Heterozygous FAM20A Mutation Causes Amelogenesis Imperfecta with Hyperplastic Dental Follicles

Amelogenesis Imperfecta (AI) is a broad classification of inherited enamel disorders with clinical and genetical heterogeneity. Mutations in nine genes (AMELX, MIM 300391; ENAM, MIM606585; MMP20,MIM604629; KLK4, MIM603767; FAM83H, MIM 611927;WDR72, MIM613214;DLX3 ,MIM600525; CNNM4,MIM607805; and FAM20A, MIM611062) have been shown to cause nonsyndromic or syndromic forms of AI. Previously, we established an ameloblast-like cell population from a sporadic AI patient’s unerupted tooth (Ped. Dentistry-UTHSCSA; IRB approval/consent) presenting with hypoplastic enamel, enlarged dental follicles, impacted and over-retained primary teeth (PLoS One 2011;6(10):e24281).  Objective: 1) Perform mutational analysis of AI candidate genes on this proband; and 2) investigate the expression/localization of the identified gene in the AI-ameloblast-like cells. Methods: Mutational analysis of AI candidate gene exons/intron boundaries by PCR amplification and bidirectional sequencing was performed. Potential alterations were compared to 90 DNA samples (30 Caucasian trios families;HapMap Project). AI and control ameloblast-like cells and protein extracts were analyzed by Western blot (WB) and immunohistochemistry using a FAM20A antibody (Sigma-Aldrich).  Results: DNA sequence revealed two heterozygous FAM20A mutations in the proband: a nonsense mutation, c.466C>T,p.R115X in exon 2 (from the father) and a missense mutation c.1354T>C,p.C452R in exon 10; both absent in control samples.  Intracellular FAM20A expression was seen in AI and normal cells, however only control protein extracts showed bands (~22kD and ~35kD) by WB. Conclusions: Our results reveal an autosomal recessive form of AI in this family caused by two novel FAM20A alterations c.466C>T, p.R115X  and c.1354T>C,p.C452R that are rare alleles in the general population.  The proband’s heterozygous mutations correlate with enamel and dental-oral tissue defects. FAM20A protein (~58kD) may be processed/degraded into two major polypeptides.  Identified FAM20A mutations are predicted to affect the kinase activity of the protein (p.C452R) or result in protein truncated (p.R115X) without the kinase domain (DUF1193), both are likely critical for proper enamel formation.