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Large-scale mutation screening in patients with dilated or hypertrophic cardiomyopathy: a pilot study using DGGE

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Abstract

Cardiomyopathies are complex myocardial diseases characterized by inappropriate ventricular hypertrophy (HCM) or dilation (DCM). Both disorders may lead to sudden death or progressive heart failure and exhibit familial aggregation with marked genetic heterogeneity. Many candidate genes were identified by linkage analysis, experimental animal studies, and expression analysis. A systematic assessment of the prevalence of different mutations in these genes requires high-throughput analyses. In this paper, we present a simple and reliable protocol for mutation screening by heteroduplex analysis which reduced costs and workload of sequencing. Employing denaturing gradient gel electrophoresis (DGGE), 11 known and 14 potential candidate genes for HCM and DCM were analyzed. DGGE assays allowed analysis of 286 of the 312 protein coding exons, performing only four alternative polymerase chain reaction protocols and only two different DGGE analysis conditions. Sensitivity for the detection of heteroduplexes proved excellent, even for GC-rich DNA fragments, which were analyzed by a combination of DGGE and constant denaturant gel electrophoresis. To confirm DGGE sensitivity in cases where no variants in our human DNA samples could be observed, we generated heteroduplexes from homologous human and chimpanzee DNA. The platform proved a valuable contribution to elucidating the genetic causes of DCM and HCM as demonstrated by the identification of 17 different known and novel mutations and 98 different polymorphisms in our setting.

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Acknowledgements

Blood from chimpanzee was kindly donated by Dr. Wolfram Ritschel and Dr. Christian Roos from the Wilhelma Zoo in Stuttgart and the Primatenzentrum Göttingen, respectively. We are thankful to Dr. Arne Pfeufer (GSF Forschungszentrum Neuherberg), Dr. Gregor Schlüter (Universität Göttingen) and Dr. Johannes Zschocke (Universität Heidelberg) for helpful discussions. The authors R. Zeller and B.T. Ivandic contributed equally. This work was supported by grant NGFN 01GS0420 (project no. NHK-S02T09) from the Bundesministerium für Bildung und Forschung.

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Authors and Affiliations

  1. Innere Medizin III, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

    Raphael Zeller, Boris T. Ivandic, Philipp Ehlermann, Oliver Mücke, Christian Zugck, Andrew Remppis, Evangelos Giannitsis, Hugo A. Katus & Dieter Weichenhan

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  1. Raphael Zeller
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  2. Boris T. Ivandic
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  3. Philipp Ehlermann
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Correspondence to Dieter Weichenhan.

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Zeller, R., Ivandic, B.T., Ehlermann, P. et al. Large-scale mutation screening in patients with dilated or hypertrophic cardiomyopathy: a pilot study using DGGE. J Mol Med 84, 682–691 (2006). https://doi.org/10.1007/s00109-006-0056-2

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  • DOI: https://doi.org/10.1007/s00109-006-0056-2

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