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

https://doi.org/10.1194/jlr.D800050-JLR200Get rights and content
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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 (rs = 0.50, P < 1 × 10−6) and with plasma Lp(a) concentration (rs = 0.30, P < 1 × 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.

cardiovascular disease
apolipoprotein(a)
quantitative real-time PCR
risk factors
genomics

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

*

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.