【作者】 王俊岭；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 神经病学， 2011， 博士

【摘要】 第一部分中国汉族人群遗传性共济失调基因诊断平台的建立第一章常染色体显性遗传和散发脊髓小脑型共济失调基因诊断平台的建立研究发现不同的种族、国家和地区,常染色体显性遗传脊髓小脑型共济失调(Spinocerebellar ataxia,SCA)不同亚型的发病率及各亚型病理和正常的核苷酸重复次数都有明显的差异,为了建立SCA致病基因的诊断平台,了解中国汉族人群SCA各亚型的分布频率特点及中国汉族人群各亚型病理和正常重复次数范围分布特点,制定一套SCA基因诊断流程,我们应用聚合酶链反应、变性聚丙烯酰胺凝胶电泳、Southern Blot等技术对来自中国汉族人群667例临床诊断为SCA的患者(430例AD遗传家系先证者,237例散发患者)进行了SCA1、SCA2、SCA3/MJD、SCA6、SCA7、SCA8、SCA10、SCA12、SCA17及DRPLA各亚型致病基因多核苷酸重复突变检测；对排除以上SCA亚型的患者应用聚合酶链反应(PCR)、变性高效液相色谱(DHPLC)、多重连接探针扩增(MLPA)结合直接测序等技术进行SCA11、SCA13、SCA14、SCA27、SCA28和SCA31亚型致病基因TTBK2、KCNC3、PRKCG、FGF14、AFG3L2和PLEKHG4点突变和SCA5、SCA11、SCA15致病基因SPTBN2、TTBK2、ITPR1大片段的插入缺失突变检测。应用T载体克隆重组DNA技术结合直接测序,对部分患者进行CAG三核苷酸病理重复次数测序分析；并应用荧光聚合酶链反应、毛细管凝胶电泳对300名中国汉族健康对照人群进行了上述SCA型三核苷酸或多核苷酸重复次数的正常范围检测和分析。结果在430例AD-SCA家系先证者中,共检测出SCA1家系25个(5.81%), SCA2家系27个(6.28%), SCA3家系267个(62.09%), SCA6家系8个(1.86%), SCA7家系8个(1.86%),SCA12家系1个(0.23%)和SCA17家系1个(0.23%),未明确基因分型SCA家系93个(21.63%)；在237例散发患者中共检测出SCA1患者6例(2.53%), SCA2患者9例(3.80%), SCA3患者23例(9.70%), SCA6患者3例(1.27%),未明确分型SCA患者196例(82.70%)；未检测出SCA8、SCA10和DRPLA亚型致病基因多核苷酸异常重复突变,SCA5、SCA11、SCA13、SCA14、SCA15/ SCA16、SCA27、SCA28和SCA31亚型传统形式的点突变及大片段的插入缺失突变。对23例SCA1患者,32例SCA2患者,305例SCA3/MJD患者,9例SCA6患者,27例SCA7患者,3例SCA12患者和2例SCA17患者进行CAG三核苷酸异常重复次数范围检测发现其范围分别是SCA1亚型39-60(平均51.09±4.88)次,SCA2型36-51(平均40.34±4.40)次,SCA3/MJD亚型49-86(,平均73.84±5.07)次,SCA6亚型23-29(平均25.56±1.94)次, SCA7亚型38-71(平均58.22±10.90)次,SCA12亚型51-52(平均51.33±0.58)次,SCA17亚型53-55(平均54.00±1.41)次；SCA1、SCA2、SCA3、SCA6、SCA7、SCA8、SCA10、SCA12、SCA17和DRPLA共10种SCA亚型致病基因正常三核苷酸或多核苷重复次数范围结果,SCA1亚型为17～35次,SCA2亚型为14～28次,SCA3/MJD亚型为13～41次,SCA6亚型为4～16次,SCA7亚型为5～17次,SCA12亚型为5～21次,SCA17亚型为23～41次,DRPLA亚型为12～33次,SCA8亚型中CTA/CTG三核苷酸的重复次数为12-43次,SCA10亚型中ATTCT五核苷酸的重复次数为9～32次。根据以上结果我们发现在中国汉族人群中SCA3/MJD为最常见的SCA亚型,其次为SCA2、SCA1、SCA7和SCA6, SCA12和SCA17比较少见,SCA8、SCA10和DRPLA罕见,其中SCA17亚型为国内首次报道。建立中国汉族人群不同SCA亚型CAG三核苷酸病理重复次数范围标准和中国汉族人群不同SCA亚型正常核苷酸重复次数范围,可为SCA的诊断提供参考标准。初步建立了SCA基因诊断平台,制定了SCA基因诊断流程。第二章常染色体隐性共济失调基因诊断平台的建立常染色体隐性遗传小脑性共济失调(autosomal recessive cerebellar ataxia, ARCA)是一大类比较罕见的、具有明显临床和遗传异质性的神经系统退行性疾病,为了建立中国汉族人群ARCA致病基因的诊断平台,了解中国汉族人群ARCA各亚型的分布频率,制定一套ARCA基因诊断流程,我们采用PCR结合DNA直接测序法对来自中国汉族人群36例临床诊断为常染色体隐性遗传先证者和60例发病年龄<20岁的散发SCA患者进行了发病率相对较高的弗里德赖希共济失调(FRDA),伴选择性维生素E缺乏的共济失调(AVED),伴动眼不能的共济失调1型(AOA1),伴动眼不能的共济失调2(AOA2)型等共四种亚型进行致病基因FXN、ATTP、APTX和SETX的相关检测分析。常规PCR难以对基因比较大外显子比较多的ARCA亚型完成突变检测,如毛细管扩张共济失调(AT),痉挛性共济失调(SACS),我们利用全基因外显子组捕获结合高通量测序技术(Exome测序)对其进行全基因外显子组测序分析。结果未发现FXN、ATTP、APTX和SETX基因致病突变,全基因外显子组测序发现一个AT患者ATM基因(c.5293 C-T)和(c.1402-1403delAA)复合杂合突变；一个SACS家系SACS基因(c.5399 G-C)和(C.788delA)复合杂合突变。S anger测序证实了该复合杂合突变的正确性,并且两个复合杂合突变在AT家系和SACS家系内分别和疾病表型共分离。本研究证实中国汉族人群ARCA家系中FRDA、AVED、AOA1和AOA2非常罕见,初步建立了ARCA基因诊断平台,制定了ARCA的基因诊断流程,以及应用Exome测序进行ARCA基因诊断的技术平台。第二部分一个常染色体显性遗传SCA家系致病基因的定位与克隆第一章一个新的常染色体显性遗传SCA家系致病基因的定位遗传性脊髓小脑型共济失调(spinocerebellar ataxia, SCA)是一类包括多种亚型在内的具有明显临床和遗传异质性的进行性神经系统退行性疾病,目前已经定位了近28个基因位点,其中22种SCA亚型致病基因已被克隆。前期工作我们收集到一个来自中国汉族的SCA家系,呈四代遗传,患者表现缓慢进展的晚发型小脑性共济失调,部分患者伴有明显的痉挛性斜颈。前期工作已经排除了已知的SCA1、SCA2、SCA3/MJD、SCA6、SCA7、SCA8、SCA10、SCA12、SCA17和DRPLA亚型致病基因三核苷酸及多核苷酸异常重复突变检测,SCA11、SCA13、SCA14、SCA27、SCA28和SCA31亚型致病基因TTBK2、KCNC3、PRKCG、FGF14、AFG3L2和PLEKHG4点突变,SCA5、SCA11、SCA15致病基因SPTBN2、TTBK2、ITPR1大片段的插入缺失突变检测和目前已定位各SCA亚型位点的连锁分析排除定位检测。通过采用HumanLinkage-12 Panels连锁分析芯片技术,对该AD-SCA家系SNP位点进行扫描,结果在第20号染色体的两个SNP rs12624577和rs674630之间,相当于20p13-12.2的区域,取得连续分布正LOD值。其中,在rs976192取得最大两点LOD值3.85,对上面区间应用微卫星遗传标记进行精细定位分析,在20号染色体上微卫星遗传标记D20S437处取得最大两点LOD值5.36,依据这两个重要的重组交换事件的发生,该家系的候选致病基因定位于D20S199和D20S917之间的区域,家系内所有临床诊断明确的患者均携带相同的单体型。该定位区间(20p13-12.2)遗传距离为18.45 cM,相当于物理距离约有8.4 Mb,区间内包含有91个候选致病基因。第二章应用Exome测序克隆一新的常染色体显性遗传脊髓小脑型共济失调致病基因-TGM6单基因病致病基因的克隆常采用传统的候选基因定位克隆技术,然而传统的定位克隆技术常常会因为家系内成员太少、基因位点的异质性、外显不全及定位区间内候选克隆基因太多等而受到限制。全基因外显子组捕获结合高通量测序一次可获得全部外显子的信息,从根本上解决了传统克隆致病基因的难题。我们联合应用连锁分析结果和Exome测序的方法去克隆一新的SCA致病基因。首先从一个呈四代遗传的SCA家系(CS)中选取4位患者进行了Exome测序,经数据分析在TGM6基因10号外显子上面发现一个错义突变c.1550T-G(L517W),该突变在家系内和疾病表型共分离,并且在500个正常对照测序分析没有发现该突变位点。信息学分析发现该错义突变在进化过程中处于高度保守区域,功能预测该突变对蛋白质的功能有明显的影响。应用连锁分析的结果,TGM6基因c.1550T-G(L517W)改变正好位于之前该家系全基因组连锁分析定位的区间(20p13-12.2)内,Exome测序和全基因组连锁定位分析的结果互相验证了TGM6基因为该家系致病基因的正确性。我们又在另外一个SCA家系(LY)发现了TGM6基因7号外显子的另外一个位点c.980A-G transition(D327G)突变,该突变LY家系内同样和疾病表型共分离,在500个正常对照测序分析没有发现该位点突变。该新的突变进一步证实了我们克隆的新的致病基因的正确性。新的致病基因TGM6的克隆说明在单基因遗传疾病中在一个家系内对部分患者进行Exome测序从而寻找致病基因是一种有效的策略,如果利用Exome测序技术和传统的定位克隆研究方法相结合,可能成为进行人类单基因遗传疾病克隆的一个更高效的新途径。第三部分应用Exome测序克隆两个新的常染色体隐性遗传共济失调致病基因--JXX和TYY在遗传性共济失调中,常染色体隐性小脑性共济失调(Autosomal recessive cerebellar ataxias, ARCA)是一大组比较罕见的具有明显临床和遗传异质性的神经系统退行性疾病,病变主要累及小脑、脊髓小脑束或/和脊髓感觉束。ARCA临床表型复杂可累及中枢神经系统、周围神经系统,部分病例可累及除神经系统之外的其它系统和器官。ARCA还包括一大批罕见的类型,根据分子发病机制、病理、自然病史和病变部位,目前ARCA中至少已划分了100多种不同的类型,其中70多个相关的致病基因已被克隆。多数的ARCA家系,因为家系成员少使得应用传统的候选基因定位克隆方法去克隆致病基因比较困难。我们应用Exome测序技术对两个ARCA家系(JX家系和TY家系)进行了全基因组外显子的捕获和测序分析,作为常染色体隐性遗传模式,我们在测序结果里主要分析纯和突变和复合杂合突变。结果我们在JX家系内发现了一个JXX基因(c.493C-T)的纯和突变,在TY家系内发现TYY基因(c.568C-T)和(c.760A-G)两突变位点的复合杂合突变,信息分析发现JXX基因(c.493C-T)和TYY基因(c.568C-T)与(c.760A-G)三个突变位点均位于高度保守区域,并且分别在JX家系和TY家系内与疾病表型共分离。500个正常对照测序分析没有发现以上三个突变位点的改变,排除了罕见多态的可能。我们又在另外一个ARCA家系(JX-NX)发现了JXX基因另外两个位点(c.389A-T和c.441G-T)的复合杂合突变,该复合杂合突变在JX-NX家系内和疾病表型共分离,在500个正常对照测序分析没有发现该两个突变位点。该新的复合杂合突变进一步证实了我们克隆的新的致病基因的正确性。新的ARCA致病基因JXX和TYY的克隆证实了在一些少见的孟德尔单基因疾病致病基因的克隆中全基因外显子组测序是一个方面而又高效的策略。更多还原

【Abstract】 Part I The establishment of genetic diagnosis platform for hereditary cerebellar ataxia of Chinese HanChapter I The establishment of genetic diagnosis platform for autosomal dominant spinocerebellar ataxiaThe incidence, expanded and normal CAG trinucleotide repeats of different subtypes of autosomal dominant Spinocerebellar ataxia had a significant difference in different races, countries and regions. To set up a genetic diagnosis platform, detect the genetic spectrum and expanded and normal CAG trinucleotide repeat numbers of SCA subtypes in Chinese Han population, thus establish a tiered-diagnostic approach in our genetic testing laboratory for SCA, the nucleotide repeat mutations of SCA 1,2,3, 6,7,8,10,12,17 and dentatorubral-pallidoluysian atrophy (DRPLA) subtypes were detected by the polymerase chain reaction (PCR), denaturing polyacrylamide gel electrophoresis and Southern Blot technique in a cohort of 667 Mainland Chinese SCA patients, including 430 families with autosomal dominant SCA and 237 sporadic forms. Subsequently, we performed the analysis of TTBK2, KCNC3, PRKCG, FGF14, AFG3L2 and PLEKHG4 gene point mutation in subtypes of SCA11,13,14,27,28 and SCA31, as well as SPTBN2, TTBK2 and ITPR1 gene insert and delete (Indel) mutations in subtypes of SCA5,11 and SCA 15 using PCR, Denature High Performance Liquid Chromatography (DHPLC), Multiplex Ligation-dependent Probe Amplification (MLPA) and DNA direct sequencing technology in patients excluded of SCA1,2,3,6,7,8,10,12,17 and DRPLA loci. The expanded CAG repeat numbers of abnormal allele of SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA 12 and SCA 17 genes were analyzed in parts of SCA patients using recombinant DNA technology by T-vector clone and direct sequencing. Meanwhile, the normal nucleotide repeat numbers of SCA1,2,3,6,7,8,10,12,17 and DRPLA were detected using fluorescence-PCR and Capillary gel electrophoresis technique in 300 healthy controls from Chinese Han.Among the 430 AD-SCA families,25 (5.81%) were positive for SCA1,27(6.28%) were positive for SCA2,267(62.09%) were positive for SCA3/MJD,8(1.86%) were positive for SCA6,8(1.86%) were positive for SCA7,1(0.23%) were positive for SCA12 and 1 (0.23%) were positive for SCA17,93(21.63%) were genetically unidentified. There were 6(2.53%) SCA1,9(3.80%) SCA2,23(9.70%) SCA3/MJD, 3(1.27%) SCA6 and 196(82.70%) were genetically unidentified in the 237 sporadic SCA patients. None expanded nucleotide repeat of SCA8, SCA10 and DRPLA mutation and traditional point and Indel mutation of SCA5. SCA11、SCA13、SCA14、SCA15/SCA16、SCA27、SCA28 and SCA31 was found. Among parts of SCA patients, the range of expanded CAG repeat number was 39 to 60 (mean=51.09±4.88) in 23 SCA1 patients,36 to 51 (mean=40.34±4.40) in 32 SCA2 patients,49 to 86 (mean=73.84±5.07) in 305 SCA3/MJD,23 to 29 (mean=25.56±1.94) in 9 SCA6 patients,38 to 71(mean=58.22±10.90) in 27 SCA7 patients,51 to 52 (mean=51.33±0.58) in 3 SCA12 patients,53 to 55 (mean=54.00±1.41) in 2 SCA17 patients. Among 300 healthy people, the range of CAG trinucleotide repeat number was 17 to 35 in SCA1,14-28 in SCA2,13-41 in SCA3/MJD,4-16 in SCA6,5-17 in SCA7,5-21 in SCA12,23-41 in SCA17, and 12-33 in DRPLA; the CTA/CTG trinucleotide repeat number on SCA8 locus were 12-43 and the ATTCT pentanucleotide repeat number on SCA 10 locus were 9-32.From the results, we concluded that the frequency of SCA3/MJD is the substantially highest subtype in patients with autosomal dominant and sporadic forms in Chinese Han SCA patients, subsequent upon SCA2, SCA1, SCA7 and SCA6; SCA12 and SCA17 are rare subtype, while SCA8, SCA10, and DRPLA were seldom found. For the first time we identified SCA 17 subtypes in China’s mainland. For the first time, we established the expanded and normal reference standard of polynucleotide repeat number of different subtypes of SCA in Chinese Han and can be used as reference criteria. Meanwhile, we established the diagnostic platform and tiered-diagnostic approach in our genetic testing laboratory for SCA of Chinese Han.ChapterⅡThe establishment of genetic diagnosis platform for autosomal recessive cerebellar ataxiaAutosomal recessive cerebellar ataxias (ARCA) constitute a large rare, clinically and genetically heterogeneous group of progressive neurodegenerative diseases. To set up the genetic diagnosis platform, detect the spectrum and establish a tiered-diagnostic approach in our genetic testing laboratory for ARCA of Chinese Han, gene mutation analysis of FXN for Friedreich’s ataxia (FRDA), ATTP for ataxia with vitamin E deficiency(AVED), APTX for ataxia plus oculomotor apraxia type 1 (AOA1) and SETX for ataxia plus oculomotor apraxia type 2 (AOA2) were carried out by polymerase chain reaction combined with DNA direct sequencing and polyacrylamide gel electrophoresis in a cohort of 96 Mainland Chinese patients affected with ataxia, including 36 index patients in families with autosomal recessive cerebellar ataxia (ARCA) and 60 sporadic patients in which the onset was less 20 years. All of these four ARCA subtypes were relatively common in worldwide. For some ARCA subtypes, such as ataxia telangiectasia (AT) and (Spastic ataxia of Charlevoix-Saguenay, SACS), it’s difficult to complete the mutation analysis by traditional PCR and direct sequencing because of too many exons in ATM gene of AT. Here, we used Whole-exome capture and massively parallel sequencing technology (Exome sequencing) to identify the pathological mutation in AT family.No mutation of FXN, ATTP, APTX and SETX were detected. A compound heterozygote mutation (c.5293 C-T) and (c.1402-1403delAA) were found in ATM gene of one AT patient and another compound heterozygote mutation (c.5399 G-C) and (C.788delA) were found in SACS gene of one SACS patient. Traditional Sanger validates the Exome sequencing results, and compound heterozygote mutation (c.5399 G-C) and (C.788delA) are absent in 500 normal unaffected individuals of matched geographical ancestry. Meanwhile, both of these compound heterozygote mutations completely co-segregate with the phenotype in each of AT and SACS family. From the results, we concluded that FRDA, AVED, AOA1 and AOA2 were seldom found subtype of ARCA in Chinese Han. We established the diagnosis platform and tiered-diagnostic approach in our genetic testing laboratory for ARCA of Chinese Han, as well as the technology of diagnosis platform using Exome sequencing for some ARCA causative genes. Part II Mapping and cloning of a novel causative gene for an SCA familyChapter I Mapping of a disease gene in a novel SCA familyAutosomal dominant spinocerebellar ataxias (ADCA) constitute a large clinically and genetically heterogeneous group of progressive neurodegenerative diseases with multiple types. To date, classical genetic studies have revealed 31 distinct genetic forms of spinocerebellar ataxias and identified 19 causative genes. A four-generation Chinese family with SCA, characterized by late onset, slow progressive gait, limb ataxia, and, in some cases, spasmodic torticollis was performed in this study. The known SCA subtypes locus and nucleotide expansion mutation of SCA1、SCA2、SCA3/MJD、SCA6、SCA7、SCA8、SCA10、SCA12、SCA17 and DRPLA, TTBK2, KCNC3, PRKCG, FGF14, AFG3L2 and PLEKHG4 gene point mutation in subtypes of SCA 11, SCA13, SCA14, SCA27, SCA28 and SCA31, as well as SPTBN2, TTBK2 and ITPR1 gene insert and delete (Indel) mutations in subtypes of SCA5, SCA11 and SCA 15 has been excluded.To localize the disease-causing gene, we carried out whole-genome genotyping using the Infinium HumanLinkage-12 Genotyping BeadChip (Illumina, San Diego, CA). Parallel inspection of the SNP data of the CS family identified a single shared region on chromosome 20p13-12.2 flanked by SNP markers rs 12624577 and rs674630 with a maximum two-point logarithm of the odds score of 3.85 (θ=0.00) at rs976192. A maximum two-point logarithm of the odds score of 5.36 (θ=0.00) was obtained at D20S437 using thirteen additional markers for fine mapping. The highest probability haplotype in the CS pedigree was reconstructed manually using the Cyrillic program. According to the key recombination, a particular single haplotype, comprising identical alleles spanned by microsatellite markers D20S199 and D20S917, was identified to co-segregate with the disease phenotype in all examined affected family members with SCA. This locus was found to span an 18.45 cM region, approximately 8.4 Mb of genomic DNA, and included 91 reference genes.ChapterⅡTGM6 identified as a novel causative gene of autosomal dominant SCA using exome sequencingPreviously, the primary means for disease gene identification has been through traditional positional cloning strategies. The power of this method, however, is limited, especially in situations where there are small family sizes, locus heterogeneity, substantially reduced reproductive fitness, and an abundance of candidate genes present in the mapped region. Here, we used a combinational strategy of exome sequencing and linkage analysis to identify a novel spinocerebellar ataxia causative gene. We sequenced the whole-exome of four patients in a Chinese four-generation spinocerebellar ataxia family, and identified a missense mutation c.1550T-G transition (L517W) in exon ten of TGM6. The mutation was completely co-segregated with the phenotype and was absent in 500 normal unaffected individuals of matched geographical ancestry. We predicted that the change had a functional impact for it was located in a highly conserved position. Meanwhile, we used linkage analysis to validate the exome results. The mutation identified using exome sequencing was located at the same region (20p13-12.2) identified by linkage analysis, which cross-validated TGM6 as the causative spinocerebellar ataxia gene in this family. We further confirmed our finding by identifying another missense mutation c.980A-G transition (D327G) in exon seven of TGM6, in an additional spinocerebellar ataxia family which also co-segregated with the phenotype and was also absent in 500 normal unaffected individuals of matched geographical ancestry. The finding of TGM6 as a novel causative gene of spinocerebellar ataxia illustrates whole-exome sequencing of affected individuals from one family as an effective and cost efficient method for mapping genes of rare Mendelian disorders and the power of a combinational method of linkage analysis and exome sequencing for further improving efficiency.PartⅢJXX and TYY identified as two novel causative genes of two autosomal recessive ataxia using Exome sequencingAmong the hereditary ataxias, autosomal recessive cerebellar ataxias (ARCA) are a heterogeneous group of rare neurological disorders that affect the cerebellum, the spinocerebellar tract and/or the sensory tracts of the spinal cord. Clinical phenotypes vary from central to peripheral nervous system, and in some case other systems and organs. This group encompasses a large number of rare diseases. According to genetic, pathological, natural history and topographical, this group can be divided into more than one hundred different subtypes. To date, classical genetic studies have identified at least fifty causative genes of ARCA.For most ARCA pedigrees, small family size is limited for traditional positional cloning strategies to find the causative gene. Here, we used Whole-exome capture and massively parallel sequencing technology to identify the causative gene of two ARCA families (JX family and TY family). We sequenced the whole-exome of two patients in each of the two Chinese ARCA families. As the autosomal recessive inherited mode, we focused on the novel homozygote and compound heterozygote SNP.At last we identified a homozygote missense mutation (c.493 C-T) in JXX gene of JX family, a compound heterozygote mutation, nonsense (c.568C-T) and missense mutation (c.760A-G), in TYY gene of TY family. Both of these homozygote and compound heterozygote mutations are at highly conserved position, completely co-segregate with the phenotype in each of JX and TY family and are absent in 500 normal unaffected individuals of matched geographical ancestry. We further confirmed our finding by identifying another compound heterozygote missense mutation, c.389 A-T and c.441 G-T, in JXX gene in an additional ARCA family (JX-NX family) which also co-segregated with the phenotype were absent in 500 normal unaffected individuals of matched geographical ancestry.The finding of JXX and TYY as novel causative genes of ARCA illustrates whole-exome sequencing as an effective and cost efficient method for mapping genes of rare Mendelian disorder.更多还原