Analysis of a recycling-impaired mutant of low density lipoprotein receptor in familial hypercholesterolemia

A mutant low density lipoprotein (LDL) receptor with abnormal ligand binding and recycling abilities was found in a patient with familial hypercholesterolemia. The molecular weights of the precursor and the mature form of the receptor were 72,000 and 115,000, respectively, which were about 45,000 smaller than those of the normal receptor. The mutant receptor was concluded to be present on the cell surface because the mature form was susceptible to Pronase digestion, and specific monoclonal antibody against the LDL receptor (IgG-C7) could bind to the cell surface. This mutant receptor could not bind LDL, but could bind other ligands for the LDL receptor, beta-migrating very low density lipoprotein, and the apolipoprotein E-lipid complex. After the receptor bound to the ligand, it disappeared from the cell surface of the mutant cells faster than that of normal cells, showing that, in the mutant cells, the receptor was not efficiently recycled back to the cell surface. Southern blotting of the genomic DNA from the patient showed a large deletion of about 12 kilobases around the epidermal growth factor precursor homology domain. For further characterization of the mutant, we cloned a 9.4-kilobase EcoRI/XbaI fragment, which was expected to contain the deletion joint. Mapping and sequencing analyses of the receptor gene showed that exons 7-14 were deleted. The nucleotide sequence suggested that this mutation may have occurred by recombination between repetitive Alu sequences in introns 6 and 14 of the receptor gene. The recombination brought about a complete deletion of the gene coding the epidermal growth factor precursor homology domain. The characteristics of the receptor protein produced by this mutation were similar to those of an artificial mutation constructed by Davis et al. (Davis, C. G., Goldstein, J. L., Südhof, T. C., Anderson, R. G. W., Russell, D. W., and Brown, M. S. (1987) Nature 326, 760-765) in which the whole gene coding this domain was deleted. The clinical phenotype of the patient having this mutation was similar to that of so-called "receptor-defective" type familial hypercholesterolemia, in which cells show detectable, but markedly reduced activity of the LDL receptor.     

Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia

This paper describes a sensitive method for study of the isoelectric point and molecular weight of immunoreactive low density lipoprotein (LDL) receptors of cultured human fibroblasts. The fibroblast receptors are solubilized with Triton X-100, partially purified by batch elution from DEAE-cellulose, and subjected to two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are transferred electrophoretically to nitrocellulose paper which is then incubated with a mouse monoclonal antibody (IgG-C7) directed against the LDL receptor, followed by an 125I-labeled antibody against mouse IgG. The receptor-bound monoclonal antibody is localized by autoradiography. By this technique, the immunodetectable LDL receptors from normal human fibroblasts migrate as a single spot with an isoelectric point of 4.3 and a Mr of approximately 160,000. In one patient with homozygous familial hypercholesterolemia whose cells fail to bind 125I-labeled IgG-C7, no immunoreactive LDL receptor spot was detected after electrophoresis. We also studied LDL receptors from three homozygotes whose cells bind 125I-IgG-C7, i.e. cross-reacting material-positive mutants. Their immunodetectable receptors were indistinguishable from normal receptors in terms of isoelectric point and molecular weight. Similarly, the receptors from one patient with the internalization-defective form of familial hypercholesterolemia showed normal electrophoretic migration. The immunoblotting technique should prove useful in analyzing structural alterations, if they exist, in LDL receptors from other subjects with cross-reacting material-positive forms of familial hypercholesterolemia.     

Mutations of the low density lipoprotein receptor in Japanese kindreds with familial hypercholesterolemia

Summary Mutations of the low density lipoprotein (LDL) receptor in 16 Japanese kindreds with homozygous familial hypercholesterolemia (FH) were studied using an anti-LDL receptor antibody. The LDL receptor mutations in Japanese FH were heterogeneous and included defects in synthesis, posttranslational processing, ligand-binding activity, and internalization of the LDL receptor. Of the 16 kindreds, 10 were receptor-negative and 5, receptor-defective types and 1 was an internalization-defective type with respect to LDL binding. The receptor-negative group was further subdivided into four groups: those with cells producing (i) no immunodetectable receptor (five kindreds); (ii) 160-kd mature receptors, which were quite scarce (two kindreds); (iii) receptors that could not be processed to the mature receptor properly (two kindreds); and (iv) receptors with an apparent molecular weight smaller than normal (one kindred). The last kindred synthesized an about 155-kd mature receptor that was rapidly degraded. This finding is compatible with the low concentration of the cell surface LDL receptors and decreased binding activity for LDL in the cells of this kindred. The receptor-defective group, which could produce a residual amount of functional receptors, exhibited a lower tendency to coronary artery disease than the receptor-negative group.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Chinese hamster ovary cell mutant with defective down-regulation of low density lipoprotein receptors

A monensin-resistant clone (Monr-31) shows a related series of differences from its parental Chinese hamster ovary (CHO) cell line in the cellular response to several ligands. The uptake and metabolism of low density lipoprotein (LDL) in the mutant cells are defective. Accumulation of fluorescent-labeled LDL as well as internalization and degradation of 125I-LDL are greatly reduced in Monr-31 cells. The receptor number for LDL on the cell surface of Monr-31 is about one-third that for CHO cells, but affinity constants for both cell lines are similar. Electrophoretic analysis shows a slightly reduced molecular weight of LDL receptor in Monr-31 cells in comparison to that in CHO cells. The internalization index (internalization plus degradation per binding) of LDL of the mutant is about one-half that of CHO cells, suggesting a failure of internalization of LDL as well as LDL binding. Hybrids (hyb-1, -2, and -3) between CHO and Monr-31 cells show LDL binding and LDL internalization activities comparable to that of CHO cells, suggesting that the altered LDL response in Monr-31 cells is recessive. Addition of exogenous LDL to culture medium down-regulates the LDL receptor activity of CHO, hyb-2, and hyb-3 cells, whereas no such down-regulation is seen in Monr-31 cells. Probably as a result of the failure of down-regulation, the prominent inhibition of sterol synthesis from acetate and 3-hydroxy-3-methylglutaryl-coenzyme A reductase observed in CHO cells is scarcely detectable in Monr-31 cells. As a correlated result, sterol synthesis from acetate is 6-fold higher in the mutant. The failure of down-regulation of LDL receptors in Monr-31 cells is discussed in relation to the altered binding and internalization of LDL.     

A missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in Sephardic Jews

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low density lipoprotein (LDL) receptor gene. Here, we characterize an LDL receptor mutation that is associated with a distinct haplotype and that causes FH in the Jewish Sephardic population originating from Safed, a town in northern Israel. The mutation was found in eight FH families originating from this community comprising 10% of heterozygote FH index cases screened in Israel. The mutation was not found in four additional FH heterozygotes whose hypercholesterolemia co-segregated with an identical LDL receptor gene haplotype. A guanine to cytosine substitution results in a missense mutation (asp¹⁴⁷ to his) in the fourth repeat of the binding domain encoded by exon 4 of the LDL receptor gene. The mutant receptor protein was synthesized in cultured cells as a 120kDa precursor form that failed to undergo normal processing to a mature cell surface form. Most of the receptor precursors were degraded in the endoplasmic reticulum. The small number of mutant receptors on the cell surface were unable to bind LDL or very low density lipoprotein. The abnormal behavior of the mutant receptor was reproduced by site-directed mutagenesis and expression of the mutant protein in CHO cells. The mutation can be diagnosed by allele-specific oligonucleotide hybridization of polymerase chain reaction amplified DNA from FH patients.     

Enhanced degradation of trypsin-treated low density lipoprotein by fibroblasts from a patient with homozygous familial hypercholesterolemia.

When 125I-labeled native low density lipoprotein was incubated with skin fibroblasts from a patient with homozygous familial hypercholesterolemia, the observed rate of degradation of the protein moiety was less than 5% the rate observed with normal fibroblasts, in agreement with previous studies. When the low density lipoprotein had been first treated with trypsin, with release of about 20% of the protein, its degradation by the patient's fibroblasts was markedly increased 8-20-fold. In contrast, the rate of degradation of the trypsin-treated lipoprotein by normal fibroblasts was, if anything, slightly reduced. In neither the normal cells nor the patient's cells was binding to the cell surface appreciably altered by trypsin treatment of the lipoprotein. Prior incubation with cholesterol and 7-ketocholesterol reduced binding of trypsin-treated low density lipoprotein to normal cells by 67% but did not affect its binding to the patient's cells. The results show that the structural modifications induced by trypsin do not interfere with binding of low density lipoprotein to its normal high affinity receptor nor its degradation by normal cells. However, the modified lipoprotein is much more readily internalized and degraded by cells from the patient with homozygous familial hypercholesterolemia.     

Characterization of two new point mutations in the low density lipoprotein receptor genes of an English patient with homozygous familial hypercholesterolemia.

Two new point mutations have been detected in the low density lipoprotein (LDL) receptor gene of a patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, in whom the mutant allele inherited from his English father has a single base substitution of A for G in exon 3, changing the codon for residue 80 in the mature protein from glutamic acid to lysine. The mutant allele inherited from his mother, who is of Irish origin, has a single base pair deletion in the codon for residue 743 in exon 15 that causes a frameshift and introduces a new stop codon in the adjacent position. The glu80 to lys mutation results in a transport-defective phenotype and a mature protein that migrates abnormally slowly on nonreduced SDS-PAGE, but normally under reducing conditions; this was confirmed by site-directed mutagenesis and expression in vitro. The deletion in exon 15 results in a null phenotype in which the putative truncated receptor protein cannot be detected in cultured skin fibroblasts and the amount of mRNA derived from the allele is reduced. The glu80 to lys mutation was found in a further five unrelated individuals in a sample of 200 FH patients from the London area and in 11 from a sample of 77 FH patients from Manchester. Haplotype analysis suggested that all the patients had inherited this allele from a common ancestor. The deletion in exon 15 was not found in the London sample, nor in any unrelated individuals in the Manchester sample.     

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.     

Haplotype analysis at the low density lipoprotein receptor locus: application to the study of familial hypercholesterolemia in Israel

Summary Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein (LDL) receptor gene. It has been shown that restriction fragment length polymorphisms (RFLPs) associated with this gene may be used for family and population studies. The present investigation is a population-based study of 19 Jewish families with hypercholesterolemia representing 9 different countries of origin. Ten RFLP sites were used to construct 24 different haplotypes from 112 chromosomes. These haplotypes vary in frequency from 0.9% to 28.6%. Five previously undescribed haplotypes, which comprise 8.1% of the sample, are reported here. The six most common haplotypes account for 70% of the sample. Segregation analysis reveals that, in Israel, distinct LDL receptor haplotypes are associated with hypercholesterolemia in 12 (63%) out of the 19 Jewish families. Five LDL receptor haplotypes co-segregate with hypercholesterolemia. Two of these haplotypes seem to be unique to specific population groups in Israel and may therefore represent founder mutations.     

Oxidized low density lipoprotein and innate immune receptors

PURPOSE OF REVIEW: Atherosclerosis is now recognized as a chronic inflammatory disease. This review discusses recent literature reporting that innate immune receptors bind oxidatively modified LDL and its many oxidized moieties and consequently modulate the atherogenic process. These innate pattern recognition receptors are known to play a central role in pro-inflammatory responses to bacteria by binding pathogen-associated molecular patterns. It is hypothesized that oxidized LDL exposes similar molecular patterns recognized by receptors of innate immunity. RECENT FINDINGS: Minimally modified LDL and its oxidized phospholipids have been found to bind to CD14 or activate Toll-like receptors on macrophages. In turn, various biological activities have been induced, including the stimulation of cytoskeletal rearrangements that alter phagocytic activity and the stimulation of cytokine secretion, such as IL-8. These findings link modified LDL with innate pattern recognition receptors, such as those involved in the lipopolysaccharide signaling pathway. Human epidemiological studies support the involvement of CD14 and TLR4 in cardiovascular diseases. Oxidized LDL has also been demonstrated to bind to C-reactive protein, an opsonic molecule activating classic complement pathway and Fcgamma receptor endocytosis. These data suggest that C-reactive protein may not only be a strong predictor of clinical disease, but may also play a role in atherogenesis. Recent data on other innate immune receptors are discussed in the context of their potential interactions with oxidized LDL and atherogenesis. SUMMARY: Recent findings suggest that oxidized forms of LDL interact with innate immune receptors. Further studies are needed to identify the role of these interactions in inflammation and atherosclerosis.     

家族性高胆固醇血症患者低密度脂蛋白受体基因新突变位点的鉴定

家族性高胆固醇血症(hypercholesterolemia familial,FH)是由于低密度脂蛋白受体(low density lipoprotein receptor,LDLR)基因突变导致的常染色体显性遗传性疾病,临床上表现为多发黄色瘤、高水平血浆LDL、早发性冠心病及有阳性家族史。本研究通过临床症状结合血脂测定诊断出一个FH家系,其纯合子FH患者的血浆总胆固醇水平高达19．05mmol／L,LDL达17．06mmol／L,并有黄色瘤;而杂合子FH患者的血浆总胆固醇水平为7．96mmol／L,LDL为5．55mmol/L,并有心绞痛症状和黄色瘤。我们对该FH家系患者LDLR基因的PCR扩增DNA片段进行测序,发现纯合子FH患者LDLR基因Exon4区域内发生了GAG683GCG突变,即编码LDLR第683位的谷氨酸被丙氨酸替换,而杂合子FH患者该位点呈现杂合突变。此基因型与临床诊断遗传谱完全一致。同时,利用获得Epstein-Barr(EB)病毒转化型人永生淋巴细胞株(EBV-Ls)与荧光探针DiI标记的LDL结合反应,再通过流式细胞仪检测结果显示,具有功能性LDLR的EBV-Ls细胞比例,在纯合子FH患者(7．02％)和杂合子FH患者(62．64％)均比健康对照者(84．69％)低,纯合子FH患者LDLR活性仅为健康对照者的8．29％、而杂合子FH患者LDLR活性约为健康对照者的73．96％,前者呈现非常显著的降低。这些EBV-Ls细胞LDLR的功能变化分析,有力地支持了该FH家系的临床诊断和DNA测序结果。经查阅最新的UMD-LDLR完全版证实,本研究发现鉴定的GAG683GCG突变是人LDLR基因的新突变位点。     

Four new mutations and polymorphic variants of the low density lipoprotein receptor in patients with familial hypercholesterolemia in Saint Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.     

Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia

HDL-associated paraoxonase type 1 (PON1) can protect LDL and HDL against oxidative modification in vitro and therefore may protect against cardiovascular disease. We investigated the effects of PON1 levels, activity, and genetic variation on high density lipoprotein-cholesterol (HDL-C) levels, circulating oxidized LDL (OxLDL), subclinical inflammation [high-sensitive C-reactive protein (Hs-CRP)], and carotid atherosclerosis. PON1 genotypes (L55M, Q192R, -107C/T, -162A/G, -824G/A, and -907G/C) were determined in 302 patients with familial hypercholesterolemia. PON1 activity was monitored by the hydrolysis rate of paraoxon, diazoxon, and phenyl acetate. PON1 levels, OxLDL, and Hs-CRP were determined using an immunoassay. The genetic variants of PON1 that were associated with high levels and activity of the enzyme were associated with higher HDL-C levels (P values for trend: 0.008, 0.020, 0.042, and 0.037 for L55M, Q192R, -107C/T, and -907G/C, respectively). In addition to the PON1 genotype, there was also a positive correlation between PON1 levels and activity and HDL-C (PON1 levels: r = 0.37, P < 0.001; paraoxonase activity: r = 0.23, P = 0.01; diazoxonase activity: r = 0.29, P < 0.001; arylesterase activity: r = 0.19, P = 0.03). Our observations support the hypothesis that both PON1 levels and activity preserve HDL-C in plasma.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

Hypercholesterolemia and atherosclerosis in low density lipoprotein receptor mutant rats

To establish low density lipoprotein receptor (LDLR) mutant rats as a hypercholesterolemia and atherosclerosis model, we screened the rat LDLR gene for mutations using an N-ethyl-N-nitrosourea mutagenesis archive of rat gene data, and identified five mutations in its introns and one missense mutation (478T>A) in exon 4. The C160S mutation was located in the ligand binding domain of LDLR and was revealed to be equivalent to mutations (C160Y/G) identified in human familial hypercholesterolemia (FH) patients. The wild type, heterozygous, and homozygous mutant rats were fed a normal chow diet or a high fat high cholesterol (HFHC) diet from the age of 10 weeks for 16 weeks. The LDLR homozygous mutants fed the normal chow diet showed higher levels of plasma total cholesterol and LDL cholesterol than the wild type rats. When fed the HFHC diet, the homozygous mutant rats exhibited severe hyperlipidemia and significant lipid deposition from the aortic arch to the abdominal aorta as well as in the aortic valves. Furthermore, the female homozygous mutants also developed xanthomatosis in their paws. In conclusion, we suggest that LDLR mutant rats are a useful novel animal model of hypercholesterolemia and atherosclerosis.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia

The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.     

Identification of the 408 valine to methionine mutation in the low density lipoprotein receptor in a German family with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is caused by different mutations in the gene encoding the low density lipoprotein receptor (LDLR). In Caucasian patients, at least three single point mutations have been identified causing FH. The asparagine₂₀₆ to glutamine, and valine₄₀₈ to methionine mutations were originally described in Afrikaners and recently identified in Dutch FH patients. The proline₆₆₄ to leucine mutations was previously identified in an FH homozygote of Asian Indian origin and later identified in patients from London. Any of these mutations can be identified using direct amplification of genomic DNA by the polymerase chain reaction (PCR) and restriction enzyme digestion of PCR products. In this study, 100 unrelated German FH patients were screened for these three mutations. The valine₄₀₈ to methionine mutation was identified in one individual and subsequently in the hypercholesterolemic child of the proband. Haplotype analysis with 7 restriction fragment length polymorphisms (RFLPs) revealed that the mutant allele carried the same haplotype as the previously described patients in South Africa and the Netherlands. Our finding supports the previous assumption of the European origin of the mutation.