A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China.
Wen'an
W
Xu
Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China.|Department of Stomatology, Zhongshan City People's Hospital, Zhongshan, Guangdong, China.
Qiuyue
Q
Chen
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China.
Cuixian
C
Liu
Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China.
Jiajing
J
Chen
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China. xiongfu@smu.edu.cn.
Fu
F
Xiong
Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China. bulingwu@smu.edu.cn.
Buling
http://orcid.org/0000-0002-5469-2861
B
Wu
D000293:Adolescent; D002973:Cleidocranial Dysplasia / Q000175:diagnosis / Q000235:genetics*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D016368:Frameshift Mutation; D005838:Genotype; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014057:Tomography, X-Ray Computed; D014070:Tooth / Q000000981:diagnostic imaging; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; D012333:RNA, Messenger; C493709:RUNX2 protein, human; D012313:RNA
Craniofacial anomalies; Haploinsufficiency; Molecular genetics; Oral systemic disease(s); RUNX2; Truncation protein