探討REST最末外顯子突變於遺傳性牙齦纖維瘤中的分子影響：轉錄體分析與功能性探討

Abstract

遺傳性牙齦纖維瘤（Hereditary Gingival Fibromatosis, HGF）為一罕見常染色體顯性遺傳疾病，臨床表現為牙齦組織對稱性增生，可導致齒列擁擠、延遲萌發與口腔功能障礙。近年研究指出 RE1-Silencing Transcription factor（REST）末端外顯子附近的截短突變與 HGF 具關聯性，但其分子致病機轉仍未明確。本研究針對一名父母均無此疾病之HGF 病患，鑑定出一新穎 frameshift 突變（c.2280delT, p.V761Lfs*3），並透過功能實驗與細胞模型加以驗證。 在 HEK293T 細胞中進行冷光素酶報導基因分析（luciferase reporter assay）顯示，此一突變型 REST蛋白（REST-Mu）喪失轉錄抑制能力，並於共表現條件下抑制 REST-WT 蛋白功能，呈dominant-negative型態。為驗證其於生理相關細胞中的功能影響，本研究建立 FLAG-REST-WT 與 REST-Mu 表現質體 ，以電穿孔法轉染人類初代牙齦纖維母細胞（primary human gingival fibroblasts, hGFs），並應用 Parse Biosciences 單細胞 RNA 定序技術（scRNA-seq）分析 FLAG-REST 野生型（H-WT）、截短突變型（H-Mu）與空載體對照（H-Mock）三組條件。結果顯示 H-Mu 與 H-WT 組細胞中 IL24、STC1、CEMIP、NEAT1 等參與細胞修復與基質重塑之基因顯著下調，且 pathway enrichment 分析指向Apelin signaling、FGF signaling、integrin signaling 與 cytoskeletal remodeling 等路徑。為進一步提升轉染效率，本研究亦成功優化電穿孔與 G418 篩選條件，在不使用病毒載體的情況下於初代 hGFs 中實現外源 FLAG-REST 基因過表現，並以 qPCR 證實轉錄表現良好。 綜合而言，本研究結合功能性分析與初代細胞單細胞轉錄層級研究，證實一新穎 REST 突變具 dominant-negative 活性，並揭示其可能透過轉錄程序障礙與訊息傳導異常導致 HGF 表型。此研究建立一可行平台以探討 REST 功能缺損在牙齦纖維母細胞中的病理角色，對理解 HGF 發病機制與後續研究具重要參考價值。

Hereditary Gingival Fibromatosis (HGF) is a rare autosomal dominant disorder characterized by symmetrical gingival overgrowth, often leading to dental crowding, delayed tooth eruption, and oral functional impairment. Recent studies have associated truncating mutations near the terminal exon of the RE1-Silencing Transcription Factor (REST) gene with HGF, although the molecular pathogenic mechanisms remain unclear. In this study, we identified a novel frameshift mutation (c.2280delT, p.V761Lfs*3) in a patient with HGF born to unaffected parents and validated its pathogenicity through functional assays and cellular modeling. Luciferase reporter assays in HEK293T cells demonstrated that this mutant REST protein (REST-Mu) lacked transcriptional repression capability and exerted a dominant-negative effect by interfering with the function of co-expressed wild-type REST (REST-WT). To assess the impact in a physiologically relevant context, FLAG-tagged REST-WT and REST-Mu expression constructs were introduced into primary human gingival fibroblasts (hGFs) via electroporation, followed by single-cell RNA sequencing (scRNA-seq) using the Parse Biosciences platform under three experimental conditions: FLAG-REST wild type (H-WT), truncation mutant (H-Mu), and empty vector control (H-Mock). Transcriptomic analysis revealed significant downregulation of key genes involved in tissue repair and extracellular matrix remodeling—including IL24, STC1, CEMIP, and NEAT1—in the REST-Mu group. Pathway enrichment analysis pointed to pathways such as Apelin signaling, FGF signaling, integrin signaling, and cytoskeletal remodeling.To further enhance transfection efficiency, this study also successfully optimized electroporation and G418 selection conditions to achieve overexpression of exogenous FLAG-REST in primary hGFs without using viral vectors, with qPCR confirming robust transcriptional expression. In conclusion, by integrating functional assays with single-cell transcriptomic analysis in primary cells, this study demonstrates that the novel REST mutation exerts dominant-negative activity and may disrupt transcriptional programs and signaling responses underlying the HGF phenotype. Our findings establish a feasible and scalable experimental platform for exploring the pathological roles of REST dysfunction in gingival fibroblasts and provide important insights for future investigations into the molecular pathogenesis of HGF.