© 1996 American Heart Association, Inc.
Articles |
A Cysteine-Containing Truncated Apo A-I Variant Associated With HDL Deficiency
the Department of Internal Medicine (K.M., J.S., Y.T., A.M., K.A.), Fukuoka (Japan) University, School of Medicine; Sasebo General Hospital (J.F.), Nagasaki, Japan; and the Department of Medicine (J.J.A.), University of Washington, Northwest Lipid Research Laboratories, Seattle.
Correspondence to Jun Sasaki, MD, Department of Internal Medicine, Fukuoka University, School of Medicine, 45-1, 7-chome Nanakuma, Jonan-Ku, Fukuoka, 814-80, Japan.
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Abstract |
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We identified a 50-year-old Japanese woman with a novel mutation in the apolipoprotein (apo) A-I gene causing high-density lipoprotein (HDL) deficiency. The patient had extremely low HDL cholesterol and apo A-I levels (0.14 mmol/L and 0.8 mg/dL, respectively) but no evidence of coronary heart disease. However, she had bilateral xanthomas of the Achilles tendon, elbow, and knee joint as well as corneal opacities. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of serum followed by immunoblotting revealed that the patient's apo A-I had a lower molecular weight (24 000) than normal apo A-I. A partial gene duplication encompassing 23 nucleotides was found by DNA sequence analysis, resulting in a tandem repeat of bases 333 to 355 from the 5' end of exon 4. This tandem repeat caused a frameshift mutation with premature termination after amino acid 207. The frameshift gives rise to a predicted protein sequence that contains two cysteines. We designated this mutant as apo A-ISasebo. Apo A-ISasebo formed heterodimers with apo A-II and apo E in the patient's plasma and was associated with both the low-density lipoprotein and HDL fractions. The patient's cholesterol esterification rate and lecithin-cholesterol acyltransferase activity were reduced to about 30% of normal, although specific enzyme activity was unaffected, suggesting that it remained functionally normal. In addition, cholesteryl ester transfer activity was reduced to about half of normal. Thus, apo A-ISasebo was associated with complex derangements of lipoprotein metabolism.
Key Words: genetics • lipoproteins • apo E • apo A-II • atherosclerosis
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Introduction |
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Apo A-I is a single polypeptide chain composed of 243 amino acid residues and has a molecular weight of 28 000.1 2 The apo A-I gene has been localized to chromosome 11 (11q 13-qter), where it forms part of a cluster with the apo C-III and apo A-IV genes.3 4 5 Apo A-I plays an important role in nascent HDL, since it promotes the efflux of cholesterol from tissues6 and serves as a cofactor for LCAT.7 The plasma HDL cholesterol concentration shows an inverse correlation with the risk of CHD,8 9 10 11 12 but the mechanism underlying the protective effect of HDL remains poorly understood. Several HDL deficiency syndromes have been studied in attempts to clarify the cardioprotective effect of this lipoprotein. A number of kindreds with hereditary analphalipoproteinemia are known to have defects of the apo A-I gene13 14 15 16 17 18 19 20 21 22 23 that result in the virtual absence of plasma HDL. In some other kindreds, the molecular defect either does not involve the apo A-I gene24 25 or is yet to be identified.26 27 28 29 Despite the almost complete absence of HDL in some kindreds, there is marked variation in the susceptibility to premature CHD. Possible reasons for this include the small number of individuals studied in each kindred, associated deficiencies of other apolipoproteins,14 15 16 17 and abnormal circulating apo A-I variants.19 20 Therefore, it is necessary to investigate individuals with isolated apo A-I deficiency to minimize potential confounding factors. In this article, we report a patient with a structural mutation of the apo A-I gene that caused isolated apo A-I deficiency associated with multiple xanthomas, corneal opacities, and partial LCAT deficiency.
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Methods |
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Proband
The proband was a 50-year-old Japanese woman who was found to have a very low plasma HDL cholesterol level on testing at Sasebo General Hospital. She first noted the presence of xanthomas when she was 9 years old. She had no history of diabetes mellitus, hypertension, or any symptoms suggesting coronary or vascular insufficiency. There was no history of cigarette smoking or alcohol use. On physical examination, she was 152 cm tall and weighed 57.5 kg (body mass index, 24.9 kg/m2). She had bilateral upper palpebral xanthomas as well as xanthomas on elbows, neck, knees, and Achilles tendons. She also had corneal opacities, but funduscopic examination revealed no retinal angiosclerosis. The fasting glucose, glycosylated hemoglobin, serum electrolytes, liver function parameters, serum protein electrophoresis, and thyroid function data were all within normal limits. Both the resting electrocardiogram and the double Master two-step test were normal. Echocardiography revealed no structural or wall motion abnormalities.
The pedigree of the proband's family is shown in Fig 1
. Neither her mother (I 2) nor her three children (III 1, III 2, and III 3) had corneal opacities or xanthomas. Her two brothers (II 1 and II 2) and younger sister (II 4) also had xanthomas by history. Her brother (II 1) died suddenly of unknown causes when he was 55 years old.
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Plasma Lipoprotein Characterization
Blood was collected into tubes containing Na2 EDTA after a 12-hour fast. Lipoproteins were isolated by sequential ultracentrifugation to obtain the following density fractions: VLDL (density, <1.019 g/mL), LDL (1.019 to 1.063 g/mL), and HDL (1.063 to 1.21 g/mL).30 Plasma cholesterol and triglyceride concentrations were determined by enzymatic methods.30 HDL cholesterol was quantified by the heparin-manganese precipitation method.30 Apo A-II, B, C-II, C-III, and E concentrations were measured by single radial immunodiffusion,31 and the ELISA was performed as described in "ELISA for Apo A-I." The plasma Lp(a) was determined by ELISA (Terumo Medical Co).32 The apo E phenotype was determined by isoelectric focusing followed by immunoblotting, as described previously.33 The apo E genotype was determined using Hha I digestion of the apo E gene after amplification by PCR, as described previously.33 Gender- and age- matched normolipidemic healthy volunteers were used as normal control subjects.
ELISA for Apo A-I
The ELISA for apo A-I was based on the assay developed by Koren et al.34 Microtiter plates (Dynatech Laboratories Inc) were first coated with 100 µL of a rabbit anti-human apo A-I antibody (50 µg/mL) by incubation for 18 hours at 25°C. After the coating buffer was removed, 300 µL of 5% (wt/vol) bovine serum albumin in 10 mmol/L PBS (pH 7.4) was added, and incubation was done overnight at 4°C. Then calibrators, controls, and samples (100 µL) were added to the blocked wells and incubated for 18 hours at 4°C, after which the wells were washed four times with 300 µL of PBS containing 0.1% Tween 20. Next, 100 µL of horseradish peroxidase–labeled rabbit anti-human apo A-I (The Binding Site Ltd) diluted 5000-fold in phosphate buffer was added to each well, and incubation was done for 5 hours at 25°C. The wells were washed, color was developed by adding o-phenylenediamine, and a standard curve was generated by plotting the absorbance at 492 nm versus the apo A-I concentration.
Electroimmunoblot Analysis
Isoelectric focusing–gel electrophoresis was performed by the one-step screening method,35 after which apo A-I was identified by immunoblotting.36 SDS-PAGE of serum was performed according to the standard technique.37 To identify mutant apo A-I heterodimers, SDS-PAGE under nonreduced conditions was also done. After electroblotting onto nitrocellulose membranes (Hybond-ECL, Amersham Life Science), an anti–apo A-I monoclonal antibody (O.E.M. Concepts Inc) and an anti–apo A-II monoclonal antibody (Shybayagi Co) were bound to the immobilized antigens. ECL Western blotting detection reagents (Amersham Life Science) were used for visualization.
DNA Amplification by PCR
Genomic DNA was isolated from 100 µL of peripheral blood according to a previously described method.36 Synthetic oligonucleotide primers were produced with a DNA synthesizer (model 380B, Applied Biosystems, Inc) by the phosphoramide method according to the manufacturer's instructions. The PCR was performed by using a modification of the protocol published by Perkin-Elmer Cetus Instruments. The reaction mixture consisted of 20 µL of genomic DNA, 10 µL of 10x reaction buffer (100 mmol/L Tris-HCl [pH 8.3], 500 mmol/L KCl, 15 mmol/L MgCl2, and 0.1% [wt/vol] gelatin), 16 µL of 1.25 mmol/L dNTPs (dATP, dCTP, dTTP, and dGTP), and 20 pmol of each primer in a volume of 100 µL. The mixture was incubated at 95°C for 10 minutes and centrifuged briefly, after which 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus) was added, and the mixture was layered under 100 µL of mineral oil. Fig 2 shows a diagram of the apo A-I gene with the locations and directions of the three pairs of primers used in the PCR: 1 and 2 (255 bp), 3 and 4 (508 bp), and 5 and 6 (762 bp). Amplification was performed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 2 minutes, and extension at 72°C for 1.5 minutes in a Perkin-Elmer Cetus Thermocycler (model PJ 1000).
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DNA Sequence Analysis
The PCR-amplified DNA was purified with Geneclean (Bio 101), and a 1-µL aliquot of the first reaction mixture was reamplified as described above with a 100:1 ratio of the same primers.38 Then the single-stranded DNA from the second PCR was sequenced by the dideoxynucleotide chain termination method using Sequenase (United States Biochemical Co). Regions with strong secondary structures were sequenced after being subcloned into the vectors pUC118 and pUC119, with 12 clones being sequenced by the above-mentioned method. All sequences were determined on both strands, and the region containing the mutation was sequenced in duplicate with separately amplified DNAs.
Identification of the Apo A-I Mutation
PCR was performed to identify the apo A-I mutation in the proband's family. Genomic DNA was amplified with two oligonucleotide primers: 5' GCCCTACCTGGACGACTTC 3' (bases 1841 to 1859) and 5' TCAGGAAGCTGACCTTGAAGCTC 3' (bases 2216 to 2238). Nucleotide numbers are based on the published sequence for apo A-I DNA.39 The PCR was performed for 35 cycles using Taq DNA polymerase (Perkin-Elmer Cetus) under the following conditions: 30 seconds at 96°C, 30 seconds at 65°C, and 1 minute at 72°C. Then PCR products were purified using Geneclean (Bio 101) and sequenced as described above. A 5 µL aliquot of the purified PCR product was subjected to electrophoresis on 5% agarose gel and visualized with ethidium bromide staining.
Plasma Distribution of Apo A-ISasebo
Plasma from normal control subjects and the proband (200 µL) was subjected to chromatography on a Superose 6HR10/30 column (Pharmacia Fine Chemicals) equilibrated with PBS containing 1 mmol/L EDTA and 0.02% NaN3 (pH 7.4) and eluted at a flow rate of 0.5 mL/min. Fifty fractions of 0.5 mL were collected, and the cholesterol content of each fraction was measured enzymatically.30 Electrophoresis of lipoproteins from plasma density fractions was performed using 12.5% SDS-polyacrylamide gels,37 and the distributions of normal apo A-I and apo A-ISasebo were visualized with an ECL Western blotting (Amersham Life Science).
Nondenaturing Gradient PAGE
The HDL particle size was determined by nondenaturing gradient PAGE using precast 4% to 30% gels (Pharmacia LKB Biotechnology).40 Plasma with a density of <1.20 g/mL was obtained by centrifugation at 100 000 rpm for 4 hours (Beckman TL-100 ultracentrifuge). To visualize the particles, 10 to 15 mg of protein was applied to the gel. Protein was stained with 0.05% Coomassie brilliant blue G-250, and the gel was scanned with a Personal Densitometer SI (Molecular Dynamics, Inc). The gel was calibrated with a standard protein mixture (HMW calibration kit, Pharmacia LKB Biotechnology) consisting of thyroglobulin (hydrated particle size, 17.0 nm), ferritin (12.2 nm), lactate dehydrogenase (8.16 nm), and bovine serum albumin (7.1 nm). HDL particle size was calculated from the calibration curve of the peak migration distance versus that of the standard.
Determination of LCAT Activity and CER
Serum LCAT activity was determined with proteoliposomes used as a substrate, as described previously.41 Egg yolk phosphatidylcholine/cholesterol liposomes were prepared by ethanol injection. Each assay mixture contained 2.25 nmol of 7
-[3H]cholesterol (22 Ci/mmol) (Du Pont-New England Nuclear) and 4.5 µg of purified human apo A-I. The molar ratio of cholesterol to phosphatidylcholine was 1:6. The esterification rate was measured by incubation of plasma (0.039 mL) with a mixture of 0.7 mmol/L EDTA, 4 mmol/L ß-mercaptoethanol, and 1% bovine serum albumin (essentially fatty acid free) for 2 hours at 37°C in a final volume of 0.0625 mL. The reaction was stopped by addition of 0.625 mL of a chloroform/methanol (2:1) mixture, followed by incubation for 2 hours at room temperature in order to extract the lipids. Labeled cholesterol ester was separated by thin-layer chromatography on silica gel plates (E. Merck) using a mobile phase consisting of petroleum ether/diethyl ether/acetic acid (20:0.6:0.2), and the radioactivity was counted by liquid scintillation spectrometry.
The CER was defined as the rate of esterification of cholesterol contained within the endogenous lipoproteins of plasma and was comparable to the total plasma LCAT activity. Plasma was equilibrated with 7-[3H]cholesterol at 4°C in a precooled 24-well plate42 43 and then incubated for 18 hours at 4°C to achieve radiolabeling. The radiolabeled plasma was then incubated for 6 hours at 37°C, and the reaction was stopped by adding 0.45 mL of a chloroform/methanol mixture (2:1 [vol/vol]). Labeled cholesterol and cholesteryl ester were separated by thin-layer chromatography, as described above, and the radioactivity associated with cholesterol and cholesteryl ester was determined with a liquid scintillation counter.
LCAT Mass
The LCAT mass was measured by radioimmunoassay using a polyclonal antibody and 125I-labeled LCAT, as described previously.44
Measurement of CETP Activity
Plasma CETP activity was analyzed by the method of Tollefson and Albers.45 Radiolabeled HDL3 was prepared from the plasma fraction (density, >1.125 g/mL) isolated from a normolipidemic control subject by incubation with [14C]cholesterol (58.0 mCi/mmol) (Du Pont-New England Nuclear) for 18 hours at 37°C. The [14C]cholesterol ester/HDL3 substrate was then isolated at a density of 1.21 g/mL by ultracentrifugation (40 000 rpm for 40 hours at 4°C). The contents of the upper one third of each tube were harvested by slicing the tube, and the solution was dialyzed. The reisolated HDL3 contained >90% of the radioactivity in the cholesteryl ester fraction, as determined by thin-layer chromatography. CETP activity was measured using a 10 µL aliquot of plasma with 50 µg of radiolabeled HDL3 and 250 µg of acceptor lipoproteins (density, <1.060 g/mL plasma fraction). The mixture was diluted to a final volume of 600 µL with 150 mmol/L NaCl and 10 mmol/L Tris-HCl (pH 7.4) and incubated for 18 hours at 37°C. Then 0.125 mL of heparin/MgCl2 reagent was added to aliquots of the mixture, and 750 µL of each supernatant (HDL) was transferred to 20-mL plastic counting vials. After 6 mL of scintillant (Insta-gel, Packard Instrument Co Inc) was added, the samples were counted in a liquid scintillation counter.
Statistical Analysis
Lipoprotein, apolipoprotein, and lipid values of control subjects and heterozygous members of the kindred were analyzed for significant differences with the paired t test.
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Results |
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Lipoprotein Analysis
Table 1
summarizes the serum lipoprotein and apolipoprotein concentrations in the proband, her mother, her sons, and her daughter. The proband had severe deficiency of HDL cholesterol and apo A-I. Her mother, her daughter, and her two sons had reduced plasma total cholesterol concentrations and plasma HDL concentrations (47% to 62% of the level in normal control subjects), and their plasma apo A-I concentrations were about 50% of normal. The proband also had a markedly reduced plasma apo A-II concentration, whereas the other family members had slightly reduced plasma apo A-II levels. Both the proband and the other family members had reduced plasma apo C-II concentrations. The mean values of total cholesterol, HDL cholesterol, apo A-I, and apo C-II concentrations in her mother, her sons, and her daughter were significantly lower compared with normal control values. No blood samples from the proband's two brothers (II 1 and II 2) and younger sister (II 4) were available to analyze their serum lipoprotein and apolipoprotein concentrations.
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Electroimmunoblot Analysis
Agarose gel electrophoresis of plasma from the proband showed the complete absence of an -migrating band (data not shown). Isoelectric focusing of plasma at a pH gradient of 4 to 6 followed by immunoblotting revealed no detectable apo A-I band in the proband. In contrast, samples from her mother, daughter, and two sons showed bands of proapo A-I (A-I 2) and apo A-I 4 and A-I 5 (Fig 3). SDS-PAGE of serum followed by immunoblotting showed that the proband's apo A-I had a lower molecular weight (24 000) than native apo A-I and that the other family members exhibited apo A-I at both normal and lower molecular weights (Fig 4).
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DNA Sequence Analysis
Each DNA sequence encompassing an exon and exon-intron boundary of the apo A-I gene was amplified with the primers shown in Fig 2
. The amplified DNAs were used to generate single-stranded DNA from the second PCR or were cloned in vectors pUC118 and pUC119. Amplified DNA of exon 4 was cloned in both vectors, with 12 independent clones being isolated and sequenced. A partial gene duplication encompassing 23 nucleotides was found in all 12 clones, which produced a tandem repeat of bases 333 to 355 from the 5' end of the exon 4 (Fig 5). The tandem repeat generated a frameshift and a stop codon (TGA) at amino acid position 208 (Fig 6). No other substitutions were found.
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Determination of the Apo A-ISasebo Status
Fig 7 shows representative agarose gel electrophoresis patterns of the PCR products from the proband with apo A-ISasebo (lane 2) and her family members (lanes 3 to 6). The PCR product of the proband had a band with a higher molecular weight (421 bp) than that of the normal control subject (398 bp), whereas PCR products from the other family members had bands of both the higher and normal molecular weights. Sequence analysis of the abnormal band in samples from the family members revealed a 23-nucleotide partial gene duplication that produced a tandem repeat of bases 333 to 355 from the 5' end of exon 4, as described above. These results indicated that the proband was homozygous and her family members were heterozygous for the apo A-ISasebo mutation. No blood samples from the proband's two brothers (II 1 and II 2) and younger sister (II 4) were available to determine the apo A-ISasebo status.
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X174/HinfI digest.
Detection of Apo A-ISasebo/Apo A-II and Apo A-ISasebo/Apo E Complexes
Apo A-ISasebo contained 207 instead of 243 amino acids, with residues 162 to 207 differing from the sequence of normal apo A-I. The molecular weights of normal apo A-I and apo A-ISasebo were 28 000 and 24 000, respectively, on the basis of the results of immunoblotting. Apo A-ISasebo was found to contain two cysteine residues that were absent from normal apo A-I (Fig 6), and cysteine residues can mediate association with other cysteine-containing proteins. To investigate whether the mutant apo A-I formed complexes with other proteins, SDS-PAGE under nonreduced conditions was followed by immunoblotting with an anti–apo A-I monoclonal antibody, and proteins of a higher molecular weight were identified (Fig 8A). The molecular weight of the thicker band was consistent with an apo A-ISasebo/apo A-II/apo A-II heterotrimer, whereas the less intense band just underneath it was consistent with an apo A-ISasebo/apo A-II heterodimer, neither of which was observed in bands from normal control subjects. SDS-PAGE under nonreduced conditions followed by immunoblotting with an anti–apo E antibody showed that specimens from the homozygous proband contained a band consistent with an apo A-ISasebo/apo E heterodimer that was not observed in normal control subjects (Fig 8B).
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Plasma Distribution of Apo A-ISasebo
Because the plasma concentration of apo A-ISasebo was very low but present in the proband, the distribution of this mutant apo A-I in the various lipoprotein fractions was determined. As shown in Fig 9, apo A-I from a normal control subject was mainly associated with the HDL fraction, whereas apo A-ISasebo was found in both the LDL and HDL fractions. This shift of apo A-ISasebo into the LDL fraction could have important metabolic consequences.
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Nondenaturing Gradient PAGE
Gradient gel electrophoresis of HDL particles separated by ultracentrifugation followed by scanning with a Personal Densitometer SI (Molecular Dynamics, Inc) is shown in Fig 10. In the homozygous proband, the HDL fraction could barely be observed. In the heterozygous family members, the HDL particle size was smaller than in the normal control subject.
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LCAT Activity, LCAT Mass, CER, and CETP Activity
Table 2 shows the LCAT activity, LCAT mass, CER, and CETP activity in plasma from the proband (homozygote), four family members (heterozygotes), and normolipidemic healthy Japanese subjects. In the proband, the LCAT activity and the CER were reduced to about 30% of the control values; the LCAT mass was 37% of that in the normal control subjects. In the four family members, the LCAT activity and LCAT mass were reduced to about 70% of normal, and the CER was reduced to half of normal.
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Plasma CETP activity data for the proband, her family members, and normolipidemic healthy Japanese subjects are also shown in Table 2. In the proband, CETP activity was reduced to about half of the control level. However, the four family members showed only a slight decrease of CETP activity.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We identified a Japanese family with a novel 23-bp insertion in the apo A-I gene, which we designated as apo A-ISasebo. The homozygous proband had markedly decreased but detectable levels of apo A-I and HDL cholesterol, whereas the other family members (who were heterozygous for the defect) had intermediate apo A-I and HDL cholesterol levels. These findings suggest that the genetic trait was transmitted in an autosomal-dominant fashion.
LCAT activity for the proband (homozygote) was reduced to about 30% of normal, and it was about 70% of normal in the heterozygous family members. In addition, the proband had a CER that was about 25% of normal. However, the magnitude of these differences suggests that the markedly reduced serum apo A-I and HDL cholesterol concentrations found in the proband are not the result of familial LCAT deficiency.41 46 47 48 Clinically, the proband showed no hyperplastic orange tonsils, splenomegaly, or relapsing neuropathy, which are the major clinical signs of Tangier disease.29
On physical examination, the proband of apo A-ISasebo had corneal opacities and extensive xanthomas. Although genotypes were not yet examined, her two brothers (II 1 and II 2) and younger sister (II 4) also had xanthomas. Previous reports have indicated that there is some heterogeneity in the phenotype of apo A-I deficiency. Patients with apo A-I deficiency as a result of a 1-base insertion in exon 3 of the apo A-I gene22 or patients with apo A-I Q[-2]x23 have both corneal opacities and xanthomas like the proband, but a patient with a 1-base deletion (G at position 202) developed only corneal opacities,19 and a nonsense mutation caused CHD and xanthomas in another patient.18 Possible explanations for this heterogeneity may include the influence of environmental factors and/or interactions with other unknown genetic defects.
Most patients with apo A-I deficiency, including our proband, do not show an increased susceptibility to the development of CHD, despite previous epidemiological evidence that the plasma HDL concentration is inversely correlated with the risk of CHD.8 9 10 11 12 Although our patient had a slightly elevated Lp(a) concentration (49 mg/dL), her serum LDL cholesterol level was near the median value in age- and sex-matched normal Japanese individuals. No other known risk factors for CHD were present in this patient. The serum LDL cholesterol concentrations reported in other patients with apo A-I deficiency vary widely over the range from 3.8 to 5.2 mmol/L. Nondenaturing gradient PAGE revealed that the heterozygous relatives of our proband had HDL particles that were smaller than normal. If the proband also had small HDL particles, this could provide a possible explanation for the lack of CHD, along with enhanced catabolism of mutant apo A-I. This hypothesis is supported by a recent report that the fractional catabolic rates of recombinant carboxy-terminal truncation mutants are much faster than those of recombinant intact apo A-I and native human apo A-I.49 The reason for the absence of CHD in most of the patients with apo A-I deficiency is still not clear.
Some other genetic disorders also cause severe HDL deficiency.13 14 15 16 The biochemical profiles and clinical manifestations of combined apo A-I/apo C-III deficiency and apo A-I/apo C-III/apo A-IV deficiency resemble those of our proband, except for the presence of CHD. However, it is unclear why the addition of apo C-III and/or apo A-IV deficiency to apo A-I deficiency leads to an increase of CHD.
The mechanism leading to HDL deficiency in association with apo A-ISasebo was analyzed. The smaller molecular weight (24 000) apo A-I demonstrated by SDS-PAGE was compatible with the findings of DNA sequence analysis. The 46 amino acids of apo A-ISasebo created by the 23-bp insertion (amino acid residues 162 to 207) are not part of native apo A-I, and this insertion could cause significant alterations of protein conformation that could destabilize and inactivate the apo A-I molecule. Apo A-IMilano is associated with a low HDL cholesterol level50 51 52 and features the substitution of a cysteine for arginine at amino acid 173, a change that introduces a cysteine into a protein previously devoid of this residue. Apo A-IMilano forms homodimers as well as apo A-IMilano/apo A-II and apo A-IMilano/apo E complexes,53 and in vivo studies have revealed hypercatabolism of this mutant apo A-I.54 Mutant apo A-I/apo A-II heterodimers have also been observed in apo A-I deficiency.19 Thus, the formation of apo A-ISasebo/apo A-II/apo A-II, apo A-ISasebo/apo A-II, and apo A-ISasebo/apo E complexes, as noted in our family, could significantly alter HDL metabolism; it is still unclear just how these complexes are metabolized in the plasma. Moreover, the distribution of apo A-ISasebo into the LDL size range, which were likely to be HDL1 particles, was found. This shift of apo A-ISasebo into the LDL fraction could have important metabolic consequences, including apo A-ISasebo/apo E heterodimer formation.
The LCAT activity, LCAT mass, and CER were reduced in our proband, and similar findings have been reported in individuals with apo A-I/C-III deficiency15 and other types of apo A-I deficiency.19 One of the mechanisms underlying a reduced LCAT mass could be that apo A-I plays an important role in LCAT production and/or stability of the substrate for LCAT. A subclass of small HDL particles is reported to be the preferred substrate for LCAT.55 The apo A-ISasebo mutation was associated with a decrease in the number and size of HDL particles, and the reduced number of HDL particles may have provided a poor substrate for LCAT.
The CETP activity in our proband with apo A-ISasebo was about half of normal. In contrast, the transfer of preexisting and newly formed cholesteryl ester from HDL to apo B–containing lipoproteins is reported to be accelerated in apo A-I/apo C-III deficiency.56 This discrepancy may be due to different methods of determining CETP activity, but the specific CETP activity in apo A-ISasebo and the mechanism by which a decrease occurs remain to be determined.
In conclusion, we found that isolated apo A-I deficiency caused by a structural mutation in the apo A-I gene was associated with xanthomas, corneal opacities, and partial LCAT and CETP deficiency. Characterization of the molecular defect in this patient may help to provide a better understanding of apo A-I structure-function relationships and the clinical manifestations in patients with apo A-I deficiency.
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Acknowledgments |
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This study was partly supported by grants from the Ministry of Health and Welfare (No. 00627437 to Dr Sasaki) and from the National Institutes of Health (HL-30086 to Dr Albers). We wish to thank Drs Yoshio Misumi and Shigenori Ogata (Fukuoka University) for their helpful technical advice. We also wish to thank Kayo Nishi for excellent technical assistance and Yuki Hashimoto for manuscript preparation.
Received March 4, 1996;
revision received May 3, 1996;
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References |
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