Finn Heath

A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein.

In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.

Two mutations in the same low-density lipoprotein receptor allele act in synergy to reduce receptor function in heterozygous familial hypercholesterolemia.

Mutations in genes are not necessarily pathogenic. Expression of mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low‐density lipoprotein (LDL) receptor gene: a missense Asn543‐His mutation (N543H) in exon 11, and an in‐frame 9‐bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected. Hum Mutat 9:437–444, 1997.

Allele‐specific measurement of low‐density lipoprotein receptor transcript levels

We have developed an assay for allele‐specific determination of low‐density lipoprotein receptor (LDLR) mRNAs. Transcript levels are measured by reverse transcription (RT), PCR, and electrophoresis on an automatic DNA sequencer using fluorescence‐labeled primers and direct quantitation of the allele‐specific RT‐PCR products. The discrimination between the allelic products is based on the use of DNA polymorphisms located in the coding regions of the gene as markers for the individual alleles. Using this method on LDLR mRNA from heterozygous patients with familial hypercholesterolemia (FH) due to a defective LDLR protein, it is possible to relate the expression of the mutant allele directly to the expressed amounts of the normal allele, thus overcoming the problems of using artificial internal standards in the PCR. To validate the method we have measured (1) the range of normal LDLR allele transcript levels, and (2) the transcript levels in patients heterozygous for different types of mutant LDLR alleles associated with FH. The method is general in principle and can be applied in the allele‐specific analysis of transcripts from all enes harbouring DNA polymorphisms in their coding regions.

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

BACKGROUND Studies indicate that human peripheral blood mononuclear cells mirror low-density lipoprotein (LDL) receptor activity of other cells in the body. To measure LDL receptor activity in patients with heterozygous familial hypercholesterolemia (FH), we prepared peripheral blood mononuclear cells from individuals with molecularly verified LDL receptor defective (Trp66-Gly mutation, n = 18) or receptor negative (Trp23-stop mutation, n = 17) heterozygous FH and from healthy individuals (n = 24). METHODS The cells were stimulated to express maximum LDL receptor by preincubation in lipoprotein-free medium. They were then incubated at 4 degrees or 37 degrees C with fluorescently conjugated LDL (DiI-LDL). T-lymphocytes and monocytes were identified by fluorescently conjugated monoclonal antibodies. DiI-LDL bound (at 4 degrees C) or internalized (at 37 degrees C) by the cells was measured using flow cytometry. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of our functional assay with the DNA diagnosis. RESULTS The diagnostic accuracy did not allow our assay to be used for diagnosis of individual cases of heterozygous FH. CONCLUSIONS We suggest that our two-color fluorescence flow cytometry assay can be used to characterize functionally gene mutations causing LDL receptor dysfunction in patients with heterozygous FH.

Functional characterization of two low density lipoprotein receptor gene mutations by fluorescence flow cytometric assessment of receptor activity in stimulated human T-lymphocytes.

We report a functional characterization of the W23X and W66G low density lipoprotein (LDL) receptor gene mutations. The authors used two‐color fluorescence flow cytometry to measure LDL receptor activity in stimulated T‐lymphocytes, prepared from patients heterozygous for the W23X or W66G mutation, and compared the results with measurements of LDL receptor activity in stimulated T‐lymphocytes prepared from unrelated healthy control subjects. It was found that the W23X mutation significantly reduced LDL receptor expression and LDL binding and internalization, and that the W66G mutation significantly reduced LDL receptor expression and LDL binding. LDL internalization in patients heterozygous for the W66G mutation was not significantly reduced. The data support the concepts that the W23X mutation prevents production of LDL receptors (class I) and that the W66G mutation produces LDL receptors unable to recycle normally in cells (class V).

Phenotypic characterization of a patient homozygous for the D558N LDL receptor gene mutation

We describe the clinical, biochemical, and genetic features of a patient with true homozygous familial hypercholesterolemia due to the D558N low‐density lipoprotein receptor gene mutation, previously designated FH Cincinnati‐4. Functional flow‐cytometric analysis of the LDL receptorR protein on upregulated EB V‐transformed lymphocytes indicated reduction of the number of receptors on the cell surface by 87% and reduction of receptor activity by 89% compared to control cells. With drugs and a portacaval shunt operation, performed when the patient was 15 years old, serum cholesterol was reduced from about 28 to about 15 mmol/l. He died at the age of 32 of a myocardial infarction. The autopsy showed generalized atherosclerosis, especially in the coronary arteries, which were severely stenosed proximally. A rare finding was a large intracranial xanthoma that apparently had been asymptomatic.