4 NOVEL PARTIAL DELETIONS OF LDL-RECEPTOR GENE IN ITALIAN PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA

In this study, we report four new partial deletions of the LDL-receptor (LDL-R) gene discovered during a survey of 326 Italian patients with familial hypercholesterolemia (FH). All deletions were found in FH heterozygotes whose LDL-R activity in skin fibroblasts ranged from 52% to 43% of the values found in control cells. The size and boundaries of the deletions were defined by Southern blotting and, in some cases, by polymerase chain reaction (PCR) amplification of genomic DNA. The sequence of the deletion joint was performed after the reverse transcription and PCR amplification of the appropriate regions of LDL-R mRNA. FHMassa is a 12-kilobase deletion spanning from intron 2 to intron 10. RT-PCR showed that the mutant allele is transcribed into one major and two minor mRNAs. In the most abundant mRNA species, exon 2 joins exon 11, as expected from DNA analysis. In one minor mRNA, which was sequenced, exon 2 joins exon 13, with exons 11 and 12 skipped as a result of an alternative splicing. FHGenova is a 4-kb deletion spanning from intron 10 to intron 12 and eliminating exons 11 and 12. FHRoma is a 4.7-kb deletion spanning from the 5' end of intron 12 to the middle of intron 14 and eliminating exons 13 and 14. This deletion differs in size from the previously described deletion (FHChieti/Macerata), which is located in the same region of the LDL-R gene but is smaller (3.7 kb). In both FHRoma and FHChieti/Macerata, the mutant LDL-R mRNA is present in a minute amount, suggesting that the deletion of exons 13 and 14 may increase mRNA degradation. FHPadova-2 is a 2-kb deletion spanning from intron 15 to intron 16 and eliminating the sole exon 16. All deletions except FHPadova-2 produce a shift in the reading frame, leading to either a very short peptide or a truncated protein. In FHPadova-2, elimination of exon 16 does not change the reading frame but is predicted to produce a receptor protein of 513 amino acids, lacking 18 amino acids of the O-linked sugar and 8 amino acids of the transmembrane domain. Ligand blot experiments with rabbit I-125-beta VLDL indicate that half the amount of LDL-receptor is present in FHPadova-2 fibroblasts, suggesting that the in-frame deletion of 26 amino acids may disrupt the intracellular transport and/or the insertion of the receptor in the plasma membrane or may increase its degradation.