Detection of the Low Density Lipoprotein Receptor Gene Pvuii Intron 15 Polymorphism Using the Polymerase Chain Reaction: Association With Plasma Lipid Traits in Healthy Men and Women

We have used anchored PCR to amplify and sequence 1400bp of the 15th intron of the Low Density Lipoprotein (LDL) receptor gene, and have determined oligonucleotides and conditions for the genotyping of the previously reported Pvull polymorphism. The cutting site (CAGCTG) is created by the transition of a CpG to a TpG within the sequence CAGCCG at a position roughly 600bp 5' from the splice acceptor site of exon 16. Genotype was determined in three populationbased samples of healthy individuals. In a group of 318 men and women from Iceland the frequencies of the Intron-15 T (cutting) allele was 0.23 (95% CI, 0.19-0.28) and was similar in men and women. In two groups of men from England (n=385) and Scotland (n=320), the frequency was similar, being 0.23 (0.19-0.27) and 0.25 (0.22-0.28) respectively. Individuals who were homozygous for the T allele had lower levels oftotal-cholesterol triglycerides and apolipoprotein B, than those with other genotypes, and in the combined group of UK men this effect reached statistical significance; compared to the CIC group, the TIT group had 6% lower cholesterol (p=0.02) and 15% lower triglycerides (p=0.03). The lowering effect associated with the TIT genotype was greater in men who were in the lowest terti Ie of body mass index (<25kg/m2) and for the trait of apoB levels, this genotype x obesity interaction was statistically significant (p=0.01). We thus confirm the association between this allele and lower levels of plasma lipid levels previously reported. The availability of a PCR-based method to detect this polymorphism will facilitate further investigation of the impact of LDL-receptor gene variation in determining lipid levels.

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Effect on plasma lipid levels of different classes of mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia.

Arteriosclerosis and Thrombosis A Journal of Vascular Biology ◽

10.1161/01.atv.14.11.1717 ◽

1994 ◽

Vol 14 (11) ◽

pp. 1717-1722 ◽

Cited By ~ 47

Author(s):

V Gudnason ◽

I N Day ◽

S E Humphries

Keyword(s):

Familial Hypercholesterolemia ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Plasma Lipid ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Receptor ◽

Lipid Levels ◽

Low Density Lipoprotein Receptor ◽

Density Lipoprotein Receptor

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Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3268-3277.1986 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3268-3277

Author(s):

R D Sege ◽

K F Kozarsky ◽

M Krieger

Keyword(s):

Gamma Irradiation ◽

Cho Cells ◽

Gamma Ray ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Ldl Receptors

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

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Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Clinical Chemistry ◽

10.1093/clinchem/44.5.966 ◽

1998 ◽

Vol 44 (5) ◽

pp. 966-972 ◽

Cited By ~ 6

Author(s):

Bent Raungaard ◽

Finn Heath ◽

Jens Uffe Brorholt-Petersen ◽

Henrik Kjærulf Jensen ◽

Ole Faergeman

Keyword(s):

Flow Cytometry ◽

T Lymphocytes ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Receptor Expression ◽

Low Density ◽

Flow Cytometry Assay ◽

Stop Mutation

Abstract We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

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Control of low density lipoprotein receptor gene expression in steroidogenic cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y89-153 ◽

1989 ◽

Vol 67 (8) ◽

pp. 968-973 ◽

Cited By ~ 9

Author(s):

Koichiro Takagi ◽

Jerome F. Strauss III

Keyword(s):

Gene Expression ◽

Cyclic Amp ◽

Granulosa Cells ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Steroidogenic Cells ◽

Receptor Gene Expression

Low density lipoprotein (LDL)-carried cholesterol is a primary substrate for steroid hormone synthesis by luteinized human granulosa cells. Chorionic gonadotropin and 8-bromo-cAMP both increase LDL receptor levels in granulosa cells by stimulating accumulation of the receptor mRNA. LDL and 25-hydroxycholesterol reduce LDL receptor expression, but this suppressive effect is partially overcome by 8-bromo-cAMP. Using fusion gene constructs containing the LDL receptor gene promoter transfected into JEG-3 cells, a cyclic AMP responsive enhancer could not be identified in the LDL receptor gene upstream promoter in transfection studies. We suggest that the LDL receptor gene in human steroidogenic cells is under negative control by a sterol effector, but that a cyclic AMP triggered process overcomes, to some extent, the sterol-mediated suppression. The detailed mechanisms by which sterol and cyclic AMP modulate LDL receptor gene expression remain to be elucidated.Key words: low density lipoproteins, low density lipoprotein receptors, cholesterol, steroidogenesis, gonadotropins.

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