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Journal of Lipid Research, Vol. 44, 254-264, February 2003
Copyright © 2003 by Lipid Research, Inc.

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Serena Altilia^*, Livia Pisciotta, Rita Garuti^*, Patrizia Tarugi^*, Alfredo Cantafora, Laura Calabresi^**, Jacopo Tagliabue, Sergio Maccari, Franco Bernini^***, Ilaria Zanotti^***, Carlo Vergani, Stefano Bertolini and Sebastiano Calandra^1,**

^* Department of Biomedical Sciences, University of Modena and Reggio Emilia
Department of Internal Medicine, University of Genoa
National Institute of Health, Rome
^** Department of Pharmacological Sciences, Center "E. Grossi Paoletti," University of Milan
Ospedale Maggiore-IRCCS, Milan
Sant'Anna Hospital, Castelnuovo Monti, Reggio Emilia
^*** Departments of Pharmacological, Biological Sciences and Applied Chemistries, University of Parma, Italy

¹ To whom correspondence should be addressed. e-mail: sebcal{at}unimo.it

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.

Supplementary key words ABCA1 minigenes • in vitro splicing • donor splice site mutation

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