携带线粒体tRNA~(Thr)15910C>T突变和12S rRNA 1555A>G突变的非综合征型耳聋家系的线粒体功能研究
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摘要
该研究旨在探讨一个非综合征型耳聋(nonsyndromic hearing impairment,NSHI)家系中线粒体t RNAThr 15910C>T和12S r RNA 1555A>G基因突变共同作用对线粒体功能的影响。该研究建立了同时携带线粒体t RNAThr 15910C>T和12S r RNA 1555A>G基因突变(双突变组)、仅携带12S r RNA 1555A>G基因突变(单突变组)和对照组的永生化淋巴细胞,这3组细胞系的线粒体DNA单体型均属于R单体型。对该家系的临床资料进行分析,当包括使用氨基糖苷类抗生素(aminoglycoside antibiotics,Am An)的药物性耳聋家系成员时,此家系耳聋外显率为37.5%;当排除用药的耳聋成员时,耳聋外显率是25.0%;相比之下,在用药和未用药的情况下,已报道的14个m.1555A>G的耳聋家系的平均外显率只有12.8%和6.1%。通过对双突变组、单突变组和对照组永生化细胞系的线粒体功能进行研究,结果发现与对照组相比,双突变和单突变组细胞系的ROS水平分别上升了19.08%(P=0.005 4)和9.05%(P=0.003 7);ΔΨm水平分别下降了47.78%(P=0.006 3)和35.39%(P=0.0245);复合体II活力分别下降了8.26%(P=0.721 1)和19.48%(P=0.004 9),复合体IV活力分别下降了32.75%(P=0.033 5)和27.44%(P=0.180 5)。m.1555A>G与m.15910C>T共同作用,导致ROS生成量升高,ΔΨm水平下降以及线粒体呼吸链复合体IV活力降低等线粒体功能缺陷,提示m.15910C>T可能是m.1555A>G导致耳聋的继发性突变。
In this study, we investigated the mitochondrial function of 12 S r RNA 1555A>G and mitochondria t RNAThr 15910C>T mutations in a nonsyndromic hearing loss(NSHL) family. We established three groups of lymphoblastoid cell lines in this study, including the double mutations group, the single mutation group and the controls, which all belong to the Eastern Asian haplogroup R. The double mutations group are 3 subjects carried both m.1555A>G and m.15910C>T. The single mutation group are 3 subjects carried m.1555A>G mutation only. The controls are 3 subjects with normal auditory sense. To analyze the clinical data of the family, the penetrance of deafness is 37.5% or 25.0% which contains or excludes the family members who have used Am An drugs; while the penetrance of deafness is 12.8% or 6.1% which contains or excludes the family members who have used Am An drugs in the 14 reported deafness families with m.1555A>G mutation. Compared with controls, the ROS level in the double-mutations group has been raised 19.08%(P=0.005 4) and the single-mutation group only raised 9.05%(P=0.003 7). Compared with controls, the mitochondrial membrane potential decreased 47.78%(P=0.006 3) and 35.39%(P=0.024 5) in the double-mutations group and the single-mutation group, respectively. It showed that 8.26%(P=0.721 1) and 19.48%(P=0.004 9) decrease in the activity of respiratory chain complex II while 32.75%(P=0.033 5) and 27.44%(P=0.180 5) decrease in the activity of respiratory chain complex IV in the double-mutations group and the single-mutation group compared with controls. These results showed that m.1555A>G and m.15910C>T led to mitochondrial dysfunction, included ROS raise up, mitochondrial membrane potential and the activity of respiratory chain complex IV decreased. Thus, m.15910C>T worsens the mitochondrial function associated with m.1555A>G, and is probably a secondary mutation of m.1555A>G caused deafness.
In this study, we investigated the mitochondrial function of 12 S r RNA 1555A>G and mitochondria t RNAThr 15910C>T mutations in a nonsyndromic hearing loss(NSHL) family. We established three groups of lymphoblastoid cell lines in this study, including the double mutations group, the single mutation group and the controls, which all belong to the Eastern Asian haplogroup R. The double mutations group are 3 subjects carried both m.1555A>G and m.15910C>T. The single mutation group are 3 subjects carried m.1555A>G mutation only. The controls are 3 subjects with normal auditory sense. To analyze the clinical data of the family, the penetrance of deafness is 37.5% or 25.0% which contains or excludes the family members who have used Am An drugs; while the penetrance of deafness is 12.8% or 6.1% which contains or excludes the family members who have used Am An drugs in the 14 reported deafness families with m.1555A>G mutation. Compared with controls, the ROS level in the double-mutations group has been raised 19.08%(P=0.005 4) and the single-mutation group only raised 9.05%(P=0.003 7). Compared with controls, the mitochondrial membrane potential decreased 47.78%(P=0.006 3) and 35.39%(P=0.024 5) in the double-mutations group and the single-mutation group, respectively. It showed that 8.26%(P=0.721 1) and 19.48%(P=0.004 9) decrease in the activity of respiratory chain complex II while 32.75%(P=0.033 5) and 27.44%(P=0.180 5) decrease in the activity of respiratory chain complex IV in the double-mutations group and the single-mutation group compared with controls. These results showed that m.1555A>G and m.15910C>T led to mitochondrial dysfunction, included ROS raise up, mitochondrial membrane potential and the activity of respiratory chain complex IV decreased. Thus, m.15910C>T worsens the mitochondrial function associated with m.1555A>G, and is probably a secondary mutation of m.1555A>G caused deafness.
引文
1 Morton CC.Genetics,genomics and gene discovery in the auditory system.Hum Mol Genet 2002;11(10):1229-40.
2 Guan MX.Mitochondrial 12S r RNA mutations associated with aminoglycoside ototoxicity.Mitochondrion 2011;11(2):237-45.
3 Helm M,Brule H,Friede D,Giege R,Putz D,Florentz C.Search for characteristic structural features of mammalian mitochondrial t RNAs.RNA 2000;6(10):1356-79.
4 Guan MX.Molecular pathogenetic mechanism of maternally inherited deafness.Ann N Y Acad Sci 2004;1011:259-71.
5 Fischel-Ghodsian N.Mitochondrial deafness mutations reviewed Hum Mutat 1999;13(4):261-70.
6 Zhao H,Li R,Wang Q,Yan Q,Deng JH,Han D,et al.Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial12S r RNA gene in a large Chinese family.Am J Hum Genet2004;74(1):139-52.
7 Zheng J,Ji Y,Guan MX.Mitochondrial t RNA mutations associated with deafness.Mitochondrion 2012;12(3):406-13.
8 Chen B,Sun D,Yang L,Zhang C,Yang A,Zhu Y,et al.Mitochondrial ND5 T12338C,t RNA(Cys)T5802C,and t RNA(Thr)G15927A variants may have a modifying role in the phenotypic manifestation of deafness-associated 12S r RNA A1555Gmutation in three Han Chinese pedigrees.Am J Med Genet A2008;146A(10):1248-58.
9 Andrews RM,Kubacka I,Chinnery PF,Lightowlers RNTurnbull DM,Howell N.Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nat Genet 1999;23(2):147.
10 Yu J,Zheng J,Zhao X,Liu J,Mao Z,Ling Y,et al.Aminoglycoside stress together with the 12S r RNA 1494C>T mutation leads to mitophagy.PLo S One 2014;9(12):e114650.
11 Mukhopadhyay P,Rajesh M,Hasko G,Hawkins BJ,Madesh M,Pacher P.Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy.Nat Protoc 2007;2(9):2295-301.
12 Gong S,Peng Y,Jiang P,Wang M,Fan M,Wang X,et al.Adeafness-associated t RNAHis mutation alters the mitochondrial function,ROS production and membrane potential.Nucleic Acids Res 2014;42(12):8039-48.
13 Garner DL,Thomas CA,Joerg HW,DeJ arnette JM,Marshall CEFluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.Biol Reprod 1997;57(6):1401-6.
14 Birch-Machin MA,Turnbull DM.Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues.Methods Cell Biol 2001;65:97-117.
15 Kirby DM,Thorburn DR,Turnbull DM,Taylor RW.Biochemical assays of respiratory chain complex activity.Methods Cell Biol2007;80:93-119.
16 Brown MD,Zhadanov S,Allen JC,Hosseini S,Newman NJAtamonov VV,et al.Novel mt DNA mutations and oxidative phosphorylation dysfunction in Russian LHON families.Hum Genet 2001;109(1):33-9.
17 Zhou X,Qian Y,Zhang J,Tong Y,Jiang P,Liang M,et al.Leber’s hereditary optic neuropathy is associated with the T3866C mutation in mitochondrial ND1 gene in three Han Chinese Families.Invest Ophthalmol Vis Sci 2012;53(8):4586-94.
18 Ying ZB,Zheng J,Cai ZY,Liu L,Dai Y,Yao J,et al.Mitochondrial haplogroup B increases the risk for hearing loss among the Eastern Asian pedigrees carrying 12S r RNA 1555A>Gmutation.Pro Cell 2015;6(11):844-8.
19 Tang XW,Li ZY,Lu JX,Zhu Y,Li RH,Wang JD,et al Mitochondrial t RNAThr G15927A mutation may influence the phenotypic manifestation of deafness-associated 12S r RNAA1555G mutation.Yi Chuan 2008;30(10):1287-94.
20 Jiang P,Jin X,Peng Y,Wang M,Liu H,Liu X,et al.The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-t RNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber’s hereditary optic neuropathy-associated mitochondrial DNA mutation.Hum Mol Genet 2016;25(3):584-96.
21 Young WY,Zhao L,Qian Y,Wang Q,Li N,Greinwald JH Jr et al.Extremely low penetrance of hearing loss in four Chinese families with the mitochondrial 12S r RNA A1555G mutation Biochem Biophys Res Commun 2005;328(4):1244-51.
22 Tang XW,Yang L,Zhu Y,Liao ZS,Wang JD,Qian YP,et al Very low penetrance of hearing loss in seven Han Chinese pedigrees carrying the deafness-associated 12S r RNA A1555Gmutation.Gene 2007;393(1/2):11-9.
23 Dai P,Liu X,Han D,Qian Y,Huang D,Yuan H,et al.Extremely low penetrance of deafness associated with the mitochondrial12S r RNA mutation in 16 Chinese families:Implication for early detection and prevention of deafness.Biochem Biophys Res Commun 2006;340(1):194-9.
24 Young WY,Zhao L,Qian Y,Li R,Chen J,Yuan H,et al.Variants in mitochondrial t RNAGlu,t RNAArg,and t RNAThr may influence the phenotypic manifestation of deafness-associated12S r RNA A1555G mutation in three Han Chinese families with hearing loss.Am J Med Genet A 2006;140(20):2188-97.
25 Yang AF,Zhu Y,Lu JX,Yang L,Zhao JY,Sun DM.Mitochondrial DNA G7444A mutation may influence the phenotypic manifestation of the deafness-associated 12S r RNA A1555Gmutation.Yi Chuan 2008;30(6):728-34.
26 Zhao LD,Wang QJ,Qian YP,Li RH,Cao JY,Hart LC,et al Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss.Biochem Biophysic Res Commun 2005;336(3):967-73.
27 de Andrade PB,Rubi B,Frigerio F,van den Ouweland JMMaassen JA,Maechler P.Diabetes-associated mitochondrial DNAmutation A3243G impairs cellular metabolic pathways necessary for beta cell function.Diabetologia 2006;49(8):1816-26.
28 Lenaz G,Baracca A,Carelli V,D’Aurelio M,Sgarbi G,Solaini G.Bioenergetics of mitochondrial diseases associated with mt DNAmutations.Biochim Biophys Acta 2004;1658(1/2):89-94.
29 St-Pierre J,Buckingham JA,Roebuck SJ,Brand MD.Topology of superoxide production from different sites in the mitochondrial electron transport chain.J Biol Chem 2002;277(47):44784-90.
30 Scheffler IE.Mitochondrial disease associated with complex I(NADH-Co Q oxidoreductase)deficiency.J Inherit Metab Dis2015;38(3):405-15.
31 Addabbo F,Montagnani M,Goligorsky MS.Mitochondria and reactive oxygen species.Hypertension 2009;53(6):885-92.
32 Hayashi G,Cortopassi G.Oxidative stress in inherited mitochondrial diseases.Free Radic Biol Med 2015;88(Pt A):10-7.
33 Eirin A,Lerman A,Lerman LO.Mitochondria:A pathogenic paradigm in hypertensive renal disease.Hypertension 2015;65(2):264-70.
34 Raimundo N,Song L,Shutt TE,McK ay SE,Cotney J,Guan MX,et al.Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell 2012;148(4):716-26.
35 Rybak LP,Whitworth CA,Mukherjea D,Ramkumar V.Mechanisms of cisplatin-induced ototoxicity and prevention.Hear Res 2007;226(1/2):157-67.
36 Riva C,Donadieu E,Magnan J,Lavieille JP.Age-related hearing loss in CD/1 mice is associated to ROS formation and HIF target proteins up-regulation in the cochlea.Exp Gerontol 2007;42(4):327-36.
37 Leis JA,Rutka JA,Gold WL.Aminoglycoside-induced ototoxicity.CMAJ 2015;187(1):E52.
38 Hirose K,Hockenbery DM,Rubel EW.Reactive oxygen species in chick hair cells after gentamicin exposure in vitro.Hear Res1997;104(1/2):1-14.
39 Henderson D,Bielefeld EC,Harris KC,Hu BH.The role of oxidative stress in noise-induced hearing loss.Ear Hear 2006;27(1):1-19.
2 Guan MX.Mitochondrial 12S r RNA mutations associated with aminoglycoside ototoxicity.Mitochondrion 2011;11(2):237-45.
3 Helm M,Brule H,Friede D,Giege R,Putz D,Florentz C.Search for characteristic structural features of mammalian mitochondrial t RNAs.RNA 2000;6(10):1356-79.
4 Guan MX.Molecular pathogenetic mechanism of maternally inherited deafness.Ann N Y Acad Sci 2004;1011:259-71.
5 Fischel-Ghodsian N.Mitochondrial deafness mutations reviewed Hum Mutat 1999;13(4):261-70.
6 Zhao H,Li R,Wang Q,Yan Q,Deng JH,Han D,et al.Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial12S r RNA gene in a large Chinese family.Am J Hum Genet2004;74(1):139-52.
7 Zheng J,Ji Y,Guan MX.Mitochondrial t RNA mutations associated with deafness.Mitochondrion 2012;12(3):406-13.
8 Chen B,Sun D,Yang L,Zhang C,Yang A,Zhu Y,et al.Mitochondrial ND5 T12338C,t RNA(Cys)T5802C,and t RNA(Thr)G15927A variants may have a modifying role in the phenotypic manifestation of deafness-associated 12S r RNA A1555Gmutation in three Han Chinese pedigrees.Am J Med Genet A2008;146A(10):1248-58.
9 Andrews RM,Kubacka I,Chinnery PF,Lightowlers RNTurnbull DM,Howell N.Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nat Genet 1999;23(2):147.
10 Yu J,Zheng J,Zhao X,Liu J,Mao Z,Ling Y,et al.Aminoglycoside stress together with the 12S r RNA 1494C>T mutation leads to mitophagy.PLo S One 2014;9(12):e114650.
11 Mukhopadhyay P,Rajesh M,Hasko G,Hawkins BJ,Madesh M,Pacher P.Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy.Nat Protoc 2007;2(9):2295-301.
12 Gong S,Peng Y,Jiang P,Wang M,Fan M,Wang X,et al.Adeafness-associated t RNAHis mutation alters the mitochondrial function,ROS production and membrane potential.Nucleic Acids Res 2014;42(12):8039-48.
13 Garner DL,Thomas CA,Joerg HW,DeJ arnette JM,Marshall CEFluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.Biol Reprod 1997;57(6):1401-6.
14 Birch-Machin MA,Turnbull DM.Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues.Methods Cell Biol 2001;65:97-117.
15 Kirby DM,Thorburn DR,Turnbull DM,Taylor RW.Biochemical assays of respiratory chain complex activity.Methods Cell Biol2007;80:93-119.
16 Brown MD,Zhadanov S,Allen JC,Hosseini S,Newman NJAtamonov VV,et al.Novel mt DNA mutations and oxidative phosphorylation dysfunction in Russian LHON families.Hum Genet 2001;109(1):33-9.
17 Zhou X,Qian Y,Zhang J,Tong Y,Jiang P,Liang M,et al.Leber’s hereditary optic neuropathy is associated with the T3866C mutation in mitochondrial ND1 gene in three Han Chinese Families.Invest Ophthalmol Vis Sci 2012;53(8):4586-94.
18 Ying ZB,Zheng J,Cai ZY,Liu L,Dai Y,Yao J,et al.Mitochondrial haplogroup B increases the risk for hearing loss among the Eastern Asian pedigrees carrying 12S r RNA 1555A>Gmutation.Pro Cell 2015;6(11):844-8.
19 Tang XW,Li ZY,Lu JX,Zhu Y,Li RH,Wang JD,et al Mitochondrial t RNAThr G15927A mutation may influence the phenotypic manifestation of deafness-associated 12S r RNAA1555G mutation.Yi Chuan 2008;30(10):1287-94.
20 Jiang P,Jin X,Peng Y,Wang M,Liu H,Liu X,et al.The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-t RNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber’s hereditary optic neuropathy-associated mitochondrial DNA mutation.Hum Mol Genet 2016;25(3):584-96.
21 Young WY,Zhao L,Qian Y,Wang Q,Li N,Greinwald JH Jr et al.Extremely low penetrance of hearing loss in four Chinese families with the mitochondrial 12S r RNA A1555G mutation Biochem Biophys Res Commun 2005;328(4):1244-51.
22 Tang XW,Yang L,Zhu Y,Liao ZS,Wang JD,Qian YP,et al Very low penetrance of hearing loss in seven Han Chinese pedigrees carrying the deafness-associated 12S r RNA A1555Gmutation.Gene 2007;393(1/2):11-9.
23 Dai P,Liu X,Han D,Qian Y,Huang D,Yuan H,et al.Extremely low penetrance of deafness associated with the mitochondrial12S r RNA mutation in 16 Chinese families:Implication for early detection and prevention of deafness.Biochem Biophys Res Commun 2006;340(1):194-9.
24 Young WY,Zhao L,Qian Y,Li R,Chen J,Yuan H,et al.Variants in mitochondrial t RNAGlu,t RNAArg,and t RNAThr may influence the phenotypic manifestation of deafness-associated12S r RNA A1555G mutation in three Han Chinese families with hearing loss.Am J Med Genet A 2006;140(20):2188-97.
25 Yang AF,Zhu Y,Lu JX,Yang L,Zhao JY,Sun DM.Mitochondrial DNA G7444A mutation may influence the phenotypic manifestation of the deafness-associated 12S r RNA A1555Gmutation.Yi Chuan 2008;30(6):728-34.
26 Zhao LD,Wang QJ,Qian YP,Li RH,Cao JY,Hart LC,et al Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss.Biochem Biophysic Res Commun 2005;336(3):967-73.
27 de Andrade PB,Rubi B,Frigerio F,van den Ouweland JMMaassen JA,Maechler P.Diabetes-associated mitochondrial DNAmutation A3243G impairs cellular metabolic pathways necessary for beta cell function.Diabetologia 2006;49(8):1816-26.
28 Lenaz G,Baracca A,Carelli V,D’Aurelio M,Sgarbi G,Solaini G.Bioenergetics of mitochondrial diseases associated with mt DNAmutations.Biochim Biophys Acta 2004;1658(1/2):89-94.
29 St-Pierre J,Buckingham JA,Roebuck SJ,Brand MD.Topology of superoxide production from different sites in the mitochondrial electron transport chain.J Biol Chem 2002;277(47):44784-90.
30 Scheffler IE.Mitochondrial disease associated with complex I(NADH-Co Q oxidoreductase)deficiency.J Inherit Metab Dis2015;38(3):405-15.
31 Addabbo F,Montagnani M,Goligorsky MS.Mitochondria and reactive oxygen species.Hypertension 2009;53(6):885-92.
32 Hayashi G,Cortopassi G.Oxidative stress in inherited mitochondrial diseases.Free Radic Biol Med 2015;88(Pt A):10-7.
33 Eirin A,Lerman A,Lerman LO.Mitochondria:A pathogenic paradigm in hypertensive renal disease.Hypertension 2015;65(2):264-70.
34 Raimundo N,Song L,Shutt TE,McK ay SE,Cotney J,Guan MX,et al.Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell 2012;148(4):716-26.
35 Rybak LP,Whitworth CA,Mukherjea D,Ramkumar V.Mechanisms of cisplatin-induced ototoxicity and prevention.Hear Res 2007;226(1/2):157-67.
36 Riva C,Donadieu E,Magnan J,Lavieille JP.Age-related hearing loss in CD/1 mice is associated to ROS formation and HIF target proteins up-regulation in the cochlea.Exp Gerontol 2007;42(4):327-36.
37 Leis JA,Rutka JA,Gold WL.Aminoglycoside-induced ototoxicity.CMAJ 2015;187(1):E52.
38 Hirose K,Hockenbery DM,Rubel EW.Reactive oxygen species in chick hair cells after gentamicin exposure in vitro.Hear Res1997;104(1/2):1-14.
39 Henderson D,Bielefeld EC,Harris KC,Hu BH.The role of oxidative stress in noise-induced hearing loss.Ear Hear 2006;27(1):1-19.