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Journal of Lipid Research, Vol. 50, 768-772, April 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

Determination of lipoprotein(a) kringle repeat number from genomic DNA: copy number variation genotyping using qPCR^*

Matthew B. Lanktree^*, Chandheeb Rajakumar^*, J. Howard Brunt, Marlys L. Koschinsky, Philip W. Connelly^** and Robert A. Hegele^1,*

^* Robarts Research Institute, Departments of Medicine and Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada
School of Nursing, University of Victoria, Victoria, British Columbia, Canada
Faculty of Science, Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario, Canada
^** St Michael's Hospital, University of Toronto, Toronto, Ontario, Canada

^* This work was supported by a team grant from the CIHR (CTP-79853, MOP-37854), the Heart and Stroke Foundation of Ontario, and Genome Canada through the Ontario Genomics Institute. Mr. Lanktree is supported by the Canadian Institutes of Health Research MD/PhD Studentship Award.

Published, JLR Papers in Press, December 5, 2008.

¹ To whom correspondence should be addressed. e-mail: hegele{at}robarts.ca

Plasma lipoprotein(a) [Lp(a)] concentration is related to risk of cardiovascular disease. The defining protein component of Lp(a) particles, apolipoprotein(a) [apo(a)], is encoded by the LPA gene. Apo(a) is extremely heterogeneous in size due to a common copy number variation, leading to a variable number of kringle-IV type 2 (KIV2)-like domains. Alleles with fewer KIV2 repeats, encoding smaller apo(a) isoforms, are associated with higher plasma Lp(a) concentrations. Two principal methods to detect variation in KIV2 repeat number are electrophoresis with immunoblotting to detect apo(a) protein isoforms or pulse-field electrophoresis of unamplified genomic DNA to detect the variation of the LPA gene. Both methods are technically challenging, laborious, and time consuming. Here, we report a rapid method to determine the number of KIV2 repeats in LPA from genomic DNA using quantitative real-time polymerase chain reaction (qPCR). With qPCR, we found KIV2 repeat number was correlated with both apo(a) isoform size as determined by immunoblotting (r_s = 0.50, P < 1 x 10^–6) and with plasma Lp(a) concentration (r_s = 0.30, P < 1 x 10^–6). The qPCR technique permits rapid evaluation of apo(a) size from genomic DNA, and thus would provide an adjunctive genomic variable, in addition to LPA single nucleotide polymorphisms, for evaluating the genetic determinants of plasma Lp(a) concentration in genetic epidemiology studies of cardiovascular disease outcomes.

Supplementary key words cardiovascular disease • apolipoprotein(a) • quantitative real-time PCR • risk factors • genomics

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