Printer Friendly

Genetic variation in KLK2 and KLK3 is associated with concentrations of hK2 and PSA in serum and seminal plasma in young men.

Prostate-specific antigen (PSA) [10] 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. PSA and hK2 have documented physiological roles 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 demembraned 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 noncatalytic pro-PSA zymogen can be converted in vitro 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 noncatalytic and bound in complex with protein C inhibitor {encoded by SERPINA5 [serpin peptidase inhibitor, clade A (a-1 antiproteinase, antitrypsin), member 5]}, [11] whereas the majority of PSA is in free, catalytically active form (fPSA), with <5% of PSA being complexed to SERPIN-type antiproteases (cPSA) (9). The sum of fPSA and cPSA closely corresponds to total PSA (tPSA). Both PSA and hK2 are noncatalytic in blood, with a relationship of free to bound forms that is opposite 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 antiproteases 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 g/L (13), and the retrograde release of PSA into the blood occurs with a frequency of <1 molecule per million secreted PSA molecules. hK2 is found in both seminal plasma and serum but at approximately 1% of the concentration of PSA (7, 14).In men affected by prostate cancer, PSA and hK2 concentrations in blood become increased 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 concentrations in men younger than 30 years. Concentrations of fPSA, but not cPSA, in serum correlate with PSA concentrations in seminal plasma in young men (18). Low concentrations 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, kallikrein-related peptidase 3) (19-24) and hK2 (KLK2, kallikrein-related peptidase 2) (25-29). Several SNPs in these genes have been associated with serum PSA and hK2 concentrations (22,24-26,29). The study cohorts have, however, been largely confined to older men, in whom the concentrations of these biomarkers may be affected by prostate cancer, benign prostatic hyperplasia, or prostatitis, all of which become more prevalent with age. Because measurement of PSA in serum is widely used for identifying individuals 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 concentrations of hK2 and PSA in serum and seminal plasma by use of a population-based cohort of young men in whom prostate conditions are very rare.

Materials and Methods

PARTICIPANTS

A total of 305 men under compulsory medical examination for military service in Sweden were enrolled into a study of reproductive function in the year 2000 (30). This group can be considered as representative for the Swedish general population of adolescent men, since at that time >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 h (SD 57; range 12-504 h). All men participated after giving written informed consent according to protocols approved by the ethics 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 variables studied here.

SEMEN AND BLOOD SAMPLES

Semen samples obtained after masturbation were delivered between 0900 and 1100. A blood sample was subsequently drawn. All participating men were asked to abstain from sexual activities for at least 48 h 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 [micro]L was mixed with 50 [micro]L of 0.1 mol/L benzamidine to inhibit liquefaction. Seminal plasma was obtained by centrifugation of the semen sample at 10 000g for 10 min. Blood and seminal plasma was kept at -70 [degrees]C until analysis.

hK2 AND PSA ANALYSES

We analyzed seminal plasma samples for hK2 by use of 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 3 PSA-specific anti-PSA monoclonal antibodies (Mabs) (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 CV for hK2 measurement in seminal plasma was 12% at a mean concentration of 0.008 g/L. We measured fPSA and tPSA in seminal plasma and serum by use of the commercially available assay Prostatus[TM] PSA Free/Total kit (Delfia[R] Reagents) (32). The analysis for tPSA in seminal plasma measures the sum of fPSA (>95%), cPSA (1%-3%), and hK2 (<1%). The analysis of fPSA in seminal plasma measures the sum of active single-chain and inactive internally cleaved 2-chain fPSA. The combination of Mab H117 and H50 provides 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 g/L. The detection limit in serum was 0.05 ng/mL (CV 5% at a mean concentration of 2.3 ng/mL) for tPSA and 0.04 ng/mL (CV 5.9% at a mean concentration of 0.25 ng/mL) for fPSA. Serum concentrations 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, intraindividual variability of these measures is <10%, and we presume the same holds here.

GENOTYPE ANALYSES

We prepared genomic DNA from peripheral leukocytes by use of a QIAamp DNA Maxi Kit (Qiagen). DNA concentrations were determined by PicoGreen[TM] DNA assay (Molecular Probes), and all samples were adjusted to the same DNA concentration. We determined genotypes by Sequenom MassArray MALDITOF analysis and made the assay design using MassArray Assay Design 2.0 software (Sequenom) as previously described (33). We initially selected 9 SNPs 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 (LD). 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.

STATISTICAL ANALYSIS

We tested the observed genotype distribution for each SNP for consistency with Hardy-Weinberg equilibrium using the Fisher exact test. To estimate the strength of LD between all possible pairwise 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 concentrations 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 %fPSA, in serum). All statistical analyses were conducted by use of Stata 9.0 (Stata Corp.).

Results

Concentrations of hK2 and PSA in seminal plasma and serum were initially determined in 303 young Swedish men. The resulting data are summarized in Table 1. We subsequently evaluated 9 SNPs from a 24-kbp region encompassing the KLK2 and KLK3 genes (Table 2) for association with concentrations of hK2 and PSA in serum and seminal plasma. LD in the region was moderate, apart from one 1-kbp haplotype block in KLK2 extending from rs198977 through rs80050017 (Fig. 1).

SNPs ASSOCIATED WITH hK2 CONCENTRATIONS

SNP associations with hK2 concentrations in seminal plasma are presented in Table 3 and Supplemental Fig. 1 (which accompanies the online version of this article at http://www.clinchem.org/content/vol60/issue3); the associations for serum are presented in Table 4 and online Supplemental Fig. 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 2-group comparison, removing the rare homozygotes, in which similar results were observed (see online Supplemental Table 1). In general, individuals homozygous for the major alleles showed higher hK2 values than 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 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 concentrations, ad hoc 2-group comparisons were consistently significant, with the exception of rs80050017, for which only the comparisons between common homozygotes and heterozygotes were significant (see online Supplemental Table 2).

SNPs ASSOCIATED WITH PSA CONCENTRATIONS

SNP associations with PSA concentrations in seminal plasma and serum are presented in Tables 3 and 4, respectively. Graphical representations of selected significant results can be found in online 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 (see online Supplemental Table 1). However, the effects were less strong than observed for the KLK2 SNPs and hK2, as there were 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 amounts for the TT genotype (median tPSA amount 2.1 vs 1.6 mg for TT vs CC). Individuals with the TT genotype also tended to have higher median seminal plasma concentrations of PSA, although the difference did not reach significance (P = 0.1). The same genotype was associated with significantly higher serum concentrations 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). Although only the comparison between the TT and TC genotype of rs1058205 showed a significant difference in serum total PSA concentrations (see online Supplemental Table 2), a continuing trend for the C allele being associated with decreased total PSA concentrations in serum can be observed (see online Supplemental Fig. 2A). In addition, the KLK2 SNPs (rs198972, rs198977, and rs198978) showed highly significant associations with %fPSA in serum. In all 3 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 [less than or equal to] 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 by 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. By 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. Of the 64 tests, one expects 3.2 tests with P values <0.05 and 0.64 tests with P values <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 <0.05 are true signals.

CORRELATION BETWEEN THE SNPs

Although LD 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 ([r.sup.2]) between all pairs of SNPs on the basis of the phased haplotypes and examined pairs of SNPs for which [r.sup.2] > 0.2. The most correlated pair of SNPs is rs3760728 and rs2271094 ([r.sup.2] = 0.60); these 2 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 concentrations of hK2.

Given the strong association between several correlated SNPs and hK2 concentrations, we next asked which of the 4-marker haplotypes formed by rs198972, rs198977, rs198978, and rs80050017 are associated with hk2 concentrations. Of the 5 haplotypes with a frequency >5%, 4 are strongly associated with hK2 concentrations in blood and semen (Table 5). Of these, 2 are also associated with the ratio between free and total PSA (Table 5). Notably, the 3 common haplotypes with a T allele at rs198977 are strongly associated with decreased hK2 concentrations, consistent with what was observed for single-marker tests.

Discussion

In the current study, we examined 9 previously reported SNPs in a 24-kbp KLK2-KLK3 region (28) for associations with hK2 and PSA concentrations in serum and seminal plasma in young men without prostate disease. For hK2, all 4 KLK2 SNPs showed association with hK2 concentrations 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 concentration was observed for rs198977. Consistent with this observation, common haplotypes containing the T allele of rs198977 are associated with decreased concentrations of hK2. Whether the rs198977 SNP is causal or other SNPs in LD with this SNP are causing the observed lower concentrations of hK2 is unknown at present.

The SNP rs198977 has previously been associated with hK2 concentrations among older men (approximately 65-70 years old) with no prostate cancer diagnosis (25, 26, 29); as in this study, the T allele was consistently associated with lower concentrations. In all 3 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 concentrations 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 those by Nam and colleagues (25, 26).

Several KLK3 SNPs showed associations with concentrations 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 with higher tPSA concentration in serum. Because 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 higher tPSA concentration and lower %fPSA in serum. This decrease in %fPSA is a consequence of the increase in serum concentrations, 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 1 study (24), although subsequent studies failed to confirm this association (22,29). Moreover, we and others (22) have argued that KLK3 variants associated with higher tPSA concentrations 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 2 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 concentration or protease activity (as suggested for [Trp.sup.226]-hK2) could result in decreased mature PSA and increased pro-PSA. Because pro-PSA is unable to form complexes with the serum antiproteases 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 concentrations in blood. Several SNPs in the KLK3 promoter were reported to be associated with serum PSA concentrations (19, 20), and 1 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 concentrations of tPSA (38). In the same cohort, we have also recently shown that a MSMB-promoter SNP (rs10993994) at the genetic locus encoding [beta]-microseminoprotein ([beta]-MSP) is significantly associated with blood and semen concentrations of PSA and semen concentrations of hK2 (40). In the 3' flanking region of KLK3, rs2735839 was found to be associated with PSA concentration in 2 studies (22, 23). In contrast, our previous study found an association for this SNP with %fPSA but not PSA concentration (29).

The associations we and others have detected may have implications for PSA and hK2 testing in prostate cancer screening. For men carrying genetic variants associated with altered concentrations of PSA or hK2, prostate cancer risk models developed on the general population maybe less accurate. Conversely, the combination of biomarker concentrations 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 concentrations, but with higher cancer risk. Indeed, a model incorporating rs198977 genotype and the interaction between genotype and hK2 concentration 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 concentrations 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 concentrations 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 cutoff values in prostate cancer testing. In addition, because low concentrations of PSA are correlated with lower percentage of motile sperm in both fertile and infertile men, a better knowledge of the factors regulating the concentrations of hK2 and PSA is of importance to understanding the mechanisms behind male infertility.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: A. Giwercman, Advisory Board for Bayer AB.

Stock Ownership: H. Lilja, Arctic Partners.

Honoraria: None declared.

Research Funding: This study was supported by the Sidney Kimmel Center for Prostate and Urologic Cancers and David H. Koch through the Prostate Cancer Foundation. R.J. Klein, the National Cancer Institute (R01 CA175491); H. Lilja, the National Cancer Institute (R01 CA160816, R01 CA175491), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Program, Swedish Cancer Society project no. 11-0624, FiDIProprogram award from TEKES in Finland, Fundacion Federico SA. Expert Testimony: None declared.

Patents: H. Lilja, patents for free PSA, hK2, and intact PSA assays.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Gun-Britt Eriksson and Kerstin Hakansson for expert technical assistance with immunoassaying. The manuscript was edited by Janet Novak; this work was paid for by Memorial Sloan-Kettering Cancer Center.

References

(1.) Darson MF, Pacelli A, Roche P, Rittenhouse HG, Wolfert RL, Young CY, et al. Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker. Urology 1997;49:857-62.

(2.) Kuriyama M, Wang MC, Lee CI, Papsidero LD, Killian CS, Inaji H, et al. Use of human prostate-specific antigen in monitoring prostate cancer. Cancer Res 1981;41:3874-6.

(3.) Yousef GM, Luo LY, Diamandis EP. Identification of novel human kallikrein-like genes on chromosome 19q13.3-q13.4. Anticancer Res 1999;19:2843-52.

(4.) Lilja H. A kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J Clin Invest 1985;76:1899-903.

(5.) de Lamirande E, Yoshida K, Yoshiike TM, Iwamoto T, Gagnon C. Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process. J Androl 2001;22:672-9.

(6.) Lovgren J, Rajakoski K, Karp M, Lundwall A, Lilja H. Activation of the zymogen form of prostate-specific antigen by human glandular kallikrein 2. Biochem Biophys Res Commun 1997;238:549-55.

(7.) Deperthes D, Chapdelaine P, Tremblay RR, Brunet C, Berton J, Hebert J, et al. Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma. Biochim Biophys Acta 1995; 1245:311-6.

(8.) Deperthes D, Frenette G, Brillard-Bourdet M, Bourgeois L, Gauthier F, Tremblay RR, Dube JY. Potential involvement of kallikrein hK2 in the hydrolysis of the human seminal vesicle proteins after ejaculation. J Androl 1996;17:659-65.

(9.) Christensson A, Lilja H. Complex formation between protein C inhibitor and prostate-specific antigen in vitro and in human semen. Eur J Biochem 1994;220:45-53.

(10.) Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O, Pettersson K, Lovgren T. Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem 1991;37:1618-25.

(11.) Grauer LS, Finlay JA, Mikolajczyk SD, Pusateri KD, Wolfert RL. Detection of human glandular kallikrein, hK2, as its precursor form and in complex with protease inhibitors in prostate carcinoma serum. J Androl 1998;19:407-11.

(12.) Becker C, Piironen T, Kiviniemi J, Lilja H, Pettersson K. Sensitive and specific immunodetection of human glandular kallikrein 2 in serum. Clin Chem 2000;46:198-206.

(13.) Ahlgren G, Rannevik G, Lilja H. Impaired secretory function of the prostate in men with oligoasthenozoospermia. J Androl 1995;16:491-8.

(14.) Piironen T, Lovgren J, Karp M, Eerola R, Lundwall A, Dowell B, et al. Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum. Clin Chem 1996;42:1034-41.

(15.) Nam RK, Diamandis EP, Toi A, Trachtenberg J, Magklara A, Scorilas A, et al. Serum human glandular kallikrein-2 protease levels predict the presence of prostate cancer among men with elevated prostate-specific antigen. J Clin Oncol 2000;18:1036-42.

(16.) Lilja H, Cronin AM, Dahlin A, Manjer J, Nilsson PM, Eastham JA, et al. Prediction of significant prostate cancer diagnosed 20 to 30 years later with a single measure of prostate-specific antigen at or before age 50. Cancer 2011;117:1210-9.

(17.) Christensson A, Bjork T, Nilsson O, Dahlen U, Matikainen MT, Cockett AT, et al. Serum prostate specific antigen complexed to alpha 1antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-5.

(18.) Savblom C, Malm J, Giwercman A, Nilsson JA, Berglund G, Lilja H. Blood levels of free-PSA but not complex-PSA significantly correlates to prostate release of PSA in semen in young men, while blood levels of complex-PSA, but not free-PSA increase with age. Prostate 2005;65:66-72.

(19.) Xue WM, Coetzee GA, Ross RK, Irvine R, Kolonel L, Henderson BE, Ingles SA. Genetic determinants of serum prostate-specific antigen levels in healthy men from a multiethnic cohort. Cancer Epidemiol Biomarkers Prev 2001;10:575-9.

(20.) Cramer SD, Chang BL, Rao A, Hawkins GA, Zheng SL, Wade WN, et al. Association between genetic polymorphisms in the prostate-specific antigen gene promoter and serum prostate-specific antigen levels. J Natl Cancer Inst 2003;95:1044-53.

(21.) Xu J, Meyers DA, Sterling DA, Zheng SL, Catalona WJ, Cramer SD, et al. Association studies of serum prostate-specific antigen levels and the genetic polymorphisms at the androgen receptor and prostate-specific antigen genes. Cancer Epidemiol Biomarkers Prev 2002;11:664-9.

(22.) Ahn J, Berndt SI, Wacholder S, Kraft P, Kibel AS, Yeager M, et al. Variation in KLK genes, prostatespecific antigen and risk of prostate cancer. Nat Genet 2008;40:1032-4.

(23.) Eeles RA, Kote-Jarai Z, Giles GG, Olama AA, Guy M, Jugurnauth SK, et al. Multiple newly identified loci associated with prostate cancer susceptibility. Nat Genet 2008;40:316-21.

(24.) Pal P, Xi H, Sun G, Kaushal R, Meeks JJ, Thaxton CS, et al. Tagging SNPs in the kallikrein genes 3 and 2 on 19q13 and their associations with prostate cancer in men of European origin. Hum Genet 2007;122:251-9.

(25.) Nam RK, Zhang WW, Trachtenberg J, Diamandis E, Toi A, Emami M, et al. Single nucleotide polymorphism of the human kallikrein-2 gene highly correlates with serum human kallikrein-2 levels and in combination enhances prostate cancer detection. J Clin Oncol 2003;21:2312-9.

(26.) Nam RK, Zhang WW, Klotz LH, Trachtenberg J, Jewett MA, Sweet J, et al. Variants of the hK2 protein gene (KLK2) are associated with serum hK2 levels and predict the presence of prostate cancer at biopsy. Clin Cancer Res 2006;12:6452-8.

(27.) Chiang CH, Hong CJ, Chang YH, Chang LS, Chen KK. Human kallikrein-2 gene polymorphism is associated with the occurrence of prostate cancer. J Urol 2005;173:429-32.

(28.) Mittal RD, Mishra DK, Thangaraj K, Singh R, Mandhani A. Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population? Steroids 2007;72:335-41.

(29.) Klein RJ, Hallden C, Cronin AM, Ploner A, Wiklund F, Bjartell AS, et al. Blood biomarker levels to aid discovery of cancer-related single nucleotide polymorphisms: kallikreins and prostate cancer. Cancer Prev Res (Phila) 2010;3:611-9.

(30.) Richthoff J, Rylander L, Hagmar L, Malm J, Giwercman A. Higher sperm counts in southern Sweden compared with Denmark. Hum Reprod 2002;17:2468-73.

(31.) Vaisanen V, Eriksson S, Ivaska KK, Lilja H, Nurmi M, Pettersson K. Development of sensitive immunoassays for free and total human glandular kallikrein 2. Clin Chem 2004;50:1607-17.

(32.) Mitrunen K, Pettersson K, PiironenT, Bjork T, Lilja H, Lovgren T. Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum. Clin Chem 1995;41:1115-20.

(33.) Klein RJ, Hallden C, Gupta A, Savage CJ, Dahlin A, Bjartell A, et al. Evaluation of multiple risk-associated single nucleotide polymorphisms versus prostate-specific antigen at baseline to predict prostate cancer in unscreened men. Eur Urol 2012;61:471-7.

(34.) Herrala A, Kurkela R, Porvari K, Isomaki R, Henttu P, Vihko P. Human prostate-specific glandular kallikrein is expressed as an active and an inactive protein. Clin Chem 1997;43:279-84.

(35.) Medeiros R, Morais A, Vasconcelos A, Costa S, Pinto D, Oliveira J, et al. Linkage between polymorphisms in the prostate specific antigen ARE1 gene region, prostate cancer risk, and circulating tumor cells. Prostate 2002;53:88-94.

(36.) Beebe-Dimmer JL, Lange LA, Cain JE, Lewis RC, Ray AM, Sarma AV, et al. Polymorphisms in the prostate-specific antigen gene promoter do not predict serum prostate-specific antigen levels in African-American men. Prostate Cancer Prostatic Dis 2006;9:50-5.

(37.) Rao A, Chang BL, Hawkins G, Hu JJ, Rosser CJ, Hall MC, et al. Analysis of G/A polymorphism in the androgen response element I of the PSA gene and its interactions with the androgen receptor polymorphisms. Urology 2003;61:864-9.

(38.) Savblom C, Giwercman A, Malm J, Hallden C, Lundin K, Lilja H, Giwercman Y. Association between polymorphisms in the prostate-specific antigen (PSA) promoter and release of PSA. Int J Androl 2009;32:479-85.

(39.) Wang LZ, Sato K, Tsuchiya N, Yu JG, Ohyama C, Satoh S, et al. Polymorphisms in prostate-specific antigen (PSA) gene, risk of prostate cancer, and serum PSA levels in Japanese population. Cancer Lett 2003;202:53-9.

(40.) Xu X, Valtonen-Andre C, Savblom C, Hallden C, Lilja H, Klein RJ. Polymorphisms at the microseminoprotein-beta locus associated with physiologic variation in beta-microseminoprotein and prostate-specific antigen levels. Cancer Epidemiol Biomarkers Prev 2010;19:2035-42.

Charlotta Savblom, [1] Christer Hallden, [2] Angel M. Cronin, [3] Torbjorn Sail, [4] Caroline Savage, [3] Emily A. Vertosick, [3] Robert J. Klein, [5] * Aleksander Giwercman, [6] and Hans Lilja [1,7,8,9]

[1] Department of Laboratory Medicine, Division of Clinical Chemistry, and [6] Reproductive Medicine Centre, Lund University, Skane University Hospital, Malmo, Sweden; [2] Biomedicine, Kristianstad University, Kristianstad, Sweden; [3] Department of Epidemiology and Biostatistics, [5] Clinical Genetics Service, Department of Medicine, and Cancer Biology & Genetics Program, and [7] Departments of Medicine, Laboratory Medicine, and Surgery (Urology), Memorial Sloan Kettering Cancer Center, New York, NY; [4] Department of Cell and Organism Biology, Lund University, Lund, Sweden; [8] Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK; [9] Institute of Biomedical Technology, University of Tampere, Tampere, Finland.

[10] Nonstandard abbreviations: PSA, prostate-specific antigen; hK2, kallikrein-related peptidase 2; fPSA, free PSA; cPSA, complexed PSA; tPSA, total PSA; SNP, single nucleotide polymorphism; Mab, monoclonal antibody; LD, linkage disequilibrium; MAF, minor allele frequency.

[11] Human genes: SERPINA5, serpin peptidase inhibitor, clade A ([alpha]-1 antiproteinase, antitrypsin), member 5; KLK2, kallikrein-related peptidase 2 encoding hK2; KLK3, kallikrein-related peptidase 3 encoding PSA.

* Address correspondence to this author at: Clinical Genetics Service, Department of Medicine, Program in Cancer Biology and Genetics, Memorial SloanKettering Cancer Center, New York, NY 10065. e-mail kleinr@mskcc.org.

Received June 20, 2013; accepted November 1, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.211219

Table 1. hK2 and PSA levels in seminal plasma and serum.

                                          Interquartile
Characteristic      n     Mean   Median       range       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
  hK2, mg/L         202   7.2    6.1         4.0-9.2      4.6
  PSA, mg           293   2.2    2.0         1.3-2.9      1.3
  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
  fPSA, ng/mL       303   0.29   0.19       0.13-0.27     0.72
  tPSA, ng/mL       303   0.64   0.50       0.35-0.67     0.84

Table 2. SNP descriptions and allele frequencies in the study cohort.

                                            Genomic      Amino acid
     SNP                Location          position (a)     change

rs2271094 (c)    KLK3 exon 2              51359497       Gly16 syn
rs61752561       KLK3 exon 3              51361382       Asp-Asn 102
rs1058205        KLK3 exon 5              51363398       3'UTR
rs3760728        Intergene                51374592
rs11670728 (d)   KLK2 upstream promoter   51376489
rs198972         KLK2 exon 3              51379893       Leu124 syn
rs198977         KLK2 exon 5              51381777       Arg-Trp 250
rs198978         KLK2 exon 5              51383072       3'UTR
rs80050017       KLK2 exon 5              51383200       3'UTR

                 Major/minor             Hardy-Weinberg
     SNP         allele (b)    MAF (b)   equilibrium P

rs2271094 (c)        A/G        0.39          0.04
rs61752561           G/A        0.03          1.0
rs1058205            T/C        0.09          0.84
rs3760728            C/G        0.36          0.10
rs11670728 (d)       G/A        0.39        <0.0001
rs198972             C/T        0.31          0.24
rs198977             C/T        0.23          0.49
rs198978             G/T        0.36          0.88
rs80050017           G/T        0.08          0.14

(a) Position on chromosome 19 according to National Center for
Biotechnology Information (NCBI) dbSNP build 137.

(b) Based on the study cohort.

(c) 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.

(d) Excluded from further analysis because of deviation from
Hardy-Weinberg equilibrium.

Table 3. Association between SNPs in KLK2 and KLK3
and levels of hK2 and PSA in seminal plasma. (a)

                            hK2

 SNP and                  Amount,
 genotype     n             mg               P

rs2271094
  AA          85    0.020(0.011-0.034)    0.8
  GA          85    0.019(0.011-0.029)
  GG          30    0.019(0.011-0.027)
  Total      200
rs61752561
  GG         188    0.018(0.010-0.030)    0.01#
  GA          13    0.030 (0.020-0.040)
  Total      201
rs1058205
  TT         123    0.018(0.011-0.029)    0.7
  CT          70    0.018(0.010-0.034)
  CC           7    0.029(0.014-0.032)
  Total      200
rs3760728
  CC          90    0.018(0.010-0.034)    0.6
  CG          87    0.020(0.011-0.030)
  GG          24    0.018(0.012-0.024)
  Total      201
rs198972
  CC          99    0.024(0.012-0.034)    0.0007#
  CT          89    0.017 (0.010-0.030)
  TT          14    0.008 (0.004-0.016)
  Total      202
rs198977
  CC         118    0.027 (0.016-0.038)   <0.0005#
  CT          69    0.014(0.010-0.022)
  TT          12    0.004 (0.002-0.005)
  Total      199
rs198978
  GG          83    0.026(0.014-0.038)    <0.0005#
  TG          95    0.017 (0.011-0.029)
  TT          22    0.008 (0.004-0.015)
  Total      200
rs80050017
  GG         169    0.021 (0.012-0.032)   <0.0005#
  TG          27    0.008 (0.006-0.021)
  TT           1    0.004 (0.004-0.004)
 Total       197

                            hK2

 SNP and     Concentration,
 genotype         mg/L            P

rs2271094
  AA         6.8 (3.7-10.1)    0.8
  GA         5.8 (4.3-8.6)
  GG         5.8 (4.0-9.1)
  Total
rs61752561
  GG         5.9 (3.8-9.1)     0.004#
  GA         10.0 (7.7-11.4)
  Total
rs1058205
  TT         5.9 (4.0-9.1)     0.2
  CT         6.2 (3.7-10.1)
  CC         9.0 (5.5-13.9)
  Total
rs3760728
  CC         6.4 (3.5-10.0)    0.6
  CG         5.9 (4.3-9.1)
  GG         4.7 (3.7-8.0)
  Total
rs198972
  CC         7.1 (5.0-11.3)    <0.0005#
  CT         5.6 (3.4-8.2)
  TT         2.9 (1.0-4.9)
  Total
rs198977
  CC         7.7 (5.2-11.2)    <0.0005#
  CT         4.8 (3.2-6.8)
  TT         1.1 (0.8-1.5)
  Total
rs198978
  GG         7.7 (5.2-11.7)    <0.0005#
  TG         5.8 (3.7-8.4)
  TT         2.0 (1.0-4.1)
  Total
rs80050017
  GG         6.8(4.6-10.0)     <0.0005#
  TG         3.0 (1.4-5.9)
  TT         1.0 (1.0-1.0)
 Total

                         PSA

 SNP and               Amount,
 genotype     n          mg           P

rs2271094
  AA         117    1.8 (1.1-2.6)   0.01#
  GA         118    2.1 (1.5-3.1)
  GG          53    2.1 (1.2-3.3)
  Total      288
rs61752561
  GG         273    2.0 (1.3-2.9)   0.3
  GA          18    2.7 (1.4-3.3)
  Total      291
rs1058205
  TT         189    2.1 (1.4-3.1)   0.02#
  CT          92    1.8 (1.1-2.6)
  CC           9    1.6(0.94-2.2)
  Total      290
rs3760728
  CC         125    1.8 (1.2-2.6)   0.1
  CG         121    2.1 (1.4-3.1)
  GG          44    2.1 (1.5-3.0)
  Total      290
rs198972
  CC         136    1.9 (1.1-2.9)   0.7
  CT         133    2.0 (1.3-3.1)
  TT          24    2.0 (1.3-2.4)
  Total      293
rs198977
  CC         173    2.0 (1.4-2.9)   1
  CT          96    2.0 (1.3-2.9)
  TT          18    2.0 (1.1-3.3)
  Total      287
rs198978
  GG         120    1.9 (1.2-2.9)   0.9
  TG         132    2.0 (1.3-3.0)
  TT          36    2.0 (1.3-2.6)
  Total      288
rs80050017
  GG         245    1.9 (1.3-2.9)   0.7
  TG          37    2.2 (1.3-3.1)
  TT           3    0.8 (0.8-4.4)
 Total       285

                         PSA

 SNP and      Concentration,
 genotype         mg/mL           P

rs2271094
  AA         0.57 (0.42-0.80)   0.01#
  GA         0.65 (0.52-0.90)
  GG         0.69 (0.49-0.98)
  Total
rs61752561
  GG         0.63 (0.45-0.88)   0.03#
  GA         0.80 (0.58-1.07)
  Total
rs1058205
  TT         0.67 (0.47-0.92)   0.1
  CT         0.57 (0.44-0.84)
  CC         0.49 (0.34-0.63)
  Total
rs3760728
  CC         0.57 (0.42-0.78)   0.01#
  CG         0.66 (0.52-0.90)
  GG         0.69 (0.51-1.0)
  Total
rs198972
  CC         0.63 (0.48-0.86)   0.8
  CT         0.65 (0.45-0.90)
  TT         0.69 (0.42-0.86)
  Total
rs198977
  CC         0.61 (0.47-0.87)   0.5
  CT         0.65 (0.45-0.95)
  TT         0.67 (0.32-0.82)
  Total
rs198978
  GG         0.63 (0.48-0.87)   1
  TG         0.62 (0.45-0.91)
  TT         0.68 (0.41-0.83)
  Total
rs80050017
  GG         0.64 (0.47-0.88)   0.4
  TG         0.69 (0.43-0.99)
  TT         0.25 (0.23-0.87)
 Total

(a) Amounts and concentrations are median (interquartile range).
Bold type indicates statistical significance.

Note: Bold type indicates statistical significance
indicated with #.

Table 4. Association between SNPs in KLK2 and KLK3
and concentrations of hK2 and PSA in serum. (a)

                        hK2, ng/mL

 SNP and                  Median
 genotype     n          (IQR) (b)           P

rs2271094    297
  AA         121    0.037 (0.026-0.052)   0.4
  GA         122    0.036 (0.028-0.051)
  GG          54    0.035 (0.022-0.045)
rs61752561   301
  GG         282    0.035 (0.025-0.051)   0.3
  GA          19    0.039 (0.035-0.046)
rs1058205    299
  TT         195    0.036 (0.025-0.050)   0.8
  CT          94    0.036 (0.026-0.050)
  CC          10    0.034 (0.025-0.040)
rs3760728    299
  CC         130    0.036 (0.025-0.051)   0.2
  CG         123    0.036 (0.027-0.051)
  GG          46    0.034(0.019-0.042)
rs198972     303
  CC         138    0.040 (0.030-0.052)   <0.0005#
  CT         140    0.035 (0.026-0.050)
  TT          25    0.017 (0.008-0.023)
rs198977     296
  CC         178    0.044 (0.033-0.054)   <0.0005#
  CT         100    0.029 (0.021-0.038)
  TT          18    0.008(0.005-0.014)
rs198978     298
  GG         121    0.043 (0.033-0.054)   <0.0005#
  TG         139    0.034 (0.026-0.050)
  TT          38    0.015 (0.008-0.023)
rs80050017   294
  GG         253    0.039 (0.028-0.051)   <0.0005#
  TG          37    0.023 (0.017-0.029)
  TT           4    0.011 (0.007-0.015)

               tPSA, ng/mL                 fPSA, ng/mL

 SNP and          Median
 genotype         (IQR)           P        Median (IQR)      P

rs2271094
  AA         0.50 (0.33-0.67)   0.8      0.18(0.13-0.26)    0.3
  GA         0.50 (0.37-0.67)            0.21 (0.15-0.27)
  GG         0.52 (0.32-0.67)            0.21 (0.13-0.29)
rs61752561
  GG         0.50 (0.35-0.68)   0.02#    0.19(0.14-0.27)    0.05
  GA         0.42 (0.29-0.51)            0.15(0.12-0.21)
rs1058205
  TT         0.53 (0.37-0.76)   0.001#   0.20 (0.15-0.28)   0.06
  CT         0.41 (0.32-0.58)            0.17(0.12-0.24)
  CC         0.44 (0.30-0.53)            0.19(0.18-0.27)
rs3760728
  CC         0.48 (0.33-0.67)   0.9      0.18(0.13-0.24)    0.3
  CG         0.50 (0.36-0.65)            0.21 (0.14-0.27)
  GG         0.50 (0.32-0.70)            0.20 (0.13-0.30)
rs198972
  CC         0.52 (0.39-0.68)   0.2      0.19(0.13-0.27)    0.6
  CT         0.45 (0.32-0.67)            0.20(0.15-0.27)
  TT         0.52 (0.32-0.60)            0.18(0.13-0.24)
rs198977
  CC         0.51 (0.36-0.68)   0.5      0.19(0.13-0.26)    0.9
  CT         0.45 (0.34-0.66)            0.19(0.14-0.28)
  TT         0.52 (0.30-0.60)            0.18(0.15-0.27)
rs198978
  GG         0.52 (0.36-0.68)   0.5      0.18(0.13-0.26)    0.3
  TG         0.47 (0.35-0.67)            0.21 (0.15-0.27)
  TT         0.48(0.31-0.61)             0.18(0.13-0.27)
rs80050017
  GG         0.50 (0.35-0.67)   0.8      0.19(0.14-0.27)    0.9
  TG         0.47 (0.35-0.66)            0.18(0.15-0.25)
  TT         0.48 (0.33-0.56)            0.20(0.11-0.31)

                 %fPSA

 SNP and       Median
 genotype      (IQR)         P

rs2271094
  AA         40 (32-50)   0.8
  GA         43 (35-49)
  GG         43 (35-52)
rs61752561
  GG         42 (34-50)   0.6
  GA         44 (36-51)
rs1058205
  TT         40 (34-48)   0.01#
  CT         44 (34-51)
  CC         49 (41-61)
rs3760728
  CC         40 (32-49)   0.3
  CG         44 (35-49)
  GG         44 (37-53)
rs198972
  CC         38 (31-47)   <0.0005#
  CT         44 (37-52)
  TT         47 (27-56)
rs198977
  CC         40 (32-48)   0.007#
  CT         45 (37-53)
  TT         44 (32-55)
rs198978
  GG         37 (31-46)   <0.0005#
  TG         45 (38-52)
  TT         46 (32-55)
rs80050017
  GG         41 (35-49)   0.6
  TG         43 (32-53)
  TT         52 (37-55)

(a) Bold type indicates statistical significance.

(b) IQR, interquartile range.

Table 5. Haplotype association with hK2 levels and %fPSA.

                                     Serum

 Haplotype                      %fPSA         log10 hK2, ng/mL
 (rs198972,
 rs198977,
 rs198978,
rs80050017)    Frequency   [beta]      P      [beta]      P

CCGG             0.61       -5.0    <0.0005    0.48    <0.0005
TCTG              0.1       2.8       0.1     -0.048     0.6
TTTG             0.098      5.3      0.007    -0.38    <0.0005
TTTT             0.074      0.62      0.8     -0.60    <0.0005
CTTG             0.056      1.4       0.6     -0.71    <0.0005
All other        0.061      4.2      0.08      0.26     0.02
  haplotypes
  (frequency
  <5% each)

                          Seminal plasma

 Haplotype       log10 hK2, mg     log10 hK2, mg/L
 (rs198972,
 rs198977,
 rs198978,
rs80050017)    [beta]      P      [beta]      P

CCGG            0.19    <0.0005    0.21    <0.0005
TCTG           0.082      0.1     0.057      0.3
TTTG           -0.27    <0.0005   -0.23    <0.0005
TTTT           -0.30    <0.0005   -0.34    <0.0005
CTTG           -0.25    <0.0005   -0.24    <0.0005
All other       0.22     0.002     0.15     0.02
  haplotypes
  (frequency
  <5% each)
COPYRIGHT 2014 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Cancer Diagnostics; Prostate-specific antigen
Author:Savblom, Charlotta; Hallden, Christer; Cronin, Angel M.; Sall, Torbjorn; Savage, Caroline; Vertosick
Publication:Clinical Chemistry
Article Type:Report
Date:Mar 1, 2014
Words:7480
Previous Article:Long-term prognostic value for patients with chronic heart failure of estimated glomerular filtration rate calculated with the new CKD-EPI equations...
Next Article:Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters