Abnormal splicing of ABCA 1 pre-mRNA in Tangier disease due to a IVS 2 5 G C mutation in ABCA 1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i ) G C in intron 2 (IVS2 5G C) and ii ) c.844 C T in exon 9 (R282X). The IVS2 5G C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 5G C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2 /mRNA), exon 4 (Ex4 /mRNA), or both these exons (Ex2 /Ex4 / mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 5G C mutation, we constructed two ABCA1 minigenes encompassing Ex1–Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2 /mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations. — Altilia, S. , L. Pisciotta, R. Garuti, P. Tarugi, A. Cantafora, L. Calabresi, J. Tagliabue, S. Maccari, F. Bernini, I. Zanotti, C. Vergani, S. Bertolini, and S. Calandra. Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 5G C mutation in ABCA1 gene. J. Lipid Res. 2003. 44: 254–264. Supplementary key words ABCA1 minigenes • in vitro splicing • donor splice site mutation Tangier disease (OMIM 600046) is a rare recessive disorder characterized by a severe deficiency or absence of HDL in plasma and an accumulation of cholesteryl esters in macrophages and other reticuloendothelial cells in many tissues (1). Cholesteryl esters accumulation accounts for some of the clinical features of Tangier disease (TD), including orange-yellow tonsils, hepatosplenomegaly, peripheral nerve neuropathy, and corneal opacifications (1, 2). The biochemical features of TD reflect a defect in the efflux of cholesterol and phospholipids from the cells due to a defective function of a membrane transporter designated ABCA1 (2, 3). This transporter plays a key role in apolipoprotein A-I (apoA-I)-mediated cholesterol efflux from the cells, which represents one of the first steps in the reverse cholesterol transport (3). Mutations in the ABCA1 gene leading to the loss of function or deficiency of ABCA1 protein are the underlying defects in TD and in the codominant disorder, familial HDL deficiency (FHD) (1–3). Since the cloning of ABCA1 cDNA (GenBank no. AF285167), more than 40 mutations of this gene have been reported in patients with TD and FHD (4–17). ABCA1 gene (GenBank no. AF275948) is located on chromosome 9 (9q31) and comprises 50 exons (18). The open reading frame of the ABCA1 transcript is of 6,783 bp (18, 19), as opposed to the 6,603 described in an earlier report (20). This transcript contains an in-frame ATG codon in exon 2, predicted to be the correct translation start site. This ATG codon is in a strong context, consistent with Kozak’s rules for initiation of translation (21), in contrast with the ATG in exon 4 (a potential translation initiation site), which is in a weaker context with regard to these rules. The translation product of the full-length ABCA1 mRNA is a protein of 2,261 amino acids (18, 19), 60 amino acids more than originally reported (1). This 601 To whom correspondence should be addressed. e-mail: sebcal@unimo.it Manuscript received 26 June 2002 and in revised form 23 October 2002. Published, JLR Papers in Press, November 4, 2002. DOI 10.1194/jlr.M200248-JLR200 by gest, on N ovem er 7, 2017 w w w . j.org D ow nladed fom