1. ¹Department of Medical Genetics and Neuroscience Center, Folkhälsan Institute of Genetics, Biomedicum Helsinki, University of Helsinki, Finland
2. ²Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland
3. ³Rational Drug Design Program, Biomedicum Helsinki, Helsinki, Finland
4. ⁴Department of Neurosciences, Epilepsy Centre, Bellaria Hospital, Bologna, Italy
5. ⁵Pediatric Neurology Unit, Department of Pediatrics, Pediatric Research Center, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland

Correspondence: Dr T Joensuu, Folkhälsan Institute of Genetics, Department of Medical Genetics and Neuroscience Center, Biomedicum Helsinki, P.O. Box 63, University of Helsinki, Helsinki, Finland. Tel: +358 9 191 25081; Fax: +358 9 191 25073; E-mail: tarja.joensuu@helsinki.fi

Received 30 June 2006; Revised 21 August 2006; Accepted 23 August 2006; Published online 27 September 2006.

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G>A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168+1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5–10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.