New 19 bp deletion polymorphism in intron‐1 of dihydrofolate reductase (DHFR): A risk factor for spina bifida acting in mothers during pregnancy?

Abstract

Up to 72% of spina bifida cystica (SB) is preventable by maternal periconceptual folic acid supplementation. The C677T allele of the methylenetetrahydrofolate reductase (MTHFR) gene and some other functional polymorphisms are risk factors for SB in some populations. However, despite extensive study, the genetic risk factors for SB are incompletely understood. Polymorphic alleles that diminish bioavailability of reduced folate in the mother during pregnancy could contribute to SB in her fetus, acting in the mother as teratogenic alleles. We recently discovered a polymorphic 19 bp deletion allele (frequency 0.45) within intron-1 of dihydrofolate reductase (DHFR) that is a good candidate for such a genetic factor. Since there is precedence for intron-1 regulatory elements and the deletion allele removes a potential Sp1 transcription factor binding site, we hypothesized that the deletion allele could be functional and act in SB mothers to increase the risk of SB in her fetus. We found that homozygosity for this deletion allele was significantly more frequent in SB mothers, but not in SB fathers or patients, compared with controls and was associated with a significantly increased odds ratio (OR) (2.035) of being an SB mother compared with other genotypes. Genotype distribution obeyed the constraints of Hardy-Weinberg equilibrium in controls, SB patients and fathers, but not in SB mothers. If confirmed, these findings could lead to improved forms of folate supplementation for pregnancy. About half of dietary folates and all of folic acid supplements must be reduced by DHFR to be available for mother and fetus. Reduced folates could be preferable for supplements during pregnancy to prevent SB.

Molecular genetic analysis of the human dihydrofolate reductase gene: relation with plasma total homocysteine, serum and red blood cell folate levels

“…Association between DHFR variants and tHcy and folate Because the 9-bp repeat is located in the promoter region and the 19-bp deletion is thought to affect gene expression, 20 we only screened these potentially most important variants for their effect on tHcy, serum and RBC folate in our study population. As shown in Table 3, the 9-bp repeat was not associated with tHcy in our study population.…”

“…17 In addition, inhibition of DHFR by MTX and the antibiotic trimethoprim were shown to increase tHcy levels by 50%, 18,19 suggesting that DHFR dysfunction owing to genetic variants may affect tHcy as well. Recently, Johnson et al 20 described a 19-bp deletion that may affect gene expression.…”

“…Newly identified variants, and the 19-bp deletion in intron 1, 20 were assessed for their effect on tHcy and serum and red blood cell (RBC) folate concentrations in population-based controls.…”

“…20 In short, the PCR reaction mix (25 ml) contained 75 ng of genomic DNA, 1 Â Ampli Taq gold buffer, 1.5 mmol/l MgCl 2 , 250 mmol/l dNTPs, 1 mmol/l of each primers (FW1: 5 0 -CCACGGTCGGG GTACCTGCC-3 0 , FW2: 5 0 -ACGGTCGGGGTGGCCGACTC-3 0 and RV: 5 0 -AAAAGGGGAATCCAGTCGG-3 0 ), 1 mol/l betaine and 0.5 U Taq polymerase. PCR conditions were as follows: initial denaturation of 4 min at 941C, 35 cycles of 941C/60 s, 581C/60 s, 721C/60 s, and a final extension of 7 min at 721C.…”

Investigation of inter-individual variability of the one-carbon folate pathway: a bioinformatic and genetic review

“…40 There are three non-coding polymorphisms (in the 5 0 upstream region, intron 1 and 3 0 -UTR) that have been the subject of a number of association studies (Table 3). The 19-bp deletion polymorphism in intron 1 in the DHFR gene has been associated with a number of diseases 41 (Table 3). Individuals homozygous for the deletion (À/À) have lower mean plasma total homocysteine than those homozygous for the insertion ( þ / þ ), 40 with the (À/À) individuals also having been shown to have 4.8-fold greater DHFR mRNA expression in lymphocytes compared with ( þ / þ ) carriers.…”