ENAM突变可导致成髓细胞发育不全。

Journal of dental research Pub Date : 2024-06-01 Epub Date: 2024-05-08 DOI:10.1177/00220345241236695
Y-L Wang, H-C Lin, T Liang, J C-Y Lin, J P Simmer, J C-C Hu, S-K Wang
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引用次数: 0

摘要

釉质发育不全症(AI)是一组遗传性疾病,其特点是釉质形成的不同阶段受到干扰而导致不同表现的釉质畸形。发育不全性釉质发育不全(hypoplastic AI)是指釉质在釉质形成的分泌阶段出现异常而导致的釉质厚度缺陷,而釉质发育不全性釉质发育不全(hypomaturation AI)则是指釉质在成熟阶段出现矿化和硬度不足。ENAM编码最大的釉质基质蛋白--釉质素,ENAM的突变已被证实可导致全身性或局部性发育不全AI。在这里,我们鉴定了两个具有不同的发育不全和釉质发育不全缺陷的人工智能家族,并在ENAM的同一位置发现了两个不同的吲哚突变,即c588+1del和c.588+1dup。微型基因剪接试验表明,这两个突变分别导致了ENAM蛋白的帧移位和截断,即p.Asn197Ilefs*81和p.Asn197Glufs*25。对小鼠下颌门牙上的ENAM进行原位杂交证实,ENAM在分泌期成髓细胞中的表达受到限制,这也提示了AI发育不全的间接致病机制。硅学分析表明,这两种截短的ENAM可能会形成淀粉样结构,并通过其C端添加的异常区域导致自身和野生型蛋白聚集。同样,蛋白质分泌试验表明,截短蛋白不能正常分泌,并阻碍了野生型ENAM的分泌。此外,与野生型相比,过量表达突变体蛋白会显著增加内质网应激,并上调未折叠蛋白反应(UPR)相关基因和 UPR 控制的促凋亡基因 TNFRSF10B 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-溴化四氮唑(MTT)检测进一步表明,这两种截短蛋白,尤其是p.Asn197Ilefs*81,可诱导细胞凋亡并降低细胞存活率,这表明这两种ENAM突变是通过成髓细胞病理变化和死亡而非简单的功能缺失引起人工智能的。这项研究表明,ENAM突变可导致全身釉质发育不全,并提示蛋白病是ENAM相关性人工智能的潜在发病机制。
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ENAM Mutations Can Cause Hypomaturation Amelogenesis Imperfecta.

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.

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