The Establishment Of Genetic Diagnosis Platform For Hereditary Cerebellar Ataxia And Mapping And Cloning Of Novel Causative Genes For Hereditary Cerebellar Ataxia

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.Chapter?The 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 (?=0.00) at rs976192. A maximum two-point logarithm of the odds score of 5.36 (?=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.Chapter?TGM6 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.Part?JXX 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.