Prostate cancer exhibits substantial clinical heterogeneity, with a subset of patients progressing to metastatic disease despite similar tumour grade and treatment. While somatic alterations driving prostate cancer progression are well characterized, the contribution of rare germline variants to metastatic potential and therapeutic response remains incompletely understood. This thesis applies an extreme phenotype approach to identify and functionally characterize rare germline variants enriched in men with high-grade, treatment-naïve localized prostate cancer who later developed metastases, compared with long-term non-progressors. Whole-exome sequencing of an extreme phenotype cohort identified multiple rare germline variants enriched in metastatic cases, including several variants in chromatin regulation and DNA damage repair genes. Two candidate genes were prioritized for functional validation: KDM6B, a histone H3K27 demethylase implicated in transcriptional regulation, and BRCA2, a key mediator of homologous recombination repair and PARP inhibitor response. CRISPR-Cas9 prime editing was used to generate isogenic LNCaP prostate cancer cell lines carrying the KDM6B K973Q variant. Transcriptomic profiling revealed coordinated changes in gene expression, including enrichment of pathways related to cell migration, extracellular matrix interaction, and cell-cell adhesion. Functional assays demonstrated reduced proliferation and clonogenic capacity in edited cells, alongside increased migration and invasion in scratch and Boyden chamber assays. These findings suggest that the KDM6B K973Q variant alters the balance between proliferative and motility-associated cellular programs. In parallel, functional modeling of the BRCA2 I1962T germline variant demonstrated altered sensitivity to PARP inhibition, consistent with partial impairment of DNA repair capacity. A pathogenic BRCA1 truncating variant served as a positive control, validating the experimental framework. Collectively, this work demonstrates that rare germline variants can influence prostate cancer behavior through distinct mechanisms, including transcriptional reprogramming and altered treatment response. By integrating extreme phenotype genetics with precise genome editing and functional assays, this thesis provides a framework for interpreting rare germline variants and highlights their potential relevance to metastatic progression and precision oncology.
Hamideh Sharifi Noghabi (Thu,) studied this question.