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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (1): 9-15. doi: 10.19723/j.issn.1671-167X.2019.01.003

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Detection and functional analysis of BMP2 gene mutation in patients with tooth agenesis

Hao WANG,Yang LIU,Hao-chen LIU,Dong HAN(),Hai-lan FENG   

  1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2018-10-07 Online:2019-02-18 Published:2019-02-25
  • Contact: Dong HAN E-mail:donghan@bjmu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(81600846);Supported by the National Natural Science Foundation of China(81670949)

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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

CLC Number: 

  • R783.4

Figure 1

Sites of missing teeth, pedigree and DNA sequencing of the family members"

Figure 2

Intraoral photographs, radiographs of the patient A, maxillary dentition and the palate appearance; B, mandibular dentition; C, frontal view of dentition; D, panoramic radiograph; E, lateral cephalometric radiograph (after orthodontic treatment)."

Figure 3

Radiographs of the palate of the patient and a normal person A, B, coronal and sagittal section on cone beam CT showing abnormal palate of the patient (before orthodontic treatment); C, D, coronal and sagittal section on cone beam CT showing the normal palate."

Figure 4

Functional analysis of mutant BMP2, distribution of BMP2 protein, Western blot analysis,statistical results of relative quantification of phospho-SMAD1/5/9 protein of three replicates A, distribution of BMP2 protein with GFP tag showed no significant difference between wild type and mutant BMP2; B, Western blot analysis at 48 h post-transfection showed impaired activation of downstream phospho-SMAD1/5/9 compared with that in the normal control (representational research result); C, relative quantification of phospho-SMAD1/5/9 protein expression normalized against GAPDH by three replicates of Western blot, *P < 0.01, vs. WT-tranfected cells. BMP2, bone morphogenetic protein 2; GFP, green fluorescent protein;Phospho-SMAD1/5/9, phospho-SMAD family member 1/5/9; SMAD1+5, SMAD family member 1+5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Vec, vector; WT, wild type; R131S, p. Arg131Ser mutant."

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