Two 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 mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low-density lipoprotein (LDL) receptor gene: a missense Asn543. His mutation (N543H) in exon 11, and an in-frame 9-bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected.