Genetic Variation in KLK2 and KLK3 Is Associated with Concentrations of hK2 and PSA in Serum and Seminal Plasma in Young Men Charlotta Sävblom, 1 Christer Hallde ´ n, 2 Angel M. Cronin, 3 Torbjörn Säll, 4 Caroline Savage, 3 Emily A. Vertosick, 3 Robert J. Klein, 5* Aleksander Giwercman, 6 and Hans Lilja 1,7,8,9 BACKGROUND: Genetic variants in KLK2 and KLK3 have been associated with increased serum concentrations of their encoded proteins, human kallikrein-related pepti- dase 2 (hK2) and prostate-specific antigen (PSA), and with prostate cancer in older men. Low PSA concentra- tions in seminal plasma (SP) have been associated with low sperm motility. To evaluate whether KLK2 and KLK3 genetic variants affect physiological prostatic secretion, we studied the association of SNPs with hK2 and PSA concentrations in SP 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. We mea- sured PSA and hK2 in SP and serum using immuno- fluorometric assays. The association of genotype fre- quencies with hK2 and PSA concentrations was tested with the Kruskal–Wallis test. RESULTS: Four KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017) were strongly associated with hK2 concentrations in SP and serum, with individuals ho- mozygous for the major alleles having 3- to 7-fold higher concentrations than the intermediate concentrations found in other homozygotes and heterozygotes (all P  0.001). Three of these SNPs were significantly associated with percentage of free PSA (%fPSA) in serum (all P  0.007). Three KLK3 SNPs showed associations with PSA in SP, and the rs1058205 SNP was associated with total PSA in serum (P = 0.001) and %fPSA (P = 0.015). CONCLUSIONS: Associations observed in young, healthy men between the SP and serum concentrations of hK2 and PSA and several genetic variants in KLK2 and KLK3 could be useful to refine models of PSA cutoff values in prostate cancer testing. © 2013 American Association for Clinical Chemistry 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 sem- inal plasma. PSA is responsible for the rapid degrada- tion of the seminal vesicle–secreted proteins semeno- gelin I and II (4). Semenogelin I inhibits the motility of intact and demembraned spermatozoa and partici- pates in the capacitation of sperm by blocking motility immediately after ejaculation (5). Thus, PSA is neces- sary 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 activa- tor of PSA (7). Catalytic hK2 can also cleave semeno- gelin I and II (8). The majority of hK2 in seminal plasma is noncata- lytic and bound in complex with protein C inhibitor {encoded by SERPINA5 [serpin peptidase inhibitor, clade A (-1 antiproteinase, antitrypsin), member 5]}, 11 whereas the majority of PSA is in free, catalyti- cally active form (fPSA), with 5% of PSA being complexed to SERPIN-type antiproteases (cPSA) (9). The sum of fPSA and cPSA closely corresponds 1 Department of Laboratory Medicine, Division of Clinical Chemistry, and 6 Re- productive Medicine Centre, Lund University, Skåne University Hospital, Malmö, Sweden; 2 Biomedicine, Kristianstad University, Kristianstad, Sweden; 3 Depart- ment 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. * Address correspondence to this author at: Clinical Genetics Service, Department of Medicine, Program in Cancer Biology and Genetics, Memorial Sloan- Kettering 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 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 (-1 antiprotei- nase, antitrypsin), member 5; KLK2, kallikrein-related peptidase 2 encoding hK2; KLK3, kallikrein-related peptidase 3 encoding PSA. Clinical Chemistry 60:3 000 – 000 (2014) Cancer Diagnostics 1 http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2013.211219 The latest version is at Papers in Press. Published November 22, 2013 as doi:10.1373/clinchem.2013.211219 Copyright (C) 2013 by The American Association for Clinical Chemistry