OCCURRENCE OF MULTIPLE ABERRANTLY SPLICED MESSENGER-RNAS OF THE LDL-RECEPTOR GENE UPON A DONOR SPLICE-SITE MUTATION THAT CAUSES FAMILIAL HYPERCHOLESTEROLEMIA (FHBENEVENTO)

A novel point mutation of the LDL-receptor gene was found in an Italia n patient with homozygous familial hypercholesterolemia. The SSCP anal ysis of the promoter and of 16 out of the 18 exons of the LDL-receptor gene was negative, suggesting that the mutation might be located in t he region of the gene encompassing exons 14 and 15, a region that had not been amenable to polymerase chain reaction (PCR) amplification fro m genomic DNA. This region was amplified from cDNA by reverse transcri ption PCR (RT-PCR). RT-PCR of proband cDNA generated three fragments o f 800, 600, and 550 bp, respectively, as opposed to a single 720 bp fr agment obtained from control cDNA. The sequence of these fragments sho wed that: i) in the 800-bp fragment exon 14 continued with the 5' end of intron 15 (90 nucleotides), which in turn was followed by exon 16; ii) in the 600-bp fragment exon 14 was followed by the 5' end of exon 15 (50 nucleotides), which continued with exon 16; iii) in the 550-bp fragment exon 14 joined directly to exon 16. These abnormally spliced mRNAs resulted from a G-->A transition at the +1 nucleotide of intron 15, which changed the invariant GT dinucleotide of the 5' donor splice site. That was associated with the activation of two cryptic donor sp lice sites in intron 15 and exon 15, respectively, and the use of an a lternative splicing leading to the skipping of exon 15. Northern blot analysis showed that the overall content of these aberrantly spliced m RNAs in proband fibroblasts was one-fourth that found in control cells . These abnormally spliced mRNAs are predicted to encode three abnorma l receptor proteins: the first would contain an insertion of 30 novel amino acids; the second would be a truncated protein of 709 amino acid s; the third would be devoid of the 57 amino acids of the O-linked sug ar domain. Ligand blot experiments indicated that the amount of LDL-re ceptor present in proband's fibroblasts was approximately one-tenth th at found in control cells.