Background: In mutations of the low-density lipoprotein (LDL) receptor gene, the defect of internalization is caused by a mutation in the cytoplasmic domain of the receptor linked with exons 17 and 18, and the O-linked sugar domain linked with exon 15 has been speculated not to affect the function of the receptor. Here, we describe a novel mutation of the O-linked sugar domain of the LDL receptor gene, designated familial hypercholesterolaemia (FH)-Mishima with Japanese pedigree, which resembles but still differs from classical defective internalization cases.
Methods: LDL metabolism was examined in cultured skin fibroblasts from patients. Immunoprecipitation and immunohistochemical techniques were applied for the detection of the receptor protein size and distribution. Screening of the mutant exon(s) of the LDL receptor gene was performed using the polymerase chain reaction-single-strand conformation polymorphism technique (PCR-SSCP), and sequencing of the mutated alleles was carried out using the dideoxy chain termination method.
Results: LDL-binding activity at 4 degrees C in skin fibroblasts from patients was similar to normal, but that at 37 degrees C with the ligand decreased time dependently and was lost at 6 h, resulting in the defect of internalization and degradation of LDL. The receptor protein on the cell surface was detected at 4 degrees C by IgG-C7, an anti-LDL receptor antibody, but was not detected after incubation with LDL at 37 degrees C. The size of the receptor was 112 kD as determined by immunoprecipitation analysis. A deletion of two nucleotides in exon 15 was detected in the DNA sequence of the LDL receptor gene. The deletion results in a shift of the reading frame after Thr-713 of the mutant and makes a stop codon at amino acid 759.
Conclusion: Deletion of the two nucleotides caused novel amino acid sequences after the O-linked sugar domain, which has the ability of sorting on the cell membrane at 4 degrees C, but not at 37 degrees C in vivo, resulting in the complete cessation of activity of the LDL receptor.