Leiden Muscular Dystrophy pages


(by Judith van Deutekom; last modified on March 27, 2000)

Recent results

Adenoviral infection requires the initial attachment of the virus to the cell surface through binding of the knob-like distal end of its fiber protein to the host cell attachment receptor CAR (Wickham et al., 1993, Tomko et al., 1997 and Roelvink et al., 1998). Following attachment, the viral internalization into the host cell is mediated by the interaction between the RGD (Arg-Gly-Asp) motifs, located in the viral pentamer coat protein, and the integrin-receptors alphaV3 and alphaV5, expressed on most cell types (Wickham et al., 1993).

To determine the expression levels of the adenoviral attachment and internalization receptors at the target cells, we performed FACS analysis on series of patient-derived amniocytes, chorion villi cells, and fibroblasts (van Deutekom et al., unpublished). Using antibodies against CAR, and the alphaV3 and alphaV5 integrins, we detected variable but substantial levels of the integrin receptors at all three cell types. The expression levels of CAR were very low at both chorion villi cells and fibroblasts, and only moderate at amniocytes. To bypass the limited availability of the CAR receptor at the target cells, cell cultures were infected with relatively high multiplicities of infection (MOI 50-200 viral particles per cell). Accordingly, we obtained transduction efficiencies (as determined by the percentage of cells expressing desmin) of 70%-90% in amniocytes, 10%-80% in chorion villi cells, and 2%-10% in fibroblasts. The efficiencies varied significantly between cell types as well as individual samples (Fig.3), which is likely caused by the differential expression of the adenoviral attachment (CAR) and internalization (integrin) receptor levels at the host cells. Nevertheless, in general the adenoviral transduction efficiencies were significantly higher then those obtained earlier with the retroviral MyoD vector.

The progress of myogenic differentiation in the infected cell cultures was immunohistochemically determined by the percentage of desmin/myosin double stained cells (Fig.4). On average, 30% of transduced amniocytes and chorion villi cells co-expressed desmin and embryonic myosin, whereas in fibroblasts this percentage was 15%. The cells that expressed myosin evidently advanced into later stages of myogenesis. At that period the absence or presence of dystrophin should become detectable. Therefore, we performed double-staining analysis using antibodies against myosin and dystrophin (Fig.5). In case of negative dystrophin staining in the majority of myosin-expressing cells, a high risk for DMD was diagnosed and PTT analysis was performed to reveal the actual mutation. A low risk percentage for DMD was assigned when most myosin-expressing cells were dystrophin-positive, using antibodies raised against different parts of the dystrophin protein.

Current Research

Although we are able to achieve adenoviral MyoD transduction efficiencies that are significantly higher than those obtained earlier with the retroviral MyoD vector, the requirement for high MOIs has a serious disadvantage. Due to the high concentration of adenoviral particles, a high percentage of cell death occurs within the infected cell cultures. This effect, probably due to the toxicity of viral gene expression, increases over time and hinders long-term differentiation periods. Consequently, only a small percentage of cells enrolls late myogenesis and expresses myosin and dystrophin. Since this factor renders the diagnosis (i.e. dystrophin detection) in some cases inconclusive, we are currently studying different modified tropism adenoviral vectors to obtain higher transduction efficiencies at lower MOIs in amniocyte, chorion villi, or fibroblast cell cultures. This would reduce the percentage of cell death within the infected cell cultures and increase the allowable differentiation periods, both of which facilitates a more confident and reproducible diagnosis.


For information or requests please contact:

Dr. Judith C.T. van Deutekom
        Department of Human and Clinical Genetics, Leiden University Medical Center
        E-mail: deutekom@lumc.nl / Tel: +31 - 71 - 527 6080

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