Date of Award

9-26-2025

Department

Department of Orthodontics

First Advisor

Marie M. Tolarová

First Committee Member

Mirek Tolar

Abstract

Introduction: Orofacial clefts, including non-syndromic cleft lip and/or palate (NSCLP), are among the most common congenital anomalies affecting approximately 1 in 700 live births. The etiology of NSCLP is multifactorial involving both genetic and environmental factors. MTHFR C677T and RFC1 G80A polymorphisms belong to candidate genes affecting folate metabolism. Compromised neural crest cell proliferation and migration are known to contribute significantly to NSCLP development. This study investigates the combined effects of MTHFR and RFC1 polymorphisms on multiplication rates of human periodontal ligament stem cells (hPDLSC), which serve as a model for neural crest-derived cells. Materials and Methods: hPDLSC were isolated from extracted teeth of 8 patients and cultured for 4-5 weeks. MTHFR C677T and RFC1 G80A polymorphism genotypes were identified using real-time PCR. Cell multiplication rates were measured over 4 days and compared across different genotype combinations. Experiments were performed in sets of three. Results: Results from 8 patients demonstrated clear genotype-dependent differences in hPDLSC multiplication rates. Wild-type homozygotes (MTHFR CC/RFC1 GG) demonstrated the highest multiplication rate (4.1), while double-mutated homozygotes (MTHFR TT/RFC1 AA) showed the lowest rate (0.3), representing only 7% of the wild-type rate. Heterozygous combinations showed intermediate multiplication rates. Conclusion: MTHFR C677T and RFC1 G80A polymorphisms differentially influence hPDLSC 3 multiplication rates in a genotype-dependent manner. The dramatic reduction in cellular proliferation observed in patients with combined polymorphisms provides mechanistic insight into how folate metabolism gene variants may contribute to NSCLP development through compromised neural crest cell proliferation during embryogenesis. These findings support the importance of adequate folate supplementation, particularly for individuals carrying genetic polymorphisms that compromise folate metabolism.

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