CX26第86位氨基酸多态性与遗传性聋相关性分析
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摘要
目的本研究在细胞生物学水平,对第86位氨基酸分别为Thr,Ser,Arg的CX26蛋白的膜定位生物学功能进行鉴定,进而结合临床数据分析该位点不同多态性突变的潜在致聋可能性。方法本研究借助GFP融合型慢病毒表达系统,分别将第86位氨基酸为Thr,Ser,Arg的CX26-GFP融合蛋白在小鼠螺旋神经节细胞中进行表达,并在荧光显微镜下观察融合蛋白的细胞膜定位现象。结果第86位氨基酸发生Ser突变后,CX26蛋白仍能够正常定位于细胞膜表面,并形成间隙链接结构,这一表型与(第86位氨基酸为Thr的)野生型CX26蛋白并无明显差异;只有当第86位氨基酸突变为Arg时,导致CX26蛋白功能丧失。结论这些现象表明CX26 T86R可能为潜在致聋突变,而CX26 T86S为不影响蛋白功能的氨基酸多态性突变。该结论与临床筛查经验性结果一致。
Objective To investigate the member localization function of CX26 protein when its 86 th amino acid is Thr, Ser or Arg, and its relations to deafness. Methods CX26-GFP protein with either Thr, Ser or Arg as the 86 th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope.Results The mutated protein of CX26 T86 S was localized to cell membrane and form gap conjunction structures, showing no difference to the wild type CX26 protein(with Thr as the 86 th amino acid). However, the gap conjunction structure disappeared when the mutation was CX26 T86 A. Conclusion These results indicate that the CX26 T86 R mutation may be a cause of hearing loss, but the CX26 T86 S as a non-pathogenic polymorphism mutation does not affect functions of the CX26 protein. The results are in accordance with the results of clinical screening.
Objective To investigate the member localization function of CX26 protein when its 86 th amino acid is Thr, Ser or Arg, and its relations to deafness. Methods CX26-GFP protein with either Thr, Ser or Arg as the 86 th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope.Results The mutated protein of CX26 T86 S was localized to cell membrane and form gap conjunction structures, showing no difference to the wild type CX26 protein(with Thr as the 86 th amino acid). However, the gap conjunction structure disappeared when the mutation was CX26 T86 A. Conclusion These results indicate that the CX26 T86 R mutation may be a cause of hearing loss, but the CX26 T86 S as a non-pathogenic polymorphism mutation does not affect functions of the CX26 protein. The results are in accordance with the results of clinical screening.
引文
1 Shou-Xia Li,Ding-Li Chen,Su-Bin Zhao,et al.Cordblood-Based High-Throμghput Screening for Deafness Gene of 646 Newborns in Jinan Area of China.Clinical and Experimental Otorhinolaryngology.2015,8(3):211-217.
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3 Propst EJ,Stockley TL,Gordon KA,et al.Ethnicity and mutations in GJB2(connexin 26)and GJB6(connexin 30)in a mμlti-cμltural Canadian paediatric Cochlear Implant Program.Int J Pediatr Otorhinolaryngol.2006,70(3):435-44.
4 Qinjun Wei,Youfuo Liu,Shuai Wang,et al.A novel compound heterozygous mutation in the GJB2 gene causing non-syndromic hearing loss in a family.INTERNATIONAL JOURNAL OFMOLECΜLAR MEDICINE 2014,33(2):310-316,5 Zelante L,Gasparini P,Estivill X,et al.Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness(DFNB1)in Mediterraneans.Hμm Mol Genet.1997,6(9):1605-1609.
6 Petersen MB,Willems PJ.Non-syndromic,autosomal-recessive deafness.Clin Genet.2006,69(5):371-392.
7 Soo-Young Choi,Park HJ,Lee KY,et al.Different Functional Consequences of Two Missense Mutations in the GJB2 Gene Associated with Nonsyndromic Hearing Loss.Hum Mutat.2009,30(7):E716-27.
8 Van Laer L,Mc Guirt WT,Yang T,et al.Autosomal dominant nonsyndromic hearing impairment.Am J Med Genet.1999,89(3):167-74.
9 Welch KO,Marin RS,Pandya A,et al.Compound heterozygosity for dominant and recessive GJB2 mutations:effect on phenotype and review of the literature.Am J Med Genet A.2007,143A(14):1567-1573.
10 Yan D,Ke X,Blanton SH,et al.A novel locus for autosomal dominant non-syndromic deafness,DFNA53,maps to chromosome14q11.2-q12.J Med Genet.2006,43(2):170-4.
11 Kenneson A,Van Naarden Braun K and Boyle C:GJB2(connexin
26)variants and nonsyndromic sensorineural hearing loss:a Hμg Ereview.Genet Med.2002,4(4):258-274.
12 Estivill X,Fortina P,Surrey S,et al.Connexin-26 mutations in sporadic and inherited sensorineural deafness.1998,351(9100):394-8.
13 Morell RJ,Kim HJ,Hood LJ,et al.Mutations in the connexin 26gene(GJB2)among Ashkenazi Jews with nonsyndromic recessive deafness.N Engl J Med.1998,339(21):1500-1505.
14 Abe S,Usami S,Shinkawa H,et al.Prevalent connexin 26 gene(GJB2)mutations in Japanese.J Med Genet.2000,37(1):41-43.
15 Sobe T,Vreμgde S,Shahin H,et al.The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli popμlation.Hμm Genet.2000,106(1):50-57.
16 Dai P,Yu F,Han B,et al.GJB2 mutation spectrμm in 2,063 Chinese patients with nonsyndromic hearing impairment.J Transl Med.2009,7:26.
17刘德胜,王金丽,撒亚莲,等.傣族、汉族非综合征型耳聋患者GJB2基因分析.昆明医科大学学报2012,(10):49~52
18 HILGERT N,SMITH R J,VAN CAMP G.Forty-six genes causing nonsyndromic hearing impairment:which ones shoμld be analyzed in DNA diagnostics.Mutat Res,2009,681(2-3):189-196.
19 Lee S.W.,Tomasetto C.,Paμl D.L.,et al.Transcriptional downregμlation of gap-junction proteins blocks junctional communication in hμman mammary tμmor cell lines.J.Cell Biol.1992,118(5):1213-1221.
20郑文波,罗建红,郦云,等.中国人语前非综合征性耳聋患者GJB2基因的突变分析中华儿科杂志.2000,38(10).
2 Ouyang XM,Yan D,Yuan HJ,Pu D,Du LL,et al.The genetic bases for non-syndromic hearing loss among Chinese.J Hμm Genet.2009,54(3):131-40.
3 Propst EJ,Stockley TL,Gordon KA,et al.Ethnicity and mutations in GJB2(connexin 26)and GJB6(connexin 30)in a mμlti-cμltural Canadian paediatric Cochlear Implant Program.Int J Pediatr Otorhinolaryngol.2006,70(3):435-44.
4 Qinjun Wei,Youfuo Liu,Shuai Wang,et al.A novel compound heterozygous mutation in the GJB2 gene causing non-syndromic hearing loss in a family.INTERNATIONAL JOURNAL OFMOLECΜLAR MEDICINE 2014,33(2):310-316,5 Zelante L,Gasparini P,Estivill X,et al.Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness(DFNB1)in Mediterraneans.Hμm Mol Genet.1997,6(9):1605-1609.
6 Petersen MB,Willems PJ.Non-syndromic,autosomal-recessive deafness.Clin Genet.2006,69(5):371-392.
7 Soo-Young Choi,Park HJ,Lee KY,et al.Different Functional Consequences of Two Missense Mutations in the GJB2 Gene Associated with Nonsyndromic Hearing Loss.Hum Mutat.2009,30(7):E716-27.
8 Van Laer L,Mc Guirt WT,Yang T,et al.Autosomal dominant nonsyndromic hearing impairment.Am J Med Genet.1999,89(3):167-74.
9 Welch KO,Marin RS,Pandya A,et al.Compound heterozygosity for dominant and recessive GJB2 mutations:effect on phenotype and review of the literature.Am J Med Genet A.2007,143A(14):1567-1573.
10 Yan D,Ke X,Blanton SH,et al.A novel locus for autosomal dominant non-syndromic deafness,DFNA53,maps to chromosome14q11.2-q12.J Med Genet.2006,43(2):170-4.
11 Kenneson A,Van Naarden Braun K and Boyle C:GJB2(connexin
26)variants and nonsyndromic sensorineural hearing loss:a Hμg Ereview.Genet Med.2002,4(4):258-274.
12 Estivill X,Fortina P,Surrey S,et al.Connexin-26 mutations in sporadic and inherited sensorineural deafness.1998,351(9100):394-8.
13 Morell RJ,Kim HJ,Hood LJ,et al.Mutations in the connexin 26gene(GJB2)among Ashkenazi Jews with nonsyndromic recessive deafness.N Engl J Med.1998,339(21):1500-1505.
14 Abe S,Usami S,Shinkawa H,et al.Prevalent connexin 26 gene(GJB2)mutations in Japanese.J Med Genet.2000,37(1):41-43.
15 Sobe T,Vreμgde S,Shahin H,et al.The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli popμlation.Hμm Genet.2000,106(1):50-57.
16 Dai P,Yu F,Han B,et al.GJB2 mutation spectrμm in 2,063 Chinese patients with nonsyndromic hearing impairment.J Transl Med.2009,7:26.
17刘德胜,王金丽,撒亚莲,等.傣族、汉族非综合征型耳聋患者GJB2基因分析.昆明医科大学学报2012,(10):49~52
18 HILGERT N,SMITH R J,VAN CAMP G.Forty-six genes causing nonsyndromic hearing impairment:which ones shoμld be analyzed in DNA diagnostics.Mutat Res,2009,681(2-3):189-196.
19 Lee S.W.,Tomasetto C.,Paμl D.L.,et al.Transcriptional downregμlation of gap-junction proteins blocks junctional communication in hμman mammary tμmor cell lines.J.Cell Biol.1992,118(5):1213-1221.
20郑文波,罗建红,郦云,等.中国人语前非综合征性耳聋患者GJB2基因的突变分析中华儿科杂志.2000,38(10).