Differences in the Phenotype Between Children With Familial Defective Apolipoprotein B-100 and Familial Hypercholesterolemia
Abstract Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited genetic disorder resulting from a point mutation in the apolipoprotein (apo) B gene and is associated with significantly elevated plasma total and LDL cholesterol levels. Despite numerous descriptions outlining the phenotype of children with familial hypercholesterolemia (FH), no study has described the biochemical and clinical phenotype in a cohort of children with FDB. The phenotypes of FH and FDB, therefore, have not been compared in children. We have studied a cohort of 38 Dutch children (all <20 years old) with FDB from 21 different families. Lipid and lipoprotein levels and the clinical phenotype were compared with 97 age-matched FH heterozygotes, as defined by molecular analysis, and with age-matched non-FDB, non-FH control subjects. Female FDB carriers (n=23) had significantly lower total cholesterol (P<.001), LDL cholesterol (P=.001), total cholesterol:HDL ratio (P<.001), and apoB levels (P=.001) than age-matched female FH heterozygotes (n=50). Similar results were noted in male FDB carriers (n=15) compared with male FH heterozygotes (n=47; P=.005, P=.007, P=.014, and P=.074, respectively). Within the FDB group, female FDB heterozygotes had higher LDL cholesterol (P=.038) and a trend to higher total cholesterol levels (P=.165) than age-matched males. Both male and female FDB carriers had significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio than age- and sex-matched control subjects, which was evident even in children <10 years of age, providing additional evidence that this mutation is penetrant in early life. These results provide evidence for a milder biochemical phenotype in children with FDB than in children with FH. The phenotype observed is intermediate between that of control subjects and FH heterozygotes matched for age and sex. As the incidence of coronary artery disease is related to both the extent and duration of cholesterol elevation, our findings might explain in part the lower incidence of clinical atherosclerosis seen in adults with this condition than in adults with FH.
- Received June 7, 1996.
- Accepted August 9, 1996.
An increase of LDL cholesterol in plasma can result from either increased synthesis or decreased clearance of LDL. FH and FDB are two common genetic conditions that cause significant defects in LDL clearance. Defective LDLR activity secondary to defects in the LDLR gene underlies FH,1 whereas in FDB the defect resides in the major ligand for the LDLR, apoB-100.2
Various mutations have been described that disrupt the ligand-binding properties of apoB to the LDLR.3 4 5 Of these, the mutation most frequently observed is the B-3500 mutation resulting from a G-to-A substitution at codon 3500 in exon 26 of the apoB gene, resulting in the substitution of a glutamine for arginine. This mutation has been reported with a frequency of between 1 in 500 and 1 in 700 in individuals of European and North American descent.6 7 A high prevalence of FDB in Switzerland (1 in 210 individuals) suggests the origins of this mutation to be in central Europe, illustrating a possible founder effect.8 Defective apoB-100 has been shown to reduce significantly the binding of apoB-100 to the LDLR,2 9 10 which results in elevated LDL cholesterol levels in carriers of this mutation.6 It has been suggested previously that as a clinical entity, FDB is indistinguishable from FH, with similar lipoprotein phenotypes and a similar reported incidence of atherosclerotic disease observed in these conditions.11 12 13 However, recently it has been demonstrated that FH and FDB are possibly distinct both clinically and biochemically, with a milder phenotype observed in adult carriers of the apoB mutation. This milder phenotype has been suggested to result from compensatory mechanisms leading to increased uptake of VLDL remnants via an apoE-mediated LDLR pathway and decreased production of apoB-defective LDL particles in FDB subjects, resulting in lower LDL cholesterol compared with subjects with FH.8 Furthermore, these compensatory mechanisms may be age dependent. Older FDB subjects may lose the ability to compensate by enhanced apoE-mediated clearance of IDL. This would predict a greater age-related increase in cholesterol levels in FDB subjects than that seen in FH heterozygotes.8
Despite the numerous reports in the literature detailing the biochemical and clinical phenotype of FH in children, no study thus far has described the phenotype of a cohort of children with FDB. An important question is how penetrant FDB is in children and whether or not FDB is expressed before puberty. Herein we report on a cohort of 38 Dutch children with FDB (all <20 years old) and show significantly higher levels of total cholesterol, LDL cholesterol, apoB, and total cholesterol:HDL cholesterol ratio in FDB carriers than in matched non-FH, non-FDB control subjects, which demonstrates that like FH, FDB is penetrant in early life. However, significantly lower total cholesterol, LDL cholesterol, apoB, and total cholesterol:HDL cholesterol ratio were evident in children with FDB than in children with molecularly defined FH matched for age, sex, and ancestry. As hypercholesterolemia has a dose- and time-dependent effect on atherosclerotic disease,1 the finding of lower cholesterol levels in FDB children than in children with FH may explain in part the reduced incidence of coronary artery disease observed in adults with FDB compared with adults with FH.
Thirty-eight children with FDB from 21 families were studied. Informed consent was obtained from subjects between 18 and 20 years old and from parents/guardians of children <18 years old. Lipid and lipoprotein levels and clinical phenotype were compared with those from a cohort of age- and sex-matched molecularly defined FH heterozygotes and age- and sex-matched control subjects. As no significant differences in lipid/lipoprotein levels between FH heterozygotes who carried null alleles or missense mutations were noted, these individuals were pooled. Between group comparisons were made for children <10 years old and also for children between 10 and 20 years old. As the precise pubertal status for most of the children was not known, groups were divided into these age categories to define a mostly prepubertal group (<10 years old) and another group including predominantly pubertal and postpubertal children (10 to 20 years old).
All study subjects were of Dutch ancestry. FDB and FH heterozygotes were ascertained by family studies of adult FDB and FH probands identified from the Amsterdam Medical Centre affiliated lipid clinics. Adults heterozygous for FDB and FH were referred to the lipid clinics with underlying hypercholesterolemia and were diagnosed with FH on the basis of the following clinical and biochemical criteria: LDL cholesterol in index cases >95th percentile for age and sex compared with the Lipid Research Clinics Prevalence Study criteria and/or presence of tendon xanthomas in index cases, and pediatric relative (<18 years of age) with LDL cholesterol >95th percentile for age and sex or with known tendon xanthomas. FDB and FH heterozygotes were thereafter identified by molecular analysis. Control subjects were unaffected family members of the FDB and FH heterozygotes. FDB was excluded in both FH heterozygotes and control subjects. No subject had any disease known to affect lipoprotein metabolism, including diabetes mellitus, kidney, liver or thyroid disease, nor was any subject taking medication known to affect lipid metabolism (except for oral contraceptives). Height and weight were recorded and BMI (weight in kilograms divided by the square of the height in meters) was calculated.
After a 12-hour overnight fast, blood samples were collected by venipuncture in EDTA tubes. Total plasma cholesterol and triglycerides were measured by using enzymatic methods previously described.14 15 HDL cholesterol was determined after precipitation of the apoB-containing lipoproteins as published previously.16 The LDL cholesterol was then calculated according to the formula of Friedewald et al.17 ApoA1 and apoB were determined by an immunological rate-nephelometric procedure using a polyclonal goat anti-human antiserum.18
After blood was extracted, cells and plasma were separated by centrifugation at 2000 rpm for 10 minutes at 4°C. Genomic DNA was isolated from the buffy coat from venous samples by previously described techniques.19 The apoB-3500 mutation was determined by the mutagenic PCR technique described by Defesche et al.13 To summarize, a 477–base pair fragment of exon 26 of the apoB gene, from nucleotide position 10 675 to 11 151 was amplified, using PCR primers as specified. In the presence of the wild-type sequence, this creates an Msp I site, whereas no site is created in the mutant sequence. These alleles are then resolved after Msp I digestion.
ApoE genotyping was performed as described by Reymer et al.20 ApoE genotyping was not performed on control subjects. Seventeen different known LDLR mutations and two new variants detected by DGGE and segregating with the FH phenotype in these families were identified in the FH cohort by molecular techniques previously described21 22 23 (Table 1⇓).
Means and SDs were calculated by using conventional methods. Differences in sex ratio among the three groups were determined by the χ2 test. Fisher's exact two-tail probability test was used to examine the frequency difference of tendon xanthomas between the groups and the difference between the FDB, FH, and control groups in overlap of LDL cholesterol above and below the 95th percentile. To determine the difference in the age distribution between the FDB, FH, and control groups, age categories were established as follows: <5, 5 to 10, 10 to 15, and 15 to 20 years. Fisher's exact two-tail probability test was employed to detect differences in the number of FDB, FH, and control children falling within each age category. As groups were not independent, within-group and between-group comparisons of means were determined by the nonparametric Mann-Whitney U test. Comparisons are made for males and females separately. For statistics on lipid parameters within the FDB and FH group by apoE genotype, when gender was shown to have a significant effect, it was included as a covariate in an ANOVA. When the influence of gender was not shown to have a significant effect, the nonparametric Mann-Whitney U test was performed to detect differences in lipid/lipoprotein levels by apoE genotype. Correlations between age and LDL cholesterol and between BMI and LDL cholesterol were calculated by using the Pearson correlation matrix. Statistics on triglyceride were performed on log-transformed data, as these data were not uniformly distributed. Statistical analyses were performed using Systat statistical software package (Version 5.2 for the Macintosh, UBC). Significance level was determined as P<.05.
Demographic and Clinical Characteristics
Age distributions for FDB, FH, and control groups are illustrated in Table 2⇓. No significant differences in number of children falling within each age category were noted between the FDB, FH, and control cohorts, with 39.5% of FDB, 42.3% of FH, and 34.3% of control subjects <10 years of age. As indicated in Table 2⇓, groups were matched for age and BMI. In addition, there was no significant difference in the sex distribution between the three groups. Two subjects with FDB and two with FH had evidence of peripheral tendon xanthomas, and in both FDB and FH groups the presence of tendon xanthomas did not correlate with LDL and total cholesterol levels (data not shown). No children in the FDB or FH groups had evidence of either arcus cornealis or xanthelasma.
Baseline plasma lipid and lipoprotein values are presented for females and males in Table 3⇓. As expected, total cholesterol, LDL cholesterol, and total cholesterol:HDL cholesterol ratio were significantly higher in FDB male and female heterozygotes than in control subjects (P<.001 for all three variables by sex). Triglycerides were lower in both female (P=.03) and male (P=.05) FDB heterozygotes than in sex-matched control subjects.
In female children, significantly lower levels of total cholesterol (P<.001), LDL cholesterol (P=.001), apoB (P=.001), and total cholesterol:HDL cholesterol ratio (P<.001) were noted in FDB than in FH heterozygotes. Similar results were observed in males (P=.005, P=.007, P=.07, and P=.01 for the four variables).
Overlap in LDL cholesterol was evident between FDB heterozygotes and control subjects (Figure⇓, panel A) and between FH heterozygotes and control subjects (Figure⇓, panel B). Four of 38 FDB carriers (10.53%) and 11 of 97 FH heterozygotes (11.34%) had an LDL cholesterol level below the 95th percentile for age and sex according to Lipid Research Clinics criteria. One of 73 control subjects (1.73%) had an LDL cholesterol above the 95th percentile for age and sex. No significant difference was noted in the percentage of FDB and FH children with LDL cholesterol falling below the 95th percentile for age and sex (data not shown).
Significantly higher LDL cholesterol levels (P=.04) were observed in female FDB carriers than in age-matched males with FDB. This sex difference was also noted in the FH cohort (Table 4⇓) but was not seen in control subjects.
Lipid Levels and ApoE Genotype
No significant difference in the apoE allele frequencies between the FDB and FH groups was noted (data not shown), and results were similar to what would be expected from a general population sample. Furthermore, no significant difference in the sex-adjusted lipid levels between the apoE genotypes in FDB or FH heterozygotes was noted (data not shown).
Lipid Levels by Age
Lipid and lipoprotein levels in female and male subjects <10 years old and in female and male subjects aged 10 to 20 years in FDB, FH, and control groups are shown in Table 5⇓. Females with FDB <10 years of age had significantly lower total cholesterol (P<.001), LDL cholesterol (P=.003), total cholesterol:HDL ratio (P=.008), and apoB (P=.03) than age-matched females with FH. For males <10 years old, a trend for lower total cholesterol, LDL cholesterol, total cholesterol:HDL ratio, and apoB levels in FDB heterozygotes was evident compared with males <10 years old with FH. Power to detect significance was limited by the small size of the FDB group (n=5). These results indicate that differences in lipoprotein values are evident between FDB and FH at an early age before puberty. Significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio were observed in FDB children than in control children <10 years of age. This was true for both males and females, illustrating that penetrance of FDB is evident at a very early age, as is well documented in young children with FH.1
In the older children (10 to 20 years), both males and females with FDB had significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio than age-matched control subjects. In this age cohort, both males and females with FDB showed a lower total cholesterol, LDL cholesterol, and total cholesterol:HDL cholesterol ratio than age-matched males and females with FH (Table 5⇑).
In FDB, FH, and control groups, no significant correlation was observed between age and LDL cholesterol levels (Figure⇑).
Lipid Levels by BMI
No significant correlation existed between BMI and LDL cholesterol levels in any of the groups studied (P=.77 for FDB, P=.92 for FH, and P=.34 for control subjects).
FDB is an autosomal dominant condition resulting from mutations in the gene for apoB, the major ligand for the LDLR.2 Previously it had been suggested that this condition was indistinguishable from FH both biochemically and clinically.11 12 13 It had been postulated that both FDB and FH resulted in similar elevations in plasma total and LDL cholesterol and a similar high incidence of premature atherosclerotic disease. A recent report, however, noted important differences between these two conditions, with a milder phenotype observed in FDB than in FH heterozygotes.8 These authors included a detailed meta-analysis of FDB and FH cohorts previously reported in the literature as well as describing their own FDB cohort.
It has previously been shown that homozygotes for FDB present with a significantly milder phenotype than the phenotype observed in FH homozygotes. Total and LDL cholesterol levels of 8.6 mmol/L and 6.9 mmol/L were reported in one adult FDB homozygote.10 These findings are supported by another report describing total cholesterol levels of 7.7 to 8.6 mmol/L in another FDB homozygote,24 cholesterol levels more in keeping with those seen in heterozygous FH. Furthermore, both FDB homozygotes (aged 54 and 31 years, respectively) were free of atherosclerotic disease, contrary to what is observed in homozygotes with FH, who frequently die of atherosclerotic-related disease in their teenage years.1
Reduced production or enhanced catabolism of VLDL remnants and/or LDL particles may be responsible for the milder biochemical phenotype in FDB, which in particular may be age related.8 Normal LDLR activity has been shown in subjects with FDB.6 It has also been shown previously that homozygotes with FDB retain 20% of normal binding of LDL.10 This residual binding of LDL was shown to occur in the large, low-density subfraction (1.019 to 1.034 kg/L) due to the apoE associated with these particles. The abnormal apoB itself was also shown to retain some residual binding in the intermediate-density subfraction (1.034 to 1.040 kg/L). The fact that certain LDL subfractions may bind to the normal LDLR in FDB and the possibility of enhanced apoE-mediated clearance of large LDL particles and VLDL remnants in subjects with FDB may in part explain the lower levels of LDL cholesterol in FDB compared with FH heterozygotes. These mechanisms would be impaired in LDLR-deficient subjects. In addition, possible upregulation of LDLR activity, in particular in younger subjects with FDB, may be a mechanism underlying the milder biochemical phenotype noted in FDB. This LDLR upregulation may also clear more efficiently the apoB produced by the normally functioning alleles.
In this report we have directly compared children <20 years old with molecularly defined FDB and FH, and our findings support the above hypotheses. We show significantly lower total and LDL cholesterol levels in children <20 years of age with FDB than in a cohort of age-, sex-, and ancestry-matched children with FH. In addition, a lower total cholesterol:HDL cholesterol ratio, a biochemical indicator of atherosclerotic disease,25 was noted in heterozygotes with FDB than in those with FH. Lower triglyceride levels were noted in male and female FDB children than in sex-matched control subjects (P=.05 and P=.03 for male and female, respectively) and a trend to lower triglycerides was also noted compared with children with FH (P=.26 and P=.06 for males and females, respectively). Similar findings were made by Miserez and Keller,8 whose study included a small number of individuals <20 years of age. On the contrary, studies in FH children have shown significantly higher triglyceride levels in FH subjects than in control subjects.26 Our finding of reduced triglyceride levels in FDB may support the hypothesis of an enhanced clearance of apoE-containing lipoproteins via normal functioning LDLRs in these individuals. The ability to compensate for defective apoB may be aided by upregulation of LDLRs in younger FDB subjects, whereas this LDLR-mediated clearance may not be possible to the same degree in FH heterozygotes. In addition, Innerarity et al6 and Raugh et al11 did not find evidence of reduced triglyceride levels in adults with FDB compared with non-FDB control subjects, suggesting that adults with FDB may have less ability to upregulate the clearance of apoB- and apoE-containing lipoproteins including VLDL remnants. Hansen et al,27 in fact, found higher triglyceride levels in a Danish cohort of FDB carriers than in age-matched family members without the apoB-3500 mutation, but it is unclear from this study how triglyceride levels were affected by age in FDB carriers. It is also unclear what hormonal differences were present in our FDB subjects as opposed to FH and control cohorts. Hormonal influences such as steroid or growth hormones, particularly through puberty, may significantly influence triglyceride metabolism and may be a factor predisposing to the lower triglyceride levels in children with FDB in our study.
Despite a milder biochemical phenotype in young children with FDB compared with FH heterozygotes, FDB still clearly expresses itself at an early age, as illustrated by the significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio in young FDB children (aged <10 years) compared with age-matched control subjects in our study. Penetrance, as defined by the ratio of carriers showing the trait (LDL cholesterol >95th percentile) to the total number of carriers, was 89.5% in FDB children, much like what was observed in our FH cohort (88.7%).
The milder biochemical phenotype observed in children with FDB compared with children with FH corresponds to observations of a milder clinical phenotype in adults with FDB compared with FH. As the incidence of atherosclerotic disease is determined by both the extent and duration of cholesterol elevation,1 lower cholesterol levels from a younger age would support a decreased incidence of vascular disease in adults with FDB compared with adults with FH. Interestingly, the significantly milder lipoprotein phenotype observed in FDB compared with FH heterozygotes was evident even at a very young age (<10 years).
In addition to the significant between-group differences, variation in LDL cholesterol levels existed within both the FDB and FH cohorts. Four (10.53%) of 38 and 11 (11.34%) of 97 FDB and FH heterozygotes, respectively, had LDL cholesterol values <95th percentile, as defined by Lipid Research Clinics Prevalence Study criteria for age and sex. This variability in cholesterol levels and overlap with control subjects for both total and LDL cholesterol existed among a cohort of subjects carrying the same apoB-3500 mutation. This has been previously observed in a small cohort of young FDB subjects compared with age-matched non-FDB relatives.27 Environmental factors known to affect lipids were examined as a possible cause of within-group variability in our study. Cigarette smoking, alcohol intake, drug ingestion (including oral contraceptives), and abnormal dietary habits were virtually nonexistent in the FDB cohort studied, and these particular environmental influences are therefore unlikely to account for this variability.
Sex differences may be one factor underlying the variability of lipid levels within the FDB group. A significant sex difference in FDB heterozygotes was noted with higher LDL cholesterol (P=.038) and a trend to higher total cholesterol (P=.165) levels evident in females with this condition compared with age-matched males. Similar sex differences have also been observed in adults with FDB8 and FH28 and children with FH.29 It has been suggested that these sex-specific differences may be a result of a referral bias rather than a real effect.29 However, as study subjects were collected by family studies of adult probands rather than by subject referral, referral bias would seem an unlikely explanation for these findings in our population, and the reason for this effect remains unclear. Whatever the underlying mechanism for this sex-specific effect, higher LDL and total cholesterol levels in females with FDB may account partly for the higher incidence of atherosclerotic disease observed in adult females with FDB than in age-matched males, as has been noted previously by other investigators.11 27 These sex differences noted in our cohorts of FDB and FH did not account for the overlap in LDL cholesterol levels between these groups and control subjects. In particular, of the 4 FDB subjects with LDL cholesterol <95th percentile, 2 were male and 2 were female. Similarly, of the 11 FH subjects with LDL cholesterol <95th percentile, 7 were male and 4 were female.
Age may be another factor underlying the within-group variability, as cholesterol levels have been shown to decrease at puberty in a “normolipidemic” population.30 In our study, however, no correlation was observed between age and baseline total or LDL cholesterol in either FDB or FH groups. A similar lack of correlation between age and LDL cholesterol was also observed in a group of FDB heterozygotes studied by Rubinsztein et al,31 although it should be noted that this group comprised only six related individuals. Similar findings were also noted in a group of FH children studied by Assouline et al.26
BMI was also not correlated with LDL cholesterol levels and not found to be a significant cause of overlap in lipid levels between the groups studied. These results conflict with reports in FH children, in which body fat has been postulated to be a significant predictor of LDL cholesterol and apoB levels.29
ApoE genotype was not a significant predictor of baseline lipid/lipoprotein levels, with no significant differences noted between apoE genotypes within the FDB group. These findings are in keeping with a recent study of young FH heterozygotes from Norway, which also did not show significant differences in lipids according to apoE genotype.29 As has been suggested for adults with FH, perhaps apoE-mediated influences on lipid/lipoprotein levels that are seen in “control” populations are unable to be detected due to a major underlying genetic disorder such as FDB.28
Unlike adults with FH and FDB, peripheral evidence of lipid deposition in children with these disorders is rare, particularly under 10 years of age. In previous studies in which a small number of FDB children were included, no evidence of peripheral lipid deposition was noted.11 31 In studies of FH children <20 years old, tendon xanthomas have been found to occur with frequencies between 0% and 7.9%.26 28 The similar incidence of tendon xanthomas in our FDB and FH cohorts, together with findings of other investigators above, would indicate that clinical stigmata would not differentiate FDB from FH in children.
From these data, it seems clear that FDB represents a significantly milder biochemical phenotype compared with age- and sex-matched children with FH. Despite these group differences, however, in view of wide variation of lipid levels within each group, lipid levels alone cannot be used to discriminate between FH and FDB. The precise mechanisms for the reduced cholesterol levels observed in FDB children are unclear but may be due to enhanced clearance of apoB and E lipoproteins via an LDLR pathway. Our findings may in part explain more recent reports indicating a lower incidence of atherosclerotic disease reported in adults with FDB than in those with FH.8
Selected Abbreviations and Acronyms
|DGGE||=||denaturing gradient gel electrophoresis|
|FDB||=||familial defective apoB-100|
This work was supported by a grant from the Netherlands Heart Foundation and MRC (Canada). Dr Kastelein is a Clinical Investigator of the Netherlands Heart Foundation. Dr Hayden is an established investigator of the BC Children's Hospital. We thank Lisette Feuth for her support in managing the data and Drs L.M. Havekes and M.P.R. Lombardi of the Gaubius Laboratory in Leiden, The Netherlands, for the characterization of the LDLR mutations.
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