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Clinical Chemistry 45: 1026-1038, 1999;
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(Clinical Chemistry. 1999;45:1026-1038.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Mutations in the Apolipoprotein (apo) B-100 Receptor-binding region: Detection of apo B-100 (Arg3500->Trp) Associated with Two New Haplotypes and Evidence That apo B-100 (Glu3405->Gln) Diminishes Receptor-mediated Uptake of LDL

Eva Fisher1, Hubert Scharnagl3, Michael M. Hoffmann3, Klaus Kusterer2, Daniela Wittmann1, Heinrich Wieland3, Werner Gross1 and Winfried März3,a

1 Gustav Embden-Centre of Biological Chemistry and
2 Department of Internal Medicine, Johann Wolfgang Goethe-University, Theodor Stern-Kai 7, 60590 Frankfurt am Main, Germany.

3 Division of Clinical Chemistry, Department of Medicine, Albert Ludwigs-University, Hugstetter Strasse 55, 79098 Freiburg, Germany.
a Author for correspondence. Fax 49-761-270-3444; e-mail maerz{at}mzl200.ukl.uni-freiburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Ligand-defective apolipoprotein (apo) B-100 is a major cause of hypercholesterolemia. For many years, apo B-100 (Arg3500->Gln) has been the only mutation known to cause ligand-defective apo B-100.

Methods: Using temperature gradient gel electrophoresis, we screened 297 unrelated individuals with LDL-cholesterol >1.55 g/L and triglycerides <2.0 g/L for sequence variants of the putative LDL receptor-binding domain of apo B-100.

Results: We found apo B-100 (Arg3500->Gln) in 21 individuals (7.1%). When extrapolated to the general population, this corresponds to the highest prevalence of apo B-100 (Arg3500->Gln) reported to date. Furthermore, we identified three unrelated carriers (1%) of a silent substitution (CTG->CTA) affecting the codon for leucine3350, four carriers (1.3%) of apo B-100 (Glu3405->Gln), and two subjects (0.7%) with apo B-100 (Arg3500->Trp). apo B-100 (Arg3500->Trp) was assigned to two different, previously unknown haplotypes. The binding, uptake, and degradation of apo B-100 (Arg3500->Trp) was lower than that of normal LDL, but higher than with apo B-100 (Arg3500->Gln), implying that the substitution of Trp3500 for Arg may cause less severe reduction of binding than the substitution of Gln. LDL from individuals heterozygous for apo B-100 (Glu3405->Gln) bound to LDL receptors at the same rate as normal LDL, but was taken up and degraded at significantly reduced rates, suggesting that domains of apo B-100 involved in binding and uptake do not completely overlap.

Conclusions: In Germany, apo B-100 (Arg3500->Gln) may be more frequent than previously assumed. Both apo B-100 (Arg3500->Trp) and apo B-100 (Glu3405->Gln) may contribute to the phenotype of ligand-defective LDL. These variants will be missed if screening is confined to apo B-100 (Arg3500->Gln) only.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nearly two-thirds of all circulating cholesterol is transported by LDL particles. The receptor-mediated catabolism of LDL is an important determinant of the concentration of LDL-cholesterol (LDL-C) in the plasma (1). Apolipoprotein (apo)1 B-100, the major protein constituent of LDL, mediates the binding of LDL to the LDL receptor (2)(3). The domain of apo B-100 that interacts with the LDL receptor has been defined using several approaches (4)(5)(6)(7). The proposed model of this binding region, comprising two clusters [A (3147–3157) and B (3359–3367)] of basic amino acids that are linked through a disulfide bond between residues 3167 and 3297 (4)(7), has been further expanded through the discovery of familial ligand-defective apo B-100 (FDB) (8)(9). This has led to the general view that residues 3130–3630 are important for the binding of apo B-100 to the LDL receptor (10). FDB is an autosomal dominantly inherited disorder in which the cellular uptake of LDL from the blood is diminished because of mutations within the apo B-100 receptor-binding domain. The biochemical and clinical characteristics of FDB are moderately to severely increased LDL-C, tendon xanthoma, arcus lipoides, and premature coronary artery disease (CAD).

To date, several point mutations of the putative receptor binding domain of apo B-100 have been identified (9)(11)(12)(13)(14)(15)(16). Only three of these mutations have been shown to produce binding-defective apo B-100 by appropriate genetic and functional investigations (9)(11)(12). The first substitution to be discovered, and apparently the most frequent one, is apo B-100 (Arg3500->Gln) (9). The other two substitutions, apo B-100 (Arg3500->Trp) (11) and apo B-100 (Arg3531->Cys) (12), occur less frequently. The Arg3531->Cys mutation has been identified in two families of different ethnic origin (12); in four CAD patients from the Great Salt Lake Basin area of the US, all of Caucasian origin (14); in two families in the United Kingdom (17); and two French individuals (18). Compared with apo B-100 (Arg3500->Gln), the mutation at codon 3531 is associated with a smaller increase in LDL-C (12)(17). Consistently, LDL that contained apo B-100 (Arg3531->Cys) exhibited less reduction of LDL receptor binding in vitro than did LDL endowed with apo B-100 (Arg3500->Gln) (12). To date, apo B-100 (Arg3500->Trp) has been described in just one family of European origin (11), twice in a mixed Chinese and Malayan hypercholesterolemic cohort (15), in one family of Asian descent living in the Glasgow region (11), and in another nine unrelated individuals from Taiwan (19). The receptor binding of apo B-100 (Arg3500->Trp) is considered similar to that of apo B-100 (Arg3500->Gln) (11).

The apo B-100 (Arg3500->Gln) mutation is observed with an approximate frequency of 1 in 500 to 1 in 700 in populations of European (Caucasian) origin (20)(21)(22). Although primary hypercholesterolemia is a common metabolic disorder in the middle European population, no additional frequent molecular reasons for binding-defective apo B-100 have yet been discovered. Hence, many investigators have relied on methods specifically tailored to detect the Arg3500->Gln substitution (21)(23)(24)(25)(26)(27)(28)(29)(30) to diagnose FDB. We examined a 2121-bp (codons 3131–3837) portion of the apo B gene, including the putative receptor binding region, using PCR and temperature gradient gel electrophoresis (TGGE) in 297 unrelated individuals with primary hypercholesterolemia to search for new genetic variants that affect the receptor binding of apo B-100. Among these, 21 carriers of apo B-100 (Arg3500->Gln) and 2 carriers of apo B-100 (Arg3500->Trp) were identified, which indicates a high prevalence of FDB in our area. Another three individuals revealed a silent substitution that changes the codon for Leu3350 from CTG to CTA, and four carriers of apo B-100 (Arg3405->Gln) were identified.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
subjects
Two hundred ninety-seven consecutive individuals with LDL-C concentrations >1.55 g/L and triglycerides <2.0 g/L referred to the Frankfurt University Hospital outpatient clinics for differential diagnosis of hyperlipoproteinemia (HLP) between 1992 and 1997 participated in the study. Individuals were included regardless of age, sex, or clinical history of cardiovascular or other diseases (142 males and 155 females; mean age, 41.4 ± 19.1 year; range, 4–84 years). The lipoprotein concentrations reported here were obtained on one single occasion, usually when the patients presented for the first time at the study site. Ten patients (3.4%) had hypothyreosis, and 17 patients (5.7%) had type 2 diabetes mellitus. Eleven patients (3.7%) presented with tendon xanthoma and/or arcus lipoides. Four males and one female below 40 years suffered from CAD. Above that age, 17 of 73 males (23%) and 16 of 84 females (19%) had CAD. HLP was not specifically treated in 227 patients (76.4%), neither with dietary recommendations nor with lipid-lowering drugs. Sixty-two patients received lipid-lowering drugs at the time of blood sampling; among these, 17 (5.7%) reported adherence to a lipid modified diet. In eight cases, records indicated that dietary advise was given by the primary care physician. All procedures were in accordance with the Helsinki Declaration of 1975, as revised in 1983. Informed consent was obtained from all study participants, and the study design was approved by the ethics review board at the Johann Wolfgang Goethe-University, Frankfurt.

blood samples
Blood was drawn into tubes containing potassium EDTA (final concentration, 3.8–5.0 mmol/L). The blood was centrifuged (1500g for 30 min at 10 °C). The supernatant plasma was used for the analysis of lipids and lipoproteins; the white blood cells were used to extract genomic DNA.

lipids, lipoproteins, and apolipoproteins
Cholesterol and triglycerides were measured enzymatically with reagents from Roche Diagnostics HDL-cholesterol (HDL-C) was measured after precipitation of apo B-containing lipoproteins (31). LDL-C was calculated according to Friedewald et al. (32), and apo B was measured by kinetic nephelometry, using the Protein Array System (Beckman Instruments). Lipoprotein(a) was measured using a commercial immunoradiometric assay (Mercodia Diagnostics). Between-day CVs were <=3.5% for total cholesterol, total triglycerides, and apo B; <=4% for LDL-C; and <=5% for HDL-C for the duration of the study.

dna preparation and oligonucleotides
Genomic DNA was prepared from white blood cells using "blood PCR" DNA isolation cartridges (Diagen), according to the manufacturer's instructions. Oligonucleotides were synthesized using the phosphoramidite method at MWG Biotech. The sequences of the oligonucleotides and their positions within the amplified region are given in Table 1 and Fig. 1 , respectively.


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  		Table 1. Positions and sequences of primers used for TGGE PCRs and RFLP typing.



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  		Figure 1. Map of the amplified regions within exon 26 of the apo B gene.

Lengths and nucleotide positions of PCR fragments are indicated. The relative positions of base changes detectable by our TGGE method are shown by vertical arrows.

pcr
apo B gene fragments were prepared by PCR in a final volume of 50 µL containing 0.1–0.2 µg of genomic DNA, 60 pmol of oligonucleotide primers, 62.5 mmol/L KCl, 12.5 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl2, 50 µmol/L each dNTP, and 1.5 U of Taq DNA polymerase (Amersham Pharmacia). Three different PCR cycle conditions were used with respect to different annealing temperatures of primers. Fragments A, B, C, D, G, and H were amplified with an initial denaturation step of 94 °C for 1 min, followed by annealing at 55 °C for 1 min and finally extension at 72 °C for 3 min. Fragment F was amplified with an annealing temperature of 58 °C for 1 min and extension at 72 °C for 3 min, and fragment E was amplifies with extension at 74 °C for 2 min. Each PCR was initiated by hot start at 95 °C for 3 min, and Taq polymerase was added at 80 °C before starting cycles.

tgge
Parallel TGGE was performed in a horizontal gel electrophoresis apparatus from Qiagen. PCR product (4 µL) was mixed with 4 µL of denaturing buffer [8 mol/L urea, 400 mmol/L 2-(N-morpholino)propanesulfonic acid, pH 8.0, 20 mmol/L EDTA, 0.1 g/L bromphenol blue, 0.1 g/L xylene xyanol FF]. Before loading, samples were denatured for 5 min at 95 °C and renatured for 15 min at 50 °C to allow the formation of heteroduplex molecules; 6µL of each sample was loaded on the gel. The gels contained 80 g/L acrylamide (at a ratio of acrylamide to N,N'-methylenebisacrylamide of 60:1), 8 mol/L urea, 20 mmol/L 2-(N-morpholino)propanesulfonic acid, pH 8.0, 1 mmol/L EDTA, and 20 g/L glycerol. The running time was 3 to 4.5 h (300 V), depending on the length of the amplified product. Silver staining was performed as described (33). The temperature gradient was 35 °C to 55 °C (or 50 °C, respectively) and was oriented parallel to the direction of migration.

dna sequencing
Homoduplex mutant bands were excised from the stained polyacrylamide gels, which had not been fixed in that case. Gel slices were incubated in diffusion buffer (0.5 mol/L ammonium acetate, 10 mmol/L magnesium acetate, 1 mmol/L EDTA, pH 8.0, 1 g/L sodium dodecyl sulfate) at 60 °C for 20 min, followed by 95 °C for 5 min, and DNA was extracted with a QIAEX II polyacrylamide gel extraction kit from Qiagen. The purified DNA (5 µL) was reamplified using Pfu DNA polymerase (Stratagene), buffers recommended by the manufacturer, and the previously applied PCR conditions. The reamplification products were electrophoresed in a 20 g/L agarose gel and cleaned with QIAEX II (Qiagen), according to the manufacturer's instructions. Sequencing was performed using the ThermoSequenase cycle sequencing kit (Amersham) with fluorescently labeled primers, according to the manufacturer's instructions. DNA was electrophoresed in an ALF DNA Sequencer (Pharmacia Biotech Europe).

apo B GENE HAPLOTYPING
Five apo B gene polymorphisms [signal peptide insertion (ins) or deletion (del), XbaI (ACT->ACC, Thr2488->Thr) restriction fragment length polymorphism (RFLP), MspI (CGG->CAG, Arg3611->Gln) RFLP, EcoRI (AAA->GAA, Lys4154->Glu) RFLP, and the 3' hypervariable region (3'VNTR)], were analyzed. The methods have been described previously (33)(34), except for the PCR conditions we used to determine the EcoRI and the MspI RFLPs. In these cases we used the primer sequences given in Table 1Up and conditions as follows: The reaction volumes were 20 µL and contained 10 pmol of each primer, 80 µmol/L each dNTP, 20–40 ng of genomic DNA, and 0.5 U of Taq DNA polymerase. Thirty-five cycles were performed (denaturation at 95 °C for 0.30 min, annealing at 60 °C for 1 min, and elongation at 72 °C for 0.15 min). The PCR products were incubated at 37 °C for 3 h with 10 U of the respective restriction enzyme and were subsequently analyzed by electrophoresis in 20 g/L agarose gels.

detection of point mutations by restriction typing
After the sequencing of those DNA fragments producing irregular patterns on TGGE, we confirmed the presence of the suspected alleles with PCR and subsequent restriction fragment analysis. The methods for Arg3500->Gln and Arg3500->Trp are described elsewhere (25)(35). The G->A transition (Leu3350->Leu) creates a PstI restriction site in fragment C (Table 1Up ). Glu3405->Gln was determined by digesting fragment D, amplified using the primer pair D3-D2. D3 contains a mismatch (AGtC) that introduces a TaqI site in the mutant allele.

binding, uptake, and degradation of 125i-labeled ldl
LDL (1.019 < d < 1.063 kg/L) was isolated using preparative ultracentrifugation (36) and iodinated using the iodine monochloride method(37). The human skin fibroblasts were from skin biopsies of normolipidemic individuals. Cells were grown in 24-well polystyrene plates, and before the experiments, the cells were preincubated for 40 h in medium containing 100 mL/L human lipoprotein-deficient serum to up-regulate LDL receptors. The binding, uptake, and degradation of 125I-labeled LDL were measured as described by Goldstein et al. (38) with slight modifications (39). To measure cell surface binding, lipoproteins were incubated with the cells for 1 h at 4 °C in DMEM containing 10 mmol/L HEPES. To measure uptake (surface binding plus internalization) and degradation, cells were incubated for 4 h at 37 °C with 125I-labeled LDL in DMEM containing 24 mmol/L bicarbonate, pH 7.4. The amount of 125I-labeled material associated with the cells (binding and internalization) was determined after lysis in 0.3 mmol/L NaOH. Proteolytic degradation was determined as 125I-labeled trichloroacetic acid-soluble (non-iodine) material in the conditioned medium.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In our survey, we wished to examine the potential importance of mutations of apo B-100 to the development of hypercholesterolemia. We established a TGGE method to screen for sequence variations within the region of the apo B gene that included the putative receptor-binding domain of the molecule. Oligonucleotide primers (Table 1Up ) were designed to amplify eight overlapping fragments (A through H) covering the cDNA sequence of amino acid residues 3131–3837 of the apo B gene (see Fig. 1Up ). PCR products were 222–451 bp in length. To increase the sensitivity of our assay, artificial high-melting domains were incorporated into amplification products B, C, D, G, and H by the use of GC-clamped oligonucleotide primers. For each fragment, the melting behavior was predicted using a previously described algorithm (40)(41) and software supplied by Qiagen. Fragment F (codons 3490–3638) included the two mutations affecting codon 3500. These mutations were predicted to shift the melting temperature by 0.83 °C (Arg3500->Gln) and 1.03 °C (Arg3500->Trp), respectively, suggesting that they could be distinguished merely by their TGGE pattern. A common MspI polymorphism that changes amino acid 3611 from Arg (CGG) to Gln (CAG) (42), and the Arg3531->Cys mutation at codon 3531 altered the calculated melting temperature of this fragment as well. Nonetheless, TGGE was applied in combination with heteroduplex analysis; i.e., each PCR-amplified product was de- and renatured to allow homo- and heteroduplex formation. Heteroduplex strands could be easily distinguished from homoduplex strands.

To evaluate the actual sensitivity of our assay, we sought documentation of its ability to detect known sequence variations. As expected, apo B-100 (Arg3500->Gln), apo B-100 (Arg3500->Trp), apoB-100 (Arg3531->Cys), and the MspI polymorphism at codon 3611 could be distinguished (Fig. 2 ). Samples heterozygous for a particular mutation produced a characteristic four-band pattern, the faster migrating pair of bands representing the homoduplexes of the mutant and wild-type strands, respectively, and the slower migrating pair of bands corresponding to heteroduplexes of the two alleles. A single homoduplex band halted at aberrant positions in the gel was observed with DNA from an individual homozygous for apo B-100 (Arg3500->Gln). Downstream and upstream sequencing of purified and reamplified mutant homoduplex bands confirmed the respective nucleotide substitutions.



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  		Figure 2. TGGE of PCR fragments C, D, and F of exon 26 of the apo B gene.

Electrophoresis was carried out in 80 g/L polyacrylamide gels, using a temperature gradient from 35 °C to 55 °C. Run times were 3 h (fragments C and D) and 4.5 h (fragment F) at 250–300 V. Fragment F: lane 1, subject without mutation; lane 2, apo B-100 (Arg3500->Gln) homozygous individual; lane 3, apo B-100 (Arg3500->Gln) heterozygous individual; lane 4, apo B-100 (Arg3500->Trp) heterozygous individual; lane 5, apo B-100 (Arg3531->Cys) heterozygous individual; lane 6, apo B-100 (Arg3611->Gln) heterozygous individual, MspI RFLP. Fragment D: lane 1, subject without mutation; lane 2, apo B-100 (Glu3405->Gln) heterozygous individual. Fragment C: lane 1, subject without mutation; lane 2, heterozygous transition at the codon for Leu3350 (CTG->CTA).

We studied samples from 297 unrelated individuals with LDL-C >1.55 g/L and triglycerides <2.0 g/L. In each of the samples exhibiting irregular melting behavior, the presence of the suspected mutation was confirmed through restriction fragment analysis and DNA sequencing where appropriate. Using this strategy, we identified 21 carriers of apo B-100 (Arg3500->Gln) and 2 carriers of apo B-100 (Arg3500->Trp). Four patients exhibited aberrant migration of fragment D (Fig. 2Up ); these subjects were heterozygous for apo B-100 (Glu3405->Gln). In three other unrelated individuals, we detected an abnormality of fragment C (Fig. 2Up ) attributable to a silent transition that changed the codon for leucine at position 3350 from CTG to CTA. There was no carrier of the Arg3531->Cys mutation detected in our sample. Two of the four mutations, apo B-100 (Arg3500->Gln) and apo B-100 (Arg3500->Trp), recently have been unequivocally linked to the development of hypercholesterolemia (11)(20). The frequency of heterozygous apo B-100 (Arg3500->Gln) carriers was 21 in 297 (7.1%) in the entire patient group and 7 in 128 (5.5%) when we considered only individuals 40–65 years of age. A recent cross-sectional survey performed at our institutions revealed the prevalence of type IIa HLP (as defined by LDL-C >1.55 g/L and triglycerides <2.0 g/L) to be at least 25% among clinically healthy individuals between the ages of 40 and 65 years (M.A. Nauck et al., unpublished results). If we assume that mutant forms of apo B-100 produce type IIa HLP only (which is a conservative estimate because in some instances patients with FDB exhibit type IIb HLP), the calculated prevalence of heterozygous ligand-defective apo B-100 would be at least 1.4% (1 in 71) among healthy individuals in the Rhein-Main area.

We were able to recruit 19 family members of 9 of the 21 unrelated apo B-100 (Arg3500->Gln) carriers. Among these subjects, 18 were heterozygous and 1 was homozygous for apo B-100 (Arg3500->Gln). Using five polymorphic markers of the apo B gene, we deduced partial haplotypes in all apo B-100 (Arg3500->Gln) subjects. Consistent with previous reports (43), they exhibited a consensus haplotype designated as ins, XbaI-, MspI+, EcoRI-, 3'VNTR-49 haplotype 194, according to the binary nomenclature of Ludwig and McCarthy (43). Haplotyping of carriers of the apo B-100 (Glu3405->Gln) mutation and of the silent transition at codon 3350 revealed the common genotypes del, XbaI+, MspI+, EcoRI+, 3'VNTR-37 and ins, XbaI-, MspI+, EcoRI+, 3'VNTR-35, respectively (Table 2 ). Because family members were not available for further analyses, we tentatively considered these haplotypes to be associated with the respective mutations. Family segregation analysis in the two families with apo B-100 (Arg3500->Trp) revealed two new haplotypes unequivocally associated with the mutant (ins, XbaI+, MspI+, EcoRI+, 3'VNTR-37 and del, XbaI+, MspI+, EcoRI+, 3'VNTR-37, respectively, see Table 3 and Fig. 3 ). These two haplotypes are distinct from those found previously in one Scottish family (XbaI+, MspI-, EcoRI+) and in Asian patients with apo B-100 (Arg3500->Trp) (XbaI-, MspI+, EcoRI+, 3'VNTR-35).


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  		Table 2. Lipids, lipoproteins, and apo B-100 haplotypes in subjects with apo B-100 (Glu3405->Gln) and apo B-100 (Leu3350->Leu).


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  		Table 3. Lipids, lipoproteins, and apo B haplotypes in the two apo B-100 (Arg3500->Trp) subjects and their relatives.



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  		Figure 3. Pedigrees of two families with apo B-100 (Arg3500->Trp).

Family members heterozygous for apo B-100 (Arg3500->Trp) are indicated by . LDL-C concentrations (g/L) are given below each symbol. Additional clinical information on each individual is provided in Table 3Up . Haplotypes were constructed by analysis of the following five apo B gene polymorphisms: signal peptide insertion (ins) or deletion (del), XbaI (ACT->ACC, Thr2488->Thr) RFLP, MspI (CGG->CAG, Arg3611->Gln) RFLP, EcoRI (AAA->GAA, Lys4154->Glu) RFLP, and the 3'VNTR region of the apo B gene. Construction of haplotypes was based on the assumption that there had been no recombination within the apo B gene. Dotted symbols represent family members who were not studied. +, presence of restriction site; -, absence of restriction site; circles, females; squares, males.

The lipid, lipoprotein, and apolipoprotein concentrations in those subjects carrying different apo B mutations are shown in Tables 2 through 4Up Up . The screening strategy produced increased cholesterol, LDL-C, and apo B concentrations in all patient groups. Three of the 21 apo B-100 (Arg3500->Gln) carriers suffered from coronary heart disease, five were on lipid-lowering drugs, and only the apo B-100 (Arg3500->Gln) homozygote revealed small xanthelasmas of the upper eyelids and a mild arcus lipoides corneae. apo B (Glu3405->Gln) was detected in three subjects with mild hypercholesterolemia (LDL-C, 1.56–1.69 g/L) and one subject with a LDL-C concentration of 2.43 g/L who were not being treated with lipid-lowering drugs. One of the apo B (Glu3405->Gln) carriers, a 52-year-old male with a LDL-C concentration of 1.57 g/L, had CAD. We studied the interaction of LDL from this patient with cultured fibroblasts. Confirming a recent report (44), the binding of LDL from the apo B-100 (Glu3405->Gln) carrier was identical to LDL from normolipidemic donors. Unexpectedly, this was not matched by cellular uptake nor lysosomal degradation. Instead, both uptake and degradation of the mutant LDL were decreased to 85% and 87.5%, respectively, with the differences reaching statistical significance (P <0.05) at the highest concentration of radiolabeled 125I-LDL assayed (Fig. 4 ).


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  		Table 4. Lipid and lipoprotein concentrations (means ± SD, g/L) of the hyperlipidemic individuals heterozygous for mutations in the apo B-100 receptor-binding domain.



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  		Figure 4. Interaction of LDL from a heterozygous carrier of apo B-100 (Glu3405->Gln) with cultured human skin fibroblasts.

LDL samples were prepared by ultracentrifugation and labeled with 125I as described in Materials and Methods. Healthy human skin fibroblasts were grown in DMEM with 100 mL/L fetal calf serum. Forty hours before the experiment, the cells were switched to medium containing 100 mL/L human lipoprotein-deficient serum. Cells then received 125I-labeled LDL from a 52-year-old heterozygous carrier with apo B-100 (Glu3405->Gln) ({blacksquare}) and from a pool of normolipidemic donors (•). Binding, uptake, and degradation were determined as described in Materials and Methods. Each data point represents the mean of two experiments, each performed in triplicate. *, P <0.05 vs control. The error bars correspond to standard deviations.

We were also interested in determining the phenotypic consequences of the apo B-100 (Arg3500->Trp) mutation. The two index patients heterozygous for apo B-100 (Arg3500->Trp) had no clinical signs of arterial occlusive disease nor any symptoms frequently associated with FDB. Two members of the GR-098 family carried the mutant allele. GR-098/I-2, the index patient, had a LDL-C concentration of 1.61 g/L while being treated with 20 mg of pravastatin daily. If we assume that this treatment produces a 25% reduction of LDL-C, his pretreatment LDL-C should have been 2.15 g/L, which is close to the concentration seen in GR-098/II-3 (2.33 g/L), his 13-year-old daughter who carried the mutation as well. Although still slightly increased, the LDL-C concentration was <2.0 g/L in both unaffected family members (GR-098/I-1 and GR-098/II-4). A history of HLP on both maternal sides of the immediately preceding generation was quoted in the GR-098 family, but no samples from the mother of the index patient or from the mother of his wife (GR-098/I-1) were available for analysis. This positive family history for HLP may, however, explain why GR-098/I-1 and GR-098/II-4 had increased LDL-C in the absence of apo B-100 (Arg3500->Trp).

The other index patient (L-1193/II-7) was a 41-year-old male who had been first diagnosed with HLP type IIa in 1987. In June 1996, his LDL-C was 2.84 g/L. Since then, he had taken a hydroxymethylglutaryl-CoA reductase inhibitor (10 mg of simvastatin once daily). In response to treatment, he experienced an ~40% reduction of plasma LDL-C. In the L-1193 kindred, assessment of the phenotypic consequences of the apo B (Arg3500->Trp) mutation was complicated because only one unaffected individual was available and because there was some interindividual variation of LDL-C among the mutation carriers. The 66-year-old mother (L-1193/I-5) of the index patient had been treated with micronized fenofibrate (200 mg once daily) for the last 10 years, which might explain why her LDL-C concentration was only 1.73 g/L. Her father died of myocardial infarction at the age of 62. In 1995, she had been diagnosed as having diabetes mellitus, which was under treatment as well. The two affected sons of L-1193/I-5, 13-year-old dizygous twins, had low LDL-C, which was, however, well above the 90th percentile for that age. Furthermore, it is in line with the assumption that apo B (Arg3500->Trp) causes hypercholesterolemia that the LDL-C concentration in the only unaffected adult member (L-1193/I-6) was lower than in the two other adult carriers of the mutation.

We analyzed the interaction of LDL from two unrelated individuals heterozygous for apo B-100 (Arg3500->Trp) with LDL receptors compared with the interaction of LDL pooled from normolipidemic individuals and LDL from an apo B-100 (Arg3500->Gln) heterozygous individual. At each of the concentrations studied, the binding, internalization, and degradation of LDL from the apo B-100 (Arg3500->Trp) heterozygotes were substantially lower than normal (on average, 58% of the values obtained for normal LDL) but consistently higher compared with LDL from an apo B-100 (Arg3500->Gln) heterozygote (44% of the values obtained for normal LDL); the differences between apo B-100 (Arg3500->Trp) LDL and normal as well as apo B-100 (Arg3500->Gln) LDL were statistically significant at all concentrations except the three lowest ligand concentrations in the assay for binding at 4 °C (Fig. 5 ). These observations were reproducible in three experiments, all of which were conducted independently, including labeling. However, we wished to rule out that the differences were caused by different degrees of denaturation of the LDL during the iodination procedure. We therefore studied the interaction of mutant and normal LDL with fibroblasts in culture, utilizing them as unlabeled competitors for iodinated normal LDL. Again, we performed three entirely independent experiments, all of which completely reproduced the differences seen in the direct binding, uptake, and degradation studies (Fig. 6 ).



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  		Figure 5. Interaction of LDL from heterozygous carriers of apo B-100 (Arg3500->Gln), heterozygous carriers of apo B-100 (Arg3500->Trp), and normal LDL with cultured human skin fibroblasts.

LDL samples were prepared by ultracentrifugation and labeled with 125I as described in Materials and Methods. Normal human skin fibroblasts were grown in DMEM with 100 mL/L fetal calf serum. Forty hours before the experiment, the cells were switched to medium containing 100 mL/L human lipoprotein-deficient serum. Cells then received 125I-labeled LDL from two apo B-100 (Arg3500->Trp) heterozygotes, GR-098/I-2 ({blacksquare}) and L-1193/I-5 ({blacktriangleup}); from an apo B-100 (Arg3500->Gln) heterozygote (•); and from a pool of normolipidemic donors ({circ}). Binding, uptake, and degradation were determined as described in Materials and Methods. Each data point represents the mean of three experiments, each performed in triplicate. *, P <0.05 vs control.



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  		Figure 6. Competition of LDL from heterozygous carriers of apo B-100 (Arg3500->Gln) and apo B-100 (Arg3500->Trp) compared with normal LDL for binding, uptake, and degradation of 125I-labeled LDL.

LDL samples were prepared by ultracentrifugation and labeled with 125I as described in Materials and Methods. Normal human skin fibroblasts were grown in DMEM with 100 mL/L fetal calf serum. Forty hours before the experiment, the cells were switched to medium containing 100 mL/L human lipoprotein-deficient serum. Cells then received 125I-labeled LDL at a protein concentration of 5 mg/L and unlabeled LDL from two apo B-100 (Arg3500->Trp) heterozygotes, GR-098/I-2 ({blacksquare}) and L-1193/I-5 ({blacktriangleup}); from an apo B-100 (Arg3500->Gln) heterozygote (•); and from a pool of normolipidemic donors ({circ}) at concentrations indicated on the y-axis. Binding, uptake, and degradation were determined as described in Materials and Methods. Each data point represents the mean of three experiments, each performed in triplicate. *, P <0.05 vs control.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Defective binding of LDL to the LDL receptor is a major cause of hypercholesterolemia. Alterations of the lipid composition of the LDL core aside, the three-dimensional structure of the receptor-binding domain of apo B-100 is subject to modification resulting from genetically determined changes in the primary structure of apo B-100. The best understood genetic abnormality of that kind is FDB attributable to a substitution of Arg by Gln at position 3500 of apo B-100. In Germany, apo B-100 (Arg3500->Gln) previously had been estimated to occur at a frequency of 1 in 700 (22). Individuals who have inherited one mutant allele develop moderate hypercholesterolemia; the few homozygous carriers of the mutation described to date have LDL-C concentrations similar to those observed in heterozygous LDL-receptor deficiency (39)(45)(46).

For many years, the replacement of Arg by Gln at position 3500 was the only mutation known to produce ligand-defective apo B-100. Reports of other apo B-100 variants (Arg3531->Cys and Arg3500->Trp) unequivocally linked to hypercholesterolemia have appeared only twice to date (11)(12), whereas several detailed investigations of the apo B-100 gene sequence that codes for its receptor-binding function failed to detect any relevant mutation beyond apo B-100 (Arg3500->Gln) (10)(13)(47)(48)(49).

Nevertheless, when commencing this study, we hypothesized that other binding-defective species of apo B-100 functionally comparable to these mutations existed in the human population. To identify such mutations, we designed a TGGE-based procedure that enabled us to search for sequence variations in the region coding for the putative receptor-binding domain of apo B-100. We chose TGGE because it affords a high sensitivity (>95%) for point mutations if experimental conditions are optimized. As demonstrated in Fig. 2Up , within a 445-bp segment, four different mutations could be detected simultaneously. Even two transitions at codon 3500, apo B-100 (Arg3500->Gln) and apo B-100 (Arg3500->Trp), were distinguishable by virtue of the 0.2 °C difference in their melting temperatures. We are convinced that TGGE represents a valid and convenient alternative to direct sequencing (10)(47), single-strand conformation polymorphism (SSCP) (13)(48), and denaturing gradient gel electrophoresis (15)(47)(49), which have been used to investigate the sequence encoding the putative receptor binding of apo B-100. Compared with SSCP, TGGE has the advantage that substantially larger DNA fragments can be analyzed without diminishing sensitivity. Showing that SSCP failed to identify two of nine samples heterozygous for apo B-100 (Arg3500->Gln), Henderson et al. (50) questioned the use of SSCP as a reliable method of detecting unknown apo B-100 point mutations. Compared with denaturing gradient gel electrophoresis, TGGE offers more technical simplicity (homogeneous gels instead of gradient gels) and shorter run times. Another easy and high-throughput approach to test simultaneously for the three FDB mutations has been heteroduplex analysis (51). This technique does not distinguish between the various types of mutations and does not allow identification of homozygosity for mutant and wild-type DNA.

We detected two sequence abnormalities with uncertain pathobiochemical relevance. The first one was a silent mutation in the third position of codon 3350, which was also detected recently by Ludwig et al. (14) and Gaffney et al. (16). The second abnormality was a known variation (52) that leads to the incorporation of the negatively charged amino acid glutamic acid in the place of glutamine at codon 3405. In our study, heterozygous carriers of this variant occurred at a relative frequency of 1.3%. This percentage is strikingly similar to the prevalence of 1.4% observed by Gaffney et al. (16) in a sample of 928 hyperlipidemic individuals. Haplotyping of that particular locus revealed the same, previously assigned haplotype (del, XbaI+, MspI+, EcoRI+, 3'VNTR-37) in all carriers (four subjects), suggesting a common ancestral origin of the Glu3405->Gln substitution. Studies conducted with LDL from a heterozygous carrier of this mutation suggested that the binding of the mutant LDL to LDL receptors was the same as for normal LDL. Unlike the binding, however, the receptor-mediated internalization and the degradation of the mutant LDL were lower compared with pooled LDL from healthy donors, the differences attaining statistical significance at the highest ligand concentration. The implications of this finding are difficult to determine at present. Pullinger et al. (44) and Ludwig et al. (14) communicated that the binding of apo B-100 (Glu3405->Gln) to the LDL receptor was the same as that of normal LDL. In contrast, Gaffney et al. (16) claimed that the ability to promote the growth of U937 cells of LDL from nine heterozygotes for apo B-100 (Glu3405->Gln) with normal triglycerides was, on average, 87% of normal. Our findings are consistent with both unimpaired binding on the one hand and impaired cellular uptake on the other hand. A speculative explanation for that divergence of binding and uptake might be that residue 3405 is not involved in the binding of apo B-100 to the LDL receptor but plays a role in mediating the conformational change of the receptor that precedes invagination, also suggesting that those domains of the LDL receptor mediating binding do not completely overlap with those triggering uptake of the complex of the receptor and the ligand.

Among the hypercholesterolemic subjects studied, 21 individuals were found to be heterozygous for apo B-100 (Arg3500->Gln), corresponding to a frequency of apo B-100 (Arg3500->Gln) of 7.1%, or 1 in 14 in that selected group. If individuals below the age of 40 and above the age of 65 were disregarded, the relative frequency of the mutation was 5.5%. If we assume a prevalence of at least 25% for type IIa HLP (defined as LDL-C >1.55 g/L and triglycerides <2.00 g/L) in the general population 40 to 65 years of age, this suggests a frequency of apo B-100 (Arg3500->Gln) of 1.4% in the Rhein-Main area. It is not likely that this estimate is biased by the inclusion of individuals with CAD, type 2 diabetes, or hypothyroidism in our survey. The prevalence of CAD in the screened population 40–65 years of age was approximately twofold higher than in the general population. This is, however, not unexpected because we selected for increased LDL-C. The prevalence of both hypothyreosis and type 2 diabetes were within the ranges observed in Western societies, indicating that the subjects that we screened were representative for the general population with regard to these characteristics. Our frequency estimate may be conservative for two reasons. First, LDL-C concentrations exceeding 1.55 g/L may actually be more frequent in middle-aged individuals than assumed here. For examples, >40% of healthy males and females 45 or older had LDL-C concentrations >1.55 g/L (155 mg/dL) in the Münster Heart Study (PROCAM), the largest prospective study of coronary heart disease risk factors in Germany (53). Second, we supposed that apo B-100 (Arg3500->Gln) is not present in individuals with other types of HLP, an assumption that may not apply in general because we sporadically observe carriers of apo B-100 (Arg3500->Gln) with increased triglycerides. Thus, apo B-100 (Arg3500->Gln) appears to be more prevalent in the Rhein-Main area than in Switzerland, where it has been estimated to occur at a frequency of 1 in 209 in the general population (54), the highest prevalence reported in the literature to date. The high frequency of FDB in this area may explain the first discovery of a patient homozygous for apo B-100 (Arg3500->Gln) in Frankfurt in 1992 (39), when FDB genotyping became established in the routine analysis of HLP in our laboratory.

We identified two families that contained six carriers of apo B-100 (Arg3500->Trp). The LDL-C concentrations presented in Table 3Up are compatible with the assumption that apo B-100 (Arg3500->Trp) causes hypercholesterolemia. However, definite assessment of the phenotypic consequences of this mutation was not possible for several reasons: First, two of the six affected individuals were on lipid-lowering drugs. Second, there was only one unaffected individual in the L-1193 kindred. Third, another genetic factor for increased LDL-C may have been present in the GR-098 kindred. apo B-100 (Arg3500->Trp) was initially described in one Scottish and one Asian family (11), and then became evident only in Asian HLP patients (15)(19). The screening of large samples of HLP patients in North America (n >800) (14), England (n = 562) (35), and Scotland (n = 412) (17) did not reveal any additional carriers of that variant. Therefore, we were surprised to detect two unrelated apo B-100 (Arg3500->Trp) heterozygotes, both of Caucasian descent, in our sample. Even more surprising was the finding that the mutation was associated with two apo B haplotypes different from the ones reported earlier in the Scottish family and the common haplotype shared by Asian HLP patients carrying apo B-100 (Arg3500->Trp). The apo B-100 (Arg3500->Trp) substitutions might, therefore, have arisen independently in our area. The binding, uptake, and degradation of apo B-100 (Arg3500->Trp) LDL was higher compared with apo B-100 (Arg3500->Gln) LDL. This stands in contrast to the first report of the apo B-100 (Arg3500->Trp) mutation (11), in which the two mutations affecting codon 3500 were indistinguishable in an assay that relied on the ability of LDL to promote the growth of U937 cells. The reason for this disagreement may lie in the techniques used to study LDL function. An advantage of the U937 cell proliferation assay over the classic binding and uptake method proposed by Goldstein et al. (38) is that it does not involve manipulation of the LDL particles during the iodination procedure. However, we obtained essentially identical results when we used the two mutant LDLs as unlabeled competitors for labeled wild-type LDL, essentially ruling out that the observed differences were related to modifications of LDL during labeling. In normal apo B-100, Arg3500 interacts with Trp4369. Mutation of Arg3500 causes the carboxy terminus of apo B-100 to interfere with the binding of the positively charged cluster of residues at positions 3358–3370 to the LDL receptor (55). The functional difference between apo B-100 (Arg3500->Trp) and apo B-100 (Arg3500->Gln) may thus indicate that Trp3500 still weakly interacts with Trp4369 through aromatic–aromatic interaction (bond strength of 1–2 kcal/mol), whereas this interaction is entirely disrupted in the presence of Gln at position 3500.

In conclusion, our observations confirm that apo B-100 (Arg3500->Gln) is the most prevalent cause of ligand-defective LDL. However, they also demonstrate that FDB may be more heterogeneous than previously assumed. This has considerable bearing on the diagnosis of ligand-defective LDL at the molecular level. Detection methods designed solely for apo B-100 (Arg3500->Gln) have been applied in many laboratories to date, whereas the possibility that other variants exist has largely been disregarded. We therefore strongly recommend the use of screening strategies, such as TGGE, capable of detecting all known functionally relevant sequence variations of the apo B-100 receptor-binding domain.


   Acknowledgments
 
A portion of this study was supported by a grant from the Center of Clinical Research II (cardiovascular diseases) at the Albert Ludwigs-University, Freiburg to W.M. Dr. Evelyn S.C. Koay from the National University Hospital in Singapore generously supplied a DNA sample with the apo B-100 (Arg3500->Trp) mutation. The DNA sample with the apo B-100 (Arg3531->Cys) mutation was a gift from Clive R. Pullinger, Cardiovascular Research Institute, University of California, San Francisco, CA. We thank Angela Eser for excellent laboratory assistance.


   Footnotes
 
1 Nonstandard abbreviations: apo B, apolipoprotein B; LDL-C, LDL-cholesterol; FDB, familial defective apo B-100; CAD, coronary artery disease; TGGE, temperature gradient gel electrophoresis; HLP, hyperlipoproteinemia; HDL-C, HDL-cholesterol; RFLP, restriction fragment length polymorphism; and SSCP, single-strand conformation polymorphism.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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