Altered spacing of promoter elements due to the dodecamer repeat expansioncontributes to reduced expression of the cystatin B gene in EPM1

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; MIM 2 54800) is an autosomal recessive disorder characterized by seizures, myoclo nus and progression to cerebellar ataxia, EPM1 arises due to mutations in t he cystatin B (CSTB) gene which encodes a cysteine proteinase inhibitor. On ly a minority of EPM1 alleles carry point mutations, while the majority con tain large expansions of the dodecamer CCCCGCCCCGCG repeat which is present at two to three copies in normal individuals. The dodecamer repeat is loca ted in the 5' flanking region of the CSTB gene, presumably in its promoter, The pathological repeat expansion results in a reduction in CSTB mRNA, whi ch may be cell specific. To elucidate the mechanism of this reduction of ge ne expression, we have studied the putative CSTB promoter in vitro. A 3.8 k b fragment, containing the putative promoter with a 600 bp repeat expansion , showed a 2- to 4-fold reduction in luciferase activity compared with an i dentical fragment with a normal repeat; this reduction was observed only in certain cell types. Introduction of heterologous DNA fragments of 730 and 1000 bp into the normal promoter, instead of the repeat expansion, showed s imilarly reduced activity. Terminal deletions of the promoter implicate a p utative AP-1 binding site, upstream of the repeat, in CSTB transcription ac tivation. We propose that a novel mechanism of pathogenesis, the altering o f the spacing of transcription factor binding sites from each other and/or the transcription initiation site due to repeat expansion, is among the cau ses of reduction in CSTB expression and thus EPM1.