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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1376-1381.)
© 1997 American Heart Association, Inc.

Articles

Skipping of Exon 14 and Possible Instability of Both the mRNA and the Resultant Truncated Protein Underlie a Common Cholesteryl Ester Transfer Protein Deficiency in Japan

Takanari Gotoda; Makoto Kinoshita; Shun Ishibashi; Toshimori Inaba; Kenji Harada; Masako Shimada; Jun-ichi Osuga; Tamio Teramoto; Yoshio Yazaki; ; Nobuhiro Yamada

From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo (T.G., S.I., T.I., K.H., M.S., J.O., Y.Y., N.Y.), and the First Department of Internal Medicine, Teikyo University School of Medicine (M.K., T.T.), Tokyo, Japan.

Correspondence to Takanari Gotoda, MD, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113, Japan. E-mail tknrgtd-tky{at}umin.ac.jp

	Abstract

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Abstract Among the Japanese population, a G-to-A mutation at the beginning of intron 14 of the human cholesteryl ester transfer protein (CETP) gene is a frequent cause of CETP deficiency characterized by markedly increased HDL cholesterol. The resulting abnormalities responsible for null CETP deficiency were studied in detail. The CETP mRNA transcripts amplified by polymerase chain reaction from the monocyte-derived macrophages of two homozygous patients were both found to be normal except for the whole deletion of exon 14. The deletion causes a shift of reading frame and introduces a premature termination codon downstream. Examination of the macrophage RNA from heterozygotes suggested the increased instability of the abnormal mRNA in the cytoplasm, because the amount of the aberrant transcript was nearly one third that of a normal transcript in the cytoplasm, while they were equal in the nucleus. Although this indicated the synthesis of a mutant CETP that lacks about 15% at its carboxy terminus, immunoblot analysis demonstrated that the abnormal CETP was virtually absent in both the media and cell lysates of transfected COS-1 cells, which massively expressed the mutant CETP mRNA. These results elucidate the primary abnormality due to the common CETP splicing mutation to be the exon skipping of mRNA, which decreases the level of mRNA and produces a truncated protein that should be rapidly degraded intracellularly.

Key Words: high-density lipoproteins • macrophages • splicing mutation • hyperalphalipoproteinemia • exon skipping

	Introduction

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The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and regulates plasma HDL cholesterol levels.¹ The genetic deficiency of CETP was first described in 1985 in Japanese patients with complete absence of plasma CETP activity and markedly increased levels of HDL cholesterol.^{2 3} The initial probands were shown in 1989 to be homozygous for a G-to-A substitution at the first nucleotide of intron 14 of the human CETP gene,^{4 5} and subsequently, the same point mutation was also found in the CETP gene of many other Japanese patients, suggesting the causative effect of the donor splice site mutation.^{6 7 8 9 10} However, the exact mechanisms whereby the mutation causes CETP deficiency have remained to be elucidated, although the mutation was assumed to have a null allelic effect because all homozygotes had no detectable levels of CETP mass and activity in the plasma.^{4 5 6 7} The mutant allele is relatively common among the Japanese population, possibly by a founder effect,⁶ and would be present in about 1% of the normal Japanese population.^{8 9 10}

Similar donor splice site mutations have been reported in other human genetic diseases, while the resultant changes were quite varied in terms of mRNA and protein levels.^{11 12 13 14 15} The abundance of the corresponding mRNA ranges from markedly decreased^{11 12} to normal levels.^{13 14 15} The pattern of aberrant splicing includes the activation of nearby cryptic splice sites,^{11 12 15} skipping of the preceding exon,^{13 14 15} and retention of an unspliced intron in the mature transcripts.¹⁵ The amounts of the protein mass, which might reflect the synthesis, stability, and/or secretion of the abnormal product, are also diverse.

In the present study, we clarify the molecular consequences of the common CETP splicing mutation at both the mRNA and protein levels. The results provide an insight into mRNA processing and suggest the importance of the carboxy terminus of CETP in the maintenance of the overall structure of the molecule.

	Methods

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Patients
Two unrelated Japanese subjects were newly diagnosed as CETP deficient during examination of severe hyperalphalipoproteinemia and were shown to have total deficiency of plasma CETP activity and mass. Their HDL cholesterol levels were 4.45 and 6.44 mmol/L, respectively (normal: 1.03 to 1.81 mmol/L). The patients showed no abnormality on physical examination and had no familial history of consanguinity. The study was approved by an institutional review committee and the study subjects gave informed consent.

Gene Amplification by Polymerase Chain Reaction (PCR)
Human CETP is composed of 476 amino acids encoded by 16 exons.^{16 17} Gene fragments containing the exon 14–intron 14 boundary were amplified by PCR from white blood cell DNA as done previously.¹⁸ A pair of oligonucleotide primers (primer J: 5'-AGCAGCTCCGAGTCCATCCAG-3' and primer K: 5'-AGTTTCCCCTCCAGCCCACA-3') were synthesized according to the published data¹⁷ to amplify the fragments that covered from 73 bp upstream to 24 bp downstream of the G-to-A substitution site.

mRNA Amplification by Reverse Transcription–PCR (RT-PCR)
Monocyte-derived macrophages were prepared by culturing human monocytes isolated from peripheral blood cells as described elsewhere.¹² Total cellular RNA was isolated from differentiated macrophages, reverse transcribed, and amplified by PCR as described previously.¹² The reverse transcription was performed with 15 pmol of 3'-outer primer (primer M: 5'-ACACCAGGGTTCCAGCTGTGA-3'). The total products were subjected to the first PCR with 15 pmol of 5'-outer primer (primer A: 5'-GGGCCACTTACACACCAC-3'). To amplify the target regions, 1/1000 of the first PCR products was subjected to the second PCR, together with an appropriate pair of internal primers. Four cDNA fragments (nucleotide positions 112 to 790, 737 to 1143, 1080 to 1249, and 1202 to 1659)¹⁶ that covered the entire coding region of human CETP cDNA were amplified for each patient. Nuclear and cytoplasmic RNAs were isolated separately from monocyte-derived macrophages of two heterozygotes according to the method reported previously.¹⁹

DNA Sequencing
DNA sequencing was performed in principle by direct sequencing of DNA fragments amplified by asymmetrical PCR.¹⁸ Several fragments were also sequenced after subcloning into the plasmid vector Bluescript II (Stratagene).

Plasma CETP Activity and Mass
Plasma CETP activity was assayed as the rate of transfer of [¹⁴C]cholesteryl ester from discoidal bilayer particle to LDL as described previously.²⁰ Plasma CETP mass was measured by radioimmunoassay with monoclonal antibody against human CETP (LT-A4) as described by Fukasawa et al.²¹

Construction of Expression Plasmids
A 1559-bp fragment (nucleotides 101 to 1659) encompassing the entire coding sequence of human CETP cDNA was amplified from normal macrophage RNA by the first PCR with Pfu DNA polymerase (Stratagene). In the second PCR, 10 cycles of amplifications were performed with mismatched primers A' and L' (primer A': 5'-GGGAAGCTTACACACCACTGCCTGATA-3' and primer L': 5'-GTGCTTGCCTTCTTCTAGAAGCCC-3'), which introduced restriction sites for HindIII and Xba I, respectively, at each end of the DNA fragment. The fragment was digested with HindIII and Xba I and transferred to the HindIII/Xba I sites of the pRc/CMV vector (Invitrogen). Subsequently, within this wild-type construct, a 686-bp Sph I–Xba I segment (nucleotides 960 to 1643) was replaced by the corresponding segment amplified from the patient's macrophage RNA. The integrity of these wild-type and mutant expression plasmids was verified by DNA sequencing before transfection into COS-1 cells.

In Vitro Expression of CETP in COS-1 Cells
COS-1 cells (8.0x10⁶ cells) were transfected with 20 µg of purified plasmid DNA by lipofection with Lipofectin (GIBCO-BRL) according to the manufacturer's instruction. The cells were incubated in 8 mL of serum-free medium Opti-MEM (GIBCO-BRL) for 72 hours, and the medium was collected. The cells were harvested and disrupted by sonication according to the procedure described by Wang et al²² and resuspended in 50 µL of PBS containing 1 mmol/L PMSF.

Immunoblot and Northern Blot Analyses
Each culture medium (5 mL) or cell lysate (50 µL) was incubated for 18 hours at 4°C with a mixture of four separate monoclonal antibodies (5 µg protein per milliliter each), including LT-A4, LT-F1, and LT-J1,²¹ as well as 2A-21, which was raised against a synthetic peptide corresponding to amino acids 361 to 379 of human CETP.²³ Pansorbin was then added to the mixture to form immune complex as described previously.²⁴ After centrifugation, the precipitates were washed three times with PBS, resuspended in SDS–polyacrylamide gel electrophoresis sample buffer, heated to 95°C for 5 minutes, and then subjected to SDS–polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. CETP on the membrane was reacted as described previously²⁰ with monoclonal antibody 2A-21 or LT-A4. Northern blot analysis was performed as described previously,¹² with a probe of human CETP cDNA fragment (737 to 1143) amplified by PCR.

	Results

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The newly identified two patients had severe hyperalphalipoproteinemia, and their plasma contained no detectable levels of CETP activity and mass. Nucleotide sequence analysis around the exon 14–intron 14 boundary confirmed that the two patients were homozygous for a G-to-A substitution at the first nucleotide of intron 14 (data not shown). The point mutation disrupts the invariant GT profile of the eukaryotic 5'-donor splice site, which is essential for normal splicing of mRNA.²⁵

To investigate the resultant abnormality introduced into CETP mRNA, monocyte-derived macrophages were employed as a source of CETP mRNA.²⁶ The mRNA was examined in detail by RT-PCR. Among a series of experiments, the amplifications of the 3'-portions of cDNA yielded distinctive results for normal samples and the patients' samples (Fig 1). Electrophoretic analysis of the normal PCR products showed bands of the expected sizes on ethidium bromide–stained agarose gel. In contrast, the same PCRs in the two patients either gave rise to a 73-bp-shorter fragment or failed in the amplification (Fig 1A). The results indicated an identical aberrant splicing event in the CETP mRNA of the patients. DNA sequencing of the 73-bp-shorter fragments revealed the complete absence of exon 14 in the abnormal transcript, which was the result of the direct splicing from exon 13 to exon 15 (Fig 2). No other alteration was found in the entire coding sequence of the CETP cDNA obtained from the patients. Thus, it could be concluded that the skipping of exon 14 is a major structural abnormality introduced into the CETP mRNA by the common intron 14 donor splice site mutation.

View larger version (62K): [in this window] [in a new window]   Figure 1. Detection of aberrant splicing in the CETP mRNA of the patients. A, PCR amplifications of three regions of CETP cDNA are comparatively shown for a normal subject and the two patients. n indicates normal; P1, patient 1; and P2, patient 2. Reaction products were analyzed on 1.5% agarose gels with DNA molecular size standards (molecular size standards [M], øX174/Hae III). B, The locations of the six PCR primers (primers H through M) used for amplifications are represented schematically on the structure of the human CETP gene (primer H: 5'-AATTCTTCAGTGATGGTGAA-3', primer I: 5'-CAACATTCTGTAGCTTACAC-3', primer J: 5'-AGCAGCTCCGAGTCCATCCAG-3', primer K: 5'-AGTTTCCCCTCCAGCCCACA-3', primer L: 5'-GTGCTTGCCTTCTGCTACAA-3', and primer M: 5'-ACACCAGGGTTCCAGCTGTGA-3').

View larger version (73K): [in this window] [in a new window]   Figure 2. Comparison of the cDNA sequences coding for normal and patient CETP. The autoradiograms represent the nucleotide sequences of CETP cDNA from a normal subject and a patient. The cDNA sequence from the patient revealed a whole deletion of the 73 nucleotides that precisely correspond to exon 14.

The relative abundance of the abnormal transcript was examined by the competitive PCR method.²⁷ Since both the normal 413-bp and the abnormal 340-bp fragments can be coamplified with the same set of primers (primers I and L), and their sizes vary only slightly, coamplification of the two fragments is thought to occur in a concentration-dependent manner. As shown in Fig 3A, the similar levels of the two fragments were obtained when RNAs of the normal subject and either patient were mixed in the ratio of 1:3, implying that the abundance of the abnormal transcript is about one third of that of the normal. In a parallel experiment (Fig 3B), the abundance of CETP mRNA in human monocyte-derived macrophages was estimated to be about 1/30 of that in the human liver, a principal source of CETP.¹ Independently of these experiments, the CETP mRNA was also examined in heterozygous subjects (Fig 4). The result confirmed that the level of the aberrantly spliced transcript is nearly one third of that of the normal (Fig 4A) and also showed that the recently identified Asp⁴⁴²-to-Gly mutation underlying partial CETP deficiency^{7 28 29} hardly influences mRNA levels in vivo in contrast with the intron 14 splicing mutation (Fig 4B).

View larger version (63K): [in this window] [in a new window]   Figure 3. Quantitation of CETP mRNA by the competitive PCR method. A, RNAs isolated from monocyte-derived macrophages of a normal subject and each patient were mixed in a series of ratios as indicated, and the mixtures (1 µg RNA) were amplified by RT-PCR with primers I and L. The relative amount of the two products (normal 413-bp and abnormal 340-bp bands) reflects the original contents of the normal and mutant CETP mRNAs. n indicates normal; P1, patient 1; and P2, patient 2. B, RNA isolated from the liver of a normal subject was mixed in a series of ratios with that from monocyte-derived macrophages of patient 1. The mixtures were coamplified as described above.

View larger version (13K): [in this window] [in a new window]   Figure 4. Simultaneous amplification of both normal and mutant CETP mRNA transcripts in heterozygotes. CETP mRNA transcripts were amplified by RT-PCR from monocyte-derived macrophages of two subjects who were heterozygous for the intron 14 donor splice site mutation (A) or for the Asp442-to-Gly mutation (B). In each panel, schematic representation of the predicted structure of the normal and mutant CETP cDNAs is given on the left, and electrophoretic analysis on 1.5% agarose gels is shown on the right. Molecular size standards (M) are øX174/Hae III. He indicates heterozygotes. The PCR products in the heterozygote for the Asp442-to-Gly mutation (B) were amplified using a mismatched primer (primer N: 5'-AGCAAAGGCGTGAGCCTCGTC-3'), together with primer L, and were subjected to digestion with restriction enzyme Sal I.

To investigate the reason for the mRNA reduction, RNA was isolated separately from nucleus and cytoplasm of monocyte-derived macrophages of two unrelated individuals heterozygous for the intron 14 splicing mutation. The result with the same RT-PCR method as in Fig 4A demonstrated that in both subjects, the amount of the abnormal transcript was equal to that of the normal transcript in the nucleus, although they differed significantly in the cytoplasm (Fig 5). This observation provides evidence that the mRNA reduction is not due to a reduced level of transcription but probably to the increased instability of the mutant transcript in the cytoplasm.

View larger version (35K): [in this window] [in a new window]   Figure 5. Amplification of CETP mRNA transcripts from nuclear and cytoplasmic RNAs of heterozygotes. CETP mRNA transcripts were amplified by RT-PCR from RNAs isolated separately from nucleus and cytoplasm of monocyte-derived macrophages of two unrelated heterozygotes for the intron 14 donor splice site mutation. The normal 458-bp and the mutant 385-bp bands were coamplified by the same RT-PCR as in Fig 4A. Molecular size standards (M) are øX174/Hae III. H1 indicates heterozygote 1 and H2, heterozygote 2.

Fig 6 shows the schematic representation of the exon-skipping event, as well as the predicted amino acid sequence of the mutant CETP. The skipping of exon 14 (73 bp) causes a shift of reading frame and introduces a premature termination codon (TAG) three residues downstream. These changes lead to the production of a mutant protein with a loss of 74 amino acid residues at its carboxy terminus and with alterations in two of its last three residues. Coupled with the observed expression of the skipped mRNA (Figs 3 through 5), this implies the synthesis of a significant level of the truncated CETP in patients.

View larger version (29K): [in this window] [in a new window]   Figure 6. Exon skipping in patient CETP mRNA leading to a shift of reading frame on translation. A, Diagram of the partial genomic structure (exons 12 to 16) of the normal and patient CETP genes. Exons are indicated by open boxes. The GT-to-AT change in the donor splice site of intron 14 results in the ligation of exon 13 to exon 15 during processing of the patient CETP mRNA. B, Comparison of nucleotide sequences and predicted amino acid sequences for the normal and mutant CETP. In the nucleotide sequence of the patient cDNA, a TAG stop codon (underlined) is introduced into position 403, due to the whole deletion of exon 14 and the concomitant shift of reading frame. The amino acid residues altered by the frameshift are marked with asterisks.

To elucidate the reason for the observed complete lack of CETP mass in the patients' plasma, plasmids harboring the normal human CETP cDNA or the mutant CETP cDNA lacking exon 14 were transiently expressed in COS-1 cells, and the culture media and cell homogenates were studied by immunoblot analysis with monoclonal antibody 2A-21 (Fig 7). The epitope of 2A-21 exists within the region encoded by exons 11 and 12 and thus upstream of the skipped exon 14. The cells expressed comparable levels of normal and mutant CETP mRNA transcripts (Fig 7A). However, immunoblot analysis showed that both the media and the cell homogenates from cells transfected with the mutant plasmid contained no detectable level of immunoreactive protein, while those from cells with the normal plasmid had significant levels of signals for CETP, with approximate molecular weight of 66 kD (Fig 7B). Similar results were also obtained with another antibody, LT-A4 (data not shown). These results establish that the truncated mutant CETP molecule resulting from the skipping of exon 14 appears neither inside nor outside cells, most compatible with the extreme instability of the mutant CETP polypeptide.

View larger version (71K): [in this window] [in a new window]   Figure 7. CETP expression in COS-1 cells transfected with the normal (N) and mutant (M) expression vectors. A, Northern blot analysis of total RNA from the transfected cells. The mutant transcript is slightly smaller than the normal one, due to lack of exon 14. B, Immunoblot analysis of CETP in culture media (left) and cell homogenates (right) of the transfected cells. The signals for CETP with approximate molecular weight of 66 kD were detected in both the medium and cell homogenates of COS-1 cells transfected with the normal expression vector. Nonspecific bands were also seen in the bottom of the blots.

	Discussion

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Four separate CETP gene mutations have been published previously. Of the four mutations, the intron 14 donor splice site mutation⁴ and the Asp⁴⁴²-to-Gly mutation in exon 15²⁸ are both known to be very common in the Japanese population.^{7 8 9 10 29} The other two mutations, the Gln³⁰⁹-to-Stop mutation in exon 10²⁶ and a 1-bp insertion in intron 14,⁷ are probably sporadic mutations. Except for the Asp⁴⁴²-to-Gly mutation that causes partial CETP deficiency, the other three seemed to have null allelic effects, although exact mechanisms underlying the null effects have not been studied. In the present study, we demonstrated that the primary abnormality due to the intron 14 donor splice site mutation is the exon skipping of mRNA, which decreases the level of mRNA and produces a truncated protein that should be degraded intracellularly. These observations clearly explain the molecular basis of the complete CETP deficiency found not only in patients with the common intron 14 splicing mutation but also in a patient with the Gln³⁰⁹-to-Stop mutation that leads to a larger truncation of the carboxy terminus of CETP.

There is increasing evidence that nonsense and frameshift mutations that prematurely terminate mRNA translation decrease the corresponding mRNA levels by poorly understood mechanisms.³⁰ We previously showed that the level of the mutant CETP mRNA transcript with the Gln³⁰⁹-to-Stop mutation in exon 10 was about 1/10 that of the normal transcript.²⁶ In this study, we found a less severe reduction in the abundance of the mutant mRNA transcript with skipping of exon 14, indicating that the introduction of a premature stop codon at different positions of the same mRNA transcripts could have different effects on the levels of mRNA. This result also seems consistent with the notion that the closer a mutation is to the 3' end of a gene the higher the levels of abnormal mRNA.³¹ Decrease in the cytoplasmic mRNA pool can be caused by reduction of any of transcription, nuclear mRNA stability, mRNA transport from nucleus to cytoplasm, and cytoplasmic mRNA stability. The mutant CETP mRNA with skipping of exon 14 would most likely have a defect in the cytoplasmic stability, because its level was reduced only in the cytoplasm and was normal in the nucleus. The presence of the cytoplasmic pathways for the degradation of mRNA containing nonsense codons has been shown in yeast.³⁰

The functional importance of the carboxy terminus of CETP was first indicated by the observation that a monoclonal antibody bound to the last carboxy-terminal 26 amino acids of human CETP neutralizes the cholesteryl ester transfer activity.³² Recently, deletional and site-directed mutagenesis of the carboxy terminus of CETP showed that amino acid residues 470 to 475 are directly involved in neutral lipid binding and are essential for catalysis of cholesteryl ester transfer.^{22 33} Thus, even if normally processed, the mutant CETP with a deletion of residues 403 to 476 and alterations in residues 400 and 402 in the patients would never be active in cholesteryl ester transfer. Our results also indicated that the carboxy-terminal region was important to secure the stability of CETP, because the mutant CETP was virtually absent in the media and cell lysates of transfected COS-1 cells. Interestingly, it was previously shown that a mutant CETP lacking residues 411 to 476 is efficiently expressed and stable in transfected insect cells.³⁴ Taking these results into consideration, it is conceivable that amino acids within residues 400 to 410 are involved in the maintenance of the overall structure of the CETP molecule.

In summary, our results demonstrated that the intron 14 donor splice site mutation prevalent among Japanese patients with CETP deficiency causes skipping of exon 14 and creation of a premature stop codon in the mRNA transcript, whose abundance is significantly reduced. The absence of the resulting truncated protein in the transfected cells that massively expressed the mutant CETP mRNA provided a molecular basis of complete CETP deficiency caused by this common CETP mutation.

	Acknowledgments

 
We are indebted to Drs Masayoshi Fukasawa, Hiroyuki Arai, and Keizo Inoue for kindly providing anti-human CETP monoclonal antibodies, Dr Shin Ohnishi for the kind gift of human liver RNA, Dr Koichi Kozaki for technical assistance, and Drs Minoru Ohkubo and Toshio Murase for referring patients.

Received October 3, 1995; accepted November 1, 1996.

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