title: Evaluating the effect of spastin splice mutations by quantitative allele-specific expression assay.
abstract: BACKGROUND
  mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP.
  
  
  METHODS
  a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed.
  
  
  RESULTS
  we have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414-1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%).
  
  
  CONCLUSIONS
  our finding supports a "threshold-effect-model" for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.
authors.affiliation: Department of Neurology, University Medical Center, Johannes Gutenberg University, Mainz, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Klimpe
authors.forename: A
authors.initials: A
authors.lastname: Zibat
authors.forename: U
authors.initials: U
authors.lastname: Zechner
authors.forename: B
authors.initials: B
authors.lastname: Wellek
authors.forename: M
authors.initials: M
authors.lastname: Shoukier
authors.forename: S M
authors.initials: SM
authors.lastname: Sauter
authors.forename: D V K
authors.initials: DV
authors.lastname: Pantakani
authors.forename: A U
authors.initials: AU
authors.lastname: Mannan
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D000328:Adult; D000483:Alleles; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015419:Spastic Paraplegia, Hereditary / Q000235:genetics*; D000074181:Spastin
chemical_list: D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human