PURPOSE: Genome-wide association studies have shown association of the atonal homolog 7 (ATOH7) and raftlin lipid raft linker 1 (RFTN1) genes with glaucoma-related optic disc parameters. ATOH7 and RFTN1 sequence variations were investigated in patients with primary open-angle glaucoma (POAG) and their relationships with vertical cup-to-disc ratio (VCDR) and central corneal thickness (CCT) were determined. METHODS: In 289 unrelated controls and 142 patients with adult-onset POAG, including 117 with high-tension glaucoma (HTG) and 25 with normal-tension glaucoma (NTG), the single exon of ATOH7 was sequenced by direct sequencing. Additional single-nucleotide polymorphisms (SNP) at upstream ATOH7 (rs1900004 and rs3858145) and an RFTN1 SNP (rs690037) were genotyped. Quantitative trait and disease associations were analyzed by linear and logistic regression respectively, controlling for sex and age. RESULTS: ATOH7 rs61854782 was associated with VCDR (P = 0.004) in controls and RFTN1 rs690037 was associated with CCT in combined POAG (HTG+NTG; P = 0.026). No coding mutation was detected in POAG, and no SNP was associated with POAG (P between 0.441 and 0.996). However, ATOH7 rs3858145 showed significant interaction with RFTN1 rs690037 in NTG and combined POAG (P = 0.026 and 0.013 respectively). ATOH7 rs3858145 GG combined with RFTN1 rs690037 TT conferred risk for glaucoma in HTG, NTG, and combined POAG (odds ratio = 2.11, 8.44, and 2.69, respectively). CONCLUSIONS: Coding mutations of ATOH7 were unlikely to be involved in POAG. But combination of ATOH7 and RFTN1 SNPs increased risk to POAG, indicating their diversified effects in the complex genetics of glaucoma.