Peptide fingerprinting of folate-responsive proteins in human B lymphoblasts and orofacial clefting.

BACKGROUND: Maternal periconceptional use of folic acid contributes to the prevention of neural crest-related congenital malformations including orofacial clefts. The underlying biological pathways affected by folic acid,however, are still not clarified. In an explorative study, we identify folate-responsive proteins and pathways by advanced proteomic techniques and their possible role in orofacial development in young children. MATERIALS AND METHODS: At 15 months of age, we obtained B lymphoblasts from 10 children with and 10 children without an orofacial cleft. Folate-responsive protein expression was determined in folate-free B-lymphoblast cultures, supplemented with 5-methyltetrahydrofolate to reach the target concentration 30 nM. Folate-associated differences of peptide and protein expressions were assessed by analysing samples before and after folate addition. Samples were trypsin digested and measured by nano-liquid chromatography coupled online to a LTQ-Orbitrap mass spectrometer. Significantly differentiating peptides were determined using a McNemar's test, and correlations with proteins and existing pathways were visualized using Ingenuity Pathway Analysis. RESULTS: We found 39 folate-responsive peptides that were assigned to 30 proteins. Those proteins consisted of histones, ribosomal and heat shock proteins (HSP), and proteins involved in antioxidant reactions, cytoskeleton,glycolysis, energy production, protein processing, signal transduction and translation. CONCLUSIONS: Histones, ribosomal and HSP were mainly found in the case group, and we confirm that almost 60% of these proteins were also found in a subset of the samples in our previous study using microarray on folate-responsive gene expression. The proteins were compared with known biological pathways and matched with recent relevant literature.

Comparable levels of folate-induced aneusomy in B-lymphoblasts from oral-cleft patients and controls.

BACKGROUND: Peri-conceptional use of folic acid contributes to protection against congenital malformations, such as neural tube defects and cleft lip with or without cleft palate (CL/P). Previous studies showed that low folate levels cause DNA damage, leading to chromosomal instability and aneusomy. This study seeks to confirm this finding and investigates whether the in vitro sensitivity towards aneusomy of chromosome 17 and 21 in the folate-deficient state differs between CL/P patients and controls. METHODS: Epstein-Barr virus-immortalized B-lymphoblasts derived from 15 CL/P children and 15 controls, were cultured in medium with high and low concentrations - approximately 40nM and 5nM - of 5-methyltetrahydrofolate, respectively. Fluorescence in situ hybridization was used to detect specific fluorescence signals for chromosomes 17 and 21. RESULTS: A significant increase in aneusomy of chromosomes 17 (2.3% vs 7.6%; p ≤ 0.001) and 21 (2.5% vs 7.0%; p ≤ 0.001) was observed after 10 days of culturing in low folate. These results were comparable in cell lines from patients and controls. Interestingly, for chromosome 17 the folate deficiency mainly resulted in an increase of monosomy (6%, p ≤ 0.001), while for chromosome 21 the increase of trisomy was larger (4.9%, p ≤ 0.001). CONCLUSIONS: These data suggest that folate deficiency is a significant risk factor in the development of aneusomy and may affect the distribution of chromosomes during cell division. The comparable aneusomy frequencies in CL/P and in controls suggest that other folate-related processes are involved in the pathogenesis of CL/P, and additional investigations are needed to identify the causal mechanisms.

Maternal medication use, carriership of the ABCB1 3435C > T polymorphism and the risk of a child with cleft lip with or without cleft palate.

Gene-environment interactions in the periconceptional period play an increasing role in the pathogenesis of birth defects, including cleft lip and/or cleft palate (CL/P). The P-glycoprotein, encoded by the ABCB1 gene, is suggested to protect the developing embryo from medication and other xenobiotic exposures. Furthermore, maternal medication use during early pregnancy is a significant risk factor for CL/P offspring. Therefore, the aim of this study is to investigate the association between the maternal and child's functional ABCB1 3435C > T polymorphism, periconceptional medication exposure, and the risk of a child with CL/P. A case-control study was performed among 175 mothers and 98 of their children with CL/P and 83 control mothers and their 65 children. Information on medication and folic acid use was collected. Mothers carrying the 3435TT genotype and using medication showed a 6.2-fold (95% CI = 1.6-24.2) increased risk of having a child with CL/P compared to mothers carrying the 3435CC genotype and not using medication. Periconceptional folic acid use reduced this risk by approximately 30% (OR = 3.9, 95% CI = 0.9-18.0). Mothers carrying the 3435TT genotype, using medication and not taking folic acid showed the highest risk estimate (OR = 19.2, 95% CI = 1.0-369.2). These data suggest that mothers who carry the ABCB1 3435C > T polymorphism are at significantly increased risk for having offspring with CL/P, especially mothers using medication in the periconceptional period.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.