Clinical Phenotype Of Hereditary Spastic Paraplegia Type 4 And SPAST Gene Novel Mutations And Protein Function Studies

Objective:To analyze the genotype and clinical features of Hereditary Spastic Paraplegia 4?SPG4?patients and to study the SPAST gene and protein function.Methods:The clinical data of patients were collected,and the peripheral blood DNA of patients was extracted by phenol-chloroform method.Multiplex Linker Probe Amplification?MLPA?detects SPAST gene rearrangement mutations.The Next Generation Sequencing analysis of MLPA-negative samples was carried out,and the results were verified by polymerase chain reaction?PCR?and Sanger sequencing.The SPAST gene mutation recombinant plasmid was constructed,and the expression and function of the recombinant plasmid spastin protein were analyzed by immunoblotting and immunofluorescence confocal microscopy.Minigene technology detects the effect of splicing mutations on transcription levels.Results:A total of 40 patients with SPG4 were diagnosed by clinical phenotypic review and genetic analysis,including 30 male patients and 10 female patients.All patients met the clinical diagnostic criteria for hereditary spastic paraplegia.The age of onset was between 0-66 years?mean 30.9±14.8 years?and the course of disease was 3-35 years?mean 15.5±7.6 years?.The clinical manifestations are simple HSP,mostly with difficulty in walking,instability and/or weakness of both lower limbs.Physical examination shows that the patient has scissor gait,increased muscle tension in both lower extremities,hyperreflexia,bilateral ankle and/or patellar clonus positive and pathological sign positive.Three patients were accompanied by pes cavus.Based on MLPA,Whole-exom Sequencing and Sanger sequencing results,we identified 31 SPAST gene sequence variants?77.50%?and 9 SPAST gene rearrangement variants?22.50%?in 40 SPG4 patients involving 34 different SPAST genotype.Among them,24 mutations have been reported,of which 10 are SPAST novel mutations?c.280delG,c.766G>T,c.1094delC,c.885dupA,c.517₅18delAG,c,1479T>A,c.908dupC,c.1536+2T>G,c.1413+1₁413+4delGTAA,c.1729-1G>A?.Non-splicing of SPAST gene mutations to construct recombinant plasmid protein levels revealed that the expression of five mutant spastin proteins was truncated,and the two mutant proteins were normal in size.Immunofluorescence confocal microscopy showed that the spastin cutting tubulin function of the mutant group was significantly abnormal compared to the wild control group.Minigene technology detected that c.1413+1₁413+4deIGTAA and c.1729-1 G>A caused abnormal splicing and could not produce normal spastin protein.The c.1536+2T>G mutation had no effect on splicing.Conclusion:SPG4 is highly clinically and genetically heterogeneous.It is clinically characterized by progressively higher muscle tension,muscle weakness,hyperreflexia and spastic gait.Some patients are accompanied by scissors gait and pes cavus.Through the combination of the Next Generation Sequencing and MLPA technology,we identified 40 patients with SPG4,and 10 novel SPAST gene mutations have not been reported in previous studies at home and abroad.The phenotype and mutation spectrum of SPG4 were expanded.Most of the SPAST gene mutations are sequence variants?75-80%?,but 20-25%of them are large fragment deletions or duplications,and the Whole Exome Sequencing technique is difficult to detect.For patients with autosomal dominant paraplegia without a clear pathogenic site in the whole exome sequencing test,further testing with MLPA should be considered.Though immunoblotting,immunofluorescence confocal and Minigene technology detects,we found that 7 novel non-splicing SPAST gene pathogenic mutations can lead to loss of spastin protein aggregation and function and affect tubulin cutting.Abnormal M1 spastin protein and M87 spastin protein can cause disease in the development of SPG4 disease,and the pathogenic mechanism tends to lose function and function simultaneously.The M1 spastin protein has two mechanisms through functional gain and loss of function,whereas the M87 spastin protein causes disease only through a mechanism of loss of function.The splicing mutations c.1413+1₁413+4de1GTAA and c.1729-1G>A resulted in aberrant splicing and did not produce normal spastin protein.The c.1536+2T>G mutation had no effect on splicing.Figure[8]table[5]reference[76]...