NEW-TYPE OF THE INTERNALIZATION-DEFECTIVE LOW-DENSITY-LIPOPROTEIN RECEPTOR OWING TO 2-NUCLEOTIDE DELETION (2199DELCA OR 2201DELCA) IN JAPANESE PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA

Background In mutations of the low-density lipoprotein (LDL) receptor gene, the defect of internalization is caused by a mutation in the cyt oplasmic 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 af fect the function of the receptor. Here, we describe a novel mutation of the O-linked sugar domain of the LDL receptor gene, designated fami lial hypercholesterolaemia (FH)Mishima with Japanese pedigree, which r esembles but still differs from classical defective internalization ca ses. 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 distri bution. Screening of the mutant exon(s) of the LDL receptor gene was p erformed using the polymerase chain reaction-single-strand conformatio n polymorphism technique (PCR-SSCP), and sequencing of the mutated all eles was carried out using the dideoxy chain termination method. Resul ts LDL-binding activity at 4 degrees C in skin fibroblasts from patien ts was similar to normal, but that at 37 degrees C with the ligand dec reased time dependently and was lost at 6 h, resulting in the defect o f internalization and degradation of LDL. The receptor protein on the cell surface was detected at 4 degrees C by IgG-C7, an anti-LDL recept or antibody, but was not detected after incubation with LDL at 37 degr ees C. The size of the receptor was 112 kD as determined by immunoprec ipitation analysis. A deletion of two nucleotides in exon 15 was detec ted 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 nucleo tides: caused novel amino acid sequences after the O-linked sugar doma in, 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 cessati on of activity of the LDL receptor.