RESUMO
A novel point mutation of the LDL-receptor gene was found in an Italian patient with homozygous familial hypercholesterolemia. The SSCP analysis 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 the region of the gene encompassing exons 14 and 15, a region that had not been amenable to polymerase chain reaction (PCR) amplification from genomic DNA. This region was amplified from cDNA by reverse transcription PCR (RT-PCR). RT-PCR of proband cDNA generated three fragments of 800, 600, and 550 bp, respectively, as opposed to a single 720 bp fragment obtained from control cDNA. The sequence of these fragments showed 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 splice sites in intron 15 and exon 15, respectively, and the use of an alternative splicing leading to the skipping of exon 15. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was one-fourth that found in control cells. These abnormally spliced mRNAs are predicted to encode three abnormal receptor proteins: the first would contain an insertion of 30 novel amino acids; the second would be a truncated protein of 709 amino acids; the third would be devoid of the 57 amino acids of the O-linked sugar domain. Ligand blot experiments indicated that the amount of LDL-receptor present in proband's fibroblasts was approximately one-tenth that found in control cells.