PARTIAL DUPLICATION OF THE EGF PRECURSOR HOMOLOGY DOMAIN OF THE LDL-RECEPTOR PROTEIN CAUSING FAMILIAL HYPERCHOLESTEROLEMIA (FH-SALERNO)

A novel mutation of low density lipoprotein (LDL)-receptor gene was fo und in an Italian familial hypercholesterolemia (FH) patient during a screening of 300 FH patients. The proband as well as her daughter were found to be heterozygotes for the mutation. Binding, internalization, and degradation of I-125-labeled LDL by the proband's fibroblasts wer e reduced to approximately 50% compared to values found in control cel ls. DNA analysis by Southern blotting showed that the mutant allele wa s characterized by an insertion of about 10 kb, which resulted from a duplication of exons 9-14 of the LDL-receptor gene. In addition, North ern blot analysis of the proband's RNA showed, besides the normal-size d LDL-receptor mRNA (5.3 kb), an additional mRNA of about 6.2 kb. The junction between exon 14 and the duplicated exon 9 was amplified by po lymerase chain reaction (PCR) from the cDNA. The sequence of the ampli fied fragment showed that exon 14 joined the duplicated exon 9 without changing the reading frame. The derived amino acid sequence indicated that the mutated receptor protein had a partial duplication of the EG F precursor homology domain. Ligand and immunoblotting revealed that p roband's fibroblasts contained one-half of the normal amount of LDL-re ceptor protein (molecular mass 130 kDa) and an abnormally large recept or of approximately 160 kDa. The amount of this abnormal receptor as d etected by two monoclonal antibodies (10A2 and 4B3) was found to be ap proximately 30% that of the normal LDL-receptor present in the same ce lls. Treatment of the proband's cells with pronase greatly reduced the amount of both normal and abnormal receptors detected, indicating tha t both receptors were present on the cell surface. Pulse-chase experim ents using [S-35]methionine indicated that the receptor was processed to the mature form (195 kDa), although at a rate slightly slower than the normal receptor (160 kDa) present in the same cells. However, the mature abnormal receptor was degraded much more rapidly (half life 4.6 h) than the normal receptor present in the proband's cells (half life 11.9 h) or in normal cells (half life 12.4 h). jlr In conclusion, the mutant allele present in our proband produces an abnormally large rec eptor protein that is normally processed to the mature form but is deg raded more rapidly than the normal counterpart. As the proband's famil y originated from the city of Salerno, in southern Italy, the mutation was named FHSalerno.