先天性缺牙患者中BMP2基因突变检测及功能分析

doi:10.19723/j.issn.1671-167X.2019.01.003

摘要/Abstract

摘要：

目的:在先天性缺牙患者中检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP2)基因突变, 记录BMP2基因突变相关的临床表型, 并进行突变功能分析以评估突变致病性。方法:选取18名先天性缺牙患者, 进行病史采集、临床检查、X线检查, 采集血液样本提取DNA, 全外显子测序,选取和牙颌面发育相关或骨骼系统遗传性疾病相关的基因进行分析,筛选可能致病的基因突变, 选取可能有功能影响的BMP2突变, 分析患者临床表现,进行突变功能试验。构建野生型和突变型BMP2质粒转染人胚胎肾293T细胞, 激光扫描共聚焦显微镜下观察蛋白质在细胞内分布, 蛋白质印迹实验检测突变型BMP2磷酸化激活下游SMAD1/5/9分子(SMAD family member 1/5/9, SMAD1/5/9)的情况。结果:在1例先天性缺牙患者中检测到可能有功能影响的BMP2突变NM_001200.3:c.393A>T(p.Arg131Ser), rs140417301, 患者父母不携带此突变,患者父亲牙齿正常,母亲先天缺失1颗前磨牙,父母上颌形态均正常。患者同时具有上颌骨前后向及水平向发育不足、上腭形态发育异常, 及继发的错颌畸形, 同时对腰椎、髋部骨的X线检查提示骨密度减低。功能试验显示该突变对骨形态发生蛋白(bone morphogenetic protein, BMP)通路活性有影响,该突变导致BMP2蛋白质磷酸化激活下游SMAD1/5/9蛋白质的能力减弱(3次重复实验分别下降32%、22%、27%), 结合家族共分离等判断该突变为“可能致病的”。结论:BMP2突变c.393A>T(p.Arg131Ser)对BMP通路活性具有一定影响,可使得突变型BMP2蛋白质对于下游SMAD1/5/9蛋白质激活能力减弱,在临床上可能导致先天性缺牙、上颌骨发育不足、上腭发育异常、错颌畸形及骨密度降低。

关键词: BMP2基因突变, 先天性缺牙, 功能分析, 骨缺陷

Abstract:

Objective:To screen for BMP2 mutation with functional impact in patients with congenital tooth agenesis and to make oral and skeletal phenotype record and functional analysis with in vitro experiments. Methods: We enrolled eighteen patients with congenital tooth agenesis. The medical and dental history was collected,and clinical and dental examinations including the X-ray examination of oral-facial and skeletal bone were performed for the phenotypic analysis. Blood samples were collected to extract DNA and whole exome sequencing was conducted. The genes involved in oral-facial development and congenital skeletal diseases were investigated for mutation screening. The mutations with functional impact were then investigated. In one patient, the BMP2 mutation with putative functional impact was selected for functional analysis. Wild type and mutant BMP2 plasmids with green fluorescent protein (GFP) tag were constructed and transfected into HEK293T cells. Subcellular protein distribution was observed under laser scanning confocal microscope. The activation of downstream SMAD1/5/9 phosphorylation by BMP2 was detected by Western blotting to investigate the functional impact and genetic pathogenicity. Results: BMP2 mutation NM_001200.3:c.393A>T(p.Arg131Ser), rs140417301 was detected in one patient with congenital tooth agenesis, while for other genes involved in oral-facial development and congenital skeletal diseases, no functionally significant mutation was found. The proband’s parents didn’t carry this mutation. The father had normal dentition, while the mother lacked one premolar, and both the parents showed normal palate and maxilla. The patient also had maxillary hypoplasia in both sagittal and coronal planes, palatal dysmorphology, and malocclusion, and was diagonsed with osteopenia after the X-ray examnination of his skeletal bone. Functional analysis showed this mutation had normal subcelluar localization but reduced phosphorylation of SMAD1/5/9 (reduction by 32%, 22%, and 27% in three independent replicates). Taken together with family co-segregation, this mutaion was considered as “likely pathogenic”. Conclusion: BMP2 mutation c.393A>T (p. Arg131Ser) affects bone morphogenetic protein signaling activity, and may affect the number of teeth, growth of maxilla and palate, and bone mineral density.

Key words: BMP2 gene mutation, Tooth agenesis, Function alanalysis, Bone defect

中图分类号:

R783.4

王皓,刘洋,刘浩辰,韩冬,冯海兰. 先天性缺牙患者中BMP2基因突变检测及功能分析[J]. 北京大学学报（医学版）, 2019, 51(1): 9-15.

Hao WANG,Yang LIU,Hao-chen LIU,Dong HAN,Hai-lan FENG. Detection and functional analysis of BMP2 gene mutation in patients with tooth agenesis[J]. Journal of Peking University(Health Sciences), 2019, 51(1): 9-15.

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