ALLELE-SPECIFIC MEASUREMENT OF LOW-DENSITY-LIPOPROTEIN RECEPTOR TRANSCRIPT LEVELS

We have developed an assay for allele-specific determination of low de nsity lipoprotein receptor (LDLR) mRNAs. Transcript levels are measure d by reverse transcription (RT), PCR, and electrophoresis on an automa tic DNA sequencer using fluorescence-labeled primers and direct quanti tation of the allele-specific RT-PCR products. The discrimination betw een the allelic products is based on the use of DNA polymorphisms loca ted in the coding regions of the gene as markers for the individual al leles. Using this method on LDLR mRNA from heterozygous patients with familial hypercholesterolemia (FH) due to a defective LDLR protein, it is possible to relate the expression of the mutant allele directly to the expressed amounts of the normal allele, thus overcoming the probl ems of using artificial internal standards in the PCR. To validate the method we have measured (1) the range of normal LDLR allele transcrip t levels, and (2) the transcript levels in patients heterozygous for d ifferent types of mutant LDLR alleles associated with FH. The method i s general in principle and can be applied in the allele-specific analy sis of transcripts from all genes harbouring DNA polymorphisms in thei r coding regions. (C) 1996 Wiley-Liss, Inc.