L.G. Jensen

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.

The Trp23-Stop and Trp66-Gly mutations in the LDL receptor gene: common causes of familial hypercholesterolemia in Denmark

Mutations in the gene for the low density lipoprotein (LDL) receptor cause the autosomal dominant disease familial hypercholesterolemia (FH), the prevalence of which is about 0.2% in most populations. By PCR-SSCP analysis and direct sequencing, we identified the receptor-negative Trp23-Stop LDL receptor mutation (FH Cincinnati-5) in 10 of 63 FH probands and the receptor-defective Trp66-Gly LDL receptor mutation (FH French Canadian-4) in another 10 of the 63 FH probands. These two mutations thus account for 30% of diagnosed FH families in Denmark. Comparison of the mean lipid concentrations (unadjusted and adjusted for age), including serum total cholesterol and LDL-cholesterol, showed no significant differences between the two groups of FH heterozygote probands (cholesterol: 10.7 mmol/l vs. 10.7 mmol/l) and between the probands and 16 and 22 non-proband family members with the Trp23-stop (cholesterol: 10.1 mmol/l) ad Trp66-Gly (cholesterol: 10.7 mmol/l) mutations, respectively.

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.

A G‐1‐to‐A acceptor splice site LDLR mutant allele leads to reduced relative transcript levels in patients with heterozygous familial hypercholesterolemia

A plethora of different mutations in the gene for the low density receptor (LDLR) are responsible for the autosomal dominant inherited disorder familial hypercholesterolemia (FH). However, only a few splice site mutations have been identified in this gene. We here report a defect presumably affecting the splicing of precursor mRNA, resulting from a novel mutation, a G to A transition at the terminal nucleotide of intron 12, of the LDLR gene detected in three unrelated families with heterozygous FH. This mutation markedly reduced the steady‐state transcript level of the mutant LDLR allele as compared to the corresponding normal LDLR allele in heterozygous FH patients as measured by a fluorescence based, allele‐specific quantitation technique. In the FH families, the acceptor splice site mutation cosegregates with hypercholesterolemia, and it is associated with onset of ischemic heart disease in the fifth and sixth decade of life.

An Iranian-Armenian LDLR frameshift mutation causing familial hypercholesterolemia

We used polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) analysis to detect a mutation in the low density lipoprotein receptor (LDLR) gene in a family of Iranian‐Armenian origin. The mutation, designated FH Yrmeih, deletes two nucleotides from exon 10 of the LDLR gene, which causes a translational frameshift, whereby a truncated LDLR protein of the first 471 residues of the LDLR with an additional 41 abnormal residues and a premature stop codon would be created. The deletion was detected in a father and son with clinical features of heterozygous FH. To our knowledge this is the first pathogenetic LDLR mutation identified in FH patients of Iranian‐Armenian ancestry.

Complexity of molecular genetics of dyslipidemia in a family highly susceptible to ischemic heart disease.

In a Danish family highly susceptible to ischemic heart disease, hyperlipidemia did not simply cosegregate with a previously undescribed 10 bp deletion in the LDL receptor gene causing heterozygous familial hypercholesterolemia (FH). This mutation, designated as FH DK‐4, deletes 10 nucleotides from ex on 4 coding for the third cysteine‐rich repeat of the ligand‐binding domain. The resulting translational frameshift and stop codon corresponding to amino acid position 181 in the LDL receptor cDNA is predicted to result in a truncated LDL receptor protein. Several family members had hyperlipidemia and early onset of ischemic heart disease not due to the 10 bp deletion, and several family members had unexpectedly high serum lipoprotein(a) contributing to high concentrations of serum LDL cholesterol. The study illustrates important limitations and possibilities of molecular genetic diagnosis.

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.