先天性缺牙患者中BMP2基因突变检测及功能分析
|
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 member1/5/9,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。
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 HEK293 T 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. 393 A > T(p. Arg131 Ser),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. 393 A > T(p. Arg131 Ser) affects bone morphogenetic protein signaling activity,and may affect the number of teeth,growth of maxilla and palate,and bone mineral density.
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 HEK293 T 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. 393 A > T(p. Arg131 Ser),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. 393 A > T(p. Arg131 Ser) affects bone morphogenetic protein signaling activity,and may affect the number of teeth,growth of maxilla and palate,and bone mineral density.
引文
[1] Wozney JM. The bone morphogenetic protein family and osteogenesis[J]. Mol Reprod Dev,1992,32(2):160-167.
[2] Zhang YD,Chen Z,Song YQ,et al. Making a tooth:growth factors,transcription factors,and stem cells[J]. Cell Res,2005,15(5):301-316.
[3] Nishinakamura R,Sakaguchi M. BMP signaling and its modifiers in kidney development[J]. Pediatr Nephrol,2014,29(4):681-686.
[4] Neubuser A,Peters H,Balling R,et al. Antagonistic interactions between FGF and BMP signaling pathways:a mechanism for positioning the sites of tooth formation[J]. Cell,1997,90(2):247-255.
[5] Yuan G,Yang G,Zheng Y,et al. The non-canonical BMP and Wnt/beta-catenin signaling pathways orchestrate early tooth development[J]. Development,2015,142(1):128-139.
[6] Zhao M,Harris SE,Horn D,et al. Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation[J]. J Cell Biol,2002,157(6):1049-1060.
[7] de Coster PJ,Marks LA,Martens LC,et al. Dental agenesis:genetic and clinical perspectives[J]. J Oral Pathol Med,2009,38(1):1-17.
[8] Yin W,Bian Z. The gene network underlying hypodontia[J]. J Dent Res,2015,94(7):878-885.
[9] Kantaputra P,Sripathomsawat W. WNT10A and isolated hypodontia[J]. Am J Med Genet A,2011,155(5):1119-1122.
[10] Wong S,Liu H,Bai B,et al. Novel missense mutations in the AXIN2 gene associated with non-syndromic oligodontia[J]. Arch Oral Biol,2014,59(3):349-353.
[11] Mues GI,Griggs R,Hartung AJ,et al. From ectodermal dysplasia to selective tooth agenesis[J]. Am J Med Genet A,2009,149(9):2037-2041.
[12] Wong S,Liu H,Han D,et al. A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia[J].Mutagenesis,2014,29(5):319-323.
[13] Stockton DW,Das P,Goldenberg M,et al. Mutation of PAX9 is associated with oligodontia[J]. Nat Genet,2000,24(1):18-19.
[14] Yu P,Yang W,Han D,et al. Mutations in WNT10B are identified in individuals with oligodontia[J]. Am J Hum Genet,2016,99(1):195-201.
[15] Liu X,Gao L,Zhao A,et al. Identification of duplication downstream of BMP2 in a Chinese family with brachydactyly type A2(BDA2)[J]. PLo S One,2014,9(4):e94201.
[16] Styrkarsdottir U,Cazier JB,Kong A,et al. Linkage of osteoporosis to chromosome 20p12 and association to BMP2[J]. PLo S Biol,2003,1(3):E69.
[17] Sahoo T,Theisen A,Sanchez-Lara PA,et al. Microdeletion20p12. 3 involving BMP2 contributes to syndromic forms of cleft palate[J]. Am J Med Genet A,2011,155(7):1646-1653.
[18] Williams ES,Uhas KA,Bunke BP,et al. Cleft palate in a multigenerational family with a microdeletion of 20p12. 3 involving BMP2[J]. Am J Med Genet A,2012,158(10):2616-2620.
[19] Mu Y,Xu Z,Contreras CI,et al. Phenotype characterization and sequence analysis of BMP2 and BMP4 variants in two Mexican families with oligodontia[J]. Genet Mol Res,2012,11(4):4110-4120.
[20]邹川,高清平,Bakrv HH,等.单纯性先天缺牙患者BMP2/BMP4基因位点分析[J].上海口腔医学,2015,24(1):83-88.
[21] Lu Y,Qian Y,Zhang J,et al Genetic variants of BMP2 and their association with the risk of non-syndromic tooth agenesis[J]. PLo S One,2016,11(6):e0158273.
[22] Prasad MK,Geoffroy V,Vicaire S,et al. A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement[J]. J Med Genet,2016,53(2):98-110.
[23] Clark GR,Duncan EL. The genetics of osteoporosis[J]. Br Med Bull,2015,113(1):73-81.
[24] Richards JB,Zheng HF,Spector TD. Genetics of osteoporosis from genome-wide association studies:advances and challenges[J]. Nat Rev Genet,2012,13(8):576-588.
[25] Hsu YH,Kiel DP. Clinical review:Genome-wide association studies of skeletal phenotypes:what we have learned and where we are headed[J]. J Clin Endocrinol Metab,2012,97(10):E1958-E1977.
[26] Richards S,Aziz N,Bale S,et al. Standards and guidelines for the interpretation of sequence variants:a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[J]. Genet Med,2015,17(5):405-424.
[27] Tan TY,Gonzaga-Jauregui C,Bhoj EJ,et al. Monoallelic BMP2variants predicted to result in haploinsufficiency cause craniofacial,skeletal,and cardiac features overlapping those of 20p12 deletions[J]. Am J Hum Genet,2017,101(6):985-994.
[28] Timberlake AT,Choi J,Zaidi S,et al. Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles[J]. Elife,2016,5(4 suppl):1.
[29] Radio FC,Majore S,Aurizi C,et al. Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP,BMP2,FTL and SLC40A1 genes[J].Blood Cells Mol Dis,2015,55(1):71-75.
[30] Breitfeld J,Martens S,Klammt J,et al. Genetic analyses of bone morphogenetic protein 2,4 and 7 in congenital combined pituitary hormone deficiency[J]. BMC Endocr Disord,2013,13:56.
[31] Xu XL,Lou J,Tang T,et al. Evaluation of different scaffolds for BMP2 genetic orthopedic tissue engineering[J]. J Biomed Mater Res B Appl Biomater,2005,75(2):289-303.
[2] Zhang YD,Chen Z,Song YQ,et al. Making a tooth:growth factors,transcription factors,and stem cells[J]. Cell Res,2005,15(5):301-316.
[3] Nishinakamura R,Sakaguchi M. BMP signaling and its modifiers in kidney development[J]. Pediatr Nephrol,2014,29(4):681-686.
[4] Neubuser A,Peters H,Balling R,et al. Antagonistic interactions between FGF and BMP signaling pathways:a mechanism for positioning the sites of tooth formation[J]. Cell,1997,90(2):247-255.
[5] Yuan G,Yang G,Zheng Y,et al. The non-canonical BMP and Wnt/beta-catenin signaling pathways orchestrate early tooth development[J]. Development,2015,142(1):128-139.
[6] Zhao M,Harris SE,Horn D,et al. Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation[J]. J Cell Biol,2002,157(6):1049-1060.
[7] de Coster PJ,Marks LA,Martens LC,et al. Dental agenesis:genetic and clinical perspectives[J]. J Oral Pathol Med,2009,38(1):1-17.
[8] Yin W,Bian Z. The gene network underlying hypodontia[J]. J Dent Res,2015,94(7):878-885.
[9] Kantaputra P,Sripathomsawat W. WNT10A and isolated hypodontia[J]. Am J Med Genet A,2011,155(5):1119-1122.
[10] Wong S,Liu H,Bai B,et al. Novel missense mutations in the AXIN2 gene associated with non-syndromic oligodontia[J]. Arch Oral Biol,2014,59(3):349-353.
[11] Mues GI,Griggs R,Hartung AJ,et al. From ectodermal dysplasia to selective tooth agenesis[J]. Am J Med Genet A,2009,149(9):2037-2041.
[12] Wong S,Liu H,Han D,et al. A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia[J].Mutagenesis,2014,29(5):319-323.
[13] Stockton DW,Das P,Goldenberg M,et al. Mutation of PAX9 is associated with oligodontia[J]. Nat Genet,2000,24(1):18-19.
[14] Yu P,Yang W,Han D,et al. Mutations in WNT10B are identified in individuals with oligodontia[J]. Am J Hum Genet,2016,99(1):195-201.
[15] Liu X,Gao L,Zhao A,et al. Identification of duplication downstream of BMP2 in a Chinese family with brachydactyly type A2(BDA2)[J]. PLo S One,2014,9(4):e94201.
[16] Styrkarsdottir U,Cazier JB,Kong A,et al. Linkage of osteoporosis to chromosome 20p12 and association to BMP2[J]. PLo S Biol,2003,1(3):E69.
[17] Sahoo T,Theisen A,Sanchez-Lara PA,et al. Microdeletion20p12. 3 involving BMP2 contributes to syndromic forms of cleft palate[J]. Am J Med Genet A,2011,155(7):1646-1653.
[18] Williams ES,Uhas KA,Bunke BP,et al. Cleft palate in a multigenerational family with a microdeletion of 20p12. 3 involving BMP2[J]. Am J Med Genet A,2012,158(10):2616-2620.
[19] Mu Y,Xu Z,Contreras CI,et al. Phenotype characterization and sequence analysis of BMP2 and BMP4 variants in two Mexican families with oligodontia[J]. Genet Mol Res,2012,11(4):4110-4120.
[20]邹川,高清平,Bakrv HH,等.单纯性先天缺牙患者BMP2/BMP4基因位点分析[J].上海口腔医学,2015,24(1):83-88.
[21] Lu Y,Qian Y,Zhang J,et al Genetic variants of BMP2 and their association with the risk of non-syndromic tooth agenesis[J]. PLo S One,2016,11(6):e0158273.
[22] Prasad MK,Geoffroy V,Vicaire S,et al. A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement[J]. J Med Genet,2016,53(2):98-110.
[23] Clark GR,Duncan EL. The genetics of osteoporosis[J]. Br Med Bull,2015,113(1):73-81.
[24] Richards JB,Zheng HF,Spector TD. Genetics of osteoporosis from genome-wide association studies:advances and challenges[J]. Nat Rev Genet,2012,13(8):576-588.
[25] Hsu YH,Kiel DP. Clinical review:Genome-wide association studies of skeletal phenotypes:what we have learned and where we are headed[J]. J Clin Endocrinol Metab,2012,97(10):E1958-E1977.
[26] Richards S,Aziz N,Bale S,et al. Standards and guidelines for the interpretation of sequence variants:a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[J]. Genet Med,2015,17(5):405-424.
[27] Tan TY,Gonzaga-Jauregui C,Bhoj EJ,et al. Monoallelic BMP2variants predicted to result in haploinsufficiency cause craniofacial,skeletal,and cardiac features overlapping those of 20p12 deletions[J]. Am J Hum Genet,2017,101(6):985-994.
[28] Timberlake AT,Choi J,Zaidi S,et al. Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles[J]. Elife,2016,5(4 suppl):1.
[29] Radio FC,Majore S,Aurizi C,et al. Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP,BMP2,FTL and SLC40A1 genes[J].Blood Cells Mol Dis,2015,55(1):71-75.
[30] Breitfeld J,Martens S,Klammt J,et al. Genetic analyses of bone morphogenetic protein 2,4 and 7 in congenital combined pituitary hormone deficiency[J]. BMC Endocr Disord,2013,13:56.
[31] Xu XL,Lou J,Tang T,et al. Evaluation of different scaffolds for BMP2 genetic orthopedic tissue engineering[J]. J Biomed Mater Res B Appl Biomater,2005,75(2):289-303.