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Method: A cohort study was carried out for a year to evaluate the presence of G-to-A transition in 5′-untranslated region of ankylosis human (ANKH) gene in Indian Khatri patients (closely resembling Europeans of primary knee osteoarthritis (OA), residing in Lucknow, India. Results: In the total participants, 25 were Khatri primary knee OA patients (cases) residing in Lucknow and 101 were random blood donors' samples (controls) collected from a blood bank. All were studied for the abovementioned mutation using real-time polymerase chain reaction (RT-PCR). GG genotype was present in 72.3% of controls and 76% of Khatri knee OA patients. The studied G-to-A mutation was found to be positive in 24.8% of controls and 16% of cases, odds ratio (95% confidence interval) being 0.6 (0.19–1.98, P = 0.42). The frequency of AA (D) genotype found around 3% (cases) and 8% (controls) with P value of 0.70. The combined frequency of both homozygous and heterozygous mutation (GA and AA) in the studied population was 28 (27.7%) in controls and 6 (24%) in cases with the odds ratio (OD) ratio of 0.82 (0.29–2.27, P = 0.70). No significant differences were observed at both genotype and allelic level in the distribution of ANKH-4 G-to-A gene polymorphism in studied subjects. Conclusion: This study did not show any significant G to A mutation in the studied subjects. [ABSTRACT FROM AUTHOR]