Analysis of two duplications of the LDL receptor gene affecting intracellular transport, catabolism, and surface binding of the LDL receptor

Two novel mutations of the low density lipoprotein (LDL)-receptor gene were found in two Italian familial hypercholesterolemia (FH)-heterozygotes. The first mutation was an 18 nucleotide duplication in exon 8 which is preceded by an A-->T transversion. The translation product of the mutant allele...

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Bibliographic Details
Published in:Journal of lipid research 1998-07, Vol.39 (7), p.1466-1475
Main Authors: Patel, D D, Lelli, N, Garuti, R, Li Volti, S, Bertolini, S, Knight, B L, Calandra, S
Format: Article
Language:English
Subjects:
Adult
Amino Acid Sequence
Base Sequence
Cell Membrane - metabolism
DNA Transposable Elements
ErbB Receptors - genetics
Exons
Female
Heterozygote
Humans
Hypercholesterolemia - genetics
Kinetics
Male
Middle Aged
Multigene Family
Nuclear Family
Point Mutation
Polymerase Chain Reaction
Protein Biosynthesis
Receptors, LDL - genetics
Receptors, LDL - metabolism
Repetitive Sequences, Nucleic Acid
Restriction Mapping
src Homology Domains
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Summary:Two novel mutations of the low density lipoprotein (LDL)-receptor gene were found in two Italian familial hypercholesterolemia (FH)-heterozygotes. The first mutation was an 18 nucleotide duplication in exon 8 which is preceded by an A-->T transversion. The translation product of the mutant allele was predicted to be a receptor with an in-frame insertion of 6 amino acids in repeat B of the epidermal growth factor precursor homology domain. Analysis of LDL-receptor activity in the proband's fibroblasts showed a 50% reduction of 125I-labeled LDL binding and pulse-chase studies suggested that little, if any, of the mutant protein was processed to the mature form. The second mutation was a 7 kb duplication (from intron 2 to intron 6) of exons 3 through 6, predicted to encode an elongated receptor with the duplication of repeats 2-7 of the ligand binding domain. The elongated receptor was processed slightly more slowly than the normal receptor, but was converted to a mature form of the expected size. This mature, mutant receptor was degraded more rapidly than the normal receptor. On ligand blotting the elongated receptor bound twice as much LDL or beta-very low density lipoprotein (betaVLDL) as the normal receptor. In contrast, maximum binding of LDL to proband's cells was decreased to approximately 70% of the normal cells with a significant increase in apparent affinity. Cell association at 37 degrees C, internalization, and degradation showed a similar reduced maximum. Thus these mutations demonstrate that duplications of amino acid sequences in the low density lipoprotein LDL-receptor may disrupt the LDL-receptor pathway at different levels.
ISSN:0022-2275