PURPOSE: Although primary congenital glaucoma (PCG)-associated CYP1B1 mutations in the heterozygous state have been evaluated for association with primary open-angle glaucoma (POAG) in several small studies, their contribution to the occurrence of POAG is still controversial. The present study was conducted to determine whether heterozygous functionally characterized CYP1B1 mutations are associated with the disease in a large cohort of German patients with POAG. METHODS: The frequency of CYP1B1 variants on direct sequencing of the entire coding region was compared in 399 unrelated German patients with POAG (270, POAG; 47, JOAG; and 82, NTG) and 376 control subjects without any signs of glaucoma on ophthalmic examination. In vitro functional assays were performed and relative enzymatic activity of the CYP1B1 variants embedded in their respective background haplotypes and not previously unambiguously classified were determined, to assess their possible causative role. RESULTS: Apart from known polymorphic variants, 11 amino acid substitutions in CYP1B1 reported before, both in PCG and POAG cases, were identified. After in vitro functional assay, variants P52L and R368H showed marked reduction of activity, confirming their role as loss-of-function mutations similar to previously determined variants G61E, N203S, and G329V. In contrast, variants G168D, A443G, and A465V showed no relevant effects and were thus classified as polymorphisms. Overall, seven functionally impaired variants were present in 13 (3.6%) patients and in 1 (0.2%) control subject (P = 0.002, OR = 5.4). Reanalysis of previous studies reporting CYP1B1 mutations in patients with POAG based on updated functional validation showed a significant excess of carriers among patients compared to controls (OR = 3.85; P = 2.3 x 10(-7)). CONCLUSIONS: Heterozygous CYP1B1 mutations with absent or reduced relative enzymatic activity can be considered a risk factor for POAG.