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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1866-1874.)
© 1995 American Heart Association, Inc.

Articles

A New Case of ApoA-I Deficiency Showing Codon 8 Nonsense Mutation of the ApoA-I Gene Without Evidence of Coronary Heart Disease

Presented in part at the 10th International Symposium on Atherosclerosis, Montreal, Canada, October 11, 1994.

Kouki Takata; Keijiro Saku; Takao Ohta; Mie Takata; Huai Bai; Shiro Jimi; Rui Liu; Hikaru Sato; Goro Kajiyama; Kikuo Arakawa

From the Department of Internal Medicine (K.T., M.T.), Hiroshima Railway Hospital, Hiroshima; the Departments of Internal Medicine and Pathology (K.S., H.B., S.J., R.L., K.A.), Fukuoka University School of Medicine, Fukuoka; the Department of Pediatrics (T.O.), Kumamoto University School of Medicine, Kumamoto; the Department of Cardiology (H.S.), Hiroshima City Hospital, Hiroshima; and the Department of Internal Medicine (G.K.), Hiroshima University School of Medicine, Hiroshima, Japan. The first two authors contributed equally to this work.

Correspondence to Keijiro Saku, MD, Department of Internal Medicine, Fukuoka University School of Medicine, 45-1-7 Nanakuma Jonanku, Fukuoka 814-01, Japan.

	Abstract

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Abstract We report a 39-year-old Japanese man with HDL and apoA-I deficiency as well as data from members of his family. Corneal opacity and a stomatocyte were found but not tonsillar hypertrophy, xanthomas, or splenomegaly. His serum HDL cholesterol, apoA-I, apoA-II, and LDL cholesterol levels were 6 mg/dL, <3 mg/dL, 6 mg/dL, and 175 mg/dL, respectively. Plasma triglyceride, phospholipid, apoB, apoC-III, and apoE levels were all within normal limits. Lecithin:cholesterol acyltransferase activity was half of normal, while lipoprotein lipase and hepatic triglyceride lipase activities were within normal limits. ApoA-I deficiency was confirmed by combined isoelectric focusing and sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by an immunoblotting method. We surveyed the apoA-I gene of the patient and five of his family members by direct sequencing after amplification by polymerase chain reaction and found a codon 8 nonsense mutation (TGGTAG, Trpstop) in exon 3 of the apoA-I gene. The results of a pedigree analysis by DNA sequencing and restricted fragment length polymorphism (Sty I) were consistent with an autosomal codominant trait. Coronary angiography was performed to evaluate coronary atherosclerosis, but no significant luminal narrowing was detected. An intracoronary ultrasound study showed mild intimal hyperplasia in segment 6. In summary, this is a case of apoA-I deficiency without evidence of coronary heart disease.

Key Words: atherosclerosis • coronary heart disease • gene • HDL-deficient syndrome • intravascular ultrasound imaging

	Introduction

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Many epidemiological and clinical studies have established that an inverse relation exists between plasma HDL levels and the risk of CHD.^{1 2 3 4 5} Since HDL and its major protein, apoA-I, may be involved in reverse cholesterol transport,^{6 7 8 9} minor reductions in plasma HDL or apoA-I levels can be potentially atherogenic. Therefore, from a clinical perspective, even minor changes in these lipoprotein parameters should be evaluated during treatment, especially in diseases that require lifelong treatment by either drugs or nonpharmacological therapy.^{10 11}

HDL can be subclassified into various types by density or particle size.^{12 13 14 15} Two major subclasses, LpA-I and LpA-I/A-II,^{5 16 17} have received considerable attention in investigations of the metabolic aspects of HDL. In vitro experiments show that both LpA-I and LpA-I/A-II can remove cellular cholesterol from cholesterol-loaded cells.^{18 19} However, clinical studies have shown that low HDL-C levels are closely linked to low LpA-I levels, and a strong inverse relation exists between plasma LpA-I concentrations and the risk of CHD,^{5 20} while plasma levels of LpA-I/A-II are fairly constant or may show reduced LpA-I/A-II levels in CHD patients.²¹ From the perspective of basic science, studies in human apoA-I transgenic mice and double transgenic mice expressing human apoA-I and apoA-II clearly indicate that LpA-I may be the antiatherogenic lipoprotein fraction within HDL.^{22 23} All these reports emphasize the importance of LpA-I as an antiatherogenic substance.

Human apoA-I is synthesized as a preproapoA-I with a 24–amino acid NH₂-terminal extension that undergoes intracellular cotranslational proteolytic cleavage into proapoA-I.^{24 25 26} In humans, proapoA-I is converted into mature forms extracellularly. To date, five cases of HDL deficiency due to apoA-I mutation^{27 28 29 30 31} and several cases of HDL deficiency due to apoA-I/C-III/A-IV deficiency and apoA-I/C-III deficiency^{32 33 34} have been reported. Here, we report a new case of HDL deficiency due to a nonsense mutation of codon 8 of the apoA-I gene without evidence of CHD.

	Methods

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Subjects
The patient (proband) is an otherwise healthy 39-year-old male bus driver who showed an extremely low level of HDL in an annual mass medical check-up in a rural area of Hiroshima prefecture, Japan, in 1991. He sought medical attention at the Hiroshima Railway Hospital solely for this HDL deficiency and not because of any symptoms. Physical examination revealed a normally developed Japanese male, height 174 cm, weight 72 kg, blood pressure 126/74 mm Hg, and heart rate 64 beats per minute and regular. Corneal opacity, which was visible upon physical examination, and a stomatocyte were found in the proband, but not tonsillar hypertrophy, xanthomas, or splenomegaly. Neurological examination was within normal limits. There was no consanguinity matching, and his parents have no significant disease. He has a brother, two sons, and one daughter (Fig 1). The patient did not suffer from any signs of CHD, and neither a resting electrocardiogram nor an exercise stress treadmill electrocardiogram revealed any signs of coronary insufficiency. Family history was not indicative of any increased prevalence of myocardial infarction. He drinks two cups of Japanese sake and one bottle of beer per day, and he is a nonsmoker. His serum HDL-C, apoA-I, apoA-II, and LDL cholesterol levels were 6 mg/dL, <3 mg/dL, 6 mg/dL, and 175 mg/dL, respectively (Table 1). The proband's liver and renal functions were all within normal limits, fasting blood sugar was 103 mg/dL, and HbA1c was 5.2%. All the studies described here were performed with the informed consent of the proband and members of his family.

View larger version (25K): [in this window] [in a new window]   Figure 1. Pedigree of a Japanese family with apoA-I deficiency. Arrow indicates the proband; circles, women; and squares, men.

View this table: [in this window] [in a new window]   Table 1. Lipoprotein Profile of the Kindred

Quantification of Serum Lipid, Lipoprotein, and Apolipoprotein Levels and LCAT and Postheparin Lipolytic Activities
Blood samples were obtained in the morning after an overnight fast from the proband and members of his family. Serum TC and TG levels and lipids in each lipoprotein fraction were measured by using enzymatic methods.^{35 36} Lipoprotein fractions were separated by standard sequential preparative ultracentrifugation techniques.³⁷ HDL-C was also measured by the heparin-Ca²⁺ precipitation method.³⁸ ApoA-I, apoA-II, apoB, apoC-II, apoC-III, and apoE were determined by the single radial immunodiffusion³⁹ and/or turbidity immunoassay⁴⁰ methods. All apolipoproteins were assayed within 48 hours. Serum lipoprotein(a) levels were measured by an enzyme-linked immunosorbent assay by using Tint Eliza Lp(a) (Biopool Co).^{41 42} LCAT activity in serum was determined by the dipalmitoyl lecithin–substrate method.⁴³ Postheparin plasma lipolytic activities of lipoprotein lipase and hepatic triglyceride lipase were measured by the modified method of Krauss et al⁴⁴ and Saku et al.⁴⁵

Electrophoretic Procedures
Lipoprotein fractions of serum from the proband and members of his family were initially separated by agarose gel electrophoresis.⁴⁶ To analyze the apoA-I and other apolipoproteins associated with HDL, combined IEF and SDS–polyacrylamide gel slab electrophoresis was performed. IEF gel electrophoresis was performed essentially as reported.^{26 45 47} Either the HDL fraction (1.063<d<1.21 g/mL) from 400 µL serum or plain serum was delipidated with acetone/ether (1:1, vol/vol) and then dissolved in buffer (8 mol/L urea and 1.6% ampholine, pH 4 to 6) at 18°C to 20°C. This mixture was applied to a 200x130-mm IEF gel containing 3.8% acrylamide, 0.26% bisacrylamide, and 2% ampholine, pH 4 to 6. Focusing was conducted for a total of 6000 to 7000 Volt-hours. IEF gels were equilibrated in 0.002 mol/L ethylmorpholine, 0.2% SDS, 0.1% ß-mercaptoethanol, 0.1% bromophenol blue, and 40% sucrose for 60 minutes and then subjected to 15% SDS–polyacrylamide gel slab electrophoresis according to the method of Laemmli.⁴⁸ Electrophoresis was performed for 4 to 5 hours at a constant current of 30 mA per gel.^{26 47} The bands and spots of apoA-I were identified on SDS–polyacrylamide gel electrophoretic gels or two-dimensional electrophoretic gels by the immunoblotting method with goat anti-human apoA-I (Dai-ichi Pure Chemicals) and horseradish-peroxidase–conjugated rabbit anti-goat IgG.⁴⁷

Gene Sequencing
Blood for leukocyte isolation and subsequent DNA preparation was collected in EDTA-containing tubes (final concentration, 50 mmol/L).⁴⁹ For subjects in whom sequencing was performed, we sequenced the exons, the splice donor and acceptor sites of the apoA-I gene, and the upstream sequence of the 5' noncoding region. Eight 21- to 30-bp-long oligonucleotides were used as primers for PCR amplification^{50 51} (Table 2). Primers were synthesized in a DNA synthesizer (Applied Biosystems). Tandem and inverse repeats as well as homology between primers were avoided. Amplification primers were also used as sequence primers. The amplification reaction was performed in 100 µL of the buffer recommended by the supplier of the Taq polymerase (Cetus), 0.5 to 1 µg DNA, final concentrations of 200 µmol/L of each dNTP, and 0.1 µmol/L of each primer. Initial denaturation at 100°C for 10 minutes was followed by the addition of 2 to 5 U Taq polymerase and 35 cycles of denaturation for 1 minute at 96°C, annealing for 1 minute at 60°C, and extension for 1 minute at 72°C by using a Perkin-Elmer-Cetus thermocycler (model PJ 2000). The PCR DNA product was purified by using Centricon X-100 tubes (Amicon) and three ultrafiltration centrifugation runs (950g for 20 minutes per run with the tubes refilled with water to 2 mL between runs). Direct sequencing was performed in an ABI DNA sequencer (model 373A) with a dye-terminator kit following the protocol of the kit manufacturer.^{50 51}

View this table: [in this window] [in a new window]   Table 2. Sequences of Oligonucleotides Used As Primers

Genotype Determination
Sty I restriction endonuclease was used to determine the genotypes among family members by using PCR-amplified DNA. The TGGTAG mutation at codon 8 led to the formation of another enzyme cutting site (CCTGGGCCTAGG) in the presence of the mutation that caused the apoA-I deficiency. A 454-bp fragment of apoA-I exon 3 was amplified for restriction analysis. Reaction conditions for PCR were unchanged. The Sty I restriction analysis was performed by using 16 U Sty I at 37°C for 2.5 hours with each PCR-amplified product. Following electrophoresis on 1% agarose NA/3% NuSieve GTG agarose gel, the digestion products were visualized by using ethidium bromide.^{50 51}

Assay for Cholesterol Esterification in Plasma and VLDL- and LDL-Depleted Plasma
VLDL- and LDL-depleted plasma was prepared by precipitating VLDL and LDL with phosphotungstate–magnesium chloride. The esterification of cholesterol was measured by using endogenous substrates.^{52 53 54} [³H]FC was incorporated onto polystyrene tissue-culture wells. One hundred microliters of plasma samples (to determine MER plasma) or VLDL- and LDL-depleted plasma samples (to determine FER HDL) in 400 µL phosphate-buffered saline was then added to each well, and [³H]FC was equilibrated with the FC in each sample by incubation. [³H]FC-labeled plasma or apoB-depleted plasma samples were then incubated at 37°C for 1 hour. The enzyme reaction was stopped, and lipid extraction and separation of FC and cholesteryl esters by thin-layer chromatography were performed.^{52 53 54}

Coronary Angiography
Coronary angiography was performed by using the Judkins technique (using a 7.5F sheath and a 6F Judkins catheter), and multiple views of all vessels in the left anterior oblique, right anterior oblique, and posteroanterior views were recorded after the intracoronary injection of isosorbide dinitrate (2.5 mg).^{55 56}

IVUS
The sheath was placed in the femoral artery, and a 7F right or left Judkins large-lumen guiding catheter was advanced into the coronary ostium. After the 0.014-inch guidewire was withdrawn, an ultrasound imaging catheter with a 30-MHz transducer (CVIS Inc) at its tip was advanced over another, finer (0.014-inch) guidewire through the guiding catheter into the coronary area, allowing us to record the coronary artery lesion. The intracoronary ultrasound image was displayed under fluoroscopy.^{57 58} The IVUS images were recorded on super VHS videotape.

	Results

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Serum Lipid, Lipoprotein, and Apolipoprotein Levels and Enzyme Activities
Table 1 shows the serum lipid and apolipoprotein concentrations in the proband, members of his family, and normal control subjects. The proband showed an extremely low HDL-C level (6 mg/dL) as measured by the heparin-Ca²⁺ precipitation procedure and an undetectable level of apoA-I (<3 mg/dL) as measured by both the single radial immunodiffusion and turbidity immunoassay methods. ApoA-II and apoC-II were decreased, and LDL cholesterol (175 mg/dL), as calculated by the method of Friedewald et al,⁵⁹ was increased. Other plasma apolipoprotein and lipid levels were within normal limits. The serum apoA-IV level (14 mg/dL) was higher than normal (8.4 to 13.4 mg/dL). However, his parents, brother, and children showed no significant reductions in HDL-C or apoA-I. The proband's lipoprotein fractions were separated by using an ultracentrifugation method. Cholesterol and TG levels in HDL fractions were lower than those in normal control subjects, and HDL deficiency was again confirmed by this technique. HDL-C subfractions were also severely reduced (HDL₂-C, 1.3 mg/dL and HDL₃-C, 2.3 mg/dL). Lipoprotein lipase and hepatic TG lipase activities were within normal limits (11.64 [normal, 10.58±2.82] and 16.80 [15.04±4.18] µmol free fatty acids · mL^-1 · h^-1 at 37°C, respectively; mean±SD), while LCAT activity was apparently half of the normal value (Table 1). ApoE phenotypes showed E3/3 in the proband.

Electrophoretic Procedures
Agarose gel electrophoresis showed a clear deficiency of -lipoprotein in the proband. His parents, brother, and children showed no significant reductions in HDL, which was confirmed by this electrophoresis. Fig 2 shows IEF (pH 4 to 6) of human purified apoA-I (mature forms), the delipidated HDL fraction of a control subject, and the patient's delipidated HDL fraction (1.063<d<1.21 g/mL). Proteolytic processing occurred during purification of human apoA-I, and more acidic isoforms (apoA-I4 and apoA-I5) were predominant, while apoA-I3 was dominant in fresh serum. Fig 3 shows combined IEF and SDS–polyacrylamide gel electrophoresis. In control delipidated HDL, isoproteins apoA-I3 and apoA-I4 were nicely separated at a molecular mass of about 28 000 D, while no spots of apoA-I isoproteins were observed in the patient's HDL fraction. IEF and SDS-gel electrophoresis were combined and electrophoretically transferred onto a Problott membrane (Applied Biosystems) at 6 V for 15 hours. Human apoA-I isoproteins were detected by sequential incubation with rabbit anti-human apoA-I antiserum for 2 hours and perioxidase-conjugated goat anti-rabbit IgG for 1 hour at room temperature with 4-chloro-1-naphthol in 20 mmol/L Tris-HCl (pH 7.4) as substrate.^{26 47} However, no apoA-I was detected in the proband by this method.

View larger version (72K): [in this window] [in a new window]   Figure 2. IEF gel electrophoresis (pH 4 to 6). Lane a, human purified apoA-I (mature forms); b, normal control subject delipidated HDL; and c, proband's delipidated HDL fraction.

View larger version (87K): [in this window] [in a new window]   Figure 3. Combined IEF and SDS–polyacrylamide gel electrophoresis. a, Delipidated HDL from a normal control subject; b, delipidated HDL fraction from the proband.

Direct Sequencing of Amplified DNA
Fig 4 illustrates the genomic organization of the apoA-I gene, the PCR-amplified regions, and the positions of the base substitutions detected in the proband's gene. For sequence analysis, pairs of oligonucleotides were used to amplify the apoA-I gene by PCR. Direct sequence analysis, which gives simultaneous information for both alleles, showed a homozygous GA exchange in the second base of codon 8 (Figs 4 and 5). All other analyses yielded the expected wild-type sequence of apoA-I. This mutation results in the replacement of the tryptophan residue (TGG) at position 8 with a stop codon (TAG). Analysis of DNA from members of the proband's family for the presence or absence of this mutation yielded a genotype that in all cases corresponded with the inherited biochemical phenotype. A structural analysis of all of the exons and large portions of the consensus regulatory sequences revealed no further deviation from the wild-type sequence.

View larger version (16K): [in this window] [in a new window]   Figure 4. Diagram showing how the single-nucleotide exchange in the third exon of the apoA-I gene is the underlying defect in apoA-I deficiency. An enlarged view of the third exon of the apoA-I gene (bottom) shows the site and nature of the nucleotide exchange in codon 8.

View larger version (2K): [in this window] [in a new window]   Figure 5. Partial sequences were obtained from (a) II-4 in Fig 1 (normal), (b) III-3 in Fig 1 (heterozygous apoA-I deficiency), and (c) proband (II-3 in Fig 1; homozygous apoA-I deficiency).

Genotype Determination
Genotype determination in members of the proband's family was performed by using a Sty I restriction endonuclease. The presence of a TGGTAG mutation created a new restriction site and led to the detection of characteristic 220- and 90-bp fragments instead of the normal 310-bp fragment alone (Fig 6). The genotypes of all other family members (Fig 1) and 92 control subjects from the general population were determined by the same procedure (data not shown). Family analysis indicated that the proband was homozygous for a vertically transmitted gene defect in which the heterozygous phenotype shows almost normal serum HDL-C and apoA-I levels.

View larger version (79K): [in this window] [in a new window]   Figure 6. Example of genotype determination in family members by using a Sty I restriction endonuclease. The presence of the Trpstop mutation formed another restriction site and led to the detection of characteristic 220- and 90-bp fragments instead of the normal 310-bp fragment alone. The other fragment at 118 bp has no significance in genotype determination. Far left lane represents molecular size markers, and far right lane is from a normal control subject (II-4). The middle three lanes from left to right refer to the proband and his two sons, respectively (II-3, III-3, and III-4 in Fig 1). The genotypes of the other family members and the 92 control subjects from the general population were determined by the same procedure (data not shown).

Coronary Angiography and IVUS
Even though the treadmill stress electrocardiogram showed no ST changes, the patient consented to undergo coronary angiography, which showed a nonstenotic coronary angiography in both the left anterior oblique and right anterior oblique views. While segment 6 was angiographically normal, mild eccentric intimal hyperplasia was detected by IVUS (Fig 7). However, this may not affect coronary flow in this patient.

View larger version (89K): [in this window] [in a new window]   Figure 7. Two IVUS views show mild eccentric intimal hyperplasia (arrows) in the proximal region of segment 6, which was angiographically normal.

	Discussion

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The clinical characteristics of the six cases of apoA-I deficiency that have been reported to date, including the present case, are summarized in Table 3. Funke et al²⁷ report a single base (G) deletion at codon 202 that resulted in a frameshift mutation in apoA-I in a 42-year-old German man who showed corneal opacity without any evidence of CHD, hepatosplenomegaly, or xanthomas. Plasma TC was 243 mg/dL, and TG was 196 mg/dL. LCAT activity was 44% of that in normal control subjects. Matsunaga et al²⁸ report a nonsense mutation at codon 84 in a 60-year-old Japanese woman who had a planar xanthoma and severe CHD without corneal opacity. This case showed remarkable coronary stenosis by coronary angiography. Her plasma TC was 154 mg/dL, and TG was 84 mg/dL. LCAT activity was not determined, but consanguinity matching was present. Lackner et al²⁹ report a 7-year-old Turkish girl in whose family consanguinity matching was also positive. This case was initially reported at the 8th International Symposium on Atherosclerosis at Rome in 1988,⁶⁰ but was not published until 1993.²⁹ This girl showed a codon 5 single-base (C) insertion in which a nonsense peptide was encoded that terminated at amino acid 33. She showed corneal opacity, xanthoma, and hepatomegaly. However, there were no clear indications of coronary atherosclerosis, perhaps because she is still a child. The oldest living heterozygotes are in their late 40s and early 50s and show no symptoms of CHD. Her plasma TC was 222 mg/dL, and TG was 107 mg/dL. Total serum LCAT activity was not determined, but LCAT was confirmed to exist by proteoliposome assay of gel-filtration fractions of serum. Her plasma apoA-IV level was 16 mg/dL, which is within the normal range, as assessed by their method of sandwich-type enzyme-linked immunosorbent assay. Ng et al³⁰ report a 34-year-old Canadian woman with HDL deficiency by the nonsense mutation at apoA-I codon -2 (CAGTAG, Glnstop). She was a product of a consanguineous marriage and showed exudative proliferative retinopathy, cataracts, spinocerebellar ataxia, and tendon xanthoma without evidence of CHD. Plasma TC was 222 mg/dL, and TG was 220 mg/dL. Among her first-degree relatives, her father died suddenly due to myocardial infarction at the age of 64 years, and one sister (of five homozygotic siblings) suffered a myocardial infarction due to coronary atherosclerosis at the age of 34 years; none of the other siblings showed signs of CHD at the time of the report. Römling et al³¹ have found HDL deficiency by the homozygous nonsense mutation in codon 32 (CAGTAG, Glnstop) of the apoA-I gene in a 31-year-old Italian woman who, like heterozygous family members, showed no clinical sign of CHD. The parents of the homozygous proband are first cousins; her serum TC was 108 mg/dL, and TG was 45 mg/dL.

View this table: [in this window] [in a new window]   Table 3. ApoA-I Deficiency Related to the ApoA-I Gene Locus

In the 39-year-old man reported here, there was no consanguinity matching. Corneal opacity and hepatomegaly were prominent, TC was 202 mg/dL, and TG was 104 mg/dL. LCAT activity was 46% of that in normal control subjects. He regularly drinks a moderate amount of alcohol (ethanol, 44 mL/d), and liver function and GTP levels were all within normal limits. Both HBs-Ag and HCV-Ab were negative. Clinically, hepatomegaly may not be associated with apoA-I deficiency but could be due instead to alcohol intake. Ultrasound study showed fatty liver. He is a nonsmoker, and the results of coronary angiography were normal. However, IVUS showed mild eccentric hyperplasia in the proximal region of segment 6 (Fig 7). To the best of our knowledge, this is the first report of intracoronary intimal hyperplasia in apoA-I deficiency, although in a clinically nonstenotic artery. Patients who have been reported to have apoA-I/C-III/A-IV^{32 33} and apoA-I/C-III deficiency³⁴ are also summarized in Table 3. Both cases had lower serum TC and TG levels, and both had apparent CHD, compared with apoA-I deficiency alone. The lower plasma TC and TG levels observed in these apoA-I/C-III/A-IV and apoA-I/C-III deficiency patients could be linked to accelerated atherosclerosis through the function of apoC-III and/or apoA-IV deficiency. The apoA-IV level in our case (14.0 mg/dL) was higher than normal (range, 8.4 to 13.4 mg/dL); apoC-III (4.2 mg/dL) was in the normal range (Tables 1 and 3). In addition, we could not find any reasons why the heterozygotes considered here showed normal HDL-C and apoA-I levels, although in other cases of apoA-I deficiency reported by Funke et al,²⁷ Lackner et al,²⁹ Ng et al,³⁰ and Römling et al,³¹ heterozygotes showed 50% to 75% of the normal control subjects' apoA-I levels. Reduced plasma apoA-II level is common in familial HDL-deficient syndrome (Table 3), while the mechanism of lower apoC-II in our patient remains unclear.

Our laboratory has studied the metabolic turnover of HDL apoA-I to obtain a better understanding of the mechanism of low and high serum HDL and apoA-I levels in humans and rabbits.^{26 61} Since HDL is thought to be important in reverse cholesterol transport,^{6 7 8 9} knowledge regarding the mechanisms by which drugs, exercise, or other factors increase HDL is important. Stimulation of HDL synthesis (and therefore transport) would be associated with an increased transport of cholesterol, assuming that HDL was involved in the efflux of cholesterol from tissue to plasma for excretion. Materials that elevate plasma HDL by decreasing the rate of HDL removal are not expected, theoretically, to result in a net efflux of body-tissue cholesterol. These considerations have important implications in our understanding of the basic mechanisms by which lipid regulation affects the atherosclerotic process. However, in our case, serum apoA-I was totally absent, and a stop codon was found at codon 8. Even in the homozygous proband no abnormal banding pattern for apoA-I was observed at different charge points by using our immunoreactive technique. Although this would suggest that no circulating apoA-I was present and that apoA-I was not being synthesized, no critical condition was observed in this case. Five of the six reported patients with apoA-I deficiency had no symptoms of CHD. Therefore, if other apolipoproteins (eg, apoA-IV, apoE) play a positive role in reverse cholesterol transport, then we can readily explain why patients with apoA-I/C-III and apoA-I/C-III/A-IV deficiencies are likely to be associated with CHD.^{32 33 34} Cheung et al⁶² report a case of HDL deficiency in which the plasma contained unusual particles (lipoprotein containing apoA-II but no apoA-I) that promoted cholesterol efflux in vitro, but the function of these apoA-I–deficient particles in vivo remains unclear.

Once cholesterol is esterified in association with LCAT, cholesteryl ester may be transferred from HDL to apoB-containing lipoproteins through the action of cholesteryl ester transfer proteins.^{63 64} Cholesteryl ester–rich lipoproteins (LDL) are then efficiently cleared by LDL receptors in the liver. The plasma concentration of LpA-I, which is assumed to be the antiatherogenic fraction of HDL, is inversely correlated with MER plasma and the rate of cholesterol esterification in VLDL- and LDL-depleted plasma (FER HDL).⁶⁵ These phenomena may be associated with the antiatherogenic nature of LpA-I, possibly by regulating the efflux of cholesteryl esters to LDL and its subsequent oxidation. In our patient with apoA-I deficiency, the fractional esterification rate was determined. FER plasma, FER HDL, and MER plasma were 1.17% per hour, 2.17% per hour, and 14.65 nmol · mL^-1 · h^-1, respectively, values that were 12% to 19% of those in control subjects. A similar tendency of severe reductions in these parameters was observed in the patient with cholesteryl ester transfer protein and LCAT deficiencies.^{52 53} FER HDL in subjects with angiographically proven CHD (n=320) is significantly greater than that in angiographically proven nonstenotic subjects (n=70) (K.S. et al, unpublished data, 1995), which is consistent with the findings of Dobiasova et al.⁶⁶ We believe that the mechanisms that have prevented serious atheromatous changes in the proband's coronary artery may be related to the fact that FER HDL is also lower in LCAT- and cholesteryl ester transfer protein–deficient patients. These data suggest that reduced cholesteryl ester formation and the subsequent reduced formation of cholesteryl ester–rich apoB-containing lipoproteins, which are easily oxidized and related to atherosclerosis, may be important factors in preventing coronary atherosclerosis compared with the initial process of reverse cholesterol removal from peripheral cells. The theory involved in the kinetic study mentioned above also indirectly supports this hypothesis in our proband, who lacks circulating apoA-I. A lack of apoA-I synthesis, which is expected to be linked to coronary atherosclerosis but is not found in our patient, strongly suggests that cholesteryl ester formation and subsequent processes may play a predominant role.

Von Eckardstein et al,⁶⁷ who report the electrophoretic variants of apoA-I in a screening of 32 000 dried blood samples, found that substitutions are overrepresented in the first tandem repeat of the apoA-I protein structure. Three cases of apoA-I deficiency, including the present case, have shown an insertion or nonsense mutation at the beginning of the proapoA-I or apoA-I structure. However, in the two other cases, mutations were present at codons 84 and 202. Since the cholesterol esterification rate and LCAT activity are less in our case and that of Funke et al,²⁷ LCAT activation properties are not necessarily confined to the apoA-I carboxyterminal region only. It is also unlikely that our patient's mutation merely represents a marker that is in linkage disequilibrium with a functionally active mutation. The possibility that it represents a common polymorphism that is of no clinical or biochemical significance was excluded by our inability to identify this mutation in 92 randomly selected DNA samples.

IVUS can show coronary atherosclerosis in a coronary artery that is judged intact by angiography.^{57 58} In our case, a small elevated region was found by IVUS. While we are uncertain whether this finding is significant, this stenotic, angiographically silent region apparently did not affect coronary flow. One possibility for the lack of coronary stenosis in this patient is that the patient is simply too young to have developed CHD. To discuss the prevalence of CHD in men of this age, Mabuchi et al⁶⁸ have calculated regression lines between the severity of coronary atherosclerosis and age in familial hypercholesterolemia (in both homozygotes and heterozygotes) in Japanese men with and without symptoms and signs of CHD. In men 39 years old, atheromatous lesions were clearly detected in heterozygous familial hypercholesterolemia patients with and without angina. In contrast, neither lesions nor symptoms were found in our case. LDL receptor deficiency and HDL apoA-I deficiency are essentially different, and the former may be more likely to produce conditions suitable for the development of atheromatous lesions. Longitudinal IVUS studies in this patient may help to elucidate the relation between HDL-deficient syndrome and atherosclerosis.

	Selected Abbreviations and Acronyms

 

CHD = coronary heart disease FC = free cholesterol FER HDL = fractional esterification rate in HDL HDL-C = HDL cholesterol IEF = isoelectric focusing IVUS = intravascular ultrasound LCAT = lecithin:cholesterol acyltransferase LpA-I = lipoprotein containing apoA-I only LpA-I/A-II = lipoprotein containing both apoA-I and apoA-II MER plasma = molar rate of cholesterol esterification in plasma PCR = polymerase chain reaction SDS = sodium dodecyl sulfate TC = total cholesterol TG = triglycerides

	Acknowledgments

 
This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (Nos. 02671114, 04671503, 06670809, and 07670827), by research grants from the Ministry of Health and Welfare, and by research grants from the Fukuoka University Research Fund.

Received April 18, 1995; accepted July 11, 1995.

	References

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