EC Dental Science

Objective: The folate cycle mediates one-carbon metabolism, which is essential for DNA replication and cell division. Compromised neural crest cell proliferation and migration contribute to the development of nonsyndromic cleft lip and/or palate (NSCL ± P), while folic acid periconceptional supplementation can prevent it. This study investigates if the multiplication of human periodontal ligament stem cells (hPDLSC) is affected by folate cycle gene polymorphisms (methylene tetrahydrofolate reductase, MTHFR C677T, and reduced folate carrier 1, RFC1 G80A) that belong to candidate genes for NSCL ± P. Can intracellular folate availability modify the proliferation of hPDLSC that developed from neural crest cells?

Material and Methods: hPDLSCs were isolated from extracted teeth of nine patients and cultured in alphaMEM without nucleosides with 10% fetal bovine serum (IRB approval 2021-80). Genotypes of MTHFR C677T (rs1801133) and RFC1 G80A (rs1051266) polymorphisms were identified using real-time PCR (Taqman kit, Thermo Fisher Scientific). The CyQuantTM LDH test (Thermo Fisher Scientific) was used to quantify cells in culture. Daily multiplication rates were calculated for nine possible genotype combinations.

Results: All combinations of MTHFR C677T and RFC1 G80A genotypes showed clear genotype-specific differences in hPDLSC multiplication rates. Double wild-type homozygotes (MTHFR 677CC/RFC1 80GG) demonstrated the highest multiplication rate (4.1-fold/day), while double-mutated-allele homozygotes (MTHFR 677TT/RFC1 80AA) showed the lowest rate (0.3-fold/day), representing only 7% of the wild-type rate. Heterozygous combinations showed intermediate multiplication rates.

Conclusion: Combined MTHFR C677T and RFC1 G80A polymorphisms differentially affected hPDLSC proliferation in a genotype-dependent manner. Our results suggest that intracellular availability of active folate may similarly alter neural crest cell proliferation and influence the probability of NSCL ± P development. This human primary cell culture model can be utilized in future studies on metabolic disturbances caused by folate deficiency. With a mechanistic understanding of specific genetic influences at the cellular level, we can move closer to personalized prevention for patients at risk of non-syndromic orofacial clefts.

Keywords: Folate; Human Periodontal Ligament Stem Cells; MTHFR C677T; RFC1 G80A; Cell Multiplication

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