Dissertation
Dissertation > Biological Sciences > Anthropology > Human Genetics

Preliminary Study of hereditary spastic paraplegia causative gene positioning its REEP1 ZFYVE27 gene function

Author LiuShiGuo
Tutor MaXu;WuErRuo;JiaMengChun
School Peking Union Medical College , China
Course Genetics
Keywords Hereditary spastic paraplegia HSP disease genes The relationship between genotype and phenotype Linkage analysis Gene mapping Zebrafish reep1 zfyve27 morpholino knockdown mRNA in situ hybridization Immunohistochemical analysis of Genetic spasm paraplegia
CLC Q987
Type PhD thesis
Year 2008
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Part I: hereditary spastic paraplegia home Department of gene positioning and mutation analysis research background: hereditary spastic paraplegia (Hereditary Spastic Paraplegias, HSP) is a group of sports neurodegenerative disease, which is characterized by lower extremity progressive spastic, by motor axons of the corticospinal tract degeneration and dysplasia. Clinical manifestations can be divided into simple and complex, simple type only showed progressive lower extremity increased muscle tone and weakness, and spinal cord outside damage, such as muscular dystrophy, mental retardation, ataxia, polyneuropathy, optic atrophy , retinitis pigmentosa, deafness, and extrapyramidal symptoms such as complex type. The mode of inheritance is divided into autosomal dominant (ADHSP), autosomal recessive (ARHSP) X chain the the recessive genetic (XRHSP). Has a strong hereditary spastic paraplegia heterogeneity, so far, has found 33 HSP the disease gene related sites, but only one of the 15 disease genes have been cloned. Objective: To study from Shandong of 5 ADHSP ARHSP pedigrees, the causative gene mapping and mutation screening, and analysis of the relationship of the various types of HSP pedigree genotype and phenotype research methods: the use of allele sharing analysis, genome-wide scan linkage analysis, positional candidate cloning strategy, the causative gene mapping and mutation screening. Family all six patients, family 2, two patients, the clinical phenotype analysis, electrophysiological and the spinal cord and brain MR scan. Results: 1. Pedigree 1, linkage analysis show microsatellite molecular markers D15S817 and D15S541 set up a chain of relations with disease genes, located in the the SPG6 (NIPA1 genes). Mutation screening NIPA1 gene exon 3 316 bp wild-type G mutation into C (G → C) by Gly → Arg 106 codon inflicted. The neurophysiological examination revealed the majority of patients with motor nerve tibial nerve and common peroneal nerve the mixed muscle action potential (CMAP) amplitude decreased. Tibial nerve sensory nerve action potential (SNAP) most no leads. All patients MEP tibialis anterior muscle (AT) and the first phalanx muscles (FMI), central motor conduction time (CMCT) or no leads or significantly longer. Spinal MRI showed that the spinal cord showed different degrees of atrophy, the corresponding segments of the spinal cord gray matter boundaries clear, gray matter on the horizontal axis bit T2WI was clear boundary, punctate or dot-like high signal symmetrical, continuous longitudinal sagittal The high signal of a strip line. Gray matter involvement, subarachnoid expand. Pedigree 2 linkage analysis show microsatellite molecular markers D2S2951 and D2S2333 with disease genes, to set up a chain of relations, positioning in the SPG31 (REEP1 gene). Mutation screening found REEP1 gene exon 5 receptor bit 4171 base by the wild-type G mutation A new splice site mutation. The neurophysiological examination revealed a right peroneal nerve CMAP amplitude decreased. Motor evoked potential (MEP) AT and FMI CMCT was significantly prolonged. MR shows the thoracic spinal cord from T1 to T10 showed varying degrees of atrophy, but showed no abnormal signal in the spinal cord. Pedigree 3 allele sharing and mutation screening method SPAST gene exon 8 1168 base, by the wild-type A mutated into G 386 codon from methionine becomes Valerian acid. Pedigree 4 analysis and genome-wide scan and linkage analysis based on allele sharing, targeted at the family SPG19 (9q33.1-9q34.11), compared with the the known SPGl9 sites and narrow the targeted area, narrowing the scope of the clones SPG19 candidate genes. Pedigree 5, the method according to the method of analysis of allele sharing and linkage analysis excluded known a simple ARHSP all disease loci, genes that cause narrowing the scope to find the cause of the family. Conclusion: Our results support NIPA1 REEP1, SPAST gene mutation can cause ADHSP, further evidence of the HSP genetic heterogeneity. 2. Elaborated in SPG6, SPG31, SPG4 in genotype and phenotype, abundant HSP pathogenic gene mutation spectrum. Our results suggest that some SPG subtype electrophysiological techniques and MRI can be used as early diagnostic indicators. By genome-wide scan to of a ADHSP pedigree positioning SPG19 HSP pedigrees for the first time discovered the pathogenic sites, and narrowed the candidate gene region, provide a basis for the discovery of cloning SPG19 candidate disease genes. 4 according to the method of allele sharing and linkage analysis, in a ARHSP exclude known simplex ARHSP all the disease locus, and to provide a basis for the discovery new ARHSP disease locus. Second part: REEP1 and ZFYVE27, preliminary studies of gene function research purposes: Hereditary spastic paraplegia is a group of sports neurodegenerative diseases, which is characterized by the gradual spasms in the lower extremities, by the corticospinal tract motor axonal degeneration and dysplasia caused. The SPG31 (REEP1) and SPG33 (ZFYVE27), gene mutations can cause autosomal dominant hereditary spastic paraplegia. Zebrafish as an animal model, to study reepl and the zfyve27 zebrafish embryos, morpholino knock down reepl, paraplegia and zfyve27 build zebrafish model of hereditary spastic paraplegia pathogenesis discussed for the study of hereditary the spastic paraplegia pathogenesis foundation. Research methods: 1. The zebra the fish reepl and zfyve27 gene cloning. 2.mRNA situ hybridization and RT-PCR method to determine the the zebrafish embryos reepl and zfyve27 the temporal and spatial expression. 3 Design morpholino-targeted antisense oligonucleotides by the the microinjection technology import fertilized egg combined use of microscopy technique and RT-PCR confirmed reepl zfyve27 morpholino specificity and in vivo efficacy. Observed morpholino knockdown whether caused phenotypic defects. Results: 1.RT-PCR results showed that reepl gene is expressed in the one-cell stage; gradual increase in the expression of zygotic gene from somite stage expression 24 to 30hpf when, persistently high, moderate expression levels thereafter, rebounded to 5dpf . express high level, after descending to the pick-up in 50% of the outsourcing period, there are high levels of expression to the bud stage; After descending, moderate and stable expression levels within the 14 somite stage to 4dpf 5dpf expression levels rebounded zfyve27 in the 1-cell stage . 2 the in situ hybrid display reepl of gene expression in somite stage in the sarcomere, to 24hpf visible when the high expression; gradually transferred from the somites to the brain tissue the somites 2dpf when there is a small amount of expression; 5dpf the gene expression in brain tissue and spinal cord, no significant expression in the deposit in the body section. zfyve27 from lcell to somites period has been pan-expression, 24hpf expression outside the notochord, after expression parts gradually concentrated in the head and endoderm is, 5dpf expression in the the head nerve tissue, organs of the endoderm, notochord. Show after 3.reepl and zfyve27 morpholino knock down the pericardial cavity to expand, tail curled, was seriously damaged the phenotype of normal zebrafish swimming ability, the entire phenotype is mainly divided into three categories according to the severity. Normal, deformity (tail slightly curly and severe curling), death. The phenotype significantly 3dpf wild-type zebrafish tail is straight, injection reepl morpholino in 339 fish, 165 (48.67%) of malformations (tail slightly curled and serious curl), 77 (22.71%) died. Injection zfyve27 morpholino 233 fish, 108 (46.35%) of the deformity, 26 (11.16%) died. Injection of morpholino the fish with injection controlmorpholino fish compared to the phenotypic group distribution significant differences (P <0.001). Immunohistochemistry with on anti TUBLIN staining analysis of embryonic axons, we found that the abnormal development of spinal motor neurons. Injection zfyve27 morpholino motor axons short and normal branch structure between the spinal cord neurons are also different, indicating the lack of zfyve27 gene seems to hinder the growth of axons. Conclusion: 1.reepl during embryonic development of the zebrafish, zygotic gene somite stage begin to express the 24 to 30hpf time, continued high expression, after which the expression level is moderate, to 5dpf when picked up, in situ hybridization showed reepl gene from somite to 24hpf major sarcomere expression; then gradually transferred from the somites to the brain tissue, 5dpf the genes expressed in the brain tissue and spinal cord and no significant expression in the somites. The spatial and temporal expression pattern reepl specific instructions reepl may play an important role during the embryonic development of the zebrafish. 2.zfyve27 high level expression from the 1-cell stage, after descending. The express rebounded to 50% of outsourcing period, expressed to 4dpf level is moderate and stable, 5dpf expression levels rebounded. Situ hybridization showed zfyve27 from lcell to somite stage has been pan-expression, 24hpf expression outside the notochord, after expression the area gradually concentrated in the head and endoderm, 5dpf expression in the the head nerve tissue, organs of the endoderm, notochord. zfyve27 specific temporal and spatial expression pattern the zfyve27 may play an important role during the embryonic development of the zebrafish. 3.reepl zfyve27 morpholino knockdown zebrafish motor neuron growth is not normal, tail curling, swimming in an impaired ability to phenotype. Our results illustrate reepl and zfyve27 necessary to promote axonal growth and development. The first time we built with reepl zfyve27 morpholino knockdown zebrafish paraplegia model. The disease phenotype for the system to lay the foundation for a detailed analysis of the pathological mechanisms of hereditary spastic paraplegia.

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