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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 6 2167-2174
Copyright © 1998 by The Endocrine Society

Original Studies

Delayed Low Density Lipoprotein (LDL) Catabolism Despite a Functional Intact LDL-Apolipoprotein B Particle and LDL-Receptor in a Subject with Clinical Homozygous Familial Hypercholesterolemia

Hartmut H.-J. Schmidt, Manfred Stuhrmann, Robert Shamburek, C. Knud Schewe, Margit Ebhardt, Loren A. Zech, Carsten Büttner, Matthias Wendt, Ulrike Beisiegel, H. Bryan Brewer, Jr., and Michael P. Manns

Abteilung Gastroenterologie und Hepatologie (H.H.-J.S., C.B., M.W., M.P.M.) and Abteilung Humangenetik (M.S., M.E.), Medizinische Hochschule Hannover, Hannover, Germany; Medizinische Poliklinik (C.K.S.), Ludwig-Maximilians-Universität, München Medizinische Klinik I (U.B.), Universitäts-Klinik Eppendorf, Hamburg, Germany; and Molecular Disease Branch (R.S., L.A.Z., H.B.B.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Hartmut H.-J. Schmidt, Abteilung Gastroenterologie und Hepatologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We identified a 38-yr-old male patient with the clinical expression of homozygous familial hypercholesterolemia presenting as severe coronary artery disease, tendon and skin xanthomas, arcus lipoides, and joint pain. The genetic trait seems to be autosomal recessive. Interestingly, serum concentrations of cholesterol responded well to diet and statins. We had no evidence of an abnormal low density lipoprotein (LDL)-apolipoprotein B (apoB) particle, which was isolated from the patient using the U937 proliferation assay as a functional test of the LDL-binding capacity. The apoB 3500 and apoB 3531 defects were ruled out by PCR. In addition, we found no evidence for a defect within the LDL-receptor by skin fibroblast analysis, linkage analysis, single-strand conformational polymorphism and Southern blot screening across the entire LDL-receptor gene. The in vivo kinetics of radioiodinated LDL-apoB were evaluated in the proband and three normal controls, subsequently. The LDL-apoB isolated from the patient showed a normal catabolism, confirming an intact LDL particle. In contrast the fractional catabolic rate (d-1) of autologous LDL in the subject and the normal controls revealed a remarkable delayed catabolism of the patient’s LDL (0.15 vs. 0.33–0.43 d-1). In addition, the elevation of LDL-cholesterol in the patient resulted from an increased production rate with 22.8 mg/kg per day vs. 12.7–15.7 mg/kg per day. These data indicate that there is another catabolic defect beyond the apoB and LDL-receptor gene causing familial hypercholesterolemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FAMILIAL hypercholesterolemia (FH) is characterized by an autosomal dominant inheritance, excessive hypercholesterolemia, premature onset of atherogenesis, xanthomas, xanthelasms, and arcus lipoides (1, 2). Mutations within the apolipoprotein B (apoB) gene and the low density lipoprotein (LDL)-receptor gene are responsible for this disease entity. ApoB is the primary ligand of the LDL particle interacting with the LDL-receptor as the initial step for its cellular uptake. In vivo LDL kinetics and in vitro binding studies established the functional roles of apoB and the LDL-receptor. Molecular genetic analysis revealed two mutations within the apoB gene and more than 160 mutations within the LDL-receptor gene causing FH (3, 4, 5). Although several genes may also be candidates for causing FH, no other genetic locus has been detected so far. Interestingly, there is accumulating evidence of at least one additional site of genetic defect causing FH: Nora et al. (6) and Bilheimer et al. (7) reported a family in which some obligate FH heterozygotes had both normal cholesterol levels and normal LDL production rate. Schuster et al. (3) also reported two pedigrees with unusual genetic traits of severe hypercholesterolemia. Zuliani et al. (8) reported an unusual Italian pedigree with severe hypercholesterolemia without any evidence of a linkage with the LDL-receptor and the apoB gene locus, suggesting for the first time an autosomal recessive trait for an unknown new defect causing FH.

We report here for the first time an autosomal recessive inheritance of the clinical phenotype of homozygous FH, which is characterized by delayed LDL catabolism. Our data demonstrate that the underlying defect is not caused by abnormalities of the apoB gene or the LDL-receptor gene. In addition, there is no evidence of ß-sitosterolemia, abnormal apoE phenotype, or increased levels of Lp(a).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Case report

We report a 38-yr-old male Turkish patient with severe hypercholesterolemia (weight 58 kg, height 163 cm). He began to notice planar xanthomas at the knees and elbows when he was 7 yr old. He started military service in 1979, at which time he first noted arthritis pain, especially in both knees. The joint pain was migratory without any signs of reddish or swollen joints. He had pain attacks for up to several weeks with moments of not being able to walk. The first time cholesterol was determined, it ranged between 600–800 mg/dL. He was started on low-fat, low-cholesterol diet and several lipid-lowering drugs. He moved to Germany in 1983. Since then he was on different fibrates until 1989 without any effective cholesterol-lowering effect. He started Lovastatin in 1989. The highest cholesterol level was 1050 mg/dL off medicine. In 1991 he was referred to our clinic. At that time he had multiple tendon and cutaneous xanthomas (Fig. 1Go), symmetrically mild arcus lipoides, but no xanthelasms. He came without any prescribed lipid-lowering drug because of ineffective drug response. He was immediately started on Cholestyramine (stepwise, reaching a maximum tolerated dose at 24 g) plus 80 mg Lovastatin, which brought his cholesterol down to 300–350 mg/dL. Since then he never complained about joint pain any more. He never presented with clinical signs of coronary artery disease; however an exercise stress test elicited exertional coronary ischemia. Cardiac catheterization revealed severe diffuse coronary atherosclerosis with 70–80% stenosis of the ramus interventricularis anterior, ramus circumflexus, and right coronary artery. Coronary artery bypass graft for four vessels was performed in July 1991. Three months later he was started on weekly LDL apheresis. Lovastatin was replaced with 40 mg simvastatin without any further cholesterol-lowering effect. Currently, the patient presents no discomfort; he can swim and jog for 3–4 miles without chest pain or shortness of breath.



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  		Figure 1. Skin and tendon xanthomas of affected patient. A, B, and C, Tuberous skin xanthomas of both knees and achilles tendon xanthomas.

 
The family history is relatively unremarkable in terms of premature atherosclerosis or xanthomas. He has two healthy sons, a 7-yr-old and a 14-yr-old. In addition, he has seven healthy siblings. His parents are from the same village. Consanguinity is not known. Figure 2Go illustrates the family pedigree with lipid values. The individual percentile of age- and sex-adjusted LDL-cholesterol (P) are depicted for each family member in this figure, demonstrating that no other subject has severe hypercholesterolemia. Only his brother A.T. has a mild hypercholesterolemia with an LDL-cholesterol of 170 mg/dL. The genetic trait seems to exclude a mutation with autosomal dominant inheritance causing the phenotype of FH.



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  		Figure 2. Pedigree analysis. Affected patient is shown as a filled box. Initials of names; ages; and serum concentrations of total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL), HDL-cholesterol (HDL), and individual percentile of age- and sex-adjusted LDL-cholesterol (P) are depicted.

 
LDL-binding analysis

Determination of LDL-binding was performed using the U937 proliferation assay as previously described (9, 10). Because of a defect in the pathway of cholesterol synthesis, the human myelomonocytic tumor cell line U937 lacks the ability to synthesize cholesterol, which makes proliferation of these cells dependent on the presence of exogenous LDL-cholesterol in the cell culture medium (11, 12). LDL-cholesterol is taken up via the apoB, E receptor. LDL particles with a binding defect of the apoB-100 cannot be taken up by the cells, resulting in a reduction of the proliferation rate. U937 cells were incubated with LDL from the index patient, one patient with familial defective apoB-100 (FDB, positive control), two healthy normocholesterolemic individuals (NC, negative controls), and four patients with FH because of a defective LDL-receptor (FH, negative controls). U937 cells were grown in RPMI 1640 supplemented with glutamine and 10% FCS (GIBCO BRL, Glasgow, UK) at 37 C in an atmosphere of 5% C02. Fresh medium was added three times a week, and cells were kept at a density between 1–8 x 105 cells/mL. The viability of cells as assessed by the trypan blue exclusion test, and the size distribution curve of a computerized cell counter analyzer system (CASY-I, Schärfe System GmbH, Reutlingen, Germany) was above 85%. Before incubation with LDL U937 cells were washed three times in PBS and cultivated for 24 h in RPMI 1640 without FCS to achieve intracellular cholesterol depletion. Serum-starved cells were seeded at a defined density (2 x 105 cells/mL) in multiwell tissue culture plates (24 wells; Costar, Cambridge, MA) containing RPMI 1640 with the addition of LDL-fractions at LDL-cholesterol concentrations of 5 µg/mL from patients and controls. Each individual incubation was performed in four different wells at the same time. After 4 days, 6-fold counts were performed from each well, and the average cell number, including standard deviation, was calculated with a computer-assisted electronic cell counter system (CASY-I).

ApoB 3500 and apoB 3531 defect

Presence for the arginine3500->glutamine and arginine3531->cysteine mutations were investigated by PCR, followed by digestion with the restriction enzymes MspI and NsiI, respectively (5, 13).

LDL-receptor mutation analysis

Using single-strand conformation polymorphism (SSCP) and Southern blotting, we screened the entire coding region of the LDL-receptor gene of the index patient for aberrant band patterns indicative for disease-causing mutations. SSCP analysis was performed according to Hobbs et al. (14), and Southern blotting and hybridization with a LDL-receptor complementary DNA probe were performed according to standard procedures.

LDL-receptor linkage analysis

For linkage analysis, isolated DNA from the patient and his family members was amplified by PCR (exons 8, 12, 13, and 15) using the primers previously published by Hobbs et al. (14). The PCR products were digested with the restriction enzymes StuI, HincII, AvaII, and MspI, respectively, for the detection of restriction fragment length polymorphisms within the corresponding exons (15).

Microsatellite analysis was also performed using 13 different markers routinely applied for paternity testing to confirm the given pedigree information (16).

LDL-receptor-binding

LDL-receptor analysis was done by a modification of the method by Goldstein and Brown (17, 18). Skin fibroblasts were incubated with I125-labeled LDL in 24-well plates (10,000 cells/well). Lipoprotein-deficient serum was obtained by recalcified human plasma from normal volunteers.

Metabolic study

The reported index patient and three normal controls participated in a metabolic study. They were hospitalized for the study in the Clinical Center of the NIH. All subjects had no hepatic, hematological, or renal abnormalities. The subjects were not on any medications from 2 weeks before initiation of the study to the end of the study. All subjects gave informed consent. The study protocol was approved by the Human Use Research Committee of the National Heart, Lung, and Blood Institute of NIH.

Isolation of LDL

LDL was isolated between d = 1.019 g/mL and d = 1.063 g/mL by ultracentrifugation, purified by recentrifugation for 22 h at d = 1.070 g/mL, and subsequently dialyzed and concentrated against 50 mM sodium phosphate-100 mM saline (pH 7.4) (19).

Iodination of LDL

The prepared samples were sterilized by filtration through a 0.22-µm filter, radioiodinated by a modification of the iodine monochloride method (20, 21). One milliliter of the prepared LDL solution was added to a 1 mL solution of 1 M glycine (pH 10). Five microcuries I125 and I131, respectively, were added to each solution, followed by the slow addition of iodine monochloride. The quantity of iodine monochloride added was calculated to yield 1 mol iodine monochloride. Each sample was dialyzed against 50 mM sodium phosphate-100 mM saline (pH 7.4), sterilized by filtration through a 0.22-µm filter, and tested for pyrogens before injection into the study subjects.

Study protocol

The study subjects were placed on an iso-weight diet. Caloric intake was 42% carbohydrate, 42% fat, 16% protein, 200 mg cholesterol/1000 kcal, and a polyunsaturated/saturated fat ratio of 1:3, resulting into a limited variation in plasma cholesterol, triglyceride, and apolipoprotein concentrations. Two days before injection, the subjects were started on potassium iodide (1200 mg/day). Subjects were injected iv with both 25 µCi I131 and 15 µCi I125. Blood samples were obtained at 10 min; 1, 3, 6, 12, 24, and 36 h; and on days 2, 3, 4, 5, 7, 8, 9, 10. Samples were collected into tubes containing EDTA at a final concentration of 0.01%, stored at 4 C and centrifuged (2000 rpm, 30 min) at 4 C. Aprotinin and Na-azide were added to each plasma sample at a final concentration of 0.05% and 200 kallikrein inhibiting units/mL. The plasma lipoproteins were isolated by ultracentrifugation; the radioactivities in plasma and the lipoprotein subfractions were quantitated in a Packard auto gamma spectrometer, and their apoB concentration determined.

Analytical methods

Plasma cholesterol and triglycerides were quantitated on an enzymic analyzer (Gilford System 3500). High density lipoprotein (HDL)-cholesterol was determined in plasma after dextran sulfate precipitation (22). The remaining lipid and lipoprotein analyses were performed by the methods of the Lipid Research Clinics (23). ApoB and apoA-I concentrations were determined by a competitive enzyme-linked immunosorbent assay, using polyclonal antibodies directed against apoB and apoA-I, respectively. Lipoproteins were isolated by sequential ultracentrifugation for the composition analysis (24). The following density fractions were obtained: very low density lipoprotein (VLDL) (d < 1.006 g/mL), IDL (1.006 g/mL < d < 1.019 g/mL), LDL (1.019 g/mL < d < 1.063 g/mL), HDL2 (1.063 g/mL < d < 1.121 g/mL), HDL3 (1.121 g/mL < d < 1.210 g/mL), and very high density lipoprotein (VHDL) (1.210 g/mL < d <1.250 g/mL). Phospholipids were assayed chromatographically as described (25).

The residence time (1/fractional catabolic rate) was calculated from the area under the multiexponential plasma decay curve by a multiexponential computer curve-fitting technique, using the SAAM Manual (26). The production rate was determined by dividing the pool size by the residence time. The pool size equals the apolipoprotein concentration multiplied by the plasma volume per kilogram body weight. The plasma volume is determined by dividing the total quantity injected by the radioactivity per unit volume determined in the sample obtained 10 min after injection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We report an unusual case with the clinical signs of severe FH. All studied relatives including the parents and the children had no remarkable hypercholesterolemia (Fig. 2Go). The 7-yr-old son had a LDL-cholesterol of 105 mg/dL, a VLDL-cholesterol of 13 mg/dL, and a HDL-cholesterol of 51 mg/dL. The 15-yr-old son had a LDL-cholesterol of 102 mg/dL, a VLDL-cholesterol of 11 mg/dL, and a HDL-cholesterol of 53 mg/dL. The lipid profile of the index patient 2 weeks after discontinuation of his lipid-lowering treatment is shown in Table 1Go. His apoE phenotype was 3/3, ß-sitosterol was 0.50 mg/dL, campesterin 0.69 mg/dL, lathosterin 1.27 mg/dL, and cholestanol 0.66 mg/dL. Therefore, there was also no evidence of type III hyperlipidemia, ß-sitosterolemia, and cerebrotendinous xanthomatosis. Another interesting finding was the excellent response to diet and statins. Initially, when we first saw the patient, his total serum cholesterol was 820 mg/dL (LDL-cholesterol, 752 mg/dL); 3 weeks later on 80 mg Lovastatin and 16 g Cholestyramine his total cholesterol was 463 mg/dL (LDL-cholesterol, 400 mg/dL). Additional dietary counseling resulted in a further decrease of the serum concentration of total cholesterol to 332 mg/dL (LDL-cholesterol, 239 mg/dL). We also evaluated the composition of the lipoprotein subfractions in the index patient (Table 1Go), reflecting a normal composition of the VLDL, IDL, HDL2, HDL3, and VHDL fraction, whereas the LDL fraction showed an increase of all studied parameters, especially total cholesterol and phospholipids.


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  		Table 1. Lipoprotein composition analysis of index patient (IP)

 
We tested the possibility of a binding defect of apoB-100 in the index patient to further elucidate the genetic defect of this unusual case. The U937 proliferation assay as a functional test of the LDL-binding capacity was performed. The proliferation rate of U937 cells after incubation with LDL from the index patient was within the normal range, indicating normal binding of the LDL particles from the index patient (Fig. 3Go). In addition, we excluded the apoB 3500 defect and the apoB 3531 defect (data not shown).



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  		Figure 3. LDL-binding analysis using U937 proliferation assay. Average cell number per microliters and SD (6-fold counts/, 4 wells/individual) are illustrated after 4 days incubation in RPMI 1640 medium alone (RPMI), RPMI 1640 medium containing 5.0 µg/mL LDL-cholesterol from index patient (IP), one patient with familial defective apoB-100 (FDB, positive control), four patients with FH (FH, negative control), and two normocholesterolemic controls (NC, negative control). Reduced proliferation rate seen in incubation with LDL from FDB patients in contrast to index patient and both normal and hypercholesteremic controls indicate normal binding of LDL of studied patient.

 
Because we had no evidence of a ligand-defective apoB, we subsequently elucidated the LDL-receptor. The binding of iodinated LDL isolated from a normolipemic control was studied using skin fibroblasts of the patient. Figure 4Go illustrates the normal binding and uptake expressed in nanomoles per milligrams of the patient compared with skin fibroblasts from a normal control subject. In addition, we obtained normal degradation of internalized LDL by skin fibroblasts derived from the patient. The additional SSCP and Southern blot analysis of all exons of the LDL-receptor followed by direct sequencing revealed no disease-causing mutation. However, we detected already-known polymorphisms in the studied patient. We evaluated the family for these polymorphisms. Figure 5BGo depicts the StuI polymorphism within exon 8, which results in a 175-bp fragment in contrast to the commonly seen 136-bp fragment, whereas Fig. 5CGo illustrates the AvaII polymorphism within the exon 13 causing an additional restriction site generating a 140-bp fragment. We confirmed these findings by testing the HincII polymorphism for exon 12 and the MspI polymorphism for exon 15. This linkage analysis demonstrated that the unaffected siblings nos. 5, 6, and 9 of the illustrated pedigree (Fig. 5AGo) carry the same paternal LDL-receptor allele as the index patient (no. 10). One brother (no. 8) had inherited the same maternal LDL-receptor allele as the proband (no. 10). The probands nos. 4 and 7 are discordant to the patient in regard to both parental LDL-receptor alleles. Thus none of the siblings have the identical LDL-receptor genotype compared with the index patient. None of the siblings nos. 5, 6, 8, and 9 who share one parental allele with the index patient had clinical and biochemical signs of FH heterozygosity. The microsatellite analysis confirmed the given pedigree information.



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  		Figure 4. LDL-receptor binding analysis. Fibroblasts were obtained by in vitro culture of a skin biopsy from index patient and a normal control subject. Binding and uptake of I125-labeled normal LDL of skin fibroblasts from both studied subjects are illustrated. Data represent mean of quadruplicate assays. In addition, we observed normal degradation (data not shown).

 


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  		Figure 5. Linkage analysis of LDL-R gene. A, Patient’s pedigree, confirmed by microsatellite analysis. B and C, Cosegregation analysis at two polymorphic restriction sites within LDL-R gene. +, Presence of specific restriction sites; -, absence for absence of specific restriction sites. B, StuI polypmorphism within exon 8 of LDL-R gene. Father (no. 3) transferred by inheritance the 175-bp fragment to his children nos. 5, 6, 9, and 10 (index patient). C, AvaII polymorphism within exon 13 of LDL-R gene. Mother (no. 2) transferred by inheritance the 140-bp fragment to her children no. 8 and 10 and to her grandchildren nos. 11 and 12 (index patient). None of patients siblings carry identical LDL-R genotype compared with patient. This pedigree differs from Fig. 2Go, because we were not able to obtain from all relatives EDTA-blood.

 
Because both the apoB and the LDL-receptor were not abnormal, we performed in vivo LDL kinetics to characterize the metabolic etiology of this disease. The characterization of the kinetically studied subjects is illustrated in Table 2Go. LDL from the patient (A.H.) and from normolipemic controls were radioiodinated and injected simultaneously into the patient and the control subjects. Figure 6Go illustrates the radioactivity decay curves of serum samples after injection of normal LDL and LDL isolated from the patient into a control subject. Both curves were superimposable, reflecting that the LDL particle from the patient was kinetically normal. Figure 7Go depicts the comparison of the radioactivity decay curves of the patients’ LDL injected into the patient and normal LDL injected into the normal control subject. The patients’ LDL was catabolized at a much slower rate. Figure 8Go reflects the radioactivity decay curves after injection of normal LDL and LDL isolated from the patient into the patient. The radioactivity curves are superimposable and turned over at a much slower catabolic rate, confirming both a normal metabolized LDL particle of the patient and a remarkable decreased catabolism of normal and patients’ LDL in the patient. Table 3Go presents the kinetic parameters of the LDL metabolism. The residence time of LDL was 6.7 days in the patient compared with 2.3–3.0 days in the control subjects. The calculated production rate was 22.8 mg/kg per day in the patient compared with 12.7–15.7 mg/kg per day in the control subjects, demonstrating an upregulation of the synthesis rate of the patients’ LDL. This oversynthesis of LDL because of decreased catabolism is well established in FH patients.


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  		Table 2. Characterization of study subjects

 


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  		Figure 6. Metabolism of both normal and patient’s LDL after injection into a normal subject. Plasma decay curves as percentage of injected dose after simultaneous injection of I125-labeled LDL from a normal control ({blacktriangleup}) and I131-labeled LDL from patient ({blacksquare}) into a normal control subject is illustrated. Almost superimposable decay curves reflect physiologically intact LDL-apoB particle of studied patient.

 


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  		Figure 7. Metabolism of autologous LDL in a normal control subject and in index patient. Plasma decay curves as percentage of injected dose after injection of autologous patient’s I131-labeled LDL into patient ({blacksquare}) and normal I125-labeled LDL into a normal control subject ({blacktriangleup}) are illustrated. This experiment demonstrates slower catabolism of LDL after injection into patient. Thus defect of metabolism in patient is on catabolic site.

 


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  		Figure 8. Metabolism of both normal and patient’s LDL after injection into patient. Plasma decay curves as percentage of injected dose after simultaneous injection of I125-labeled LDL from a normal control ({blacktriangleup}) and I131-labeled LDL from patient ({blacksquare}) into index patient is illustrated. Almost superimposable decay curves reflect physiolocally intact LDL-apoB particle of studied patient, whereas both radiolabeled particles were cleared at a reduced rate from plasma as shown in Fig. 7Go.

 

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  		Table 3. Kinetic parameters of LDL-apoB in studied subjects

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The clinical phenotype of FH is caused by impaired catabolism of LDL either because of a defective binding of LDL to the apoB, E receptor or to mutations within the LDL-receptor gene. There have been two defects within the receptor binding region of the apoB-100, which is also called familial defective apoB, and more than 160 different mutations of the LDL-receptor gene identified that cause FH (1, 3, 4, 5). However, no other genes have been shown so far to be involved in the phenotypical expression of FH. We present an unusual case of clinical homozygous FH with a presumably autosomal recessive inheritance and onset of xanthomas as early as in childhood. Similar cases already have been reported as pseudo-FH in the literature (27). However, pseudo-FH patients seem to have a less pronounced cholesterol level and no tendon xanthomas (28). Another interesting finding is the excellent response to diet and especially to statins in our patient, which is not seen in patients with homozygous LDL-receptor deficiency, but in patients with pseudo-FH (27, 28). This reported case had severe joint pain independent from the localization of xanthomas, which disappeared after lowering his cholesterol. This finding already has been already reported occasionally (29). However, no exact mechanism for the association of high serum cholesterol and joint pain has been found so far. We excluded an abnormal LDL-apoB binding and uptake by the U937 proliferation assay. The U937 proliferation assay reliably detects defective binding of LDL because of the Arg3500->Gln mutation (9, 10, 30, 31). The reduction of the proliferation rate of U937 cells after incubation with LDL from patients with familial defective apoB-100 is 31–88% (9, 10, 30). The sensitivity of the U937 assay is not influenced by the patients’ age, sex, treatment, apoE genotype, cholesterol, Lp(a), and triglyceride levels (10, 30). The sensitivity of the U937 proliferation assay to detect defective binding because of other mutations in the apoB gene such as the newly discovered Arg3531->Cys has yet to be defined. However, because uptake of LDL-cholesterol by U937 cells is LDL-receptor mediated, another mutation in the apoB gene that causes a significant reduction of LDL binding, should also be expected to result into a reduced proliferation rate. Binding affinity of LDL particles with the Arg3531->Cys mutation is only 27% as compared with normal LDL. This is comparable with the reduction of the binding affinity in patients with the Arg3500->Gln mutation (36%) (5). In addition, we excluded the apoB 3500 and apoB 3531 gene defects and confirmed a metabolically normal LDL-apoB particle by in vivo kinetics. We had no evidence for a defect within the LDL-receptor gene from the analysis of skin fibroblasts, SSCP and Southern blot analysis, and cosegregation analysis. Although none of the siblings had the identical LDL-receptor genotype as the patient, the four siblings with one identical allele had no clinical signs of FH heterozygosity. Additional evidence against mutations within the LDL-receptor gene comes from the clinical presentation of both normolipemic children from the patient’s, who otherwise should present clinical and biochemical signs of heterozygous FH. Because the so-far-known genetic defects causing FH were extremely unlikely, we studied the metabolic fate of LDL to characterize the metabolic etiology of this disease. We obtained a remarkable prolonged residence time and an upregulated synthesis of the patients LDL-apoB particle compared with normal controls. The upregulation of LDL-apoB synthesis in the presence of a catabolic defect is well established for defects within the LDL-receptor gene blocking the binding of both IDL and LDL (1). Therefore, our data suggest that the underlying cause of severe hypercholesterolemia in the reported case is the catabolic site of LDL metabolism.

Lestavel-Delattre et al. (32) reported 41 heterozygous FH patients, identifying 9 of them as having neither an apoB 3500 defect nor a LDL-binding defect using skin fibroblast analysis and an indirect immunocytofluorimetric assay on lymphocytes (32). The pedigree analysis was not mentioned in their study. Because the immunocytofluorimetric approach is very unreliable in our hands, we used skin fibroblast analysis and excluded a cosegregation with the LDL-receptor gene by linkage analysis. Zuliani et al. (8) characterized an Italian pedigree with autosomal recessive inheritance and clinical homozygous FH. Using linkage analysis, they excluded the LDL-receptor and the apoB genes as the underlying defect. This confirms our observation that there are defects causing homozygous FH that are different from the LDL-receptor and the apoB genes. We extended the analysis of this new type of severe hypercholesterolemia, revealing the catabolic site of LDL metabolism as the underlying cause.

The genetic characterization of patients with FH revealed that subjects with the same gene defect may present clinically very variable (1, 3, 33). One of the proposed reasons is gene-gene interactions influencing the LDL-receptor activity, resulting in different levels of serum cholesterol. However, it is very unlikely that this would explain our observation, because the LDL-cholesterol levels differed extremely. Other potential candidate genes such as an abnormal apoE, increased Lp(a), decreased HDL-cholesterol, increased IDL-cholesterol, and postprandial lipemia were excluded in this reported case. In addition, defects in enzymes involved in lipid metabolism such as lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, and cholesterol ester transfer protein can also be excluded. Recently, the identification of the LDL-receptor-related protein and the VLDL-receptor revealed new insights into additional receptor-dependent pathways involved in cholesterol metabolism (34, 35). However, no defects have been reported in these genes so far. Potentially, a defect within the VLDL-receptor may result into an accumulation of VLDL-cholesterol and chylomicrons, because this receptor binds triglyceride-rich particles in vitro mediated by apoE and lipoprotein lipase (36, 37, 38). In contrast, our case had no evidence of increased levels for VLDL and chylomicrons. A defect within the LDL-receptor-related protein would presumably also result into an accumulation of triglyceride-rich lipoprotein particles (39, 40). As mentioned above, our index patient had no increase of the chylomicron or VLDL fraction. Unfortunately, we identified only one affected subject in this family. Therefore, we were not able to perform linkage analysis to reveal the defect on the molecular level. More affected subjects are required to address this question. Another explanation for our case could be an immunological response against the LDL-receptor resulting in competitive inhibition of the LDL particle. A similar phenomenon was already identified in a subject with chylomicron syndrome because of antibodies against lipoprotein lipase (41). However, we had no clinical or biochemical signs of an underlying autoimmune disease in this patient. In this context there is also the possibility of a protein that specifically inhibits the ligand binding of the LDL-receptor such as the interaction of the 39-kDa receptor associated protein with the LDL-receptor-related protein/{alpha}2-macroglobulin receptor and the VLDL-receptor (42, 43, 44). We currently address these different options for identifying the underlying defect. In conclusion, our case provides the first evidence of a catabolic defect beyond the apoB and LDL-receptor gene causing FH. The primary defect has yet to be defined.


   Acknowledgments
 
We thank Professor K. von Bergmann of the Department of Clinical Pharmacology in Bonn for gas chromatographic analysis for exclusion of ß-sitosterolemia and cerebrotendinous xanthomatosis.

Received December 3, 1997.

Revised January 28, 1998.

Accepted February 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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