Identification and expression analysis of spastin gene mutations in hereditary spastic paraplegia

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterize d primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AA A family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations ident ified are in noninvariant splice-junction sequences. Reverse transcription- PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or part ially penetrant; that is, the mutant allele produced both mutant (skipped e xon) and wild-type (full-length) transcripts. This phenomenon was reproduce d in in vitro splicing experiments, with a minigene splicing-vector constru ct only in the context of the endogenous splice junctions flanking the spli ce junctions of the skipped exon. In the absence of endogenous splice junct ions, only mutant transcript was detected. The existence of at least one le aky mutation suggests that relatively small differences in the level of wil d-type spastin expression can have significant functional consequences. Thi s may account, at least in part, for the wide ranges in age at onset, sympt om severity, and rate of symptom progression that have been reported to occ ur both among and within families with SPG linked to SPG4. In addition, the se results suggest caution in the interpretation of data solely obtained wi th minigene constructs to study the effects of sequence variation on splici ng. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.