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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3433-3441

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3433-3441.)
© 1997 American Heart Association, Inc.

Articles

Deficiency of Cholesteryl Ester Transfer Protein

Description of the Molecular Defect and the Dissociation of Cholesteryl Ester and Triglyceride Transport in Plasma

Andreas Ritsch; Heinz Drexel; Franz W. Amann; Christa Pfeifhofer; ; Josef R. Patsch

From the Departments of Medicine, University of Innsbruck, Innsbruck, Austria (A.R., C.P., J.R.P.), and the University Hospital Zürich, Zürich, Switzerland (H.D., F.W.A.).

Correspondence to Josef R. Patsch, MD, Department of Medicine, University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria.


*    Abstract
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 *Abstract
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Abstract A patient is described who exhibited, despite excessively high postprandial triglyceride levels, high levels of HDL cholesterol. Measurement of CETP activity and mass in the patient's plasma showed values of less than 5% and 2%, respectively, of a normolipidemic plasma pool. The CETP cDNA of the patient exhibited a mutation (T -> G), turning codon 57 (TAT) of exon 2 into a stop codon (TAG) and abolishing a, XcmI restriction site. Digestion of directly amplified CETP cDNA from the patient with XcmI indicated the exclusive presence of CETP cDNA containing the mutation. Analysis of the corresponding region of the CETP gene indicated the patient to be heterozygous for the nonsense mutation at codon 57, a finding that can only be explained by the presence of a null allele in addition to the allele with the nonsense mutation. The combination of TG intolerance of uncertain cause, together with CETP deficiency due to a novel mutation, produced the paradoxical constellation—high levels of HDL cholesterol (172 mg/dL) associated with a high postprandial lipemia of 1460 mg triglycerides/dL.8 hours—and provided further insight into the role of CETP as mediator between pools of triglycerides and cholesteryl esters in plasma.


Key Words: HDL • cholesteryl ester transfer protein • hyperalphalipoproteinemia • mutations • triglycerides


*    Introduction
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 *Introduction
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Transport of TG and CE in plasma can be viewed as taking place via two major groups of lipoproteins, the TG-rich lipoproteins (chylomicrons and VLDL) on one hand and the cholesterol-rich lipoproteins (LDL and HDL) on the other. Usually, however, these two avenues for TG and CE transport are not completely separated. Rather, they are linked by exchange processes catalyzed mostly by a plasma glycoprotein, LTP-I1 or CETP.2 3 4 5 To our knowledge, six molecular defects causing CETP deficiency have been elucidated thus far, five of them completely preventing synthesis of CETP,6 7 8 9 10 and one resulting in reduced activity.8 Affected family members have in their plasma high levels of HDL cholesterol6 7 8 known to be a negative risk factor for CAD.11 12 13 At least for one of the molecular defects with CETP deficiency, absence of atherosclerosis and longevity was reported.14

In frank hypertriglyceridemia HDL cholesterol and HDL2 levels are low.13 HDL2 levels are also low in individuals with normal fasting but high postprandial triglyceride levels.15 Cause and effect for this inverse relationship between fasting or postprandial triglyceride levels and HDL2 have been elucidated;16 for example, immunological blockage of LPL in the chicken is followed by immediate accumulation of TG-rich lipoproteins in plasma, which in turn is followed by a continuous reduction of HDL cholesterol and the replacement of large HDL particles by smaller, denser HDL.17 Another example is heterozygous LPL deficiency in man due to the amino acid exchange of Gly188->Glu, which can be associated with normal TG-levels in the postabsorptive state but, postprandially, is accompanied by pronounced and prolonged lipemia. For this condition the term "TG-intolerance" was coined in order to stress the fact that normotriglyceridemia is maintained in the unchallenged postabsorptive state while hypertriglyceridemia surfaces in the postprandial state of challenge.18 TG intolerance is associated with characteristic alterations in lipoprotein composition and distribution, namely, high levels of CE-enriched VLDL and IDL, small dense LDL, and low HDL cholesterol with decreased levels of HDL2 enriched with TG. These steady-state lipoprotein characteristics supplement the condition of TG intolerance to what was called the "syndrome of TG intolerance."18 The evidence from experimental and genetic deficiency of LPL—the key enzyme for TG catabolism—clearly demonstrates that the cause for the inverse relationship between triglyceride concentrations and HDL levels is the metabolism of TG and that the effect is the HDL level.

The relationship between TG and HDL (and the other lipoproteins) is mediated by CETP, and its deficiency could provide the opportunity to further elucidate the role of CETP for this relationship. In the absence of the action of CETP, even high postprandial TG levels would be expected to fail to leave marks on HDL2 and the other lipoproteins. We discovered such a constellation; we now report on a patient with TG intolerance who paradoxically displayed in her plasma—because of CETP deficiency due to a novel mutation—extremely high HDL2 levels and large buoyant LDL.


*    Methods
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 *Methods
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Study Subject
A 65-year-old woman was referred to Zürich University Hospital because of arterial hypertension and hypercholesterolemia. She was of Chinese origin and had lived in Europe since 1950. The patient's mother and two of the patient's four siblings had hypertension. No coronary disease was known in the patient's family. The patient had no signs of atherosclerotic disease and no clinical symptoms except exertional dyspnea (but not angina) of stage II according to the NYHA classification. The patient was in menopause since age 51, did not smoke, and consumed an average Western diet with about 40% calories originating from fat. Her height was 159 cm and her body weight was 53 kg and had been constant during the last years (body mass index 20.96 kg/m2). Blood pressure recordings ranged from 160/85 to 200/95 mm Hg. All results of the clinical examinations were normal including normal results of fundoscopy. ECG and chest x-ray also gave normal results. In particular, no signs of left ventricular hypertrophy were detectable. Blood chemistry revealed normal liver, kidney and thyroid function, and complete blood count was normal. The Ethics Committee of Zürich University Hospital approved the study, and informed consent by the patient was obtained.

Plasma Lipids, Lipoproteins, and Apolipoproteins
Plasma was collected after an overnight fast into vials containing EDTA to give a final concentration of 1 mg/mL. Cholesterol and triglycerides were measured in plasma by enzymatic methods.19 20 Quantification of HDL, HDL2, and HDL3 cholesterol included the same enzymatic methods in combination with a stepwise precipitation procedure to remove apoB-containing lipoproteins and HDL2, respectively.21 22 Plasma apoA-I and apoB were quantified by automated colorimetric and turbidimetric procedures, respectively (Böhringer Mannheim, Germany). ApoE phenotype was determined by isoelectric focusing of delipidated plasma, Western blotting, and immunostaining.23

An aliquot of postabsorptive plasma was subjected to rate zonal ultracentrifugation in a Ti-14 rotor (Beckman Instruments) under conditions to isolate VLDL, IDL, and LDL.24 25 A second aliquot was used to isolate HDL2 and HDL3, respectively.24 25 Zonal rotor fractions were analyzed for protein,26 phospholipids,27 total and unesterified cholesterol, and triglycerides (Böhringer Mannheim). Stokes diameters of LDL and HDL subfractions were estimated after electrophoresis under nondenaturing conditions on 2.5% to 16% and 4% to 30% polyacrylamide gradient gels (Isophore, Isolab Inc.), respectively.28 29

Postprandial Lipemia
Immediately after sampling of postabsorptive plasma, the patient ingested a liquid fatty meal described in detail previously.18 Briefly, the test meal contained per meter square of body surface area 65.0 g fat with a P/S ratio of 0.06, 24 g carbohydrate, 4.75 g protein, and 240 mg cholesterol. Triglycerides were determined 0, 2, 4, 6, 8, and 10 hours postprandially. The magnitude of postprandial lipemia was quantified by two methods as (1) the maximal postprandial triglyceride increase that represents half the sum of the two highest postprandial triglyceride values minus the fasting triglyceride value,15 and (2) as the area under the postprandial TG curve normalized to the fasting level.15

CETP Mass and Activity
Cholesteryl ester transfer activity was determined as described by Groener et al30 using a substrate-independent isotope assay that measures radiolabeled cholesteryl ester transfer from exogenous LDL to exogenous HDL, mediated by a fraction of the patient's fasting plasma from which VLDL and LDL had been removed before the assay procedure. Radiolabeling of exogenous LDL was performed according to the method of Morton and Zilversmit.31 CETP mass was quantified using an immunoradiometric assay employing a polyclonal antibody.32

LPL and HL Activity
The patient was injected in the postabsorptive state intravenously with 2,280 U/m2 heparin (Novo Industri A/S) to release LPL and HL into the circulation, and postheparin plasma was collected after 10 minutes. Measurements of LPL activity and HL activity were performed as described.18

Amplification of DNA and mRNA Fragments
Isolation of genomic DNA and total RNA from mononuclear cells of the patient's blood was performed according to standard procedures.33 34 For amplification of DNA fragments PCR was performed in 10 mmol/L Tris (pH 9.0, 25°C), 50 mmol/L KCl, 15 mmol/L MgCl2, 0.1% gelatin, and 1% Triton X-100, supplemented with corresponding quantities of dNTPs, primers, and Taq DNA polymerase (Böhringer Mannheim). Long- range PCR was performed with ELONGASE Enzyme Mix (Life Technologies) according to the user's manual. The whole CETP gene was amplified in two overlapping fragments using primers F2 and R1 (fragment D, {approx}12 kB), as well as primers F3 and R2 (fragment E, {approx}12 kB), respectively. Primers for amplification and sequence analysis of all CETP exons with adjacent intron sequences had been synthesized according to published sequence data (accession numbers M32992 and J02898, EMBL + GenBank Release 82) or as described7 and are listed in Table 1Down.


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  		Table 1. Sequences of Primers Used for PCR, RT-PCR, and Sequence Analysis

Reverse transcriptase PCR was performed in 25 µL of 50 mmol/L Tris (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, supplemented with 5 nmol of each dNTP, 5 U of RNAse-inhibitor from human placenta (Böhringer Mannheim), 15 pmol of primer R3, and 12 U of Avian Myeloblastosis Virus Reverse Transcriptase (Promega). Total RNA isolated from mononuclear cells from blood according to the guanidinium thiocyanic acid method34 was used as template. The whole reverse transcription reaction was subjected to the first PCR using primers F1 and R3. Using aliquots of the first PCR as templates, two overlapping fragments of the CETP cDNA were amplified using primers F2 and R1 (fragment A, 864 bp), as well as primers F3 and R2 (fragment B, 799bp), respectively. To amplify a shortened fragment designated fragment A' (469 bp) reverse transcription was performed using primer R1, followed by PCR using primers F2 and Ex 4/5. Primers for amplification and sequence analysis were synthesized according to published sequence data (accession numbers M32992 and J02898, EMBL + GenBank Release 82) or as described7 and are listed in Table 1Up.

All amplification reactions were performed in 0.5 mL polypropylene microtubes (Treff) with an overlay of Paraffin liquid (Merck) in a TRIO-Thermoblock (Biometra). XcmI digestion of PCR fragments was performed after the user's manual (New England Biolabs). Amplified cDNA and genomic DNA fragments were subcloned into pUC18 for sequence analysis or were sequenced directly. Manual DNA sequencing was performed with Sequenase Version 2.0 DNA Sequencing Kit (Amersham) according to the user's manual. Direct sequencing of PCR products was performed with Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham) using Automated DNA Sequencer 2000 L (LI-COR Inc.).

Cell Transfection and CAT Assays
HepG2 cells were cultured in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, at 37°C in 5% CO2 and 95% air. One day before the transfection procedure cells were plated in MultiWell plates (9.6 cm2/well, Falcon). Transfection was performed using the Lipofectin reagent (Gibco BRL, Life Technologies) as described by Felgner.35 Twenty-four hours after transfection extracts of cells were prepared and subjected to analysis for CAT activity.36 Plasmids pBLCAT3 (without promoter) and pBLCAT2 containing the herpes simplex virus TK promoter were used as negative and positive control of transfection, respectively.37


*    Results
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 *Results
down arrowDiscussion
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On routine lipid analysis, the high total plasma cholesterol level of patient M.Y. turned out to be due to an extremely high concentration of HDL cholesterol while LDL cholesterol was low. Most of HDL cholesterol was contained in the fraction precipitated with HDL2.22 In agreement with this lipoprotein lipid distribution, concentration of apoA-I was very high, and that of apoB was low (Table 2Down). After confirming the conspicuous lipid profile in repeated measurements careful analysis of lipoproteins and lipid-modifying enzymes was begun.


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  		Table 2. Fasting Plasma Lipid and Apolipoprotein Levels of Patient M.Y.

Zonal ultracentrifugal analysis showed an excessively high lipoprotein peak within the elution volume range of HDL2 (Fig 1Down, I, B). The exact peak elution volume was only 100 mL as opposed to 120 mL to 130 mL for typical HDL224 suggesting that the "HDL2" of patient M.Y. was of somewhat lower density and larger size than typical HDL2. Indeed, nondenaturing gradient gel electrophoresis showed a mean Stokes diameter of 12.5 nm compared to 10.5 nm of typical HDL2.38



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  		Figure 1. Magnitude of postprandial lipemia and parameters associated with the syndrome of impaired TG tolerance. (A) Postprandial plasma TG values after an oral fat meal in M.Y. (I), 8 carriers of LPL deficiency (II), and 8 controls (III). (B, C) Zonal ultracentrifugal analysis of HDL subfractions (B), and lipoproteins with density <1.063g/mL (C), in M.Y. (I) and one representative subject of carriers of LPL deficiency (II) and of controls (III), respectively. The core compositions of HDL2 and IDL are shown schematically in sector diagrams, where black-and-white sectors denote weight percentages of cholesteryl esters and TG, respectively. Data except those of M.Y. are derived from Miesenböck et al.18

Zonal ultracentrifugal analysis for apoB-containing lipoproteins25 showed a very small LDL peak consistent with the low LDL cholesterol level of 83 mg/dL plasma (Table 2Up). This LDL peak displayed a high degree of heterogeneity with at least three subpopulations (Fig 1Up, I, C). Heterogeneity of LDL in CETP deficiency has been described before.39 This finding was evident not only on zonal ultracentrifugation but also on gradient gel electrophoresis. Here, the patient's LDL displayed a major peak with a median Stokes diameter of 26.8 nm, and a minor peak with an even larger diameter of 28.3 nm. Thus, LDL of patient M.Y. showed an overall larger size than what has been described for LDL pattern A.40

Compositional data on lipoprotein fractions are summarized in Table 3Down. VLDL and IDL contained TG as their major core lipid. LDL contained mostly CE. The two HDL subfractions, HDL2 and HDL3, contained almost exclusively CE in their core and only trace amounts of TG.


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  		Table 3. Composition of Lipoprotein Fractions of Patient M.Y.

Since high HDL2 levels in the postabsorptive state are associated with small postprandial triglyceride elevations,15 we were anxious to find out how patient M.Y. with her extremely high plasma levels of HDL2 would react to our standard oral fat load test. Instead of the expected attenuated postprandial rise of triglycerides, M.Y. showed a very pronounced lipemia with a maximal TG increase of 280 mg/dL and the unusually large calculated postprandial lipemia of 1460 mg TG/dL plasma.8 hours (Fig 1Up, I, A). The high postprandial lipemia was not caused by reduced activities of HL or LPL in the patient's plasma, which were 269 mU/mL and 314 mU/mL, respectively.

The lipoprotein profile, particularly the extremely high levels of large, virtually TG-free HDL2 particles and the lack of the usual reciprocal relationship between HDL2 levels and the magnitude of postprandial lipemia15 pointed to a potential problem with neutral lipid transfer and its major catalyst, CETP. Measurement of CETP mass and activity with our CETP IRMA32 and a specific CETP activity assay30 gave, in repeated measurements, less than 2% of CETP mass concentration and less than 5% of CETP activity of a standard normolipidemic plasma pool.32

To elucidate the molecular cause of the apparent CETP deficiency we examined the coding sequence of the CETP gene. CETP mRNA of M.Y. was isolated through reverse transcriptase PCR. As source of the CETP transcript we used total RNA from mononuclear cells obtained from the blood of patient M.Y. The CETP mRNA was amplified in two overlapping fragments, A and B, as described by others.7 Both fragments appeared to be of the same length when compared to the corresponding fragments of a control subject. Sequence analysis of fragment A revealed a point mutation (T -> G) in codon 57 of exon 2 turning the codon for tyrosin (TAT) into a stop-codon (TAG) (Fig 2Down).



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  		Figure 2. Sequence analysis of the CETP cDNA at codon 57 of exon 2 of patient M.Y. and a control subject. Exchange of T for G at the third position of codon 57 in the CETP cDNA of patient M.Y. lead to the formation of a stop codon (Stop) instead of the codon for tyrosin (Tyr).

The mutation abolished one of two XcmI restriction sites of fragment A, ie, the one at position 603, leaving the other at position 653 unchanged. XcmI digestion of fragment A amplified directly from total RNA clearly indicated the presence of the XcmI site at position 653 and the absence of the one at position 603 (Fig 3Down). This result indicates that the entire CETP cDNA material that we were able to amplify and isolate contained the point mutation at codon 57 of exon 2. Synthesis of any functional CETP appeared to not take place because we could not detect any of a correct CETP mRNA in patient M.Y.



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  		Figure 3. Amplification and analysis of a CETP cDNA fragment in patient M.Y. and a control subject. (A) Diagram of the amplified region of CETP cDNA. The first half of CETP cDNA (denoted as fragment A) was amplified through reverse transcriptase-PCR as described by Gotoda et al.7 Total RNA (1 µg) isolated from monocyte-derived macrophages was incubated 1 hour at 37°C in 25 µL reverse transcriptase PCR buffer supplemented with 15 pmol primer R3, 5 nmol of each dNTP, 40 U of ribonuclease inhibitor, and 12 U of avian myeloblastosis virus reverse transcriptase. The whole reaction was supplemented with 25 µL of a solution containing 50 mmol/L KCl, 10 mmol/L Tris (pH 8.3), 0.01% gelatin, 5 nmol of each dNTP, 5 µL DMSO, 15 pmol of primer F1, and 2.5 µL of Taq DNA polymerase. Twenty-five cycles of PCR 1 included 1 minute at 94°C, 2 minutes at 50°C, and 3 minutes at 72°C, respectively. One microliter of amplified CETP cDNA was subjected to the second PCR, using primers F2 and R1 for amplification of fragment A. The second PCR included 35 of the cycles performed in PCR 1. XcmI restriction sites are indicated by vertical lines. XcmI digestions of fragment A of M.Y. and a control, respectively, are shown schematically in the lower half of the diagram. The XcmI restriction site abolished by the mutation is marked by an x. (B) Electrophoretic analysis of fragment A amplified from total RNA of M.Y. and a control subject (C), respectively, before (-) and after (+) digestion with restriction enzyme XcmI. Fragment A of the control was cut at both XcmI sites into three fragments of 579 bp, 235 bp, and 50 bp, respectively. The 50 bp fragment is too small to be detected on this agarose gel. Fragment A of M.Y. was cut only at XcmI site 653 into two fragments of 579 bp and 285 bp, respectively.

To confirm the point mutation observed in the CETP mRNA of M.Y., we analyzed the corresponding region of the CETP gene. For this purpose, a fragment of the CETP gene containing exon 2 and the adjacent intron sequences was amplified through PCR using as template genomic DNA isolated from mononuclear cells (Fig 4ADown). Digestion of this fragment, designated fragment C in Fig 4Down, indicated that M.Y. was heterozygous for the point mutation at codon 57 (Fig 4BDown).



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  		Figure 4. Amplification and analysis of exon 2 of the CETP gene. (A) Diagram of the amplified region of the CETP gene. Exon 2 with the adjacent intron sequences (denoted as fragment C) was amplified using primers IN1 and IN2R synthesized according to published sequences (described in the EMBL + GenBank Release 82, accession numbers M32992 and J02898). One microgram of genomic DNA isolated from mononuclear cells was used as a template. Thirty cycles of PCR included 30 seconds at 94°C, 15 seconds at 96°C, 1 minute at 57°C, and 30 seconds at 72°C, respectively. The XcmI restriction site of exon 2 is indicated by a vertical line. (B) Electrophoretic analysis of exon 2 with adjacent sequences amplified from genomic DNA of M.Y. and a control, respectively, before (-) and after (+) digestion with restriction enzyme XcmI. Fragment C of the control was cut completely by enzyme XcmI into two fragments of 325 bp and 85 bp. The 85 bp fragment is too small to be detected on this agarose gel. Only half of fragment C of M.Y. was digested by XcmI into the same fragments, whereas the other half remained undigested.

In considering the results depicted in Figs 3Up and 4Up, one could argue that amplification of the CETP cDNA encoded by the allele not containing the mutation in exon 2 was not possible because of a mutation in the target sequence of the oligonucleotide R3 used for RT. Therefore, primer R1, located in exon 9, was employed alternatively for reverse transcription. Primers F2 and Ex4/5 were used for subsequent PCR amplification leading to the synthesis of a shorter fragment A, denoted as fragment A' (469 bp). Restriction analysis of fragment A' with XcmI clearly showed that we were again only able to isolate a CETP-cDNA fragment containing the mutation in exon 2.

Another possibility for our not finding the second allele at the level of cDNA was that a defective promoter prevented transcription of the second allele. Therefore, we analyzed the 5' region of the CETP gene of patient M.Y. upstream to position -360. This was performed by amplification of the corresponding fragment using genomic DNA as template, subcloning into the reporter plasmid pBLCAT3,37 and transfection into HepG2 cells known to produce CETP in vitro. This fragment of M.Y. lead to the same level of transcription of the reporter gene as the corresponding fragment of a control (Fig 5Down). On direct sequencing of the amplified PCR product two deviations from the published sequence were found,41 a transition (A->T) at -309 and an insertion of an additional A into a sequence of 5 A's at -270 to -266 to make it 6 A's at -271 to -266. However, these deviations were found also in the CETP promoter of a control subject with normal CETP mass and activity.



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  		Figure 5. Examination of the CETP promoter of M.Y. (A) Diagram of the amplified region of the CETP promoter. A 360 bp CETP promoter fragment (fragment P) of M.Y. and a control were amplified using primers Pro1 and Pro2, and 1 µg genomic DNA as a template, respectively. Thirty cycles of PCR included 30 seconds at 94°C, 15 seconds at 96°C, 1 minute at 50°C, and 30 seconds at 72°C, respectively. (B) Electrophoretic analysis of fragment P of M.Y. (Y) and a control (C), respectively. (C) Transfection studies with the CETP promoter of M.Y.: HepG2 cells were transfected with CAT plasmids containing no promoter (pBLCAT3),37 the Herpes simplex virus TK promoter (pBLCAT2),37 the CETP promoter of the control subject, and the CETP promoter of M.Y., respectively. Values shown are means±SDs from CAT activity measurements of three transfection experiments.

To examine the CETP gene structure of both alleles of M.Y. we isolated the CETP-gene adopting the technique of long-range PCR using the same primers as for amplification of CETP cDNA but employing genomic DNA as template. PCR with primer pairs F2-R1 and F3-R2 resulted in fragments of about 12 kB in size containing complete exons 1 to 8 (fragment D) and 9 to 16 (fragment E), respectively in Fig 6Down. Restriction analysis of these fragments showed the same restriction pattern as described41 and as seen by analysis of the corresponding fragments of a control (Fig 6Down). Fragment D containing exons 1 to 8 was used as template for amplification of a smaller fragment containing exon 2. Only half of this fragment was cut in restriction analysis with XcmI showing that both alleles of the CETP gene were amplified in the long-range PCR.



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  		Figure 6. Amplification and analysis of the CETP gene in patient M.Y. and a control subject. (A) Diagram of the amplified region of the CETP gene. The whole CETP gene was amplified in two overlapping fragments using primers F2 and R1 (fragment D, {approx}12 kB), as well as primers F3 and R2 (fragment E, {approx}12 kB), respectively. Long-range PCR was performed with ELONGASE Enzyme Mix (Life Technologies) according to the user's manual. Thirty cycles of PCR included 10 seconds at 94°C, 30 seconds at 60°C, and 10 minutes at 72°C, respectively. After 10 cycles the elongation time was elongated for 20 seconds/cycle. Exons are shown in black. Restriction sites of enzymes EcoRI (E) and BgIII (B) are indicated. (B) Electrophoretic analysis of fragments D and E amplified from genomic DNA of a control (C) and M.Y. (Y), before and after digestion with restriction enzymes EcoRI and BgIII, respectively; M=marker.

As an additional possibility for failing to produce CETP cDNA derived from the allele not containing the mutation in exon 2, we considered a splicing defect leading to a truncated or unstable CETP mRNA. Therefore, all exon-intron boundaries of the CETP gene were analyzed by sequence analysis. Ten fragments of the CETP gene containing all exons and adjacent intron sequences were amplified by PCR and analyzed by direct sequencing (Table 4Down). No deviations from the published sequence were found except the mutation at codon 57 in exon 2, as well as a silent mutation at codon 270 in exon 9 coding for phenylalanine (TTC->TTT), both seen only in one allele, respectively.


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  		Table 4. Primers for Amplification and Sequencing of 10 PCR Fragments Containing All Exons of the CETP Gene


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
High HDL cholesterol levels usually signal a metabolic situation with excellent TG clearing capacity.15 However, our patient showed—despite high HDL cholesterol and extremely high HDL2 levels—in the standardized fat load test a very pronounced postprandial lipemia. Fig 7Down illustrates the outlier nature of patient M.Y. with respect to the generally observed strong negative correlation between HDL cholesterol and the magnitude of postprandial lipemia.15 This finding indicated to us that we were confronted with a very unusual patient who was TG-intolerant yet with the "wrong" HDL distribution.



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  		Figure 7. Relationship between levels of HDL cholesterol and magnitude of postprandial lipemia in 28 healthy study volunteers ({circ})15 and patient M.Y. ({bullet}).

As a cause for the mismatch between HDL cholesterol and postprandial lipemia we considered CETP deficiency. Indeed, patient M.Y. displayed CETP deficiency on measurement of both CETP mass and activity. At this point, two questions arose: (1) what was the molecular defect causing CETP deficiency, and (2) is the TG-intolerance hypothesis18 correct? If TG-intolerance indeed causes the syndrome of TG-intolerance with all its marks on VLDL, IDL, LDL, and HDL,18 then CETP deficiency should abolish all these marks.

The first question could be answered by examining the patient's CETP cDNA and CETP gene. After amplifying her CETP cDNA only cDNA containing the mutation in codon 57 of exon 2 was found suggesting that synthesis of full-length CETP did not take place. On the genomic level, however, patient M.Y. displayed heterogeneity with respect to the mutation. We interpret these data to suggest the presence of a null allele in addition to the allele containing the point mutation at codon 57. We undertook major efforts to detect the second allele at the level of cDNA by using an alternative primer for RT to exclude a mutation in the target sequence. We feel that artifacts for the subsequent PCR can also be discounted because the same primers used for the procedure on genomic material invariably allowed us to amplify both alleles of the CETP gene.

For the synthesis of correct mRNA to be prevented completely or to be reduced to nondetectable levels several possibilities can be envisioned; one reason could be a mutation in regulatory sequences in adjacent noncoding regions or in intron sequences of the gene. Analysis of the 5'- region of the CETP gene of patient M.Y. to position -360 displayed no sequence deviation when compared to a control with normal CETP mass and activity. As additional reasons for the absence of CETP-cDNA encoded by the second allele rearrangement of the CETP gene as well as mutations causing defective splicing of pre-mRNA or leading to the synthesis of an unstable mRNA would have to be considered. The overall CETP gene structure of both alleles appeared to be intact, as seen by amplification and restriction analysis of two overlapping CETP gene fragments. The presence of a splicing defect is unlikely, since by sequence analysis of all exons and exon-intron boundaries only one silent mutation was found in one allele altering codon 270 in exon 9 from TTC to TTT.

Potential causes for the manifestation of the null allele other than those mentioned above are rather difficult to identify, especially those involving unstable mRNA. Nevertheless, the exclusive presence of CETP cDNA containing the nonsense mutation at codon 57 is in agreement with the total lack of activity and mass of CETP in the patient's plasma.

The second question we set out to answer, ie, regarding the validity of the TG intolerance hypothesis and the crucial role of CETP for completing TG intolerance to the full syndrome, was answered clearly. In patients heterozygous for LPL deficiency, a paradigm of TG intolerance,18 postprandial lipemia is pronounced. These individuals not only display impaired triglyceride tolerance but, associated with it, a typical fasting lipoprotein constellation adding to what we termed "the syndrome of impaired TG tolerance."18 This syndrome includes cholesterol enriched VLDL and IDL, high IDL levels, small dense LDL particles (pattern B), and low HDL cholesterol due to low levels of HDL2 particles enriched with TG.18 Recently, another mutation in the LPL gene causing low HDL2 was described.42 Our patient M.Y. exhibited only TG intolerance but none of the typical stigmata on HDL2 or any of the other lipoprotein classes. Instead, she had CE-poor VLDL and IDL, low IDL levels, large LDL of low density, HDL particles containing almost no TG and extremely high levels of HDL2 and even larger less dense HDL particles. Thus, M.Y. exhibited impaired TG tolerance without any of the lipoprotein stigmata typical for the syndrome of impaired TG tolerance (Fig 1Up). On the contrary, the lipoprotein profile of M.Y. was reminiscent of TG "supertolerance" observed with IDDM patients with pancreas grafts.43 These patients have very attenuated postprandial lipemia due to high LPL activity caused by peripheral hyperinsulinemia. They also have high levels of HDL2, large LDL, and very low concentrations of VLDL and IDL. Our patient's chimeric phenotype of TG-intolerance yet with the lipoprotein characteristics of excellent TG metabolic capacity finds its explanation in the absence of CETP stressing the eminent role of CETP as link between the avenues of cholesterol and triglyceride transport.

TG-intolerance is most likely caused by a variety of factors. Demonstrated reasons include heterozygous LPL-deficiency,18 insulin resistance,44 NIDDM,45 and apoE isoforms.46 Patient M.Y. had no deficiency in LPL or HL activity, she had no NIDDM, and her hypertension was not part of the polymetabolic syndrome. However, she exhibited apoE3/E2-phenotype, which conceivably could have caused or at least contributed to her TG-intolerance. As an additional cause, CETP deficiency itself comes to mind. Generally speaking, when a mediator between pools is not functional and, as a consequence, the size of one pool increases, an increase of the second pool would not be entirely unexpected. If HDL cholesterol rises because of the lack of CETP action, levels of triglycerides, the second reactant of the exchange process, could also rise. CETP deficiency could conceivably contribute to TG intolerance, if only with a second handicap such as an apoE2 allele. Further experiments including postprandial studies in other cases of CETP deficiency could help to shed light on this question.

If CETP-deficiency did not contribute to the TG intolerance of patient M.Y., it provided a fortunate situation for us insofar as only in this way the chimeric phenotype was possible with TG intolerance on one hand and the lipoprotein pattern of TG supertolerance on the other. This situation allowed us to not only report a molecular defect of the CETP gene, not described so far, but also to demonstrate that absence of CETP action prevents the development of the full-blown syndrome of TG intolerance even when TG metabolic capacity is seriously impaired.


*    Selected Abbreviations and Acronyms
 
TG = triglycerides
CE = cholesteryl esters
CETP = cholesteryl ester transfer protein
CAD = coronary artery disease
LPL = lipoprotein lipase
IDL = intermediate density lipoprotein
HL = hepatic lipase
LTP-I = lipid transfer protein
CAT = chloramphenicol acetyltransferase
NIDDM = non–insulin-dependent diabetes mellitus


*    Acknowledgments
 
This work was supported by grants S-4606 (J.R.P.) and S07106-MED (J.R.P) of the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung.

Received June 2, 1997; accepted August 4, 1997.


*    References
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