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1、三核苷酸重复与人类疾病三核苷酸重复与人类疾病Trinucleotiderepeatsandhumandisorder简介简介（INTRODUCTIONINTRODUCTION ）1.三核苷酸重复是生物体内常见的微卫星序列（Trinucleotide repeats(TNRs)are microsatellite Trinucleotide repeats(TNRs)are microsatellite sequencessequences）2.三核苷酸不稳定重复导致疾病是突变重要且独特的形式（Disease-causing repeat instability is an Disease-ca
2、using repeat instability is an important and unique form of mutationimportant and unique form of mutation）3.三核苷酸重复和40多种神经肌肉和神经退行性疾病有关（linked to more than 40 neurological,linked to more than 40 neurological,neurodegenerative and neuromuscular disorders.neurodegenerative and neuromuscular disorders.）比
3、如 亨廷顿氏病（Huntingtons diseaseHuntingtons disease）；肌强直性营养不良（myotonic dystrophymyotonic dystrophy）；脆性X染色体（fragile X syndromefragile X syndrome）三核苷酸重复带来人类疾病特点三核苷酸重复带来人类疾病特点characteristicsofdisorderscausedbytrinucleotiderepeatcharacteristicsofdisorderscausedbytrinucleotiderepeat1.不稳定重复突变既有体细胞也有性细胞 (the th
4、e mutant repeats show both somatic and germline instability)mutant repeats show both somatic and germline instability)2.表现为发病年龄越来越早（an earlier age of onsetan earlier age of onset）发病程度越来越重（and increasing severity of and increasing severity of phenotype in subsequent generationsphenotype in subsequent
5、 generations）即所谓的遗传早发现象（anticipation）3.双亲的来源影响遗传早发现象，三核苷酸通过父亲比母亲遗传更多（the parental origin of the the parental origin of the disease allele can often influence anticipationdisease allele can often influence anticipation）3.重复在非编码区域降低转录和翻译（Repeats in non-coding regions-reduced transcription or translati
6、on）当然，理解症状的分子机制是开发每种疾病治疗方法的根本（Understanding the molecular mechanisms behind the symptoms specific to each disease will allow new therapies to be developed）1.重复在蛋白质编码区域导致毒蛋白（Repeats in protein coding sequences-toxic proteins）2.重复在RNA编码区域改变RNA功能（Repeats in RNA coding regions-altered RNA function）三核苷酸重
7、复带来疾病的机制三核苷酸重复带来疾病的机制DiseaseDiseaseMutation unitMutation unitGene name Gene name(protein(protein product)product)Putative Putative functionfunctionNormal Normal repeat lengthrepeat lengthPathogenic Pathogenic repeat lengthrepeat lengthDM1DM1强直性肌营强直性肌营强直性肌营强直性肌营养不良养不良养不良养不良(CTG)n(CTG)nDMPKDMPK (DMPK)
8、(DMPK)RNA mediatedRNA mediated5-375-3750-100050-1000DM2DM2强直性肌营强直性肌营强直性肌营强直性肌营养不良养不良养不良养不良(CCTG)n(CCTG)nZNF9ZNF9(ZNF9)(ZNF9)RNA mediatedRNA mediated10-2610-2675-11,00075-11,000FXATASFXATAS(CGG)n(CGG)nFMR1 FMR1(FMRP)(FMRP)RNA mediatedRNA mediated6-606-6060-20060-200重复在RNA编码区域改变RNA功能DiseaseDiseaseMuta
9、tion unitMutation unitGene name Gene name(protein(protein product)product)Putative Putative functionfunctionNormal Normal repeat lengthrepeat lengthPathogenic Pathogenic repeat lengthrepeat lengthHDHD亨廷顿氏病亨廷顿氏病(CAG)n(CAG)nHD HD(Huntingtin)(Huntingtin)Signaling,Signaling,transport,transport,transcrip
10、tiontranscription11-3411-3440-12140-121SCA1SCA1脊髓小脑共脊髓小脑共济失调济失调(CAG)n(CAG)nSCA1SCA1(ataxin (ataxin 1)1)TranscriptionTranscription6-396-3940-8240-82SCA2SCA2脊髓小脑共脊髓小脑共济失调济失调(CAG)n(CAG)nSCA2SCA2(ataxin (ataxin 2)2)RNA RNA metabolismmetabolism15-2415-2432-20032-200SCA3SCA3脊髓小脑共脊髓小脑共济失调济失调(CAG)n(CAG)nSCA
11、3SCA3(ataxin (ataxin 3)3)De-De-unquitylating unquitylating activityactivity13-3613-3661-8461-84重复在蛋白质编码区域导致毒蛋白重复在非编码区域改变RNA功能DiseaseDiseaseMutation unitMutation unitGene name Gene name(protein(protein product)product)Putative Putative functionfunctionNormal Normal repeat lengthrepeat lengthPathogeni
12、c Pathogenic repeat lengthrepeat lengthFRDAFRDA弗里德赖希弗里德赖希共济失调共济失调(GAA)n(GAA)nFRDAFRDA (frataxin)(frataxin)Mitochondrial Mitochondrial iron iron metabolismmetabolism6-326-32200-200-17001700FRAXAFRAXA脆性脆性X X染色染色体体(CGG)n(CGG)nFMR1FMR1 (FMRP)(FMRP)Translational Translational regulationregulation6-606-60
13、200200FRAXEFRAXE脆性脆性X X染色染色体体(CCG)n(CCG)nFMR2FMR2 (FMR2)(FMR2)TranscriptionTranscription4-324-32200-900200-900三核苷酸重复种类三核苷酸重复种类（按照其相对位置）（按照其相对位置）（按照其相对位置）（按照其相对位置）Categoryofthetrinucleotiderepeat(basedontherelativelocation)Categoryofthetrinucleotiderepeat(basedontherelativelocation)1.第一种是三核苷酸重复在基因非编码
14、区域，常见有 六种遗传病（Repeats in non-Repeats in non-coding sequences for six diseases coding sequences for six diseases）2.第二种是三核苷酸重复在基因编码区域，常见有七种遗传病，经常表现为多聚谷氨酸束（code for code for polyglutaminepolyglutamine tracts tracts）重复序列在非编码区域的疾病重复序列在编码区域的疾病三核苷酸重复在非编码区三核苷酸重复在非编码区（Repeatsinnon-codingsequencesRepeatsinnon-
15、codingsequences）不编码的三核苷酸重复带来疾病的特点不编码的三核苷酸重复带来疾病的特点NON-CODINGTRINUCLEOTIDEREPEATDISORDERSNON-CODINGTRINUCLEOTIDEREPEATDISORDERS1.大量和可变的重复扩展导致多种组织功能丧失和功能退化（Large and variable repeat Large and variable repeat expansions that result in multiple tissue dysfunction or expansions that result in multiple ti
16、ssue dysfunction or degenerationdegeneration）2.疾病症状也随重复多少不同，即所谓体细胞异质性（Phenotypic manifestations within a Phenotypic manifestations within a disease are variabledisease are variable，Degree of somatic Degree of somatic heterogeneityheterogeneity）脆性脆性X染色体染色体FragileXsyndrome脆性脆性X染色体基因型种类染色体基因型种类genotype
17、ofFragileXsyndrome1.FRAXA 位于Xq27.32.FRAXE 位于Xq283.FRAXF 位于Xq27.3-q28 以上三种临床症状和发病机制类似脆性X染色体Fragile X syndrome(FRAXA)Fragile X syndrome(FRAXA)1.Fragile X syndrome(1.Fragile X syndrome(脆性脆性X X综合症综合症):):与与X X染色体相关染色体相关的缺陷，男性中的缺陷，男性中1/20001/2000和女性中和女性中1/40001/4000的几率，产生的几率，产生遗传性的智力缺陷遗传性的智力缺陷2.2.基因座：基因座：
18、Xq27.3Xq27.3；Xq28;Xq27.3Xq28;Xq27.3 q28q28，叶酸敏感，叶酸敏感的脆性位点的脆性位点 (在缺乏叶酸和在缺乏叶酸和thymidinethymidine的条件下，染色体的条件下，染色体某些区域在有丝分裂期间不能正常地形成染色质某些区域在有丝分裂期间不能正常地形成染色质)3.fragile X mental retardation 13.fragile X mental retardation 1；2 2；(FMR1(FMR1；2 2；)基因：基因：RNARNA结合蛋白质，在胚胎和成年的组织中表达，结合蛋白质，在胚胎和成年的组织中表达，在大脑和睾丸中高表达在大
19、脑和睾丸中高表达4.4.与蛋白质的翻译及与蛋白质的翻译及mRNAmRNA的转运有关，调控突触的的转运有关，调控突触的可塑性可塑性Fragile X syndrome(FRAXA)1.1.脆性脆性X X综合症的分子机理：综合症的分子机理：FMR1FMR1的的55非翻译区非翻译区CGGCGG的多样的多样性性 ,FMR2,FMR2的的55非翻译区非翻译区CCGCCG的的多样性多样性 2.2.正常情况，正常情况，FMR1FMR1存在存在6-526-52个个CGGCGG重复片段重复片段3.3.脆性脆性X X失调综合症失调综合症 (轻微症状轻微症状)：52-20052-200个个CGGCGG重复片段重复片
20、段4.FRAXA:4.FRAXA:超过超过52-20052-200个个CGGCGG重复片段重复片段5.CGG5.CGG重复片段发生重复片段发生de novo de novo methylationmethylation，从而抑制了，从而抑制了FMR1FMR1的转录的转录 Fragile X syndrome(FRAXA)l l1.CGG1.CGG重复片段的不稳定性导致异常的重复片段的不稳定性导致异常的DNADNA甲基化甲基化l l2.2.正常情况的正常情况的CGGCGG重复片段在重复片段在CpGCpG岛中，不被甲基化岛中，不被甲基化l l3.CGG3.CGG重复片段很多的时候，发生重复片段很多
21、的时候，发生de novo de novo methylationmethylation，使得基因沉默，使得基因沉默叶酸敏感的脆性区域1.Folate-sensitive fragile sites.1.Folate-sensitive fragile sites.2.FRAXA2.FRAXA基因座是基因组上叶酸最敏感的区域，是其基因座是基因组上叶酸最敏感的区域，是其它区域的它区域的2020倍倍3.3.其他位点其他位点/基因：基因：FRAXE,FRAXF,FRA10A,FRAXE,FRAXF,FRA10A,FRA11B and FRA16AFRA11B and FRA16A4.FRAXE4.F
22、RAXE与人疾病有关与人疾病有关5.5.基因组的结构与基因组的结构与FMR1FMR1相似，都是相似，都是CGGCGG重复片段的重复片段的扩增扩增 FRA10AFMRP通过结合核糖体阻止蛋白质的合成弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)Friedreichataxia(FRDA)1863年Friedreich首先报道，常染色体隐性遗传，发病率1/50000。9q1321.1，GAA异常扩增常染色体隐性（autosomal recessiveautosomal recess
23、ive）唯一没有遗传早发现象（the only triplet repeat the only triplet repeat disorder that does not show anticipationdisorder that does not show anticipation）共济失调（Ataxia(loss of voluntary muscular coordination)Ataxia(loss of voluntary muscular coordination)屈筋反射減退（Diminished reflexesDiminished reflexes）心肌疾病Cardiom
24、yopathy(heart enlargement)Cardiomyopathy(heart enlargement)糖尿病Diabetes Diabetes 脊髓退化Degeneration in the spinal cordDegeneration in the spinal cord 九号染色体图谱9q1321.1l l由于由于GAAGAA在内含子内重复扩增造成，正常人在内含子内重复扩增造成，正常人7-207-20，患者患者200-900200-900次重复次重复（FRDA is caused by a large intronic FRDA is caused by a large
25、intronic GAA repeat expansion GAA repeat expansion）l l基因位于基因位于9 9号染色体，基因产物为号染色体，基因产物为frataxinfrataxin，210aa210aa located on chromosome 9(Gene:X25/Potein:located on chromosome 9(Gene:X25/Potein:frataxinfrataxin)l l导致基因产物为导致基因产物为frataxinfrataxin表达减少表达减少（which leads to which leads to reduced reduced ge
26、negene expression expression）l l推测是由于富含推测是由于富含ATAT序列导致表达减少序列导致表达减少（The expanded The expanded AT-rich sequence most probably causes AT-rich sequence most probably causes）l l GAA/TTCGAA/TTC自我配对导致自我配对导致DNADNA呈现三链结构呈现三链结构（self-self-association of the GAA/TTC tract,which stabilizes the DNA in a associati
27、on of the GAA/TTC tract,which stabilizes the DNA in a triplex structuretriplex structure）弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)Friedreichataxia(FRDA)弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)Friedreichataxia(FRDA)GAA/TTC自我配对导致DNA呈现三链结构如何抑制mRNA的合成GAAGAA在内含子内重复在内含子内重复扩扩增增1.Frataxin.Frataxin 蛋白在人细胞线粒
28、体中发现，蛋白在人细胞线粒体中发现，右图是通过右图是通过GFPGFP定位的定位的frataxinfrataxin，功，功能不明能不明（frataxin is found in the mitochondria of frataxin is found in the mitochondria of humanhuman）2.在酵母中的类似蛋白在酵母中的类似蛋白 YFH1YFH1控制离子控制离子水平和呼吸功能水平和呼吸功能（there is a very similar there is a very similar protein in yeast,YFH1,protein in yeast,Y
29、FH1,YFH1 is involved in controlling,iron levels and YFH1 is involved in controlling,iron levels and respiratory function)respiratory function)3.通过研究通过研究YFH1YFH1可以帮助我们了解可以帮助我们了解FrataxinFrataxin的功能的功能 （Frataxin and YFH1 are so Frataxin and YFH1 are so similar,studying YFH1 may help us understand simil
30、ar,studying YFH1 may help us understand the role of frataxin in FRDA the role of frataxin in FRDA）弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)Friedreichataxia(FRDA)基因转录减少导致frataxin 减少（decreases decreases frataxin levelsfrataxin levels）导致frataxin 功能的部分丧失（a partial loss of a partial loss of frataxin func
31、tion frataxin function）干扰酵母X25同源基因结果是线粒体离子异常累积，线粒体DNA丢失，多种铁硫蛋白依赖酶活性丧失，被氧化敏感性提高等，（Disruption of the yeast Disruption of the yeast X25X25 homolog(YFH1):abnormal homolog(YFH1):abnormal accumulation of mitochondrial ironaccumulation of mitochondrial iron；loss of mtDNAloss of mtDNA；multiple multiple iron
32、sulfur-dependent enzyme deficienciesironsulfur-dependent enzyme deficiencies；increased sensitivity increased sensitivity to oxidative stressto oxidative stress）弗里德赖希共济失调弗里德赖希共济失调 Friedreichataxia(FRDA)Friedreichataxia(FRDA)Frataxin可能的作用机制疾病的研究和可能的治疗方案强直性肌营养不良强直性肌营养不良 Myotonicdystrophy(DM)强直性肌营养不良强直性
33、肌营养不良【Myotonicdystrophy(DM)Myotonicdystrophy(DM)】基因定位于19q1312，以肌强直和进行性肌无力、萎缩为特征常染色体显性遗传多系统异常（multisystem disordermultisystem disorder）差别迥异的表型（highly variable phenotypeshighly variable phenotypes）遗传性早发 （AnticipationAnticipation）肌强直无力，张力减退（Myotonia/muscle weakness/Myotonia/muscle weakness/HypotoniaHyp
34、otonia）发育异常和智力缺陷（Developmental Developmental abnormalities/mental handicapabnormalities/mental handicap）有两种类型DM1/DM21.DM基因编码一种周期性依赖ATP丝氨 酸、苏氨酸蛋白激酶.2.在该基因的3端非翻译区含CTG三核 苷酸重复3.正常人此序列高度多态,含重复527 次，而DM 患者此序列重复50 次以上强直性肌营养不良机制强直性肌营养不良机制【Myotonicdystrophy(DM)Myotonicdystrophy(DM)】强直性肌营养不良机制强直性肌营养不良机制【Myoto
35、nicdystrophy(DM)Myotonicdystrophy(DM)】CTG重复在3-UTR区域强直性肌营养不良分子机制强直性肌营养不良分子机制【Myotonicdystrophy(DM)Myotonicdystrophy(DM)】三核苷酸重复在编码区三核苷酸重复在编码区（RepeatsincodingsequencesRepeatsincodingsequences）三核苷酸重复在基因编码区域，常见有七种遗传病，经常表现为多聚谷氨酸束（code for polyglutamine tracts）脊髓小脑性共济失调脊髓小脑性共济失调 Spinocerebellarataxia(SCA)S
36、pinocerebellarataxia(SCA)常染色体显性遗传，SCA SCA 存在遗传异质性存在遗传异质性,经连锁分析定位经连锁分析定位SCA1(6p22223)SCA1(6p22223)、SCA2(SCA2(12q2322411)12q2322411)、SCA3/JD(14q241323212)SCA3/JD(14q241323212)、SCA4(16q242qter)SCA4(16q242qter)、SCA5(11q13)SCA5(11q13)进行性共济失调（progressive ataxia）小脑萎缩（cerebellar atrophy）快速反应减缓（decreased bri
37、sk reflexes）基因在大脑中表达（SCA8 is expressed primarily in the brain）由于CAG重复在外显子扩张所致【is caused by an expanded CTG repeat exon】亨廷顿舞蹈症亨廷顿舞蹈症Huntingtonsdisease亨廷顿舞蹈病 Huntingtons Disease 1.1.又称慢性进行性舞蹈病又称慢性进行性舞蹈病2.2.常染色体显性遗传，该基因定位于常染色体显性遗传，该基因定位于4p16.34p16.33.3.致病基因编码致病基因编码HuntingtinHuntingtin，在，在ORF5ORF5端有一个端有
38、一个多态性的多态性的 CAGCAG三核苷酸重复序列，正常值一般三核苷酸重复序列，正常值一般在在11-3411-34之间，而在之间，而在HDHD患者则明显增加患者则明显增加(n40)(n40)，通，通常常CAGCAG重复扩增越多，发病越早重复扩增越多，发病越早4.4.病理改变病理改变:脑萎缩，基底节萎缩为主，以尾状核脑萎缩，基底节萎缩为主，以尾状核最为明显，苍白球及壳核也受累，并有间脑、大最为明显，苍白球及壳核也受累，并有间脑、大脑皮层等广泛受累的神经元变性脱失。脑皮层等广泛受累的神经元变性脱失。亨廷顿舞蹈症亨廷顿舞蹈症HuntingtonsdiseaseCAG重复在1号外显子，一般有 35组下图就是测序结果亨延顿素主要功能是线粒体的能量代谢亨延顿素主要功能是线粒体的能量代谢亨廷顿素中谷氨酸重复次数决定了成淋巴细胞中ATP/ADP的比例亨廷顿舞蹈症亨廷顿舞蹈症Huntingtonsdisease从图中可以看出正常人比亨廷顿舞蹈病人ATP/ADP比值要高，CAG重复也就是说谷氨酸重复越多ATP/ADP越小，带来的危害越大多聚谷氨酸影响蛋白质的构像，最终通过蛋白蛋白相互作用影响蛋白的溶解性，通过泛素途径等影响机体功能亨廷顿舞蹈症亨廷顿舞蹈症Huntingtonsdisease