Analysis of two duplications of the LDL receptor gene affectingintracellular transport, catabolism, and surface binding of the LDLreceptor

Abstract

Two novel mutations of the low density lipoprotein (LDL)-receptor genewere found in two Italian familial hypercholesterolemia (FH)-heterozygotes. Thefirst mutation was an 18 nucleotide duplication in exon 8 which is preceded byan A→T transversion. The translation product of the mutant allele waspredicted to be a receptor with an in-frame insertion of 6 amino acids in repeatB of the epidermal growth factor precursor homology domain. Analysis ofLDL-receptor activity in the proband's fibroblasts showed a 50% reduction of125I-labeled LDL binding and pulse-chase studies suggested thatlittle, if any, of the mutant protein was processed to the mature form. Thesecond mutation was a 7 kb duplication (from intron 2 to intron 6) of exons 3through 6, predicted to encode an elongated receptor with the duplication ofrepeats 2–7 of the ligand binding domain. The elongated receptor wasprocessed slightly more slowly than the normal receptor, but was converted to amature form of the expected size. This mature, mutant receptor was degraded morerapidly than the normal receptor. On ligand blotting the elongated receptorbound twice as much LDL or beta-very low density lipoprotein (βVLDL) asthe normal receptor. In contrast, maximum binding of LDL to proband's cells wasdecreased to approximately 70% of the normal cells with a significant increasein apparent affinity. Cell association at 37°C, internalization, anddegradation showed a similar reduced maximum. Thus thesemutations demonstrate that duplications of amino acid sequences in the lowdensity lipoprotein LDL-receptor may disrupt the LDL-receptor pathway atdifferent levels.—Patel, D. D., N. Lelli, R. Garuti, S. Li Volti, S.Bertolini, B. L. Knight, and S. Calandra. Analysis of two duplications of theLDL-receptor gene affecting intracellular transport, catabolism, and surfacebinding of the LDL receptor. J. Lipid Res. 1998. 39:1466–1475.

Keywords

• familial hypercholesterolemia
• amino acid sequence