Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)–related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM-associated AI.

中文翻译：

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ENAM 突变可能导致成熟度低、釉质生成不完善


釉质生成不全症 (AI) 是一组多样化的遗传性疾病，其特征是由于牙釉质形成的不同阶段的干扰引起的牙釉质畸形的各种表现。发育不全的AI表明牙釉质厚度缺陷是由于牙釉质生成的分泌阶段的畸变造成的，而低成熟AI则表明牙釉质矿化和成熟阶段建立的硬度不足。 ENAM 编码最大的牙釉质基质蛋白牙釉质，其突变已被证明会导致全身或局部发育不全的 AI。在这里，我们表征了 2 个具有不同发育不全和低成熟牙釉质缺陷的 AI 家族，并在 ENAM 的同一位置鉴定了 2 个不同的 indel 突变：c588+1del 和 c.588+1dup。小基因剪接分析表明，它们分别引起 ENAM 蛋白 p.Asn197Ilefs*81 和 p.Asn197Glufs*25 的移码和截短。 Enam 在小鼠下颌切牙上的原位杂交证实了其在分泌期成釉细胞中的限制性表达，并提示了低成熟 AI 的间接致病机制。计算机分析表明，这 2 个截短的 ENAM 可能形成淀粉样蛋白结构，并通过在其 C 末端添加的异常区域导致蛋白质自身聚集以及与野生型蛋白质聚集。一致地，蛋白质分泌测定表明截短的蛋白质不能正确分泌并且阻碍野生型ENAM的分泌。此外，与野生型相比，突变蛋白的过度表达显着增加了内质网应激，并上调了未折叠蛋白反应（UPR）相关基因和TNFRSF10B（一种UPR控制的促凋亡基因）的表达。 Caspase、末端脱氧核苷酸转移酶 UTP 缺口末端标记 (TUNEL) 和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴化物 (MTT) 分析进一步表明，这两种截短的蛋白质，尤其是 p.Asn197Ilefs*81，诱导细胞凋亡并降低细胞存活率，表明 2 个 ENAM 突变通过成釉细胞病理学和死亡导致 AI，而不是通过简单的功能丧失。这项研究表明，ENAM 突变可导致普遍的牙釉质发育不全缺陷，并表明蛋白质病是 ENAM 相关 AI 的潜在发病机制。