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

Abstract

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.

Fig. 1.

A: Gene structure and linkage disequilibrium (LD) plot of LPA locus generated using HapMap CEPH (Centre d'Etude du Poymorphisme Humain) data. Red boxes indicate areas of complete LD, white boxes indicate areas of recombination, and blue boxes are uninformative areas. Exons are indicated by black boxes with every fifth exon labeled in top track. Physical position and exon numbers are taken from National Centre for Biotechnology Information reference sequence build 36.3. Kringles with sequence homology with the fourth plasminogen kringle (KIV) and fifth plasminogen kringle (KV) are indicated. Each kringle is encoded by two exons. B: Amino acid alignment showing sequence homology between kringle repeats.

Fig. 2.

Correlation observed between the two probes found within the kringle-IV type 2 (KIV2) repeat: Δcycle thresholds (C_T) C_T4, the probe found in exon 4, and ΔC_T5, the probe found in exon 5.

Fig. 3.

The relationship between apolipoprotein(a) [apo(a)] isoform as identified by PAGE and immunoblotting and from quantitative real-time polymerase chain reaction (qPCR).

Fig. 4.

The relationship between plasma of lipoprotein(a) [Lp(a)] concentration and from qPCR.