A novel type of familial hypercholesterolemia: double heterozygous mutations in LDL receptor and LDL receptor adaptor protein 1 gene

Background

Autosomal recessive hypercholesterolemia (ARH) is an extremely rare inherited hypercholesterolemia, the cause of which is mutations in low-density lipoprotein (LDL) receptor adaptor protein 1 (LDLRAP1) gene.

Methods

A total of 146 heterozygous familial hypercholesterolemic (FH) patients with a mutation in LDLR gene were screened for genes encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1.

Results

Among the 146 subjects, we identified a 79-year-old Japanese female with double mutations in LDLR gene (c.2431A > T) and LDLRAP1 gene (c.606dup). Two other relatives with double mutations in those genes in her family were also identified. Although the proband exhibited massive Achilles tendon xanthoma and coronary and aortic valvular disease, serum LDL-C level of subjects with double mutations was similar with that of subjects with single LDLR mutation (284.0 ± 43.5 versus 265.1 ± 57.4 mg/dl).

Conclusion

Additional mutation in LDLRAP1 may account for severer phenotype in terms of xanthoma and atherosclerotic cardiovascular disease in FH patients.     

Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1

Scavenger receptor BI (SR-BI) is a multiligand cell-surface receptor that plays a central role in high density lipoprotein homeostasis in rodents. To investigate a role for SR-BI in atherosclerosis, mice with attenuated SR-BI expression were crossed with low density lipoprotein (LDL) receptor-deficient mice. Compound-homozygous mutants showed increased plasma cholesterol, surprisingly due primarily to increased LDL cholesterol and apolipoprotein B levels. LDL turnover studies showed that this resulted from increased LDL cholesterol production rather than decreased LDL catabolism. Atherosclerotic lesion size was significantly increased in male compound-mutant mice relative to LDL receptor-deficient controls (93 427+/-16 079 versus 34 448+/-5 331 microm(2), respectively; P=0.003). The proatherogenic effect of attenuated SR-BI expression may in part be due to increased LDL cholesterol levels. These findings suggest that upregulation of the receptor could have therapeutic potential for the treatment of atherosclerosis.     

Combined effects of cholesterol reduction and apolipoprotein A-I expression on atherosclerosis in LDL receptor deficient mice

Reduction of total and LDL cholesterol reduces atherosclerosis and clinical cardiovascular events. High density lipoprotein (HDL) cholesterol levels have a strong inverse association with atherosclerosis, and overexpression of apolipoprotein A-I (apoA-I), the major protein component of HDL, reduces atherosclerosis in hypercholesterolemic animals. However, little is known about the potential for additive or synergistic effects between cholesterol reduction and apoA-I overexpression on atherosclerosis. In the current study, we tested the hypothesis that significant reduction of plasma cholesterol combined with overexpression of apoA-I would reduce atherosclerosis to a greater extent than either one alone. We used somatic gene transfer of the LDL receptor (to induce cholesterol reduction) and apoA-I in LDL receptor deficient mice fed a Western type diet and compared the combination to expression of each gene alone and to controls. Atherosclerosis was quantitated using two independent methods, by en face analysis of the entire aorta and by cross-sectional analysis of the aortic root. Although the reduction of cholesterol was transient, expression of the LDL receptor alone significantly reduced atherosclerosis by 45% in the aorta and 44% in the aortic root compared with controls. Overexpression of human apoA-I alone reduced atherosclerosis by 42% in the aorta and 44% in the aortic root compared with controls. Co-expression of the LDL receptor with apoA-I resulted in significantly higher levels of apoA-I than expression of apoA-I alone. Although co-expression of the LDL receptor and apoA-I reduced atherosclerosis by 37% in the aorta and 32% in the aortic root compared with controls, the reduction in atherosclerosis was no different than that seen with expression of the LDL receptor alone or apoA-I alone. In summary, in this relatively short-term murine model, simultaneous reduction of cholesterol and expression of apoA-I was associated with higher levels of apoA-I than expression of apoA-I alone but did not result in greater reduction in atherosclerosis compared with either one alone.     

Expression of LDL receptor and uptake of LDL in mouse preimplantation embryos

OBJECTIVES: The aim of this study is to address the role of low-density lipoprotein (LDL) on mouse preimplantation embryos. METHODS: The temporal expression of low-density lipoprotein receptor (LDLR) and side-chain cleavage cytochrome P450 (P450scc) mRNAs in mouse oocytes and preimplantation embryos up to the hatched blastocyst stage were analyzed by RT-PCR and nested PCR techniques. Simultaneously, the expression of LDLR in the protein level was analyzed by fluorescent immunohistochemistory at oocyte, 4-cell embryo and blastocyst. Uptake of LDL was analyzed using the LDL labeled with the fluorescent probe DiO. RESULTS: LDLR mRNA was detected at oocyte, 8-cell, morula, blastocyst and hatched blastocyst stages. P450scc mRNA was detected at oocyte, 1-cell, 2-cell, 4-cell, blastocyst and hatched blastocyst stages. LDLR protein was detected in blastocyst. P450scc protein was detected in oocyte, 4-cell, and blastocyst. LDL-DiO was taken into embryo at blastocyst stage and this uptake was competitively suppressed by excess unlabeled LDL. CONCLUSIONS: It is suggested from the present study that LDLR may play an essential role in uptake of exogenous LDL into blastocyst stage and cholesterol derived from LDL may be the source of steroid hormone synthesis.     

Familial hypercholesterolaemia and LDL receptor mutations.

Inherited defects in the gene for the low density lipoprotein (LDL)-receptor give rise to familial hypercholesterolaemia (FH), a disorder in which defective catabolism of LDL causes a marked increase in its concentration in plasma. As a result, there is excessive deposition of cholesterol in the arterial wall leading to accelerated atherosclerosis and premature coronary heart disease in most patients, although there are differences in its severity. Many different mutations have been found in the LDL receptor genes of FH patients, and although this heterogeneity has provided information about the relationship between structure and function in different domains of the protein, it makes simple DNA-based diagnosis of the disease impossible. When sufficient groups of patients with defined mutations are available it will be possible to determine the relative importance of any particular mutation compared with other genetic or environmental factors in relation to the severity of their symptoms or their response to treatment.     

Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro.

Familial hypercholesterolemia is caused by a genetic deficiency of the low density lipoprotein (LDL) receptor. The Watanabe heritable hyperlipidemic (WHHL) rabbit, which is also defective in LDL receptor activity, provides an excellent animal model of homozygous familial hypercholesterolemia. As a step toward development of effective gene therapy for familial hypercholesterolemia, we have constructed a transmissible retroviral vector containing a full-length human cDNA for the LDL receptor. WHHL fibroblasts infected in vitro expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies. Infected fibroblasts bound and degraded a monoclonal antibody specific for the human LDL receptor (IgGC7) in a manner comparable to that seen with normal human fibroblasts. Human LDL was also degraded by infected WHHL cells and promoted cholesterol esterification to the same degree as seen in normal human fibroblasts. Although technical problems remain to be solved, these studies show that, in principle, gene therapy may be possible for familial hypercholesterolemia patients.     

Attenuated expression of profilin-1 confers protection from atherosclerosis in the LDL receptor null mouse

Atherosclerosis-related events are a major cause of morbidity and death worldwide, but the mechanisms underlying atherogenesis are not fully understood. We showed in previous studies that the actin-binding protein profilin-1 (pfn) was upregulated in atherosclerotic plaques and in endothelial cells (ECs) treated with oxidized low-density lipoproteins (oxLDL). The present study addressed the role of pfn in atheroma formation. To this end, mice with heterozygous deficiency of pfn, Pfn(+/-), were crossed with Ldlr(-/-) mice. After 2 months under a 1.25% cholesterol atherogenic diet, Pfn(+/-)Ldlr(-/-) (PfnHet) exhibited a significant reduction in lesion burden compared with Ldlr(-/-) control mice (PfnWT), whereas total cholesterol and triglyceride levels were similar in the 2 groups. Relevant atheroprotective changes were identified in PfnHet. When compared with PfnWT, aortas from PfnHet mice showed preserved endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO)-dependent signaling, and reduced vascular cell adhesion molecule (VCAM)-1 expression and macrophage accumulation at lesion-prone sites. Similarly, knockdown of pfn in cultured aortic ECs was protective against endothelial dysfunction triggered by oxLDL. Finally, bone marrow-derived macrophages from PfnHet showed blunted internalization of oxLDL and oxLDL-induced inflammation. These studies demonstrate that pfn levels modulate processes critical for early atheroma formation and suggest that pfn heterozygosity confers atheroprotection through combined endothelial- and macrophage-dependent mechanisms.     

Oxidized LDL and expression of monocyte adhesion molecules

Accumulation of substantial numbers of monocyte/macrophages, as well as activated T lymphocytes, in focal areas of arterial intima appears to be a hallmark of atherogenesis. Our report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins, such as oxidized LDL and remnants lipoproteins in diabetic and type III hyperlipidemic patients, can upregulate adhesion molecules for monocytes and T lymphocytes, and growth factors, such as heparin-binding epidermal growth factor-like growth factor and PDGF-A and B chains. Recently we identified the novel receptor for oxidized LDL, named Lox-1. Therefore in this paper we summarize the importance of the interaction between oxidized LDL and its receptor, LOX-1 in terms of early stage of atherogenesis.     

Diverse roles for the LDL receptor family.

The low-density lipoprotein (LDL) receptor family consists of several related scavenger receptors that not only function as important cargo transporters, but also inform the cell of changes in its environment by mediating signaling responses. The LDL receptor was the first family member to be characterized and its function seems to be restricted to lipoprotein metabolism. By contrast, lipoprotein metabolism does not appear to be the exclusive function of the other characterized LDL receptor family members. It is now apparent that cargo transport by members of the LDL receptor family is closely associated with regulation of cellular physiology and cellular signaling events. Here, we focus on the diverse biological activities of certain members of this family.     

Interaction between LDL receptor and lipoproteins containing apo B]

The physiocochemically defined lipoproteins such as VLDL, LDL are comprised of subpopulations with different lipid and apolipoprotein composition. In order to determine the respective roles of different apolipoproteins (B, C-III, E) in their metabolism, four species (LpB, LpB:E, LpB: C-III and LpB: C-III: E) have been separated by sequential immunoaffinity chromatography. We examined the binding characteristics of each lipoprotein to HeLa cells and expressed the results in relation to the number of moles of apo B. LpB particles which contained apo B as their sole apolipoprotein had lower affinity for the LDL receptor that did total LDL but an apparently higher number of binding sites. The presence of apo E of phenotype E3 or E4 on one particle increased the affinity for the receptor. The apparent number of binding sites decreased probably due to the fact that a particle containing multiple copies of apo E bound to more than one molecule of receptor. Interaction with several LDL receptors would also explain the higher binding affinity which we observed. When the apo E phenotype was E2/E2, the LpB: E particle did not bind to the receptor. We showed also that apo C-III, when present, diminished the binding of apo B containing lipoproteins. These data suggest that apolipoproteins E and C-III impaired the interaction of apo B with the LDL receptor. It is likely that in LpB: E only apo E (in the case of E3 or E4 phenotype) participates in the LDL receptor binding.     

Low density lipoprotein (LDL) binding affinity for the LDL receptor in hyperlipoproteinemia.

We measured the binding affinity of low density lipoprotein (LDL) for the LDL receptor in patients with various types of hyperlipoproteinemia and investigated the effects of LDL lipid composition and particle size on receptor affinity. LDL (1.019 < d < 1.063) was isolated by sequential ultracentrifugation from the serum of normolipidemic controls and patients with hyperlipoproteinemia. Patients with type IIa hyperlipoproteinemia had LDL with a similar receptor affinity to that of normal LDL. However, patients with hypertriglyceridemia (type IIb and type IV hyperlipoproteinemia) had LDL with a low receptor affinity, and the degree of the reduction in affinity paralleled the severity of the hypertriglyceridemia. The LDL of hypertriglyceridemic patients was rich in protein and triglycerides, had a low content of cholesterol and phospholipids, and was smaller than normal, thus resembling the atherogenic lipoprotein known as small, dense LDL. These abnormalities were observed even in patients with mild hypertriglyceridemia regardless of their serum cholesterol levels. The degree of alteration in LDL lipid composition and particle size was strongly associated with the reduction of LDL receptor affinity. We also examined the effects of two lipid-lowering agents (bezafibrate and probucol) on the characteristics of LDL. LDL receptor affinity was only improved when the lipid composition and particle size were normalized by drug therapy. Although it has been reported that decreased cholesteryl ester transfer protein (CETP) activity results in the formation of small LDL, plasma CETP activity was normal in the hyperlipoproteinemic patients and the normalization of LDL characteristics by drug therapy was not accompanied by an increase of CETP activity. Our results suggested that an abnormal lipid composition and/or small particle size might cause a decrease in the receptor affinity of LDL. These structural and functional abnormalities were reversed by drug therapy, underlining the importance of treating hypertriglyceridemia for the prevention of atherosclerosis.