Original Article

The apolipoprotein B R3531C mutation: characteristics of 24 subjects from 9 kindreds

	ABSTRACT

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Familial ligand-defective apolipoprotein B (apoB) is a group of disorders caused by mutations in the apoB gene. In this report the R3531C mutation is characterized further using a monoclonal antibody MB19/dynamic laser light scattering technique to measure ratios of Cys³⁵³¹ to normal low density lipoprotein (LDL) particles. All six subjects studied showed a preferential accumulation of particles carrying the defective apoB allotype. We determined binding properties of LDL from R3531C heterozygotes by measurement of high-affinity binding to LDL receptors on fibroblasts and its ability promote growth of U937 cells. LDL from R3531C heterozygotes, compared to normal LDL, had 49.3% of the binding affinity and was 74% as effective in a U937 cell proliferation assay. To identify new probands, we screened 2570 subjects for the R3531C mutation. Nine probands were found with 15 affected relatives. Of the seven haplotypes we uncovered, two were novel, while five were identical to one initially reported as associated with Cys³⁵³¹. Three silent mutations were detected also: T3540T, N3542N and T3552T. Analysis of lipid profiles of R3531C families showed, as with the R3500Q mutation, variable expression of the phenotype, modulated by environmental and other genetic factors. Both mutations tend to produce lower plasma levels of LDL in affected subjects than do defects of the LDL receptor (familial hypercholesterolemia, FH).

This study shows that the Cys³⁵³¹ LDL particles are not only defective at binding to the LDL receptor, as determined by two separate methods, but that in all cases they accumulate preferentially compared to the normal allotype.—Pullinger, C. R., D. Gaffney, M. M. Gutierrez, M. J. Malloy, V. N. Schumaker, C. J. Packard, and J. P. Kane. Apolipoprotein B R3531C mutation: characteristics of 24 subjects from 9 kindreds. J. Lipid Res. 1999. 40: 318–327.

Supplementary key words: apolipoprotein B, atherosclerosis, hypercholesterolemia, haplotypes, genetic mutation

	INTRODUCTION

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Familial ligand-defective apolipoprotein B (apoB) is a group of autosomal-dominant inherited disorders caused by mutations of the low density lipoprotein (LDL) receptor ligand domain in apoB (1). A somewhat less severe form of hypercholesterolemia is associated with these disorders than seen with mutations of the LDL receptor in familial hypercholesterolemia (FH), as would be expected, a priori, because the affinity of very low density lipoprotein (VLDL) remnants for the LDL receptor is not affected as clearance of these remnants is mediated by apoE (1). Three mutations of apoB have been reported that cause reductions in binding affinity for the LDL receptor. These are the R3500Q (2), R3531C (3) (4), and R3500W (5) mutations.

Because each LDL particle carries a single molecule of apoB-100, it would be expected, a priori, that such defects would lead to the selective accumulation in plasma of those particles bearing the defective apoB allotype. We have identified nine additional R3531C probands and have in a number of cases measured the mass ratios of the defective Cys³⁵³¹ LDL to normal Arg³⁵³¹ LDL particles using a monoclonal antibody MB19/dynamic laser light scattering technique. This approach determines specifically whether an apoB mutation results in a change in lipoprotein metabolism. It is based on the pioneering work of Young and his co-workers (6). They showed that the monoclonal antibody MB19 against human apoB could be used to detect a polymorphism of apoB with LDL designated MB19₁/MB19₁ binding with high affinity, and MB19₂/MB19₂ with low affinity. LDL from heterozygotes had an intermediate binding affinity. This polymorphism was shown to be an ApaLI restriction fragment length polymorphism (RFLP) in codon 71, a Thr'Ile substitution (7), and represents the Ag(c/g) locus on apoB (8), with Ile⁷¹ being the high binding and Thr⁷¹ the low binding allotype. The polymorphism itself has no influence on plasma levels of apoB or lipoproteins (9). A solid-phase MB19 radioimmunoassay assay (6) was used with patients in kindreds with familial hypobetalipoproteinemia to show different mass ratios of the two apoB allotypes in plasma (10) (11). It has also been used with R3500Q subjects, who are also Thr⁷¹/Ile⁷¹ heterozygotes, to determine the percentage of defective LDL in plasma (12) (13). A refinement of the method using dynamic scattering of laser light from LDL-MB19 complexes (14) has been used in the present study to generate more precise estimates of allelic ratios. A maximum of two LDL particles can be bound by the bivalent antibody. MB19 will promote substantial dimer formation with Ile⁷¹ apoB LDL, but little with Thr⁷¹ apoB LDL. When compared to a calibration curve, constructed from mixtures of Ile⁷¹/Ile⁷¹ and Thr⁷¹/Thr⁷¹ apoB LDL, the amount of dimer formed with LDL isolated from heterozygous Thr⁷¹/ Ile⁷¹ individuals yields an estimate of the ratio of the two apoB allotypes.

The ability of LDL prepared from R3531C subjects to promote growth of a cell line with a growth requirement for LDL was compared with that of normal LDL. In individual cases, the effect of the mutation on the binding affinity for the LDL receptor was measured using both conventional displacement and dual-label fibroblast assays. We have also assessed the inter-individual variation in the expression of this mutation in terms of its effects on the levels of blood lipids and lipoproteins in seven kindreds.

The serendipitous discovery of three novel apoB gene mutations as a result of these studies is reported.

	METHODS

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Preparation of genomic DNA
Genomic DNA was routinely prepared from whole blood obtained from patients in the lipid clinics of University of California, San Francisco (UCSF) and the Glasgow Royal Infirmary as previously described (4) (5). DNA was also prepared from volunteers who took part in the Determinants of Coronary Disease in Women study at UCSF.

Identification of R3531C probands
To detect the apoB R3531C mutation, two approaches were used. First, DNA prepared from 1372 patients and unselected volunteers attending the Lipid Clinic of the University of California, San Francisco, was screened using a previously described method (4). The PCR product spanning nucleotides 9561 to 11142 was digested with NsiI prior to agarose electrophoresis. The R3531C mutation is detected as the gain of an NsiI site due to the change at nucleotide 10800.

Second, DNA prepared from 1171 patients attending the Lipid Clinic of the Glasgow Royal Infirmary was subjected to amplification by polymerase chain reaction (PCR) followed by analysis by denaturing gradient gel electrophoresis (DGGE). Primers were chosen for PCR using the Melt 87 programs (written by Dr. Leonard Lerman, 1988, copyright with Massachusetts Institute of Technology, Cambridge, MA). A region of apoB spanning the 3531 codon was amplified using buffer, primer, and enzyme concentrations as recommended by the manufacturer (MBI Fermentas, Tyne and Wear, UK). The primers used ( Table 1) flank 207 base pairs of sequence from codon 3512 to codon 3581 (Table 1, fragment I). Thirty five cycles of PCR were performed on a Hybaid Touchdown thermal cycler (Hybaid, Teddington, Middlesex, UK), consisting of denaturation (95°C for 5 min initially and 1 min thereafter), annealing (66°C for 1 min), and extension (72°C for 1 min). After the final extension step the products were heated to 98°C for 12 min and cooled slowly. The products of the PCR amplification were subjected to DGGE on an 8% polyacrylamide gel at 60°C for 16 h at 55 volts. The gradient was 35% to 60% denaturant as defined by Myers, Maniatis, and Lerman (15). Other conditions were as previously described (5). To confirm the pattern observed on DGGE with each R3531C patient (J.K., H.M., J.C., and J.R.) the PCR product was synthesized as for DGGE, but with rapid cooling to encourage homoduplexes. After digestion with NsiI the samples were run on a nondenaturing 8% polyacrylamide gel. The absence of an NsiI site resulted in an uncut band of 286 bp, (normal allele and normal controls) while its presence (R3531C mutation) resulted in the two additional bands on the gel of 206 and 80 bp.

Other DGGE analysis
As a result of routine screening of LDL prepared from patients attending the UCSF lipid clinic, a number of patients were identified whose LDL had a binding affinity for the LDL receptor that was below the normal range. A fibroblast dual-label equilibrium binding assay was used for this purpose as described previously (4) (16). DNA prepared from 27 of these patients and from individuals with known apoB variants (Q3405E, R3480P, R3500Q, R3500W, and R3531C) was subjected to three separate PCR amplifications yielding overlapping fragments II, III, and IV (Table 1). These fragments were digested with the appropriate restriction enzyme (Table 1) and subjected to DGGE analysis on 7.5% acrylamide (2.7% BIS) gels containing either a 20–50% (fragments II and III) or a 30–50% (fragment IV) denaturing gradient (15). The gels were stained with SYBR Green DNA stain (17) and photographed using 254 nm epi-illumination.

Cloning and sequencing strategies
Fragment I PCR products from subjects that exhibited novel DGGE banding patterns were cloned using the T vector system (Northumbria Biologicals Ltd., Cramlington, UK) and sequenced using the Sequenase Version 2.0 kit (Amersham Life Science, Buckinghamshire, UK) as described previously (5). By using sequencing primers in both orientations from the plasmid vector, and using the sense PCR primer (Table 1) the entire amplified segment was sequenced. In the case of fragment IV, a novel banding pattern was investigated by sequencing using asymmetric PCR as previously described (18) with the primers listed in Table 1 and a Thermo Sequenase cycle sequencing kit (Amersham Life Science Ltd., Arlington Heights, IL). DNA from the four patients identified by PCR and NsiI digestion as having the R3531C mutation (A.M., S.L., L.T., A.P., and J.A.) was sequenced as previously described (4) to confirm the presence of this mutation.

Percentage mass ratio of defective to normal LDL
LDL (1.021–1.055 g/ml) were prepared from 6 individuals heterozygous for the R3531C mutation and 5 control subjects who had all been determined as heterozygous for the Thr⁷¹/Ile⁷¹ polymorphism, detected as an ApaLI RFLP as previously described (4) (7). In all six cases the mutant Cys³⁵³¹ allele is associated with the Thr⁷¹ allele (presence of an ApaLI site at nucleotide 416) as determined by haplotype analysis (see below). The mass ratio of the two apoB allelic products was measured by dynamic laser light scattering (4) (14). Briefly, the diameters of LDL and LDL–antibody complexes were measured using a NICOMP Model 270 particle sizer (Particle Sizing Systems, Santa Barbara, CA) equipped with an argon ion laser (514.5 nm wavelength). Monoclonal antibodies MB19 and MB47 have been described previously (6) (19) (20). MB47 (the binding of which is not influenced by the Ile⁷¹/Thr⁷¹ polymorphism) was used as a control to take account of other factors causing variations in binding (4) (14). LDLs from apoB (Ile⁷¹/Ile⁷¹) and apoB (Thr⁷¹/Thr⁷¹) homozygotes were mixed at known ratios to construct a calibration curve. With these LDL mixtures, at concentrations of 15 nM, the mean diameters were measured in the absence of antibody, with an equivalent amount of MB19 and with MB47. The relative percentage diameter increase was plotted against the percentage of strongly binding apoB (Ile⁷¹) and fitted using a second-order polynomial. For the LDL from apoB (Ile⁷¹/Thr⁷¹) heterozygotes, the percentage Ile⁷¹ apoB LDL was calculated as the mean of at least five separate measurements performed on each sample.

Fibroblast LDL receptor binding assay
Competitive dual-label (¹²⁵I/¹³¹I) equilibrium binding assays were carried out on radiolabeled LDL, isolated in the density range 1.021–1.055 g/ml, as previously described (4) (16). By way of comparison to R3531C patients and normal subjects, binding affinities were measured on LDL prepared from three patients with the R3500Q mutation. These were identified by differential oligonucleotide hybridization (4). A competitive displacement binding assay was also performed using ¹²⁵I-labeled LDL as described by Goldstein, Basu, and Brown (21).

LDL-induced U937 cell growth assay
The binding properties of LDL from R3531C subjects were tested using the LDL-induced growth assay as previously described (5). For this purpose LDL (1.021–1.055 g/ml) were isolated from 17 subjects heterozygous for the R3531C mutation and from 11 normal individuals.

ApoB gene haplotype analysis
The genotypes of eight apoB gene markers were determined as previously described (4) (5). The 3' VNTR was assessed, in terms of numbers of repeats, according to Ludwig and co-workers (22).

	RESULTS

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NsiI and DGGE screening
Using PCR plus digestion with NsiI followed by agarose gel electrophoresis or PCR with DGGE ( Figure 1) we screened a total of 2570 individuals for the presence of the apoB R3531C mutation. All mutations were confirmed, either by sequencing or by restriction digestion with NsiI. The result of these studies was the identification of nine new R3531C heterozygous probands. Five were patients at lipid clinics and four were unselected volunteers. One of the volunteers (A.P. II-1) had a family history of heart disease.

View larger version (69K): [in this window] [in a new window]   Figure 1. DGGE analysis of part of exon 26 of the apoB gene coding for sequences that affect binding to the LDL receptor. The four fragments examined (I to IV) are as described in Table 1. A: Detection of the R3531C mutation by analysis of codons 3512 to 3581(fragment I). B: Detection of novel conserved mutations, N3542N and T3540T in fragment I. C: Analysis of fragment III during screening of patients with LDL of low binding affinity revealed only the presence of the Q3405E polymorphism. D: Analysis of fragment IV could detect several mutations including the novel, conserved, T3552T.

Three silent mutations were discovered by DGGE analysis. These were a T for G transition at nucleotide 10829 (T3540T), a T for C transition at nucleotide 10835 (N3542N), and a T for C transition at nucleotide 10865 (T3552T).

Laser light scattering
In the six heterozygous R3531C individuals who were also heterozygous for the apoB MB19 polymorphism (Ile⁷¹/Thr⁷¹) 42.6% of LDL was bound strongly to the antibody, i.e., carried the Ile⁷¹ site ( Table 2). The mean percentage mass ratio of defective Cys³⁵³¹ (Thr⁷¹ apoB) to normal LDL (Ile⁷¹ apoB) particles was 57:43; a result significantly different (P = 0.001) from the ratio of 50:50 observed for the LDL from five normal MB19 heterozygotes. This showed that the defective binding affinity for the LDL receptor of the mutant apoB Cys³⁵³¹ LDL particles leads to their accumulation in the plasma and that the R3531C mutation has an effect on lipoprotein metabolism in affected individuals. These results are similar to those obtained previously on three other individuals with this genotype, when the mean ratio was found to be 59:41 (4).

Receptor-binding affinity of LDL
As measured using a dual-label assay, the affinities of LDL prepared from four heterozygous R3531C subjects was in each case considerably lower than the reference LDL ( Table 3). The mean value of 49.3% is somewhat lower than we observed in our previous study where LDL was prepared from eight individuals and the mean affinity was 60.3% (4). The mean affinity of LDL from the three R3500Q individuals was 37.2%, similar to the value of 34.3% that we found previously (4) and also to that reported by others (13) (23). In a competitive binding assay ( Figure 2) LDL from two individuals heterozygous for the apoB R3531C mutation and from two control subjects were compared. In agreement with the dual-label assay results, LDL from the Cys³⁵³¹ subjects (IC₅₀ 4.3 and 3.2 µg/ml) was a less effective competitor than normal LDL (IC₅₀ 2.3 and 1.6 µg/ml).

View larger version (22K): [in this window] [in a new window]   Figure 2. Comparison of the ability of LDL from two individuals heterozygous for the apoB R3531C mutation (LT II-1 and J.C. II-3) and two control subjects and ) to compete with 125I-labeled normal LDL for high-affinity binding to human skin fibroblasts at 4°C.

T3552T was the only mutation found on DGGE screening of the 3127–3584 region in a group of patients whose LDL had defective binding affinities as measured using the dual-label fibroblast assay. The mean binding affinity of the LDL prepared from these 27 individuals was 62.8 ± 1.2% (mean ± SE) of that of the reference LDL. Thus, mutations in this region of apoB do not seem to account for the defective binding seen in these assays. It is possible that some mutations will not have been detected using the DGGE approach although this method has been acknowledged to detect nearly all mutations (24).

R3531C family studies
The pedigrees of seven of the R3531C probands are shown in Figure 3. Table 4 displays the plasma lipid profiles of the probands together with those of family members. Table 5 shows the clinical histories of the probands and, in addition, age- and sex-adjusted lipid values. No one had tendon xanthomas. Plasma levels of total cholesterol and LDL cholesterol in affected individuals were 32 mg/dl and 21 mg/dl higher, respectively, than the levels observed in their unaffected relatives (23 mg/dl and 16 mg/dl, when age and sex adjusted), consistent with our earlier findings (4). However, in the present study these differences in lipids were not statistically significant.

View larger version (30K): [in this window] [in a new window]   Figure 3. Pedigrees of seven of the probands studied; and indicate males and females, respectively, who are heterozygous for the presence of the apoB R3531C mutation. Beneath the symbols are the plasma levels of total cholesterol (in mg/dl).

The plasma lipid profiles of the individuals with the three novel silent mutations found by DGGE analysis are presented in Table 6.

ApoB haplotype analysis
Eight apoB gene markers (the signal peptide insertion/deletion polymorphism, the 3' VNTR, and six RFLPs) were used to construct haplotypes of the Cys³⁵³¹ alleles. For six of the probands, it was possible to deduce the haplotype of the allele carrying the Cys³⁵³¹ codon. These are presented in Table 7 together with the ancestries of these individuals. This table also includes, by way of comparison, the 3531C haplotypes of two families (L.S. and A.C.) that we reported previously (4). The 3531C haplotype present in the H. M. family is unique. The partial, but clearly distinct haplotype of another proband (J.A.) is also included in this table. The A.M., J.K., L.T., J.C., and A.P. families all had the same pattern as the L.S. kindred, which is of Celtic and Native American ancestry. Hence, we have now detected a total of four different haplotypes associated with Cys³⁵³¹ alleles.

U937 cell assays
LDL were prepared from 17 of the individuals who were heterozygous for the R3531C mutation, from 9 of their unaffected relatives and from 2 other normal subjects. This LDL was examined for its ability to promote growth of the cell line U937. These cells have an absolute requirement for extracellular LDL cholesterol for growth and have been used previously to identify LDLs with defective binding (5) (26) (27). The mean growth rate of the cells, relative to a standard reference LDL, was 74.1 ± 4.0% (mean ± SE) for LDL from the R3531C subjects and 96.6 ± 5.1% for the normal subjects (P = 0.0018). The relative growth rates using LDL from heterozygous R3500Q and R3500W subjects was found previously to be about 50% that of normal LDL (5) (28). Thus, our data presented here, which show that the LDL from individuals with the R3531C mutation is 74% as effective at promoting growth as normal LDL, are in line with the binding affinity and mass ratio data presented here and previously (4).

	DISCUSSION

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We report here the discovery of nine additional unrelated probands heterozygous for the apoB R3531C mutation as a result of screening 2570 individuals. Thus, together with our previous report, we have found 11 such cases out of 4130, a frequency of one in 375. The frequency in the general population is no doubt less, as most subjects screened were pre-selected for lipid abnormalities. By comparison, the R3500Q mutation is present at 1 in 500 in populations of European descent (23). Other workers have recently reported the detection of additional individuals with the R3531C mutation. Four probands were found among a group of patients with coronary artery disease (CAD), with no family members available for cosegregation analysis (29); two families were identified by Wenham and co-workers (30) each with a history of atherosclerosis; and two probands, both with severe hypercholesterolemia, were reported in a French population (31).

LDL from a total of nine R3531C heterozygotes in this and a previous study (4) have been assayed using the MB19/dynamic laser light scattering method to measure the apoB allelic mass ratios. Ligand-defective Cys³⁵³¹ LDL particles accumulated and comprised a mean of 58 ± 1% of the total particles present. Radioimmunoassays have previously been used to show that the defective apoB Gln³⁵⁰⁰ allotype accumulates in the plasma of patients with the apoB R3500Q mutation (12) (13). In these studies the mass ratio of Gln³⁵⁰⁰ to Arg³⁵⁰⁰ LDL was 73:27 (12). Comparable results are seen with laser light scattering, the percentage of Gln³⁵⁰⁰ LDL was found to be 87% and 74% in two R3500Q heterozygotes (14). The greater accumulation of mutant particles with R3500Q relative to R3531C is presumably due to the fact that Cys³⁵³¹ LDL particles possess more residual affinity for the receptor. We have shown previously (4) and again in this study that LDL from R3531C heterozygotes is defective in binding to the LDL receptor. In the total of 12 heterozygous subjects who have now been tested, the overall binding affinity compared to reference LDL was 57 ± 4%. This value is for total LDL, being a weighted average of the normal and defective LDL present in the circulation. Defective apoB Cys³⁵³¹ LDL itself has been calculated to have 27% of normal affinity (4) compared with less than 10% for Gln³⁵⁰⁰ particles (4) (12) (23). In line with these differences in the ratio of apoB allotypes and in binding activity, LDL prepared from R3531C heterozygotes exhibited an ability to promote U937 cell growth that was intermediate between that of R3500Q LDL and normal LDL.

Myant (32) has noted that, in all cases examined, LDL from R3500Q heterozygotes with normal levels of cholesterol had defective binding. In the present study one R3531C heterozygote, despite a normal level of cholesterol (J.C. II-3), had LDL with the expected defective binding. She, and another affected individual with a normal level of cholesterol (A.M. III-10), had an accumulation of the defective Cys³⁵³¹ LDL particles. One R3531C subject reported by Wenham and co-workers (30) was normolipidemic, as was one of the eight affected individuals in the initial study by Pullinger et al. (4). LDL prepared from this latter subject had 48% of normal binding affinity. Thus, in evaluating any specific mutation of apoB, measurement of LDL binding affinity and, more significantly, determining whether there is an accumulation of the mutant allotype, is more revealing than the plasma level of cholesterol.

As with the R3500Q mutation (32) (33), there would appear to be variable expression of the R3531C phenotype, which is modulated by environmental and other genetic factors. This variable penetrance makes it difficult to compare the clinical significance of this mutation with the R3500Q mutation and with FH. A recent study of 76 families identified 129 individuals with FH and 28 with the R3500Q mutation (34). The R3500Q mutation had the effect of raising total cholesterol by 93 mg/dl (42%) and FH 174 mg/dl (79%) above normal levels. Another study of 34 R3500Q subjects reported an increase of 67 mg/dl (35%) compared to unaffected relatives (29). In the present report, we found that the R3531C mutation was associated with an increase of 32 mg/dl or 16%, though this was not statistically significant. However, when the present data on our 24 individuals with the R3531C mutation was combined with that on the original 8 subjects (4) and with that on 12 other individuals (29) (30) (31), the mean cholesterol level was 253 mg/dl (n = 44) compared to 202 mg /dl (n = 29) for unaffected relatives, an increase of 51 mg/dl or 25% (P < 0.0003). LDL cholesterol levels were 171 mg/dl for 40 R3531C heterozygotes and 135 mg/dl for 25 unaffected relatives, an increase of 36 mg/dl or 27% (P < 0.01).

The original two families with the R3531C mutation had apoB haplotypes associated with the mutant allele that were different from one another (4). The first was of Celtic and Native American background. This haplotype is associated with the Cys³⁵³¹ allele in five of the probands reported here, three of whom have Scottish or Irish ancestry. There is one new haplotype (H.M., Table 7) and one partial haplotype that is also clearly different from all the others (J.A., Table 7). In nine of the probands for which the analysis is meaningful there are four different haplotypes. This evidence, given that recombination events in this region of the apoB gene have not been detectable (35), points to multiple independent R3531C mutations. This is in contrast to the R3500Q mutation where one haplotype predominates in several populations in Europe and North America (36) (37), though two unique haplotypes, one of German (38) the other of Chinese ethnic origin (39), have been reported. The initial two probands reported with the apoB R3500W mutation had different haplotypes (5). The first was of Scottish and the second of Asian ancestry. Two other R3500W probands, a Chinese and a Malay, had this second haplotype (40).

We discovered three novel silent mutations of apoB. One (T3552T) was found by DGGE screening of 27 patients whose LDL had defective binding. However, no other mutations were detected among this group although we were able to detect all other known mutations in the region examined. Thus, mutations of apoB in the generally accepted receptor-binding domain do not seem to account for the defective binding affinity of the LDL here.

At the time of completion of this paper a report was published describing screening for the R3500Q, R3531C, and R3500W mutations of 9255 subjects from a general population in Denmark together with 948 patients with ischemic heart disease (41). Seven persons with the R3531C mutation were found in the general population (prevalence of 0.08%) and one among those with heart disease. The heterozygous R3531C carriers did not have a higher level of plasma cholesterol than the general population. The authors concluded that their results suggest that the R3531C mutation is not sufficient to cause hypercholesterolemia, is not associated with increased risk of ischemic heart disease, and that other additional factors are possibly required. It is difficult to project conclusions from only 7 patients compared to the 44 presented here and elsewhere (4) (29) (30) (31), particularly as the average age of the R3531C Danish people was 73 so they may represent a survivor population. The individual lipid values for the 7 subjects were not reported. The general population in this study had a mean total cholesterol of 235 mg/dl, and LDL cholesterol of 154 mg/dl, which is similar to our 24 R3531C carriers (234 mg/dl and 152 mg/dl, respectively, Table 4) and somewhat higher than the 18 unaffected relatives (202 mg/dl and 131 mg/dl, Table 4). This suggests that this Danish population is relatively hypercholesterolemic compared to the families we studied. The different conclusions of this study may reflect other determinants of cholesterol levels in the Danish population. Several key elements determine LDL levels in individuals including the production rate of VLDL and whatever determines the extraction rate of VLDL remnants from plasma. Receptor ligand defects may have a smaller impact when the subjects are relatively hypercholesterolemic and there is a partial diminution in LDL receptor activity.

	FOOTNOTES

¹ Both authors contributed equally to this study.

	ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grants HL14237 (Arteriosclerosis Specialized Center of Research), HL50782, and HL50779, by a gift from Donald and Susan Schleicher, and by a gift from the Joseph Drown Foundation. These studies were carried out in part in the General Clinical Research Center, Moffitt Hospital, University of California, San Francisco, with funds provided by the National Center for Research Resources, 5 MO1 RR-00079, U.S. Public Health Service. D. G and C. J. P. would like to thank the British Heart Foundation for support (grant number PG/96013) and Dr. Keith Vass of the Beaton Institute for Cancer Research, Glasgow, UK, for computer DNA analysis.

Manuscript received August 18, 1998; and in revised form October 9, 1998.

Abbreviations: apoB, apolipoprotein B; CAD, coronary artery disease; DGGE, denaturing gradient gel electrophoresis; FH, familial hypercholesterolemia; HDL, high density lipoprotein; LDL, low density lipoprotein; RFLP, restriction fragment length polymorphism; TC, total plasma cholesterol; TG, total plasma triglyceride; VLDL, very low density lipoprotein; VNTR, variable number of terminal repeats

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TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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