Zdeněk Lukáš

Novel dystrophin mutations revealed by analysis of dystrophin mRNA: alternative splicing suppresses the phenotypic effect of a nonsense mutation

The complete dystrophin mRNA sequence has been analyzed in 20 Duchenne muscular dystrophy and Becker muscular dystrophy patients. In 13 cases, deletions in mRNA were detected using reverse transcription-polymerase chain reaction and in another seven cases, point mutations were found using the protein truncation test. Sixteen patients diagnosed with Duchenne muscular dystrophy showed the presence of deletions or of nonsense point mutations. From four patients with the Becker muscular dystrophy phenotype, three cases were associated with deletions conserving the translational frame and one was associated with a nonsense mutation E1110X. In the case of the E1110X mutation, an alternative splicing of dystrophin mRNA (3485-3640del) was detected in this patient which included the E1110X mutation site (nucleotide 3536) and did not change the translation reading frame. Individual nonsense point mutations were characterized by sequence analysis, which showed five novel mutations with respect to those reported in the Cardiff Human Gene Mutation Database http://uwcm.web.cf.ac.uk/uwcm/mg/hgmd0.html and the Leiden muscular dystrophy pages http://www.dmd.nl/.

Sequestration of MBNL1 in tissues of patients with myotonic dystrophy type 2

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-resolution 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 endothelia, 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.

The screening for X-linked Emery-Dreifuss muscular dystrophy amongst young patients with idiopathic heart conduction system disease treated by a pacemaker implant.

The X‐linked Emery–Dreifuss muscular dystrophy (X‐EDMD) is a hereditary muscle disorder associated with cardiac involvement. Sinus node dysfunction and atrioventricular conduction defects, typical of X‐EDMD, occur in both males and females and may result in sudden cardiac death unless treated by permanent pacing. The objective of the study was to determine the frequency and relevance of X‐EDMD in heart conduction system disease in young individuals treated with a pacemaker implant. The medical history of 3450 paced individuals in the region of South Moravia, Czech republic, was reviewed. Thirty‐five patients, 20 males and 15 females, with idiopathic heart conduction disease of onset before age 40 were identified and screened for X‐EDMD. Within these 35 individuals, only one male was found to carry a mutation in X‐EDMD gene. We conclude that the clinical relevance of X‐EDMD in heart conduction system disease is very low. It should, however, be included into the diagnostic work‐up of young male individuals with idiopathic cardiac conduction disturbances.

P5.6 Insulin receptor splicing in human adipose tissue of patients with DM2

called Photovoice will be tested to explore the experience of individuals with DM1. To describe the experiences of individuals with DM1, to identify barriers and facilitators to their health, to assess the feasibility of using Photovoice with this population, and to develop a list of themes and health concerns that may be used to generate future research questions. Photovoice is a qualitative research method that gives participants cameras to document their experiences. Photovoice was developed based on concepts from feminist theory, documentary photography, and the work of educator Paulo Freire. The goal of Photovoice is to give those outside of the traditional power structure the ability to capture their experiences and share them with policy makers. Participants will be given cameras and asked to “take pictures of what it is like to live with DM1”. They will also be asked to take pictures of barriers and facilitators to their health. Their photographs will guide individual interviews and a focus group. There is a need to add patient-centered research to the DM1 literature while exploring new methods for data collection. In this study, we assume that those living with DM1 are experts about their condition, and that Photovoice may be an appropriate research method to use to capture their experiences.

G.P.7 04 Development of neurogenic and myogenic conditions in critical illness neuromuscular disorders: follow-up histopathological study

Histopathological analysis of 52 biopsies from 43 adult critically ill patients was focused on the course of critically ill patients with intensive care polyneuropathy (ICP) or myopathy (ICM). Needle or open biopsies were processed using a set of conventional histological and histochemical methods. The results were compared with neurophysiologic findings and the clinical state of the patients. ICM and ICP develop soon after the beginning of the disease. The longest interval between the onset of the disease and findings of necrotizing myopathy (MN) was 6 weeks (42 days) after the beginning of the disease, thereafter only ICP of mild forms of ICM were recorded. In nine patients, the biopsy examination was repeated and the histological picture of the lesion changed in all cases. When the second biopsy was performed 11-31 months after the 1st one (7 samples), all myogenic features i.e. picture of simple or necrotizing myopathy disappeared and either recovery with normal histopathological finding (five patients) or a picture of a persistent neurogenic lesion (two patients) was found. On the other hand, electrophysiological evidence of chronic denervation was recorded in all seven patients and did not reflect the clinical improvement of the patients condition.

P.P.2 07 Molecular genetic diagnostics of myotonic dystrophy and facioscapulohumeral muscular dystrophy in Czech patients

DNA diagnostics of MD2 was performed in 100 patients with suspicion of MD2. In case of 48 patients, MD2 was confirmed. DNA diagnostics of FSHD was performed in 32 patients with suspicion of FSHD and confirmed in case of 19 patients.