The project aimed at establishing if genomic copy number aberrations contribute to the mutational spectrum underlying hereditary spastic paraplegia (HSP). In a collaboration with MRC-Holland (The Netherlands) we started out by developing a MLPA probe mix which targets SPG3A (atlastin) and SPG4 (spastin). This novel diagnostic tool was subsequently validated using control DNAs and DNAs from HSP patients carrying a known multi-exonic SPG4 deletion. We then analysed a cohort of 65 index patients from families with autosomal dominant (AD) HSP, all of which had been found SPG4 mutation negative by conventional approaches. We obtained evidence for the presence of differing SPG4 deletions in 12 cases. By segregation analysis, cDNA amplification, and breakpoint mapping we confirmed these data and proved the causative nature of the defects. Copy number aberrations affecting SPG3A were not observed.
From our findings we estimate partial SPG4 deletions to underlie approximately 10% of all ADHSP cases. Since this significant kind of mutation can not be detected by conventional mutation screening approaches, implementation of our MLPA assay in the routine diagnostic procedure is highly recommended. Our data also represent an additional argument for the pathogenic mechanism of SPG4-HSP to be haploinsufficiency rather than a dominant negative effect as the deletions involving the 5' end of the gene should result in neither transcript nor protein expressed from the mutant allele.
Our study has been published in Neurology (Beetz et al., "High frequency of partial SPAST deletions in autosomal dominant hereditary spastic paraplegia", accepted 2006/07/28). The assay introduced here is commercially available as "MLPA mix P165" from MRC-Holland (www.mlpa.com).