Abstract
Background
Beta-microseminoprotein (MSP) is one of the three most abundantly secreted proteins of the prostate, and has been suggested as a biomarker for prostate cancer risk. A common variant, rs10993994, in the 5’ region of the gene which encodes MSP (MSMB), has recently been identified as a risk factor for prostate cancer.
Methods
We examined the association between rs10993994 genotype and MSP levels in a sample of 500 prostate cancer-free men from four racial/ethnic populations in the Multiethnic Cohort (European Americans, African Americans, Latinos, and Japanese Americans). Generalized linear models were used to estimate the association between rs10993994 genotype and MSP levels.
Results
We observed robust associations between rs10994994 genotype and MSP levels in each racial/ethnic population (all P<10−8) with carriers of the C allele having lower geometric mean MSP levels (ng/mL) (CC/CT/TT genotypes: European Americans, 28.8/20.9/10.0; African Americans, 29.0/21.9/10.9; Latinos, 29.2/17.1/8.3; and Japanese Americans 25.8/16.4/6.7). We estimated the variant accounts for 30–50% of the variation in MSP levels in each population. We also observed significant differences in MSP levels between populations (P=3.5×10−6), with MSP levels observed to be highest in African Americans and lowest in Japanese Americans.
Conclusions
Rs10993994 genotype is strongly associated with plasma MSP levels in multiple racial/ethnic populations.
Impact
This supports the hypothesis that rs10993994 may be the biologically functional allele.
Keywords: MSMB, beta-microseminoprotein, prostate, genetic, multiethnic
Introduction
Beta-microseminoprotein (MSP) has been implicated as a biomarker of prostate cancer risk, detection and prognosis (1, 2). MSP, also called prostate secretory protein of 94 amino acids (PSP94), is one of the most highly secreted proteins by the prostate gland (3). Aside from an abundant occurrence of MSMB-transcripts and MSP-protein in the prostate, abundant expression has also been found in trachea and the bronchi in the respiratory tract, and in the gastric mucosa, which may help to explain why circulating levels are quite comparable in women and in men below age 40 (4–7). MSP may have a physiologic role in fertility as it has been shown to bind sperm and inhibit the acrosome reaction (8). In the prostate, MSP expression has been observed to be higher in benign than in cancerous tissue (9). Serum MSP levels have been found to be lower in men with aggressive prostate cancer (1) as well as in men with a prostate cancer recurrence after radical prostatectomy (2, 10). Studies in vivo and in vitro have also implicated MSP as having a functional role in regulating cellular growth in the prostate through apoptosis (11).
Genome-wide association studies (GWAS) of prostate cancer have identified a common risk variant (rs10993994) in the 5’ region of MSMB on chromosome 10q11.2 (the gene that encodes MSP) (12, 13). We recently replicated the association of this variant with prostate cancer risk in a multiethnic population (14). The variant, rs10993994, is located 57 bp upstream of the first exon of MSMB with the C allele (hypothesized low risk allele) found to preferentially bind the CREB transcription factor and to be associated with higher MSMB gene expression in tumor cell lines (15, 16). A recent study among Chinese men, in which 60 of 251 cases and 30 of 258 controls were evaluated for the potential association between MSP levels and MSMB genotype, reported serum MSP levels to be lower in those who carried the CT or TT genotype than in those with the CC genotype among the 60 cases (17). These genetic data, together with the clinical and laboratory observations, are consistent with the hypothesis that lower MSMB expression is associated with prostate cancer development.
In an attempt to clarify the biological mechanism underlying the association of genetic variation at the MSMB locus and prostate cancer risk, we examined the association between rs10993994 genotype and MSP levels in the blood plasma from prostate cancer-free men from four racial/ethnic populations (European Americans, African Americans, Latinos, and Japanese Americans). We also examined known and putative risk factors for prostate cancer as potential contributors to inter-individual variability of circulating levels of MSP in the study population.
Methods
Study Subjects
This study consisted of male participants of the Multiethnic Cohort (MEC). The MEC includes 96,810 men and 118,441 women and is comprised mainly of African Americans, Japanese Americans, Latinos, Native Hawaiians and European Americans (18). Between 1993 and 1996, adults between 45 and 75 years old were enrolled by completing a 26-page, self-administered questionnaire asking detailed information about demographic factors, personal behaviors, dietary habits and history of prior medical conditions. Potential cohort members were identified through Department of Motor Vehicles drivers’ license files, voter registration files and Health Care Financing Administration data files. Between 1995 and 2006, blood specimens were collected from ~67,000 MEC participants for genetic and biomarker analyses.
Blood plasma MSP and PSA levels and rs10993994 genotype were measured in 500 men from four racial/ethnic groups (n=125 of each European Americans, African Americans, Latinos, and Japanese Americans). Blood samples of the men included in this study were collected between 1994 and 2004, and were cancer free, as determined through linkage of the cohort with SEER Cancer Registries in California and Hawaii, as of January 1, 2009. Men were randomly selected from the control group of the nested prostate cancer case-control study in the MEC (19). We limited the study to men in the age range of 50–69 years old at the time of blood draw as this is the age when men are typically PSA screened for prostate cancer (20). We also limited the study to men with a self-reported BMI (kg/m2) between 19 and 35 in order to avoid subjects with extremely high or low BMI levels where the effects on MSP are not known. Four hundred and twelve (82%) of the men had fasted 8 or more hours before blood collection. The Institutional Review Boards at the University of Southern California and University of Hawaii approved the study protocol.
Lab Assays
Archived EDTA anti-coagulated blood plasma samples (stored frozen for 3–14 years at −80°C after initial processing within 4 hours from venipuncture) were retrieved and shipped frozen on dry ice to Malmö, Sweden in fall of 2009. Analyses of free and total PSA, and MSP were performed blinded to ethnicity or genotyping data in Dr. Lilja’s laboratory at the Wallenberg Research Laboratories, Department of Laboratory Medicine, Lund University, Skåne University Hospital in Malmö, Sweden during 2009 – 2010. Production and purification of the polyclonal rabbit anti-MSP antibody and seminal plasma MSP have been described elsewhere (21). Biotinylation and Europium labeling of the anti-MSP antibody were done as previously described (22). Streptavidin coated microtiter plates were from Kaivogen Oy, Turku, Finland. Assay buffer and wash solution have been described previously (21).
The MSP immunoassay of plasma MSP levels, which have previously been shown to not be significantly different from serum MSP levels (4), was done with the AutoDelfia 1235 automatic immunoassay system (Perkin-Elmer Life Sciences, Wallac, Turku, Finland). First, 150 ng of the biotinylated MSP-antibody was attached to the streptavidin coated wells in 100 µl of assay buffer during a 30 min incubation. After two washes, 100 µl of assay buffer and 10 µl of standard or sample material were added to the wells, incubated 1 h and washed twice. The Eu-labelled anti-MSP antibody (50 ng) was added in 200 µl of assay buffer. After 1 h incubation, the wells were washed six times before adding 200 µl of the Delfia enhancement solution (Perkin-Elmer Life Sciences, Wallac, Turku, Finland) which was then incubated for 5 min at room temperature with shaking before the time-resolved measurement of the Eu-fluorescence. To measure free and total PSA, we used the dual-label DELFIA Prostatus® total/free PSA-Assay (Perkin-Elmer, Turku, Finland) (23), which is calibrated against the WHO 96/670 (PSA-WHO) and WHO 68/668 (free PSA-WHO) standards. Samples were run in 8 batches. Mean coefficients of variation of 7.4%, 11.8%, and 7.8% were observed for MSP, total PSA, and free PSA respectively among 20 blinded duplicate samples (5 per ethnic group).
Genotyping
Genotyping of rs10993994 and other GWAS-validated risk alleles for prostate cancer was performed using the Taqman allelic discrimination, as previously described (14, 19, 24). The DNA for genotyping was extracted from buffy coat samples using a QIAamp® 96 DNA blood kit (Qiagen, Valencia, CA, USA). Of the ~5% duplicate samples used to assess genotyping reproducibility, there was 100% concordance for rs10993994.
Statistical analysis
MSP and PSA levels were log transformed to better normalize the distributions and mean levels are presented as least squares geometric means. Generalized linear models were used to examine the association between rs10993994 and MSP, adjusted for age (continuous), BMI (continuous), laboratory batch, and race/ethnicity (pooled analysis). Laboratory batch was not significantly associated with MSP levels. We also adjusted for PSA levels, as PSA was found to be modestly correlated with MSP levels. Adjustment for either total, free PSA (ng/mL) or percent free PSA provided similar results. The least squares geometric mean MSP levels and the percent change relative to the wild-type (i.e. low risk) rs10993994 genotype (CC) was calculated in ethnic-specific analyses. The effect of global European ancestry on the association between MSP levels and rs10993994 was examined in the admixed racial/ethnic populations (African Americans and Latinos) using ancestry estimates from a previous study (19). Fasting time before blood collection was not included in the analysis because it was not significantly associated with MSP levels and had no effect on the association between rs10993994 and MSP.
As the determinants of MSP levels are largely unknown, we also examined other known and suspected risk factors for prostate cancer in relation to MSP levels including demographic factors, dietary factors, and established common risk variants from GWAS. More specifically we examined age at the time of blood draw, BMI (kg/m2), weight (kg), height (cm), total PSA (ng/mL), free PSA (ng/mL), percent free PSA, physical activity (hours of vigorous work and exercise), and intake of saturated fat (percentage of calories from saturated fat), red meat (calorie adjusted intake per day; g/kcal/day), lycopene (calorie adjusted intake per day; 100mcg/kcal/day), calcium (calorie adjusted intake per day; mg/kcal/day), vitamin D (calorie adjusted intake per day; IU/kcal/day), and alcohol (grams per day). We tested the association between an increase of 1 unit of each factor and the percent change in geometric mean MSP levels. Age, BMI, weight, and height were coded as continuous variables. A Bonferroni corrected p-value ≤ 5.0×10−3 (based on ~10 independent tests) was used to determine statistical significance in the analysis of each risk factor and MSP levels. We also examined 26 other validated genetic risk variants for prostate cancer in association with MSP levels (12, 13, 19, 24–27). Coefficients of determination (r2) were estimated in multivariable models in order to assess the variance of MSP levels explained by rs10993994 genotype and other covariates.
Results
The mean ages at the time of blood draw were well matched between racial/ethnic groups; African Americans were slightly younger on average (mean age, 60.2 years) and Japanese Americans slightly older (mean age, 61.5 years; Table 1). There were significant differences in mean BMI among racial/ethnic groups (p=1.9×10−3), with Japanese Americans having the lowest mean BMI (25.0 kg/m2) and Latinos the highest (26.5 kg/m2). There were also significant differences in both weight and height among the groups. We observed significant differences in geometric mean MSP levels adjusted for age, BMI, laboratory batch, and free PSA across racial/ethnic groups (P=2.0×10−4). On average, European Americans had the highest levels (20.4 ng/mL), followed by Latinos (19.7 ng/mL) and African Americans (19.0 ng/mL) with the lowest levels observed for Japanese Americans (14.8 ng/mL). Untransformed mean MSP levels were highest in European Americans (mean 24.2 ng/mL; standard deviation 15.9) and lowest in Japanese Americans (mean 19.2; standard deviation 20.4) (Supplemental Table 1).
Table 1.
Descriptive characteristics by ethnicity (n=500).
| European Americans |
African Americans |
Latinos | Japanese Americans |
p value | |
|---|---|---|---|---|---|
| N | 125 | 125 | 125 | 125 | |
| Age (yr) | 61.1 ± 5.2 | 60.2 ± 5.5 | 60.6 ± 4.7 | 61.5 ± 5.4 | 0.21* |
| BMI (kg/m2) | 26.1 ± 3.3 | 26.3 ± 3.5 | 26.5 ± 3.4 | 25.0 ± 3.0 | 1.9×10−3* |
| Weight (kg) | 84 ± 12 | 86 ± 14 | 82 ± 12 | 72 ± 10 | 1.0×10−16* |
| Height (cm) | 177 ± 7 | 179 ± 7 | 175 ± 7 | 169 ± 6 | 3.5×10−29* |
| MSP (ng/mL)‡ | 20.5 | 18.6 | 19.2 | 15.1 | 1.0×10−3* |
| MSP (ng/mL)§ | 20.4 | 19.0 | 19.7 | 14.8 | 2.0×10−4† |
| MSP (ng/mL)‖ | 17.8 | 19.3 | 16.4 | 14.1 | 3.5×10−6† |
| PSA – total (ng/mL)‡ | 1.10 | 0.98 | 1.01 | 0.94 | 0.48* |
| PSA – total (ng/mL)** | 1.09 | 0.99 | 1.02 | 0.92 | 0.40† |
| PSA – free (ng/mL)‡ | 0.33 | 0.29 | 0.28 | 0.28 | 0.21* |
| PSA – free (ng/mL)** | 0.33 | 0.29 | 0.28 | 0.28 | 0.28† |
| rs10993994 Frequency of T allele |
0.41 | 0.55 | 0.36 | 0.48 |
Global test, ANOVA.
Global test, ANCOVA
Unadjusted crude least squares geometric means.
Least squares geometric means adjusted for laboratory batch, free PSA, age, and BMI.
Least squares geometric means adjusted for laboratory batch, free PSA, age, BMI, and rs10993994 genotype.
Least squares geometric means adjusted for laboratory batch, MSP, age, and BMI.
The risk allele at rs10993994 (T allele) was common in all populations and ranged in frequency from 0.36 in Latinos to 0.55 in African Americans. The variant, rs10993994, was strongly and highly statistically associated with MSP levels in each racial/ethnic group (all global P values < 4 × 10−9; Figure 1). Compared to men with the CC genotype, men with the CT genotype had a percent change in geometric mean PSA levels between −24.3% (African Americans) and −41.5% (Latinos) (all P<0.05), and men with the TT genotype had changes ranging from −62.3% in African Americans to −74.2% in Japanese Americans (all P< 10−8) (Table 2). We observed little evidence of heterogeneity of the association of genotype and MSP levels between racial/ethnic groups (P=0.37; Table 2). The associations were similar when adjusting for global European ancestry in the admixed African American and Latino populations (Supplemental Table 2). Differences in the frequency of rs10993994 genotype could not explain the ethnic differences in MSP levels (global P = 3.5×10−6; Table 1). Following adjustment for genotype, MSP levels were highest in African Americans (19.3 ng/mL) and lowest in Japanese Americans (14.1 ng/mL; Table 1). We did not observe evidence that age, BMI, or PSA modified the association between MSMB genotype and geometric mean MSP levels.
Figure 1.
The least squares geometric mean of log MSP levels (ng/mL) for each racial/ethnic group (European Americans, African Americans, Latinos, and Japanese Americans) by the genotype of rs10993994 (n=500). The error bars represent the upper and lower 95% confidence limits of the mean. The values are adjusted for age (continuous), BMI (continuous), free PSA level (ng/mL), and laboratory batch. P values are from a global ANCOVA test (Type III sum of squares F-test).
Table 2.
Least squares geometric mean MSP levels (ng/mL) and percent change by rs10993994 genotype.
| rs10993994 Genotype |
European Americans (n=125) |
African Americans (n=125) |
Latinos (n=125) |
Japanese Americans (n=125) |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | Mean* % change |
P value | n | Mean* % change |
P value | n | Mean* % change |
P value | n | Mean* % change |
P value |
|
| CC | 44 | 28.8 Ref |
ref | 23 | 29.0 ref |
ref | 52 | 29.2 ref |
ref | 32 | 25.8 ref |
ref |
| CT | 60 | 20.9 –27.6% |
1.0 × 10−3 | 66 | 21.9 –24.3% |
0.041 | 55 | 17.1 –41.5% |
1.4 × 10−9 | 66 | 16.4 –36.4% |
1.3 × 10−5 |
| TT | 21 | 10.0 –65.2% |
5.0 × 10−12 | 36 | 10.9 –62.3% |
6.6 × 10−9 | 18 | 8.3 –71.5% |
9.2 × 10−19 | 27 | 6.7 –74.2% |
8.8 × 10−20 |
| r2† | 0.32 | 0.30 | 0.49 | 0.50 | ||||||||
| r2‡ | 0.44 | 0.39 | 0.60 | 0.61 | ||||||||
Values adjusted for age, BMI, free PSA, and laboratory batch.
Univariate model with rs10993994 (categorized as genotype class) as independent variable.
Multivariable model with rs10993994 (categorized as genotype class), age, BMI, and laboratory batch as independent variables.
We found no significant association between age and MSP levels (P=0.10), however the youngest 5-year age group (50–54 years) was noted as having lower least squares geometric mean levels than the older groups (55–69 years; Table 3). We observed inverse associations of MSP levels with BMI (P=7.4×10−3) and weight (P=3.6×10−3) and a suggestive inverse association with height (P=0.072). Least squares geometric mean MSP levels by quintiles of these factors are shown in Table 3. Family history of prostate cancer was positively associated with MSP levels in African Americans (P=0.023), but not in the pooled population (P=0.34). We observed modest, yet, statistically significant positive correlations between MSP levels and free (P=3.2×10−9), and total (P=4.2×10−5) PSA, with Pearson correlation coefficients of 0.15 (0.07–0.21 among racial/ethnic groups) and 0.13 (0.08–0.19 among racial/ethnic groups), respectively. Least squares geometric mean MSP levels by quintiles of the PSA measures are shown in Table 3. Aside from age and height in Latinos the direction of the associations with these factors was consistent across populations.
Table 3.
The association of geometric mean MSP levels with age, anthropometic variables and PSA levels.
| European Americans (n=125) |
African Americans (n=125) |
Latinos (n=125) |
Japanese Americans (n=125) |
Pooled (n=500) |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Group* | n | MSP (ng/mL) |
n | MSP (ng/mL) |
n | MSP (ng/mL) |
n | MSP (ng/mL) |
n | MSP (ng/mL) |
|
| Age (yrs)† | 50–54 | 18 | 14.8 | 25 | 16.5 | 11 | 17.4 | 15 | 12.3 | 69 | 14.9 |
| 55–59 | 26 | 20.7 | 28 | 18.4 | 39 | 17.0 | 27 | 13.8 | 120 | 17.2 | |
| 60–64 | 39 | 18.1 | 44 | 19.9 | 47 | 15.6 | 35 | 14.2 | 165 | 16.8 | |
| 65–69 | 42 | 18.5 | 28 | 21.0 | 28 | 15.3 | 48 | 14.9 | 146 | 17.3 | |
| P value‡ | 0.16 | 0.45 | 0.65 | 0.58 | 0.18 | ||||||
| BMI (kg/m2)§ | Quintile 1 | 21 | 17.9 | 21 | 24.7 | 22 | 20.7 | 36 | 14.2 | 100 | 18.2 |
| Quintile 2 | 26 | 18.7 | 29 | 20.0 | 16 | 15.4 | 29 | 14.4 | 100 | 17.0 | |
| Quintile 3 | 31 | 19.2 | 20 | 17.4 | 24 | 16.9 | 25 | 15.9 | 100 | 17.6 | |
| Quintile 4 | 18 | 15.7 | 30 | 17.2 | 33 | 14.4 | 19 | 13.4 | 100 | 15.5 | |
| Quintile 5 | 29 | 18.3 | 25 | 18.1 | 30 | 14.9 | 16 | 11.8 | 100 | 15.6 | |
| P value‡ | 0.72 | 0.25 | 0.026 | 0.36 | 0.076 | ||||||
| Weight (kg)§ | Quintile 1 | 17 | 22.6 | 14 | 29.8 | 22 | 18.1 | 53 | 14.7 | 106 | 19.6 |
| Quintile 2 | 26 | 16.3 | 27 | 20.7 | 20 | 17.0 | 33 | 13.9 | 106 | 16.5 | |
| Quintile 3 | 26 | 21.5 | 26 | 19.6 | 30 | 15.4 | 21 | 15.1 | 103 | 17.6 | |
| Quintile 4 | 31 | 14.5 | 17 | 13.8 | 30 | 15.1 | 14 | 11.7 | 92 | 13.9 | |
| Quintile 5 | 25 | 18.6 | 41 | 17.7 | 23 | 15.3 | 4 | 12.6 | 93 | 16.0 | |
| P value‡ | 5.8×10−3 | 6.8×10−3 | 0.48 | 0.49 | 5.1×10−5 | ||||||
| Height (cm)§ | Quintile 1 | 18 | 22.4 | 13 | 24.4 | 27 | 16.0 | 66 | 15.2 | 124 | 18.7 |
| Quintile 2 | 13 | 19.2 | 24 | 21.0 | 33 | 16.5 | 37 | 12.9 | 107 | 17.0 | |
| Quintile 3 | 25 | 16.6 | 13 | 19.7 | 21 | 14.8 | 9 | 13.2 | 68 | 15.4 | |
| Quintile 4 | 36 | 19.0 | 31 | 17.1 | 25 | 17.0 | 12 | 13.6 | 104 | 16.4 | |
| Quintile 5 | 33 | 16.3 | 44 | 17.6 | 19 | 15.2 | 1 | 14.9 | 97 | 15.2 | |
| P value‡ | 0.16 | 0.35 | 0.81 | 0.49 | 0.036 | ||||||
| Total PSA‖ (ng/mL) |
Quintile 1 | 19 | 19.1 | 28 | 17.1 | 26 | 15.1 | 29 | 13.5 | 102 | 15.6 |
| Quintile 2 | 27 | 15.7 | 26 | 17.5 | 26 | 14.9 | 19 | 9.9 | 98 | 14.4 | |
| Quintile 3 | 22 | 14.6 | 23 | 18.5 | 26 | 15.0 | 29 | 14.5 | 100 | 16.2 | |
| Quintile 4 | 31 | 19.2 | 24 | 20.5 | 18 | 16.7 | 27 | 18.6 | 100 | 18.4 | |
| Quintile 5 | 26 | 23.9 | 24 | 21.8 | 29 | 18.5 | 21 | 15.0 | 100 | 19.5 | |
| P value‡ | 6.2×10−3 | 0.56 | 0.4 | 4.3×10−4 | 7.0×10−5 | ||||||
| Free PSA‖ (ng/mL) | Quintile 1 | 23 | 14.1 | 33 | 15.8 | 30 | 12.9 | 27 | 12.6 | 113 | 13.8 |
| Quintile 2 | 18 | 19.2 | 18 | 16.7 | 29 | 15.0 | 32 | 12.3 | 97 | 15.5 | |
| Quintile 3 | 27 | 17.1 | 24 | 23.4 | 23 | 17.2 | 20 | 14.4 | 94 | 17.6 | |
| Quintile 4 | 28 | 16.4 | 26 | 19.2 | 20 | 16.3 | 26 | 16.3 | 100 | 16.9 | |
| Quintile 5 | 29 | 25.4 | 24 | 21.5 | 23 | 20.3 | 20 | 17.5 | 96 | 21.2 | |
| P value‡ | 5.0×10−4 | 0.074 | 0.012 | 0.042 | 3.2×10−8 | ||||||
Aside for age, the groups showed are quintiles based in the pooled sample. For some covariates, the number of subjects in each quintile is not the same because many subjects share the same value. The cutpoints used are: BMI (<23.13 kg/m2, 23.13–24.92, 24.93–26.77, 26.78–28.58, 28.59–34.99; weight (<156 kg, 156–170, 171–185, 186–204, ≥205); height (<67 cm, 67–68, 69, 70–71, ≥72), total PSA (<0.54 ng/mL, 0.54–0.77, 0.78–1.15, 1.16–1.84, ≥1.85), and; free PSA (<0.18 ng/mL, 0.18–0.25, 0.26–0.34, 0.35–0.51, ≥ 0.52).
Least squares geometric mean of MSP adjusted for BMI, free PSA, laboratory batch, rs10993994, and race/ethnicity (pooled).
Global test, ANCOVA.
Least squares geometric mean of MSP adjusted for age, free PSA, laboratory batch, rs10993994, and race/ethnicity (pooled).
Least squares geometric mean of MSP adjusted for age, BMI, laboratory batch, rs10993994, and race/ethnicity (pooled).
We found no strong associations between suspected risk factors for prostate cancer and MSP levels (Supplemental Table 3). Of the dietary variables, we observed a −0.44% decrease in MSP levels per 1 unit (100mcg/kcal/day) increase in lycopene intake which was only nominally statistically significant (p=0.047; see Methods). In testing 26 validated risk variants (12, 13, 19, 24–27) for association with MSP levels, we observed a nominally significant inverse association with only two variants, rs721048 on chromosome 2p15 (EHBP1 gene locus; −11.5% change in MSP (ng/mL) per allele P=0.013), and rs7931342 on chromosome 11q13 (7.2% change in MSP per allele P=0.041).
In unadjusted models, rs10993994 genotype explained between 30% (African Americans) and 50% (Japanese Americans) of the variation in log MSP levels. Together with age, BMI, free PSA, and laboratory batch, between 39% (African Americans) and 61% (Japanese Americans) of the variation in log MSP levels could be explained. In ethnic-pooled analysis, rs10993994 explained 38% of the variance in log MSP levels, while 48% of the variance could be explained when including age, BMI, free PSA, ethnicity, and laboratory batch.
Discussion
In this study, we found that rs10993994 genotype is an important and highly statistically significant predictor of circulating MSP levels. The association of genotype and MSP levels was observed in all racial/ethnic groups which supports fine-mapping studies pinpointing rs10993994 as the most strongly associated prostate cancer risk variant in the region (15, 16). We also found MSP levels to vary significantly among racial/ethnic groups, and after conditioning on genotype, African Americans were observed to have the highest levels and Japanese the lowest.
The variant is located in a putative CREB transcription factor binding site and the C to T change at this position has been shown to influence CREB binding, with preferential binding observed with the low risk C allele. In cell lines, the C allele has also been associated with increased MSMB expression (16). A recent study in a Chinese Han population found significantly lower circulating serum MSP levels in men with prostate cancer and the CT or TT genotype (16.32 µg/L) compared to men with the CC genotype (19.33 µg/L) (P=0.022) (17). They also noted a similar but non-significant association in 30 unaffected men. In Japanese Americans we observed geometric mean levels of 6.7, 16.4, and 25.8 (ng/mL) in men with the TT, CT, and CC genotypes respectively. While our findings are similar in direction, it is difficult to compare the absolute MSP values between the studies since MSP values reported by our new sandwich-type immunoassay are markedly higher (close to three-fold) compared to the competitive assay-design used in prior studies (21), although MSP-values reported by the two different assay designs have excellent correlation (r2>0.90). Our observation among unaffected men from multiple populations provides further support for the hypothesis that variant rs10993994 is biologically functional, and that the risk for prostate cancer may be conferred by altering expression of MSP.
Numerous studies have investigated the genetic basis underlying inter-individual variation in circulating levels of biomarkers with relevance to prostate cancer including PSA, SHBG, testosterone, 3α-androstanediol-glucoronide, and 17β-estradiol (28, 29). The genetic variants identified to date in association with these biomarkers however explain only a small fraction of the variation in circulating levels in any population. In contrast, the variant at the MSMB locus alone may account for 30% (African Americans) to 50% (Japanese Americans) of the variation in levels in any one population. This finding supports those of Xu et al. in a population of 18–21 year old Swedish men (paper co-submitted). While rs10993994 does explain a large portion of the variance in MSP levels, a portion of the remaining variance may be explained by day-to-day inter-individual variation, environmental risk factors as well as genetic variation both local to the MSMB region and genome-wide. Extensive sequencing and fine-mapping efforts of the MSMB region (30, 31) have identified additional local variation that will need to be examined for association with MSP levels in future experiments.
In previous analyses of the MEC data (32), we did not replicate reports (33, 34) that high lycopene intake or blood measurements may be protective for prostate cancer, and we note here that the suggestive weak inverse association between MSP and lycopene intake is not consistent with such protection. However, the association with lycopene seen here (p=0.047) should be interpreted cautiously, as this is not significant after adjusting for multiple tests (Bonferroni corrected α=5.0×10−3). The geometric mean MSP levels were lower in men with more risk alleles for rs721048 which lies in an intron of the EHBP1 gene on chromosome 2, and MSP levels were higher in men with more risk alleles of rs7931342 located in a gene desert on chromosome 11. These associations also do not survive correction for multiple comparisons and will require replication.
In summary, we observed a strong association between rs10993994 genotype and plasma levels of MSP in a multiethnic sample of men without prostate cancer. The association was robust and statistically significant in all four racial/ethnic groups. The variant explained a large proportion of the variability in plasma MSP levels in all groups. Examining MSP in a prospective study will be necessary to determine whether the prostate cancer risk association with rs10993994 is mediated through influence on MSP levels, as well as the population risk associated with MSP levels.
Supplementary Material
Acknowledgements
We thank Gun-Britt Eriksson and Mona Hassan Al-Battat for expert assistance with immunoassays.
The MEC was supported by National Cancer Institute grants CA63464 and CA54281. This research was supported in part by the National Cancer Institute [P30 CA008748 to MSKCC]; National Cancer Institute Specialized Programs of Research Excellence [P50-CA92629]; Swedish Cancer Society [08-0345]; Swedish Research Council [Medicine: K2009-54X-20095-04-3]; Sidney Kimmel Center for Prostate and Urologic Cancers, and David H. Koch through the Prostate Cancer Foundation, Academy of Finland (Project 206690), and Fundación Federico SA.
Footnotes
Conflict of interest statement: Dr. Hans Lilja holds patents for free PSA assays.
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