40 Chinese Journal of Otology Vol. 8 No.1 2010 DNA A1555G - ( 030001) 2 12 68 DNA (polymerase chain reaction PCR) DNA A1555G 7 DNA 12S rrna 1555 A G A1555G ; DNA A1555G ; ; ; R764.433 R342.4 A 1672-2922(2010)01-040-06 1555 G mutation detect for the mitochondrial DNA in a pedigree with maternally inherited hearing loss and sporadic non-syndromic deafness ZHAO Fang ZHANG Qin-na ENT Department Shanxi Medical University Taiyuan 030001 China Abstract Objective To study the molecular etiology of a Chinese pedgree with maternally inherited amino - glycoside antibiotics-induced deafness based on DNA microarray. Methods Twelve members in a Chinese pedigree with maternally inherited aminoglycoside antibiotic- induced deafness and 68 sporadic non-syndromic deafness were included in this study. Their genomic DNA samples were extracted from isolated leukocytes. The mitochondrial DNA target fragments were amplified by polymerase chain reaction (PCR) for detecting the mitochondrial DNA 1555 G mutation with the DNA microarray. Results Seven individuals in this pedigree carried A G mutation at the 1555th bp of mitochondrial 12S rrna. No 1555 G mutation was found in sporadic cases. Conclusion The mitochondrial DNA 1555G mutation may be one of the major factors resulting in aminoglycoside antibiotic-induced deafness in this pedigree and the 1555 G carriers were mainly distributed in the pedigree with maternally inherited deafness. Key words Hearing loss; Mitochondrial DNA; Aminogloside antibiotics; DNA microarray T7511C ) [4-6] (aminoglycoside antibiotics AmAn) mtdna 12S [1] 1 000 1 [2] rrna A1555G 1% ~ 2% [3] AmAn 12S rrna (A1555G C1494T) trna ser (UCN ) (A7445G 7472insC T7510C : 200807 : : Email: zf9669@163.com : Email: Zqn0667@yahoo.com AmAn AmAn A1555G 50 2 ~ 50
2010 8 1 41 507 ; 2 3 136 ; 7 17 ; 2 [7-4 6 7 11] ; 5 2 : (1) [12] ; (2) 12 68 13; 9 15 1 ; 1 2 3 [7] 1.1 1.1.1 ( 1) 17 18 3 1.1.2 68 19 2 16 : 14 ( ) 13 10 3 24 2 2 ( ) 3 180 ; 2 6 ; 8 I1 5
42 Chinese Journal of Otology Vol. 8 No.1 2010 1.2 DNA PCR (tag sequence) ( ); : DYY-8B ; : Centrifuge 5415R Eppendorf ; ABI 9700 PCR (polymerase chain reaction) ; ( 1.4.2 PCR ) HZQ-C 11 11 7Mix R LuxScan TM 10K / B 4Mix PCR 15 μl 100 ng DNA 10 hot- DNA star buffer 2.5 mmol / L dntp 25 mmol / L MgCl 2 1.3 DNA 3 ~ 4 ml 30 s 72 45 s 32 70 45 min 60 10 / DNA ( DNA) DNA 70% 1% 94 C 55 C; 0.2 C / s 55 C -20 70 C PCR DNA TE(pH 8.0) 1.4.3 BECKMAN DU800 PCR 95 5 min [12] 1.4 PCR Mix 5U / μl hotstar ddh 2 O PCR : 95 15 min 96 1 min 94 30 s 55 min 0.5 C / s 3 min 2.5 μl PCR 10 μl 1.4.1 50 Cy3 1 h
2010 8 1 43
44 Chinese Journal of Otology Vol. 8 No.1 2010 1 W - 1.4.4 42 2 min 42 DNA A1555G 2 min ATP 1 200 r / min 2 min 1.4.5 A1555G : LuxScan R TM 10K / B 90 532 nm 1.4.6 ( 3 1) 2 80 : 1 ( 12 7 ( 6 13) ) 5 ; ; 2 3 5 68 7 1 7 2 4 17 1 mtdna A1555G 58.3%; 7 mtdna A1555G 6 AmAn 85.7% 4 ( ) 2 12 68 10 SLC26A4 ( 7 14.7% ) 8 GJB2 ( 11.8% ); 1 DNA A1555G 7 GJB2 SLC26A4 ; 1 GJB2 5 ; mtdna A1555G 0 mtdna A1555G 9) 7 A1555G 6 3 [13] A1555G 1996 M - ( ) [14] Prezant A1555G 12S rrna 12S rrna 12S rrna [15] DNA [16] A1555G A1555G ( 1 ( 13) 1993 Prezant 3 AmAn AmAn mtdna 12S (5 / 6) rrna 1555 A G
2010 8 1 45 hot spot for mutations associated with aminoglycoside induced and non -syndromic hearing loss. Biochem Biophys Res Commun mtdna 2007 361(1): 133-139. A1555G [17] 961 DNA1555G 1998 33 [18] [19] C A1555G (2): 67-70. [20] 8 DNA 1555A-G. 1999 14 (2): 67-68. 0% ~ 17% 9. 3% ~ 29%. 2001 15( ): : 1 53-58. 42.9% (9 / 21) 10 47.6% (10 / 21) 7... 29. mtdna. 2001 23(5): 401-405. 11. A1555G. A1555G 2004 (6): 21-23. 12 2008 6(1): 61-66. 13 Prezant TR Agapian JV Bohlman MC et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic induced and non-syndromic deafness. Nat Genet 1993 4 (3): 289-294. 14 DNA. 1996 18(6): 3-5. ( ) 15 Zhao H Li R Wang Q et al. Maternally inherited amino - glycoside-induced and nonsyndromic deafness is associated with mtdna A1555G the novel C1494T mutation in the mitochondrial 12S rrna gene in a large Chinese family. Am J Hum Genet 2004 Jan 74(1): 139 DNA -152. 16 Usami S Koda E Tsukamoto K et el. Molecular diagnosis of deafness: impact of gene identification. Audiol Neuro-Otol 2002 7(3): 185-190.... 1 Bitner -Glindzicz M. Hereditary deafness and phenotyping in hu - mans. Br Med Bull 2002 63: 73-94. 17 Pandya A Xia XJ Erdenetungalag R et al. Heterogenous point mutations in the mitochondrial trna Ser (UCN) precursor co- existing 2 with the A1555G mutation in deaf students from Mongolia. Am J Toriello HV Reardon W Gorlin RJ.. 2 [M].. : Hum Genet 1999 65(6): 1803-1806. 2006: 10. 18 Li R Xing G Yan M et al. Cosegregation of C -insertion at 3. DNA. 2008 32(4): 210-212. position 961 with the A1555G mutation of the mitochondrial 12SrRNA gene in a large Chinese family with maternally inherited 4 Li RH Ishikawa K Deng JH et al. Maternally inherited non - hearing loss. Am J Med Genet 2004 124A (2): 113-117. syndromic hearing loss is associated with the T7511C mutation in ser (UCN) the mitochondrial trna gene in a Japanese family. Biochem Biophys Res Commun 2005 328(1): 32-37. 19 Yoshida M Shintani T Hirao M et al. Aminoglycoside induced hearing Loss in a patient with the 961 mutation in mitochondrial DNA. ORL J Otorhinolaryngol Relat Spec 2002 64(3): 219-222. 5 Hutchin TP Parker MJ Young ID et al. A novel mutation in the mitochondrial trna Ser(UCN) gene in a family with non-syndromic sensorineural hearing impairment. J Med Genet 2000 37 (9): 692-20 Tang XW Li Y Zhu Y et al. Very low penetrance of hearing loss in seven Han Chinese pedigrees carrying the deafness-associated 12S rrna A1555G mutation. Gene 2007 393(1-2 ): 11-19. 694. 6 Jin LJ Yang AF Zhu Y et al. Mitochondrial trna Ser(UCN) gene is the ( :2009-7-10)