title: Large-scale mutation screening in patients with dilated or hypertrophic cardiomyopathy: a pilot study using DGGE.
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.
authors.affiliation: Innere Medizin III, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.
authors.forename: Raphael
authors.initials: R
authors.lastname: Zeller
authors.forename: Boris T
authors.initials: BT
authors.lastname: Ivandic
authors.forename: Philipp
authors.initials: P
authors.lastname: Ehlermann
authors.forename: Oliver
authors.initials: O
authors.lastname: Mücke
authors.forename: Christian
authors.initials: C
authors.lastname: Zugck
authors.forename: Andrew
authors.initials: A
authors.lastname: Remppis
authors.forename: Evangelos
authors.initials: E
authors.lastname: Giannitsis
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Katus
authors.forename: Dieter
authors.initials: D
authors.lastname: Weichenhan
mesh_terms: D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002311:Cardiomyopathy, Dilated / Q000235:genetics*; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D010865:Pilot Projects