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Abnormal phenotypes in cloned pigs can be partly due to changes in epigenetic modifications such as methylation levels of promoter CpG islands. Neuronatin is an imprinted gene, conserved in human, pig, cattle and mouse, which is expressed exclusively from the paternal allele. Three CpG islands located in the promoter region of the porcine neuronatin gene have the potential to regulate the gene expression by cytosine methylation. To illustrate whether neuronatin was differentially expressed among nuclear transfer macroglossia–positive and nuclear transfer macroglossia–negative pigs and in vitro-fertilized pigs, we detected its expression level by qRT-PCR and further quantified methylation levels by pyrosequencing DNA from the liver. The results showed that neuronatin was expressed at a significantly higher level in livers of nuclear transfer macroglossia-positive pigs compared with normal cloned and in vitro-fertilized pigs. Livers of nuclear transfer macroglossia-positive pigs also had a significantly lower methylation level at CpG island 2 and CpG island 3 in the promoter region.