Our laboratory has identified a Nuclear factor I-C (NFI-C) mutation associated with a novel autosomal recessive form of Radicular Dentin Dysplasia (AR-RDD). NIF-C belongs to a family of proteins which act as transcription factors and adenovirus DNA replication factors. NFI-C, located on chromosome 19p33, is encoded by 11 exons that are alternatively spliced producing multiple isoforms. Molars of
Nfic-/- mouse lack roots demonstrating NFI-C is a critical for normal root formation.
Objectives: To establish primary periodontal ligament (PDL) cells from an AR-RDD patient; characterize their growth rate and gene expression profile; determine alterative splicing patterns in dental cells; and determine the functional significance of the NFI-C mutation.
Methods: A mandibular unerupted molar from an AR-RDD patient, removed for clinical treatment, was obtained with signed consent and used to establish PDL cells associated with the limited root dentin. Cells were grown in DMEM medium and expanded. PDL cells (AR-RDD and unaffected age- and sex-matched) were grown, total RNA isolated using RNA STAT-60 kit and cDNA synthesized using the TaqMan RT-PCR kit. PCR amplification was used to determine NFI-C alternative spliced transcripts. Quantitative real-time PCR was performed using SYBR Green chemistry to investigate mRNA expression levels of various tooth associated proteins as compared to unaffected cells with transcript abundance correlated to h18sRNA.
Results: The AD-RDD PDL cells showed normal morphology, mRNA expression levels were altered such as a significant decrease in tuftelin (p<0.05). NFI-C mRNA expression was 4.3 fold lower in AR-RDD PDL cells than controls (p<0.05) while the cell growth rate was higher.
Conclusion: The identified AR-RDD mutation dramatically decreased NFI-C mRNA expression levels suggesting the disease phenotype is hypomorphic. The human AR-RDD PDL cells offer a unique resource to study NFI-C's function in root formation and identify downstream target genes. Support: UTHSCSA-PREF,UAB-IOHR & ProDoc-CAPES(ACA; Brazil).