Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Raungaard, Bent; Heath, Finn; Brorholt-Petersen, Jens Uffe; Jensen, Henrik Kjærulf; Færgeman, Ole

doi:10.1002/(sici)1097-0320(19990501)36:1<52::aid-cyto7>3.0.co;2-1

Cited by 13 publications

(10 citation statements)

References 34 publications

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“…Interestingly, this reduction was observed only with lower concentrations of LDL, but not with higher levels of LDL. However, it is in agreement with data, showing that lower concentrations of LDL in medium might stronger stimulate LDLR activity [7, 9]. Relatively low decrease of binding and internalization could result from the fact, that examined probands were heterozygotes and 50% of receptors retained functional activity.…”

Section: Discussionsupporting

confidence: 91%

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

et al. 2011

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The purpose of this investigation was to characterize a new mutation in the LDL-receptor (LDLR) gene in three families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all probands. The novel mutation c986G>T (Cys308Phe) in the exon 7 of LDLR gene was found in three apparently unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% versus healthy control) of the fluorescent label in the lymphocytes of patients heterozygous for this mutation. Concentrations of serum LDL-C in probands before treatment were between 9.5 and 10.5 mmol/l. All patients had corneal arcus and tendon xanthoma. Clinically, families were characterized by premature coronary artery disease. This mutation occurred relatively frequently in our group of patients with FH, but this could be explained by a founder effect since we demonstrated their common ancestors.

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Section: Discussionsupporting

confidence: 91%

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

et al. 2011

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“…Together, our data indicate that the complex of LpR and HDLp remains intact during its intracellular itinerary, which is in complete agreement with the occurrence of ligand recycling [14,16–18], and may provide a vital determinant of the ligand‐recycling capacity of LpR. In several studies, flow cytometry has been used to quantify lipoprotein binding and uptake [29,53–59]. In most cases, the experiments were performed on blood cells.…”

Section: Discussionsupporting

confidence: 67%

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

et al. 2008

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The insect low‐density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high‐density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for resecretion in a transferrin‐like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand‐binding and ligand‐dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR–HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp‐binding receptors, containing either the β‐propeller or both the β‐propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand‐binding domain of LpR. The LpR–HDLp complex additionally appeared to be EDTA‐resistant, excluding a low Ca2+ concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand‐binding repeats 1–7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA‐2–7. It is therefore proposed that the remarkable stability of the LpR–HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.

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“…The main problem of these functional assays was the bad separation between residual LDLR activity from FH patients and controls. The separation of different leukocyte populations obtains more accurate results ( 8 ); the selection of viable lymphocytes by stimulation with a mitogen or by fl ow cytometry gating improves the discrimination between patients and controls although it does not allow complete discrimination of FH heterozygote patients from healthy controls ( 9,10 ).…”

Section: Discussionmentioning

confidence: 69%

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

Romano

Taranto²,

Mirabelli

et al. 2011

Journal of Lipid Research

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Supplementary key words LDL receptor • familial hypercholesterolemia • EBV-transformed B-lymphocytes • mitogen stimulated T-lymphocytes • functional activityFamilial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevated plasma LDL cholesterol, tendon xanthomas, and premature coronary heart disease. FH is mostly caused by mutations within the low density lipoprotein receptor (LDLR; MIM# 143890) gene, or in its ligand apoB-100 (MIM# 107730), or in the proprotein convertase subtilisin/kexin type 9 (PCSK9; MIM# 607786) gene ( 1 ).At present, more than 1,100 variants of LDLR gene have been listed in the LDLR databases underlying a high genetic heterogeneity of LDLR mutations. These are distributed across the 18 exons, introns, and the promoter region of the LDLR gene and include point mutations, insertions, deletions, and major rearrangements ( 2 ).Although LDLR defects are primarily identifi ed by genetic methods, it is extremely important to demonstrate a defi ciency in the LDLR function of FH suspect patients through functional studies. Receptor assays reported so far include measurement of radiolabeled-LDL or fl uorescently-labeled LDL binding and/or uptake in skin fi broblasts Abstract The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous EpsteinBarr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fl uorescent LDL followed by fl ow cytometry analysis. Residual LDLR activity was calculated normalizing fl uorescence for the mean fl uorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes. CEINGE -Biotecnologie Avanzate,* Napoli, Italy ; Dipartimento di Biochimica e Biotecnologie Mediche, † and Dipartimento di Medicina Clinica e Sperimentale, † † Università degli Studi di Napoli Federico II, Napoli, Italy ; Istituto di Ricovero e Cura a Carattere Scientifi co Fondazione SDN, § ...

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Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Cited by 13 publications

References 34 publications

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

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