Diagnosis Of Hereditary Spastic Paraplegia In China Using Targeted Next-generation Sequencing And Gene Functional Analysis

Background and objective: Hereditary spastic paraplegia(HSP)refers to a group of the most clinically and genetically heterogeneous neurodegenerative diseases characterized by progressive weakness and spasticity of lower limbs.Historically,HSP is distinguished as pure or complicated forms according to the clinical symptoms.All modes of inheritance have appeared,including autosomal dominant(AD),autosomal recessive(AR),x-linked and mitochondrial inheritances.The objective of this study was to improve the molecular diagnosis of cases affected by HSP and understand the gene mutation characteristics of HSP patients in China.Methods: Patients with suspected HSP were recruited from 2009 to 2016.In total,55 unrelated Chinese HSP pedigrees were enrolled,comprising 39 autosomal dominant(AD),8 autosomal recessive(AR)and 8 sporadic cases.In addition,500 normal individuals without history of neurological disorders were involved as controls.Firstly,a panel of 62 genes relevant to spastic paraplegia(SPG)was designed and 55 HSP probands were subject to targeted next-generation(NGS)for mutations of HSP causing genes.Secondly,the data of targeted NGS were analyzed,filtered and validated by Sanger sequencing.Then,the variants identified were screened in the families and 500 controls.Subsequently,the classification of novel variants is based on the American College of Medical Genetics and Genomics(ACMG)standards and guidelines.Finally,cells functional researched were conducted to clear the pathogenicity of ambiguous novel variants,further defining the molecular diagnosis.Results:1.We made a genetic diagnosis in 60%(33/55)of families and identified 32 mutations,including 13 previously reported mutations and 19 novel mutations,of which one was de novel mutation(NIPA1: c.G316A).The cells functional findings were suggestive of the pathogenicity of the novel BSCL2(c.G1309C)variant.2.Altogether,21 families were diagnosis as SPG4(21/39,53.8%),4 as SPG3(4/39,10.3%),2 as SPG11,2 as SPG6,1 as SPG31,1as SPG17,1 as SPG30 and 1as SPG5.Conclusions:1.Our findings broaden the mutation spectrum of HSP.SPAST(SPG4)mutations are the most common causes of Chinese AD-HSP followed by ATL1(SPG3).SPG6 is possibly more frequent in China with a percentage of 5.6% in AD-HSP(1/18)and 11.1% in sporadic HSP(1/9)negative for SPAST mutation.2.We identified the third BSCL2(SPG17)mutation(c.G1309C)related to HSP and first reported the KIF1A(SPG30)mutation(c.G304A)in China.3.Targeted next-generation sequencing(NGS),a high-throughput and cost-effective technology,has shown salient superiority in the diagnosis of HSP with clinical and genetical heterogenicity.It can strikingly improve the efficiency and diagnosis rate.Nonetheless,the function research is warranted for variants with ambiguous properties.