Genetics of the quantitative Lp(a) lipoprotein trait

Summary Lp(a) glycoprotein exhibits an apparent size polymorphism that is associated with genetically controlled Lp(a) lipoprotein concentrations in plasma (Utermann et al. 1988). We have tested the hypothesis that this polymorphism is genetically controlled by studying 15 matings with a total of 44 offspring. This confirmed our conclusion that Lp(a) types are controlled by a series of codominant alleles Lp^F, Lp^B, Lp^S1, Lp^S2, Lp^S3 and Lp^S4 and by a null allele Lp^o. Together with the data from the accompanying paper this indicates that the structural gene for the Lp(a) protein is the major gene locus determining Lp(a) lipoprotein concentrations in plasma.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Apolipoprotein(a) [apo(a)] is a large serum glycoprotein with several genetically determined isoforms differing in their apparent molecular weight. We determined the effects of the apo(a) isoforms on total cholesterol, high-density lipo-protein (HDL)-cholesterol, lipoprotein(a), and triglyceride levels in a sample of 473 unrelated Tyrolean adults. Average lipoprotein(a) and total cholesterol levels were significantly different among apo(a) types. These significant differences were found among the 13 apo(a) isoform patterns observed in this sample and among several logical subsets of the isoform patterns (e.g. considering only the single band types). The data suggest that the effects of apo(a) alleles on Lp(a) levels are additive. The effects of apo(a) on total cholesterol levels cannot be entirely explained by the cholesterol fraction estimated to be contained in the lipoprotein(a) particle. We estimate that the apo(a) glycoprotein polymorphism accounts for 41.9% and 9.6% of the variability in lipoprotein(a) and total cholesterol levels, respectively. This is the strongest effect of a single polymorphic gene on plasma lipid and lipoprotein levels reported so far.     

Lp(a) lipoprotein level and longevity.

Lp(a) lipoprotein forms a distinct class of serum lipoproteins. Its unique immunochemical properties are caused by the Lp(a) polypeptide chain which is attached to apolipoprotein B (apoB) by a disulfide bridge. The level of Lp(a) lipoprotein is under strict genetic control. It is well established that a high level of Lp(a) lipoprotein is a genetic risk factor for atherosclerotic disease, particularly coronary heart disease (CHD). Since cardiovascular disease is one of the major causes of death there should be a shortage of people with genetic determinants of cardiovascular disease in people who are very old and still have adequate physical and mental capacities. The authors have studied Lp(a) lipoprotein levels in 102 persons who were 83 years or older when blood samples were drawn. This study group was a subpopulation of a series comprising 456 persons who had been 80 years or older at intake in an intervention study of old people living at home. Only those without physical or mental incapacities were included in the present study. There was a striking shortage of persons with an Lp(a) lipoprotein level higher than the 75th percentile of the general population in this series of people who had achieved successful ageing. The highest value observed among the old people corresponds to the 88th percentile of the general population. It is highly unlikely that the present observations reflect chance events or fall in Lp(a) lipoprotein levels in people who had higher levels at a younger age. The most likely explanation of our finding is that a sizeable fraction of people with high Lp(a) lipoprotein levels die before reaching a very high age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay of human plasma Lp(a) lipoprotein.

A quantitative immunodiffusion assay demonstrated Lp(a) lipoprotein in 91% (911 of 1000) of subjects. In order to quantitate Lp(a) in all plasma, a sensitive and specific double antibody radioimmunoassay was developed. The between-assay coefficient of variation was 8%. Lp(a) levels by radioimmunoassay were highly correlated with those obtained by the less sensitive radial immunodiffusion method (r = 0.98, n = 51). All but one of the 89 Lp(a) "negative" subjects by immunodiffusion had detectable levels of Lp(a) by radioimmunoassay. The one subject without detectable Lp(a) had abetalipoproteinemia (without detectable apolipoprotein B by radioimmunoassay). Furthermore, Lp(a) was detected in all three non-human primates examined: patas monkey, baboon, and pig-tail monkey. Quantitation of Lp(a) levels in 90 male myocardial infarction (MI) survivors and their spouses showed that the distribution of Lp(a) levels of MI survivors was significantly higher above the 50th percentile cut-point (P < 0.02) and exceeded that of the spouses. Furthermore, the Lp(a) distribution at and above the 50th percentile for the MI survivors who had an MI at age <50 (n = 36) was shifted to values higher than those having an MI at age >50. Thus, high levels of Lp(a) may be associated with premature coronary disease. We conclude that Lp(a) is present in all individuals with apolipoprotein B and that apolipoprotein B appears necessary for the plasma transport of the Lp(a) lipoprotein. Consistent with this hypothesis, quantitative immunochemical precipitation of (125)I-Lp(a) indicated that essentially all individual molecules of six purified Lp(a) preparations contain both the Lp(a) antigen and apolipoprotein B.     

Quantitative genetic studies of the human plasma Lp(a) lipoprotein

Lp(a) lipoprotein [Lp(a)] was quantified in 1251 adults, including 300 mother-father-offspring triplets, by a sensitive radial immunodiffusion assay. Lp(a) was not correlated with age, sex, or cholesterol or glyceride concentrations. Significant correlations were found between mother-offspring (r=0.34), father-offspring (r=0.40), and midparent-offspring (r=0.52), whereas no correlation was found between husband-wife pairs (r=0.02). Analysis of triplets separated on the basis of midparent Lp(a) concentrations indicated a resemblance of midparent to offspring regardless of midparent concentration. Bimodality was not detected in any of the offspring distributions. These data are compatible with the hypothesis that the observed quantitative Lp(a) variation is determined by a polygenic model of inheritance. The possibility of major gene effects is not ruled out.     

Studies on the structure of the Lp(a) lipoprotein: Isolation and partial characterization of the Lp(a)-specific antigen

Lp(a), a polymorphic lipoprotein of human serum, has been isolated from strongly positive normal sera. The described procedure utilizes a serum pool of several liters. The purified lipoprotein has been investigated in its intact and totally delipidized form. Protein constituents could be characterized as apolipoprotein-B, apolipoprotein-AIII, and the Lp(a)-specific, dissociable polypeptide. Albumin could only be found in some of the preparations. The Lp(a)-specific polypeptide could be separated and characterized by its amino acid composition.     

Protein composition of Lp(a) lipoprotein from human plasma

The apolipoprotein composition of purified human Lp(a) lipoprotein was investigated by SDS--polyacrylamide gel electrophoresis and immunochemically. The lipoprotein contains two different polypeptides. One is identical by its app. Mr of approximately 250 000 and immunologically with apolipoprotein B of LDL (B-100). The other polypeptide has a higher app. Mr (approximately 350 000) and stains strongly with the periodate-Schiff's reagent. This high-Mr glycoprotein contains the specific Lp(a) immunoreactivity but does not react with antibodies against apo B. Apo B and Lp(a)-protein seem to be linked by disulfide bonds in the native lipoprotein. The unreduced detergent delipidized protein moiety from Lp(a) lipoprotein shows a single band of Mr approximately 700 000 in SDS--polyacrylamide gel electrophoresis and the immunoprecipitates formed against anti-Lp(a) and anti-apo B by the unreduced protein show a reaction of immunological identity.     

Linkage between the loci for the Lp(a) lipoprotein (LP) and plasminogen (PLG)

Summary A locus, LP, that determines quantitative variation of Lp(a) lipoprotein phenotypes is linked to the plasminogen (PLG) locus (peak lod score =12.73). This linkage relationship assigns a locus with alleles that have an affect on risk for coronary artery disease to the long arm of chromosome 6.     

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a)

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.     

Latex immunoassay of human serum Lp(a+) lipoprotein

A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108).     

Purification of a beta lipoprotein fraction rich in Lp(a)]

A Lp (a) rich fraction was prepared from heparin/manganese chloride precipitated beta-lipoproteins, by using DEAE cellulose chromatography and gel filtration on Sepharose 4 B.     

Estimation of the contribution of quantitative trait loci (QTL) to the variance of a quantitative trait by means of genetic markers

The estimation of the contribution of an individual quantitative trait locus (QTL) to the variance of a quantitative trait is considered in the framework of an analysis of variance (ANOVA). ANOVA mean squares expectations which are appropriate to the specific case of QTL mapping experiments are derived. These expectations allow the specificities associated with the limited number of genotypes at a given locus to be taken into account. Discrepancies with classical expectations are particularly important for two-class experiments (backcross, recombinant inbred lines, doubled haploid populations) and F₂ populations. The result allows us firstly to reconsider the power of experiments (i.e. the probability of detecting a QTL with a given contribution to the variance of the trait). It illustrates that the use of classical formulae for mean squares expectations leads to a strong underestimation of the power of the experiments. Secondly, from the observed mean squares it is possible to estimate directly the variance associated with a locus and the fraction of the total variance associated to this locus (r _l ² ). When compared to other methods, the values estimated using this method are unbiased. Considering unbiased estimators increases in importance when (1) the experimental size is limited; (2) the number of genotypes at the locus of interest is large; and (3) the fraction of the variation associated with this locus is small. Finally, specific mean squares expectations allows us to propose a simple analytical method by which to estimate the confidence interval of r _l ² . This point is particularly important since results indicate that 95% confidence intervals for r _l ² can be rather wide:2–23% for a 10% estimate and 8–34% for a 20% estimate if 100 individuals are considered.     

Serum Lp(a) lipoprotein concentrations in insulin dependent diabetic patients with microalbuminuria.

OBJECTIVE--To compare the serum concentrations of lipoproteins and apolipoproteins in insulin dependent diabetic patients with and without microalbuminuria. DESIGN--Cross sectional study. SETTING--Paediatric and medical outpatient clinic at a university hospital. PATIENTS--76 insulin dependent diabetic patients: 41 with microalbuminuria (20 males, 21 females) and 35 controls without microalbuminuria (18 males, 17 females). The two groups were similar with respect to age, duration of disease, and haemoglobin A1c concentrations before the study. MAIN OUTCOME MEASURES--Serum concentrations of Lp(a) lipoprotein, total cholesterol, high density lipoprotein cholesterol, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, and apolipoproteins A-I, A-II, and B. RESULTS--Median serum Lp(a) lipoprotein concentration was 10.0 mg/100 ml in the microalbuminuric group and 4.9 mg/100 ml in the control group (p = 0.007). 17 (41%) of the microalbuminuric patients and five (14%) of the control patients had Lp(a) lipoprotein values above the upper quartile of a normal population. Median serum triglycerides concentrations in the microalbuminuric and control groups were 1.15 mmol/l and 0.88 mmol/l respectively (p = 0.03). Median very low density lipoprotein cholesterol concentration was 0.52 mmol/l in the microalbuminuric group and 0.40 mmol/l in the control group (p = 0.03). No significant differences in serum concentrations of total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, or apolipoproteins A-I, A-II, and B were found between the groups. CONCLUSIONS--Serum concentrations of Lp(a) lipoprotein are twice as high in insulin dependent diabetic patients with microalbuminuria as in those without microalbuminuria. Increased concentrations of Lp(a) lipoprotein might partly explain the increased morbidity and mortality from cardiovascular disease observed among patients with diabetic nephropathy.