A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.
A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.
复制标题
一种新型复杂的 RUNX2 基因突变导致锁骨颅骨发育不良
DOI：
10.1186/s12881-017-0375-x
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发表时间：
2017-02-07
影响因子：
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通讯作者：
Wu B
中科院分区：
文献类型：
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作者：
Xu W;Chen Q;Liu C;Chen J;Xiong F;Wu B
已知 runt 相关转录因子 2 (RUNX2) 基因的单倍体不足会导致锁骨颅骨发育不良 (CCD)。在这里，我们研究了一个中国 CCD 家族中复杂的杂合 RUNX2 基因突变以及与该突变相关的发病机制。从先证者、她的父母和 3 个兄弟姐妹以及 150 名正常对照者的外周静脉血中提取基因组 DNA。通过PCR扩增和Sanger测序对其各自的RUNX2基因序列进行分析。通过生物信息学、实时 PCR、蛋白质印迹分析和亚细胞定位研究，研究了与 RUNX2 突变相关的发病机制。我们鉴定了 2 个复杂的杂合突变，涉及 RUNX2 基因外显子 3 中的 c.398-399 insACAGCAGCAGCA 插入和 c.411-412 insG 移码突变。移码突变改变了RUNX2蛋白的结构，但不影响其mRNA水平的表达。与野生型蛋白相比，用表达RUNX2变体的质粒转染HEK293T细胞降低了变体RUNX2蛋白的分子量。亚细胞定位测定显示突变蛋白定位于细胞核和细胞质，而野生型蛋白定位于细胞核。我们的研究结果表明，新的 c.398–399insACAGCAGCAGCA 突变与 c.411–412insG 移码突变同时发生，导致 RUNX2 截短。 RUNX2 单倍体不足与 CCD 发病机制相关。这些结果扩展了 RUNX2 基因的已知突变谱，并表明新突变在 CCD 发病机制中的功能作用。
Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations. Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies. We identified 2 complex heterozygous mutations involving a c.398–399 insACAGCAGCAGCAGCA insertion and a c.411–412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus. Our findings demonstrated that the novel c.398–399insACAGCAGCAGCAGCA mutation occurred alongside the c.411–412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.