Preliminary Study Of Hereditary Spastic Paraplegia Disease-causing Gene Is Located And Its Reep1 And Zfyve27 Gene Function

Backrounds:Hereditary spastic paraplegia(HSP) is a collection of neurological disorders characterised by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity.Clinically,HSP are divided into pure forms(symptoms confined to lower extremity weakness,bladder disturbance,and to a lesser extent impaired position sense in the legs) or complicated forms when additional neurologic deficits are present,such as optic neuropathy,retinopathy,extra pyramidal,disturbance,dementia, ataxia,ichthyosis,mental retardation,deafness,and epilepsy.HSP can be inherited in an autosomal dominant(ADHSP),an autosomal recessive(ARHSP) or an X-chromosome-linked(XHSP) manner.HSP is genetically heterogeneous,and 15 genes have been cloned although the 33 disease loci have been identified.Objects:To describe five Shandong province kindred with ADHSP and ARHSP and detect which gene mutation involved and analyze whether there are genotype-phenotype correlation in clinical character respectively.Methods:Linkage analysis and mutation detection were performed.And then,clinical analysis, electrophysiological examination and magnetic resonance imaging(MRI) of brain and spinal cord were also performed in all affected individual of the family 1 and two affected of family 2.Results:1.Linkage analysis confined family family 1 to SPG6(N1PA1) with positive Iod scores obtained for the D15S817 and D15S541.Every effected individual had a c316(G106R) mutation in the NIPA1 gene.Neurophysiological examination revealed in most of patients the decreased amplitude of compound muscle action potentials(CMAP) to the tibialis and perineus nerve in motor nerve.Sensory nerve action potential(SNAP) to the tibialis nerve was not elicited in most of patients.Central motor conduction time(CMCT) to abductor pollicis brevis muscle(APB) and the first metatarsal interosseus muscle(FMI) and the anterior tibial muscle(TA) after by transcraniai magnetic stimulation were either absent or clearly prolonged in all patients.MRI of spinal cord showed different levels of atrophy was found in every affected individual.In significant atrophy segments of spinal cord,all the patients revealed distinct grey and white matter boundary.The lesions present clear boundary,median zygomorphic,spot or patch increased signal on transverse axis T2W1 and continuous longitudinal strip increased signal on the anteroposterior axis.2.Linkage analysis confined family 2 to SPG31(reepl) with positive iod scores obtained for the D2S2951 and D2S3333.A novel splice-site mutation(REEPI c417+1g＞a) was identified.Neurophysiological examination revealed in two patients the decreased amplitude of CMAP to the right perineus nerve in motor nerve.Central motor conduction time to the first metatarsal interosseus and anterior tibial muscles were clearly prolonged.MR showed that thoracic cord atrophy was found from T1 to T10 with no abvious abnormal signal.3.A known mutation,namely SPAST c1168(M3861),was identified by allele sharing and mutation detection in the family 3.4.Family 4 was confined to SPGI9(9q33.1-9q34.11) by allele sharing and linkage analysis and a genome-wide scan.We curtailed the scope of cloning candidate gene of SPG19 compared with known SPG19 locus.5.The reported disease locus relative to pure ARHSP had been excluded by allele sharing and linkage analysis.Therefore,we curtailed the scope of gene mapping in this family.Conclusions:1.Our study supports evidence that mutations in NIPA1,reepl and SPAST cause ADHSP and demonstrates further allelic heterogeneity.2.We analyzing genotype-phenotype correlation of SPG6,SPG31 and SPG4.It also suggests that electrophysiological techniques and MR1 could be used to diagnose subtype of HSP early.3.We maped an ADHSP to SPG19(9q33.1-9q34.11) by a genome-wide scan.We found SPG19 locus in Chinese family firstly and curtailed the scope of cloning candidate gene of SPG 19.4.We exclude the reported disease locus relative to pure ARHSP by allele sharing and linkage analysis in the family 5.Therefore,we curtailed the scope of gene mapping in this family and lay basis on identifying a novel pure ARHSP locus. Objects:Hereditary spastic paraplegia(HSP) is a collection of neurological disorders characterised by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity.Mutations in the SPG31(reepl) and SPG33(ZFYVE27) can cause autosomal dominant hereditary spastic paraplegia.To explore the developmental role of reepl and zfyve27 using the zebrafish model and construct the model of spastic paraplegia with morpholino knocking down reepl and zfyve27 and lay the basis on study of the pathogenesy of hereditary spastic paraplegia.Methods:1.To clone reepl and zfyve27 ofzebrafish.2.To determine expression pattern of reepl and zfyve27 in the development of zebrafish embryo by mRNA insitu-hybridization and RT-PCR.3.To design morpholino-modified antisense oligonucleotides and micro-inject into zygote of zebrafish together with confirming specificity and efficiency in vivo of reepl and zfyve27 morpholino.We observe weather phenotype associated with disease exist after morpholino knockdown.Results:1.RT-PCR of reepl indicated expression in 1-cell stage;zygocyte gene began to express in somite stage,then turned to increase expression gradually and kept high in 24～30hpf.After medium expression,it came to increase in 5dpf.RT-PCR of zfyve27 indicated high expression in 1-cell stage;it began to decrease expression after 1-cell stage and began to increase in 50%epiboly stage,to bud stage zfyve27 showed high expression and begin to decrease progressively after then.The zfyve27 had stable and medium expression from 14 somite stage to 4dpf,and began to increase expression in 5dpf.2.In situ hybridization of reepl mRNA indicated the myocomma expression domains in somite stage,high expression in 24hpf;and then expression gradually transfered from somite to brain,reepl showed small expression in somite in 2dpf,while no clear expression existed in somite in 5dpf when reepl mainly expressed in brain tissue and notochord.In situ hybridization of zfyve27 mRNA indicated the general expression domains from 1 cell stage to somite stage.It expressed widespread in vivo except notochord in 24hpf,then turned to concentrate gradually expression in brain and endoblast;zfyve27 expressed in nerve tissue of brain,endoblast and notochord in 5dpf.3.Morpholino oligonucleotide knockdown of the reepl and zfyve27 protein ortholog in zebrafish resulted in an enlarged heart cavity,along with a curly-tail phenotype that severely impaired the ability of the fish to swim properly.The overall phenotype ranged in severity and was classified in three major groups:normal,deformation,(slightly curly and severely curly),and death.This phenotype was clearly visible at 3 day post fertilization(dpf) when wildtype zebrafish are with a straight tail.Injection of the reepl morpholino resulted in 165 (48.67%) of 339 fish with deformation and 77(22.71%) with a death.Injection of the zfyve27 morpholino resulted in 108(46.35%) of 233 fish with deformation and 26(11.16%) with a death.The reepl and zfyve27 morpholino fish had a significantly different distribution of phenotypic groups compared with those with control morpholino injections (P＜0.001).4.On histochemical analysis of the embryos by use o f an antiacetylated tubulinstain for growing axons,we found that the motor neurons in the spinal cord did not develop normally. Motor neuron axons in fish injected with zfyve27 gene alone were shorter and showed abnormal branching.The structure of interneurons in the spinal cord was also different.The absence of the zfyve27 gene or mutations in this gene during early development thus seemed to hamper axonal outgrowth.Conclusions:1.reepl zygocyte gene began to express in somite stage,kept high in 24～30hpf.After medium expression,it came to increase in 5dpf.In situ hybridization of reepl indicated myocomma expression domains frome somite stage to 24hpf and then expression gradually transfered from somite to brain.While no clear expression existed in somite in 5dpf when reepl mainly expressed in brain tissue and notochord.Expression pattern of reepl reveal requirement development of nerve system during embryonic development 2.zfyve27 indicated high expression in 1-cell stage;it began to decrease expression after 1-cell stage and began to increase in 50%epiboly stage,to bud stage zfyve27 showed high expression and begin to decrease progressively after then.The zfyve27 had stable and medium expression from 14 somite stage to 4dpf,and began to increase expression in 5dpf. In situ hybridization of zfyve27 indicated the general expression domains from 1 cell stage to somite stage.It expressed widespread in vivo except notochord in 24hpf,then turned to concentrate gradually expression in brain and endoblast;zfyve27 expressed in nerve tissue of brain,endoblast and notochord in 5dpf.Expression pattern of zfyve27 reveal requirement development of nerve system during embryonic development.3.reepl and zfyve27 morpholino knockdown result in phenotypes such as curly-tail,the impaired ability to swim and abnormal development of motor neuron.Our results reveal a critical requirement for reepl and zfyve27 to promote axonal outgrowth during embryonic development.4.We firstly construct HPS model by zebrafish with reepl and zfyve27 morpholino knockdown.The zebrafish embryo was used as a novel model system in which to dissect the pathogenetic mechanisms underlying HSP.