(by Judith van Deutekom; last modified on September 21, 2001)
Diagnosis of muscular dystrophies is often hampered by the absence of muscle tissue from the patient. We have developed the MyoD-system, an in vitro muscle differentiation system, as a way to bypass this problem (Sancho et al., 1993 and Roest et al. 1993). Although we are primarily applying this method for diagnosis of difficult cases of DMD (Roest et al. 1996a / 1996b / 1999 / PhD thesis), it may also be useful for the diagnosis (or research) of other neuromuscular disorders. Our strategy is based on the retroviral or adenoviral delivery of the MyoD gene to patient-derived fibroblasts, amniocytes or chorionic villi cells (Fig.1). Expression of the introduced MyoD gene induces differentiation of the cells into myogenic cells which, within a few days, start to express early muscle proteins like desmin. Through serum deprivation, the cells further differentiate and fuse into multi-nucleated myotubes. A time-dependent range of muscle-specific proteins becomes expressed, including myosin, titin and dystrophin.
For the diagnosis of DMD, the myo-differentiated cell cultures are screened for the absence or presence of dystrophin by immunohistochemical analysis. In case of DMD-patients, no dystrophin is present. Subsequent RNA-analysis by PTT (Roest et al., 1993a / 1993b / PhD thesis) is then performed to reveal the actual mutation. In case of DMD-carriers, the myoD-technique can be used to induce muscle-specific expression, thereby facilitating PTT-analysis performed on RNA isolated from the myo-differentiated cell cultures. An immunohistochemical analysis of myoD-converted these carrier-derived cells is difficult; although 50% of the cells can be expected to be dystrophin-negative (they carry the dystrophin mutation), the differentiating cells fuse with their neigbours and consequently all fibers become dystrophin-positive. Accordingly, the myoD-mediated induction of myogenesis enables prenatal diagnosis and point mutation detection for DMD within a time-period of three weeks (Roest et al. 1993 / 1996a /1996b / 1999 / PhD thesis).
The MyoD-gene is one of the muscle determination factors (MDFs) belonging to a group of muscle-specific basic helix-loop-helix (bHLH) transcription factors. In a complex regulatory network with myf-5, myogenin, and MRF4, MyoD plays a key role in the determination and differentiation of all skeletal muscle lineages (reviewed by Weintraub et al., 1991, Arnold & Braun, 1996 and Arnold & Winter, 1998). Following isolation of the MyoD gene by Davis et al. (1987), it was shown that the transfection and subsequent forced expression of the MyoD gene in a variety of differentiated non-muscle cell types (fibroblast, fat, melanoma, neuroblastoma, chondroblast, liver, and retinal pigmented epithelial cell lines) can initiate the process of myogenesis (Davis et al., 1987; Lin et al., 1989, Weintraub et al., 1989, Choi et al., 1990). This phenomenon facilitates the expression analysis of muscle proteins in non-muscle cells, which was first applied to prenatal and postnatal diagnosis of DMD by Sancho et al., 1993. In this study a retroviral vector was used to deliver the MyoD gene to both control and patient-derived fibroblasts, amniocytes and chorion villi cells. This retroviral vector (Weintraub et al., 1989) not only contained the murine MyoD cDNA under transcriptional control of the Moloney murine sarcoma virus long terminal repeat, but also a neomycin phosphotransferase gene driven by the Simian virus 40 early promoter enabling selection for transduced cells. Following myogenic differentiation, immunohistochemical analysis of the MyoD-converted cells from controls and patients revealed the presence or absence of dystrophin in all cases.
In our diagnostic protocol, we initially used the same retroviral vector to deliver the MyoD gene to patient-derived fibroblasts, amniocytes, and chorion villi cells. Notwithstanding several successful applications especially in fibroblasts (Roest et al. 1996a / 1996b), the retroviral delivery of the MyoD gene enclosed a few restrictions. First, despite the neomycin-selection for retroviral transduction, merely 25% of cells apparently expressed the MyoD gene and, hence, enrolled early myogenesis (as determined by desmin-expression). Furthermore, only a small percentage (2%) of these cells subsequently proceeded into late-myogenesis expressing myosin and dystrophin. These relatively low final efficiencies hindered straightforward application of this technique for diagnostic purposes. Another restriction of the retrovirus-based MyoD protocol included the neomycin-selection. Due to this step, the entire procedure was time-consuming (4-5 weeks) which may be inappropriate for prenatal diagnostic inquires. These restrictions motivated us to consider adenovirus as an alternative vehicle for the delivery of the myoD gene.
The adenoviral vector (kindly provided by C. Murry; Murry et al., 1996) that we now use for MyoD gene transfer was derived from an E1-deleted Ad5 strain and carries the 2.0 kb murine MyoD-cDNA under the control of the Rous sarcoma virus long terminal repeat. The protocol has been adjusted and optimized for the adenoviral vector and includes infection of the target cells, immediately followed by serum-deprivation. Myotube formation occurs within 5 days, and cell cultures are immunohistochemically analyzed at 8 to 10 days post-infection. The entire procedure currently takes about two to three weeks (Fig.2).
Current MyoD-research, directed at further improvement of the methodology.
We apply the MyoD-induced myogenesis as a supplementary tool for the diagnosis of DMD. In about 65% of patients (and families) affected with Duchenne/Becker muscular dystrophy (DMD/BMD), a mutation is found using conventional analytical methods such as multiplex PCR or Southern blot analysis. In the remaining 35% of the cases no mutation can be identified. For those cases, haplotype analysis is performed to enable risk estimation for potential carriers in the affected family or a point mutation screening is performed (PTT-analysis or DGGE-screen). When these analyses are inconclusive, or not feasible, we implement the MyoD-technique using fibroblast cell samples or, in case of a prenatal diagnosis, using amniocyte or chorion villi cell sample.
Provided that routine DNA analyses are inconclusive, we perform (in collaboration with our DNA-diagnostic department - headed by dr. Bert Bakker) the MyoD-technique trying to answer DMD diagnostic requests. We are also offering this analysis of foreign samples (preferably fibroblast samples) at a cost of EURO 550 per sample. However, due to the research setting, risk estimations should be considered cautiously (see current MyoD-research). For research purposes, we are currently particulary interested in cell lines from patients with Limb-Girdle muscular dystrophy (LGMD).
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