| Objective:Hereditary spastic paraparesis(HSP)is a heterogeneous group of disorders characterized by weakness and spasticity of lower limbs.Complex HSP may combine with additional symptoms such as cognitive impairment,ataxia,optic nerve atrophy and hearing loss.HSP is transmitted as an autosomal dominant,autosomal recessive or X-linked trait.With the constant development of sequencing technology,an increasing number of genes are related to HSP.In addition,some HSP subtypes are rare.We intend to perform the genetics analysis in the enrolled pedigrees with HSP,and describe the clinical and genetic characteristics of HSP patients in China and further carry out functional studies for the rare subtypes,which is meaningful for the comprehension of the feature of HSP and is conducive to exploring the pathogenic mechanisms.Methods:Whole exome sequencing(WES)combined with Sanger sequencing were performed in all the HSP probands.Multiplex ligation-dependent probe amplification(MLPA)assays were carried out for patients without positive findings in known HSP-related genes.Immunofluorescence was used to analyze the cellular changes caused by novel mutations detected in our study in RTN2(SPG12)and CAPN1(SPG76)gene.A Minigene assay was performed to explore the influence of the novel splicing mutation in ERLIN1 gene on the splice site.Results:A total of 150 HSP probands were recruited,including 93 males and 57 females.The average age of onset was 25.46 ± 15.66 years and the mean disease duration was 12.47± 10.15 years.110(73.3%)patients were pure HSP,while 40(26.7%)patients were complex.Dysarthria was the most common additional symptom.Cognitive involvement,ataxia,muscular atrophy,tremor,developmental delay,visual symptoms,peripheral neuropathy,epilepsy,and dysphagia were also observed in our cohort.Among 77(51.3%)patients with positive family history,autosome dominant is the most common mode of inheritance(n=50).17 subtypes were detected in this study,of which,SPG4(SPAST),SPG3A(ATL1)and SPG11(SPG11)are the most common subtype.The alteration of subcellular localization of truncated RTN2 and CAPN1 were shown by immunofluorescence.Truncated RTN2 was distributed in the cytoplasm diffusely,and truncated CAPN1 aggregated abnormally in the perinuclear compartment.Splicing variant c.504+1G>A would cause a complete skip of exon 7 in ERLIN1 gene.Conclusions:Pathogenic or likely pathogenic variants in known HSP-related genes were identified in 73(49%)probands by WES combined with MLPA assay,of which 13(9%)patients carried rearrangement mutations.94%of autosomal dominant hereditary spastic paraplegia(AD-HSP)and 70%of autosomal recessive hereditary spastic paraplegia(AD-HSP)were pure HSP.Mutant RTN2 would lead to SPG12.Till now,2 dominant pedigrees and 2 sporadic SPG12 patients have been detected.We report the first case of SPG12 in China.This patient carried a frameshift mutation,which caused a lack of a conserved domain(RHD domain)and a disruption of co-localization with the endoplasmic reticulum.The variant may act as a haploinsufficiency mechanism.Further experiments are required to explore whether truncated mutations could cause additional toxic gain of function or not.Mutations in ERLIN1 gene may cause SPG62.Currently,only 3 pedigrees have been reported.We detected a homozygous splicing mutation in ERLIN1 gene resulting in a complete deletion of exon7 which may cause a disruption of the conserved PHB domain,which is the first case of SPG62 in China.Calpains encoded by CAPN1 gene belongs to calcium-dependent proteases,which are related to synaptic plasticity,axon maturation and maintenance.We identified a novel frameshift variant in CAPN1 gene which could cause a premature termination and disrupt the conserved PEF domain of the protein family.The co-localization of calpains and tubulin was destroyed by the mutation,which may affect the interaction between calpain-1 and microtubules.This study summarizes the clinical characteristics of HSP patients in China and expands the mutation spectrum,which may improve the efficiency of clinical diagnosis.Functional experiments for the rare HSP subtype were carried out,which is beneficial to the exploration of the pathogenic mechanism and is meaningful for the investigation of individualized treatment in the future. |
