Isolation of CAG/CTG repeat from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPC4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogen eous neurodegenerative disorder of the central nervous system clinically ch aracterized by progressive spasticity mainly affecting the lower limbs. Thr ee distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variab ility suggestive of anticipation and evidence has been provided that CAG/CT G repeat expansions may be involved. To isolate CAG/CTG repeat containing s equences from within the SPG4 candidate region, a novel approach was develo ped. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (greater than or equal to 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing s equences from four YACs spanning the SPG4 candidate region between D2S400 a nd D2S367. A total of four CAG/CTG containing sequences were isolated of wh ich three were novel. However, none of the four CAG/CTG repeats showed expa nded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method cou ld be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on c hromosomes 17 and is, respectively. Also, the RED expansions in six SPG fam ilies could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.