Genetics of the low density lipoprotein receptor:

Fibroblast low density lipoprotein (LDL) plasma membrane receptor activity, measured as 125I-LDL association (plasma membrane binding plus intracellular accumulation) and degradation was determined in cell strains from 14 monozygotic (MZ) and 21 like-sexed dizygotic (DZ) normolipidemic twin pairs. The twins were between 57 and 62 years old and had liver apart for an average of 38 years (range 0-60). The intrapair differences were significantly smaller in MZ than in DZ twin pairs in fibroblast 125I-LDL association as well as degradation assays (P less than 0.05). These findings suggest a genetic influence on normal variation in LDL receptor activity in vitro. In two MZ pairs discordant for psoriasis, the psoriatic twin had markedly lower LDL receptor activity than the cotwin.     

Very low density lipoprotein and low density lipoprotein isolated from patients with hepatitis C infection induce altered cellular lipid metabolism

Several abnormalities of lipid metabolism, including hypo-beta-lipoproteinemia and liver steatosis are associated with infection by hepatitis C virus (HCV). The aim of this study was to determine whether circulating lipoproteins of patients with HCV infection could directly cause alterations of lipid cellular metabolism. To this end the metabolic response of human monocyte-derived macrophages (HMDM) to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), measuring the cholesteryl ester (CE) and triglyceride (TG) production was analyzed. Lipoproteins were isolated from 18 patients infected with hepatitis C virus (HCV-VLDL and HCV-LDL) and from normal healthy donors (ct-VLDL and ct-LDL). In comparison to ct-lipoproteins, HCV-lipoproteins induced significant differences in HMDM CE and TG production. HCV-VLDL decreased CE and TG production; while HCV-LDL induced an increased TG synthesis. The present findings suggest that HCV infection modifies VLDL and LDL molecular composition, affecting cellular lipid metabolism, thus promoting intracellular lipid accumulation and hypo-beta-lipoproteinemia.     

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.     

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).     

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.     

内源性高甘油三酯血症患者血浆VLDL、LDL及HDL对血凝的影响

目的：研究内源性高甘油三酯血症(HTG)患血浆极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)是否发生了氧化修饰及其对血凝的影响。方法：对2l例内源性高甘油三酯血症患与2l例年龄性别相近的正常人的血脂、脂质过氧化物进行了分析。用一次性密度梯度超速离心法分离血浆VLDL、LDL及HDL，测定这三种脂蛋白的234nm光吸收、相对电泳迁移率(REM)和硫代巴比妥酸反应物质(TBARS)，分别将这三种脂蛋白加入由正常人新鲜混合血浆构成的反应系统中，按试剂盒分别测定凝血酶原时间(PT)及活化部分凝血酶原时间(APIT)。结果：内源性HTG患血浆TG含量平均升高2．73倍，HDLC下降l.7l倍，同时LPO升高1．22倍;HTG组VLDL、LDL及HDL的REM、234nm光吸收值、TBARS含量均较对照组显增加(P＜0．01)，表明内源性HTG患血浆VLDL、LDL及LDL均发生了氧化修饰生成Ox—VLDL、Ox-LDL.PT及APTT在分别加入HTG组的VLDL、LDL及HDL后均比加入相应正常组脂蛋白明显缩短(P均＜0．05)。相关分析表明，HTG组血浆VLDL及HDL相对电泳迁移率(REM)与PT呈负相关(P＜0．01)。结论：HTG患血浆VLDL、LDL及HDL发生了氧化修饰，并使PT及APTT明显缩短。     

A 9.6 kilobase deletion in the low density lipoprotein receptor gene in Norwegian familial hypercholesterolemia subjects

Haplotype analysis of the low density lipoprotein receptor (LDLR) gene was performed in Norwegian subjects heterozygous for familial hypercholesterolemia (FH). Southern blot analysis of genomic DNA, using an exon 18 specific probe and the restriction enzyme NcoI, showed that two out of 57 unrelated FH subjects had an abnormal 3.6 kb band. Further analyses revealed that this abnormal band was due to a 9.6 kb deletion that included exons 16 and 17. The 5' deletion breakpoint was after 245 bp of intron 15, and the 3' deletion breakpoint was in exon 18 after nucleotide 3390 of cDNA. Thus, both the membrane-spanning and cytoplasmatic domains of the receptor had been deleted. A polymerase chain reaction (PCR) method was developed to identify this deletion among other Norwegian FH subjects. As a result of this screening one additional subject was found out of 124 subjects screened. Thus, three out of 181 (1.7%) unrelated Norwegian FH subject possessed this deletion. The deletion was found on the same haplotype in the three unrelated subjects, suggesting a common mutagenic event. The deletion is identical to a deletion (FH-Helsinki) that is very common among Finnish FH subjects. However, it is not yet known whether the mutations evolved separately in the two countries.     

低密度脂蛋白受体相关蛋白5基因的基因组结构

目的 确定2低密度脂蛋白受体相关蛋白5（low density lipoprotein receptor related protein,5 LRP5）的基因组结构。方法 以LRP5基因的cDNA序列为线索，采用计算机杂交方法首先通过对比分析该基因的cDNA序列和基因组序列，初步确定LRP5基因的基因组结构，按分析得到的基因组结构设计引物，扩增并测定外显子序列和外显子内含子接头序列，确定该基因的基因组结构。结果 LRP5基因的基因组DNA全长为131.6kb，含4有23个外显子和22个内含子；在编码序列中检测到3个单核苷酸多态，即位于第2外显子的A459G，位于第10外显子的C2220T和位于第21外显子的G4416C；LRP5基因含有4个已知的短串联重复序列，即D11S1917，D11S4087，D11S1337和D11S4178，它们分别位于该基因的5'端和第1，4，13内含子内。结论 LRP5基因的基因组结构的确定，为分析该基因突变和功能研究奠定了基础。     

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus

Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03–1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (E1/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the E1 gene product (amino acids 222–336) and the C-terminal part of the E2 protein (amino acids 523–809). Lipoproteins did not bind to recombinant HCV core protein. Received: 22 December 1999     

Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes

Low density lipoprotein receptors (LDLR), capable of internalizing LDL, are expressed in polymorphonuclear neutrophils (PMN). The expression was assessed using anti-LDLR antibody by flow cytometry. The internalization of LDL was assessed by: (i) quantification of the uptake of labelled LDL with 1,1′-dioctadecyl-3,3,3′,3′ tetramethyl-indocarboxycyanine perchlorate (DiI) by flow cytometry; and (ii) the binding of LDL-¹²⁵I. In fresh purified cells, Lineweaver–Burk analysis of LDL binding (LDL-DiI) revealed that the calculated Kd (internalized LDL) for PMN (15.0 × 10⁻⁹ m) is lower than the Kd for monocytes (1.1 × 10⁻⁷ m) and the Kd for lymphocytes (3.2 × 10⁻⁷ m). Scatchard analysis (LDL-¹²⁵I) revealed 25 000 binding sites and a Kd of 9.6 × 10⁻⁹ m for PMN. The interaction of LDL with its receptor caused a two-fold fast (peak at 1 min) and transient increase in the oxidative burst, measured by the formation of 2′,7′ dicholoflurescein (DCF) by flow cytometry. This effect was not observed in monocytes or lymphocytes, and it was blocked by anti-LDLR antibody. The stimulation of LDL was optimal at 10 μg of protein/ml. LDL was able to suppress DCF formation induced by phorbol myristate acetate (PMA) and PMA was unable to further stimulate LDL-treated cells, suggesting protein kinase-C (PKC) involvement in LDL effects. Using a PKC assay, LDL was shown to induce a two-fold increase in PKC translocation to the membrane. Thus, LDL increases PMN oxidative burst through a PKC-dependent pathway.     

乙型肝炎病毒感染者血清HBV DNA水平与HDL-C的相关性

目的分析乙型肝炎病毒（HBV）感染者血清HBV DNA和高密度脂蛋白胆固醇（HDL-C）水平,探讨血清HBV DNA拷贝数与HDL-C水平的相关性。方法收集116例HBV感染者血清,通过实时荧光定量PCR法测定血清HBV DNA拷贝数,通过选择性抑制均相测定法分析血清HDL-C值;计算HBVDNA拷贝数与HDL-C的相关系数,并对相关系数进行显著性检验。结果在乙型肝炎病毒感染者中,血清HBV DNA拷贝数对数均值为（4.18±1.77）/ml,分布范围为（1.38～7.85）/ml;血清HDL-C浓度均值为（1.30±0.29）mmol/L,分布范围为（0.66～2.01）mmol/L。血清HBVDNA拷贝数与HDL-C水平存在负相关（r=-0.5346,P=0.0023）。结论 HDL-C对乙型肝炎病毒的复制有抑制作用。     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

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.     

动脉粥样硬化性脑梗塞与低密度脂蛋白受体基因NcoI、AvaⅡ多态性的关系

目的 探讨动脉粥样硬化性脑梗死（atherosclerotic cerebral infarction,ACI)与低密度脂蛋白受体（low density lipoprotein receptor,LDL-R)基因NcoI、AvaⅡ多态性的关系。方法 用聚合酶链反应技术检测113名辽宁藉汉族健康人和77例ACI患者的LD－R基因NcoI、AvaⅡ多态性及血脂、载脂蛋白的含量。结果 LDL－R基因NcoI、AvaⅡ等位基因频率健康人N^ 为0．667、A^ 为0．230；ACI组N^ 为0．662、A^ 为0．125。A^-A^-与N^ N^ 联合存在时ACI的发病相对风险率（RR）为5．56（P＜0．001），引起血清TG、TC、LDL－C、LP（a)升高的相对风险率依次为4．29、7．67、9．33、3．09（P＜0．05）。结论 LDL－R基因A^-A^-与N^ N^ 联合存在影响血脂、脂蛋白的含量，与ACI密切相关。     

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.     

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。     

两个家族性高胆固醇血症纯合子家系低密度脂蛋白受体基因分析

目的：分析家族性高胆固醇血症（familial hypercholesterolemia,FH)低密度脂蛋白受体（low density lipoprotein receptor,LDLR)的基因突变。方法：提取5个彼此无亲缘关系临床诊断为FH的纯合子患儿及其家系成员的基因组DNA,用聚合酶链反应－单链构象多态性分析方法,对LDLR基因的启动子和全部18个外显子进行突变检测,并对结果异常者进行DNA测序。结果：在两个家系分别发现A606T和C263R两种突变。结论：LDLR基因在以上两位点的突变可引起FH,中国FH患者的LDLR基因可能存在特有的突变位点。     

低密度脂蛋白诱导下调的新基因cDNA的克隆及组织表达

用改进的mRNA差异显示．PCR技术分析ECV304在低密度脂蛋白的诱导下表达水平明显差异的cDNA。获得-差异表达的265bp新EST；以该EST为探针，从人主动脉cDNA文库中筛选到一个1726bp的cDNA克隆，其748-1266bp之间的519个碱基构成一个完整的开放阅读框架，编码172个氨基酸组成的蛋白质。其羧基端94-172区间氨基酸序列中含有三个重复的C2H2型锌指蛋白基序CX2CX3FX5LX2HX3H，Northen Blot证实两组ECV304中均出现一条1．7kb的区带，LDL诱导后其表达降低了2．2倍。与差异显示-PCR的结果一致，该基因被定名为LRZFG。LRZFG是多组织表达的基因，其在动脉粥样硬化早期病理反应的作用有待于进一步研究。