Applicant:

Christian Beetz, Dr. rer. nat., Institut für Klinische Chemie und Laboratoriumsdiagnostik Universitätsklinikum der Friedrich-Schiller-Universität Jena Erlanger Allee 101  07749 JENA

e-mail: christian.beetz@med.uni-jena.de

Hereditary spastic paraplegia (HSP) is genetically highly heterogeneous but frequent involvement is observed for only a few specific loci. Regarding autosomal dominant HSP (AD-HSP), conventional sequencing identifies mutations in SPG3A (atlastin) or SPG4 (spastin) in ~50% of cases. A number of pertinent studies, however, failed to reveal a causative alteration in either of these genes despite suggestive linkage data. The identification of large genomic SPG4 deletions in two such pedigrees argues for copy number aberrations to account for an hitherto underestimated fraction in the mutational spectrum leading to HSP.

We intend to develop a simple assay allowing standardised screening for copy number aberrations affecting SPG3A or SPG4. We chose multiplex ligation-dependent probe amplification (MLPA), a novel PCR-based quantitative method, as a potentially appropriate methodology. In collaboration with MRC-Holland (The Netherlands), MLPA probes targeting all relevant exons are designed, tested and combined in a single kit. In a first round of investigation, this kit will be applied to DNAs previously collected at the Institut für Klinische Chemie und Laboratoriumsdiagnostik at the FSU Jena. These samples have been extensively analysed for HSP-associated mutations and one of them, containing a multi-exonic SPG4 deletion, will serve as a positive control during establishment of the assay. Long range PCR and/or Southern blotting will be applied to validate and further characterise positive MLPA findings. In order to enable the analysis of DNAs from additional HSP patients, we are planning to collaborate with other HSP centres at a later stage of the project.

Our study should (i) establish the incidence of HSP-associated copy number aberrations affecting SPG3A and SPG4, (ii) depending on the very findings, help to further elucidate the pathomechanism leading to HSP, and (iii) guide a potential implementation of the assay in routine molecular diagnosis of AD-HSP.

Final report

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).