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 lo tv 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 tvas 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 I-125-labeled LDL binding and pulse-chase studies suggested that Little, if an); 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 lipoproteiu (beta VLDL) 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