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For instance, research indicating hypomethylation of genes like DKK1, LGR6, and PRICKLE1 in patients with AS has demonstrated encouraging diagnostic potential, evidenced by moderate area under the curve values in ROC analyses ( 3.4 Inherent biological and methodological challenges In addition to technical and financial obstacles, the clinical application of DNA methylation biomarkers in AS faces considerable biological and methodological challenges.Cellular heterogeneity is a major biological concern