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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1719-1729

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1719-1729.)
© 1995 American Heart Association, Inc.

Articles

Differences in the Phenotypic Characteristics of Subjects With Familial Defective Apolipoprotein B-100 and Familial Hypercholesterolemia

André R. Miserez; Ulrich Keller

From the Departments of Research and Internal Medicine, University Hospital, Basel, Switzerland.

Correspondence to A.R. Miserez, MD, Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas TX 75235-7200.


*    Abstract
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 *Abstract
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Abstract Familial defective apolipoprotein B-100 (FDB) is a recently identified autosomal-dominantly inherited disorder caused by a point mutation in the apolipoprotein (apo) B gene. To determine whether the phenotypic characteristics in FDB subjects are similar to those in subjects with familial hypercholesterolemia (FH), 76 kindreds fulfilling the clinical criteria for heterozygous FH/FDB were characterized using molecular biological techniques. Allele-specific polymerase chain reaction (PCR) at the apoB locus was used for diagnosis or exclusion of FDB. PCR-based methods for detection of two point mutations (V408M and P664L) at the LDL receptor (LDLR) locus, cosegregation analysis using eight restriction fragment length polymorphisms (RFLPs) at the LDLR locus, or the exclusion of FDB confirmed the clinical diagnosis of FH. Three kindreds were not included because of a missing cosegregation between a particular haplotype and the FH phenotype. We predicted that a similar number of kindreds would be detected in the two groups, assuming comparable prevalences of the diseases in our population and similar phenotypic characteristics. However, only nine kindreds were identified with the FDB mutation compared with 64 kindreds with FH (P<.0001). From these 73 kindreds, 28 FDB heterozygotes and 129 FH heterozygotes were compared using multivariate analysis. There were no differences between these two groups with respect to age, sex, and apoE genotype distribution, lipoprotein(a) concentrations, body mass index, blood pressure, and smoking habits. However, FDB subjects demonstrated significantly lower concentrations of total cholesterol (8.1 versus 10.2 mmol/L, P<.001), LDL cholesterol (6.3 versus 8.2 mmol/L, P<.001), and triglycerides (1.3 versus 1.8 mmol/L, P=.025) and higher concentrations of HDL cholesterol (1.4 versus 1.2 mmol/L, P=.015) than subjects with FH. In contrast to FH, female FDB subjects tended to have higher concentrations of total cholesterol (8.9 versus 7.5 mmol/L, P=.032) and LDL cholesterol (7.1 versus 5.7 mmol/L, P=.026) than FDB males. The same results regarding total and LDL cholesterol and sex differences were observed when individual data of 238 FDB and 415 FH subjects from the literature were compared. In addition, FDB subjects showed much larger total cholesterol fluctuations than FH subjects (median of intraindividual coefficients of variation: FDB, 14.5%; FH, 5.3%; P<.001). In summary, these results demonstrate that FDB subjects tend to have a milder form of hyperlipoproteinemia than FH subjects and that only a part of the subjects with FDB fulfill the established criteria for identifying FH.


Key Words: apolipoprotein B-100 • LDL receptor • mutations • genotype • phenotype


*    Introduction
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 *Introduction
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LDL particles are catabolized primarily via the LDLR pathway, mediated by the interaction between the apoB molecules, the apolipoprotein moieties of the LDL particles, and the hepatic LDLRs. ApoB molecules are synthesized in hepatocytes and secreted in VLDL, where they function as ligands for the LDLR after conversion of VLDL to LDL. Binding of apoB to the LDLR results in cellular internalization of the LDL particles and their lysosomal degradation.1 Hence, on one hand, defects of the receptor as observed in FH or, on the other hand, defects of the ligand as observed in FDB result in increased LDLC concentrations.1 2 Defective binding of LDL particles to their receptors due to structural defects of apoB was recently identified in patients with moderately increased LDLC concentrations.3 4 5 This metabolic disorder was designated as FDB. A markedly reduced (20% to 32% of normal) binding of LDL particles isolated from subjects with FDB to LDLRs of cultured fibroblasts and a subsequent decrease of the clearance of LDL in turnover studies have been demonstrated in this disorder.5 6 FDB is caused by a single base substitution (G to A) at nucleotide 10 7087 in exon 26 of the apoB gene, creating an arginine-to-glutamine substitution at the codon for amino acid 3500.8 A high prevalence of FDB was found in the United States and in several European countries, in particular north of the Alps (prevalence in Switzerland, 1/210), suggesting that the origin of this point mutation is in central Europe.9 In a review published in 1990, the average TC concentration of the 41 FDB subjects described was considerably lower than that reported in the literature for subjects with FH.2 However, other reports investigating the clinical features of FDB in comparison to FH did not provide evidence for differences in the phenotypic characteristics between these two disorders.10 11 12 Thus, FDB and FH have been regarded as clinically indistinguishable.11 If this conclusion is correct and if the prevalences of the two disorders are similar in a given population, then the number of kindreds with FDB and the number with FH in a sample of kindreds presenting clinically with FH (FH/FDB) would be expected to be similar. To test this hypothesis, families clinically characterized as FH/FDB were identified and studied for the presence of defects in the apoB and the LDLR gene. Unexpectedly, the frequency of kindreds with FDB was much lower in our sample than that of kindreds with FH. Further analyses presented in this article confirmed our suggestion that differences between the phenotypic characteristics of FDB and FH subjects exist.


*    Methods
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 *Methods
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Subjects
The analysis of individuals with FDB or FH in this study was based on a sample of 76 unrelated white kindreds from the German- and French-speaking parts of Switzerland north of the Alps. Most of the families were of autochthonous Swiss origin; no families from the Italian-speaking part (south of the Alps, approximately 290 000 inhabitants) of Switzerland were included. In a first step, kindreds were identified by lipid specialists from our lipid clinic and from other Swiss university hospitals according to the classic criteria for primary hypercholesterolemia: a family history of hypercholesterolemia (type IIa hyperlipoproteinemia: fasting pretreatment LDLC exceeding the 95th percentile for the Swiss population corrected for age and sex,13 14 normal fasting TG) present in three or more members from at least two generations and inherited in an autosomal-dominant fashion; a family history of premature atherosclerosis; and family members with tendon xanthomas, xanthelasmas, or arcus lipoides corneae. All kindreds were screened for FDB. For confirmation of FH, all FDB-negative kindreds were screened for two point mutations at the LDLR locus. Except in two kindreds with a LDLR point mutation, cosegregation analysis using RFLPs at the LDLR locus was performed. Kindreds with a missing cosegregation between a particular haplotype and the FH phenotype were excluded. In a second step, we included all individuals from the remaining families who had been characterized as affected by FDB (confirmed by allele-specific PCR) or FH (confirmed by PCR-based methods for direct mutation detection, by cosegregation analysis, or by the exclusion of the FDB mutation) or as not affected by both disorders (control subjects). Exclusion criteria for the individuals were secondary causes of hypercholesterolemia (diabetes mellitus, hypothyroidism, kidney or liver diseases) or missing data. To verify the results from our sample of FDB and FH subjects, 37 articles were retrieved from MEDLINE, and individual data for 653 subjects characterized as affected either by FDB or by a particular LDLR defect were collected and compiled in a database.

Lipoprotein Analyses
LDLC was determined from overnight fasting blood samples after precipitation with heparin, and HDLC was determined after precipitation with phosphotungstic acid and magnesium ions. Lipid concentrations were measured using enzymatic colorimetric methods (Boehringer Mannheim). Pretreatment values only (no lipid-lowering diet or drugs) were used for further analyses.

DNA Analyses
Total genomic DNA extraction from white blood cells was performed as described previously.9 A combined allele-specific and asymmetric PCR-based method was used for screening for the apoB defect at nucleotide 10 708 causing FDB.15 PCR amplification and subsequent digestion with Nla III or Pst I, respectively, were performed for screening for the presence of the V408M and P664L mutations in the LDLR gene.16 17 Eight RFLPs (assessed by Southern blots or by PCR amplification and subsequent digestion) at the restriction sites of Taq I, Stu I, Hinc II, Ava II, ApaLI (5'), Pvu II, Nco I, and ApaLI (3') were used for cosegregation analysis at the LDLR locus as described elsewhere.18 The RFLPs extended from intron 4 to the noncoding region, 14.6 kb downstream to the 3' end of exon 18 of the LDLR gene and spanning a total distance of approximately 43.3 kb. Restriction isotyping of human apoE was performed by gene amplification and cleavage with Cfo I (isoschizomere of Hha I).19

Statistical Methods
Interactions of different variables were studied with MANOVA using the SUPERANOVA program. Independent parameters in MANOVA were age; sex; pretreatment concentrations of TC, LDLC, HDLC, VLDLC, and TG; lipoprotein(a); apoE genotypes; body mass index; diastolic and systolic blood pressure; smoking; the presence of xanthomas, xanthelasmas, or arcus lipoides; coronary artery disease (assessed by coronary angiography or defined by the presence of myocardial infarctions); cerebrovascular disease (defined by the presence of strokes); and peripheral vascular disease (assessed by angiography or defined by the presence of intermittent claudication). Differences of means within the three groups (FH, FDB, and control) of normally distributed values (TC, LDLC, HDLC, VLDLC, body mass index, and blood pressure) were assessed with ANOVA and Scheffé's F tests and differences of not normally distributed values (age, TG, lipoprotein[a]) by the Kruskal-Wallis test. Relative frequencies of clinical features (sex; apoE genotypes; frequencies of obesity, hypertension, smoking, xanthomas, xanthelasmas, and arcus lipoides; and coronary artery, cerebrovascular, and peripheral vascular disease) within the three groups were assessed by {chi}2 tests for homogeneity (Brandt-Snedecor). Significant differences between the groups stratified by sex were assessed by unpaired t tests for normally distributed values, by U tests (Mann-Whitney) for not normally distributed values, and by Fisher's exact tests for frequencies. Regression analysis was performed to assess age dependency of the different variables. Univariate analyses and regression analyses were studied using the STATVIEW II program. Normally distributed data are presented as mean±SD, not normally distributed data as median (range), and frequencies as percentages (ratios of absolute numbers). The probability P to observe zFDB or less kindreds with FDB from a sample of n kindreds (n=zFDB+zFH where zFDB is the number of kindreds with FDB, and zFH is the number of kindreds with FH; fFDB,obs=zFDB/n is the observed frequency of kindreds with FDB, and fFH,obs=zFH/n is the observed frequency with FH) was calculated using

The ratio of the expected frequencies of FDB and FH (fFDB,exp/fFH,exp) in the sample n, with fFDB,exp=1-fFH,exp, was based on the ratio of the estimated prevalences of FDB and FH (gFDB/gFH) in the Swiss population.


*    Results
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 *Results
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From the 76 kindreds, there were nine kindreds with the G-to-A substitution at the apoB locus (FDB) and 64 kindreds with LDLR defects (FH). In these 64 kindreds, two with a mutation in exon 9 (V408M, FH-Afrikaner 2; three FH subjects included) and two kindreds with a mutation in exon 14, in the growth factor repeat C of the receptor (P664L, FH-Gujerat; 6 FH subjects), were detected. In 50 kindreds (110 FH subjects), FH was confirmed by cosegregation analysis, including two kindreds (five FH subjects) that were confirmed by direct detection of the point mutation as well. In 12 kindreds, FH was confirmed by exclusion of FDB (15 FH subjects). Three kindreds were not included because of a missing cosegregation of a particular haplotype with the FH phenotype. The observed frequency of kindreds with FDB in the sample was 9/73 or approximately 0.12, with a 99% confidence interval of 0.05 to 0.27 (or 4/73 to 19/73). The expected frequency based on the assumed prevalences of FDB and FH in Switzerland was 51/73 or approximately 0.70. The probability value to find a frequency of nine or fewer kindreds with FDB in the sample (fFDB,obs <=0.12 or <=9/73 with fFDB,exp=0.70) was P<.0001.

A total of 263 individuals (comprising 28 heterozygous FDB subjects, 129 heterozygous FH subjects, and 106 unaffected family members serving as control subjects) were included.

No differences were observed between FH, FDB, and control groups with respect to age, sex, apoE genotype distribution, body mass index, systolic and diastolic blood pressure, or smoking habits (Table 1Down). Differences in lipoprotein(a) concentrations in a subset of FDB and FH subjects were not statistically significant.


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  		Table 1. Comparison of Clinical and Genetic Characteristics Potentially Influencing Lipoprotein Metabolism and Atherogenesis in FH, FDB, and Control Subjects

Differences between FDB and FH groups were detected in our sample (Table 2Down) for mean TC (Fig 1Down), LDLC, HDLC, VLDLC, TG concentrations, and the TC-HDLC ratio but not for the relative frequencies of tendon xanthomas, xanthelasmas, arcus lipoides, and coronary artery, cerebrovascular, and peripheral artery disease. Differences between males and females regarding mean TC and LDLC concentrations were detected in FDB but not in FH. In all three groups, there was a significant age-dependent increase in TC and LDLC but not in HDLC concentrations (Table 2Down). In contrast to FH, there was an age-dependent increase in the TC-HDLC ratio in FDB subjects. In contrast to FH and control, there was no age-dependent increase in VLDLC concentrations in FDB subjects. In FH, TG concentrations were significantly higher than in FDB and control groups, and HDLC concentrations were significantly lower than in FDB and control (Table 2Down). Serial measurements of TC levels in FDB subjects revealed striking fluctuations (up to 40%), in particular in young FDB subjects aged between 15 and 40 years. The median of intraindividual coefficients of variation (CV) was higher in FDB than in FH subjects (FDB: n=14, CV=14.5%; FH: n=23, CV=5.3%; P=.0005; Fig 2Down).


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  		Table 2. Comparison of Lipid Concentrations and Clinical Characteristics Typical of Familial Forms of Hypercholesterolemia in FH, FDB, and Control Subjects



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  		Figure 1. Bar graphs show TC concentrations of subjects with FH and FDB and in control subjects stratified according to sex and different age groups.



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  		Figure 2. Plot shows intraindividual coefficients of variance of repeatedly measured TC concentrations in 23 FH subjects and in 14 FDB subjects. {circ} indicates FDB subjects >30 years; {bullet}, subjects <=30 years; {square}, FH subjects >30 years; and {blacksquare}, FH subjects <=30 years. The horizontal bars denote the medians of the respective groups.

In the compiled data from the literature including 238 FDB and 415 FH subjects, the differences between FDB and FH regarding mean TC and LDLC concentrations were confirmed (TC, P<=.0047; LDLC, P<=.0078), except in FH-Gujerat (TC, P=.0213; LDLC, P=.13; Table 3Down). The differences between males and females regarding mean TC (P=.0001) and LDLC (P=.0004) concentrations in FDB but not in FH were also confirmed by the compiled data (Table 3Down). The significant age-dependent increases in TC and LDLC but not in HDLC concentrations were detected in the compiled data as well: in the FDB sample and in the FH samples of 26 or more subjects (n>=26, P<=.0079; Table 3Down).


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  		Table 3. Comparison of Clinical and Laboratory Characteristics in Subjects With Different LDLR Mutations Causing FH and in Subjects With FDB From the Literature

The frequency of xanthomas in the compiled data from the literature was significantly lower in FDB than in FH when the samples of FH subjects for comparison were large enough (n>=44, P<=.0107; Table 3Up) but not in our sample of only 28 FDB subjects (Table 2Up; P=.09). There was a predominance of xanthomas in females with FDB, which was significant in the compiled data from the literature including 65 FDB subjects (P=.0249) but not in our sample of only 28 FDB subjects (Table 2Up; P=.07). In FH subjects from our sample, there was a predominance of coronary artery disease in males (n=129, P=.007) that was not found in FDB subjects from our sample (n=28, P=.24) or by analyzing a larger number of FDB subjects from the compiled data characterized for the presence or absence of coronary artery disease (n=79, disease present in FDB males: 47.5%, present in FDB females: 51.3%; P=.74).

In addition to these differences between FDB and FH, two further associations were detected. In FH subjects with xanthomas, there was an absolute but also relative predominance of a homozygosity for the apoE {varepsilon}3 allele (E3/3; P=.003) compared with the heterozygous apoE genotypes (E2/3, E2/4, and E3/4). In FDB subjects suffering from strokes, there was a predominance of the apoE {varepsilon}4 allele (E2/4, E3/4; P=.025).


*    Discussion
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 *Discussion
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The present study revealed quantitative differences between FDB and FH with respect to mean pretreatment lipoprotein concentrations and differences with respect to the modulation of the phenotype by independent factors. There are three lines of evidence for quantitative differences between the two disorders. First, significantly lower mean TC and LDLC concentrations in FDB heterozygotes compared with FH heterozygotes were detected in our sample (Table 2Up). Second, significantly lower mean TC and LDLC concentrations in FDB heterozygotes compared with FH heterozygotes also were detected in a compilation of 238 FDB and 415 FH subjects from the literature, even when FDB was individually compared with each of the LDLR mutations (Table 3Up). Third, an unexpectedly (if one assumes no differences between FDB and FH) low number of nine FDB kindreds was found in a sample of 73 (FDB and FH) kindreds selected according to the clinical characteristics of FH/FDB. The selection of the kindreds for this study was based on the clinical criteria for familial forms of hypercholesterolemia and was blinded with respect to the differential diagnosis of FDB versus FH. The ratio of the observed frequencies of the two disorders (fFDB,obs/fFH,obs) in the sample should reflect (after exclusion of kindreds not affected by either FDB or FH, thus fFH,obs=1-fFDB,obs) the proportion of the prevalences of the two disorders (gFDB/gFH) in a given population if the chance of selection were identical for kindreds with FDB and FH, ie, the subjects from the FDB and FH kindreds would fulfill the same clinical criteria. If we assume that FDB and FH cause exactly the same phenotypic features and the prevalences of FDB and FH are the same in a given population (gFDB=gFH), then the expected frequencies of the two disorders (fFDB,exp and fFH,exp) in the study sample should be identical also. Furthermore, if FDB and FH cause exactly the same features but the prevalence of FDB is approximately 1/210, as has been estimated for the Swiss population,9 and the prevalence of FH is approximately 1/500, as it is assumed to be for white populations without a founder effect,1 then we would expect to find even more kindreds with FDB than with FH in our study sample. However, the observed frequency of FDB kindreds (fFDB,obs=0.12 or 9/73) was much lower than expected (fFDB,exp=0.70 or 51/73). On the other hand, the expected frequency fFDB,exp was clearly above the upper 99% confidence limit of fFDB,obs. Even if one assumes an equal prevalence for FDB and FH (fFDB,exp=fFH,exp=0.50), this would still be the case. Similar ratios of observed and expected frequencies in samples of subjects clinically diagnosed as FH were also found in other populations studied (Table 4Down). Hence, the frequencies of FDB kindreds observed in our study sample and in all these other countries were unexpectedly low. Since this discrepancy is very unlikely to have occurred by chance alone (P<.0001), the clinical criteria defined above appear to be much more appropriate to identify kindreds with FH due to LDLR defects than those with FDB. This indicates that affected subjects from FH families better fulfill the clinical criteria than those from FDB families. Hence, from this observation, there must also exist differences in the phenotypic characteristics of these two disorders. An exception might be FH-Gujerat (P664L) (see Reference 1111 and Table 3Up), where TC but not LDLC concentrations were significantly higher in FH subjects. In our data, there were only two of 64 families with this particular mutation.


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  		Table 4. Expected and Observed Frequencies of Subjects With FDB in Samples of Subjects Clinically Diagnosed as FH1

Three kindreds in which the clinical characteristics of FH (at least LDLC concentrations >95th percentile) did not cosegregate with a particular LDLR haplotype were not included in our study. It is possible that a missing cosegregation in a family could be due to a normocholesterolemic LDLR mutation carrier. However, the probability that a subject carrying an LDLR defect does not fulfill our inclusion criteria because of an LDLC concentration below the 95th percentile is small. Since LDLC concentrations are normally distributed, this probability can be roughly estimated by z=(r95GP-µFH)/SDFH. The FH subjects in Table 3Up were studied using molecular biological techniques; thus, normocholesterolemic FH carriers or FDB subjects would have been detected. Using the overall mean LDLC concentration of these 242 FH subjects (µFH=7.8, SDFH=±1.6 mmol/L) and an average 95th percentile from the Lipid Research Clinics study data20 (r95GP=4.6 mmol/L), z is -2.0 and thus P<.03. Therefore, a sample defined by our inclusion criteria should represent more than 97% of the subjects carrying an LDLR mutation in a population. In line with this expected low frequency (3%), there was only one subject (in one of the excluded families) who was normocholesterolemic but shared with her hypercholesterolemic family members the haplotype corresponding to the LDLR defect. The other two families have been excluded because there was in each family a hypercholesterolemic subject with a haplotype that could not be detected in her or his mother or in any of the brothers or sisters. Extensive studies using further DNA markers suggested recombinational events in these latter two families. To rule out the question of influence on the results of possible normocholesterolemic LDLR mutation carriers, an extended analysis was performed. However, the inclusion of all the potentially (clinically and/or molecular genetically) affected individuals from these three families (eight hypercholesterolemic subjects, one normocholesterolemic subject) did not change our results.

In 1986, FDB was detected in subjects who had been investigated because of an only moderate hypercholesterolemia (TC, 6.5 to 7.8 mmol/L) and the lack of the classic clinical features of FH. Thus, this first report described the disorder as a cause for moderate hypercholesterolemia in comparison to FH.4 There are further findings that are in line with our observation of quantitative differences in the phenotypic characteristics between FDB and FH:

In FDB, a gene dosage effect, as it has been described in FH,1 appears to be missing. In both subjects homozygous for FDB reported so far, TC was only mildly increased6 21 compared with subjects homozygous for FH (TC in FH homozygotes, 16.8 to 25.9 mmol/L).22 23 In the case reported by März et al,6 the TC and LDLC concentrations in the FDB homozygote (mmol/L: TC, 8.6; LDLC, 6.9) were similar to those of the heterozygous members of the same family (mmol/L: TC, 7.6 to 8.6; LDLC, 6.0 to 7.1; in subjects of the same generation). In the case reported by Funke et al,21 the TC concentrations in the FDB homozygote (7.7 to 8.6 mmol/L) were between those of her heterozygous father (9.2 to 11.1 mmol/L) and her heterozygous mother (6.5 mmol/L). In both FDB homozygotes, TC and LDLC concentrations were similar to those in the 238 FDB heterozygotes whose data are compiled in Table 3Up. This is clearly in contrast to FH, where LDLC concentrations are usually sixfold to 10-fold increased in homozygotes compared with only a twofold to threefold increase in heterozygotes.1 24 Furthermore, both FDB homozygotes described hitherto, a 54-year-old man and a 31-year-old woman, did not show any evidence for coronary or peripheral artery atherosclerosis. This is also in clear contrast to FH homozygotes, in whom coronary artery atherosclerosis frequently has its clinical onset before the age of 10 years. FH homozygotes usually succumb to the complications of coronary artery disease before age 20 years.24 In addition, in both FDB homozygotes, treatment with HMG-CoA reductase inhibitors caused a pronounced decrease of TC and LDLC concentrations (as reported by März et al,6 by 26% and 31%, respectively) in contrast to FH homozygotes, who are usually resistant to HMG-CoA reductase inhibitors.24 In FDB homozygotes, binding or uptake of LDL is greatly diminished but not completely abolished. The residual binding activity of LDL from an FDB homozygote was approximately 20% of normal.6 The residual binding of the LDL to the receptor has been demonstrated on the one hand to be due to a receptor-mediated uptake of the LDL subfractions of the lowest densities (1.019 to 1.034 kg/L) in this article by the remaining amounts of apoE on these particles. On the other hand, in the LDL subfractions of intermediate density (1.034 to 1.040 kg/L), the defective apoB displayed some residual receptor binding by itself.6 The apoB molecule is modeled as a belt of about 2.0x5.4 nm with an average length of about 58.5 nm, which surrounds the LDL particle.25 Arginine 3500 is suggested to stabilize two clusters of amino acids (3147 to 3157 and 3359 to 3367) that have been assumed to ensure the binding of the apoB to the LDL receptor.6 Since the radius of the LDL particle may vary considerably and, according to this model, also that of the apoB,25 the stabilization of the two amino acid clusters may be of importance to maintain the binding affinity of the apoB to the LDL receptor. In FDB, the two amino acid clusters may be disrupted in LDL particles smaller than 9.6 nm and larger than 10.1 nm (LDL subfractions of 1.040 to 1.063 kg/L and of 1.019 to 1.034 kg/L), as suggested by experiments of März et al.6 After removal of the apoE-containing particles, these subfractions showed no receptor binding. On the other hand, LDL particles of 9.6 to 10.1 nm (subfractions of intermediate density or 1.034 to 1.040 kg/L) might be of the optimal size to allow a residual receptor-mediated binding despite the loss of the stabilizing amino acid arginine at position 3500 or, in other words, despite the FDB mutation. LDL particles smaller than 9.6 nm were the only subfractions in FDB homozygotes with completely abolished binding and uptake.6 In contrast to these results, defective LDL receptors of FH homozygotes often show a total or near total inability to bind or take up LDL particles. In a review by Goldstein and Brown22 of 57 FH homozygotes, there were 31 subjects showing a functional LDLR activity below 2% of normal.

Although differences between FDB and FH are quite obvious in homozygotes, they are much more difficult to detect in heterozygous subjects. A major problem is the contribution of the normal allele to the phenotypic characteristics of a heterozygote. Another reason could be that the expression of the hyperlipoproteinemia in heterozygotes may be modulated by environmental factors and also may be dependent on age. Both appear to be the case in FDB rather than in FH. Differences between FDB and FH may therefore be difficult to detect if only adult individuals are included in the analysis. This might be a reason why previous studies investigating the phenotypic features of FDB10 11 12 did not provide evidence for differences in phenotype between FDB and FH. Thus, a study design with inclusion of individuals from all age groups, exclusion of individuals for whom the clinical diagnosis was not consistent with the molecular biological methods, and use of multivariate analysis including other factors known to modulate the lipoprotein levels was performed.

In FDB, moderate expression or absence of hypercholesterolemia has been observed by several authors.2 9 12 21 26 27 Numerous FDB subjects with TC and LDLC concentrations within the normal range have been described.

In FH, variable expression has been described as well.22 23 However, FH is highly penetrant at all ages,22 and individuals with LDLR defects and LDLC concentrations below the 95th percentile of the general population are rare, as demonstrated above. In some of these cases, the missing elevation of LDLC has been suggested to be caused by an interaction with other genes. In a family including normocholesterolemic subjects with LDLR defects, a dominant gene suppressing the hypercholesterolemia has been demonstrated to be responsible for the absence of elevated levels in some of these individuals.28

In the present study, a large number of unrelated families was studied. Thus, a lipid-lowering or a lipid-increasing effect of another gene present in one of these families is not expected to bias our results, as could be the case if the same number of subjects had been collected from a few unrelated families only.

Besides quantitative differences between FDB and FH regarding mean lipoprotein concentrations, there was evidence for at least two further differences with respect to the modulation of the phenotype by independent factors.

First, in FDB, there were differences between males and females (higher mean TC and LDLC concentrations in FDB females, Table 2Up). These differences were confirmed by an independent sample of 238 FDB subjects from the literature showing significant differences as well (Table 3Up). The predominance of xanthomas in FDB females was significant in the compiled data from the literature including 65 FDB subjects but not in our sample of only 28 FDB subjects. Both sex-specific differences were not detected in FH subjects from our sample (Table 2Up) or in FH subjects from the compiled data.

A lipid disorder (familial dysbetalipoproteinemia) affecting apoE, the other ligand known to bind to the LDLR, results in a markedly reduced binding of apoE-containing VLDL remnants to the LDLR. This disorder is characterized by lower TC and LDLC concentrations in women than in men, onset of hyperlipidemia approximately 10 years later in women, and a distinct influence of estrogens on hyperlipoproteinemia.29 In contrast to this disorder, sex-specific LDLC-lowering effects appear to be absent in women with FDB. In FDB subjects of the present study, TC and LDLC concentrations were significantly higher in women than men, the onset of hyperlipidemia was not different between women and men, and female FDB subjects showed the highest TC and LDLC concentrations (Fig 1Up). The differences between the two disorders, each characterized by functionally impaired binding either of apoB or apoE, suggest a different role for the two apolipoproteins in the sex-specific mechanisms modulating the LDLC levels. Furthermore, because of higher LDLC concentrations, females with FDB potentially lose their advantage with respect to atherosclerosis after the age of 41 to 60 years (Fig 1Up). Thus, the predominance of coronary artery disease in males with FH, which has been detected in our sample and in other studies,24 is expected to be less distinct in FDB than in FH. This conclusion is consistent with the compiled data of 79 subjects, which revealed no sex-specific difference in FDB with respect to coronary artery disease.

The second difference between FDB and FH was the presence of marked spontaneous cholesterol fluctuations (including normal and hypercholesterolemic values) in the FDB subjects available for repeated measurements. The median of the intraindividual coefficient of variation in FDB subjects (14.5%) in the present study was larger than in subjects with FH (5.3%) but also larger than that in subjects with type III hyperlipoproteinemia (5.1%)30 or healthy subjects (4.9% to 7.1%).31 32 33 In contrast to these observations, in FH heterozygotes the elevated levels can often be diagnosed at birth and persist throughout life,22 a lipid-lowering diet usually reduces the TC concentrations only by 10% to 15%.24

The striking fluctuations and the distinct increase in early adulthood (15 to 40 years, Figs 1Up and 2Up) support the hypothesis that there are compensatory mechanisms either reducing the production rate of the apoB-containing particles or increasing the catabolism of apoB-defective LDL particles (eg, by an increased uptake of apoE-containing VLDL remnants and large LDL particles or by an improved binding and uptake of LDL particles of intermediate size).6 In heterozygotes, differences between FDB and FH in lipoprotein concentrations may be caused by a decreased uptake of apoE-containing particles by the defective LDLRs in FH subjects.34 This hypothesis is supported by the significantly higher TG and VLDLC concentrations in FH compared with FDB subjects in our sample (Table 2Up). Increased TG concentrations have been previously reported in approximately 10% of the subjects with FH.1 In an animal model for FH (the WHHL rabbit), differences between LDLR-defective animals and controls with respect to LDLR-mediated uptake of apoE-containing particles have been demonstrated.35 Hence, these observations suggest that LDLR defects may be associated with an impaired clearance of apoE-containing particles. On the other hand, on the assumption that the clearance of apoE-containing TG-rich VLDL remnants and large LDL particles is unaffected in FDB because both the LDLR and apoE are intact, this mechanism would explain the lower TG and VLDLC concentrations found in FDB when compared with FH. In our sample, the TG and VLDLC concentrations in FDB subjects were not higher than those in the control group (Table 2Up). These observations are consistent with the finding that, in FDB, the uptake of apoE-containing particles is unimpaired.6 Furthermore, since VLDL remnants are the precursors of LDL, the apoE-mediated binding and uptake of apoE-containing VLDL remnants may contribute to the only moderate hyperlipoproteinemia found in FDB compared with FH. In FDB heterozygotes, upregulation of hepatic LDLRs may enhance the residual binding of large LDL particles and the clearance of the apoE-containing VLDL remnants before they are converted to LDL. In FDB heterozygotes, an upregulation of LDLRs results in the production of 100% functional active LDLRs. Thus, an upregulation is probably more effective with respect to the apoE-mediated uptake than an upregulation of LDLRs in FH heterozygotes to the same extent, which is expected to result in production of only about 50% functional active LDLRs. This is consistent with the observation in FDB subjects that an upregulation of LDLRs by HMG-CoA reductase inhibitors reduces the TC and LDLC concentration quite considerably, even in FDB homozygotes.6 21

However, the spontaneous increase in TC concentrations of young FDB subjects suggests that such mechanisms may decompensate rapidly with increasing age and that a large percentage of individuals with the FDB mutation develop hypercholesterolemia. Regarding the marked fluctuations detected in FDB subjects, these compensatory mechanisms appear to be very susceptible to environmental factors. As long as the VLDL production is normal, and predominantly large LDL particles are produced that can be cleared by the LDL receptors, the hyperlipoproteinemia may remain moderate. Dietary change, such as to a carbohydrate-rich diet, results in the formation of large VLDL particles that are converted to smaller LDL particles.6 25 26 Since the size of the LDL particles has been demonstrated to be of importance for the binding of the defective apoB to the LDLR,6 dietary changes may therefore cause a change in the binding affinity of the apoB. In FH heterozygotes, however, the apoB is stabilized by arginine at position 3500; thus, smaller LDL particles generated under these conditions are still able to bind to the functional active LDLRs, and the LDLC concentrations in FH subjects are less susceptible to these factors.

In summary, the present study reveals several lines of evidence for differences in the phenotypic characteristics between subjects with FDB and FH. In FDB, mean pretreatment TC and LDLC concentrations were lower than in FH. In contrast to FH, there were higher TC and LDLC concentrations in FDB females than in FDB males. Significantly larger fluctuations in subjects with FDB than in subjects with FH suggest differences between FDB and FH in ability to compensate for metabolic changes (eg, those caused by environmental factors). However, once decompensated, heterozygous individuals with FDB may be hardly distinguishable from heterozygous individuals with FH. Thus, the inclusion of other (in particular, pediatric) members of the patient's family may be helpful in distinguishing between these two lipid disorders.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
FDB = familial defective apolipoprotein B
FH = familial hypercholesterolemia
HDLC = HDL cholesterol
HMG-CoA = 3-hydroxy-3-methylglutaryl–coenzyme A
LDLC = LDL cholesterol
LDLR = LDL receptor
PCR = polymerase chain reaction
RFLP = restriction fragment length polymorphism
TC = total cholesterol
TG = triglycerides
VLDLC = VLDL cholesterol


*    Acknowledgments
 
This work was supported by the Postgraduate Course for Experimental Biology and Medicine of the University of Zürich and by the "Wissenschaftlicher Kredit" of the University Hospital of Basel, Switzerland. We are most grateful to numerous physicians in our country who provided excellent anamnestic data and helped us collect blood samples from FDB and FH subjects and their relatives. The technical help of N. Chiodetti is gratefully acknowledged.

Received October 14, 1994; accepted July 25, 1995.


*    References
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