Molekularne patologicka diagnostika dystrofinopatii se zamerenim na identifikaci prenasecek.

Abstract

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive neuromuscular disorders caused by dystrophin gene (DG) mutanions. The incidence of these disorders is very high (of about one DMD in 3500 newborn males, of about one BMD in 17000 newborn males) due to the extreme size of the DG. In approximately two-thirds of affected males the DG is disrupted by deletions not evenly distributed but concentrated in major and minor hot spots arround exons 45-47 and 7-9 respectively. Duplications are revealed in about 5-8% of cases and point mutation are reported to cause about one-third of total DMD/BMD cases. Discovery of the DG and its gene product led to the development of many diagnostical methods working on the protein or gene levels. Complex diagnosis of muscular distrophies has been provided in a limited extent in our country and some diagnostical methods useful in a diagnosis of preclinical stages of these diseases as well as in an identification of carriers of DMD/BMD have been put into practice in our institution. The combination of a clinical examination, muscle biopsy and the mutation analysis of mRNA using RT-PCT (reverse transcription-polymerase chain reaction) with a protein truncation test (PTT) brings the most effective results in a diagnosis of DMD/BMD. Histological examination of a muscle biopsy completed by an immunohistochemically detected deficient sarcolemmal expression of the dystrophin proved to be very reliable in a diagnosis of DMD. Normal or partially deficient sarcolemmal expression of the dystrophin in patients with a suspected BMD indicates the Western blot examination and the DG mutation analysis. RT-PCR and PTT enable the detection of deletions, duplications as well as point mutations in the DG and encompasse a large diagnostical scope in comparison with examinations on DNA level using the multiplex PCR (mPCR) which enables an identification of deletions and duplications in DG only. mPCR plays an important role especially in families with multiple incidence of DMD/BMD and with an identified and described deletion or duplication in the DG. Skipping of invasive muscular biopsies is reasonable in these cases. One of the major problems in DMD/BMD genetic counceling is still the definition of the carrier status in "at risk" females. The dissertation presents a diagnostical strategy using the method of fluorescence in situ hybridisation for an identification of DMD/BMD carriers. In females the detection of deletions is complicated by the presence of a second X-chromosome, carrying a normal DG. FISH allows the two X-chromosomes to be examined individually and constitutes an excellent alternative for diagnosis deletions in DMD/BMD heterozygotes. We have used a set of six cosmid probes for the detection of the most frequently deleted areas of the DG from the Department of Human Genetics, Leidel Medical Center, Nederlands. This optimized six cosmid sets shoud detect a deletion in 78% of all deletion carriers and i. e. 52% of all Duchenne/Becker muscular dystrophy cases. Data obtained in our study confirmed results of other authors in that FISH can be used to detect the deletions in the DG. FISH analysis of peripheral blood lymphocytes appears to be an effective and direct method for establishing the DMD/BMD carrier status of females. We suggest FISH to metaphase or interphase chromosomes for a rapid screening of DMD/BMD carriers.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi