1. ¹Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark
2. ²The Heart Hospital, University College London, London, UK

Correspondence: Dr PS Andersen, Department of Clinical Biochemistry, Statens Serum Institut, 5 Artillerivej, Copenhagen S DK-2300, Denmark. Tel: +45 3268 8190; Fax: +45 3268 3878; E-mail: psa@ssi.dk

³Present address: Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark

Received 17 October 2007; Revised 22 January 2008; Accepted 7 February 2008; Published online 12 March 2008.

Abstract

Hypertrophic cardiomyopathy is primarily caused by mutations in genes encoding cardiac sarcomere proteins. Large screening studies identify mutations in 35–65% of the diagnosed patients and 15–30% of these are discovered within the MYBPC3 gene encoding the cardiac myosin binding protein C. The aim of this study is to determine whether intronic variation flanking the three micro-exons in MYBPC3 is disease-causing. Two hundred and fifty unrelated patients with hypertrophic cardiomyopathy were genotyped in MYBPC3, using automated single-strand conformation polymorphism, and sequenced for confirmation. Mutations located in the flanking introns of the MYBPC3 micro-exons were examined using in silico methods. Ectopic expression of mRNA in blood leukocytes in the respective patients was examined using reverse transcription-PCR. A total of seven mutations were discovered in the introns flanking the two micro-exons 10 and 14, but none were found in introns flanking exon 11. Functional studies together with co-segregation analysis indicate that four mutations are associated with HCM, in the respective patients. All four mutations result in premature termination codons, which suggests that haploinsufficiency is a pathogenic mechanism of this type of mutation. It is demonstrated that the use of in silico methods together with RNA studies on peripheral blood leukocytes is a useful tool to evaluate the potential effects of mutations on pre-mRNA splicing.