The relative electrophoretic mobility of apo(a) isoforms depends on the gel system: proposal of a nomenclature for apo(a) phenotypes

doi:10.1016/0021-9150(96)05844-3

Atherosclerosis

Volume 125, Issue 1, 23 August 1996, Pages 53-61

https://doi.org/10.1016/0021-9150(96)05844-3 Get rights and content

Abstract

Genetic apo(a) isoforms were originally defined according to their relative mobility in SDS-PAGE compared to apoB-100 and were designated as F, B or S1–S4 isotypes. This widely accepted nomenclature does not accommodate the broad spectrum of apo(a) isoforms (>30) detected by high resolution SDS-agarose gel electrophoresis. Moreover we here show that the relative mobilities of apo(a) isoforms depend on the SDS-gel system used. Comparison of the SDS-PAGE system originally used for phenotyping with SDS-agarose gel electrophoresis and two commercial SDS-PAGE systems (PhastGel, Pharmacia, Sweden and NOVEX, USA) demonstrated marked differences in resolving power and resulted in very different R_f values for identical isoforms. Hence phenotyping results from laboratories using different systems are not comparable. We therefore propose a nomenclature of apo(a) isoforms which reports the number of kringle IV repeats in the apo(a) allele (e.g. apo(a) K-IV²⁰ would designate an isoform with 20 K-IV repeats). This is achieved by using standards in which the number of kringle IV repeats has been determined by pulsed field gel electrophoresis of genomic DNA. The proposed nomenclature (i) accounts for the increased resolution of apo(a) phenotyping methods; (ii) is flexible to the introduction of smaller or larger isoforms; (iii) allows to report data from systems with lower resolution as ‘binned’ isoform categories; (iv) allows the comparison of phenotyping results between different investigators; and (v) can be applied on DNA as well as on protein based apo(a) phenotyping.

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Cited by (46)

Lipoprotein(a) beyond the kringle IV repeat polymorphism: The complexity of genetic variation in the LPA gene
2022, Atherosclerosis
Citation Excerpt :
Genetic variants associated with certain Lp(a) concentration ranges were already in the past very helpful to support causality between Lp(a) and outcomes. Lp(a) was the first use case for Mendelian randomization studies in the 1990s [33,34], long before this term was coined (see review [35] in this series). Nevertheless, the causality of Lp(a) has been debated for a long time [36] until numerous genetic studies underscored the causality of Lp(a) concentrations using genetic variants strongly associated with high Lp(a) concentrations and subsequent cardiovascular disease [37–44].
High lipoprotein(a) [Lp(a)] concentrations are one of the most important genetically determined risk factors for cardiovascular disease. Lp(a) concentrations are an enigmatic trait largely controlled by one single gene (LPA) that contains a complex interplay of several genetic elements with many surprising effects discussed in this review. A hypervariable coding copy number variation (the kringle IV type-2 repeat, KIV-2) generates >40 apolipoprotein(a) protein isoforms and determines the median Lp(a) concentrations. Carriers of small isoforms with up to 22 kringle IV domains have median Lp(a) concentrations up to 5 times higher than those with large isoforms (>22 kringle IV domains). The effect of the apo(a) isoforms are, however, modified by many functional single nucleotide polymorphisms (SNPs) distributed over the complete range of allele frequencies (<0.1% to >20%) with very pronounced effects on Lp(a) concentrations. A complex interaction is present between the apo(a) isoforms and LPA SNPs, with isoforms partially masking the effect of functional SNPs and, vice versa, SNPs lowering the Lp(a) concentrations of affected isoforms. This picture is further complicated by SNP-SNP interactions, a poorly understood role of other polymorphisms such as short tandem repeats and linkage structures that are poorly captured by common R² values. A further layer of complexity derives from recent findings that several functional SNPs are located in the KIV-2 repeat and are thus not accessible to conventional sequencing and genotyping technologies. A critical impact of the ancestry on correlation structures and baseline Lp(a) values becomes increasingly evident.
This review provides a comprehensive overview on the complex genetic architecture of the Lp(a) concentrations in plasma, a field that has made tremendous progress with the introduction of new technologies. Understanding the genetics of Lp(a) might be a key to many mysteries of Lp(a) and booster new ideas on the metabolism of Lp(a) and possible interventional targets.
Lack of association of rs3798220 with small apolipoprotein(a) isoforms and high lipoprotein(a) levels in East and Southeast Asians
2015, Atherosclerosis
Citation Excerpt :
However, minor allele frequencies (MAFs) of this SNP range from only approximately 1%–2% in the general European population. Therefore, genotyping for the variant allele as a surrogate for the laborious laboratory assessment of apo(a) isoform size by pulsed-field gel [4,5] or SDS gel electrophoresis [25] captures only a small proportion of the individuals with small apo(a) isoforms and increased risk [26]. Nevertheless, rs3798220 has found commercial application in genetic risk evaluation for CAD [27].
The variant allele of rs3798220 in the apolipoprotein(a) gene (LPA) is used to assess the risk for coronary artery disease (CAD) in Europeans, where it is associated with short alleles of the Kringle IV-2 (KIV-2) copy number variation (CNV) and high lipoprotein(a) (Lp(a)) concentrations. No association of rs3798220 with CAD was detected in a GWAS of East Asians. Our study investigated the association of rs3798220 with Lp(a) concentrations and KIV-2 CNV size in non-European populations to explain the missing association of the variant with CAD in Asians.
We screened three populations from Africa and seven from Asia by TaqMan Assay for rs3798220 and determined KIV-2 CNV sizes of LPA alleles by pulsed-field gel electrophoresis (PFGE). Additionally, CAD cases from India were analysed. To investigate the phylogenetic origin of rs3798220, 40 LPA alleles from Chinese individuals were separated by PFGE and haplotyped for further SNPs.
The variant was not found in Africans. Allele frequencies in East and Southeast Asians ranged from 2.9% to 11.6%, and were very low (0.15%) in CAD cases and controls from India. The variant was neither associated with short KIV-2 CNV alleles nor elevated Lp(a) concentrations in Asians.
Our study shows that rs3798220 is no marker for short KIV-2 CNV alleles and high Lp(a) in East and Southeast Asians, although the haplotype background is shared with Europeans. It appears unlikely that this SNP confers atherogenic potential on its own. Furthermore, this SNP does not explain Lp(a) attributed risk for CAD in Asian Indians.
Familial lecithin-cholesterol acyltransferase deficiency: Biochemical characteristics and molecular analysis of a new LCAT mutation in a Polish family
2006, Atherosclerosis
Familial LCAT deficiency (FLD) is a rare genetic disorder associated with corneal opacities, anaemia and proteinuria with renal failure. Here we report detailed analyses on plasma lipids, lipoproteins, and the molecular defect in two siblings from a Polish family presenting classical symptoms of FLD and their family members with newly discovered Val309Met mutation in exon 6 of LCAT gene. Both patients displayed low total (2.19 and 2.94 mmol/l) and HDL-cholesterol concentrations (0.52 and 0.48 mmol/l), low percentage of cholesteryl esters (CE) (11.1 and 12%), and decreased apo AI and apo AII serum levels. Low LDL-cholesterol, apo B and Lp(a) levels, and increased oleate/linoleate ratios in CE could be of importance in the development of atherosclerosis in these patients with low HDL-cholesterol.
LCAT activity was 10% of normal, α-LCAT activity was 0, and LCAT concentration was undetectable by immunoassay. Plasma CETP activity was at lower limits of normal.
PCR and sequence analysis of DNA from the proband and affected brother revealed a novel G → A mutation in exon 6 of LCAT gene, which resulted in an amino acid substitution of valine for methionine (Val309Met). The proband and affected brother were both homozygous carriers, while the mother, siblings and children of patients were heterozygous carriers of a newly discovered mutation. This is the first LCAT mutation described in the Slavic population.
Stimulation of vascular smooth muscle cell proliferation and migration by apolipoprotein(a) is dependent on inhibition of transforming growth factor-β activation and on the presence of kringle IV type 9
2004, Journal of Biological Chemistry
Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor β (TGF-β) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-β; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.
Electrophoretic measurement of lipoprotein(a) cholesterol in plasma with and without ultracentrifugation: Comparison with an immunoturbidimetric lipoprotein(a) method
2004, Clinical Biochemistry
Objectives: Elevated plasma lipoprotein(a) [Lp(a)] is a significant risk factor for vascular disease. Standardization of Lp(a) mass measurement is complicated by the heterogeneity of apolipoprotein(a) [apo(a)]. We investigated whether Lp(a) cholesterol measurement, which is not influenced by apo(a) size, is a viable alternative to measuring Lp(a) mass.
Design and methods: Plasma Lp(a) cholesterol was measured electrophoretically, with and without ultracentrifugation, and results were compared to each other and to immunoturbidimetrically measured Lp(a) mass in 470 subjects.
Results: Ultracentrifuged and whole plasma Lp(a) cholesterol levels demonstrated high correlation (R = 0.964). All samples with detectable (≥2.0 mg/dl) Lp(a) cholesterol had Lp(a) mass >30 mg/dl (the clinically relevant cutpoint), while 59 samples with Lp(a) mass >30 mg/dl did not have detectable Lp(a) cholesterol.
Conclusions: Lp(a) cholesterol can be measured in whole plasma without interference from VLDL lipoproteins. The relative clinical merits of measuring Lp(a) cholesterol vs. Lp(a) mass or both in combination deserves investigation.
Analysis of the apo(a) size polymorphism in Asian Indian populations: Association with Lp(a) concentration and coronary heart disease
2003, Atherosclerosis
Citation Excerpt :
The size of the genomic fragments from which the number of K-IV repeats was estimated was determined by comparison with a commercial DNA size marker (Midrange II PFG marker, New England Biolabs, Beverly, MA). Apo(a) phenotyping was performed by SDS-agarose gel electrophoresis (SDS-AGE) and immunoblotting with an apo(a) specific antibody as described [20]. As standard a combination of five isoforms containing 15, 19, 22, 27, and 34 K-IV repeats, respectively, was used and apo(a) isoforms were designated according to their number of K-IV repeats [20].
Most studies aiming to detect associations of genetic variation with common complex diseases, e.g. coronary heart disease (CHD) have been performed in populations with a western lifestyle but it is unclear whether associations detected in one geographic group exist also in others. We here have determined lipoprotein(a) levels and apo(a) K-IV-2 repeat genotypes in CHD patients (N=254) and controls (N=480) from two Asian Indian populations (Tamil Nadu and New Delhi). In both populations and also in the pooled dataset median Lp(a) levels were significantly elevated in the patients (27.4 mg/dl) compared with the controls (17.6 mg/dl). Apo(a) K-IV-2 allele frequencies were not different between the CHD patients and controls and thus did not explain the increased Lp(a) levels in CHD patients. Contrary to what has recently been observed in Black and White men short (K-IV≤22) alleles associated with high Lp(a) concentration were not overrepresented in the patients. Rather, short (K-IV≤22), intermediate (K-IV 23–29) and long (K-IV≥30) apo(a) alleles were all associated with higher Lp(a) levels in the patients. Accordingly relative risk (estimated as odds ratio) for CHD rose continuously with increasing Lp(a) but was independent of apo(a) allele length. Together with previous studies our results indicate that the relation between apo(a) genotypes, Lp(a) levels, and CHD may be heterogeneous across ethnic groups and that it depends on the genetic architecture of the Lp(a) trait in a given population whether an association of K-IV-2 repeat length with CHD exists or not.

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Research paperThe relative electrophoretic mobility of apo(a) isoforms depends on the gel system: proposal of a nomenclature for apo(a) phenotypes

Abstract

Atherosclerosis

J Lipid Res

J Lipid Res

Biochem Biophys Res Commun

J Biol Chem

J Lipid Res

Lp(a) glycoprotein phenotype. Inheritance and relation to Lp(a)-lipoprotein concentration in plasma

J Clin Invest

Genetics of the quantitative Lp(a) lipoprotein trait. III. Contribution of Lp(a) glycoprotein phenotypes to normal lipid values

Hum Genet

Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations

J Clin Invest

The apolipoprotein(a) gene — a transcribed hypervariable locus controlling plasma lipoprotein(a) concentration

Hum Genet

Lipoprotein(a) concentrations and apolipoprotein(a) phenotypes in Caucasians and African Americans — The CARDIA Study

Arterioscler Thromb

Research paper
The relative electrophoretic mobility of apo(a) isoforms depends on the gel system: proposal of a nomenclature for apo(a) phenotypes