Two Mutations in the Caprine <i>MTHFR</i> 3'UTR Regulated by MicroRNAs Are Associated with Milk Production Traits

Background 5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism by irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, a predominant circulating form of folate. Folate is reportedly important for milk protein synthesis, and MTHFR may be a key regulatory point of folate metabolism for milk protein synthesis in mammary epithelial cells. Prior to this study, polymorphisms of the MTHFR gene were not associated with milk production traits from a breeding perspective. Single nucleotide polymorphisms (SNPs) at microRNA (miRNA) binding sites (miR-SNPs) can affect gene expression. This study aimed to identify the effects of miR-SNPs (g.2244A>G and g.2264A>G) in the caprine MTHFR 3' UTR on the milk production traits of dairy goats. Results Guanzhong dairy (GD, n = 325) goats were used to detect SNPs in the caprine MTHFR 3' UTR by DNA sequencing. Two novel SNPs (g.2244A>G and g.2264A>G) were identified in the said region. The homozygous haplotype A-G of the SNPs g.2244A>G and g.2264A>G was significantly associated with milk yield and milk protein levels in GD goats (P < 0.05). Functional assays indicated that the MTHFR 2244 A → G substitution could increase the binding activity of hsa-miR-1266 with the MTHFR 3' UTR. The MTHFR 2264 A → G substitution could decrease the binding activity of hsa-miR-616 with the MTHFR 3' UTR. In addition, we observed a significant increase in the MTHFR mRNA levels of homozygous haplotype A-G carriers relative to those of homozygous haplotype G-A carriers. These results indicated that both SNPs altered the MTHFR mRNA levels. These altered levels of MTHFR mRNA may account for the association of SNPs with milk production traits. Conclusions This study is the first to report that the g.2244A>G and g.2264A>G polymorphisms were associated with milk production traits in GD goats. Further investigations should explore the underlying miRNA-mediated mechanisms that are modified by the g.2244A>G and g.2264A>G SNPs. The current study evaluated these SNPs as potential genetic markers in goats, with potential applications in breeding programs.背景 5,10-亚甲基四氢叶酸还原酶（5,10-Methylenetetrahydrofolate reductase，简称MTHFR）可通过将5,10-亚甲基四氢叶酸不可逆转化为5-甲基四氢叶酸（一种主要的循环叶酸形式），在叶酸代谢中发挥核心作用。据报道，叶酸对乳蛋白合成至关重要，而MTHFR或可作为乳腺上皮细胞中乳蛋白合成相关叶酸代谢的关键调控节点。在本研究开展前，从育种视角来看，MTHFR基因多态性与产奶性状并无关联。微小RNA（microRNA，简称miRNA）结合位点处的单核苷酸多态性（single nucleotide polymorphisms，简称SNPs，又称miR-SNPs）可影响基因表达。本研究旨在探究山羊MTHFR基因3'非翻译区（3' UTR）内的miR-SNPs（g.2244A>G与g.2264A>G）对奶山羊产奶性状的影响。 结果 本研究以325头关中奶山羊（简称GD）为实验对象，通过DNA测序技术检测山羊MTHFR基因3' UTR区域的单核苷酸多态性。该区域共鉴定出2个全新的单核苷酸多态位点（g.2244A>G与g.2264A>G）。g.2244A>G与g.2264A>G的纯合单倍型A-G与关中奶山羊的产奶量及乳蛋白含量呈显著相关（P < 0.05）。功能实验结果显示，MTHFR基因2244位A→G的碱基替换可增强hsa-miR-1266与MTHFR基因3' UTR的结合活性；而MTHFR基因2264位A→G的碱基替换则会降低hsa-miR-616与MTHFR基因3' UTR的结合活性。此外，相较于纯合单倍型G-A的携带者，纯合单倍型A-G携带者的MTHFR mRNA水平显著升高。上述结果表明，这两个单核苷酸多态位点均可改变MTHFR基因的mRNA水平，而该水平变化或可解释单核苷酸多态性与产奶性状之间的关联。 结论 本研究首次报道了g.2244A>G与g.2264A>G多态性与关中奶山羊产奶性状的关联。后续研究应进一步探究g.2244A>G与g.2264A>G单核苷酸多态性所调控的miRNA介导的潜在分子机制。本研究证实上述两个单核苷酸多态位点可作为山羊潜在的遗传标记，有望应用于山羊育种工作中。