目的:本研究旨在确定一个具有不典型的发骨综合征(TDO)临床表现的家族中的DLX3基因突变及其对牙釉质厚度的影响,显微硬度,结构和形成。
方法:全外显子组测序检测到该家族中的DLX3突变。Micro-CT,维氏硬度计,对先证者和对照组的乳牙进行了能量色散光谱仪和扫描电子显微镜检查。体外实验初步验证了该突变对成釉细胞分化的影响,并提出了可能的分子机制。
结果:我们在该家族中发现了一个新的DLX3移码突变(NM_005220.3:c.604_605del:p.S202*)。与对照牙齿相比,突变牙釉质显示厚度显着下降,硬度和钙含量以及镁含量的增加。牙釉质结构出现紊乱。在永生成釉细胞谱系细胞(ALC)中,此突变影响成釉细胞分化并下调釉质基质蛋白(EMP)基因的表达水平(Amelx,Tuft1,Klk4,Ambn,Odam)。荧光素酶报告基因测定表明该突变显著降低DLX3对Amelx/Odam/Klk4的反式激活活性。
结论:我们在一个中国牙釉质发育不良家族中发现了一个新的DLX3突变,该突变可能通过抑制Amelx/Odam/Klk4的转录活性而影响成釉细胞分化,从而干扰牙釉质形成。我们的发现进一步扩大了变异谱,丰富了DLX3突变的分子遗传学证据。
OBJECTIVE: This study aimed to identify a DLX3 gene mutation in a family with atypical clinical manifestations of tricho-dento-osseous syndrome (TDO) and its impact on tooth enamel thickness, microhardness, structure and formation.
METHODS: Whole-exome sequencing detected DLX3 mutations in the family. Micro-CT, Vickers hardness tester, energy dispersive spectrometer and scanning electron microscopy were performed on the deciduous teeth of the proband and controls. In vitro experiments preliminarily verified the effect of this mutation on ameloblast differentiation and suggested possible molecular mechanisms.
RESULTS: We found a new DLX3 frame-shift mutation (NM_005220.3: c.604_605del: p. S202 *) in this family. Compared with control teeth, the mutant enamel showed a significant decrease in thickness, hardness and calcium content and an increase in magnesium content. The enamel structure appeared disordered. In an immortalized ameloblast-lineage cell (ALC) line, this mutation affected ameloblast differentiation and downregulated the expression levels of enamel matrix protein (EMP) genes (Amelx, Tuft1, Klk4, Ambn, Odam). A luciferase reporter assay demonstrated that this mutation significantly reduced the transactivation activity of DLX3 on Amelx/Odam/Klk4.
CONCLUSIONS: We found a new DLX3 mutation in a Chinese family with enamel dysplasia and that this mutation may affect ameloblast differentiation by inhibiting the transcriptional activity of Amelx/Odam/Klk4, thereby interfering with enamel formation. Our findings further expand the variation spectrum and enrich the evidence of molecular genetics of DLX3 mutations.