI ≥30 kg/m
2
; normal: BMI<30
kg/m
2
) and continuous variable (mean BMI). In our sample, women who were
homozygous for the wild type allele were about twice as likely to be obese (BMI ≥30
kg/m
2
) as women who carried 1 or 2 copies of the variant allele (95%CI 1.1-4.2)
(Table 3). This association was most pronounced among women who were
premenopausal (OR=4.8, 95%CI 1.3-18.1), who were less physically active
(OR=3.9, 95%CI 1.5- 9.7), or who had chemotherapy (OR=6.1, 95%CI 1.8-20.4). A
three-way interaction term for genotype, chemotherapy, and physical activity was
not statistically significant and there was no difference in moderate to vigorous
physical activity levels at the 24-month follow-up visit by history of chemotherapy.
When we examined the association between ADRB3 and mean BMI, we found mean
BMI was slightly higher for homozygous carriers of the ADRB3 wild type allele than
carriers of the variant; however, this association did not reach statistical significance
(p = 0.15) (Table 4). Similar to our findings for obesity (BMI ≥30 kg/m
2
), the
association between ADRB3 and mean BMI was statistically significant when
restricted to premenopausal women (p = 0.03), women who had a history of
chemotherapy (p = 0.04), or women who were less physically active (p = 0.01).
Change in mean BMI from 5 years prior to diagnosis to the 24-month follow-up
interview was slightly higher for homozygous carriers of the ADRB3 wild type allele
than carriers of the variant (wildtype BMI change 3.3, 95%CI 2.6-3.9 Vs. variant
BMI change 2.8, 95%CI 1.7-4.0), but this change was not statistically significant .

12

Table 3. Relative odds of being obese associated with ADRB3 genotype overall
and by menopausal status, physical activity level, and chemotherapy.
Case (%) Control (%) OR§ 95% CI** P
All patients
Trp64Trp 86 (84.31) 84 (71.79) 2.14 1.09 - 4.18 0.03
Trp64Arg, Arg64Arg 16 (15.69) 33 (28.21)
Menopausal Status
Pre Menopausal:
Trp64Trp 32 (91.43) 38 (70.37) 4.76 1.25 - 18.05 0.02
Trp64Arg, Arg64Arg 3 (8.57) 16 (29.63)
Post Menopausal:
Trp64Trp 43 (82.69) 38 (74.51) 1.51 0.58 - 3.98 0.40
Trp64Arg, Arg64Arg 9 (17.31) 13 (25.49)
P
interaction
= 0.22
MET hours/week of Moderate or Vigorous Activity
≤13.13||
Trp64Trp 48 (85.71) 33 (61.11) 3.85 1.52 – 9.74 <0.0
1
Trp64Arg, Arg64Arg 8 (14.29) 21 (38.89)
>13.13
Trp64Trp 38 (82.61) 51 (80.95) 1.12 0.42 – 3.01 0.83
Trp64Arg, Arg64Arg 8 (17.39) 12 (19.05)
P
interaction
= 0.08
Chemo Therapy
No
Trp64Trp 43 (78.18) 55 (76.39) 1.11 0.48 – 2.57 0.81
Trp64Arg, Arg64Arg 12 (21.82) 17 (23.61)
Yes
Trp64Trp 43 (91.49) 29 (64.44) 6.12 1.84 –
20.43
<0.0
1
Trp64Arg, Arg64Arg 4 (8.51) 16 (35.56)
P
interaction
= 0.03
§adjusted for age.
**confidence interval.
||median value.

13

Table 4. Age adjusted mean BMI (kg/m
2
) for each genotype across different
levels of menopausal status, physical activity, and chemotherapy.
Trp64Trp

Mean (95% CI)
Trp64Arg,
Arg64Arg
Mean (95% CI)
P
All Patients 30.01
(28.97 – 31.09)
28.42
(26.61 - 30.35)
0.15
Menopausal Status
Pre-menopausal 29.61
(28.08 – 31.22)
26.01
(23.48 – 28.81)
0.03
Post-menopausal 30.20
(28.66 – 31.82)
30.70
(27.76 – 33.95)
0.78
P
interaction
= 0.10
MET hours/week of Moderate or Vigorous Activity
≤13.13 31.96
(30.36 – 33.65)
28.17
(25.85 – 30.69)
0.01
>13.13 28.32
(27.03 – 29.68)
28.85
(26.13 –31.85)
0.74
P
interaction
= 0.06
Chemo Therapy
No 29.18
(27.85 - 30.58)
29.04
(26.6414 - 31.65)
0.92
Yes 31.17
(29.53 - 32.90)
27.55
(24.86 - 30.52)
0.04
P
interaction
= 0.13
§adjusted for age.

14

DISCUSSION
In this sample of African-American breast cancer survivors, we found the
Trp64Arg ADRB3 substitution was associated with elevated measures of weight.
Specifically, we found that women who were homozygous for ADRB3 wildtype
allele had significantly higher levels of visceral abdominal fat compared to women
who carried the variant. Further, women with the wildtype allele were more likely to
be obese. This association was strongest among premenopausal women, women
who had a history of chemotherapy, or who were less physically active; however the
numbers of women in each stratum were small. We did not find significant
associations for ADRB3 with other measures of weight, including weight gain, waist
circumference, hip circumference, or waist to hip ratio.
In previous epidemiologic studies, the association between the ADRB3
variant and obesity-related phenotypes has been investigated among individuals from
several racial/ethnic groups and nationalities, including Caucasians (Widen, Lehto et
al. 1995) , Japanese (Kadowaki, Yasuda et al. 1995; Nagase, Aoki et al. 1997;
Oizumi, Daimon et al. 2001), Pima Indians (Walston, Silver et al. 1995), Mexican
Americans (Mitchell, Blangero et al. 1998), African Americans (Lowe Jr, Rotimi et
al. 2001; Terra, McGorray et al. 2005) and Jamaicans (McFarlane-Anderson, Bennett
et al. 1998). Previous studies of white or Japanese populations found the variant
allele of the ADRB3 receptor to be associated with elevated measures of obesity
(Widen, Lehto et al. 1995), whereas two previous studies of African-Americans
reported associations with the wildtype receptor. Consistent with our data, Lowe Jr.
15

et al reported a protective association between the variant allele and BMI among
African-American women (P=0.04) (Lowe Jr, Rotimi et al. 2001). Similarly, Terra
et al reported that African-Americans who were homozygous for the wildtype allele
had higher mean BMI than variant carriers, although the association did not reach
statistical significance (Terra, McGorray et al. 2005). In contrast, McFarlane-
Anderson et al found that Jamaican women who were carriers of the variant allele
had a significantly increased BMI compared to women who were homozygous for
the wildtype allele (McFarlane-Anderson, Bennett et al. 1998).
ADRB3 is a G-coupled protein receptor (Emorine, Marullo et al. 1989) that
stimulates the mobilization of lipids from white fat cells and increases thermogenesis
in brown fat cells (Arner and Hoffstedt 1999). The tryptophan-to-arginine
substitution at codon 64 of ADRB3 was initially reported in 1995 (Clement, Vaisse et
al. 1995; Walston, Silver et al. 1995; Widen, Lehto et al. 1995). The substitution is
located in the first coding exon, at the junction of the first transmembrain domain
and the first intracellular loop of the receptor (Candelore, Deng et al. 1996; Pietri-
Rouxel, St John Manning et al. 1997). The impact of the missense change is not
known; however, it has been suggested that the substitution could impact receptor
protein folding or alter functional properties such as ligand binding or receptor
activation (Candelore, Deng et al. 1996). The biochemical evidence that the
substitution impairs receptor signaling is mixed. In vitro studies suggest the
substitution does not impact ADRB3 receptor expression, its ability to bind
andrenergic agonists, or its activation in response to selective or non-selective
16

agonists (Candelore, Deng et al. 1996). However, several studies found the
maximal activation potential of cyclic AMP in Arg variant cells was reduced,
suggesting the substitution may alter the receptor’s ability to interact with G protein
(Candelore, Deng et al. 1996; Pietri-Rouxel, St John Manning et al. 1997; Arner and
Hoffstedt 1999). In clinical studies, a significant reduction in receptor sensitivity to
a beta3-agonist was found in adipocytes of Arg carriers (Hoffstedt, Poirier et al.
1999), whereas two studies observed no association with basal metabolic rate
(Heasman, Sutherland et al. 1985; Gagnon, Mauriege et al. 1996).
The ADRB3 receptor is more abundant and active in visceral adipose tissue
than subcutaneous adipose (Emorine, Blin et al. 1994; Hoffstedt, Shimizu et al.
1995; Lonnqvist, Thome et al. 1995; Katzmarzyk, Perusse et al. 1999). The high
expression and activity of ADRB3 in visceral adipose is consistent with our observed
association between the ADRB3 variant and visceral, but not subcutaneous fat.
Findings from a previous study of Japanese women (Sakane, Yoshida et al. 1997)
also indicated a significant association between the ADRB3 Trp64Arg substitution
and visceral abdominal fat. However, in our AA cases, the positive association
between ADRB3 and obesity was found for the wildtype receptor, and in Japanese
women, the association was found for the variant. A third study (Quebec Family
Study) found no association present between the missense change and abdominal
visceral fat (Gagnon, Mauriege et al. 1996).
We found the association of ADRB3 and measures of obesity to be stronger
among premenopausal than postmenopausal women. Pasquali et al. also found an
17

association of the ADRB3 gene and weight among pre-menopausal women, but not
postmenopausal women (Pasquali, Casimirri et al. 1994). Whereas, a study by
McFarlane-Anderson et al. (McFarlane-Anderson, Bennett et al. 1998) found the
association between ADRB3 and BMI to be significantly elevated among both pre-
and postmenopausal women. The presence of a stronger association between the
ADRB3 missense change and obesity in premenopausal women may be explained by
the fact that the expression of ADRB3 is age-dependent and expression has been
shown to decrease with age (Emorine, Blin et al. 1994). Other studies have found
postmenopausal women to have significantly less fat-free mass (Poehlman, Toth et
al. 1995) and significantly higher BMI (Pasquali, Casimirri et al. 1994) than
premenopausal women suggesting possible changes in physical activity or
metabolism with age, while some studies have reported that the odds of being obese
was similar for pre- and postmenopausal women (Huang, Hamajima et al. 2001).
In our sample of breast cancer cases, the association of ADRB3 and obesity
was strongest among women with a history of chemotherapy. Higher chemotherapy
associated weight gain among breast cancer cases has been reported by HEAL
investigators using data from the Washington and New Mexico sites (Irwin, Crumley
et al. 2003; Irwin, McTiernan et al. 2004). Previous studies also have found that
women experience increasing levels of fat mass, percent body fat (Demark-
Wahnefried, Peterson et al. 2001), and body weight (Heasman, Sutherland et al.
1985; Demark-Wahnefried, Winer et al. 1993; Aslani, Smith et al. 1999) following
adjuvant chemotherapy. This increase appears to occur shortly after diagnosis,
18

typically within the first year and includes a larger increase in fat mass than weight
gain associated with normal aging. This sarcopenic weight gain is believed to be
due, at least in part, to decreased physical activity and possibly to metabolic changes
(Irwin, Crumley et al. 2003). One possible explanation is lower activity during the
first year of diagnosis due to nausea, fatigue, and general malaise following therapy.
However in our sample of African American breast cancer cases, women who were
treated with chemotherapy reported similar levels of moderate and vigorous physical
activity (Mean MET hours chemo = 20.7 ; Mean MET hours no chemo = 19.9) at the
24-month follow-up interview as women who did not take chemotherapy. We did
not collect physical activity history at the time of diagnosis and treatment, so it
remains possible that women with chemotherapy gained weight due to lower activity
at the time of their treatment, but we were not able to evaluate these differences.
Physical activity has been shown to impact levels of visceral, subcutaneous,
and total body fat (Santeusanio, Di Loreto et al. 2003; Slentz, Aiken et al. 2005).
Accumulation of visceral fat, more so than accumulation in other parts of the body,
has been of clinical concern because of its potential contribution to risk of chronic
conditions including diabetes, hyperinsulinemia, and hypertension (Warren,
Schreiner et al. 2006). It also may contribute to risk of postmenopausal breast cancer
due to the production of estrogens in adipose tissue. There is growing evidence that
physical activity can decrease the risk of breast cancer (Bernstein, Henderson et al.
1994; Gilliland, Li et al. 2001; International Agency for Research on Cancer 2002)
and suggestive evidence that physical activity after breast cancer diagnosis may
19

improve survival (Holmes, Chen et al. 2005). In the current analysis, we found the
association between the ADRB3 Trp genotype and obesity to be highest among
sedentary women. Our finding that physical activity modifies the effect of ADRB3
on obesity is supported by data from one other study of Spanish participants (Marti,
Corbalan et al. 2002). In this study, Marti et al found that risk of obesity was higher
among carriers of the ADRB3 variant when restricted to sedentary people. Together,
these data suggest that the association between the ADRB3 gene and obesity may be
altered by moderate levels of physical activity.
While we were able to detect an association between ADRB3 and measures of
weight among HEAL African-American breast cancer cases, these results may not be
generalizable to other racial/ethnic groups. The associations for our African-
American set of cases and two other African-American populations (Lowe Jr, Rotimi
et al. 2001; Terra, McGorray et al. 2005) were found with the wildtype allele,
whereas the ADRB3-weight association in other white and Japanese populations has
been described with the variant allele. While some experimental studies suggest that
carriers of the Arg variant may have reduced fat metabolism, the functional role of
the missense is not yet established, nor do we know how lifestyle or other lipid
metabolism pathway genes influence the ADRB3-weight association. Further,
nearly 46% of our population was obese, which may represent a group of individuals
prone to weight gain due to genetic background or other individual characteristics.
Another limitation of our study is that we collected weight at 24-months post
diagnosis and at various ages prior to diagnosis (e.g. 5 years prior to diagnosis, age
20

35, age 18), however we did not measure weight or physical activity at the time of
diagnosis so we could not examine changes from the time of diagnosis to the 24-
month follow-up interview.

21

CONCLUSION
Several studies have reported an association between the codon 64 missense
change of the ADRB3 gene and measures of weight. The results of the current study
suggest that the wildtype allele of the ADRB3 codon 64 substitution is associated
with measures of weight and obesity in our sample of African American breast
cancer cases. Because our association was found with the opposite allele as reported
in whites and the functionality of this change has not been clearly established, these
findings will need to be confirmed in an independent set of African-American
women. In our sample, the relationship between the ADRB3 gene and obesity was
modified by menopausal status, history of chemotherapy, and modest levels of
physical activity. The physical activity finding suggests that while some women
may be genetically predisposed to obesity, this tendency can be overcome with
modest levels of exercise.

22

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27

Abstract (if available)

Abstract Weight is an important determinant of disease-free survival in women diagnosed with breast cancer. ADRB3 is a candidate obesity gene that plays an active role in lipid metabolism in visceral fat tissue. A missense change at codon 64 of this gene has been associated with obesity-related phenotypes and may be relevant to weight and obesity among breast cancer cases. The current analysis includes 219 women from the Los Angeles component of the HEAL Study -- a population-based cohort of African-American (AA) breast cancer patients. We found AA women who were homozygous for the ADRB3 wild type allele had significantly higher mean visceral fat levels than women who carried the variant (p=0.04) and were significantly more likely to have a BMI >30 kg/m2 (OR=2.1, 95% CI 1.1-4.2). The association with BMI was most pronounced among women who were premenopausal, who had taken chemotherapy, or who were not physically active.

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Asset Metadata

Creator Li, Xuejun (author)

Core Title The ADRB3 TRP64ARG variant and obesity in African American breast cancer cases

School Keck School of Medicine

Degree Master of Science

Degree Program Biostatistics

Degree Conferral Date 2006-12

Publication Date 11/14/2006

Defense Date 10/01/2005

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

Tag ADRB3,African American women,HEAL study,OAI-PMH Harvest,obesity

Language English

Advisor McKean-Cowdin, Roberta (committee chair), Gauderman, W. James (committee member), Gilliland, Frank D. (committee member)

Creator Email xuejunal@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m130

Unique identifier UC1276611

Identifier etd-Li-20061114 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-21316 (legacy record id),usctheses-m130 (legacy record id)

Legacy Identifier etd-Li-20061114.pdf

Dmrecord 21316

Document Type Thesis

Rights Li, Xuejun

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu