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Cystatin B: Mutation Detection, Alternative Splicing and Expression in Progressive Myclonus Epilepsy of Unverricht-Lundborg Type (EPM1) Patients

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Cystatin B: Mutation Detection, Alternative Splicing and Expression in Progressive Myclonus Epilepsy of Unverricht-Lundborg Type (EPM1) Patients European Journal of Human Genetics (2007) 15, 185–193 & 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00 www.nature.com/ejhg ARTICLE Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients Tarja Joensuu*,1, Mervi Kuronen1, Kirsi Alakurtti1, Saara Tegelberg1, Paula Hakala1, Antti Aalto2, Laura Huopaniemi3, Nina Aula1, Roberto Michellucci4, Kai Eriksson5 and Anna-Elina Lehesjoki1 1Department of Medical Genetics and Neuroscience Center, Folkha¨lsan Institute of Genetics, Biomedicum Helsinki, University of Helsinki, Finland; 2Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland; 3Rational Drug Design Program, Biomedicum Helsinki, Helsinki, Finland; 4Department of Neurosciences, Epilepsy Centre, Bellaria Hospital, Bologna, Italy; 5Pediatric Neurology Unit, Department of Pediatrics, Pediatric Research Center, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland 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.149G4A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168 þ 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. European Journal of Human Genetics (2007) 15, 185–193. doi:10.1038/sj.ejhg.5201723; published online 27 September 2006 Keywords: cystatin B; progressive myoclonus epilepsy; alternative splicing; real-time PCR; expansion repeat; lysosomes *Correspondence: Dr T Joensuu, Folkha¨lsan Institute of Genetics, Depart- Introduction ment of Medical Genetics and Neuroscience Center, Biomedicum Eight mutations in the cystatin B gene (CSTB, OMIM No. Helsinki, P.O. Box 63, University of Helsinki, Helsinki, Finland. 601145) encoding a cysteine protease inhibitor have thus Tel: þ 358 9 191 25081; Fax: þ 358 9 191 25073; far been reported to associate with an autosomal recessive E-mail: [email protected] Received 30 June 2006; revised 21 August 2006; accepted 23 August 2006; neurodegenerative disorder, progressive myoclonus epi- published online 27 September 2006 lepsy of Unverricht-Lundborg type (EPM1, OMIM No. Cystatin B gene expression T Joensuu et al 186 254800).1–7 The onset of EPM1 with stimulus-sensitive five heterozygous expansion mutation carriers and three myoclonus and/or tonic-clonic epileptic seizures occurs at control individuals with no expansion, as determined by the age of 6–15 years with progressive ataxia manifesting Southern blot hybridizations. The method was applied in later.8,9 Most of the disease alleles harbour an unstable molecular diagnostics of an Italian patient with a clinical expansion of at least 30 copies of a normally polymorphic suspicion of EPM1. 12-nucleotide, dodecamer repeat located in the promoter Genomic DNA from a Finnish EPM1 patient previously region of the CSTB gene.3,5,7,10 Three reported EPM1 identified to be a heterozygous carrier of the dodecamer mutations affect splice sites (c.67-1G4C, c.168G4A, expansion mutation, as well as from an Italian patient (see c.169-2A4G), two result in amino-acid changes above) with no previously known CSTB mutations were (c.10G4C, p.G4R; c.212A4C, p.Q71P) and two predict scanned for novel mutations by sequencing. The identified truncated proteins either through creating a stop codon novel mutations were screened in a panel of 73 Finnish and (c.202C4T) or producing a frameshift (c.218_219delTC). 93 CEPH control individuals. Markedly reduced CSTB mRNA expression has been Total RNA and/or protein extracted from lymphoblastoid reported by Northern analysis in lymphoblastoid cells of cells of eight EPM1 patients of which two were homo- EPM1 patients who are heterozygous or homozygous for zygous for the minisatellite expansion and six compound the expansion mutation.1,3,4,6,11 Consequently, decreased heterozygous for the expansion and either the splice site inhibitory activity of the CSTB protein in these cells has c.67-1G4C(N ¼ 4), the p.G50E missense c.149G4A(N ¼ 1) been demonstrated.12 On the contrary, by RNase protec- or the nonsense c.202C4T(N ¼ 1) mutation were analyzed tion assay normal CSTB expression levels were detected in for the CSTB gene and protein expression. One expansion lymphoblastoid cells and fibroblasts of patients, while mutation heterozygous carrier and two non-carrier indivi- blood leukocytes showed reduced mRNA level, suggesting duals were examined as controls. CSTB and CSTB2 expres- cell-specific regulation of CSTB expression.5 Consistent sion was also examined from fibroblast RNA of two with these results, the activity of the putative CSTB expansion mutation homozygous patients and two con- promoter, which contained B50 copies of dodecamer trols. For the first-strand cDNA synthesis, total RNA was repeats, was found to be cell-specific and varied from isolated using the RNAqueoust–4PCR kit (Ambion, Austin, normal to two- to fourfold reduction.13 The repression of TX, USA). The c.168 þ 1_18del mutation was characterized transcription was proposed to result from the disrupted by RT-PCR from total RNA extracted from a fresh blood spacing of transcription factor binding sites from the using the PAXgene Blood RNA Validation kit (Qiagen, transcription initiation site. Contrary to these results, but Hilden, Germany). compatible with the Northern blot data on CSTB expres- The study has been approved by an institutional ethical sion, our in vitro study with a promoter construct contain- review board at the University of Helsinki. ing 19 copies of the dodecamer repeat in transiently transfected COS-1 cells showed a reduced promoter activity Mutation analysis of 10-fold.11 Later, the expanded dodecamer repeats have The amplification conditions across the dodecamer repeat been shown to induce stable tetraplex secondary struc- expansion were modified from the Expand Long Template tures, which has been suggested to result in the repression PCR system (Roche Diagnostics, Mannheim, Germany) and of CSTB transcription by altered chromatin structures.14 performed in a total of 20 ml reaction mix using primers 2F Here, we have characterized the CSTB gene in more (50-CCC GGA AAG ACG ATA CCA G-30) and 1R (50-GAG detail. We describe a novel direct PCR amplification GAG GCA CTT TGG CTT C-30) (Figure 1) with 15 ng protocol, which allows reliable detection of expanded genomic DNA, 0.5 mM dNTP, 0.5 mM of each primer, 0.12 U CSTB alleles from genomic DNA and describe the identi- enzyme mix, 1 X BSA, 5% DMSO, and 1.0 M GC-melt mix fication and characterization of two novel EPM1-associated (Clontech, Palo Alto, CA, USA). Following denaturation at CSTB gene mutations. We have further quantitated the 941C for 2 min, PCR was carried out in 40 cycles of 941C for CSTB expression in human tissues and in patients’ 10 s, 571C for 45 s, and 681C for 8 min. A final elongation lymphoblastoid cells by real-time PCR. Moreover, we have was at 681C for 7 min. experimentally verified alternative splicing of CSTB and For mutation scanning, 2949 bp of the CSTB gene describe the characterization of a ubiquitously expressed (GenBank AF208234), including the exons, the introns, variant, CSTB2. and the 50 and 30 untranslated regions, was amplified in overlapping fragments (Figure 1) using the following primers: 1F (50-AAA CGC AAA TTC CAC CAG AG-30) and Materials and methods 1R (50-GAG GAG GCA CTT TGG CTT C-30), 2F (50-CCC EPM1 patient and control samples GGA AAG ACG ATA CCA G-30) and 2R (50-CGG CTT CTT The novel PCR-based method to amplify the dodecamer TCG CTC CAG-30), 3F (50-GCC GAG ACC CAG CAC ATC- repeat expansion from genomic DNA was validated in 16 30) and 3R (50-CCT GTG GAC CTT TTA TGC AG-30), 4F (50- patients with homozygous mutation for the expansion, GCA AGA GGT CCC CAG TGA TA-30) and 4R (50-TGA CAC European Journal of Human Genetics Cystatin B gene expression T Joensuu et al 187 ATG STOP INT1 INT2 EXON1 EXON2 EXON3 568 bp 1F 1R 457 bp 2F 2R 675 bp 3F 3R 925 bp 4F 4R 317 bp 5F 5R 304 bp 6F 6R 421 bp 7F 7R Figure 1 Schematic structure of the CSTB gene and the localization of primers used in mutation analysis with lengths of the respective
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