Mutant transcripts of the LDL receptor gene: mRNA structure and quantity

mRNA of the low‐density lipoprotein receptor (LDLR) gene from 22 heterozygous familial hypercholesterolemic subjects possessing different mutations in this gene was analyzed by Northern blot analysis and reverse transcription‐polymerase chain reaction (RT‐PCR) in order to detect abnormally spliced transcripts. These analyses revealed abnormally spliced transcripts for the two splice‐site mutations 1359‐1G→A and 1705+1G→T. The abnormally spliced transcript for mutation 1359‐1G→A was caused by activation of a cryptic acceptor splice site in exon 10. As a result, seven nucleotides of exon 10 were deleted. For mutation 1705+1G→T, two mutant transcripts were observed. In the first transcript, exon 10 was spliced to exon 13, and in the second transcript intron 11 was retained. The relative amount of mutant transcripts from 14 of the 22 subjects was determined by use of an RT‐PCR‐based method. Quantitation of the relative amounts of mutant transcripts for five missense mutations resulted in a mean value (±SD) of 52.8% (±4.55). In comparison, quantitation of the relative amounts of mutant transcripts for five nonsense mutations resulted in a mean value of 31.8% (±6.91). This value was significantly lower than the value of 54.2% (±2.38) obtained for nine healthy subjects ( P < 0.0001). The relative amount of mutant transcripts for the 1705+1G→T mutation was 36%. Thus, transcripts from alleles containing premature stop codons are present in reduced amounts, whereas transcripts from alleles containing missense mutations are present in normal amounts. These findings underscore the importance of determining how mutations affect mRNA structure and quantity in order to understand how mutations cause disease. Hum Mutat 13:186–196, 1999. © 1999 Wiley‐Liss, Inc.