PDF(Pubmed)
Abstract：
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.
摘要：
牙釉质发育不全（AI）是一组遗传性疾病，其特征是由于牙釉质形成的不同阶段的干扰而引起的牙釉质畸形的各种表现。虽然发育不良的AI表明牙釉质厚度缺陷是由于牙釉质生成的分泌阶段的像差引起的，增生AI表明在成熟期建立的牙釉质矿化和硬度不足。ENAM突变,编码最大的釉质基质蛋白，enamelin,已被证明会导致广泛性或局部发育不良的AI。这里,我们表征了2个AI家族具有不同的发育不良和牙釉质缺损，并在ENAM的相同位置鉴定了2个不同的indel突变，c588+1del和c.588+1dup。小基因剪接实验表明，它们引起ENAM蛋白的移码和截短，p.Asn197Ilefs*81和p.Asn197Glufs*25。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表达受限，并提示了AI充血的间接致病机制。计算机模拟分析表明，这2个截短的ENAM可能形成淀粉样蛋白结构，并通过其C末端添加的异常区域与自身和野生型蛋白引起蛋白质聚集。始终如一，蛋白质分泌试验表明,截短的蛋白质不能正常分泌并阻碍野生型ENAM的分泌。此外，与野生型相比，突变蛋白的过表达显着增加内质网应激，并上调未折叠蛋白反应（UPR）相关基因和TNFRSF10B的表达，UPR控制的促凋亡基因。胱天蛋白酶,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）测定进一步揭示了两种截短的蛋白质，特别是p.Asn197Ilefs*81，诱导细胞凋亡和降低细胞存活，这表明2个ENAM突变通过成釉细胞病理和死亡而不是通过简单的功能丧失引起AI。这项研究表明，ENAM突变可导致广泛的牙釉质缺损，并提示蛋白质病是ENAM相关AI的潜在发病机制。
