FAM20A Isoforms, Localization and Gene-Expression Related to Amelogenesis Imperfecta

FAM20A mutations cause Amelogenesis Imperfecta and Gingival Fibromatosis Syndrome(AIGFS; MIM614253) and  Enamel-Renal Syndrome(ERS;MIM204690).  FAM20A, with three alternatively spliced isoforms (transcript V.1 (541 aa), transcript V.2(404 aa) and V.3 transcript(142aa; reported non-translated)), is a non-traditional secreted  kinases localize in the Golgi apparatus. AIGFS/ERS patients have varying dental features (thin enamel, pulpal calcifications, delayed tooth resorption/eruption and enlarged dental follicles), general/localized gingival hyperplasia and presence/absence of renal calcifications.  Objectives:  To determine Fam20A isoforms expression profiles, their cellular localization and differential-gene profile of ameloblast-like cells (AI-WAm) with known FAM20A mutations.  Methods:  AI-WAm and ST-03(control) cells were cultured (6-18 days) in osteogenic media. qRT-PCR, with transcript-specific (FAM20AV.1, V.2 and V.3) primers, was used to quantitate transcripts. Human-FAM20A-V.1 was cloned into pLENTI-HIS vector and transfected into HEK293T cells, conditioned media and cell lysates were harvested. Protein fractionation of AI-WAm/ST-03 cells was performed using sucrose gradient and differential speed-centrifugation. FAM20A protein was detected by Western blot and localized by immunohistocytochemistry (IHC) using FAM20A antibody.  Transcriptional profiling (AI-WAm/ST-03) was performed using the Affymetrix Human U133-Plus 2.0 Array and Ingenuity Pathways Analysis (IPA) software.  Results: FAM20A V.1 is expressed 2-fold higher in control versus AI-WAm cells, V.2 had the lowest expression, while V.1 and V.3  increased with mineralization. Western blot analysis showed FAM20A is secreted with a ~70kDa protein-(V.1) seen in transfected , ST-03 and AI-WAm (decreased levels) cells. A ~42kDa- (V.3) was seen in transfected cells and ST-03 nuclear fractions. IHC showed nuclear and Golgi FAM20A staining. Critical tooth signaling pathways, FGF-BMP-MSX-RUNX2, were dysregulated in AI-WAm ameloblasts while enamel proteins AMELX-AMBN-ENAM-TUFT were down-regulated.  IPA showed significant dysregulation of cell cycle, cell growth/proliferation and cellular assembly while 80% of top toxicity lists involved renal networks. Conclusions: FAM20A is differentially expressed and localized in ameloblasts.  Consequences of FAM20A mutations are broad, and not specific to enamel proteins.  Support:UAB-SOD-IOHR/UAB-GC-CODED.