Sequestration of MBNL1 in tissues of patients with myotonic dystrophy type 2 Z. Luka ´s ˇ a,⇑,1 , M. Falk b,⇑,1 , J. Feit a , O. Souc ˇek a , I. Falkova ´ a,b , L. S ˇ tefanc ˇı ´kova ´ b , E. Janous ˇova ´ c , L. Fajkusova ´ d , J. Zaora ´lkova ´ a , R. Hraba ´lkova ´ a a Institute of Pathology, Brno Faculty Hospital, Brno, Czech Republic b Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic c Institute of Biostatistics and Analyses, Faculty of Medicine and Faculty of Science, Masaryk University, Brno, Czech Republic d Centre of Molecular Biology and Gene Therapy, Brno Faculty Hospital, Brno, Czech Republic Received 9 January 2012; received in revised form 2 March 2012; accepted 6 March 2012 Abstract The pathogenesis of myotonic dystrophy type 2 includes the sequestration of MBNL proteins by expanded CCUG transcripts, which leads to an abnormal splicing of their target pre-mRNAs. We have found CCUG exp RNA transcripts of the ZNF9 gene associated with the formation of ribonuclear foci in human skeletal muscle and some non-muscle tissues present in muscle biopsies and skin excisions from myotonic dystrophy type 2 patients. Using RNA-FISH and immunofluorescence-FISH methods in combination with a high-res- olution confocal microscopy, we demonstrate a different frequency of nuclei containing the CCUG exp foci, a different expression pattern of MBNL1 protein and a different sequestration of MBNL1 by CCUG exp repeats in skeletal muscle, vascular smooth muscle and endo- thelia, Schwann cells, adipocytes, and ectodermal derivatives. The level of CCUG exp transcription in epidermal and hair sheath cells is lower compared with that in other tissues examined. We suppose that non-muscle tissues of myotonic dystrophy type 2 patients might be affected by a similar molecular mechanism as the skeletal muscle, as suggested by our observation of an aberrant insulin receptor splicing in myotonic dystrophy type 2 adipocytes. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Myotonic dystrophy; Transcription of ZNF9; CCUG repeat expansion; Sequestration of MBNL1 protein; Ribonuclear foci; Insulin receptor alternative splicing 1. Introduction Both myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2) share a similar pathogenetic pattern beginning with an expansion of CTG and CCTG nucleotide repeats due to mutation in the genes encoding dystrophia myotonica protein kinase (DMPK) and zinc- finger protein 9 (ZNF9), respectively. Taneja et al. [1] were the first to describe and illustrate intranuclear foci contain- ing CTG repeat expansions in skeletal muscle and fibro- blasts of DM1 patients; the most of transcripts with expanded repeats remain retained in the nuclei of cells. Consequently, DMPK protein levels are reduced [2]. Miller et al. [3] demonstrated recruitment of RNA-binding proteins to the CUG exp foci in DM1 patients. Liquori et al. [4] showed that the second form of DM2 is also caused by an expansion, concretely of a CCTG repeat in the first intron of the ZNF9 gene. Consequently, co-localization 0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.03.004 ⇑ Corresponding authors. Addresses: Department of Pathology, Brno Faculty Hospital, Jihlavska ´ 20, CZ-625 00 Brno, Czech Republic. Tel.: +420 532233837 (Z. Luka ´s ˇ); Laboratory of Chromatin Function, Damage and Repair, Dept. of Molecular Cytology and Cytometry, Institute of Biophysics, Academy of Sciences of CR, Kralovopolska 135, 61265 Brno, Czech Republic. Tel.: +420 541517165; mobile: +420 728 084060 (M. Falk). E-mail addresses: zlukas@nbox.cz, zlukas@fnbrno.cz (Z. Luka ´s ˇ), mfalk@seznam.cz (M. Falk). 1 The first and second author contributed equally. www.elsevier.com/locate/nmd Available online at www.sciencedirect.com Neuromuscular Disorders xxx (2012) xxx–xxx ARTICLE IN PRESS Please cite this article in press as: Luka ´s ˇ Z et al., Sequestration of MBNL1 in tissues of patients with myotonic dystrophy type 2, Neuromuscul Disord (2012), http://dx.doi.org/10.1016/j.nmd.2012.03.004