The MTHFD1L gene encodes the mitochondrial monofunctional enzyme with proven 10-formyltetrahydrofolate synthetase activity. MTHFD1L expression is upregulated in human colon cancer and breast cancer, and high levels of this protein are correlated with growth rate of human cancer cell lines. Genetic association studies demonstrated that MTHFD1L polymorphisms are associated with coronary artery disease, Alzheimer’s disease and neural tube defects (NTDs). Moreover, MTHFD1L knockout mice develop NTDs with 100% penetrance. This project involves the characterization of MTHFD1L with a particular focus on genetic polymorphisms and disease risk. MTHFD1L polymorphisms have been studied in relation to cleft disease and Melting Curve Analysis was developed for genotyping the Deletion Insertion Polymorphism rs3832406. MicroRNA-9 and 197 were proven to downregulate MTHFD1L at mRNA and protein level. The impact of polymorphism rs7646 on miR-197 binding suggests a mechanistic explanation of the previous association of this SNP with NTD risk. Sequence analysis was performed to discover and characterize MTHFD1 and MTHFD1L similar sequences. MTHFD1L expression was proven to be affected by a 24 hour exposure to cycloleucine in lymphoblast. Finally, HEK293 stable cell lines with either MTHFD1L downregulated or overexpressed have been generated and the resulting proteomes have been assessed by Mass Spec.