Exon 10 skipping caused by intron 10 splice donor site mutation in cholesteryl ester transfer protein gene results in abnormal downstream splice site selection

1996 
Cholesteryl ester transfer protein (CETP) defi- ciency is the most common cause of hyperalphalipoprote- inemia in Japan. However, the genetic basis of this disorder has not been fully characterized. We have studied a 49-year- old Japanese male presenting with total cholesterol, HDL- cholesterol, and apolipoprotein A-I levels of 300, 236, and 233 mg/dl, respectively, and total absence of CETP activity and mass in plasma. Sequence analysis of the patient's CETP gene revealed that the splice donor consensus GT was substi- tuted by GG in intron 10 (intron 10 splice defect) and by AT in intron 14 (intron 14 splice defect). Restriction digestion of PCR-amplified DNA using NdeI and Mae111 established that the patient was a compound heterozygote for both gene defects. Sequencing of cDNA amplified by RT-PCR from the patient's monocytederived macrophage RNA demonstrated abnormal splicing with deletion of exon 10 as well as alterna- tive splicing at a native AG site located 31 nucleotides 5' of the normal splice acceptor in intron 13. Thus, the intron 10 splice defect results in exon 10 skipping and the insertion of a 31 bp fragment between exon 13 and exon 14, which contains an in frame stop codon. The presence of abnormally spliced mRNA was further confirmed by amplification of patient cDNA using CETP specific primers. Abnormal splic- ing of exon 14 as a result of the intron 14 splice defect was not detected, indicating potential unstable CETP mRNA de- rived from that mutation. I These findings demonstrate that a novel splice site mutation in intron 10 of the CETP gene results in the skipping of exon 10, as well as disruption of downstream splicing at intron 13 identifymg a novel mechanism leading to CETP deficiency.Sakai, N., S. San- tamarina-Fojo, s. Yamashita, Y. Matsuzawa, and H. B. Brewer, Jr. Exon 10 skipping caused by intron 10 splice donor site mutation in cholesteryl ester transfer protein gene results in abnormal downstream splice site se1ection.J. Lipid Res. 1996. 37: 2065-2073. High density lipoprotein (HDL) is the major an- tiatherogenic lipoprotein with increased plasma concen- tration of HDL associated with low risk of coronary heart disease (1-4). One of several proposed mecha- nisms by which HDL may protect against the develop ment of atherosclerosis is reverse cholesterol transport (5, 6). In this process, HDL acts as a carrier lipoprotein that mediates the transfer of cholesterol from periph- eral cells to the liver (5-8). Various transfer proteins, enzymes, and receptors, such as 1ecithin:cholesterol acyl- transferase (LCAT), cholesteryl ester transfer protein (CETP), hepatic triglyceride lipase, low density lipopro- tein (LDL) receptors, remnant receptors, and putative HDL receptors may play key roles in this process of reverse cholesterol transport. CETP is a glycoprotein that mediates mainly the transfer of cholesteryl ester (CE) from HDL to lower density lipoproteins, such as very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL in exchange for triglycerides (TG) in human plasma (9-1 1). The important role of CETP-mediated lipid transfer in reverse cholesterol transport has been established by the identification and characterization of patients with CETP deficiency (12-17). CETP deficiency is the most common cause of hyperalphalipoprote-
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