Clinical And Mutation Analysis Of Two Families Associated With Hereditary Spastic Paraplegia (SPG4 And SPG31)

Objective Hereditary spastic paraplegia(HSP or SPG)is a rare group of highly clinical and inherited heterogeneous neurodegenerative disease of the nervous system.HSP can be categorized into pure and complicated HSP according to clinical characteristics.Pure HSP is characterized by progressive spastic paraplegia of the lower limbs.Besides,complicated HSP is also associated with the symptoms of damaging in nervous system or non-nervous system.The inheritance of HSP includes autosomal dominant inheritance(AD),autosomal recessive inheritance(AR),X-linked recessive inheritance and mitochondrial inheritance.At present,more than 84 pathogenic gene loci have been identified,and only 67 pathogenic genes have been cloned.In this study,two HSP pedigrees were collected.Clinical characteristics and genetic sequence were analyzed in order to improve clinicians’ understanding of HSP and make for the genetic counseling as well as prenatal diagnosis in families.Methods1.Two HSP pedigrees were enrolled including 6 patients,20 asymptomatic relatives and 40 controls.The detailed disease history inquiry and physical examination were conducted by more than 2 experienced neurologists.The family diagrams were illustrated.Besides,clinical data were collected comprised of imaging,sensory evoked potentials,magnetic stimulation motor evoked potentials,nerve conduction velocity,electroencephalogram,surface electromyography,ophthalmology,biochemical examinations and so on.2.Peripheral venous blood was collected from the patients of 2 pedigrees.The whole exome sequencing was performed after taking the genetic DNA.Sanger sequencing was also conducted for the confirmation of candidate variant from whole exome sequencing.Besides,real-time fluorescence quantitative PCR was implemented in order to validate the copy number variation(CNV).At the same time,the gene detection of the candidate variant was conducted in the asymptomatic relatives of 2 pedigrees and 40 controls.3.Functional prediction of candidate variant was conducted by bioinformatics software Poly Phen2 and SIFT.Homologous sequence comparison was implemented in order to analyze the high conservatism of the variant in the evolution of species by the software of DNAMAN.4.Homology modeling for the candidate variant was carried out by using the online bioinformatics tools of HOPE in order to predict the potential impact of amino acid residues changes caused by mutations on protein function.Results1.The family diagrams revealed that there were patients in every generations.The patients in 2 pedigrees were all inherited by AD.The initial symptoms were weakness of the lower limb and gait abnormality.The predominant clinical features were hypermyotonia,hyperreflexia,positive pathological reflex and scissors gait in the lower limb,which belonged to pure HSP.In pedigree 1,the age at onset of patients decreased in subsequent generations.All patients were associated with similar clinical presentation,and the severity of symptoms varied.2.The results of the auxiliary inspections were as follows.The examinations of cerebral magnetic resonance imaging(MRI)and diffusion tensor imaging(DTI)were normal in all patients.Spinal MRI revealed the atrophy of spinal cord,and the atrophy of thoracic cord was obvious.The spinal X-ray in one patient showed scoliosis.Additionally,the sensory evoked potentials and magnetic stimulation motor evoked potentials were abnormal in 3 patients.One patient was associated with abnormality in the examination of nerve conduction velocity.The impairment of vision was observed in 3 patients.The surface electromyography displayed the spasm was existed in the bilateral rectus femoris,semitendinosus,lateral head and medial head of gastrocnemius muscle.The inspections of electroencephalogram and biochemical examinations were normal in all patients.3.The whole exome sequencing,Sanger sequencing and real-time fluorescence quantitative PCR were all performed.The results showed that a heterozygous gene mutation(c.1037G>T,p.G346V)in exon 7 of SPAST gene was identified in pedigree 1,which was a de novo mutation.In pedigree 2,the CNV of 0.9 Mb interstitial deletion(chr2:85942693-86842693)in chromosome 2 was confirmed.The gene mutation was neither reported nor collected by the relevant databases.The clinical features of pedigree 2 were highly related to REEP1 gene involved in the interstitial deletion.Thus,the diagnosis of SPG31 was made.What’s more,the gene sequencing of the relatives and 40 controls was normal.4.The missense mutation was predicted by bioinformatics software Poly Phen2 and SIFT.The predicted fraction of Poly Phen2 was 1.000.The score predicted by SIFT was 0.000.The pathogenicity of the mutation was suggested.The result of homologous sequence comparison displayed that the genetic locus of the mutation was very conservative in the evolution of different species.5.The result of the prediction for protein structure showed the mutation(1037G>T,p.G346V)resulted in a glycine/valine transition at codon 346.The valine residue was more hydrophobic and bigger than glycine.Besides,the mutant residue was buried in the core of the protein,AAA domain.The change of the protein structure and function was suggested.Conclusions1.The main pathogenic genes of 2 pedigrees were SPAST and REEP1 respectively,which belonged to SPG4 and SPG31 respectively.The 2 pedigrees were all inherited by AD inheritance and characterized by pure HSP.The initial symptoms were weakness of the lower limb and gait abnormality.The clinical manifestation was characterized by hypermyotonia,hyperreflexia,positive pathological reflex and scissors gait in lower limb.There was the phenomenon of genetic prematurity in pedigree 1.2.The missense mutation(1037G>T,p.G346V)in exon 7 of SPAST was detected.The mutation was not reported in the literature or collected in relevant databases,which was de novo.This study expanded the mutation spectrum of SPAST.3.In this study,the CNV of 0.9 Mb interstitial deletion(chr2:85942693-86842693)involved REEP1 gene was identified in the short arm of chromosome 2.The genetic variation was a novel CNV,which was not reported in the literature.