Gene-gene interaction between the low density lipoprotein receptor and apolipoprotein E loci affects lipid levels

A restriction fragment length polymorphism (RFLP) at the low density lipoprotein receptor (LDLR) locus, detectable with the restriction enzyme PvuII has been studied in a second series of Norwegian subjects, believed to be representative of the general population. The results confirm the association of normal alleles at the LDLR locus with differences in age- and sex-corrected total and LDL cholesterol. A gene identified as one of the alleles in this RFLP appears to eliminate the effect of the apolipoprotein E4 (apoE4) gene on cholesterol (or its allele must be present for the apoE4 effect on lipid level to manifest itself). The findings in this series substantiate previous indications that normal alleles are of importance in the control of LDLR activity and that normal LDLR alleles contribute to the population variation in cholesterol. Finally, they confirm that an interaction between LDLR and apoE genes contributes to the population variation in total and LDL cholesterol.     

Normal DNA polymorphism at the low density lipoprotein receptor (LDLR) locus associated with serum cholesterol level

A restriction fragment length polymorphism (RFLP) at the low density lipoprotein receptor (LDLR) locus detectable with the restriction enzyme PvuII exhibits association with total serum cholesterol level. People who are homozygous for absence of the PvuII restriction site have a significantly higher total cholesterol level than heterozygotes (the number of homozygotes for presence of the restriction site was too small to permit meaningful comparison). This difference is significant at the 2% level. Thus, this study of sex- and age-adjusted cholesterol levels in a sample of healthy people yields additional evidence and sustains our previous proposal that normal alleles at the LDLR locus contribute to the population variation in total cholesterol levels. Absence of the PvuII site appears to confer an odds ratio of approximately 2.7 for having a cholesterol level in the top quartile of the population distribution.     

Normal genetic variation at the low density lipoprotein receptor (LDLR) locus influences cholesterol levels in children

The population of Czechoslovakia is at high risk of premature atherosclerosis. Normal DNA polymorphism at the low density lipoprotein receptor (LDLR) locus detectable with the restriction enzyme PvuII was analyzed in Czech children with a high or a low concentration of total serum cholesterol. The PvuII restriction site was found significantly more often in the low cholesterol group than in the high cholesterol group. Thus, normal genetic variation at the LDLR locus contributes to the population variation in cholesterol in children in the population studied.     

DNA polymorphism at the locus for human cholesteryl ester transfer protein (CETP) is associated with high density lipoprotein cholesterol and apolipoprotein levels

Cholesteryl ester transfer protein (CETP) is a protein involved in "reverse cholesterol transport" and it could play an important role in facilitating the removal of cholesteryl esters from peripheral tissues for transport to the liver or for transfer of cholesterol between plasma lipoprotein particles. Both functions may be relevant to susceptibility or resistance to atherosclerotic disease. We have studied 149 and 146 unrelated persons, respectively, for the A and B polymorphism at the CETP locus detectable with the restriction enzyme TaqI. The B system is by far the more polymorphic. A search for association with risk or "anti-risk" factor levels was conducted with the following quantitative parameters: total cholesterol, HDL cholesterol, triglycerides, apolipoprotein AI (apoA-I), apolipoprotein B (apoB) and Lp(a) lipoprotein levels. Highly significant differences in apoA-I concentration were found between the two categories of homozygotes in the B polymorphism. The association observed remained significant after multiplying the p value by the number of quantitative parameters used for the association tests. There was a dosage effect on the apoA-I level of genes in the B polymorphism. We conclude that the associations observed are likely to reflect true biological phenomena. The effect of CETP genes appeared to be limited to non-smokers.     

Characterization of low density lipoprotein receptor (LDLR) gene mutations in Albania

Introduction

Familial hypercholesterolaemia (FH) is a clinical syndrome characterised by elevated serum total cholesterol (TCHOL) levels due to an increase in low-density lipoprotein (LDL) cholesterol, by tendon xanthomata and clinical manifestations of ischaemic heart disease in early life. Typically, it results from mutations in the low-density lipoprotein receptor (LDLR) gene. So far, more than 800 mutations have been reported for the LDLR gene and account for FH. The nature of LDLR gene mutations varies among different ethnicities. Until now no mutations of LDLR have been reported in the Albanian population.

Material and methods

We assessed the contribution of the LDLR gene mutations as causes of FH in an Albanian population. Fifty probands with a clinical diagnosis of FH were included. We analysed all the exons and the promoter of the LDLR gene by using restriction isotyping or direct sequencing.

Results

Twenty-one patients were heterozygous for the 1646G>A mutation (FH Genoa) in exon 11 and 9 patients were heterozygous for the 81T>C mutation in exon 2 of the LDLR gene.

Conclusions

This report describes two LDLR gene mutations accounting for FH in Albania (1646G>A, 81T>C).     

Lp(a) lipoprotein enters cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor

Lp(a) lipoprotein shares the apoB antigen with low density lipoprotein (LDL). The Lp(a) antigen is unique for Lp(a) lipoprotein. Fibroblast association (i.e. plasma membrane binding plus intracellular accumulation), plasma membrane binding, intracellular accumulation and degradation of 125I-Lp(a) lipoprotein were studied in strains from subjects with or without autosomal dominant hypercholesterolemia (HC). Subjects without HC (non-HCs) have cell surface receptors for low density lipoprotein (LDL receptors). On the average, HC heterozygotes have half-normal LDL receptor activity and "receptor-negative" HC homozygous cell strains lack functional receptors. Fibroblast processing of 125I-Lp(a) lipoprotein was compared to fibroblast processing of 125I-LDL. LDL receptor-dependent processing of 125I-LDL was saturated at about 50 microgram apo 125I-LDL.ml-1 in non-HC fibroblasts. 125I-Lp(a) lipoprotein was, however, largely processed independently of receptor mechanisms by non-HC cells (highest concentration examined 150 microgram apo 125I-Lp(a) lipoprotein . ml-1). Lp(a) lipoprotein did not interfere with 125I-LDL for fibroblast association, but inhibited 125I-LDL degradation. The interference with 125I-LDL degradation was time dependent. Only slightly higher 125I-Lp(a) lipoprotein processing values were found in non-HC and HC heterozygous strains than in "receptor-negative" HC homozygous strains. However, non-HC cells had more than tenfold higher 125I-LDL processing values than "receptor-negative" HC homozygous cells.     

DNA deletions in the low density lipoprotein (LDL) receptor gene in Danish families with familial hypercholesterolemia

DNA samples from 25 unrelated Danish patients with familial hypercholesterolemia (FH) were screened by Southern blot hybridization to detect gross alterations in the low density lipoprotein (LDL) receptor gene. Three FH-patients were found to have a deletion. Two of these delete part of the cysteine rich domain, which comprises the ligand binding region of the LDL-receptor. The third deletion encompasses coding regions for the cytoplasmic part of the receptor. As two of these deletions could be equivalent to previously described LDL-receptor gene alterations, these data seem to support a notion of recombination hot spots which involve Alu-sequences.     

Genetics of the low density lipoprotein receptor:

Fibroblast association (plasma membrane binding plus intracellular accumulation) and degradation of radioiodinated low density lipoprotein (125I-LDL) index plasma membrane LDL receptor activity. Cultured fibroblasts from 23 subjects affected with familial hypercholesterolemia (HC) and from 95 subjects without HC (non-HCs) were tested for 125I-LDL association and degradation. Both LDL receptor activity indices were twice as high in non-HC and HC heterozygous cell strains. This is compatible with a major gene effect on LDL receptor activity. However, a considerable overlap between non-HC and HC heterozygous values was found in the 125I-LDL association assay [median (range) 970 (330-2500), and 450 (250-490), respectively] and in the degradation assay [median (range) 810 (280-2020), and 470 (160-790), respectively]. The values are expressed as ng 125I-LDL X mg cell protein-1 X 4.5 h-1. These great overlaps in the LDL receptor activity indices support the view that the influence of LDL receptor activity on the HC phenotype may be smaller than believed previously. Furthermore, for the diagnosis of HC, these LDL receptor activity assays are far more expensive and have less sensitivity and specificity than simple serum cholesterol determination. The LDL receptor-dependent 125I-LDL association values for the HC heterozygous individuals clustered into four groups. Family data supported the hypothesis that this variation could be due to four different LDL receptor variants, each coded for by different alleles at the LDL receptor locus. If confirmed, this finding may have implications for the understanding of the variable expression of HC and also of the genetic impact on lipoprotein metabolism and susceptibility to atherosclerosis in non-HCs.     

高脂血症APOE基因多态性与高密度脂蛋白亚类组成的关系

目的探讨高脂血症患者载脂蛋白E(apolipoproteinE,APOE)基因多态性与高密度脂蛋白(highdensitylipoprotein,HDL)亚类组成变化的关系。方法应用聚合酶链反应-限制性片段长度多态性和双向电泳-免疫印迹检测法,分析112例高脂血症患者和73名正常对照者APOE基因型、HDL各亚类组成及相对含量。结果高脂血症组和对照组APOE基因型及等位基因频率分布均以E3/3和ε3最高。高脂血症患者中等位基因ε2携带者血清APOE/C较等位基因ε3升高,而HDL3b则下降,其差异有显著性(P<0.05)。对照组中等位基因ε2携带者血清甘油三酯、apoE、apoE/C较等位基因ε3和ε4携带者升高,等位基因ε2携带者HDL3a较等位基因ε3携带者低,其差异有显著性(P<0.05)。结论APOE基因多态性可能与血清HDL部分亚类的含量变化相关。     

Characterization and geographic distribution of the low density lipoprotein receptor (LDLR) gene mutations in northwestern Greece

Familial Hypercholesterolaemia (FH) is a clinical syndrome characterised by elevated serum total cholesterol levels due to an increase in low density lipoprotein (LDL) cholesterol, by tendon xanthomata and clinical manifestations of ischaemic heart disease in early life. Typically, it results from mutations in the low-density lipoprotein receptor (LDLR) gene. So far, over 600 mutations have been reported for the LDLR gene and account for FH. The nature of LDLR gene mutations is different in various ethnicities and has also regional distribution within each ethnicity. Eleven mutations have already been described in the Greek population. This report describes seven LDLR gene mutations accounting for FH in Northwestern Greece (81T>G, 517T>C, 858C>A, 1285G>A, 1352T>C, 1646G>A and 1775G>A) and their geographic distribution. We have recently described one of these mutations (1352T>C) as a novel point mutation in a Greek family originating from Northwestern Greece. Furthermore, two previously identified mutations (81T>C, 1775G>A) were also detected in the Greek FH patients for the first time. The 1775G>A mutation was responsible for all the homozygous patients in our area, indicating a founder effect. These data will favor the development of tailed information and screening programs in Northwestern Greece for the primary prevention of cardiovascular disease in FH patients.     

DNA polymorphism at the apolipoprotein B locus is associated with lipoprotein level

A strong association has been uncovered between DNA variation at the apolipoprotein B (apoB) locus (detectable with the restriction endonuclease XbaI) and apoB level. The findings are suggestive of associations also between this DNA polymorphism and total cholesterol as well as fasting triglyceride levels, confirming recent results reported by British workers. The data suggest that lipid/apolipoprotein associations with the XbaI polymorphism are primarily caused by an effect on apoB level. In the present and in a previously reported study we found a strong association between the XbaI polymorphism and the homospecific Ag antigenic variation in low density lipoprotein (LDL) which had previously exhibited associations with lipid levels. The present data indicate that the apoB/lipid associations of the Ag and XbaI polymorphisms may reflect the same phenomenon. The associations reported could reflect variation in an apoB domain close to Ag as well as to the XbaI restriction site that is of importance for lipid binding by apoB. Alternatively, the association of apoB level with the XbaI polymorphism (which reflects a silent third base mutation in a threonine codon) could reflect phenomena related to codon usage.     

Lp(a) lipoprotein is a major risk factor for cardiovascular disease: pathogenic mechanisms and clinical significance

The results of two previous and two recent studies of middle-aged males and females are presented to exemplify the clinical importance of lipoprotein (a) (Lp(a)) as a risk factor for atherosclerosis and coronary heart disease. In these studies various conventional and recently suggested risk factors were included and different methods for Lp(a) quantification were used. Lp(a) was a significant risk factor in all four studies. In the recent prospective case-control study, Lp(a) and cholesterol were found to act synergistically and predict primary acute myocardial infarction in Swedish males. A cholesterol level above 6.5 mmol/1 increased the risk of acute myocardial infarction if the Lp(a) level was above 200 mg/1. The plasma apo A-I level was a protective factor. In the other recent case-control study, an Lp(a) level above 500 mg/1 was a highly significant risk factor in Black and White US women with myocardial infarction or advanced coronary artery disease in addition to low density lipoprotein cholesterol levels above 130 mg/dl. A high apo A-I level was a protective factor. In these studies no other factors tested reached significance in multivariate logistic regression analysis. A hypothetical association between high Lp(a) levels and intracellular infection with Chlamydia pneumoniae is discussed. The results suggest that the Lp(a) level is useful in identifying high-risk individuals. Lowering low density lipoprotein cholesterol below 100 mg/dl (7lt;2.6 mmol/1) seems to be most important in both males and females with high-risk Lp(a) levels.     

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives

Obese (BMI 26 kg/m²; n=51) and lean (BMI <26 kg/m²; n=61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a Hin cII RFLP of the low density lipoprotein receptor gene ( LDLR ). A similar analysis was performed in obese (n=28) and lean (n=68) nonmotensives. A significant association of the C allele of the T → C variant responsible for this RFLP was seen with obesity ( x ²=4.6, P =0.029) in the hypertensive, but not in the normotensive, group (odds ratio=3.0 for the CC genotype and 2.7 for CT ). Furthermore, BMI tracked with genotypes of this allele in the hypertensives ( P =0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the Hin cII RFLP and an Apa LI RFLP ( x ²=33, P <0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.     

Changes in the ratio between apolipoprotein (a) and lipids in human plasma during interaction with polyclonal sheep antibodies against lipoprotein (a)

Plasma contents of apolipoprotein (a), apolipoprotein B 100, cholesterol, triglycerides, and vitamin E were measured in 2 patients with lipoprotein (a) concentration >100 mg/dl during the interaction with the anti-lipoprotein (a) immunosorbent. Intraindividual heterogeneity of apolipoprotein (a)-containing particles in the plasma was demonstrated. Polyclonal antibodies against lipoprotein (a) immobilized on Sepharose CL-4B more effectively removed free apolipoprotein (a) than complexes containing apolipoproteins B 100, apolipoprotein (a), lipids, and vitamin E.     

家族性高胆固醇血症纯合子家系低密度脂蛋白受体功能检测及基因突变分析

目的：分析家庭性高胆固醇血症（familial hypercholesterolemia,FH）患者低密度脂蛋白受体（low density lipoprotein receptor,LDLR）的功能改变及基因突变。方法：分离FH患者外周血淋巴细胞，用流式细胞仪观察淋巴细胞结合和摄取荧光标记的低密度脂蛋白的情况。抽提FH患者外周血基因组DNA为模板，进行聚合酶链反应－单链构象多态性分析（polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP）及DNA序列分析。结果：对一家两例临床诊断为FH纯合子的患儿及其父母的外周血淋巴细胞LDLR功能进行了分析，发现均表现为低密度脂蛋白（LDL）摄取和结合障碍。进一步从基因水平进行了研究，发现LDLR基因突变是位于第6外显子编码第297位氨基酸的碱基发生缺失，导致移码突变并使得终止密码子TGA在第369位提前出现，从而不能表达正常的LDLR，体内胆固醇的代谢发生障碍。结论：对1例家庭性高胆固醇血症纯合子家系应用流式细胞仪方法初步发现LDLR功能缺陷，进一步结合PCR－SSCP方法证实其LDLR存在新的突变类型。     

Low density lipoprotein receptor (LDLR) gene mutations in Canadian subjects with familial hypercholesterolemia,but not of French descent

Heterozygous familial hypercholesterolemia (FH) is a relatively common autosomal dominant disorder, which is characterized by elevated plasma concentrations of low density lipoprotein (LDL) cholesterol and early coronary heart disease. FH results from mutations in the gene encoding the LDL receptor (LDLR). In Canada, there is a founder effect for LDLR mutations in FH among individuals of French descent, most of whom reside in the province of Quebec. However, the spectrum of mutations in other regions, specifically in the populous and predominantly English‐speaking province of Ontario, has not been studied. We sequenced the coding regions, promoter and intron‐exon boundaries of the LDLR gene in 60 Ontario FH subjects from a variety of ethnic backgrounds other than French Canadian. We found 25 LDLR mutations in 34 subjects. Eleven LDLR mutations were novel, including two in‐frame deletions of a single amino acid (one each in exons 2 and 4), two larger deletions that shifted the reading frame (one each in exons 4 and 10), five missense mutations (C42R, A370T, T413M, L561P and E760D) and two splice acceptor mutations (one each in introns 3 and 8). The results indicate that FH is more genetically diverse in Ontario than in Quebec. The results are also consistent with findings from investigations of the LDLR in FH conducted in other countries, in which PCR‐based, exon‐by‐exon sequencing uncovers small mutations in about half of the subjects screened. The gap in molecular diagnosis suggests that lesions not found by this sequencing strategy, such as larger scale LDLR mutations that cannot be amplified, may underlie a substantial number of cases of FH. Alternatively, there might be genetic heterogeneity underlying the FH phenotype, with contributions from other single or multiple genes. Hum Mutat 18:359, 2001. © 2001 Wiley‐Liss, Inc.     

Inherited quantitative DNA variation in the LPA ("apolipoprotein (a)") gene

The Lp(a) antigen resides in a polypeptide chain that is attached to apolipoprotein B (apoB) by a disulfide bridge. Recently, cDNA for this polypeptide chain (frequently referred to as the Lp(a) polypeptide chain, Lp(a) apolipoprotein or apolipoprotein (a] was cloned and extensive homology to plasminogen was uncovered. This homology creates significant difficulties in studying DNA variation in the gene (the LPA gene) for this polypeptide and the plasminogen gene. We have studied a variant 2 kilobase (kb) DNA fragment detectable after digestion with the restriction enzyme MspI, which appears to originate from the LPA gene since it is detected by LPA probes but not with plasminogen probes. It is related to the "kringle IV" region of the LPA gene since it is detected with an LPA probe that only contains "kringle IV" repeats. A proportion of people appears to lack (or have an undetectable level of) the 2 kb fragment and there are significant quantitative differences between samples from people who have the fragment. Presence and amount of this fragment appear to segregate in families as a Mendelian trait. This quantitative DNA variation is likely to reflect differences between individuals in number of "kringle IV" repeats at the LPA locus.     

A new informative Alw 26 I polymorphism in exon 10 of the human low density lipoprotein receptor gene. Application to prenatal diagnosis

Using DNA sequencing of the coding and exon flanking regions of the low density lipoprotein receptor (LDLR) gene we identified an Alw26 I site in exon 10 by a transition G1426A. The alleles are represented by one uncut fragment (A1 = 108 bp) or two fragments (A2 = 82 bp and 26 pb). Two other fragments (72 bp and 16 bp) were systematically found within the amplified product. The alleles were detected in 157 unrelated French Caucasians with A1 frequency = 0.58 and A2 = 0.42. The observed heterozygoty was 44.5%. Homozygous familial hypercholesterolemie (FH) has a severe clinical picture leading to death during childhood. Because it is very informative, the present polymorphism was very useful as genetic marker for clinical diagnosis and counseling as we described in linkage analysis at the LDLR locus for prenatal diagnosis in a fetus who could inherit two LDLR mutant alleles from FH heterozygote parents. Hum Mutat 11:483, 1998. © 1998 Wiley-Liss, Inc.     

Restriction site polymorphism at the LPA (Lp(a) apoliprotein; apoliprotein(a)) locus

A restriction site polymorphism in the Lp(a) apolipoprotein gene (the LPA gene) is reported. The basis for the polymorphism is presence or absence of an MspI restriction site that appears to be 3' to the last kringle IV structure of the gene. The "1" gene (presence of the restriction site) has a frequency of 0.316 and the "2" gene (absence of the restriction site) has a frequency of 0.684. Both members of each of 67 monozygotic (MZ) twin pairs had the same genotype and there was Mendelian segregation of the DNA variants in 40 families with a total of 75 children. There was a lower proportion of people with genotype 1-1 in the top quartile than in the 3 bottom quartiles of the population distribution of Lp(a) lipoprotein levels but the difference did not reach statistical significance.     

DNA polymorphisms at fibrinogen loci and plasma fibrinogen concentration

Associations have been reported between restriction fragment length polymorphisms (RFLPs) at fibrinogen loci and plasma fibrinogen concentration, in a British study. We have examined a series of unrelated Norwegians. We found no association between plasma fibrinogen concentration and any genotype in either of two fibrinogen polymorphisms examined (one at the α-fibrinogen locus, the other at the β-fibrinogen locus). We have also examined monozygotic twins and evaluated heritability of fibrinogen level by the intraclass correlation coefficient. We arrived at an unimpressive estimate of heritability. With such a low level of heritability, it would have been surprising if we had found an association with a single gene marker in a relatively limited series of people. The reason for the discrepancy between the British and the Norwegian study is unknown. Great care has to be exercised in interpreting disease associations, since with DNA variations being examined at an increasing number of "candidate loci", the risk of finding spurious associations increases with the number of analyses conducted.