2 MUTATIONS IN THE SAME LOW-DENSITY-LIPOPROTEIN RECEPTOR ALLELE ACT IN SYNERGY TO REDUCE RECEPTOR FUNCTION IN HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA

Mutations in genes are not necessarily pathogenic. Expression of mutan t genes in cells can therefore be required to demonstrate that mutatio ns in fact disturb protein function. This applies especially to missen se mutations, which cause an amino acid to be replaced by another amin o acid. In the present study of two families with familial hypercholes terolemia in the heterozygous form, we found two mutations in the same allele of the low density lipoprotein (LDL) receptor gene: a missense Asn(543)-His mutation (N543H) in exon 11, and an in frame 9-bp deleti on (2393del9) in exon 17. The two mutations were identified in heteroz ygous FH index patients in whom no other pathogenic mutations were det ected by SSCP analysis of the remaining 16 exons and the promoter regi on. Both mutations cosegregated with hypercholesterolemia within the f amilies. Each of these mutations had little or no effect on receptor f unction in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric mea surement of fluorescent LDL uptake in cells, was reduced by 75%. Immun ostainable receptors on the cell surface were decreased by 80% as meas ured by flow cytometry. The two mutations therefore acted in synergy t o affect receptor function, possibly during intracellular receptor tra nsport, since Northern blot analysis suggested that mRNA levels were u naffected, Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected. (C) 1997 Wiley-Liss, Inc.