Ingested folate needs to be chemically
Ingested folate needs to be chemically modified in order to become a one carbon donor for cell metabolism. Firstly, folate turns to dihydrofolic 5,7-Dichlorokynurenic acid (DHF), it is then converted into tetrahydrofolate (THF) by the dihydrofolate reductase enzyme (DHFR) and finally converted into N5, N10-methylene THF. Decreased DHFR activity can potentially lead to a lower production of the active form of folic acid – the carrier of one carbon unit – possibly compromising a variety of fundamental cell activities such as nucleotide synthesis, DNA and histone methylation, that are essential for cell proliferation and regulation of gene expression [Chen et al., 1984].
Alteration of folate metabolism appears to modify the risk of at least two congenital malformations: neural tube defects (NTDs) and orofacial clefts (OFC). These embryogenesis anomalies occur almost at the same time of development and both involve the embryo midline structures. Not surprisingly, several variants of genes of the folate pathway have been involved in both NTDs and OFC [Blanton et al., 2011, Martinelli et al., 2001, Martinelli et al., 2006, Molloy et al., 2009]. Parle-McDermott and colleagues found that mothers with a 19 bp intron deletion polymorphism mapping in DHFR gene present a reduced risk of having children affected by NTD, and that this variant correlates with increased DHFR mRNA levels [Parle-McDermott et al., 2007].
Until today, there have not been any studies evaluating the possible role of DHFR in OFC. On this occasion, a family-based association study was performed to test if DHFR polymorphisms could influence the risk of NS-CL/P.
Materials and methods
Results A family-based association approach was adopted to test the involvement of DHFR genetic polymorphisms in CL/P aetiology. Genotype frequencies in probands and parents were distributed according to the Hardy–Weinberg equilibrium law and no Mendelian errors were detected. The hypothesis of association between alleles or haplotypes and CL/P was tested with a likelihood ratio approach implemented in the UNPHASED program. Table 1 shows results of allelic association analysis. The rs1677693 provided evidence of association even if at borderline level (P value 0.049). The variant allele appeared to have a protective effect OR = 0.80 (95% C.I. 0.64–0.99). Haplotype analysis of the DHFR locus confirmed association with CL/P with a greater confidence level. Indeed, the overall association test with the combination of rs1677693 and rs1650723 provided a global P value of 0.016. Table 2 reports the association test for each haplotype of this marker combination. The haplotype rs1677693(A)-rs1650723(G) provided the strongest signal of association OR 0.64 (95% C.I. 0.47–0.86) (P value = 0.006). A permutation test, to allow multiple testing corrections over all the 33 association tests carried out in the study, was performed with 1000 replicates. The best P value for association (P = 0.006) was adjusted to P = 0.047, thus confirming the evidence of association between DHFR polymorphisms and the occurrence of NS-CL/P.
Discussion Histone and DNA methylation are epigenetic modifications involved in cell differentiation and gene expression regulation: crucial functions during embryonic development. The rapid growth and the successive fusion of maxillary processes and palatal shelves during early embryogenesis are critical steps in orofacial development, both requiring an effective methyl synthesis and availability. DHFR plays an essential role in folate pathway and in one-carbon metabolism and its activity could be essential in the maintenance of optimal DNA synthesis and epigenetic modifications. In fact, the role of DHFR is to convert dihydrofolate into tetrahydrofolate, a methyl group shuttle required not only for the de novo synthesis of thymidylic acid and certain amino acids, but especially for purine production.