Materials and Methods

Participants (n = 639) were recruited for a cross-sectional study of risk factors for menopausal hot flashes in midlife women. Detailed methods of participant recruitment are described elsewhere. Briefly, from 2000 through 2004, generally healthy women residing in Baltimore city and the surrounding counties were sent recruitment letters via mail. Potential participants had to meet the following eligibility criteria: aged 45-54 years, not taking hormonal therapy, not pregnant, and no history of cancer. Further, because the study was designed to examine the health of women undergoing the menopausal transition, women were eligible only if they reported having at least 3 menstrual periods in the last 12 months. Therefore, all women in the study were either premenopausal or perimenopausal. Eligible women were scheduled to come to the clinic during the morning hours and were instructed to fast overnight prior to the visit. During the clinic visit, each participant had their weight and height measured, and a blood sample was drawn for genetic and hormone concentrations analyses. The participants also completed the detailed study survey, which included questions regarding demographics; medical, family, and reproductive history; past exogenous hormone use; menopausal symptoms; and lifestyle habits (eg, smoking, diet). All participants in this study gave written informed consent according to procedures approved by the University of Illinois and Johns Hopkins University Institutional Review Boards.

Age and race/ethnicity were self-reported. BMI was calculated based on height and weight measurements of the participant at the clinic visit and categorized either as normal (≤24.9 kg/m ² ), overweight (25.0-29.9 kg/m ² ), or obese (≥30.0 kg/m ² ). Smoking status at the time of enrollment (current/former/never) was determined using the questions “Have you ever smoked cigarettes?” and “Do you still smoke cigarettes?” A woman’s history of ever experiencing midlife hot flashes was determined using the question “Have you ever experienced hot flashes?” Women experiencing hot flashes were further queried regarding hot flash severity (mild, moderate, or severe), frequency (daily, weekly, or monthly), and duration (number of months/years). Hot flash outcomes examined in the analyses were: ever experienced hot flashes, moderate or severe hot flashes, daily hot flashes, and hot flashes experienced for ≥1 year (long duration). For all hot flash outcomes, the comparison group was those who had never experienced hot flashes.

SNPs genotyped to address the hypotheses were: AHR (rs2066853; Arg554Lys; G>A), AHRR (rs2292596; Pro185Ala; C>G), ARNT (rs2228099; Val89Val; G>C), and CYP1B1 (rs1800440; Asn453Ser; A>G). Additionally, genotype data for the SNP CYP1B1 (rs1056836; Leu432Val; C>G) that was reported in a previously published manuscript from this study were also used in the analyses.

Genomic DNA was isolated from the donated blood samples using the GeneElute Blood Genomic DNA kit (Sigma-Aldrich, St. Louis, MO) and was amplified through polymerase chain reactions (PCR) based on published primers. Genotyping of AHR , ARNT , and AHRR SNPs was done using the allele-specific PCR method. Each reaction tube of AHR , ARNT , and AHRR contained: DNA sample 3 μL, HotStarTaq Plus Master mix 10 μL (Qiagen, Venlo, The Netherlands), 10X reaction buffer 2 μL (Qiagen), forward and reverse primers (50 mmol/L) 0.1 μL of each, allele-specific primer (50 mmol/L) 0.1 μL ( AHR ) or 0.02 μL ( AHRR , ARNT ), and RNase-free water 4.7 μL ( AHR ) or 4.78 μL ( ARNT , AHRR ). Each CYP1B1 PCR tube contained: DNA sample 3 μL, HotStarTaq Plus DNA polymerase 0.1 μL (5 U/μL) (Qiagen), 10X reaction buffer 2 μL (Qiagen), forward and reverse primers (50 mmol/L) 0.1 μL of each, RNase-free water 14.3 μL (Qiagen), and dNTPs (10 mmol/L) 0.4 μL (Invitrogen, Carlsbad, CA). PCR protocols included: activation at 95°C for 5 minutes; denaturation at 94°C for 45 seconds; annealing at 63°C ( AHR , AHRR ), 57°C ( ARNT ), or 60°C ( CYP1B1 ) for 45 seconds; elongation at 72°C for 45 seconds (denaturation to elongation steps were repeated 30 cycles [ AHR , CYP1B1 ] or 33 cycles [ AHRR , ARNT ]); final elongation at 72°C for 5 minutes; and lastly 10°C for 5 minutes. CYP1B1 PCR products were digested with restriction enzyme (MwoI; New England BioLabs, Inc., Ipswich, MA). All products were visualized by 2.5% ( AHR , ARNT , AHRR ) or 3.0% ( CYP1B1 ) agarose gel electrophoresis. Participants were categorized according to the genotype for each SNP as homozygous for the major allele, heterozygous, or homozygous for the minor allele.

Serum concentrations of estradiol, estrone, testosterone, progesterone, and SHBG were measured using enzyme-linked immunosorbent assays. All assays were performed as previously described. Briefly, the assays were run using the manufacturers’ instructions. All assays were conducted in the same laboratory by a single investigator. All samples were run in duplicate and mean values for each participant were used in the analyses. The laboratory personnel were blind with respect to any information concerning study subjects. In addition, positive controls containing known amounts of the tested hormone or SHBG were included in each batch. Further, some samples were run in multiple assays to ensure that the assay values did not dramatically shift over time. The minimum detection limits for the estradiol, estrone, testosterone, androstenedione, progesterone, and SHBG assays were 7 pg/mL, 10 pg/mL, 0.04 ng/mL, 0.03 ng/mL, 0.1 ng/mL, and 0.1 nmol/L, respectively. No estradiol, estrone, testosterone, androstenedione, or SHBG measurements were below the limit of detection. For progesterone measurements that were below the limit of detection ( n = 66), the value was set at the limit of detection (0.1 ng/mL). The average intraassay coefficient of variation was 3.3 ± 0.17% for estradiol, 4.8 ± 0.25% for estrone, 2.2 ± 0.56% for testosterone, 2.5 ± 0.60% for androstenedione, 2.1 ± 0.65% for progesterone, and 2.4 ± 0.67% for SHBG. The average interassay coefficient of variation for all assays was <5%. To assess the amount of unbound serum estradiol or testosterone, ratios of serum estradiol to SHBG and serum testosterone to SHBG were calculated as described previously to generate free serum estradiol indices and free serum testosterone indices.

The associations between the SNPs and both the categorical covariates and hot flash outcome variables were analyzed using χ ² tests. Both unadjusted and confounder-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for associations between the SNPs and the hot flash outcome variables were generated using logistic regression models. The following covariates were included as potential confounders in all of the logistic regression models, as they were associated with ever experiencing hot flashes in previous analyses from this study or in the published literature: race, BMI, smoking status, age. In addition, race-stratified analyses were conducted to examine differences in the analyzed associations between Caucasian and African American women in the sample; however, the OR estimates did not substantially differ and, therefore, we determined that race was not an effect modifier. Thus, instead of presenting the race-stratified results, we treated race as a confounder in the analyses along with BMI, age, and smoking status. Lastly, generalized linear models were used to examine the associations between the SNPs and hormone concentrations, ratios, and SHBG. Analyses were performed using SAS software (version 9.1; SAS Institute Inc, Cary, NC). A P value < .05 was considered to be statistically significant.

Results

Characteristics and genotype distributions of the study sample by hot flash status are presented in Table 1 . Women with hot flashes were significantly older and more likely to be African American, to be overweight or obese, and to smoke cigarettes compared to women without hot flashes. The percentages of women with genetic polymorphisms were not significantly different between women with and without hot flashes. We used the Hardy-Weinberg equation to estimate if the observed genotype frequencies in our study sample differed from the predicted genetic variation at equilibrium of the general population. All SNPs excluding AHR and AHRR were in Hardy-Weinberg equilibrium (data not shown), indicating that there was a stable frequency distribution of these genotypes in our study compared to the predicted variation of the general population.

In the unadjusted analyses, the AHR SNP was associated with ever experiencing hot flashes ( Table 2 ). Specifically, women homozygous for the AHR minor allele (rs2066853 A) had increased odds of any hot flashes (OR, 2.67; 95% CI, 1.13–6.33) compared to women homozygous for the major allele (rs2066853 G). However, after adjustment for age, race, BMI, and smoking status, this association was attenuated and no longer statistically significant (OR, 2.44; 95% CI, 0.99–6.01). Similarly, the combined genotypes of the AHR and CYP1B1 (rs1800440) SNPs were significantly associated with ever experiencing hot flashes only in the unadjusted statistical model (OR, 1.93; 95% CI, 1.00–3.75). The combined genotypes of the CYP1B1 SNPs (rs1800440 and rs1056836) were also associated with ever experiencing hot flashes, but only in the confounder-adjusted model (OR, 1.51; 95% CI, 1.01–2.31). None of the other SNPs or combinations of SNPs were significantly associated with ever experiencing hot flashes in the unadjusted or adjusted analyses.

The results of the analyses examining the selected SNP genotypes and the severity, frequency, and duration of hot flashes are shown in Table 3 . Hot flash severity was significantly associated only with the combined genotypes of the selected SNPs in CYP1B1. Specifically, carriers of at least 1 minor allele for both CYP1B1 SNPs (rs1800440 G and rs1056836 G) had greater odds of moderate or severe hot flashes compared to women who were homozygous for the major allele of both CYP1B1 SNPs (OR, 1.68; 95% CI, 1.00–2.82). Long-lasting hot flashes (experienced for ≥1 year) were more likely among women homozygous for minor allele of the AHR (rs2066853 A), CYP1B1 ( rs1800440 G), or combined SNPs of the CYP1B1 (rs1800440 G and rs1056836 G) compared to women who were homozygous for the major alleles; however, only the associations between the CYP1B1 SNP (rs1800440) and combined CYP1B1 SNPs (rs1800440 and rs1056836) remained statistically significant after adjustment for the other covariates. Hot flash frequency was not significantly associated with any of the SNPs or their combinations.

The associations between the selected SNPs and hormones as well as SHBG are shown in Table 4 . Women homozygous for the major allele of AHR (rs2066853 G), ARNT (rs2228099 G), and CYP1B1 (rs1800440 A) had significantly higher serum concentrations of SHBG, estrone, and estradiol, respectively, compared to carriers of the minor alleles (homozygous or heterozygous) of those genes. Additionally, significant associations were observed for the combined genotypes of AHR (rs2066853) and AHRR (rs2292596) and SHBG and free testosterone index and between AHR (rs2066853) and ARNT (rs2228099) and serum estradiol, estrone, and testosterone concentration ( Table 5 ). No other statistically significant associations were observed between the individual SNPs or their combinations and hormone concentrations, ratios, or SHBG.

TABLE 4

Associations between SNPs, hormone concentrations/ratios, and SHBG concentration

Genotype	Estradiol, pg/mL		Estrone, pg/mL		Testosterone, ng/mL		Progesterone, ng/mL		SHBG, nmol/L		Free estradiol index		Free testosterone index
	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value	Geometric mean 95% LL, UL	P value
AHR (rs2066853)
+/+ (GG)	101.28	.21	136.10	.06	0.46	.41	0.83	.38	59.62	.05	0.62	.77	2.66	.07
	93.79, 109.37		127.27, 145.54		0.43, 0.49		0.73, 0.95		56.34, 63.08		0.57, 0.68		2.44, 2.90
+/– (GA)	89.83		129.25		0.49		0.76		54.66		0.60		3.09
	79.50, 101.49		116.19, 143.78		0.44, 0.53		0.61, 0.94		49.97, 59.79		0.52, 0.70		2.69, 3.55
–/– (AA)	88.63		100.32		0.49		0.59		47.22		0.69		3.59
	66.48, 118.16		78.06, 128.94		0.29, 0.61		0.35, 0.98		38.22, 58.34		0.49, 0.97		2.59, 4.97
AHRR (rs2292596)
+/+ (CC)	95.41	.83	133.02	.80	0.45	.49	0.80	.86	59.22	.23	0.59	.29	2.65	.19
	86.50, 105.23		122.10, 144.92		0.42, 0.49		0.67, 0.94		55.10, 63.65		0.53, 0.66		2.37, 2.96
+/– (CG)	99.23		130.82		0.48		0.81		55.74		0.65		2.98
	90.99, 108.21		121.28, 141.11		0.45, 0.51		0.69, 0.94		52.30, 59.41		0.59, 0.72		2.70, 3.29
–/– (GG)	94.76		143.49		0.45		0.69		66.15		0.53		2.37
	69.18, 129.81		108.98, 188.93		0.36, 0.57		0.39, 1.20		52.49, 83.37		0.36, 0.76		1.66, 3.39
ARNT (rs2228099)
+/+ (GG)	105.59	.20	143.30	.04	0.50	.14	0.85	.44	60.75	.24	0.64	.70	2.84	.73
	94.55, 117.93		130.13, 157.79		0.46, 0.54		0.70, 1.04		56.00, 65.90		0.56, 0.73		2.51, 3.22
+/– (GC)	92.80		131.28		0.45		0.81		56.63		0.60		2.74
	84.83, 101.52		121.39, 141.97		0.42, 0.48		0.69, 0.95		53.00, 60.50		0.54, 0.67		2.474, 3.04
–/– (CC)	96.11		115.72		0.47		0.68		54.51		0.65		2.97
	82.44, 112.03		101.24, 132.28		0.42, 0.52		0.52, 0.90		48.68, 61.03		0.54, 0.78		2.49, 3.53
CYP1B1 (rs1800440)
+/+ (AA)	97.00	.04	132.17	.19	0.46	.79	0.79	.98	57.90	.92	0.62	.08	2.77	.81
	89.85, 104.71		123.61, 141.31		0.44, 0.49		0.69, 0.91		54.71, 61.26		0.56, 0.67		2.54, 3.03
+/– (AG)	103.28		136.10		0.48		0.80		56.64		0.67		2.91
	91.55-116.51		122.48, 151.26		0.44, 0.52		0.65, 0.99		51.81, 61.92		0.58, 0.77		2.54, 3.34
–/– (GG)	66.41		103.27		0.44		0.75		57.00		0.43		2.68
	48.23, 91.44		78.06, 136.61		0.35, 0.58		0.43, 1.33		49.74, 72.19		0.29, 0.62		1.86, 3.86

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