A Study On The Pathogenic Genes Of A Hereditary Spastic Paraplegia Family Basedon Second-generation Sequencing Targetedcapture Technology

Objective: Hereditary spastic paraplegias（HSPs）are a group of clinically and genetically heterogeneous neurodegenerative diseases with progressive spasticity and weakness in the lower limbs.The purpose of this study is to make accurate genetic diagnosis for patients in a Chinese HSP family by using targeted-capture next generation sequencing（TCNGS）and provide evidence for genetic counseling.Methods: A small three-generation family including 11 members were collected as research objects and the pedigree map was drawn.Peripheral anticoagulant blood samples were collected and genomic DNA（g DNA）of blood was extracted from nine of them after obtaining informed consent.Detailed medical history and physical examination were performed from all participants,and clinical examination datas including brain magnetic resonance imaging（MRI）and electromyography（EMG）were collected from three patients.Firstly,the g DNA sample of the proband（II-1）was selected for Targeted-capture Next generation sequencing.The results were compared with and filtered against Human reference genome,the 1000 Genomes project,db SNP142 database.etc,then the SIFT,Poly Phen2,FATHMM bioinformatics analysis softwares were used to predict the function of the newly discovered NS/SS/Indel variants and obtain the suspicious causative mutations.Finally,candidate gene variants were verified by Sanger sequencing in another 8 immediate family members of the family,thus clearly identifying the diagnosis of patients in this family.Results: Clinical examination results: the proband（II-1）presented with typical progressive lower extremity stiffness,weakness,and ataxia.Two sisters of him（II-3,II-4）showed similar symptoms and additional dysarthria.Brain MRI revealed cerebellar atrophy in all three patients.Genetic analysis results: All patients in this family were found to carry two mutations（c.1150₁150+1ins CTAC and c.2062C>T,p.Arg688Trp）and a single nucleotide polymorphism（c.2063G>A,p.Arg688Gln）in SPG7.And the latter two amino acid substitution mutations were proved to be at adjacent site and resulted in a stop codon mutation.Most of asymptomatic members only carry the c.1150₁150+1ins CTAC mutation.In addition,both of the two SPG7 mutation sites were newly identified.Conclusion: Our finding showed that the disease of this family was complicated hereditary spastic paraplegia type 7（SPG7）.The novel compound heterozygous SPG7 mutations（c.1150₁150-1ins CTAC;c.2062C>T,p.Arg688Trp;c.2063G>A,p.Arg688Gln）were the causative gene mutations of the HSP family.And this case proves to be the first case report of SPG7 mutation in HSPs reported in the Chinese population.Our findings not only widen the spectrum of SPG7 mutations,but also indicate that the SPG7 mutation is an important cause of adult-onset undiagnosed hereditary ataxia and should not be neglected.At the same time,it also reminded the majority of physicians and researchers that the next generation sequencing can be appropriately selected and applied for its great effect in diagnosing such rare or highly heterogeneous neurological diseases.