A dihydrofolate reductase 2 (DHFR2) variant is associated with risk of neural tube defects in an Irish cohort but not in a United Kingdom cohort

Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, Bethesda, Maryland School of Biotechnology, Dublin City University, Dublin, Ireland Department of Clinical Medicine, School of Medicine, Trinity College, Dublin, Ireland Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California Division of Intramural Population Health Research, Eunice Kennedy Shriver, National Institute for Child Health and Human Development, Bethesda, Maryland

NTD occurrence rates vary among populations with a range of 6-60 per 10,000 pregnancies worldwide (Molloy, Pangilinan, & Brody, 2017). Because NTDs are relatively common, and can be severe or fatal defects, preventing folate-responsive defects is a major public health priority (Bailey, West Jr., & Black, 2015). Voluntary or mandatory folic acid fortification programs have been implemented across the globe, with variable success (Khoshnood et al., 2015).
Given such wide public health implications, there is a need to further understand the mechanism by which folate-responsive NTDs occur.
Although NTDs are known to have both nutritional and genetic risk factors (Bailey et al., 2015), much of the genetic component remains to be discovered. An unbiased genome-wide association study is an appealing approach but has not yet been performed due to the difficulty of obtaining a sufficient number of affected participants. In contrast, candidate genes studies are feasible and have some potential advantages compared with a GWAS. First, because variant selection can use linkage disequilibrium to inform the selection of variants, a tailored approach can provide more complete variant coverage of the gene(s) in question. Second, querying this targeted search space may identify small but real association signals that would be lost in the multiple-test correction of a full-scale GWAS.
Genetic investigations have focused on genes that either metabolize or transport folate due to the well-established protective effect of preconceptional use of folic acid supplements (Czeizel & Dudas, 1992;MRC Vitamin Study Research Group, 1991). Many genetic association studies have considered variants within folate pathway genes as candidate risk factors in the search of genetic variants that may increase the population and an individual's risk of an NTD (Molloy et al., 2017).
We report here our evaluation of the folate retrogene known as dihydrofolate folate reductase 2 (DHFR2, formerly DHFRL1) (McEntee et al., 2011) as a candidate gene for NTD risk. We previously reported that a 19 bp intronic allele of dihydrofolate reductase (DHFR) may decrease risk of NTDs (Parle-McDermott et al., 2007). DHFR mediates the entry of dietary folic acid into folate metabolism. Owing to this key role, DHFR has been extensively studied as a drug target (methotrexate) and a selection tool in cell culture (McEntee et al., 2011). In addition to DHFR, humans and other primates have acquired an a second gene family member, making DHFR2 a compelling candidate for harboring variation that might alter NTD risk. Moreover, as a relatively newly identified gene, DHFR2 has not been considered as a candidate in many human disease contexts.
Our initial cohort in the investigation of DHFR2 consisted of 595 trio families that included an affected case and one or both parents plus a control sample of 1,000 individuals. This cohort has been described previously (Brody et al., 2002;Pangilinan et al., 2014;Shields et al., 1999). In brief, NTD cases and their parents were recruited throughout Ireland (1993Ireland ( -2004, and controls were randomly selected from women attending their first prenatal visit in Dublin (1986)(1987)(1988)(1989)(1990). We successfully genotyped seven single nucleotide polymorphisms (SNPs) across the DHFR2 gene region by detection of allele-specific primer extension using matrix-assisted laser desorption/ confidence intervals for mother-control, father-control, and casecontrol comparisons. While we did not observe any significant effect of the homozygous genotype for any of the seven candidate SNPs (data not shown; all genotype data is available in the Supporting Information Data File), we did observe a statistically significant association in the TDT analyses (440 complete trios). A significant overtransmission from parents to affected offspring of the more common allele was observed in three SNPs (rs7645522, rs10454213, and rs17855824) that all share high LD (D 0 = 1, r 2 ≥ .995) with each other (Table 1, Figure 1) and should be considered a single association signal. This indicates that the major allele, at a frequency of 0.87 for all three SNPs, is associated with an increase in the case risk of an NTD.
We further investigated this statistically significant DHFR2 SNP signal by considering whether it (rs10454213 and rs17855824) or other SNPs in the region (rs33944211, rs11927165, and rs6765621) could contribute to a quantitative trait of relevance to folate one carbon metabolism, that is, have an influence on circulating folate biomarkers in a cohort of young, healthy Irish university students known as the Trinity Student Study (Brosnan et al., 2018;Desch et al., 2013;Molloy et al., 2016;Shane et al., 2018). An additive model of linear We also tested whether the DHFR2 SNP rs17855824 had an impact on the gene expression level of DHFR2 mRNA (eQTL) using publicly available RNA sequencing data. The high LD with the other two NTD associated SNPs meant that it was not necessary to test all three. The RPKM (reads per kilobase of transcript per million reads mapped) for DHFR2 and genotypes for SNP rs17855824 were downloaded from the Geuvadis RNA Sequencing project (Lappalainen et al., 2013 We next used a second UK cohort to assess whether a similar significant TDT association would be replicated. This cohort has been previously described (Pangilinan et al., 2010 show a significant over-transmission of the "A" allele as observed in the Irish cohort (Table 2). In fact, a nonsignificant over-transmission of the minor allele, that is, the "G" was observed for both SNPs in the UK cohort. In the absence of a significant difference in allele transmission in the UK cohort, we conclude that this SNP does not contribute to NTD risk in this UK cohort, or the cohort was underpowered (354 complete UK trios tested compared with 440 complete Irish trios) to detect a significant difference in allele transmission.
In summary, our consideration of the human DHFR2 gene as a candidate genetic risk factor for NTD revealed mixed results. We observed an association signal in an Irish cohort but failed to replicate this in a separate, smaller UK cohort. We also assessed DHFR2 as a potential QTL for biomarkers of folate one carbon metabolism and as a eQTL. Upon correction for multiple tests, these analyses did not show any significant correlations with serum folate, red cell folate, plasma total homocysteine, plasma formate, or tissue mRNA expression levels. This is in contrast to the MTHFR 677C>T variant which was previously found to be the major genetic modifier of these biomarkers in the same cohort (Brosnan et al., 2018;Shane et al., 2018).
Research is currently ongoing to elucidate the functional role of DHFR2 and its confirmation as a genetic modifier of NTD risk requires replication in another cohort. While periconceptional folic acid supple- NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from: the GTEx Portal on 07/08/2020.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. All genotype data is available in the Supporting Information Data File.