Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia that affects ligand binding and intracellular movement of receptors.

Abstract

The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline) to CTG (leucine) and introducing a new site for the restriction enzyme Pst I. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which rwst cousin marriages occurred frequently. The amino acid change occurs in the center of growth factor repeat C in the epidermal growth factor precursor-homology domain of the protein, a region of highly conserved sequence between bovine and human LDL receptors, and results in slowed, but complete, maturation of the precursor to the mature form of the receptor and, despite its remoteness from the ligand-binding domain, in impaired binding of LDL. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding.The low density lipoprotein (LDL) receptor is a cell-surface protein that mediates the specific recognition, uptake, and degradation of plasma LDL. During receptor-mediated endocytosis, LDL receptors on the cell surface cluster into coated pits that internalize to form endocytic vesicles from which the receptor recycles to the cell surface, while the ligand is degraded in lysosomes (1). The LDL receptor is synthesized as a precursor protein, of apparent molecular weight 120,000 by gel electrophoresis, and undergoes considerable posttranslational modification by the addition and remodeling of both 0-linked and N-linked carbohydrate chains before it reaches the cell surface as a mature protein of apparent molecular weight 160,000 (2).From cDNA cloning of LDL-receptor mRNA (3) and from the intron-exon structure of the gene (4) it has been predicted that the LDL receptor is an integral membrane protein of 839 amino acid residues that comprises a mosaic of several different domains. Analysis of naturally occurring mutations in the LDL receptor gene in patients with familial hypercholesterolemia (FH) and of constructed mutations has shed some light on the function of these different domains in the process of receptor-mediated endocytosis. The first NH2-terminal 292 residues of the LDL receptor comprise the ligand-binding domain, containing seven cysteine-rich 40-amino acid repeats. Mutations in this...

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

“…These results indicate that T-lymphocytes of the K790X mutation also possess a characteristic similar to the internalization-defective allele; therefore, a conventional assay using lymphocytes would fail to measure their values accurately. On the other hand, the P664L mutation is classified as a transport-defective allele, meaning that conventional assays can measure mostly accurate values (15).…”

“…On the other hand, P664L mutation, which is classified as a transport-defective allele (15), is also one of the common mutations among Japanese FH subjects, and its frequency of occurrence is 6% (8).…”

Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia.

“…2 During the past few years, the availability of cDNA and genomic clones 3 " 5 has made it possible to characterize the mutations of the LDL receptor (LDL-R) gene at the DNA level. A large array of " 28 These mutations are scattered throughout the 45 kilobases (kb) of the LDL-R gene on chromosome 19. 29 During the past 2 years, we have undertaken a survey of Italian FH patients through a collaborative study involving several lipid clinics and aimed at investigating the frequency and features of the major structural rearrangements of the LDL-R gene.…”

Familial hypercholesterolemia in Morocco: first report of mutations in the LDL receptor gene

“…The P664L mutation has been demonstrated to be causative of FH (Soutar et al 1989). We found this mutation in one compound heterozygous proband.…”