Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient
Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient
复制标题
DOI：
10.1093/mutage/gev057
复制
发表时间：
2016-01-01
期刊：
影响因子：
2.7
通讯作者：
Tokita, Yoshihito
中科院分区：
文献类型：
--
作者：
Shibata, Akio;Machida, Junichiro;Tokita, Yoshihito
锁骨颅骨发育不良; MIM 119600）是一种常染色体显性骨骼发育不良，其特征为锁骨发育不全和/或再生障碍性锁骨、面中部发育不全、颅缝缺失或延迟闭合、中等身材矮小、恒牙列延迟萌出和多生牙。在大约三分之二的CCD患者中，RUNX 2基因的单倍不足可以解释其分子发病机制。在我们目前的研究中，我们确定了一个新的和罕见的变异的RUNX 2基因（c.181_189dupGCGGCGGCT）在日本患者与CCD的表型特征。插入导致多聚丙氨酸束中的丙氨酸三肽扩增（+3Ala）。迄今为止，具有丙氨酸十肽扩增（+10Ala）的RUNX 2变体是待报道的具有多聚丙氨酸段扩增的RUNX 2致病变体的唯一实例，而RUNX 2（+1Ala）已从健康人群中分离。因此，需要对RUNX 2（+3Ala）变体进行精确分析以澄清三肽扩增的RUNX 2是否是具有丙氨酸段扩增的第二致病突变体。因此，我们研究了突变体RUNX 2（+3Ala）的生化特性，该突变体在多聚丙氨酸区含有20个丙氨酸残基。RUNX 2（+3Ala）转染COS 7细胞24 h后形成细胞内泛素化聚集体，并在体外发挥显性负效应。在基因转染后24 h，尽管观察到RUNX 2（+10Ala）略有减少，但所有这些突变体均显著激活成骨细胞特异性元件-2，RUNX 2靶基因骨钙素启动子中的顺式作用序列。RUNX 2（+3Ala）的聚集生长明显低于RUNX 2（+10Ala）。此外，我们研究了具有不同丙氨酸段长度的其他几种RUNX 2变体，并发现聚集的阈值可能是RUNX 2（+3Ala）。我们的结论是，RUNX 2（+3Ala）是在我们目前的情况下CCD的原因，并在体外细胞内聚集体的积累与丙氨酸束的长度。
Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24 h, and exerted a dominant negative effect in vitro. At 24 h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.