Novel mutations in spastin gene and absence of correlation with age at onset of symptoms

Materials and methods.

Thirty-seven families with pure DHSP were analyzed. The locus in three families (581, 9998, and 1252) was shown to be linked chromosome 2p22.4 Three families (1093, 9997, and 840) were excluded from linkage to this region and the other families were not sufficiently large to be tested by linkage analysis. The phenotype in these families was pure HSP with established autosomal dominant inheritance as described previously.4 DNA samples from one or two affected members from each family were analyzed by single-stranded conformation polymorphism (SSCP) and then confirmed by sequencing if SSCP change was identified.

In the analysis of the age at onset of symptoms, only symptomatic patients were included; asymptomatic family members found to be affected by physical examination were excluded.

Genomic sequences of recently released bacteria artificial chromosomes (BAC) that map to the SPG4 region on chromosome 2p22 were obtained by computer-based search using the BLAST program and the previously mapped STS and EST. Each BAC sequence was analyzed using the exon search program GenScan, and putative exons were identified. Each of these putative exons was in turn tested back by homology search against the genome databases using the BLAST program. This allowed the identification of KIAA1083,8 an anonymous complementary DNA containing a 1752-bp open reading frame and predicted to code for a 584-amino-acid protein that belongs to the AAA superfamily. KIAA1083 complementary DNA was subsequently found to have 100% homology with spastin except for a 32-amino-acid segment, corresponding to exon 2 of the spastin gene, which is not present in KIAA1083. This exon is most likely alternatively spliced. We concluded that the same gene encodes for both KIAA1083 and spastin.

After identification of the boundaries of the 15 exons that constitute the KIAA1083 gene, primers that flank each of these exons were synthesized and used for SSCP and sequencing analyses as previously described.9 Given the large size of exon 1, it was analyzed in three overlapping fragments. After the release of the spastin sequence, the alternatively spliced exon 2 was also tested for mutations in our families with HSP.

Results.

Five different novel mutations in the KIAA1083 gene were identified in five of the 37 families with DHSP tested. This suggests that families with SPG4 mutations represent about 13.5% of all families with DHSP, which is lower than previously thought.4 Four families were from North America and one family (9998) was from Tunisia. The three families with mutations previously mapped to 2p224 were found to have mutations in the KIAA1083 gene. No mutation was observed in the families whose HSP was excluded from linkage to this region or in 200 chromosomes from 100 unrelated individuals.

Table 1 summarizes the five mutations identified. Two mutations were predicted to cause amino acid substitutions that affect conserved amino acids in mouse spastin proteins. Three mutations were predicted to generate a truncated protein either by creating a stop codon or by causing a frame shift (see table 1).

The mean age at onset of symptoms (MAOS) in our five families with mutations in the spastin gene was 30.8 years. Analysis of each family separately showed that the MAOS varied from 13.1 years in family 1252 to 40 years in family 9329 (table 2). There was no obvious correlation between age at onset of symptoms and mutation (figure): the MAOS in families 581 and 9803, which had mutations predicted to truncate the protein, was similar to that of family 9998, which carried a mutation predicted to cause an amino acid substitution. Also, the MAOS in family 1252 was two decades earlier than in family 581, and both had a mutation predicted to cause a truncation of the protein.

• Download figure
• Open in new tab
• Download powerpoint

Figure. Kaplan-Meier curve of age at onset of symptoms in patients with hereditary spastic paraplegia carrying mutations in spastin. Age at onset of symptoms is presented on the x axis and the percentage of affected individuals on the y axis. Green = family (Fam) 9803; yellow = Fam 9329; orange = Fam 581; pink = Fam 1252; violet = Fam 9998; black = all families.

Discussion.

We report five novel mutations in the spastin gene associated with the HSP phenotype in five different families. That these mutations cause the disease process is very likely, as no similar mutations in control subjects were identified. Our findings suggest that SPG4 mutations are less common than previously thought, as only 13.5% of all families with DHSP had mutation in spastin gene, rather than the 50% previously reported. This discrepancy suggests that either there was a recruitment bias favoring large families in the linkage analysis study, or SSCP analysis had a lower-than-expected sensitivity.

The function of spastin is unknown despite its relation to the AAA protein family, because these proteins have a wide variety of functions.7 It is tempting to speculate that spastin and paraplegin have related functions as both belong to the AAA family. However, the absence of a strong amino acid homology between the two proteins6 and the mitochondrial localization of paraplegin, which is contrary to the presumed nuclear localization of spastin,5,6⇓ do not favor a functional link between these two proteins. Other tempting hypotheses include a possible involvement of spastin in axonal transport, as only large and long axons in the spinal cord are affected in HSP, or a possible role of spastin in zinc homeostasis reminiscent forthe possible role of zinc in mutant SOD1 in familial ALS. The identification of the cellular sublocalization of spastin and the analysis of the effect of its inhibition on neurons may help determine its function.