Expression of an LDL receptor allele with two different mutations (E256K and I402T)

Aims-To investigate the disease causing event in patients with familial hyp ercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. Methods-Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the pati ents and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL recep tor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effe cts of the mutations on LDL receptor function. Results-The two mutations were located on the same allele of the LDL recept or gene. All mutant constructs resulted in the production of a detectable p rotein in CHO cells. The cells expressing only the I402T mutation, or the c ombination of I402T and E256K mutations, were seriously affected in mediati ng uptake and degradation of LDL. Contrary to initial predictions, the cell s expressing only the E256K mutation showed essentially the same binding, u ptake, and degradation of I-125 labelled LDL as cells transfected with norm al LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation , and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients stud ied. Conclusions-Despite the information available on the structure-function rel ations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitu tion of E256K alone is a rare sequence variation, without a detectable phen otype modulating effect.