Identification of functional non-coding variants associated with orofacial cleft.

Area of Science:

• Genetics
• Developmental Biology
• Genomic Medicine

Background:

• Oral facial cleft (OFC) is a common birth defect with complex genetic underpinnings.
• Genome-wide association studies (GWAS) have identified numerous genomic loci associated with OFC, but the functional variants remain largely unknown.
• Effector genes implicated in OFC are often expressed in embryonic oral epithelium.

Purpose of the Study:

• To identify functional single nucleotide polymorphisms (SNPs) associated with oral facial clefts (OFC) at known risk loci.
• To investigate the impact of these SNPs on enhancer activity and gene expression in relevant cellular models.
• To connect genetic variations in OFC to specific molecular mechanisms of pathogenesis.

Main Methods:

• Massively parallel reporter assays (MPRA) were used to screen for SNPs with allele-specific enhancer activity in a fetal oral epithelial cell line.
• Chromatin-mark data was utilized to filter candidate SNPs, prioritizing those located in active enhancers.
• Engineered induced pluripotent stem cells (iPSCs) were differentiated into embryonic oral epithelium to assess the functional impact of top candidate SNPs on gene expression and transcription factor binding.

Main Results:

• Several SNPs demonstrated allele-specific effects on enhancer activity in oral epithelial cells.
• SNPs near FOXE1, IRF6, MAFB, TFAP2A, and TP63 were validated as potential functional variants.
• In iPSC-derived oral epithelium, specific SNPs near IRF6 and FOXE1 altered effector gene expression and transcription factor binding.
• Conditional GWAS analyses indicated that two functional SNPs near IRF6 significantly contribute to cleft lip with or without cleft palate (CL/P) risk.

Conclusions:

• This study successfully identified functional SNPs associated with OFC, demonstrating allele-specific effects on gene regulation in oral epithelium.
• The findings provide a mechanistic link between genetic variations and the pathogenesis of OFC, particularly CL/P.
• The identified functional variants near IRF6 are major contributors to CL/P risk, offering targets for future research and potential therapeutic strategies.