High-Resolution Melting PCR-Based SNP Germline Genotyping of KLK3, JAZF1, and HNF1B in Prostate Cancer and Benign...

Background/Objectives: Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are highly prevalent among African men. While single-nucleotide polymorphisms (SNPs) in KLK3, HNF1B, and JAZF1 are established risk markers in Western cohorts, their specific allele frequencies and diagnostic utility remain poorly characterized in African populations. This study evaluated the diagnostic accuracy, sensitivity, and cost-effectiveness of High-Resolution Melting (HRM) PCR for germline SNP genotyping in a cohort of Nigerian men. Methods: Peripheral blood buffy coat DNA was extracted from 31 male participants attending the urology clinic at Lagos University Teaching Hospital, classified into PCa (n = 13), BPH (n = 12), and healthy controls (n = 6). Real-time PCR and HRM analysis were optimized to genotype three specific risk variants: rs2735839 (KLK3), rs4430796 (HNF1B), and rs10486567 (JAZF1). Allelic discrimination was validated using sequence-verified, synthetic positive controls for homozygous wild-type and mutant variants. HRM analysis generated reproducible amplification and melting profiles that enabled clear discrimination of wild-type, heterozygous, and variant genotypes for all three loci. Results: The risk-associated alleles of KLK3 (G), HNF1B (C), and JAZF1 (T) occurred more frequently among patients with prostate cancer than among those with benign prostatic hyperplasia. Clinically, prostate cancer patients also presented with markedly higher PSA concentrations, older age at diagnosis, and higher Gleason scores, consistent with more aggressive disease. The optimized HRM workflow required less than two hours from amplification to genotype assignment and eliminated the need for post-PCR processing, making it suitable for routine molecular testing in resource-constrained laboratories. Conclusions: HRM-based genotyping of KLK3 (rs2735839), HNF1B (rs4430796), and JAZF1 (rs10486567) provides a highly reproducible, rapid, and low-cost molecular tool for mapping prostate disease risk variants. Because it requires no post-PCR processing, this closed-tube HRM workflow is ideally suited for resource-limited clinical settings to enhance early risk stratification and differentiate malignant PCa from benign BPH.