Inactivating mutations causing Sotos syndrome have been previously associated with a specific genome-wide DNA methylation (DNAm) pattern. Sotos syndrome is characterized by phenotypic overlap with other overgrowth syndromes, and a definite diagnosis might not be easily reached due to the high prevalence of variants of unknown significance (VoUS) that are identified in patients with a suggestive phenotype. we performed microarray DNAm profiling in a set of 11 individuals with a clinical suspicion of Sotos syndrome and carrying an VoUS or previously unreported variants to solve uncertainty in defining pathogenicity of the observed variants. The impact of the training cohort size on sensitivity and prediction confidence of the classifier was assessed. The Sotos syndrome-specific DNAm signature was validated in six individuals with a clinical diagnosis of Sotos syndrome and carrying pathogenic variants. Applying this approach to the remaining 11 individuals with variants, we succeeded in confirming pathogenicity in eight subjects and excluding the diagnosis of Sotos syndrome in three. The sensitivity and prediction confidence of the classifier based on the different sizes of the training sets did not show substantial differences, though the overall performance was improved by using a data balancing strategy. The present approach solved uncertainty in cases with VoUS, further demonstrating the clinical utility of DNAm profiling.