| 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. |
