Haplotypes of the human apoprotein AI-CIII-AIV gene cluster in coronary atherosclerosis

Summary Restriction fragment length polymorphisms of the apoprotein AI-CIII-AIV gene cluster (on the long arm of chromosome 11) were investigated in a group of Caucasian survivors of myocardial infarction, using genomic hybridisation analysis. Four common haplotypes were identified at this locus, M1P1S1 (a), M2P1S1 (b), M1P2S1 (c), and M2P1S2 (d); where M1 and M2 are the common and uncommon alleles defined using the restriction enzyme MspI, P1 and P2 are the common and uncommon alleles defined by the enzyme PstI, and S1 and S2 are the common and uncommon alleles defined by the enzyme SstI. Seven genotype combinations were observed of approximate frequencies; a/a 0.70 (33/47), a/d 0.15 (7/47), a/b 0.04 (2/47), d/d 0.04 (2/47), a/c 0.02 (1/47), b/c 0.02 (1/47), and c/d 0.02 (1/47). In contrast the corresponding values for normotriglyceridaemic Caucasian controls, without a personal or family history of atherosclerotic heart disease were; 0.83 (40/48), 0.02 (1/48), 0.06 (3/48), 0, 0.04 (2/48), 0.04 (2/48), and 0. The relative incidence of the d haplotype, characterised by the presence of a cleavage site for the enzyme Sstl in the fourth exon of the ApoCIII gene was significantly higher in the patient group (P<0.01). However, because of the tight linkage between the polymorphic loci studied, it was not possible to identify haplotypes associated with any greater risk of premature atherosclerosis than when the SstI polymorphism was considered in isolation.     

DNA polymorphisms and haplotypes in the human transferrin gene

Although a large number of human serum transferrin (TF) variants have been described, only one RFLP (AvaI) has so far been found. Here we report three new RFLPs (MvaI in intron 5 and exon 7, BbvI in exon 7) and correlations between RFLPs and between RFLPs and serum TF types. There were strong, but not always complete, disequilibria between RFLP and serum protein alleles. Thus, the most common serum TF variant, C1, was heterogeneous and could be subdivided into two common haplotypes, whereas the C2, C3, and DCHI variants were completely or almost completely (C2) homogeneous. There was a total genotypic agreement between the BbvI polymorphism and the presence/absence of the TF C3 variant, and the mutation that creates the BbvI site was found to lead to a G258S amino acid substitution. Received: 15 June 1997 / Accepted: 15 November 1997     

Chromosomal localization of the human apoprotein CI gene and of a polymorphic apoprotein AII gene

Human apoprotein(apo) CI and apo AII cDNA probes have been used to analyze the segregation of the human genes in panels of human-mouse hybrids. The apo CI (APOCI) gene segregates with chromosome 19 and the apo AII (APOA2) gene with chromosome 1. Somatic cell hybrids containing chromosome translocations were used to map the apo AII gene to the 1p21-1qter region. Human APOA2 is polymorphic for the restriction endonuclease Msp I. Comparison of human and mouse chromosome 1 reveals a conserved group including apo AII, renin and peptidase genes and suggests that APOA2 will be found distal to this group on human chromosome 1. The mouse apo AII gene is closely linked with genes that regulate HDL structure. Similar HDL regulatory genes will probably be found near human APOA2.     

A DNA polymorphism of the apoprotein AII gene in hypertriglyceridaemia

Summary A polymorphism of the apolipoprotein AII gene (on chromosome 1) was investigated using genomic hybridisation analysis. The two common alleles at this locus were defined by MspI restriction fragments of 3.0 kilobase pairs (M3.0) and 3.7 kilobase pairs (M3.7) respectively. The M3.7 allele was significantly more common (P<0.02) in Caucasian subjects who were normo-lipaemic (34%, 20/59) than in those who were hypertriglyceridaemic (16%, 16/98). Serum triglyceride levels were measured in 126 Caucasian subjects with different combinations of disease-associated alleles at the Apo AII and Apo CIII gene loci. Mean serum triglyceride levels were found to be significantly higher (P<0.05) in subjects with disease-associated alleles of both the Apo CIII and Apo AII genes, compared with subjects with a disease-associated allele of one or neither locus.     

Polymorphic DNA haplotypes at the human low-density lipoprotein receptor gene locus in Koreans

Many low-density lipoprotein (LDL) receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL-receptor genes for prenatal diagnosis of familial hypercholesterolemia (FH) or to study the role of the LDL-receptor gene in polygenic hypercholesterolemia requires the use of closely linked restriction fragment lenghth polymorphisms (RFLPs). In the present study nine different RFLPs (TaqI, StuI, HincII, BstEII, AvaII, PvuII, MspIA, MspIB, and NcoI) and a sequence variation at Arg450 were used to clarify the characteristics of the LDL-receptor gene in Koreans. A total of 978 LDL-receptor alleles from 244 members of 43 different pedigrees (15 normal and 28 FH pedigrees) and 245 individuals (187 normal and 58 FH) were analyzed. Frequencies of these polymorphisms did not differ significantly between controls and FH patients. Individually, seven sites--TaqI, BstEII, AvaII, MspIA, MspIB, NcoI and Arg450--had heterozygosity indices ranging from 0.3610 to 0.4601, whereas the PvuII site displayed low levels of polymorphism and StuI was monomorphic. Haplotypes were constructed for 215 individuals of 13 normal and 24 FH pedigrees using the nine polymorphisms. Of 512 (= 2(9)) possible combinations for the nine polymorphic sites, 39 unique haplotypes were detected. The frequency distribution of individual haplotypes ranged from 1/155 (0.65%) to 40/155 (25.8%). The four most common haplotypes accounted for 59.4% of those sampled. Statistical analysis of the haplotypes indicated marked linkage disequilibrium for these 10 sites and throughout the region containing the LDL-receptor gene. Owing to the high degree of linkage disequilibrium over the entire locus, not all RFLPs were informative. We rank each RFLP according to its informativeness and present a strategy for the optimal selection of RFLPs for pedigree analysis.     

Isolation of the human HDL apoprotein A1 gene.

Apo A1 is the major apoprotein of the human plasma high density lipoprotein (HDL). We have isolated apo A1 cDNA and genomic clones and used them to study the gene organisation as defined by its restriction enzyme map. These studies showed that apo A1 is coded by a unique gene. Cross hybridisation was not observed with functionally related apoprotein genes. Increased levels of HDL have been correlated with certain protection against coronary heart disease. If there is a genetic component that contributes to the variable levels of HDL found in the population, it may be possible to correlate these differences with distinct gene organisation patterns.     

DNA polymorphism haplotypes of the human apolipoprotein APOA1-APOC3-APOA4 gene cluster

Summary The genes coding for apolipoproteins A1, C3, and A4 (APOA1, APOC3, APOA4) are closely linked and tandemly organized within a 15-kilobase (kb) DNA segment on the long arm of human chromosome 11. The nucleotide variability of a 61-kb DNA segment containing these genes and their flanking sequences was studied by restriction analysis of a sample of 18 unrelated Northern Europeans using seven different genomic DNA probes. Eleven restriction site polymorphisms located within this DNA segment were used for haplotype analysis of 129 Mediterranean and 67 American black chromosomes. Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium within the APOA1-APOC3-APOA4 gene cluster. Several haplotypes arose by recombination, and the rate of recombination within this gene cluster was estimated to be at least 4 times greater than that expected based on uniform recombination. The polymorphism information content of each of these polymorphisms, taken individually, ranges between 0.053 and 0.375, while that of their haplotypes ranges between 0.858 and 0.862. Therefore, DNA polymorphism haplotypes in the APOA1-APOC3-APOA4 gene cluster constitute a highly informative genetic marker on the long arm of human chromosome 11.     

Expression of urotensin-II in human coronary atherosclerosis

The vasoactive peptide urotensin-II (U-II) is best known for its ability to regulate peripheral vascular and cardiac contractile function in vivo, and recent in vitro studies have suggested a role for the peptide in the control of vascular remodeling by inducing smooth muscle proliferation and fibroblast-mediated collagen deposition. Therefore, U-II may play a role in the etiology of atherosclerosis. In the present study we sought to determine the expression of U-II in coronary arteries from patients with coronary atherosclerosis and from normal control subjects, using immunohistochemistry and in situ hybridization. In normal coronary arteries, there was little expression of U-II in all types of cells. In contrast, in patients with coronary atherosclerosis, endothelial expression of U-II was significantly increased in all diseased segments (P < 0.05). Greater expression of U-II was noted in endothelial cells of lesions with subendothelial inflammation or fibrofatty lesion compared with that of endothelial cells underlined by dense fibrosis or minimal intimal thickening. Myointimal cells and foam cells also expressed U-II. In most diseased segments, medial smooth muscle cells exhibited moderate expression of U-II. These findings demonstrate upregulation of U-II in endothelial, myointimal and medial smooth muscle cells of atherosclerotic human coronary arteries, and suggest a possible role for U-II in the pathogenesis of coronary atherosclerosis.     

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.     

Cytophotometric analysis of the apoprotein B content of sections of the human aorta

A cytophotometric technique to quantitate the content of apolipoprotein B (apo B) in cryostate sections of human aorta is described. The method is based on the comparison of different regions of the sections stained by indirect immunoperoxidase technique, using antibody to apo B. It has been found that the optical density of stained intimal layer was 8-22 times higher than that of the stained aortic media, which in its turn, differed from the control values. The intensity of specific staining of fibrous plaques was 58-87% of the staining intimal tissue of the apparently unaffected intima. About 25-42% of apo B content was extracted from the sections of the normal part of intima and fibrous plaque tissue during incubation of nonfixed sections in a physiologic saline.     

DNA polymorphism haplotypes of the human lipoprotein lipase gene: possible association with high density lipoprotein levels

Summary Lipoprotein lipase (LPL) plays a central role in the metabolism of lipoproteins by hydrolyzing the core triglycerides of circulating very low density lipoproteins and chylomicrons. The enzyme is encoded by a gene about 30kb in size located on the short arm of human chromosome 8. We have determined the locations of the four common DNA polymorphisms along the gene, including a polymorphism that occurred only among an American black population examined. These restriction site polymorphisms were used for haplotype analysis of Mediterranean and US black families. Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium between these sites. No correlation was observed between the level of linkage disequilibrium and the physical distance of the polymorphic sites. The polymorphism information content of the haplotypes ranged from 0.65 to 0.74, thereby constituting a relatively useful genetic marker on chromosome 8. We tested for possible associations between the polymorphisms and circulating lipoprotein phenotypes in a population of 139 Caucasians undergoing coronary arteriography and 50 of their spouses. Some possibly significant associations between LPL gene polymorphisms and levels of high density lipoprotein cholesterol (P = 0.015) and total plasma cholesterol (P = 0.025) were observed. In contrast to a previous report, we found no significant associations with the levels of plasma triglycerides.     

A selective difference between human Y-chromosomal DNA haplotypes

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] and [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] and [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] and [11] and as many as 5% of males are infertile [11] and [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.     

A DNA polymorphism of an apoprotein gene associates with the hypertriglyceridaemia of primary gout

Summary Genomic hybridization analysis has been used to investigate allelic frequencies of the genes coding for the four major apoproteins of high density lipoprotein (HDL); apoproteins AI, AII, CII and CIII, in a group of Caucasian subjects with primary gout. An uncommon allelic variant of the apoprotein CIII gene (the S2 allele) was significantly more common among the patients with gout (9/48, 19%) than among normouricaemic controls who were either randomly selected (1/41, 2%, P=0.03) or normotriglyceidaemic (0/33, 0%, P=0.013). Approximately 46% (22/48) of the subjects with gout were hypertriglyceridaemic (with a serum triglyceride >2.1 mmol/l). Of the 22 patients in this subgroup, 5 (23%) had the uncommon S1S2 genotype, which was also a significantly greater proportion than among the normotriglyceridaemic controls (P=0.015). These data suggest that the hypertriglyceridaemia associated with primary gout may have a genetic basis. In contrast, we found no differences in the frequencies of restriction fragment length polymorphisms of the genes for apoproteins AI, AII and CII.     

Analysis of gene complexes predisposing to coronary atherosclerosis

The following seven polymorphic marker loci of genes responsible for predisposition to coronary atherosclerosis (CAS) were studied: the ACE locus responsible for angiotensin-converting enzyme insertion/deletion polymorphism for the presence or absence of the Alu insertion in the gene; the F13, PLAT, and APOA1 loci, controlling the clotting factor 13, plasminogen-activating tissue factor, and apolipoprotein A, respectively; the MTHFR and AGT polymorphic loci responsible for point mutations in methylenetetrahydrofolate reductase and those in angiotensinogen, respectively, and the NOS3 locus controlling the number of tandem repeats in the nitric oxide synthase gene. These loci are located on different chromosomes and encode products involved into various metabolic pathways leading to CAS. In the populations studied, significant differences between healthy subjects and patients predisposed to cardiovascular diseases were revealed with regard to the above seven markers. The 174M allele (T174M polymorphism in the ACE gene) was significantly associated with coronary atherosclerosis. It was found that specific gene combinations are involved in the CAS development and determine variation in the pathogenetically important quantitative traits.     

Comparison of two commercial nephelometric methods for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays

Normotriglyceridemic and hypertriglyceridemic samples were analyzed for apoproteins A-I and B using the Beckman Array System and the Behring Nephelometer, and the nephelometric values were compared to values obtained by highly standardized radioimmunoassays developed at the Northwest Lipid Research Center. Although the means of the apoA-I values obtained by each method were similar, comparison of sample values by least-squares regression analysis revealed large differences (Sy = 20 mg/dl for Beckman, Sy = 18 mg/dl for Behring) (Sy = standard error of the estimate) regardless of whether the comparison included hypertriglyceridemic samples. For normotriglyceridemic samples, there was good agreement between apoB values obtained by the Behring Nephelometer and those obtained by RIA (r = 0.91, m = 1.03, Sy = 12 mg/dl). However, significantly higher apoB values were obtained on hypertriglyceridemic samples by the Behring Nephelometer. ApoB values for normotriglyceridemic samples obtained by the Beckman System and RIA showed fairly good correlation (r = 0.86, m = 0.71, Sy = 14 mg/dl). However, the nephelometric values for normotriglyceridemic samples averaged 29% lower than those obtained by RIA. This difference could largely be accounted for by the low apoprotein B value assigned to the Beckman calibrator. Significantly lower apoprotein B values were obtained on hypertriglyceridemic samples by the Beckman Nephelometer even after correction for calibration differences. Apoprotein values obtained by nephelometric methods may be inaccurate, particularly if the samples are hypertriglyceridemic.     

Comparison of commercial kits for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays performed at the Northwest Lipid Research Center

There has recently been a proliferation of commercial kits available for apoproteins A-I and B. Since reference procedures for apoproteins have not yet been established we have elected to compare apoprotein kit methods with highly standardized apoprotein B and A-I radioimmunoassays developed at the Northwest Lipid Research Center. Commercial radial immunodiffusion kits for apoproteins A-I and B were obtained from three separate companies, Calbiochem, Daiichi Pure Chemicals, and Tago, and a commercial radioimmunoassay kit for apoprotein A-I was obtained from Ventrex Laboratories. Considerable differences were observed between the commercial kit methods and the Northwest Lipid Research radioimmunoassay methods. Some of the differences between methods were related to the assigned value of the reference materials. Other differences between methods were clearly method-dependent.     

Deoxyribonucleic acid polymorphism of the apoprotein AI-CIII-AIV gene cluster and coronary heart disease in non-insulin-dependent diabetes.

The prevalence of an uncommon allelic variant (S2) of the apoprotein AI-CIII-AIV gene cluster was determined in non-insulin-dependent diabetics with or without evidence of coronary heart disease and in controls. Frequencies of the S2 allele were 14% for diabetics with coronary heart disease compared with 2% for non-diabetics with no clinical evidence of occlusive vascular disease. No subject with the S2 allele was detected among a further group of matched diabetics without clinical features of macrovascular disease. The results suggest that a genetic component contributes to the susceptibility to coronary heart disease in non-insulin-dependent diabetics. Whether the observed deoxyribonucleic acid variant is aetiological for atherosclerosis or in linkage disequilibrium with other atherogenic loci on chromosome 11 remains to be clarified.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Novel polymorphisms and haplotypes in the human coagulation factor XIII A-subunit gene

Novel polymorphic sites within the coding region of the human coagulation factor XIII A-subunit (F13A) gene and their haplotypic combinations with the other polymorphic sites thus far reported are presented. Polymorphic bands were detected in exons 2, 5, 8, 12 and 14 by using single strand conformational polymorphism analysis and antithetic forms of the polymorphic exons were linked with each other, cosegregating as distinct sequence haplotypes. In Finnish, German, and Russian populations a total of 18 haplotypes were observed of possible 72 haplotypic combinations of the 5 exons. Ten of the haplotypes detected were found to have no novel mutations but to be only combinations of preexisting mutations. No tightly associated combinations in pairwise comparisons between antithetic forms of the polymorphic exons were observed, indicating that there may be recombinational hotspots within the F13A gene region. Received: 10 November 1995 / Revised: 2 May 1996