The location of the three independent signals and their association with PCa risk, raise the possibility that these SNPs may be causally related to disease risk or affect the clinical detection of PSA, although this remains to be proven. the rs61752561 and rs266863 are solitary independent SNPs associated with PCa risk (Supplemental Number 1 derived from Locus Explorer). The overall PCa familial relative risk contributed by this region was 0.86 (95% CI 0.76, 0.98), an increase by 1.9 fold partly due to the identification of the two novel signals (3). codes for prostate specific antigen (PSA), a prostatic secretory protein and a serine protease reported to have a bi-directional role involved in both tumor suppression and progression (6, 7). PSA is the most widely used non-invasive biomarker for PCa detection and monitoring (8, 9). However, the level of sensitivity and specificity of the test for clinically significant cancer is definitely poor and thus the use of PSA like a biomarker is still controversial (10, 11). Accordingly, recent research offers focussed on novel interventions to improve the diagnostic specificity such as proPSA, the Prostate Health Index, free/total (F/T) PSA and PSA velocity. Total PSA (tPSA) comprises both the complexed (with inhibitors ?1-antichymotrypsin/ACT/SERPINA3, alpha-2-macroglobulin/A2M, protein C inhibitor/PCI, alpha Ciprofloxacin hydrochloride hydrate 1-protease inhibitor (API)) and free PSA (fPSA) forms. A lower %fPSA has been associated with higher probability of a Ciprofloxacin hydrochloride hydrate analysis of PCa, while measuring F/T PSA is considered more sensitive in discriminating PCa from benign disease (12). Association between SNPs in the region and PCa risk has been reported previously (4, 5, 13C15); however, no consistent correlation has been observed thus far between the cancer-associated SNPs in the gene and protein levels of either PSA or F/T PSA percentage. The location of the three self-employed signals and their association with PCa risk, raise the possibility that these SNPs may be causally related to disease risk or impact the clinical detection of PSA, although this remains to be verified. In an self-employed study, we showed rs17632542, which is in LD with the rs62113212 SNP, to have suggestive functional part in PCa aetiology (4, 16, 17). Here, we hypothesised that the second recently GWAS recognized non-synonymous SNP may influence PSA function and therefore its part in PCa. As a result, our current analysis focussed on characterising the practical consequences of the rs61752561 SNP, having a minor-allele rate of recurrence (MAF)=0.04, that leads to an amino acid substitution, Asp84Asn (chymotrypsin numbering) of PSA. Further we identified whether the rs61752561 SNP correlated with circulating PSA levels and affected the F/T PSA percentage. Materials and Methods Detailed Materials and Methods are explained in the Supplemental file and Supplemental Table 2. Results In silico analysis suggested differences in stability and function for the rs61752561 SNP To analyse the possible impact of the rs61752561 non-synonymous SNP on PSA protein stability, protein function and post-translational control, publicly available tools were used. I-Mutant3.0 and FoldX tools predicted the rs61752561 SNP had the potential to reduce protein stability (Supplemental Table 3). SIFT, PROVEAN and PANTHER, suggested a neutral or a marginal effect on protein function (Supplemental Table 3). NetNGlyc1.0 tool expected that rs61752561 may generate an additional glycosylation site due to generation of the consensus sequence Asn-X-Ser/Thr. rs61752561 introduces an additional glycosylation site To examine the biochemical effects on PSA of Asp84Asn substitution expected by our analysis, we indicated and purified recombinant PSA from Ciprofloxacin hydrochloride hydrate candida cells. Recombinant Asn84 PSA when resolved on SDS PAGE demonstrated a mobility shift (Supplemental Number 2A) consistent with the gain of a glycosylation site also suggested from the NetNGlyC prediction tool. A catalytically inactive mutant Ala195 PSA (Ser195Ala substitution) was also generated. To verify the mobility shift was due Ciprofloxacin hydrochloride hydrate to glycosylation, a deglycosylation assay for the recombinant adult PSA variants crazy type (WT) and Asn84 PSA was performed (Number 1A). For Asn84 PSA, the extra-glycosylated band was Fst also eliminated on PNGase treatment and aligned around 25C26 kDa (similar to the additional PSA proteins), verifying the glycosylation site created for the rs61752561-derived protein variant is an N-glycosylation (Number 1A). Open in a separate window Number 1. Biochemical analysis of the rs62752561 SNP effect on PSA.A) A representative Western blot analysis using anti-PSA shows deglycosylation of mature recombinant PSA protein variants (WT and Asn84) on PNGase treatment, aligning around 25C26 kDa. An extra glycosylated band was observed for the Asn84 PSA only which was eliminated on PNGase treatment (pink arrows) aligning to the PNGase treated WT PSA indicating an N-glycosylation. B) The Tm ideals were determined as the maximum of the first derivative of the melting curves. The fluorescence ideals are on an arbitrary level (AU). C) The 1st derivative ideals (d(fluorescence)/dT) are normalised and given in %, where 100% corresponds to the highest.