Pathogenic Gene Identification And Functional Study Of Three Families With Autosomal Dominant Spastic Paraplegia

Hereditary spastic paraplegias(HSPs)is a group of neurodegenerative disorders characterized by dysfunction of corticospinal tracts.The key diagnostic clinical finding are progressive spasticity and weakness,hypermyotonia and scissors gait of the lower limbs.To date,78 genetic loci have been located and 64 pathogenic genes have been cloned.Spastic paraplegia type 4(SPG4)is the most common autosomal dominant hereditary spastic paraplegias(AD-HSPs),which is accounting for about 40% in AD-HSPs.SPAST is the pathogenic gene of SPG4,and spastin protein encoded by SPAST plays the role of microtubule-severing.After microtubules was severed into small segments,which are involved in the synthesis of membranous organelles,transport of membrane vesicle and neuronal axonal elongation.In this study,we collected three families with autosomal dominant spastic paraplegias in Henan province.The patients in three families showed hypermyotonia,hyperactivity of tendon reflex,and scissors gait of both lower limbs.Other diseases were not examined except for spastic paraplegias.Clinical features showed that three families were pure hereditary spastic paraplegias.We found that positive linkage was identified with the microsatellite marker D2S367,and a known SPG4 pathogenic gene SPAST located at 2.05 Mb upstream of this site.Then,Sanger sequencing was performed on the exons and intron-exon boundaries of the SPAST of three probands,and a novel duplication c.985 dupA was found in SPAST.Moreover,the c.985 dupA of SPAST fully segregated with the spastic paraplegias phenotype in each of families by high resolution melting.And the c.985 dupA of SPAST was not found in 100 normal populations by high resolution melting.The mutation c.985 dupA leads to a shift in the reading frame at 329 th codon(Met329Asn)and results in a premature termination codon 3 amino acid residues downstream from the site.Thus,the mutation will produce a truncated protein p.Met329Asnfs*3 of spastin.Interestingly,the expression of the truncated spastin protein was significantly higher than wild type in HEK293 T cells via Western blotting.The immunofluorescence results revealed that the truncated spastin protein and microtubules in cells were evenly distributed around the nucleus,forming a ring.While the wild-type spastin protein and microtubules distributed scattered around the nucleus and cytoplasm.Taken together,these results suggest that truncated spastin protein may lose the function of microtubule-severing and failed to sever microtubules into small segments.Thus,they could not participate in the synthesis of membranous organelles,transport of membrane vesicle,and neuronal axonal elongation.And those results eventually resulted in the denaturation and death of axon neurons cells,and the formation of spastic paraplegias.This study not only broadens the mutant spectrum of SPAST,but also lays a theoretical foundation for the molecular pathogenesis research and prenatal diagnosis of this disease.