Study On Genetic Variation And Correlation Between Genotype And Phenotype Of Spastic Paraplegia

Background and Objective Spastic paraplegia is a group of neurodegenerative diseases,which presenting scissors gait,slow progressive hypertonicity and weakness of lower limbs.Hereditary spastic paraparesis(HSP or SPG)is the most common type of spastic paraplegia.HSP has obvious genetic heterogeneity.The inherited pattern can be autosomal dominant,autosomal recessive,X-linked recessive and mitochondrial modes.Up to now,more than 100 HSP-related pathogenic genes have been reported,of which 79 have been officially named SPG1~SPG79.In recent years,with the widespread application of next-generation sequencing(NGS)technology,more and more novel pathogenic genes of HSP have been detected.Moreover,HSP has also prominent genetic heterogeneity.With the expansion of phenotypes of known pathogenic genes,the overlap of phenotypes and genotypes,and the diversity of genetic mutations,which frequentky lead to misdiagnosis of HSP.In addition,a large number of cases with pyramidal signs of non-hereditary spastic paraplegia are reported,such as amyotrophic lateral sclerosis(ALS),charcot-marie-tooth disease(CMT),hereditary ataxia(HA),adrenomyeloneuropathy(AMN)and human T-lymphotropic virus types(HTLV-1)infection-associated tropical paraplegia(HTLV-I-associated myelopathy/ tropical spastic paraparesis,HAM/TSP).All the aspects exacerbate the difficulty to the clinical diagnosis of HSP.Therefore,the accurate clinical diagnosis of HSP relies on a proper system of screening and analysis.In this study,suspected HSP patients were first screened for dynamic mutations of spinocerebellar ataxia type 3(SCA3),and then multiplex ligation dependent probe amplification(MLPA)was used to perform copy number analysis of HSP-related pathogenic genes,finally targeted next-generation sequencing was applied to find pathogenic genes for accurate diagnosis.Additionally, there is no effective treatment for this disease currently.The underlying reason is that the specific pathogenic mechanism is unknown.In this study,the most common clinical subtypes of autosomal dominant HSP and autosomal recessive HSP,SPG4 and SPG11,were selected for the function analysis.Mutant protein stability,mitochondrial morphology,autophagosome-lysosome morphology and fusion were studied on patient-derived skin fibroblasts to elucidate the specific pathogenesis of HSP.Methods 1.For suspected HSP patients,PCR and agarose gel electrophoresis were first used to carry out the ATXN3 gene dynamic mutation screening;Secondly,MLPA screening combined with NGS were applied to find out HSP-related pathogenic genes;Finally,combined with clinical data,the relationship between their genotypes and phenotypes were recognized for further analysis.2.Construct novel point mutation SPG4 plasmid and then transfected 293 T cells,combined with cycloheximide(CHX)intervention,detection of mutant protein expression level and stability using western blot.3.Skin biopsy of SPG4 and SPG11 patients to establish fibroblast cell lines.To analyze mitochondrial distribution and functional changes in autophagy-lysosome morphology and fusion by cell immunofluorescence and western blotting.4.Combined with Sanger sequencing and family co-segregation analysis,we verified ABCD1 gene mutations and analyzed the clinical data of confirmed AMN patients.5.For patients without genetic mutation,we further screen the suspected HAM/TSP patients according to their age of onset and the duration of disease.We screen their serum HTLV-1/2 antibody and peripheral blood HTLV-1 virus nucleic acid to confirm the diagnosis by ELISA and fluorescence quantitative PCR.Results 1.In the 150 suspected HSPs families,a total of 14 probands were found to carry CAG repeats of exon 10 in ATXN3 gene,which was diagnosed as SCA3.2.HSP-related pathogenic genes were identified in 57 probands by MLPA combined with targeted second-generation sequencing.Among them,there are 25 cases of autosomal dominant HSP,including 4 cases of SPG3A(ATL1),18 cases of SPG4(SPAST),1 case of SPG8(KIAA0196),2 cases of SPG33(ZFYVE27).Among them,there are 32 cases of autosomal recessive HSP,including 20 cases of SPG5(CYP7B1),6 cases of SPG11(SPG11),1 in each of SPG28(DDHD1),SPG30(KIF1A),SPG35(FA2H),SPG43(C19orf12),SPG46(GBA2)and SPG54(DDHD2).3.Of the 20 SPG5 probands,we found that as many as 65%(13/20)of patients carry the homozygous mutation c.334 C>T(p.R112*)and this is further corroborated with the founder effect in Fujian region.4.The four new mutations in SPG4 would not only reduce the expression of spastin protein,but also weaken the stability of spastin protein.5.The distribution of mitochondria in the fibroblasts of SPG4 patients was uneven,mainly concentrated in the near cell nucleus,and the mitochondrial morphology was mainly fragmented,which was different from the long tubular morphology of healthy controls.6.The abnormal volume of lysosomes in fibroblasts of SPG11 patients were increased,and the autophagosomes were abnormally aggregated but did not affect the fusion of lysosomes and autophagosomes.7.A total of 5 AMN patients with ABCD1 mutations were found to have an abnormally high ratio of serum long-chain fatty acids(C26:0/C22:0),and brain-type patients were higher than simple-type patients.8.Based on the age of onset and short duration of disease,3 patients with suspected HAM/TSP were picked in 74 patients with negative genes.According to positive serum HTLV antibody and peripheral blood nucleic acid,a patient of HAM/TSP was diagnosed.Conclusions 1.A large proportion of patients with spinocerebellar ataxia type III(SCA3)patients may present with typical pyramidal signs and may be misdiagnosed as HSP.They should be identified.Troubleshooting of ATXN3 gene dynamic mutations by PCR can reduce misdiagnosis.In addition,MLPA can specifically detect copy number variants of target gene,which cannot be replaced by second-generation sequencing.2.Targeting second-generation sequencing can locate disease-causing mutations efficiently and accurately.In this study,57 HSP probands were diagnosed,covering 12 clinical subtypes;at the same time,25 new pathogenic mutation sites and clinical phenotypes of some rare subtypes were found,which enriched the HSP gene mutations in China and extent the database and clinical phenotype spectrum.3.SPG4 and SPG5 are the most common clinical subtypes of autosomal dominant HSP and autosomal recessive HSP in Fujian region,respectively.Among them,the CYP7B1 gene of SPG5 has a hot spot mutation c.334 C>T(p.R112*).4.Mitochondrial and autophagy-lysosome dysfunction plays an important role in the pathogenesis of HSP.5.Peroxisomal diseases represented by AMN are mainly manifested as impaired symptoms of upper motor neurons,even most of them have no imaging signs,which are easily misdiagnosed as HSP.Biochemical detection combined with gene detection and VLFAC can be used to identify them.In addition,tropical paraplegia due to the prevalence of the HTLV-1 virus infection in some regions is also easily confused with HSP.A diagnosis can be made by detecting virus antibodies and DNA sequences.