• cleft palate;
  • CpG methylation;
  • craniofacial;
  • epigenetics;
  • extra-cellular matrix;
  • promoter microarray;
  • pyrosequencing;
  • secondary palate;
  • Wnt signaling


Environmental factors contribute to the etiology of cleft palate (CP). Identification of genes that are methylated during development of the secondary palate will contribute to a better understanding of the gene-environment link contributing to CP.


Genomic DNA fragments from secondary palate tissue from gestational days (GDs) 12 to 14 were subjected to Selective Enrichment of Methylated DNA (SEMD) and used to probe NimbleGen 2.1M mouse promoter arrays. Input (control) and SEMD samples were labeled with Cy3 and Cy5, respectively, and used for array hybridization (three arrays per GD). Data were analyzed using the Bioconductor package Ringo. Gene methylation was verified by pyrosequencing analysis and expression by quantitative real-time PCR.


A total of 5577 methylated genes were identified during palate development: (1) 74% of genes were methylated on all three GDs; (2) CpG islands accounted for only 30% of methylated regions of interest (MRIs); (3) location of MRIs was more often observed in gene bodies (73%) than in promoters; (4) evaluation of MRIs on GDs 12–14 revealed no significant differentially methylated regions; (5) DAVID analysis of MRIs revealed that the cadherin and Wnt signaling pathways, as well as pathways involved in proteoglycan synthesis, were significantly enriched for methylated genes.


Our prior studies identified differentially expressed mRNAs and microRNAs in the developing palate. The current study complements these studies by identifying genes whose expression may be altered as a result of DNA methylation. Birth Defects Research (Part A) 97:171–186, 2013. © 2013 Wiley Periodicals, Inc.