Partial duplication of the EGF precursor homology domain of the LDL receptor protein causing familial hypercholesterolemia (FH-Salerno)

Abstract A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian familial hypercholesterolemia(FH) patient during a screening of 300 FH patients. Theproband as well as her daughter were found to be heterozygotesfor the mutation. Binding, internalization, and degradation of‘25I-labeled LDL by the proband’s fibroblasts were reduced toapproximately 50% compared to values found in control cells.DNA analysis by Southern blotting showed that the mutant allelewas characterized by an insertion of about 10 kb, whichresulted from a duplication of exons 9-14 of the LDL-receptorgene. In addition, Northern blot analysis of the proband’s RNAshowed, besides the normal-sized LDL-receptor mRNA (5.3kb), an additional mRNA of about 6.2 kb. The junction betweenexon 14 and the duplicated exon 9 was amplified by polymerasechain reaction (PCR) from the cDNA. The sequence of the amplifiedfragment showed that exon 14 joined the duplicated exon9 without changing the reading frame. The derived amino acidsequence indicated that the mutated receptor protein had a partialduplication of the EGF precursor homology domain. Ligandand immunoblotting revealed that proband’s fibroblasts containedone-half of the normal amount of LDL-receptor protein(molecular mass 130 kDa) and an abnormally large receptor ofapproximately 160 kDa. The amount of this abnormal receptoras detected by two monoclonal antibodies (10A2 and 4B3) wasfound to be approximately 30% that of the normal LDLreceptorpresent in the same cells. Treatment of the proband’scells with pronase greatly reduced the amount of both normaland abnormal receptors detected, indicating that both receptorswere present on the cell surface. Pulse-chase experiments using[35S]methionine indicated that the receptor was processed to themature form (195 kDa), although at a rate slightly slower thanthe normal receptor (160 kDa) present in the same cells.However, the mature abnormal receptor was degraded muchmore rapidly (half life 4.6 h) than the normal receptor presentin the proband’s cells (half life 11.9 h) or in normal cells (half life12.4 h). I In conclusion, the mutant allele present in our probandproduces an abnormally large receptor protein that is normallyprocessed to the mature form but is degraded morerapidly than the normal counterpart. As the proband‘s familyoriginated from the city of Salerno, in southern Italy, the mutationwas named FH-Salerno.