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To determine if a missense change at codon 64 of ADRB3 (Trp64Arg), a candidate obesity gene, is associated with obesity and levels of subcutaneous or visceral fat in African American breast cancer cases. Several observational studies have found that women who are overweight or obese at the time of diagnosis, as well as those who gain weight after diagnosis, are at greater risk for breast cancer recurrence and death than non-overweight women.
Prospective cohort of breast cancer cases.
219 African American breast cancer patients participating in the Los Angeles component of the Health, Eating, Activity, and Lifestyle (HEAL) Study.
ADRB3 Trp64Arg genotype, measures of weight including: body mass index (BMI), weight gain (weight 5 years prior to diagnosis compared to weight at 30 months post diagnosis), obesity (BMI ≥30 kg/m2), waist/hip circumference, and visceral or subcutaneous fat determined by magnetic resonance imaging.
African American 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 be obese (OR=2.1, 95% CI 1.1–4.2). The association with obesity was most pronounced among women who were premenopausal (OR=4.8, 95%CI 1.3–18), who received chemotherapy for their breast cancer (OR=6.1, 95% CI 1.8–20), or who were not physically active (OR=3.9, 95%CI 1.5–9.7).
The wildtype allele of the ADRB3 missense change was associated with measures of obesity in our sample of African American women. The association was modified by menopausal status, history of chemotherapy, and modest levels of physical activity. These results will need to be confirmed in an independent sample.
Breast cancer is the most common cancer in women as well as the second leading cause of cancer deaths1. One important predictor of breast cancer prognosis for women is weight2. 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 than non-overweight women2–6. 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 lower breast cancer risk, presumably because obesity is associated with menstrual cycle disturbances including secondary amenorrhea7, while postmenopausal obesity has been associated with an increased risk of breast cancer, attributed to greater estrogen biosynthesis in peripheral adipose tissue8. There is some evidence that weight control measures such as physical activity may improve survival in women diagnosed with breast cancer and therefore, genetic determinants of weight or weight gain may also be important determinants of survival in breast cancer patients.
Family studies suggest that human obesity has both environmental and genetic determinants9,10. Efforts 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. One such gene, ADRB3, has been targeted as a candidate obesity gene because of its role in the regulation of lipolysis and thermogenesis11. The ADRB3 gene is highly conserved between humans and rodents and its functional impairment may lead to obesity by altering metabolic processes of fat tissue11,12. The receptor is expressed in visceral and subcutaneous fat tissue but is more abundant and active in visceral tissue11. A missense change at codon 64 of this gene (Trp64Arg) has been identified and has been associated with measures of obesity12–14, weight gain15, insulin resistance13, and type II diabetes12.
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 patients. 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-tohip-ratio (WHR)) approximately 30 months after diagnosis of breast cancer, when most women had finished their primary cancer treatment. The analysis was completed to understand better the determinants of weight in breast cancer patients, an important predictor of disease-free and total survival.
The analysis included 219 breast cancer patients (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 analysis was restricted to Los Angeles participants because collection of magnetic resonance imaging data for determination of visceral and subcutaneous fat was exclusive to the Los Angeles site. The HEAL study includes 1,183 pre- and post-menopausal breast cancer patients from California, Washington, and New Mexico16 and was designed to evaluate the roles of hormones, genetics, diet, weight change and physical activity on breast cancer prognosis and survival17–19. 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 previously19.
Participants for the Los Angeles component of the HEAL study were 366 African American women with in-situ, localized, or regional 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 English18. Participation in the HEAL Study included completion of an in-person interview conducted 24 months following the CARE or INSITU study interviews.
The analysis was restricted to 219 African American women who had provided a blood sample for genetic analysis and who had complete anthropometric and questionnaire data. Of the original 366 women who were eligible, 105 women were excluded because they did not complete a 24-month follow-up interview; reasons included death (N=19), illness (N=2), moved out of study area (N=16), refused to participate (N=29), or never reached for a second interview (N=39). An additional 42 women who completed the interview were excluded for missing covariate history including: missing weight (N=1), missing hip and waist measurements (N=9), no blood sample (N=29), and missing fat free mass and percent body fat measurements (N=3).
The Los Angeles component of the study was performed after receiving approval by the Institutional Review Board at the University of Southern California.
Questionnaire data for HEAL were collected during in-person interviews conducted 24 months (approximately 30 months after breast cancer diagnosis) following the CARE or INSITU baseline study interviews. Baseline information was collected 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, family history of breast cancer and other diseases, history of tobacco, 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). The HEAL 24-month follow-up interview included information on tobacco and alcohol use, menopausal status, physical activity, medical problems including diabetes, anthropometric measurements and cancer treatment history. Treatment information, including chemotherapy and tamoxifen history, was obtained by self-report, by examining medications used at the time of the interview and by abstracting each patient's medical records. Los Angeles participants also were asked to undergo magnetic resonance imaging (MRI) to measure abdominal fat. Further details on measures of physical activity, anthropometrics, and MRI are described below.
The HEAL physical activity questionnaire was based on the Modifiable Activity Questionnaire developed by Kriska and colleagues20. 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. Each household or recreational 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 Activities21, where a MET is defined as the ratio of the associated metabolic rate for a specific activity divided by the resting metabolic rate (RMR).
Anthropometric measurements including weight, height, skinfold thickness (tricep, subscapular, thigh, calf), and circumference (waist, hip, midarm, midthigh, calf) were taken by trained interviewers using standardized HEAL protocols19. Weight was measured on a fully clothed respondent. 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/m2, based on the World Health Organization (WHO) definition of obesity22. Waist and hip measurements were taken at the time of each woman's magnetic resonance imaging (MRI) appointment or at the patient's home if the respondent refused the MRI. 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 buttocks19. All measurements were completed by one trained USC interviewer.
MRI was completed at the Los Angeles County-USC Imaging Science Center; single 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.
Fasting blood samples (35 ml) were obtained from each participant at the time of the HEAL 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 described23. 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 were collected and analyzed with ABI Prism 7700 Sequence Detection System version 1.6.5. All Taqman genotyping calls were confirmed by sequencing several samples of each allele.
Stage of disease is coded from medical records and retained as part of the Angeles County Cancer Surveillance Program (CSP) database. The CSP has been a National Cancer Institute-funded Surveillance Epidemiology and End Results (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.
Statistical analyses were performed on logarithmically transformed values of weight, BMI, and abdominal fat 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, weight 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/m2).
All final models were adjusted for age at baseline interview. Adjustment for other potential determinants of obesity were considered, including physical activity, menopausal status at baseline and 24-month interviews, smoking history, history of chemotherapy, tamoxifen therapy, usual weight (i.e. weight 5 years prior to diagnosis) and diabetes. These variables were not included in the final model as they did not substantially affect our results. Because ADRB3 has been associated with diabetes in some studies, a sub-analysis excluding women with a history of diabetes was completed. Stratum specific estimates were calculated using product terms to evaluate potential effect modification separately by menopausal status at baseline and 24-month interviews (premenopausal/post), history of chemotherapy (ever versus never), moderate/vigorous physical activity (above and below median MET value), smoking history (current, past, never), tamoxifen therapy (yes/no) and radiation therapy (yes/no). Wald tests were used to determine p-values for interaction for PROC LOGISTIC Procedure. T-test were used to determine p-values for interaction for PROC GLM Procedure. Calculations were performed in SAS Version 8.2 (SAS Institute, Cary, NC).
The mean age at the baseline interview (approximately 6 months after diagnosis) of the 219 breast cancer survivors in the analysis was 51years (Table 1). One hundred two women (46.6%) were classified as obese (BMI≥30kg/m2). The mean visceral and subcutaneous abdominal fat measurements were 96.6 cm2 and 392 cm2, 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 were approximately 20 per week. Among 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 genotypes were in Hardy-Weinberg equilibrium.
African-American patients who carried the ADRB3 homozygous wild type allele had significantly higher geometric mean levels of visceral abdominal fat compared to women who carried the variant allele (p = 0.04); geometric mean levels of subcutaneous abdominal fat did not differ by ADRB3 genotype (p = 0.50) (Table 2). Differences in hip circumference, waist circumference, and waist to hip ratio also did not differ by ADRB3 genotype (data not shown).
We examined the association between ADRB3 genotype and BMI, where BMI was classified obese (BMI≥30 kg/m2) or non-obese (BMI<30 kg/m2). Women who were homozygous for the wild type allele were twice as likely to be obese (BMI≥30 kg/m2) as women who carried 1 or 2 copies of the variant allele (OR=2.1; 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 been treated with chemotherapy (OR=6.1, 95%CI 1.8–20.4); the associations by menopausal status, chemotherapy and physical activity were independent. No differences in association were found by smoking, tamoxifen, or radiation therapy history.
When we examined the association between ADRB3 and geometric mean BMI, we found that mean BMI was slightly higher for homozygous carriers of the ADRB3 wild type allele than for carriers of the variant (p = 0.15) (Table 4). Similar to our findings for obesity (BMI≥30 kg/m2), the association between ADRB3 genotype and geometric mean BMI was statistically significant when restricted to premenopausal women (p = 0.03), women who had received 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, although not statistically significantly 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, p=0.53). None of our results changed after adjusting for a history of diabetes or excluding these women from the analyses [N=34].
In this sample of African-American breast cancer survivors, we found the Trp64Arg substitution in ADRB3 was associated with reduced measures of adiposity. 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 been treated with 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.
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%)24.
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 Caucasians13, Japanese12,25,26, Pima Indians24, Mexican Americans14, African Americans27,28 and Jamaicans29. Previous studies of white or Japanese populations found the variant allele of the ADRB3 receptor to be associated with elevated measures of obesity13, whereas two previous studies of African-Americans reported associations with the wildtype receptor. Consistent with our data, Lowe Jr. et al reported a protective association between the variant allele and BMI among African-American women (P=0.04)27. 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 significance28. 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 allele29.
ADRB3 is a G-coupled protein receptor30 that stimulates the mobilization of lipids from white fat cells and increases thermogenesis in brown fat cells31. The tryptophan-to-arginine substitution at codon 64 of ADRB3 was initially reported in 199513,15,24. The substitution is located in the first coding exon, at the junction of the first transmembrain domain and the first intracellular loop of the receptor32,33. The impact of the missense change is not known; it has been suggested that the substitution could impact receptor protein folding or alter functional properties such as ligand binding or receptor activation32. 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 agonists32. 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 protein31–33. In clinical studies, a significant reduction in receptor sensitivity to a beta3-agonist was found in adipocytes of Arg carriers34, whereas two studies observed no association with basal metabolic rate35,36.
The ADRB3 receptor is more abundant and active in visceral adipose tissue than subcutaneous adipose11,37–39. The high expression and activity of ADRB3 in visceral adipose tissue is consistent with our observed association between the ADRB3 variant and visceral, but not subcutaneous fat. Findings from a previous study of Japanese women40 also indicated a significant association between the ADRB3 Trp64Arg substitution and visceral abdominal fat. In our African American 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 fat36.
We found the association of ADRB3 and measures of obesity to be stronger among premenopausal than postmenopausal women. Pasquali et al. also found an association of the ADRB3 gene and weight among pre-menopausal women, but not postmenopausal women41. Whereas, a study by McFarlane-Anderson et al.29 found a strong association between ADRB3 and BMI 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 age11. Studies also have found postmenopausal women to have significantly higher BMI41 and significantly less fat-free mass41–43 than premenopausal women while others have reported the odds of being obese was similar for pre- and postmenopausal women44.
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 sites17,18. Previous studies also have found that women experience increasing levels of fat mass, percent body fat45, and body weight35,46,47 following adjuvant chemotherapy. This increase appears to occur shortly after diagnosis, 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 changes17. One possible explanation is lower activity during the first year of diagnosis due to nausea and fatigue following therapy. This difference was not observed in our sample of African American breast cancer cases, as 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.
Physical activity has been shown to impact levels of visceral, subcutaneous, and total body fat48–50. 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 hypertension51 and 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 cancer52–54 and suggestive evidence that physical activity after breast cancer diagnosis may improve survival55. 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 the study of Spanish participants56 where 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 populations27,28 were found with the wildtype allele, whereas the ADRB3-weight association in 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 35, age 18), but 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.
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 higher 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 may be overcome with modest levels of exercise.
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract Nos. N01-PC-35139, N01-PC-35139 and NIH/NCI/PC-67010. Initial data collection for the Los Angeles County patients was supported by the National Institute of Child Health and Human Development through contract N01 HD 3-3175.
The collection of California cancer incidence data used in this publication was supported by the California Department of Health Services as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885. The ideas and opinions expressed herein are those of the author, and no endorsement by the State of California, Department of Health Services is intended or should be inferred.