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

mRNA of the low-density lipoprotein receptor (LDLR) gene from 22 heterozygo us familial hyper cholesterolemic subjects possessing different mutations i n this gene was analyzed by Northern blot analysis and reverse transcriptio n-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 sp liced transcript for mutation 1359-1G-->A was caused by activation of a cry ptic acceptor spice site in exon 10, As a result, seven nucleotides of exon 10 were deleted. For mutation 1705 + 1G-->T, two mutant transcripts were o bserved. In the first transcript, exon 10 was spliced to exon 13, and in th e second transcript intron 11 was retained. The relative amount of mutant t ranscripts from 14 of the 22 subjects was determined by use of an RT PCR ba sed 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). T his value was significantly lower than the value of 54.2% (+/-2.38) obtaine d for nine healthy subjects (P < 0.0001). The relative amount of mutant tra nscripts for the 1705 + 1G-->T mutation was 36%. Thus, transcripts from all eles containing premature stop codons are present in reduced amounts, where as transcripts from alleles containing missense mutations are present in no rmal amounts. These findings underscore the importance of determining how m utations affect mRNA structure and quantity in order to understand how muta tions cause disease. Hum Mutat 13:186-196, 1999. (C) 1999 Wiley-Liss, Inc.