Molecular Genetics and Metabolism xxx (2007) xxx–xxx www.elsevier.com/locate/ymgme 1096-7192/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.01.009 ARTICLE IN PRESS Please cite this article in press as: I.J.M. van der Linden et al., Variation and expression of dihydrofolate reductase (DHFR) in relation to spina biWda, Mol. Genet. Metab. (2007), doi:10.1016/j.ymgme.2007.01.009 Variation and expression of dihydrofolate reductase (DHFR) in relation to spina biWda Ivon J.M. van der Linden a , Uyen Nguyen a , Sandra G. Heil a , Barbara Franke b , Suzanne Vloet a , Henkjan Gellekink a,c , Martin den Heijer c,d , Henk J. Blom a,¤ a Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands b The Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands c The Department of Endocrinology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands d The Department of Epidemiology and Biostatistics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Received 17 January 2007; accepted 17 January 2007 Abstract The dihydrofolate reductase (DHFR) enzyme is important for folate availability, folate turnover and DNA synthesis. The 19-bp dele- tion in intron-1 of DHFR has been associated with the risk of having spina biWda aVected oVspring, supposedly by changing DHFR gene expression. A 9-bp repeat in exon 1 of the mutS homolog 3 (MSH3) gene was recently demonstrated to be also located in the 5'UTR of DHFR and may possibly aVect DHFR gene expression as well. We examined the association between these DHFR variants and spina biWda risk and investigated their eVect on DHFR expression. Our study population, consisting of 121 mothers of a spina biWda aVected child, 109 spina biWda patients, 292 control women and 234 pediatric controls was screened for the DHFR 19-bp deletion and the DHFR 9-bp repeat. DHFR gene expression was measured in 66 spina biWda patients, using real-time PCR analysis. In this study population, the DHFR 19-bp del/del genotype was not associated with spina biWda risk in mothers and children (OR: 0.8; 95%CI: 0.4–1.5 and OR: 1.2; 95%CI: 0.6–2.2, respectively) and both the WT/del and the del/del genotype did not aVect DHFR expression relative to the WT/WT geno- type (relative expression D 0.89, p D 0.46 and relative expression D 1.26, p D 0.24, respectively). The DHFR 9-bp repeat was not associated with spina biWda risk in mothers and children. DHFR expression of the 6/6 allele was 73% increased compared to the 3/3 allele, although not signiWcantly (relative expression D 1.73, p D 0.09). We did not Wnd evidence for an eVect of the DHFR 19-bp deletion or 9-bp repeat on spina biWda risk in mothers and children. An eVect of the 6/6 repeat genotype on DHFR expression cannot be ruled out.  2007 Elsevier Inc. All rights reserved. Keywords: DHFR; Spina biWda; Real-time quantitative PCR Introduction The B vitamin folate has been associated with a variety of complications during pregnancy e.g., orofacial clefts [1], congenital heart defects [2] and neural tube defects (NTDs) [3]. The most obvious and extensively studied birth defects in relation to folate are NTDs. Nowadays it is well established that periconceptional folate supplemen- tation reduces the occurrence and recurrence risk of NTDs by 50–70 % [3–5]. The search for the molecular genetic basis of this beneWcial eVect of folic acid however continues [6]. DHFR reduces dihydrofolate (DHF) that is formed during thymidylate (dTMP) synthesis back into tetrahy- drofolate (THF) and hereby stimulates folate turnover, the production of dTMPs and hence DNA synthesis. These processes are all essential for rapid cell growth and division. An adequate folate turnover is also important for proper methylation of DNA, RNA and proteins, since folate donates its methyl group to homocysteine that is converted into S-adenosylmethionine after remethylation to methionine. S-Adenosylmethionine is one of the most * Corresponding author. Fax: +31 24 3668754. E-mail address: h.blom@cukz.umcn.nl (H.J. Blom).