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Updated: Jan 28, 2026
Methylated DNA Immunoprecipitation
Published on: January 2, 2009
A comparison of DNA methylation in newborn blood samples from infants with and without orofacial clefts
Zongli Xu1, Rolv T Lie2,3, Allen J Wilcox1
1Epidemiology Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.
Insights
This study investigated DNA methylation in infants with orofacial clefts, finding limited evidence for widespread epigenetic differences. While some specific DNA methylation sites and regions were identified, they do not fully support previous reports.
Area of Science:
- Genetics and Epigenetics
- Congenital Disorders
- Developmental Biology
Background:
- Orofacial clefts are common congenital birth defects with unknown biological mechanisms.
- Clefts are believed to result from complex genetic, environmental, and epigenetic factors.
Purpose of the Study:
- To investigate DNA methylation profiles in infants with orofacial clefts.
- To identify differentially methylated CpGs and regions associated with cleft subtypes.
Main Methods:
- Epigenome-wide association analyses (EWAS) were performed on blood samples from 747 infants.
- DNA methylation profiles of over 450,000 CpGs were analyzed.
- Comparisons were made between controls and various cleft case groups, including subtypes.
Main Results:
- No significant associations were found at the Bonferroni P value threshold of 10^-7 when comparing controls to individual cleft subtypes.
- Two significantly differentially methylated CpGs were identified after pooling case groups: cg09696939 (near BICC1) for cleft lip and palate (CLP)+cleft lip only (CLO), and cg26985354 (in CLASRP) for CLP+cleft palate only (CPO).
- A total of 56 significant differentially methylated regions (DMRs) were identified when comparing controls to individual cleft subtypes, with only one DMR shared across all three cleft groups. Numerous DMRs were also found in combined case group and case-case comparisons.
Conclusions:
- The study identified specific CpGs and DMRs associated with orofacial cleft subtypes.
- While some evidence suggests a role for DNA methylation in the etiology of orofacial clefts, widespread differences in blood DNA methylation were not supported.
- Findings contrast with previous reports suggesting broader epigenetic alterations in infants with facial clefts.
Background:
Isolated orofacial clefts are among the most common congenital birth defects. Although the underlying biological mechanisms remain largely unknown, clefts are thought to be complex disorders influenced by genetic, environmental, and potentially epigenetic factors.
Methods:
In blood samples from 2- to 3-day-old infants (n = 747) collected in a nationwide population-based study of orofacial clefts in Norway, we measured DNA methylation profiles for more than 450,000 CpGs and then conducted epigenome-wide association analyses (EWAS). We tested methylation profile difference at each CpG between controls (n = 436) and each of the cleft subtypes (92 cleft lip only, CLO; 84 cleft palate only, CPO; 132 cleft lip and palate, CLP). We also compared controls to various combinations of case groups and compared case subtypes to each other. Finally, using the EWAS results, we searched for larger differentially methylated regions (DMRs) associated with orofacial clefts.
Results:
In EWAS comparing controls to individual cleft subtypes, we found no significant associations at a Bonferroni P value threshold of 10-7. After pooling case groups, we found two significantly differentially methylated CpGs: cg09696939 near gene BICC1 is associated with CLO+CLP (P = 9.58 × 10-8); cg26985354 in gene CLASRP is associated with CPO+CLP (P = 7.38 × 10-8). In DMR analysis, we identified a total of 56 significant regions when comparing controls to individual cleft subtypes (10 for CLO, 6 for CPO, 41 for CLP). Only one DMR is shared among the three cleft groups. In combined case group analysis, we found 26 DMRs for CLP+CLO, 31 for CLP+CPO, and 37 when all subtypes are combined. Finally, in case-case comparisons of subtypes, we identified 10 DMRs when comparing CLP to CPO, 9 in CLP compared to CLO, and 13 in CLP compared to CPO.
Conclusions:
We identified two individual CpGs and multiple DMRs that differ between controls and cleft case subtypes. Although we find some evidence for the possible role of DNA methylation in etiology of orofacial clefts, our study does not support previous reports of widespread differences in blood DNA methylation between babies with and without facial clefts.
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