The Folate Cycle plays an important role in neurological development, as demonstrated by the link between folate intake and risk for NTDs. SHMT1 and SHMT2 represent an important component of the Folate Cycle (see ). Substrates and products for these enzymes are neurotransmitter receptor modulators (glycine and serine), inhibitory neurotransmitters (glycine), and precursors for monoamine neurotransmitter biosynthesis (5,10-methylene-THF). The SHMT enzymes also provide single carbon units that can be used for homocysteine remethylation (Miller 2008
). Genetic variation in SHMT1
has been associated with a wide variety of human phenotypes, including risk for neural tube defects (Relton et al. 2004
, Heil et al. 2001
), childhood acute leukemia (Vijayakrishnan & Houlston 2010
), rectal carcinoma (Komlosi et al. 2010
), and prostate cancer (Collin et al. 2009
). Most of those studies focused on the SHMT1
Leu474Phe variant allozyme. However, we failed to detect significant differences in either enzyme activity or protein quantity, as compared to WT, for any of the 9 nsSNPs in SHMT2
or the 4 nsSNPs in SHMT1
, including Leu474Phe (). Therefore, our results suggest that these 13 nsSNPs, including that encoding Lys474Phe, might not themselves be biologically relevant, a conclusion that agrees, in part, with previous substrate kinetic studies (Fu et al. 2005
). Obviously, we can not rule out the possibility that these variants might have a significant effect on other activities catalyzed by SHMT1 that we did not measure (Schirch & Szebenyi 2005
Although none of the nsSNPs that we observed was associated with significant functional consequences, we did identify very strong relationships between SHMT1 mRNA expression in LCLs and SNP genotypes (). The strongest associations were observed for rs669340 in intron 1 (p = 2.2E-14) and rs7207306 in intron 5 (p = 5.4E-13). It might be useful to point out that rs1979277, which encodes the Leu474Phe variant allozyme, was also strongly associated mRNA expression in the LCLs (p-value=6.29E-06), and that it is in LD with rs7207306, which might explain, in part, the clinical associations reported previously for this SNP (Collin et al. 2009
, Heil et al. 2001
, Komlosi et al., Relton et al. 2004
, Vijayakrishnan & Houlston 2010
). However, rs669340 and rs7207306 in introns 1 and 5, respectively, were not in LD with one another (r2
=0.03–0.20), suggesting there might be two or more SNPs that regulate SHMT1 expression. In an attempt to validate and extend associations that we observed in LCLs at the mRNA level, with a clear understanding that transcription regulation is tissue-specific, we also assayed variation in SHMT1 protein concentrations in cytosolic preparations obtained from human liver biopsy samples. Of the SNPs that were genotyped, rs669340 in intron 1 showed the strongest association with protein quantity (p=1.18E-05) ( and ). That association remained significant even after correcting for multiple comparisons. However, the intron 5 SNP (rs7207306) was not significantly associated with hepatic cytosol SHMT1 protein levels–serving to emphasize the tissue-specific nature of transcription regulation.
In an attempt to identify functional candidates that might regulate transcription and/or translation, we considered all SNPs with strong associations to either SHMT1 mRNA expression in LCLs and/or protein expression in human hepatic tissue as possible candidates for further functional follow-up. Although rs7207306 and rs669340 had the lowest p-values, 14 additional SNPs had significant associations with SHMT1 mRNA expression (p<1E-10) that could potentially be functional (). Therefore, we studied the function of rs7207306 and rs669340, the SNPs with the lowest p-values, as well as two promoter SNPs, rs638416 and rs643333. The rs638416 SNP is located (−119) bp from the site of transcription initiation and is in strong LD with rs669340, while rs643333 is located (−283) bp from the transcription start site and is in LD with rs7207306. These two SNPs, as well as approximately 1 kb of 5′-flanking sequence, a region that we had resequenced, were cloned into luciferase reporter gene constructs and expressed in HepG2 cells. Both rs638416 and rs643333 map to a region of SHMT1
that contains many experimentally characterized transcription factor binding sites based on the “ENCODE Integrated Regulation” track on the UCSC genome browser (http://genome.ucsc.edu
). For example, the rs638416 variant allele disrupts a WT1 transcription factor binding site, while the variant allele for rs643333 introduces a putative SRF transcription factor binding site (http://gene-regulation.com/pub/programs/alibaba2/index.html
). The results of our dual luciferase assays suggested that rs638416 might be functional (). However, rs643333 did not appear to be functional in HepG2 cells, although we cannot rule out the possibility that the effect of this SNP on SHMT1 transcription is specific to LCLs. In addition, the two most significant SNPs associated with mRNA expression, rs7207306 and rs669340, also failed to show a significant effect on transcriptional activity as determined by reporter gene assay (Supplementary Fig. 2
), although we cannot exclude the possibility that these variants might be functional through other mechanisms (e.g., epigenetics).
Finally, in an attempt to take a broader approach that extended beyond SHMT1 and SHMT2, we compared previously published mRNA expression data for the same LCLs that were used in our studies as well as previously published hepatic protein expression data for the same hepatic biopsy samples that we studied for four other Folate and Methionine Cycle genes, COMT, BHMT, MAT2A and MAT2B (). Strong correlations were observed between mRNA expression levels among SHMT1, SHMT2, COMT, MAT2A and MAT2B in the LCLs (). BHMT was not expressed in these cells. At the protein level, SHMT1 was significantly correlated with both COMT enzyme activity and BHMT protein levels in cytosol preparations from the same set of hepatic biopsy samples (). These observations raise the possibility that genes encoding proteins within the Folate and Methinone Cycles might be regulated in a “coordinated fashion”. These associations will obviously require replication and the mechanism(s) responsible will require functional pursuit in the course of future experiments.
In summary, the results of the present study indicate that nsSNPs—SNPs that alter the encoded amino acid—in SHMT1
may not have a major effect on the biological function of these enzymes, but multiple SNPs within SHMT1
are associated with SHMT1 mRNA expression, which could help explain some of the clinical association results that have been reported (Collin et al. 2009
, Heil et al. 2001
, Komlosi et al. 2010
, Relton et al. 2004
, Vijayakrishnan & Houlston 2010
). Finally, SHMT1
, as well as other Folate and Methionine Cycle genes, might be regulated in a coordinated but complex fashion. Therefore, the present study not only describes individual genetic variation that directly affects SHMT1 and SHMT2 activity, but may also provide insight into the overall regulation of the Folate and Methionine Cycles.