| Objective: Hereditary spastic paraplegia is a typical neurodegenerative disease with genetic heterogeneity.It is characterized by chronic progressive myasthenia and chronic spastic lower limb paralysis.Corticospinal tract length dependent axonal degeneration is the main cause.This research aims to provide basis for genetic counseling of members of a Hui family with simple HSP in Yunnan Province by carrying on the family clinical analysis,gene screening and build low pathogenic candidate genes cell type model,at the same time enrich the HSP pathogenic gene library and preliminarily figure out the possible molecular mechanism of HSP pathogenesis caused by pathogenic candidate genes.Finally,it provides new targets and treatment ideas for the precise treatment of HSP.Methods: Taking 37 family members of 4 generations in the HSP family as the research object,collecting detailed medical history data of the patients and conduct systematic and comprehensive physical examination.At the same time,the patients conducted blood routine examination,blood biochemistry,brain MRI and neuroelectrophysiology,and collected data for analysis and research.At the same time,after informing the patients and their families of relevant risks and benefits,obtaining informed consent and signing the informed consent,DNA was extracted from the patient’s peripheral blood samples and prepared into samples.After sequencing,multiple possible pathogenic candidate genes were obtained.According to the prediction results of bioinformatics analysis software,combined with family clinical symptoms and genetic analysis,the new mutation of dnajc16 was regarded as the most likely pathogenic synergistic gene in the family.The dnajc16 gene editing cell knockdown model was constructed by CRISPR / cas9 gene editing technology,and the target protein expression of the gene edited cell model was detected by Western blot technology.Results:Results of clinical examination and pedigree genetic analysis shows there were 11 patients with HSP in this family,including 6 males and 5 females.The gender ratio of men to women was about 1:1.The age range of onset was 20-33 years old,mostly with symptoms such as weakness and spasm of both lower limbs.In addition to the above manifestations,family patients had no complex HSP symptoms such as mental retardation and cognitive impairment.The MRI examination of the proband’s head showed that there was no obvious positive finding,but in the MRI examination of the spinal cord,it was found that the third generation No.10 had the sign of thinning of the thoracic spinal cord.Among the pedigree patients,neurogenic damage was found in 4 cases conducted nerve electrophysiological examination.Gene test went out that several possible pathogenic candidate genes were obtained.Among them,the new mutation of dnajc16（DNAJC16: c.718,C > T,p.Q240 X,Ref Seq NM-015291）found in the screening may play a role in promoting the occurrence and development of the disease.On this basis,dnajc16 gene knockdown cell model was successfully constructed.Western blot testing showed that the protein expression decreased after gene editing.Conclusion: The pedigrees follow the autosomal dominant inheritance pattern,and the clinical classification is simple HSP.The clinical manifestations of pedigree patients are different,and their onset age is advanced generation by generation.Several possible pathogenic candidate genes were screened in this family by gene sequencing,but the pathogenicity of pathogenic candidate genes in this family remains to be verified.Among them,dnajc16 is a possible pathogenic candidate gene.On this basis,we successfully constructed the dnajc16 knockdown cell model,and detected the constructed cell model by Western blot technology.It was found that the gene expression protein decreased after gene editing,which laid a foundation for further verification of functional mechanism. |
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