Identification and Expression Analysis of Spastin Gene Mutations in Hereditary Spastic Paraplegia

Abstract

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA 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 identified 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 partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

Screening of patients with hereditary spastic paraplegia reveals seven novel mutations in theSPG4 (Spastin) gene

“…There are almost 100 SPG4 mutations described so far, including point mutations (missense, nonsense and splice site) and small insertions and deletions, with most reported families having a unique mutation Bürger et al, 2000;Hentati et al, 2000;Lindsey et al, 2000;Svenson et al, 2001;Meijer et al, 2002;Patrono et al, 2002;Morita et al, 2002;Sauter et al, 2002;Proukakis et al, 2002]. Mutations have now been reported in all SPG4 exons, except the alternatively spliced exon 4 [Svenson et al, 2001].…”

“…Mutations have now been reported in all SPG4 exons, except the alternatively spliced exon 4 [Svenson et al, 2001].…”

A novel missense mutation (I344K) in the SPG4 gene in a Korean family with autosomal-dominant hereditary spastic paraplegia

“…Missense, nonsense, and splice-site mutations, as well as deletions and insertions, have all been observed. Although these mutations have been found scattered along the length of SPG4, the majority of the missense mutations are localized between exons 7 and 16 (Bürger et al 2000;Fonknechten et al 2000;Hazan et al 1999;Hentati et al 2000;Lindsey et al 2000;Svenson et al 2001). This region of SPG4 has been shown to correspond to a highly conserved ATPase domain, also called the AAA cassette,…”

“…Recently, Hazen et al (1999) identified the spastin (SPG4) gene, which encodes a new member of the AAA (ATPase associated with diverse cellular activities) protein family. After this discovery, more than 70 mutations of the SPG4 gene have been reported, including missense, nonsense, and splice-site mutations, as well as insertions and deletions (Bürger et al 2000;de Bantel et al 2001;Fonknechten et al 2000;Hazan et al 1999;Hentati et al 2000;Jiggins et al 2001;Lindsey et al 2000;Namekawa et al 2001;Santorelli et al 2000;Svenson et al 2001). Here, we present a novel missense (I344K) mutation in the SPG4 gene in a large Korean family with pure AD-HSP.…”

“…The importance of the AAA cassette in spastin function is suggested by its high degree of conservation among AAA protein family members and by the locations of the spastin gene mutations identified to date (Hazan et al 1999;Svenson et al 2001).…”