Genome‐wide pathway analysis of folate‐responsive genes to unravel the pathogenesis of orofacial clefting in man

Abstract

A cleft of the lip with or without the palate (CLP) is a frequent congenital malformation with a heterogeneous etiology, for which folic acid supplementation has a protective effect. To gain more insight into the molecular pathways affected by natural folate, we examined gene expression profiles of cultured B-lymphoblasts from CLP patients before and after the addition of 5-methyltetrahydrofolate (5-mTHF) to the cultures. Immortalized B-lymphoblasts from five children with CLP were cultured in folate-deficient medium for 5 days. 5-mTHF was added to a concentration of 30 nM. Gene expression patterns were then evaluated before and after supplementation using Human Genome U133 Plus 2.0 arrays. Data analysis was performed with Omniviz and the GEPAS analysis suite. Differential genes were categorized into biological pathways with Ingenuity Pathway systems. Differential expression was validated by quantitative RT-PCR. Using supervised clustering, with a false discovery rate <1%, we identified 144 and 409 significantly up-regulated and down-regulated probesets, respectively, after 5-mTHF addition. The regulated genes were involved in a variety of biological pathways, including one carbon pool and cell cycle regulation, biosynthesis of amino acids and DNA/RNA nucleotides, protein processing, apoptosis, and DNA repair. The large variety of the identified folate responsive pathways fits with the modifying role of folate via the methylation pathway. From the present data we may conclude that folate deficiency deranges normal cell development, which might contribute to the development of CLP. The role of these folate responsive genes in CLP development is intriguing and needs further investigation.