A Study On The Causative Gene Of A SPG26 Family Based On Next-generation Sequencing Technology

Objective: The hereditary spastic paraplegias(HSPs)are a group of hereditary neurodegenerative diseases characterized by progressive spasticity and weakness of the lower limbs with clinical and genetic heterogeneity.Targeted Next-generation Sequencing(NGS)was used to genetically diagnose an HSP family from Henan province in this study.And in vitro experiment was performed to confirm the effect of splicing mutations on protein function.The clinical characteristics of patients were analyzed and summarized to provide a basis for genetic diagnosis and further pathogenesis research.Methods: The clinical data of a HSP family was collected and the family map was drawn.After obtaining the informed consent of the patients and their families,the peripheral blood DNA samples were extracted and targeted second-generation sequencing was performed.We also utilize the gene database NCBI db SNP and geneme1000 to screen and exclude polymorphic sites.Then the bioinformatics software such as SIFT,Poly Phen2,and Human Splicer Finder were used to predict the effect of the mutation site on protein function.Finally,PCR was combined with Sanger Sequencing to verify the pathogenicity of the selected mutations.In the second part,we constructed constructed wild and mutant Minigene vectors including a splice mutation site in the intron region and transfected the vectors into HEK293 T cells to simulate splicing process in vitro,and extracted cellular RNA for reverse transcription analysis(RT-PCR).Finally,sequence the DNA segment to analyze whether the mutation affects m RNA splicing.Results: In an autosomal recessive HSP family,we found that two patients had poor academic performance,severe mental impairment and emotional changes in addition to the common clinical manifestations of simple HSP such as progressive lower limb weakness,gait abnormalities,and pyramidal tract signs.Through exon sequencing and Sanger sequencing verification,it was found that all the patients of this familycarried two new compound heterozygous mutations of the B4GALNT1 gene(c.1424C> T,p.Ser475 Phe and c.1002 + 2T> G).After testing the patient’s parents and two unaffected sisters,the mutation was found to be compatible with the co-segregation of the family.These two mutations are novel mutations and are predicted to be pathogenic by bioinformatics.For intron region splicing mutations(c.1002 + 2T> G),we constructed wild-type and mutant Minigene vectors to study the effect of m RNA splicing and it was found that compared to wild-type result,the mutations in intron 8 caused partial intron 8 retention,splicing products of mutant DNA are 142 bp longer than that of wild type DNA.Mutations alter the normal splicing product sequence,which may affect the structure and function of the transcription product.Conclusion: We found the first domestic SPG26 family in China.Similar to the clinical characteristics of SPG26 families reported abroad,SPG26 patients in China have rare clinical manifestations such as adolescent onset and lower limb spasticity,as well as severe intellectual impairment.The two novel mutations(c.1424C> T and c.1002 + 2T> G)expand the gene mutation spectrum of B4GALNT1 and provide a basis for the genetic diagnosis and pathogenesis of HSP.