A novel truncating variant of SPAST associated with hereditary spastic paraplegia indicates a haploinsufficiency pathogenic mechanism

Abstract

IntroductionHereditary spastic paraplegias (HSPs) are genetic neurodegenerative diseases. The most common form of pure HSP that is inherited in an autosomal dominant manner is spastic paraplegia type 4 (SPG4), which is caused by mutations in the SPAST gene. Different theories have been proposed as the mechanism underlying SPAST-HSP for different types of genetic mutations, including gain- and loss-of-function mechanisms. To better understand the mutation mechanisms, we performed genetic analysis and investigated a truncating SPAST variant that segregated with disease in one family.Objectives and methodsWe described a pure HSP pedigree with family members across four generations. We performed genetic analysis and investigated a novel frameshift pathogenic variant (c.862_863dupAC, p. H289Lfs*27) in this family. We performed reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, and quantitative RT-PCR using total RNA from an Epstein-Barr virus-induced lymphoblastoid cell line produced from the proband. We also performed Western blotting on cell lysates to investigate if the protein expression of spastin is affected by this variant.ResultsThis variant (c.862_863dupAC, p. H289Lfs*27) co-segregated with pure HSP in this family and is not registered in any public database. Measurement of SPAST transcripts in lymphoblasts from the proband demonstrated a reduction of SPAST transcript levels through likely nonsense-mediated mRNA decay. Immunoblot analyses demonstrated a reduction of spastin protein expression levels in lymphoblasts.ConclusionWe report an SPG4 family with a novel heterozygous frameshift variant p.H289Lfs*27 in SPAST. Our study implies haploinsufficiency as the pathogenic mechanism for this variant and expands the known mutation spectrum of SPAST.

New cellular imaging‐based method to distinguish the SPG4 subtype of hereditary spastic paraplegia

“…To evaluate whether this SPG4 LCL phenotype might be induced by spastin level reduction, spastin protein levels were first verified in our SPG4 LCLs. According to the type of SPG4 truncating mutations (Table 1) and literature evidence [20, 21], WB analyses showed a significant reduction of approximately 50% of spastin protein levels in the SPG4 LCLs compared to HD LCLs, whilst, as expected, the spastin truncated forms and M1 isoforms were undetectable (Figures 1b and S1d,e). Next, a reduction of spastin protein levels was induced like those of haploinsufficient SPG4 ‐HSP patients' cells in the HD LCLs.…”