Abstract
BACKGROUND
Genetic variants in KLK2 and KLK3 have been associated with increased serum levels of their encoded proteins human kallikrein-related peptidase 2 (hK2) and prostate-specific antigen (PSA), and with prostate cancer in older men. Catalytic PSA, possibly activated by hK2, cleaves semenogelin I and II in semen to release motile sperm; low PSA levels in seminal plasma are associated with low sperm motility. To evaluate whether common genetic variants in KLK2 and KLK3 affect physiological prostatic secretion, we studied the association of SNPs with hK2 and PSA levels in seminal plasma and serum of young healthy men.
METHODS
Leukocyte DNA was extracted from 303 male military conscripts (median age 18.1 years). Nine SNPs across KLK2-KLK3 were genotyped. PSA and hK2 were measured in seminal plasma and serum with immunofluorometric assays. The association of genotype frequencies with hK2 and PSA levels was tested using the Kruskal-Wallis test.
RESULTS
Four KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017) were strongly associated with hK2 levels in seminal plasma and serum, with individuals homozygous for the major alleles having 3- to 7-fold higher levels than the other homozygote and heterozygotes having intermediate levels (all P<0.001). Three of these SNPs were significantly associated with %fPSA in serum (all P<0.007). Three KLK3 SNPs showed associations with PSA in seminal plasma and the rs1058205 SNP was associated with total PSA in serum (P=0.001), and with lower %free PSA (P=0.015).
CONCLUSION
In young men without prostate disease, both the seminal plasma and serum levels of hK2 and PSA are associated with several genetic variants in KLK2 and KLK3 that could be used to refine models of PSA cut-off values in prostate cancer testing.
Keywords: prostate cancer, prostate-specific antigen, tumor markers, seminal fluid
Introduction
Prostate-specific antigen (PSA) and a closely related protease, kallikrein-related peptidase 2 (hK2), are commonly used as markers of prostate cancer (1, 2). PSA and hK2 are members of the kallikrein gene family (3) and their expression is regulated by the ligand-dependent activation of the androgen receptor. The documented physiological roles of PSA and hK2 are in seminal plasma. PSA is responsible for the rapid degradation of the seminal vesicle-secreted proteins semenogelin I and II (4). Semenogelin I inhibits the motility of intact and demembranated spermatozoa and participates in the capacitation of sperm by blocking motility immediately after ejaculation (5). Thus, PSA is necessary for the release of progressively motile sperm. The non-catalytic proPSA-zymogen can in vitro be converted to catalytically active PSA by catalytic hK2 (6) which suggests that hK2 may be a physiological activator of PSA (7). Catalytic hK2 can also cleave semenogelin I and II (8).
The majority of hK2 in seminal plasma is non-catalytic and bound in complex with protein C inhibitor (encoded by SERPINA5), while the majority of PSA is in free, catalytically active form (fPSA), with less than 5% of PSA being complexed to SERPIN-type anti-proteases (cPSA) (9). The sum of fPSA and cPSA closely correspond to total PSA (tPSA). Both PSA and hK2 are non-catalytic in blood with a relationship of free to bound forms that is opposite to that in seminal fluid. hK2 in blood occurs predominantly in free unbound forms, whereas the majority of PSA is covalently bound in complexes with extracellular anti-proteases such as alpha-1-antichymotrypsin (encoded by SERPINA3) and only 5–35% circulates in free, unbound forms (10–12). In healthy men, the PSA concentration in seminal plasma is 0.2–5 mg/mL (13) and the retrograde release of PSA into the blood occurs with a frequency of less than one molecule per million secreted PSA molecules. hK2 is found in both seminal plasma and in serum but at approximately one percent of the concentration of PSA (7, 14). In men affected by prostate cancer, levels of PSA and hK2 in blood become elevated decades before cancer diagnosis (15, 16), and the ratio of free to total PSA (%fPSA) decreases (17). Only limited data have been reported on PSA and hK2 levels in men younger than 30 years of age. Levels of fPSA, but not cPSA, in serum correlate with levels of PSA in seminal plasma in young men (18). Low levels of PSA in seminal plasma have been shown to be associated with a reduced percentage of motile sperm (13).
The risk of prostate cancer has been associated with single-nucleotide polymorphisms (SNPs) located in the genes coding for PSA (KLK3) (19–24) and hK2 (KLK2) (25–29). A number of SNPs in these genes have been associated with serum levels of PSA and hK2 (22, 24–26, 29). The study cohorts have, however, been largely confined to older men, in whom the levels of these biomarkers may be affected by prostate cancer, benign prostatic hyperplasia, or prostatitis, all of which become more prevalent with age. Since measurement of PSA levels in serum are widely used for identifying subjects to be offered diagnostic procedures for detection of prostate cancer, it is important to know whether genetic factors may influence the non-cancer related prostatic secretion of kallikreins. To address this question, we examined the relationship between SNPs in KLK2 and KLK3 and levels of hK2 and PSA in seminal plasma and in serum, using a population-based cohort of young men in whom prostate conditions are very rare.
Material and Methods
Subjects
A total of 305 men under compulsory medical examination for military service in Sweden were in the year 2000 enrolled in a study of reproductive function (30). This group can be considered as representative for the Swedish general population of adolescent men since at that time more than 95% of young men in Sweden underwent examination for military service. Their median age was 18.1 years (SD, 0.4; range, 18–21 years) and median abstinence time was 85 hours (SD, 57; range, 12–504 hours). All men participated after giving written informed consent according to protocols approved by the ethical review board at Lund University. For the present study, not enough biospecimen material was available to measure all markers in all individuals; however, we expect the availability of biospecimen to be random with respect to the variable studied here.
Semen and Blood Samples
Semen samples were obtained after masturbation and delivered between 9–11 a.m. A blood sample was subsequently drawn. All participating men were asked to abstain from sexual activities for at least 48 hours and to note the actual abstinence time. Semen volume was determined by weighing the semen sample, assuming a density of 1 g/mL. For each semen sample, 450 μl was mixed with 50 μl of 0.1 M benzamidine to inhibit liquefaction. Seminal plasma was obtained by centrifugation of the semen sample 10 000 × g for 10 minutes. Blood and seminal plasma was kept at −70°C until analysis.
hK2 and PSA Analyses
Seminal plasma samples were analyzed for hK2 using a previously reported immunofluorometric assay (12) with minor modifications. The sample volume and extent of labeling of the tracer antibody were increased and blocking of tPSA was enhanced by the use of three PSA-specific anti-PSA monoclonal antibodies (Mab) (2E9, 5F7 and 5H6) that do not cross-react with hK2. The biotinylated Mab 6H10 was used to capture hK2. Finally, hK2 was detected by use of the Mab 7G1-Eu (31). The coefficient of variation (CV) for hK2-measurement in seminal plasma was 12% at a mean concentration of 0.008 mg/mL. Measurements of free PSA (fPSA) and total PSA (tPSA) in seminal plasma and serum were performed using the commercially available assay Prostatus™ PSA Free/Total kit (Delfia® Reagents) (32). The analysis for tPSA in seminal plasma measures the sum of fPSA (> 95%), PSA in complex with protein C inhibitor (cPSA)(1–3%) and hK2 (< 1%). The analysis of fPSA in seminal plasma measures the sum of active single chain and inactive internally cleaved two-chained fPSA. The combination of Mab H117 and H50 provide equimolar detection of fPSA and cPSA but also cross-reacts with hK2, whereas fPSA is measured by the combination of Mab H117 and 5A10 with no significant cross-reactivity to cPSA or hK2. CV for PSA measurements in seminal plasma was 12% at a mean concentration of 0.66 mg/mL. The detection limit in serum was 0.05 ng/mL (CV was 5% at a mean concentration of 2.3 ng/mL) for tPSA and 0.04 ng/mL (CV was 5.9% at a mean concentration of 0.25 ng/mL) for fPSA. Serum levels of hK2, fPSA, and tPSA were analyzed in 303 men, seminal plasma tPSA in 293 men, and seminal plasma hK2 in 202 men due to missing semen and serum material. The characteristics of the study group are presented in Table 1. We note that in previous work, intra-individual variability of these measures is less than 10% and we presume the same holds here.
Table 1.
hK2 and PSA levels in seminal plasma and serum.
| Characteristic | No. | Mean | Median | IQR | SD |
|---|---|---|---|---|---|
| Semen volume (mL) | 303 | 3.2 | 3.2 | 2.3–4.0 | 1.3 |
| Seminal plasma hK2 (mg) | 202 | 0.02 | 0.02 | 0.01–0.03 | 0.02 |
| Seminal plasma hK2 (mg/L) | 202 | 7.2 | 6.1 | 4.0–9.2 | 4.6 |
| Seminal plasma PSA (mg) | 293 | 2.2 | 2.0 | 1.3–2.9 | 1.3 |
| Seminal plasma PSA (mg/mL) | 293 | 0.70 | 0.64 | 0.46–0.88 | 0.34 |
| Serum hK2 (ng/mL) | 303 | 0.04 | 0.04 | 0.03–0.05 | 0.02 |
| Serum fPSA (ng/mL) | 303 | 0.29 | 0.19 | 0.13–0.27 | 0.72 |
| Serum tPSA (ng/mL) | 303 | 0.64 | 0.50 | 0.35–0.67 | 0.84 |
IQR, inter-quartile range.
Genotype Analyses
Genomic DNA was prepared from peripheral leukocytes using a QIAamp DNA Maxi Kit (Qiagen). DNA concentrations were determined by PicoGreen™ DNA assay (Molecular Probes), and all samples were adjusted to the same DNA concentration. Genotypes were determined by Sequenom MassARRAY MALDI-TOF analysis and assay design was made using MassARRAY Assay Design 2.0 software (Sequenom) as previously described (33). A total of 9 SNPs were initially selected for analysis on the basis of our prior observation of SNP/biomarker correlation in older men (29), removing some redundant SNPs due to linkage disequilibrium. One SNP, rs11670728 in KLK2, showed a significant deviation from Hardy-Weinberg equilibrium and was excluded from further analysis. The SNPs studied are described in Table 2.
Table 2.
SNP descriptions and allele frequencies in the study cohort.
| SNP | Location | Genomic Positiona | Amino Acid Change | Major/Minor Alleleb | MAFb | HWE P-value |
|---|---|---|---|---|---|---|
| rs2271094c | KLK3 exon 2 | 51359497 | Gly16 syn | A/G | 0.39 | 0.04 |
| rs61752561 | KLK3 exon 3 | 51361382 | Asp-Asn 102 | G/A | 0.03 | 1.0 |
| rs1058205 | KLK3 exon 5 | 51363398 | 3′UTR | T/C | 0.09 | 0.84 |
| rs3760728 | Intergene | 51374592 | - | C/G | 0.36 | 0.10 |
| rs11670728d | KLK2 upstream | 51376489 | promoter | G/A | 0.39 | <0.0001 |
| rs198972 | KLK2 exon 3 | 51379893 | Leu124 syn | C/T | 0.31 | 0.24 |
| rs198977 | KLK2 exon 5 | 51381777 | Arg-Trp 250 | C/T | 0.23 | 0.49 |
| rs198978 | KLK2 exon 5 | 51383072 | 3′UTR | G/T | 0.36 | 0.88 |
| rs80050017 | KLK2 exon 5 | 51383200 | 3′UTR | G/T | 0.08 | 0.14 |
Position on chromosome 19 according to NCBI dbSNP build 137.
Based on the study cohort.
This SNP (rs2271094) is represented by rs11573 in build 137 of NCBI dbSNP. We provide the build 137 coordinates for the SNP here, but use the older nomenclature of rs2271094 to be consistent with our prior publications.
This SNP was excluded from further analysis due to deviation from Hardy-Weinberg equilibrium (HWE).
Statistical Analysis
The observed genotype distribution for each SNP was tested for consistency with Hardy-Weinberg equilibrium using Fisher’s exact test. To estimate the strength of linkage disequilibrium (LD) between all possible pair-wise combinations of SNPs, we calculated D′ using Haploview 4.0 (http://www.broad.mit.edu/mpg/haploview). The group characteristics of the seminal and serum levels of hK2, fPSA and tPSA were summarized descriptively. We used the Kruskal-Wallis test to examine the associations between genotype at each SNP and kallikrein values (concentrations and absolute amounts of hK2 and PSA in seminal plasma ; and concentrations of hK2, tPSA, fPSA and the %fPSA, in serum). All statistical analyses were conducted using Stata 9.0 (Stata).
Results
Levels of hK2 and PSA in seminal plasma and serum were initially determined in a total of 303 young Swedish men. The resulting data are summarized in Table 1. A total of 9 SNPs from a 24-kbp region encompassing the KLK2 and KLK3 genes (Table 2) were subsequently evaluated for association with levels of hK2 and PSA in seminal plasma and in serum. LD in the region was moderate, apart from one 1-kbp haplotype block in KLK2 extending from rs198977 through rs80050017 (Fig. 1).
Figure 1.
Linkage disequilibrium plot of the SNPs studied.
SNPs associated with hK2 levels
SNP associations with levels of hK2 in seminal plasma are presented in Table 3 and Supplementary Figure 1; the associations for serum are presented in Table 4 and Supplementary Figure 2. The genotypes of all 4 KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017), were strongly associated with hK2 amount and concentration in seminal plasma and with hK2 concentration in serum (all P-values < 0.001). As very few rare homozygotes were observed for rs80050017, we also performed a two-group comparison, removing the rare homozygotes, in which similar results were observed (Supplementary Table 1). In general, individuals homozygous for the major alleles showed higher hK2 values compared with individuals with the other genotypes. The effects were in all cases such that the heterozygotes had intermediate values compared with the homozygotes and the effects were more pronounced in seminal plasma compared with serum. The effects were generally quite strong; in most cases there was more than a 3-fold difference between the homozygotes. The rs198977 SNP showed the strongest effect with 4- to 7-fold differences between the homozygotes. In addition, rs61752561 in KLK3 showed an association with hK2 amount and concentration in seminal plasma, but has a low minor allele frequency (MAF) of only 0.03. Notably, for all 4 SNPs associated with hK2 levels, ad-hoc two-group comparisons were consistently significant with the exception of rs80050017 for which only the comparisons between common homozygotes and heterozygotes were significant (Supplementary Table 2).
Table 3.
Association between SNPs in KLK2 and KLK3 and levels of hK2 and PSA in seminal plasma.
| hK2 | PSA | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|||||||||||
| SNP | Genotype | No. | Amount, mg Median (IQR) | P-value | Conc., mg/L Median (IQR) | P-value | No. | Amount, mg Median (IQR) | P-value | Conc., mg/mL Median (IQR) | P-value |
| rs2271094 | AA | 85 | 0.020 (0.011, 0.034) | 0.8 | 6.8 (3.7, 10.1) | 0.8 | 117 | 1.8 (1.1, 2.6) | 0.01 | 0.57 (0.42, 0.80) | 0.01 |
| GA | 85 | 0.019 (0.011, 0.029) | 5.8 (4.3, 8.6) | 118 | 2.1 (1.5, 3.1) | 0.65 (0.52, 0.90) | |||||
| GG | 30 | 0.019 (0.011, 0.027) | 5.8 (4.0, 9.1) | 53 | 2.1 (1.2, 3.3) | 0.69 (0.49, 0.98) | |||||
| Total | 200 | 288 | |||||||||
|
| |||||||||||
| rs61752561 | GG | 188 | 0.018 (0.010, 0.030) | 0.01 | 5.9 (3.8, 9.1) | 0.004 | 273 | 2.0 (1.3, 2.9) | 0.3 | 0.63 (0.45, 0.88) | 0.03 |
| GA | 13 | 0.030 (0.020, 0.040) | 10.0 (7.7, 11.4) | 18 | 2.7 (1.4, 3.3) | 0.80 (0.58, 1.07) | |||||
| Total | 201 | 291 | |||||||||
|
| |||||||||||
| rs1058205 | TT | 123 | 0.018 (0.011, 0.029) | 0.7 | 5.9 (4.0, 9.1) | 0.2 | 189 | 2.1 (1.4, 3.1) | 0.02 | 0.67 (0.47, 0.92) | 0.1 |
| CT | 70 | 0.018 (0.010, 0.034) | 6.2 (3.7, 10.1) | 92 | 1.8 (1.1, 2.6) | 0.57 (0.44, 0.84) | |||||
| CC | 7 | 0.029 (0.014, 0.032) | 9.0 (5.5, 13.9) | 9 | 1.6 (0.94, 2.2) | 0.49 (0.34, 0.63) | |||||
| Total | 200 | 290 | |||||||||
|
| |||||||||||
| rs3760728 | CC | 90 | 0.018 (0.010, 0.034) | 0.6 | 6.4 (3.5, 10.0) | 0.6 | 125 | 1.8 (1.2, 2.6) | 0.1 | 0.57 (0.42, 0.78) | 0.01 |
| CG | 87 | 0.020 (0.011, 0.030) | 5.9 (4.3, 9.1) | 121 | 2.1 (1.4, 3.1) | 0.66 (0.52, 0.90) | |||||
| GG | 24 | 0.018 (0.012, 0.024) | 4.7 (3.7, 8.0) | 44 | 2.1 (1.5, 3.0) | 0.69 (0.51, 1.0) | |||||
| Total | 201 | 290 | |||||||||
|
| |||||||||||
| rs198972 | CC | 99 | 0.024 (0.012, 0.034) | 0.0007 | 7.1 (5.0, 11.3) | <0.0005 | 136 | 1.9 (1.1, 2.9) | 0.7 | 0.63 (0.48, 0.86) | 0.8 |
| CT | 89 | 0.017 (0.010, 0.030) | 5.6 (3.4, 8.2) | 133 | 2.0 (1.3, 3.1) | 0.65 (0.45, 0.90 ) | |||||
| TT | 14 | 0.008 (0.004, 0.016) | 2.9 (1.0, 4.9) | 24 | 2.0 (1.3, 2.4) | 0.69 (0.42, 0.86) | |||||
| Total | 202 | 293 | |||||||||
|
| |||||||||||
| rs198977 | CC | 118 | 0.027 (0.016, 0.038) | <0.0005 | 7.7 (5.2, 11.2) | <0.0005 | 173 | 2.0 (1.4, 2.9) | 1 | 0.61 (0.47, 0.87) | 0.5 |
| CT | 69 | 0.014 (0.010, 0.022) | 4.8 (3.2, 6.8) | 96 | 2.0 (1.3, 2.9) | 0.65 (0.45, 0.95) | |||||
| TT | 12 | 0.004 (0.002, 0.005) | 1.1 (0.8, 1.5) | 18 | 2.0 (1.1, 3.3) | 0.67 (0.32, 0.82) | |||||
| Total | 199 | 287 | |||||||||
|
| |||||||||||
| rs198978 | GG | 83 | 0.026 (0.014, 0.038) | <0.0005 | 7.7 (5.2, 11.7) | <0.0005 | 120 | 1.9 (1.2, 2.9) | 0.9 | 0.63 (0.48, 0.87) | 1 |
| TG | 95 | 0.017 (0.011, 0.029) | 5.8 (3.7, 8.4) | 132 | 2.0 (1.3, 3.0) | 0.62 (0.45, 0.91) | |||||
| TT | 22 | 0.008 (0.004, 0.015) | 2.0 (1.0, 4.1) | 36 | 2.0 (1.3, 2.6) | 0.68 (0.41, 0.83) | |||||
| Total | 200 | 288 | |||||||||
|
| |||||||||||
| rs80050017 | GG | 169 | 0.021 (0.012, 0.032) | <0.0005 | 6.8 (4.6, 10.0) | <0.0005 | 245 | 1.9 (1.3, 2.9) | 0.7 | 0.64 (0.47, 0.88) | 0.4 |
| TG | 27 | 0.008 (0.006, 0.021) | 3.0 (1.4, 5.9) | 37 | 2.2 (1.3, 3.1) | 0.69 (0.43, 0.99) | |||||
| TT | 1 | 0.004 (0.004, 0.004) | 1.0 (1.0, 1.0) | 3 | 0.8 (0.8, 4.4) | 0.25 (0.23, 0.87) | |||||
| Total | 197 | 285 | |||||||||
Bold type indicates statistical significance.
IQR, inter-quartile range.
Table 4.
Association between SNPs in KLK2 and KLK3 and concentrations of hK2 and PSA in serum.
| hK2, ng/mL | tPSA, ng/mL | fPSA, ng/mL | Percent fPSA | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
|
||||||||||
| SNP | Genotype | No. | Median (IQR) | P-value | Median (IQR) | P-value | Median (IQR) | P-value | Median (IQR) | P-value |
| rs2271094 | AA | 121 | 0.037 (0.026, 0.052) | 0.4 | 0.50 (0.33, 0.67) | 0.8 | 0.18 (0.13, 0.26) | 0.3 | 40 (32, 50) | 0.8 |
| GA | 122 | 0.036 (0.028, 0.051) | 0.50 (0.37, 0.67) | 0.21 (0.15, 0.27) | 43 (35, 49) | |||||
| GG | 54 | 0.035 (0.022, 0.045) | 0.52 (0.32, 0.67) | 0.21 (0.13, 0.29) | 43 (35, 52) | |||||
| Total | 297 | |||||||||
|
| ||||||||||
| rs61752561 | GG | 282 | 0.035 (0.025, 0.051) | 0.3 | 0.50 (0.35, 0.68) | 0.02 | 0.19 (0.14, 0.27) | 0.05 | 42 (34, 50) | 0.6 |
| GA | 19 | 0.039 (0.035, 0.046) | 0.42 (0.29, 0.51) | 0.15 (0.12, 0.21) | 44 (36, 51) | |||||
| Total | ||||||||||
|
| ||||||||||
| rs1058205 | TT | 195 | 0.036 (0.025, 0.050) | 0.8 | 0.53 (0.37, 0.76) | 0.001 | 0.20 (0.15, 0.28) | 0.06 | 40 (34, 48) | 0.01 |
| CT | 94 | 0.036 (0.026, 0.050) | 0.41 (0.32, 0.58) | 0.17 (0.12, 0.24) | 44 (34, 51) | |||||
| CC | 10 | 0.034 (0.025, 0.040) | 0.44 (0.30, 0.53) | 0.19 (0.18, 0.27) | 49 (41, 61) | |||||
| Total | 299 | |||||||||
|
| ||||||||||
| rs3760728 | CC | 130 | 0.036 (0.025, 0.051) | 0.2 | 0.48 (0.33, 0.67) | 0.9 | 0.18 (0.13, 0.24) | 0.3 | 40 (32, 49) | 0.3 |
| CG | 123 | 0.036 (0.027, 0.051) | 0.50 (0.36, 0.65) | 0.21 (0.14, 0.27) | 44 (35, 49) | |||||
| GG | 46 | 0.034 (0.019, 0.042) | 0.50 (0.32, 0.70) | 0.20 (0.13, 0.30) | 44 (37, 53) | |||||
| Total | 299 | |||||||||
|
| ||||||||||
| rs198972 | CC | 138 | 0.040 (0.030, 0.052) | <0.0005 | 0.52 (0.39, 0.68) | 0.2 | 0.19 (0.13, 0.27) | 0.6 | 38 (31, 47) | <0.0005 |
| CT | 140 | 0.035 (0.026, 0.050) | 0.45 (0.32, 0.67) | 0.20 (0.15, 0.27) | 44 (37, 52) | |||||
| TT | 25 | 0.017 (0.008, 0.023) | 0.52 (0.32, 0.60) | 0.18 (0.13, 0.24) | 47 (27, 56) | |||||
| Total | 303 | |||||||||
|
| ||||||||||
| rs198977 | CC | 178 | 0.044 (0.033, 0.054) | <0.0005 | 0.51 (0.36, 0.68) | 0.5 | 0.19 (0.13, 0.26) | 0.9 | 40 (32, 48) | 0.007 |
| CT | 100 | 0.029 (0.021, 0.038) | 0.45 (0.34, 0.66) | 0.19 (0.14, 0.28) | 45 (37, 53) | |||||
| TT | 18 | 0.008 (0.005, 0.014) | 0.52 (0.30, 0.60) | 0.18 (0.15, 0.27) | 44 (32, 55) | |||||
| Total | 296 | |||||||||
|
| ||||||||||
| rs198978 | GG | 121 | 0.043 (0.033, 0.054) | <0.0005 | 0.52 (0.36, 0.68) | 0.5 | 0.18 (0.13, 0.26) | 0.3 | 37 (31, 46) | <0.0005 |
| TG | 139 | 0.034 (0.026, 0.050) | 0.47 (0.35, 0.67) | 0.21 (0.15, 0.27) | 45 (38, 52) | |||||
| TT | 38 | 0.015 (0.008, 0.023) | 0.48 (0.31, 0.61) | 0.18 (0.13, 0.27) | 46 (32, 55) | |||||
| Total | 298 | |||||||||
|
| ||||||||||
| rs80050017 | GG | 253 | 0.039 (0.028, 0.051) | <0.0005 | 0.50 (0.35, 0.67) | 0.8 | 0.19 (0.14, 0.27) | 0.9 | 41 (35, 49) | 0.6 |
| TG | 37 | 0.023 (0.017, 0.029) | 0.47 (0.35, 0.66) | 0.18 (0.15, 0.25) | 43 (32, 53) | |||||
| TT | 4 | 0.011 (0.007, 0.015) | 0.48 (0.33, 0.56) | 0.20 (0.11, 0.31) | 52 (37, 55) | |||||
| Total | 294 | |||||||||
Bold type indicates statistical significance.
IQR, inter-quartile range.
SNPs associated with PSA levels
SNP associations with levels of PSA in seminal plasma and serum are presented in Tables 3 and 4, respectively. Graphical representations of selected significant results can be found in Supplemental Tables 3 and 4. The genotypes of all 3 KLK3 SNPs (rs2271094, rs61752561, and rs1058205), were associated with PSA amount or concentration in seminal plasma (Table 3). For rs1058205, the rare homozygote count was low; similar results are observed when the common homozygote group is compared to the heterozygote group (Supplementary Table 1). However, the effects were less strong than observed for the KLK2 SNPs and hK2, as there were between 0.8- to 1.3-fold differences between the homozygotes. Apart from the rs61752561 SNP with a low MAF, rs1058205 was the only KLK3 SNP showing significance for PSA in serum (Table 4). Similarly, SNP rs1058205 showed a statistically significant association with higher seminal PSA amount for the TT genotype (median tPSA amount 2.1 vs. 1.6 mg for TT vs. CC). Subjects with the TT genotype also tended to have higher median seminal plasma concentration of PSA, though the difference did not reach significance (P=0.1). The same genotype was associated with significantly higher serum concentration of tPSA (median 0.53 vs. 0.44 ng/mL for TT vs. CC genotype) and lower %fPSA (40% vs. 49% for TT vs. CC genotype). Though only the comparison between the TT and TC genotype of rs1058205 showed a significant difference in serum total PSA levels (Supplemental Table 2), a continuing trend for the C allele being associated with decreased total PSA levels in serum can be observed (Supplemental Figure 2A). In addition, the KLK2 SNPs (rs198972, rs198977, and rs198978) showed highly significant associations with %fPSA in serum. In all three cases individuals homozygous for the minor alleles showed higher values of %fPSA (rs198972, 47% vs 38% for TT vs CC; rs198977, 44% vs 40% for TT vs CC; rs198978, 46% vs 37% for TT vs GG; all P≤0.007).
Significance of the associations
The P-values presented in Tables 3 and 4 are unadjusted for the multiple testing performed. A commonly used compensation for multiple testing is the Bonferroni correction, which simply divides the significance level with the number of tests. In the present study a total of 64 tests have been performed. Thus, a global significance level of 0.05 corresponds to a significance level of 0.0008 in the individual tests. Using this criterion, 14 of the 25 tests that were significant in the individual tests remain significant. It should be noted that the Bonferroni method is very strict. Another way to assess this problem is to compare the expected number of significant results given that all null hypotheses are true to the observed number. Out of the 64 tests, one expects 3.2 tests with P-values below 0.05 and 0.64 tests with P-values below 0.01. The observed numbers are 25 and 18, respectively. Thus, both the Bonferroni correction approach and the comparison of the expected and observed numbers of significant results, indicate strongly that a majority of the cases with P-values below 0.05 are true signals.
Correlation between the SNPs
Though linkage disequilibrium appears to be moderate in this region (Fig. 1), correlation between SNPs could still mean that some of the SNPs are tagging the same functional variant and thus are redundant. To examine this further, we calculated the pairwise correlation coefficient (r2) between all pairs of SNPs based on the phased haplotypes and examined pairs of SNPs for which r2 > 0.2. The most correlated pair of SNPs is rs3760728 and rs2271094 (r2=0.60); these two SNPs are also both significantly associated with the concentration of PSA in seminal fluid. Additionally, numerous pairwise correlations between rs198972, rs198977, rs198978, and rs80050017 are observed; these SNPs are consistently associated with levels of hK2.
Given the strong association between several correlated SNPs and hK2 levels, we next asked which of the 4-marker haplotypes formed by rs198972, rs198977, rs198978, and rs80050017 are associated with hk2 levels. Of the five haplotypes with a frequency greater than 5%, four are strongly associated with hK2 levels in blood and semen (Table 5). Of these, two are also associated with the ratio between free and total PSA (Table 5). Notably, the three common haplotypes with a “T” allele at rs198977 are strongly associated with decreased levels of hK2, consistent with what was observed for single-marker tests.
Table 5.
Haplotype association with hK2 levels and percent fPSA
| Serum | Seminal plasma | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Percent fPSA | log10 hK2, ng/mL | log10 hK2, mg | log10 hK2, mg/L | ||||||
| Haplotype (rs198972, rs198977, rs198978, rs80050017) | Frequency | Beta | P-value | Beta | P-value | Beta | P-value | Beta | P-value |
| CCGG | 0.61 | −5.0 | <0.0005 | 0.48 | <0.0005 | 0.19 | <0.0005 | 0.21 | <0.0005 |
| TCTG | 0.1 | 2.8 | 0.1 | −0.048 | 0.6 | 0.082 | 0.1 | 0.057 | 0.3 |
| TTTG | 0.098 | 5.3 | 0.007 | −0.38 | <0.0005 | −0.27 | <0.0005 | −0.23 | <0.0005 |
| TTTT | 0.074 | 0.62 | 0.8 | −0.60 | <0.0005 | −0.30 | <0.0005 | −0.34 | <0.0005 |
| CTTG | 0.056 | 1.4 | 0.6 | −0.71 | <0.0005 | −0.25 | <0.0005 | −0.24 | <0.0005 |
| All other haplotypes (frequency <5% each) | 0.061 | 4.2 | 0.08 | 0.26 | 0.02 | 0.22 | 0.002 | 0.15 | 0.02 |
Discussion
In the current study, we examined 9 previously reported SNPs in a 24 kbp KLK2 - KLK3 region (28) for associations with levels of hK2 and PSA in seminal plasma and serum in young men without prostate disease. For hK2, all four KLK2 SNPs showed association with hK2 levels both in seminal plasma and serum. Three of these SNPs (rs198977, rs198978, and rs80050017) formed a haplotype block, whereas the remaining SNP (rs198972) was in moderate LD with these SNPs. The strongest effect on hK2 level was observed for rs198977. Consistent with this observation, common haplotypes containing the “T” allele of rs198977 are associated with decreased levels of hK2. Whether the rs198977 SNP is causal or if other SNPs in LD with this SNP are causing the observed lower levels of hK2 is unknown at present.
The SNP rs198977 has previously been associated with hK2 levels among older men with no prostate cancer diagnosis (~65–70 years old) (25, 26, 29); as in this study, the T allele was consistently associated with lower levels. In all three prior studies, the T allele was also associated with slightly increased risk of prostate cancer. In contrast, a smaller study of Chinese men (27) reported increased risk of prostate cancer for men carrying the C allele. This discrepancy may possibly be explained by the different populations studied, but the differences nevertheless raise questions. The C to T substitution in rs198977 corresponds to an Arg226Trp change in hK2. An in vitro study suggested that the Trp226-hK2 variant lacked protease activity, although the experimental system did not allow a definitive assessment (34). The observation that the T allele of rs198977 is associated with lower hK2 levels in multiple populations and among both young and old men suggests that it results from a basic aspect of hK2 biology resulting in lower hK2 expression. Another possibility is that the Trp226 form of hK2 (encoded by the T allele) is more rapidly degraded, or poorly detected by both of the different monoclonal antibodies each capturing and detecting hK2 through uniquely distinct epitopes in our study and that by Nam et al. (25, 26).
Several KLK3 SNPs showed associations with levels of PSA. The rs2271094 SNP was significantly associated with PSA amount and concentration in seminal plasma. For rs61752561, the GG genotype showed an association with lower PSA concentration in seminal plasma, but an association with higher tPSA concentration in serum. Since this SNP has a MAF of only 0.03, these associations are likely due to chance. For a third KLK3 SNP, rs1058205, the TT genotype showed a statistically significant association with higher PSA amount in seminal plasma and with higher tPSA concentration and lower %fPSA in serum. This decrease in %fPSA is a consequence of the increase in serum levels, which is due to the difference in mechanisms and rates of elimination of fPSA and complex-bound PSA. Our results with this SNP fit with a prior report in which the T variant was associated with higher serum tPSA in older men (22). This variant was also associated with higher cancer risk in one study (24), though subsequent studies failed to confirm this association (22, 29). Moreover, we and others (22) have argued that KLK3 variants associated with higher tPSA levels may exhibit spurious associations with prostate cancer risk if PSA testing was involved in detection of the cancers.
Three KLK2 SNPs, rs198972, rs198977, and rs198978, were significantly associated with %fPSA in serum. In each case, the allele associated with higher %fPSA was also associated with lower hK2. How variants in KLK2 might affect the %fPSA is not entirely clear; however, we propose two possible explanations. The first is a protein-protein interaction between hK2 and PSA, in which a variant hK2 might selectively stabilize fPSA, thereby increasing %fPSA. Second, if hK2 is responsible for PSA processing in vivo, then variants that reduce hK2 level or protease activity (as suggested for Trp226-hK2) could result in decreased mature PSA and increased pro-PSA. Because pro-PSA is unable to form complexes with the serum anti-proteases and therefore remains free in serum, the result would be an increase in the %fPSA. Interestingly, rs198977 was also associated with both hK2 and %fPSA in our prior study of older men (29).
Other KLK3 SNPs have also been associated with PSA levels in blood. Several SNPs in the KLK3 promoter were reported to be associated with serum PSA levels (19, 20), and one was associated with prostate cancer risk (35), but later studies failed to confirm these associations (21, 36–39). In an earlier study of the same cohort, we found that a KLK3 promoter SNP, rs266882, in combination with androgen receptor gene CAG repeat length, was significantly associated with serum levels of tPSA (38). In the same cohort we have also recently shown, that a MSMB-promoter SNP (rs10993994) at the genetic locus encoding β-microseminoprotein (β-MSP) is significantly associated with blood and semen levels of PSA and semen levels of hK2 (40). In the 3′ flanking region of KLK3, rs2735839 was found to be associated with PSA level in two studies (22, 23). In contrast, our previous study found an association for this SNP with %fPSA but not PSA level (29).
The associations others and we have detected may have implications for PSA and hK2 testing in prostate cancer screening. For men carrying genetic variants associated with altered levels of PSA or hK2, prostate cancer risk models developed on the general population may be less accurate. Conversely, the combination of biomarker levels and genotype may offer a means of increasing the accuracy of prostate cancer prediction. These considerations are particularly applicable to the KLK2 SNP rs198977, for which the TT genotype is associated with dramatically lower hK2 levels, but with higher cancer risk. Indeed, a model incorporating rs198977 genotype and the interaction between genotype and hK2 level was suggested to have higher accuracy for predicting prostate cancer than a model with biomarker data alone (29).
In conclusion, we documented the association of SNPs in KLK3 and KLK2 with levels of PSA and hK2 in young men. These results demonstrate that associations exist several decades before any substantial incidence of prostate cancer or other prostate conditions were found to influence kallikrein levels in the blood (16). Moreover, most of the variants associated with altered tPSA or hK2 concentrations in serum were associated with corresponding albeit more pronounced alterations in seminal plasma. Therefore, the causative genetic variants most likely exert their effects through global changes in expression or protein stability, rather than being related to a prostate disease process. The information from analyses of variants in the KLK2 and KLK3 could also be used to refine models of PSA cut-off values in prostate cancer testing. In addition, because low levels of PSA are correlated with lower percentage of motile sperm in both fertile and infertile men, a better knowledge of the factors regulating the levels of hK2 and PSA is of importance to understanding the mechanisms behind male infertility.
Supplementary Material
Acknowledgments
We thank Gun-Britt Eriksson and Kerstin Håkansson for expert technical assistance with immunoassaying. This work was funded by the National Cancer Institute [R01CA160816, R01 CA175491, and P50-CA92629], the Sidney Kimmel Center for Prostate and Urologic Cancers, and David H. Koch through the Prostate Cancer Foundation, the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Program, Swedish Cancer Society project no. 11-0624, a FiDIPro-program award from TEKES in Finland, and Fundacion Federico SA. The manuscript was edited by Janet Novak; this work was paid for by MSKCC.
Abbreviations
- hK2
kallikrein-related peptidase 2
- PSA
prostate-specific antigen
- fPSA
free PSA
- cPSA
complexed PSA
- tPSA
total PSA
- %fPSA
ratio of free to total PSA
- SNP
single nucleotide polymorphism
- Mab
monoclonal antibody
- LD
linkage disequilibrium
Human genes
- KLK2
kallikrein-related peptidase 2 encoding hK2
- KLK3
kallikrein-related peptidase 3 encoding PSA
- AR
androgen receptor
Footnotes
Publisher’s Disclaimer: This is an un-copyedited authored manuscript copyrighted by the American Association for Clinical Chemistry (AACC). This may not be duplicated or reproduced, other than for personal use or within the rule of ‘Fair Use of Copyrighted Materials’ (section 107, Title 17, U.S. Code) without permission of the copyright owner, AACC. The AACC disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties. The final publisher-authenticated version of the article will be made available at http://www.clinchem.org 12 months after its publication in Clinical Chemistry.
Authors Disclosures of Potential Conflicts of Interests
Hans Lilja holds patents for free PSA, hK2, and intact PSA assays
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