Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1294-1306

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tilly-Kiesi, M.
Right arrow Articles by Taskinen, M.-R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tilly-Kiesi, M.
Right arrow Articles by Taskinen, M.-R.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1294-1306.)
© 1995 American Heart Association, Inc.

Articles

ApoA-IHelsinki (Lys107->0) Associated With Reduced HDL Cholesterol and LpA-I:A-II Deficiency

Marju Tilly-Kiesi; Zhang Qiuping; Sonja Ehnholm; Juhani Kahri; Sanni Lahdenperä; Christian Ehnholm; Marja-Riitta Taskinen

From the Third Department of Medicine (M.T.-K., J.K., S.L., M.-R.T.), University of Helsinki, and the Department of Biochemistry (Z.Q., S.E., C.E.), National Public Health Institute, Helsinki, Finland.

Correspondence to Prof Marja-Riitta Taskinen, MD, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract A Finnish kindred with premature coronary heart disease and decreased HDL cholesterol levels was identified as having an apoA-I variant, apoA-I (Lys107->0), caused by a 3-bp deletion of nucleotides 1396 through 1398 in exon 4 of the apoA-I gene. These subjects (n=10) were heterozygous for this mutation. The mean serum HDL cholesterol concentration (26.7±9.7 mg/dL) of affected family members was 36% lower than that of unaffected family members (P<.05). Mean serum apoA-I and apoA-II concentrations in heterozygotes were reduced by 18% and 22%, respectively, compared with normal family members (P<.05). In heterozygotes the mean concentration of lipoprotein containing both apoA-I and apoA-II (LpA-I:A-II) was 31% lower than in those with normal apoA-I (P<.001), while the mean level of lipoproteins containing apoA-I without apoA-II was similar in the two groups. HDL density-gradient ultracentrifugation showed a lack of HDL2 and small dense HDL3 in heterozygotes compared with unaffected family members. The HDL particle size distribution, as analyzed by nondenaturing gradient gel electrophoresis of heterozygotes, revealed one major peak at 8.0 to 9.7 nm, a minor peak at 7.8 to 8.5 nm, and an absence of HDL2b and HDL2a peaks. These latter peaks were observed in unaffected family members. Serum levels of LDL cholesterol, triglycerides, VLDL, IDL, and LDL subclasses were similar in the two groups. However, in heterozygotes the cholesterol-to-triglyceride ratios in VLDL2, LDL1, LDL3, HDL2b, HDL2a, and HDL3a were 8% to 54% lower than in unaffected family members (P<.05). Cholesteryl ester transfer protein activity in heterozygotes was reduced by 25% compared with unaffected family members (P<.05), while the plasma lecithin:cholesterol acyltransferase (LCAT) activity did not differ between heterozygotes and unaffected family members. The ability of isolated variant apoA-I to serve as a cofactor for LCAT in vitro did not differ from that of normal apoA-I. Our data are consistent with the concept that a low HDL cholesterol level in subjects heterozygous for the apoA-IHelsinki mutation (Lys107->0) having normal LCAT activity is a consequence of decreased concentration of LpA-I:A-II particles and of a smaller size and reduced cholesterol content of HDL particles.


Key Words: apoA-I gene mutation • HDL deficiency • lecithin:cholesterol acyltransferase • CETP • coronary heart disease


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Decreased plasma HDL-C levels have been associated with premature CHD.1 ApoA-I is the major protein of HDL, and with apoA-II accounts for about 85% of the protein within HDL.2 3 In some studies4 5 6 7 but not in others8 9 10 plasma apoA-I concentrations provide more information about CHD risk than do HDL-C levels. The protective role of HDL against CHD has been related to its function in the transport of cholesterol from peripheral cells to the liver for excretion, the so called "reverse cholesterol transport process."11 12 Two distinct populations of HDL, LpA-I and LpA-I:A-II, have been isolated.13 14 15 Recently it has been noted that reduced LpA-I but not LpA-I:A-II levels account for the decreased HDL-C concentrations in patients with CHD.16 In other studies a reduced HDL-C level in CHD patients was associated with decreases in both types of particles.17 18 19

Some HDL-deficient syndromes, such as Tangier disease or familial apoA-I/apoC-III or apoA-I/apoC-III/apoA-IV deficiency states, are characterized by premature coronary artery disease.20 21 22 23 In contrast, apoA-I variants, including apoA-IMilano, apoA-IMarburg, and apoA-IIowa, have not been associated with premature CHD.24 25 26

In this article we describe a deletion mutation in the human apoA-I gene associated with low serum HDL-C and decreased apoA-I and apoA-II concentrations in a kindred with a high incidence of CHD. We have characterized the association of this apoA-I variant on different lipoproteins and their subclasses, on lipoprotein particle size and density distribution, and on the activities of the major enzymes involved in lipoprotein metabolism.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
*Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Subjects
A 51-year-old woman (A.U.T.) from South Finland was referred to the Lipid Research Clinic, HUCH, because of her low HDL-C concentration (0.24 mmol/L [9 mg/dL]). She had significant obesity (body mass index, 38.9 kg/m2) and had received medication for essential hypertension for 14 years. For the previous 3 years she had received a combination of captopril and indapamide. She had no history of angina pectoris or other vascular disease and had never smoked. On physical examination she had normal skin, no xanthomas, and no xanthelasmas, and the ophthalmoscopic examination showed no corneal opacification. Her liver was not enlarged, and her spleen was not palpable. Her tonsils had been removed after the delivery of her first child. The peripheral pulses were symmetrical and easily palpable. The laboratory results showed normal thyroid, liver, and renal function. Her fasting blood glucose and glycosylated hemoglobin levels were normal.

The proband's family was from southern Finland. Altogether 25 relatives participated in the study. Her mother (K.K.) was in excellent physical health and was not taking any medication. Her father had received medication for anginal symptoms for 3 years and died of cerebral hemorrhage at the age of 69 years. The father had five brothers and three sisters, of whom only one sister (A.A.S.) was still alive and in good health at age 85 years. The medical records of the proband's father, four uncles, and two aunts revealed that one aunt was diagnosed as having ischemic heart disease based on symptoms and electrocardiographic findings. She received medication for CHD symptoms and died suddenly at age 48 years. The other aunt had died at the age of 66 years of heart failure after she had three prior admissions to HUCH for acute MI. An uncle of the proband had died of his fourth acute MI at HUCH at age 54 years. The autopsy confirmed coronary atherosclerosis. One uncle had died of MI at age 63 years. The autopsy confirmed substantial atherosclerotic changes in the coronary arteries and abdominal aorta. According to medical records two other uncles died of CHD at 63 and 66 years, respectively. The latter uncle suffered his first MI at age 53 years. No HDL-C values for the proband's aunts or uncles were available in their medical records. In addition, three of the proband's cousins had died of CHD between the ages of 46 and 62 years, one of them after receiving a heart transplantation at HUCH. His HDL-C level (measured on several occasions) was below the normal range (0.80 mmol/L [31 mg/dL]).

The proband's older sister (K.A.P.) and two brothers (R.O.K. and K.K.K.) had no history of cardiovascular disease and were healthy as assessed by physical examination and laboratory tests. The proband's two sons (M.J.V. and K.P.V.) and her brothers' sons (R.K. and J.K.) were also healthy. In addition, 14 cousins participated in the study. Two of them (T.M.A. and O.E.O.) had suffered an MI, but the others had no clinical signs or symptoms of CHD. Two unaffected subjects (P.E.K. and R.E.K.) were diagnosed as having non–insulin-dependent diabetes mellitus according to the results of laboratory tests. A pedigree of the family is presented in Fig 1Down. The experimental protocol was approved by the Ethical Committee of the Third Department of Medicine of Helsinki University.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Pedigree of the family having the apoA-I (Lys107->0) deletion. Affected family members were all heterozygous for the defect. Squares indicate men; circles, women; and diagonal line, deceased.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
*Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
DNA Preparation
Genomic DNA was isolated from frozen whole blood of the proband, her family members, and a series of normal control subjects.27

Amplification of DNA
Oligodeoxynucleotide primers used in amplification and sequencing were chosen according to the published apoA-I gene structure28 and synthesized on an Applied Biosystems model 381A DNA synthesizer. The sequences and positions of these primers are shown in Table 1Down. Six fragments of the apoA-I gene of the subjects were amplified by using PCR with genomic DNA as a template. The PCR was performed in a reaction mixture containing 1 µmol/L of primers, 0.2 mmol/L each dATP, dCTP, dGTP, and dTTP, 50 mmol/L Tris-HCl (pH 8.8), 1.5 mmol/L MgCl2, 15 mmol/L (NH4)2SO4, 0.1% Triton X-100, 0.1 mg/mL gelatin, and 2.5 U Taq DNA polymerase (Promega Corp) in a final volume of 100 µL. The reaction conditions on a programmable heating block (MJ Research Inc) were as follows: 95°C for 1 minute, 58°C to 63°C for 1 minute, and 72°C for 2.5 minutes, 30 cycles.


View this table:
[in this window]
[in a new window]
  		Table 1. Sequence and Position of Oligodeoxynucleotide Primers

SSCP Analysis
Six fragments of the apoA-I gene of the subjects were amplified by PCR following the reaction conditions described above, and 2 to 3 µL [{alpha}-32P]dATP or dCTP (3000 Ci/mmol/L; Amersham) was added to each tube. The PCR product was diluted 1:1.5 with 0.1% sodium dodecyl sulfate and 10 mmol/L EDTA, denatured, and loaded on neutral 5% polyacrylamide gel for mobility-shift electrophoresis.29 Autoradiography was performed by using an intensifying screen with Kodak X-omat film at room temperature for 24 to 72 hours.

Direct Sequencing
The PCR products were purified by Qiaex (Diagen GmbH) and directly sequenced with the dideoxy chain-termination reaction by using a sequence kit (US Biochemical Corp). For sequencing of the fourth exon, an additional primer, No. 13, which is located 76 nucleotides 5' of the mutation point, was used.

Solid-Phase Minisequencing
The solid-phase minisequencing and the preparation of the batches of pooled leukocyte DNA were performed as described by Syvänen et al.30 The 127-bp fragment located 111 nucleotides downstream of the 5' end of the apoA-I gene exon 4 was amplified for minisequencing. The PCR reaction mixture and the conditions were as mentioned above except that the primers used were 2 µmol/L biotinylated primer No. 14 and 10 µmol/L primer No. 8, and the annealing temperature was 60°C. The detection-reaction mixture, which consisted of 0.2 µmol/L detection primer No. 15 and 0.4 µmol/L [3H]dTTP (34 Ci/mmol; Amersham) to detect the mutation as well as 1 U Taq DNA polymerase in 50 µL PCR buffer, was added to each well. The radioactivity was measured in a liquid scintillation counter (1210 Ultrobeta, LKB).

ApoA-I Isoform Analysis
The apoA-I isoforms of the subjects were determined by isoelectric focusing and immunoblotting.31

Lipids and Lipoproteins
Blood samples were obtained in the morning after a 12-hour fast. Serum lipoproteins were isolated by sequential ultracentrifugation in a Beckman L8-70 ultracentrifuge (Beckman Instruments) with a Beckman 50 TI rotor.32

Density-Gradient Ultracentrifugation of Lipoproteins
Density-gradient ultracentrifugation of VLDL and IDL was performed in a Beckman L8-70 ultracentrifuge with an SW40 TI swinging-bucket rotor with centrifuge tubes by using a 2-mL VLDL and IDL preparation isolated by sequential ultracentrifugation and the method described previously.33

The LDL subfractions LDL1, LDL2, and LDL3 were separated by density-gradient ultracentrifugation in a Beckman SW40 TI swinging-bucket rotor from the LDL sample obtained by sequential ultracentrifugation. The method used is slightly modified from that described by Griffin et al34 for plasma samples. Briefly, the discontinuous NaBr gradient was prepared by layering, from bottom to top, 0.5 mL d=1.1900 g/mL, 1.2 mL sample solution (0.9 mL LDL obtained from sequential ultracentrifugation in 0.3 mL NaBr, d=1.5350 g/mL), 1.5 mL d=1.0630 g/mL, 1.5 mL d=1.0560 g/mL, 1.5 mL d=1.0450 g/mL, 2.0 mL d=1.0340 g/mL, 2.0 mL d=1.0240 g/mL, and 0.7 mL d=1.0190 g/mL. The tubes were centrifuged in a Beckman L8-70 ultracentrifuge at 40 000 rpm for 24 hours at 23°C, and the rotor was allowed to stop without braking.

After ultracentrifugation the tubes were discharged from the top by using a Beckman Recovery System, Perfusor V (B. Braun) infusion pump and Maxidens solvent (Nyegaard & Co A/S). The protein-absorbance profiles of the tubes were monitored with an absorbance meter (Pharmacia), and the density gradient was controlled by using a DMA 46 density meter (Anton Paar). Three 1.5-mL fractions were collected: LDL1 (d=1.024 to 1.0330 g/mL), LDL2 (d=1.0330 to 1.042 g/mL), and LDL3 (d=1.0420 to 1.0550 g/mL).

The HDL density-gradient ultracentrifugation method was based on the method described by Groot et al35 except that first all apoB-containing lipoproteins (ie, VLDL, IDL, and LDL) were removed from the serum samples by ultracentrifugation. A serum sample of 2.0 mL in 0.3 mL NaBr solution (d=1.5350 g/mL) was overlaid with 0.7 mL NaBr solution (d=1.0600 g/mL) by using Beckman 1/2x2 polycarbonate centrifuge tubes. After ultracentrifugation in a Beckman Optima TL ultracentrifuge with a Beckman TLA 100.3 rotor at 100 000 rpm for 5 hours at 20°C, the thin yellowish supernatant layer containing VLDL, IDL, and LDL was removed by aspiration. The density of the infranatant was increased by adding 1.0 g dry NaBr, and a 2.0-mL volume of this sample solvent was transferred by pipette into the bottom of a Beckman Ultraclear 9/16x33/4 tube. The discontinuous gradient above the sample was then prepared by layering NaBr solution in the following order: 1.5 mL d=1.2500 g/mL, 6.7 mL d=1.2200 g/mL, and 2.0 mL distilled water. After ultracentrifugation in a Beckman L8-70 ultracentrifuge with an SW40 TI swinging-bucket rotor at 40 000 rpm for 18 hours at 20°C, the tubes were emptied as described for LDL separation. Five fractions of 1.3 mL each, representing HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c, were collected.

LDL Gradient Gel Electrophoresis
Nondenaturing polyacrylamide gradient gel electrophoresis of LDL was performed on serum samples with 2% to 16% gels (Pharmacia) by using the method of Nichols et al.36 The gels were stained with Sudan Black B lipid stain.37 After the gels were destained and scanned, the particle diameter of the major LDL peak was defined by comparing the mobility of the sample with that of reference LDL.37

HDL Gradient Gel Electrophoresis
The HDL gradient gel electrophoresis was performed36 with Pharmacia 4% to 30% gels. Serum was adjusted to d=1.2100 g/mL with NaBr solution (d=1.335 g/mL) and ultracentrifuged in a Beckman L8-70 ultracentrifuge at 35 000 rpm for 65 hours at 4°C in a Beckman 50 TI rotor. The supernatant (1 mL) was recovered by tube slicing and mixed with 0.25 mL of 40% sucrose. Using commercially available gels (Pharmacia) and a sample volume of 10 µL, the gradient gel electrophoresis was run in Gel Electrophoresis Apparatus GE 2/4 (Pharmacia) for 3000 Volt-hours in a buffer containing 80 mmol/L borate, 90 mmol/L Tris-HCl, 3 mmol/L EDTA, and 3 mmol/L NaN3, pH 8.35. The gels were fixed with 10% sulfosalicylic acid, stained with 3.5% Coomassie G-250 brilliant blue, destained with 5% acetic acid, and scanned at 595 nm by using a computer-assisted Cliniscan 2 (Beckman Instruments). Particle diameters of HDL fractions were assessed by comparing the mobility of the sample with the mobility of molecular-weight calibration proteins (Pharmacia).

Measurement of CETP and LCAT Activity
LDL and HDL isolated from the plasma of healthy donors were dialyzed extensively against 10 mmol/L Tris HCl, pH 7.4, containing 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.1 g/L NaN3. The LDL was labeled with cholesteryl(1-14C)oleate (Amersham) by using the lipid-dispersion method described by Morton and Zilversmit.38 The labeled LDL was ultracentrifuged again at 40 000 rpm for 24 hours at 20°C in an SW40 TI rotor. The measurement of CETP activity was accomplished by the method of Groener et al.39 The results were calculated by measuring the CETP activity of plasma samples divided by the CETP activity of the reference sample in the same assay40 and are expressed in arbitrary units. The LCAT activity was measured41 by using proteoliposome substrates containing either control apoA-I or apoA-I (Lys107->0).

Measurement of LpA-I and LpA-I:A-II Concentrations
The concentration of LpA-I particles was measured by electroimmunoassay (Sebia) as described by Parra et al.42 The concentration of LpA-I:A-II was calculated by subtracting the LpA-I concentration from the serum apoA-I concentration. The interassay variation for LpA-I particle measurements was 7.3%.

Analytical Methods
Cholesterol, free cholesterol, phospholipid, and triglyceride levels were analyzed by using enzymatic colorimetric methods with a Cobas Mira analyzer (Hoffmann-La Roche) and reagent kits (for cholesterol and triglycerides, Nos. 0715166 and 0722138, Hoffmann-La Roche; for free cholesterol, No. 310328, Boehringer Mannheim GmbH Diagnostica; and for phospholipids, No. 990-54009, Wako Chemicals GmbH). Serum apoA-I, apoA-II, and apoB concentrations were measured by using an immunoturbidometric assay (Orion Diagnostica). The serum concentrations of apolipoproteins C-II and C-III were determined by using immunochemical agarose plates according to the instructions of the manufacturer (Daiichi Pure Chemicals Co). The normal ranges of apoC-II are 3.5±1.2 and 3.3±1.2 mg/dL and those of apoC-III are 7.6±2.2 and 7.3±3.7 mg/dL for men and women, respectively. Blood glucose concentration was measured by using the glucose oxidase method (Auto-Analyzer, Technicon). Glycosylated hemoglobin (normal range, 4% to 6%) was measured by using a Diamat Analyzer System (BioRad, Clinical Division). Serum insulin levels were measured by using a radioimmunoassay after precipitation with polyethylene glycol with the Phadeseph insulin radioimmunoassay kit (Pharmacia).

Statistical Analyses
The significances of differences between affected and unaffected subjects were assessed by Mann-Whitney nonparametric tests using BMDP statistical software (University of California). Pearson's correlation coefficient analysis was used to estimate the relationship between variables.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Analysis of the ApoA-I Variant
Isoelectric focusing of the plasma of the proband (A.U.T.) and nine family members (K.K.K., R.O.K., K.P.V., T.M.A., O.E.O., P.P.V., M.V., R.K., and L.S.T.) revealed a variant apoA-I isoprotein that had a decrease of one positive charge compared with normal mature apoA-I (Fig 2Down). The relative plasma concentrations of the variant apoA-I isoform and normal apoA-I were similar as judged from the isoelectric focusing pattern. To localize the mutation that caused the net charge change of the variant isoprotein, all exons of the apoA-I genes of the propositus and her relatives were amplified by PCR and subjected to SSCP analysis. Fig 3Down shows the first fragment of exon 4 of the apoA-I gene of the proband and some of the affected family members. Compared with the DNA migration pattern of control subjects, they had two additional bands with faster mobility.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 2. Isoelectric focusing of apoA-I. Lane 1, control apoA-I; lane 2, apoA-I (Lys107->0). Arrow indicates shift mobility caused by mutation. An anode appears at the bottom.



View larger version (107K):
[in this window]
[in a new window]
  		Figure 3. PCR-SSCP analysis of exon 4 of apoA-I. Genomic DNA from leukocytes was subjected to PCR-SSCP amplification by using a pair of primers in exon 4 of apoA-I. Amplified DNA from the proband (lane 1), her brother (lane 2), and her brother's son (lane 4) was used; lanes 3, 6, and 7 show control DNA. Lane 5 contains a mixture of normal and mutated DNA. Electrophoresis was performed in nondenaturing 5% polyacrylamide gels containing 10% glycerol at room temperature for 12 hours.

Direct sequencing of the coding region of the apoA-I gene revealed that the proband and the nine other affected family members (Fig 1Up) were heterozygous for a 3-bp deletion in exon 4 from nucleotides 1396 through 1398, removing the codon (AAG) for Lys107 of the mature apolipoprotein. Exon 4 of the apoA-I gene contains six 66-bp-long homologous tandem repeats that code for six 22-amino acid segments.28 The 3-bp deletion described above occurred within repeat number one. The rest of the apoA-I gene sequence was normal.

The 3-bp deletion in the apoA-I DNA of the proband and affected family members was further confirmed by using solid-phase minisequencing, in which a detection primer, annealing immediately adjacent to the mutation site, is elongated by a DNA polymerase with a single dNTP corresponding to the nucleotide at the site of the mutation. To identify the normal sequence (TGAAGAA) and the sequence including the 3-bp deletion (TGAAGAC), [3H]dTTP is included in one minisequencing reaction and [3H]dGTP in another reaction. The ratio between the incorporated nucleotides directly reflects the ratio between the two sequences present in the PCR product.

DNA samples from the proband (A.U.T.), her two brothers (K.K.K. and R.O.K.), and her son (K.P.V.) as well as DNA samples from two unaffected family members (K.K. and K.A.P.) and two unrelated control subjects were analyzed by solid-phase minisequencing. The presence of the mutation resulted in a significant incorporation of [3H]GTP, leading to a T/G ratio of about 1, while this ratio in unaffected family members and control subjects was >10 (Table 2Down).


View this table:
[in this window]
[in a new window]
  		Table 2. Determination and Quantification of the 3-bp Deletion Causing Removal of Lys107 of ApoA-I DNA in Affected and Control Individuals by Solid-Phase Minisequencing

To determine the prevalence of the mutation apoA-I (Lys107->0) in the Finnish population, solid-phase minisequencing of pooled DNA from Finnish subjects was performed. Three separate pools containing DNA from 921, 189, and 180 individuals, respectively, were analyzed. In these population samples no subjects with the apoA-I (Lys107->0) mutation could be identified (Table 2Up).

Serum Lipoprotein and Apolipoprotein Levels and Enzyme Activities
The clinical characteristics and lipid values measured in subjects of the pedigree are presented in Table 3DownDown. The mean serum HDL-C concentration of the affected family members was clearly below the normal range and was reduced by 36% compared with the mean HDL-C value of the unaffected family members. Total serum cholesterol, LDL cholesterol, and triglyceride concentrations were similar in apoA-I (Lys107->0)–affected and unaffected subjects. The proband had moderately increased serum triglycerides, while the rest of the apoA-I (Lys107->0) heterozygotes were normotriglyceridemic.43 The fasting blood glucose, glycosylated hemoglobin, and serum insulin values were within the normal ranges for all affected subjects (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 3. Clinical Characteristics, Lipid, Lipoprotein, Apolipoprotein, LpA-I, and LpA-I:A-II Levels, and LCAT and CETP Activities of Family Members With (n=10) and Without (n=15) the ApoA-I (Lys107->0) Mutation


View this table:
[in this window]
[in a new window]
  		Table 3A. (Continued)

The mean serum apoA-I concentration of heterozygotes was reduced by 18.3% and the apoA-II concentration by 22.2% compared with unaffected family members (P<.05 and P<.01, respectively, Table 3UpUp). The mean apoC-II and apoC-III concentrations of heterozygotes were within the normal ranges and were similar to unaffected family members (data not shown).

The mean plasma LCAT activity in carriers of the mutant apoA-I gene did not differ from the LCAT activity of unaffected family members. In vitro no defect was observed in the LCAT activation properties of the isolated mutant apoA-I. However, the mean CETP activity was reduced by 25% in affected family members compared with unaffected family members (P<.05, Table 3UpUp). The mean CETP activity of the unaffected family members was similar to the level measured in our laboratory for healthy control subjects.44

Characteristics of HDL
The two most prominent characteristics of the HDL density distribution profiles of the affected subjects were the total absence of HDL2 and the shift of the HDL3 subfraction toward a higher density compared with the profiles of unaffected family members (Fig 4Down). The percentages of HDL found in the five subfractions HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c were 5.7%, 7.3%, 18.1%, 33.7%, and 35.2%, respectively, for affected family members compared with 9.4% (P<.01), 12.1% (P<.001), 19.4% (NS), 29.1% (P<.001), and 29.9% (P<.05) in unaffected family members (Table 4Down). The data indicate that in heterozygotes the HDL density distribution is altered, with more than two thirds of the HDL particles being in the two most dense HDL subfractions, HDL3b and HDL3c.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Protein absorbance profiles of HDL subclasses obtained by density-gradient ultracentrifugation. Individual curves of apoA-I (Lys107->0) carriers (top) and unaffected family members (bottom) are shown.


View this table:
[in this window]
[in a new window]
  		Table 4. Concentration and Composition of Lipoprotein Subfractions as Separated by Density-Gradient Ultracentrifugation of Related Subjects With and Without the ApoA-I Gene Mutation

The HDL particle size distribution was determined by using gradient gel electrophoresis (Fig 5Down). The HDL particle size distribution profiles of all the affected family members were characterized by a predominance of the HDL3 subclass. In the subjects with the apoA-I (Lys107->0) variant one major peak appeared at 8.0 to 9.7 nm and a minor peak at 7.8 to 8.5 nm. In unaffected family members the HDL particle profile was more polydisperse and consisted of two to five peaks with similar mobility. The major peak appeared at 8.0 to 8.9 nm and a minor peak between 7.8 to 8.0 nm, and most unaffected subjects also displayed one or two prominent peaks at 9.3 to 11.5 nm. The results indicate that the apoA-I (Lys107->0) variant is associated with a very low level of the HDL2b subclass, similar to subjects with apoA-IMilano.45



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Individual HDL gradient gel electrophoretic profiles from family members with (upper two rows) and without (lower three rows) the apoA-I (Lys107->0) gene mutation.

The mean concentrations of LpA-I particles were comparable in affected and unaffected family members, but the concentration of LpA-I:A-II particles was 31% lower in affected than in normal family members (P<.001; Table 3UpUp).

Characteristics of VLDL, IDL, and LDL
The density distribution curves of VLDL, IDL, and LDL did not show any characteristic features or specific particle subpopulations in affected compared with unaffected family members (data not shown).

In all 10 subjects with the apoA-I (Lys107->0) deletion the LDL particle size distribution revealed one major peak without clearly distinguishable minor peaks (data not shown). The computer-calculated particle diameter of the major LDL peak was 25.7 nm for both the apoA-I (Lys107->0) carriers and noncarriers. The presence of the apoA-I (Lys107->0) deletion did not seem to affect the LDL particle size distribution. In all family members the particle diameter of the major LDL peak correlated negatively with the serum triglyceride concentration (r2=.5809, P<.001), serum insulin level (r2=.2464, P<.05), and VLDL1, VLDL2, and IDL concentrations (r2=.4268, r2=.5466, and r2=.3084, respectively, P<=.05). A positive correlation was observed between the particle diameter of the major LDL peak and the concentrations of the HDL2b (r2=.1773), HDL2a (r2=.2758), HDL3a (r2=.3128), and HDL3b (r2=.2227) subfractions (all P<.05).

Composition of Lipoprotein Subclasses
In the HDL2b subfraction of affected family members the percentage of triglycerides was increased by 63%, and in HDL2a and HDL3a the percentages of cholesterol were decreased by 15% and 14%, respectively (both P<=.05). The most significant characteristic of the HDL composition of subjects with the apoA-I (Lys107->0) mutation was the low cholesterol-to-triglyceride ratio in the largest HDL subclasses (Table 4Up). In HDL2b, HDL2a, and HDL3a subfractions the cholesterol-to-triglyceride ratio was reduced by 54%, 39%, and 35%, respectively (P<=.05), compared with unaffected family members. Compositions of the two most dense HDL subfractions, HDL3b and HDL3c, were comparable in affected and unaffected subjects. Similarly, in subclasses of LDL and VLDL the heterozygotes tended to have a moderately increased content of triglycerides and a lower cholesterol-to-triglyceride ratio, with a significant difference in LDL1, LDL3, and VLDL2 (P<.05; Table 4Up).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We report an apoA-I variant detected in a Finnish kindred. Isoelectric focusing of plasma from the proband and nine other family members revealed a variant apoA-I isoprotein with a decrease of one positive charge compared with normal apoA-I. PCR-SSCP analysis, direct sequencing, and solid-phase minisequencing analysis showed that the variant apoA-I isoprotein was caused by a 3-bp deletion of nucleotides 1396 through 1398 in exon 4 of the apoA-I gene, resulting in the lack of the codon for Lys107 of apoA-I.

The mean HDL-C concentration of affected family members was reduced by one third compared with unaffected family members, while mean total and LDL cholesterol concentrations were similar in affected and unaffected subjects. An increased incidence of hypertriglyceridemia is typical of some apoA-I variants,25 46 47 but the serum triglyceride concentrations of all the apoA-I (Lys107->0) heterozygotes except one were normal. The proband had moderately increased serum triglycerides, possibly as a consequence of obesity and low lipoprotein lipase activity.48 49 Concomitant moderate hypertriglyceridemia explains the proband's exceptionally low HDL-C concentration compared with the HDL-C levels of other affected family members.50

Heterozygous subjects had mean serum apoA-I and apoA-II concentrations that were reduced by a similar extent. The mean concentrations of LpA-I particles were the same in affected and unaffected subjects, while affected family members had a 31% lower mean concentration of LpA-I:A-II particles than unaffected family members. Puchois et al16 and Barkia et al51 have shown that reduced concentrations of LpA-I but not LpA-I:A-II are associated with coronary artery disease, and in vitro studies support the assumption that LpA-I is cardioprotective.16 51 In contrast, other studies have shown that LpA-I and LpA-I:A-II are reduced in coronary artery disease or CHD patients to a similar degree.17 18 19 52

An inverse correlation between the HDL-C level and the fractional catabolic rate of apoA-I and apoA-II has been demonstrated,53 54 55 56 and in heterozygous apoA-IMilano (Arg173->Cys) and apoA-IIowa (Gly26->Arg) subjects both the mutant and the normal apoA-I are catabolized at an increased rate.26 57 In addition, apoA-II levels are decreased in subjects with apoA-I (Arg173->Cys) or apoA-I (Gly26->Arg), which suggests that apoA-II may be more rapidly catabolized in these subjects as well.26 However, apoA-II kinetics have not been studied in subjects with apoA-I variants, and no data have been reported on the metabolism of the two HDL particle populations in apoA-I–variant subjects. On the basis of the normal concentration of LpA-I particles and the decreased concentration of LpA-I:A-II particles in apoA-I (Lys107->0) Helsinki heterozygotes, we hypothesize that this mutation primarily affects the metabolism of HDL particles containing both apoA-I and apoA-II. Our preliminary data from in vivo turnover studies in two apoA-I (Lys107->0) heterozygotes show an increase in the fractional catabolic rate of both apoA-I and apoA-II (Tilly-Kiesi et al, unpublished data, 1995).

Several characteristic features of the HDL of heterozygotes were observed in comparison with the HDL of unaffected family members. Density-gradient ultracentrifugation studies demonstrated that the heterozygotes lacked HDL2, and more than two thirds of their total HDL was found in subfractions HDL3b and HDL3c, which have the highest density and the lowest cholesterol/protein ratio.2 3 These data are consistent with the observation that these subjects have a greater percentage reduction in serum HDL-C levels than in apoA-I and apoA-II concentrations. HDL particle size distribution studies revealed that heterozygotes lacked HDL peaks in the particle size range >9.7 nm, indicating a deficiency of larger HDL particles.58

The LDL subclass concentrations, density distribution, and particle size of heterozygotes resembled those of unaffected family members. The concentrations and density distribution of VLDL and IDL were also similar in affected and unaffected family members. Lipoprotein lipase and hepatic lipase, which are related to the conversion of VLDL to IDL and LDL, are also involved in the conversion of large LDL to smaller LDL particles.59 60 61 62 Similar mean postheparin lipoprotein lipase and hepatic lipase activities were found in affected and unaffected family members (data not shown), consistent with the similarity of VLDL, IDL, and LDL subclass distributions in these two groups.

In Tangier homozygotes and in subjects homozygous for the apoA-I (Glu136->Lys) mutation, the content of triglycerides in LDL is increased.63 64 In heterozygotes the cholesterol-to-triglyceride ratio was significantly reduced in VLDL2, LDL1, LDL3, HDL2b, HDL2a, and HDL3a, and in all other lipoprotein subclasses a similar trend was noted. An imbalance in the amounts of donor (HDL) and acceptor (VLDL, LDL) particles for cholesteryl ester transfer may account for the diminished cholesterol-to-triglyceride ratio in the plasma lipoproteins of heterozygotes.65 66 The reduced CETP activity of heterozygotes compared with unaffected family members may be a compensatory alteration to increase the HDL2/HDL3 ratio and to correct the HDL composition.67

ApoA-I is an important activator of the LCAT enzyme.68 69 As summarized in Table 5Down, partial LCAT deficiency due to reduced LCAT mass or to reduced ability of the variant apoA-I to activate LCAT is associated with apoA-I variants such as apoA-I (Cys173->Arg), apoA-I (Gua202->0), apoA-I (Glu146->Arg160) deletion, apoA-I (Lys107->0), and apoA-I (Pro143->Arg).47 70 71 72 73 We isolated mutant apoA-I (Lys107->0) from our proband and studied the interaction of mutant apoA-I with LCAT in vitro. No defect in the LCAT activating properties of the mutant apoA-I was observed. In addition, the mean plasma LCAT activity of affected subjects was similar to that found in unaffected subjects.


View this table:
[in this window]
[in a new window]
  		Table 5. Characteristics of Previously Described ApoA-I Variants

HDL deficiency due to Tangier disease or apoA-I/apoC-III or apoA-I/apoC-III/apoA-IV deficiency syndromes has been associated with premature CHD.20 21 22 23 In addition, apoA-I gene mutations causing a truncated apoA-I protein (Gln84->stop) and an absence of apoA-I (Q[-2]X) are associated with xanthomas and premature CHD.74 89 The majority of apoA-I variants have not been associated with low HDL-C levels or premature CHD. Of the 23 reported apoA-I variants that we reviewed (Table 5Up), only seven are clearly associated with reduced plasma HDL-C and apoA-I concentrations, and the mean age of the 69 subjects was 33 years, a very low age to make accurate estimations of any association with CHD. Interestingly, in three apoA-I variants causing HDL deficiency the ability of variant apoA-I to activate LCAT and/or the plasma concentration of LCAT is markedly reduced.47 71 73 In contrast, apoA-I (Lys107->0) heterozygotes had HDL deficiency and normal LCAT activity. It should be noted that markedly premature CHD has not been observed in LCAT deficiency or fish-eye disease despite strikingly low levels of HDL-C.12 Therefore, normal LCAT activity may be necessary for low HDL-C to be a CHD risk factor.

In kindred studies it is hard to actually assess associations of mutations with CHD because of the limited number of subjects and the strong association of CHD with age and gender. Pedigree analysis indicated that the proband received the mutation from her father. Medical-record review revealed that the father and six of his siblings had CHD; in four of these siblings CHD was present before the age of 55 years. These six siblings died of CHD, while the father died of cerebral hemorrhage. The mean age of death in these seven subjects at 61 years is earlier than that observed in the normal population. The one remaining sibling of the father was an unaffected subject and healthy at age 84 years. Among the seven heterozygotes (mean age, 56 years) of the proband and her generation, two (T.M.A.) and (O.E.O.) had CHD at age 65 and 63 years, respectively, without any other risk factors except for a low HDL-C level. In addition, three other subjects of this generation, including T.M.A.'s sister and brother, had died of CHD at the ages of 46, 55, and 62 years. It was not possible to determine whether they were heterozygotes. None of the unaffected family members had symptoms or signs of CHD.

The combined data indicate that the apoA-IHelsinki mutation (Lys107->0) results in decreased serum HDL-C, apoA-I, apoA-II, and LpA-I:A-II levels, reduced CETP activity, normal lipolytic enzyme and LCAT activities, and may be associated with premature CHD.


*    Selected Abbreviations and Acronyms
 
CETP = cholesteryl ester transfer protein
CHD = coronary heart disease
HDL-C = HDL cholesterol
HUCH = Helsinki University Central Hospital
LCAT = lecithin:cholesterol acyltransferase
LpA-I = lipoprotein with apoA-I but no apoA-II
LpA-I:A-II = lipoprotein with both apoA-I and apoA-II
MI = myocardial infarction
PCR = polymerase chain reaction
SSCP = single-strand conformation polymorphism


*    Acknowledgments
 
This study was supported by grants from the Paavo Nurmi Foundation and the Sigrid Juselius Foundation Helsinki, Finland, the Finnish State Medical Research Council and the University of Helsinki. We are indebted to the proband and to all the family members for their interest and participation. Dr Jorma Viikinkoski is kindly thanked for referring the proband to our center. The authors are grateful to Drs Ernst J. Schaefer and J. Ordovas at the Lipid Metabolism Laboratory, US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Mass for their expert comments on the manuscript. We thank Leena Lehikoinen, Sirpa Rannikko, and Sirkka-Liisa Runeberg for their excellent technical assistance, and the competent personnel of the Metabolic Ward of the Third Department of Medicine, HUCH.

Received September 14, 1994; accepted June 20, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Gordon DJ, Probstfield JL, Garrison RJ, Neaton JD, Castelli WP, Knoke JD, Jacobs DR, Bangdiwala S, Tyroler HF. High-density lipoprotein cholesterol and cardiovascular disease: four prospective American studies. Circulation. 1989;79:5-15.
  2. Patsch JR, Gotto AM. Metabolism of high density lipoproteins. In: Gotto AM, ed. Plasma Lipoproteins. New York, NY: Elsevier Science Publishers BV; 1987:221-259.
  3. Eisenberg S. High density lipoprotein metabolism. J Lipid Res. 1984;25:1017-1058. [Medline] [Order article via Infotrieve]
  4. Avogaro P, Bittolo-Bon BG, Cazzolato G. Plasma levels of apolipoprotein A-I and apolipoprotein B in human atherosclerosis. Artery. 1978;4:385-394.
  5. Avogaro P, Bittolo-Bon G, Quici GB. Are apolipoproteins better discriminators than lipids for atherosclerosis? Lancet. 1979;1:901-903. [Medline] [Order article via Infotrieve]
  6. Maciejko JJ, Holmes DR, Kottke BA, Zinsmeister AB, Dink DM, Mao SJT. Apolipoprotein A-I as a marker of angiographically assessed coronary artery disease. N Engl J Med. 1983;309:385-389. [Abstract]
  7. Buring JE, O'Connor GT, Goldhaber SZ, Rosner B, Herbert PN, Blum CB, Breslow JL, Hennekens CH. Decreased HDL2 and HDL3 cholesterol, apoA-I, and apoA-II, and increased risk of myocardial infarction. Circulation. 1992;85:22-29. [Abstract/Free Full Text]
  8. Ishikawa T, Fidge N, Thelle DS, Førde OH, Miller NE. The Tromsø Heart Study: serum apolipoprotein AI concentration in relation to future coronary heart disease. Eur J Clin Invest. 1978;8:179-182. [Medline] [Order article via Infotrieve]
  9. Stampfer MJ, Sacks FM, Salvini S, Willett WC, Hennekens CH. A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. N Engl J Med. 1991;325:373-381. [Abstract]
  10. Lyu L-C, Shieh M-J, Ordovas JM, Lichtenstein AH, Wilson PWF, Schaefer EJ. Plasma lipoprotein and apolipoprotein levels in Taipei and Framingham. Arterioscler Thromb. 1993;13:1429-1440. [Abstract/Free Full Text]
  11. Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968;9:155-163. [Abstract]
  12. Norum KR, Gjone E, Glomset JA. Familial lecithin:cholesterol acyltransferase deficiency, including fish eye disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Inc; 1989:1181-1194.
  13. Cheung MC, Albers JJ. Distribution of high density lipoprotein particles with different apolipoprotein composition: particles with A-I and A-II and particles with A-I but no A-II. J Lipid Res. 1982;23:747-753. [Abstract]
  14. Atmeh RF, Shepherd J, Packard CJ. Subpopulations of apolipoprotein A-I in human high-density lipoproteins, their metabolic properties and response to drug therapy. Biochim Biophys Acta. 1983;75:175-188.
  15. Cheung MC, Albers JJ. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. J Biol Chem. 1984;259:12201-12209. [Abstract/Free Full Text]
  16. Puchois P, Kandoussi A, Fievet P, Fourrier JL, Bertrand M, Koren E, Fruchart JC. Apolipoprotein A-I containing lipoproteins in coronary artery disease. Atherosclerosis. 1987;68:35-40. [Medline] [Order article via Infotrieve]
  17. Parra HJ, Arveiler D, Evans AE, Cambou JP, Amouyel P, Bingham A, McMaster D, Schaffer P, Douste-Blazy Ph, Luc G, Richard JL, Ducimetière P, Fruchart JC, Cambien F. A case-control study of lipoprotein particles in two populations at contrasting risk for coronary heart disease: the ECTIM Study. Arterioscler Thromb. 1992;12:701-707. [Abstract/Free Full Text]
  18. Coste-Burel M, Mainard F, Chivot L, Auget JL, Madec Y. Study of lipoprotein particles LpAI and LpAIAII in patients before coronary bypass surgery. Clin Chem. 1990;36:1889-1891. [Abstract]
  19. Genest JJ, Bard JM, Fruchart JC, Ordovas JM, Wilson PWF, Schaefer EJ. Plasma apolipoprotein A-I, A-II, B, E and C-III containing particles in men with premature coronary artery disease. Atherosclerosis. 1991;90:149-157. [Medline] [Order article via Infotrieve]
  20. Schaefer EJ, Ordovas JM. Diagnosis and management of high density lipoprotein deficiency states. In: Miller NE, Tall AR, eds. High Density Lipoproteins and Atherosclerosis, III. New York, NY: Elsevier Science Publishers BV; 1992:235-251.
  21. Schaefer EJ. Clinical, biochemical, and genetic features in familial disorders of high-density lipoprotein deficiency. Arteriosclerosis. 1984;4:303-322. [Abstract/Free Full Text]
  22. Norum RA, Lakier JB, Goldstein S, Angel A, Goldberg RB, Block WD, Noffze DK, Dolphin PJ, Edelglass J, Bogorad DD, Alaupovic P. Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. N Engl J Med. 1982;306:1513-1519. [Abstract]
  23. Ordovas JM, Cassidy DK, Civeira F, Bisgaier CL, Schaefer EJ. Familial apolipoprotein A-I, C-III, and A-IV deficiency with marked high density lipoprotein deficiency and premature atherosclerosis due to a deletion of the apolipoprotein A-I, C-III, and A-IV gene complex. J Biol Chem. 1989;264:16339-16342. [Abstract/Free Full Text]
  24. Franceschini G, Sirtori CR, Capurso A, Weisgraber KH, Mahley RW. The AIMilano apoprotein: decreased high density lipoprotein cholesterol levels with significant lipoprotein modifications and without clinical atherosclerosis in an Italian family. J Clin Invest. 1980;66:892-900.
  25. Utermann G, Steinmetz A, Paetzold R, Wilk J, Feussner G, Kaffarnik H, Mueller-Eckhardt C, Seidel D, Vogelberg K-H, Zimmer F. Apolipoprotein AIMarburg: studies on two kindreds with a mutant of human apolipoprotein AI. Hum Genet. 1982;61:329-337. [Medline] [Order article via Infotrieve]
  26. Rader DJ, Gregg RE, Meng MS, Schaefer JR, Zech LA, Benson MD, Brewer HB Jr. In vivo metabolism of a mutant apolipoprotein, apoA-IIowa, associated with hypoalphalipoproteinemia and hereditary systemic amyloidosis. J Lipid Res. 1992;33:755-763. [Abstract]
  27. Higuchi R. Simple and rapid preparation of samples for PCR. In: Erlich HA, ed. PCR Technology: Principles and Applications for DNA Amplification. New York, NY: Stockton Press; 1989:31-39.
  28. Karathanasis SK, Zannis VI, Breslow LL. Isolation and characterization of the human apolipoprotein A-I gene. Proc Natl Acad Sci U S A. 1983;80:6147-6151. [Abstract/Free Full Text]
  29. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics. 1989;5:874-879. [Medline] [Order article via Infotrieve]
  30. Syvänen AC, Ikonen E, Manninen T, Bengström M, Söderlund H, Aula P, Peltonen L. Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland. Genomics. 1992;12:590-595. [Medline] [Order article via Infotrieve]
  31. Ehnholm C, Lukka M, Rostedt J, Harper K. Monoclonal antibodies specific for different regions of human apolipoprotein A-I: characterization of an antibody that does not bind to a genetic variant of apoA-I(Glu136 - Lys). J Lipid Res. 1986;27:1259-1264. [Abstract]
  32. Havel JR, Eder HA, Bragdon JH. Distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
  33. Räisänen-Sokolowski A, Tilly-Kiesi M, Ustinov J, Mennander A, Paavonen T, Tikkanen MJ, Häyry P. Hyperlipidemia accelerates allograft arteriosclerosis (chronic rejection) in the rat. Arterioscler Thromb. 1994;14:2032-2042. [Abstract/Free Full Text]
  34. Griffin BA, Caslake MJ, Yip B, Tait GW, Packard CJ, Shepherd J. Low density lipoprotein subfractions. Atherosclerosis. 1990;83:59-67. [Medline] [Order article via Infotrieve]
  35. Groot PHE, Scheek LM, Havekes L, van Noort WL, van't Hooft FM. A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor. J Lipid Res. 1982;23:1342-1353. [Abstract]
  36. Nichols AV, Krauss RM, Musliner TA. Nondenaturing polyacrylamide gradient gel electrophoresis. In: Segrest JP, Albers JJ, eds. Methods in Enzymology: Plasma Lipoproteins. London, England: Academic Press Inc; 1986;128:417-431.
  37. Tilly-Kiesi M, Syvänne M, Kuusi T, Lahdenperä S, Taskinen M-R. Abnormalities of low density lipoproteins in normolipidemic type II diabetic and nondiabetic patients with coronary artery disease. J Lipid Res. 1992;33:333-342. [Abstract]
  38. Morton RE, Zilversmit DB. A plasma inhibitor of triglyceride and cholesteryl ester transfer activities. J Biol Chem. 1981;256:11992-11995. [Abstract/Free Full Text]
  39. Groener JEM, Pelton RW, Kostner GM. Improved estimation of cholesteryl ester transfer/exchange activity in serum or plasma. Clin Chem. 1986;32:282-286.
  40. Barter PJ, Jones ME. Rate of exchange of esterified cholesterol between human plasma low and high density lipoproteins. Atherosclerosis. 1982;34:67-74.
  41. Jauhiainen M, Dophin PJ. Human plasma lecithin-cholesterol acyltransferase: an elucidation of the catalytic mechanism. J Biol Chem. 1986;261:7032-7043. [Abstract/Free Full Text]
  42. Parra HJ, Mezdour H, Ghalim N, Bard JM, Fruchart JC. Differential electroimmunoassay of human LpA-I lipoprotein particles on ready-to-use plates. Clin Chem. 1990;36:1431-1435. [Abstract/Free Full Text]
  43. Adult Treatment Panel II. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults. JAMA. 1993;269:3015-3023. [Medline] [Order article via Infotrieve]
  44. Kahri J, Groop P-H, Viberti GC, Elliot T, Taskinen M-R. Regulation of apolipoprotein A-I containing lipoproteins in insulin dependent diabetes mellitus. Diabetes. 1993;42:1281-1288. [Abstract]
  45. Franceschini G, Calabrisis L, Tosi C, Sirtori CR, Fragiacomo C, Noseda G, Gong E, Blanche P, Nichols AV. Apolipoprotein A-IMilano: correlation between high-density lipoprotein subclass distribution and triglyceridemia. Arteriosclerosis. 1987;7:426-435. [Abstract/Free Full Text]
  46. Weisgraber KH, Bersot TP, Mahley RW. A-IMilano apolipoprotein: isolation and characterization of a cysteine-containing variant of the A-I apolipoprotein from human high density lipoproteins. J Clin Invest. 1980;66:901-907.
  47. Funke H, von Eckardstein A, Pritchard PH, Karas M, Albers JJ, Assmann G. A frameshift mutation in the human apolipoprotein A-I gene causes high density lipoprotein deficiency, partial lecithin:cholesterol-acyltransferase deficiency, and corneal opacities. J Clin Invest. 1991;87:371-376.
  48. Taskinen M-R, Nikkilä EA. Lipoprotein lipase activity in adipose tissue and in postheparin plasma in human obesity. Acta Med Scand. 1977;202:399-408. [Medline] [Order article via Infotrieve]
  49. Eckel RH. Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases. N Engl J Med. 1989;320:1060-1068. [Abstract]
  50. Nikkilä EA. HDL in relation to the metabolism of triglyceride-rich lipoproteins. In: Miller NE, Miller GJ, eds. Clinical and Metabolic Aspects of High Density Lipoproteins. New York, NY: Elsevier Science Publishers BV; 1984:217-245.
  51. Barkia A, Puchois P, Ghalim N, Torpier G, Barbaras R, Ailhaud G, Fruchart JC. Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells. Atherosclerosis. 1991;87:135-146. [Medline] [Order article via Infotrieve]
  52. Montali A, Vega GL, Grundy SM. Concentrations of apolipoprotein A-I–containing particles in patients with hypoalphalipoproteinemia. Arterioscler Thromb. 1994;14:511-517. [Abstract/Free Full Text]
  53. Schaefer EJ, Zech LA, Jenkins LL, Aamodt RA, Bronzert TJ, Rubalcaba EA, Lindgren FT, Brewer HB Jr. Human apolipoprotein A-I and A-II metabolism. J Lipid Res. 1982;23:850-862. [Abstract]
  54. Brinton EA, Eisenberg S, Breslow JL. Elevated high density lipoprotein cholesterol levels correlate with decreased apolipoprotein A-I and A-II fractional catabolic rate in women. J Clin Invest. 1989;84:262-269.
  55. Brinton EA, Eisenberg S, Breslow JL. Increased apoA-I and apoA-II fractional catabolic rate in patients with low high density lipoprotein-cholesterol levels with or without hypertriglyceridemia. J Clin Invest. 1991;87:536-544.
  56. Horowitz BS, Goldberg IJ, Merab J, Vanni TM, Ramakrishnan R, Ginsberg HN. Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol. J Clin Invest. 1993;91:1743-1752.
  57. Roma P, Gregg RE, Meng MS, Ronan R, Zech LA, Franceschini G, Sirtori CR, Brewer HB Jr. In vivo metabolism of a mutant form of apolipoprotein A-I, apo A-IMilano, associated with familial hypoalphalipoproteinemia. J Clin Invest. 1990;91:1445-1452.
  58. Cheung MC, Nichols AV, Blance PJ, Gong EL, Franceschini G, Sirtori CR. Characterization of A-I-containing lipoproteins in subjects with A-I Milano variant. Biochim Biophys Acta. 1988;960:73-82. [Medline] [Order article via Infotrieve]
  59. Deckelbaum RJ, Eisenberg S, Oschry Y, Butbul E, Sharon I, Olivecrona T. Reversible modification of human low density lipoproteins toward triglyceride-rich precursors. J Biol Chem. 1982;257:6509-6517. [Free Full Text]
  60. Auwerx JH, Marzetta CA, Hokanson JE, Brunzell JD. Large buoyant LDL-like particles in hepatic lipase deficiency. Arteriosclerosis. 1988;9:319-325. [Abstract/Free Full Text]
  61. Connelly PW, Maguire GF, Lee M, Little JA. Plasma lipoproteins in familial hepatic lipase deficiency. Arteriosclerosis. 1990;10:40-48. [Abstract/Free Full Text]
  62. Levy E, Deckelbaum RJ, Thibault RL, Seidman E, Olivecrona T, Roy CC. In vitro remodelling of plasma lipoproteins in whole plasma by lipoprotein lipase in primary and secondary hypertriglyceridemia. Eur J Clin Invest. 1990;20:422-431. [Medline] [Order article via Infotrieve]
  63. Heinen RJ, Herbert PN, Frederickson DS, Forte T, Lindgren FT. Properties of the plasma very low and low density lipoproteins in Tangier disease. J Clin Invest. 1978;61:120-132.
  64. Rall SC, Weisgraber KH, Mahley RW, Ehnholm C, Schamaun O, Olaisen B, Blomhoff JP, Teisberg P. Identification of homozygosity for a human apolipoprotein A-I variant. J Lipid Res. 1986;27:436-441. [Abstract]
  65. Chait A, Eisenberg S, Steinmetz A, Albers JJ, Bierman EL. Low-density lipoproteins modified by lipid transfer protein have altered biological activity. Biochim Biophys Acta. 1984;795:314-325. [Medline] [Order article via Infotrieve]
  66. Gambert P, Bouzerand-Gambert C, Athias A, Farnier M, Lallemant C. Human low density lipoprotein subfractions separated by gradient gel electrophoresis: composition, distribution, and alterations induced by cholesteryl ester transfer protein. J Lipid Res. 1990;31:1199-1210. [Abstract]
  67. Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y, Mabuchi H, Tall AR. Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation. N Engl J Med. 1990;323:1234-1238. [Abstract]
  68. Cheung MC, Wolf AC, Lum KD, Tollefson JH, Albers JJ. Distribution and localization of lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity in A-I-containing lipoproteins. J Lipid Res. 1986;27:1135-1144. [Abstract]
  69. Cheung MC, Wang D, Lum KD, Albers JJ. Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma. Biochim Biophys Acta. 1988;962:258-264. [Medline] [Order article via Infotrieve]
  70. Utermann G, Haas J, Steinmetz A, Peatzold R, Rall SC, Weisgraber KH. Apolipoprotein A-IGiessen (Pro143-Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase. Eur J Biochem. 1984;144:325-331. [Medline] [Order article via Infotrieve]
  71. Franceschini G, Calabresi L, Sirtori CR, Cheung MC. Apolipoprotein A-IMilano: partial lecithin:cholesterol acyltransferase deficiency due to low levels of a functional enzyme. Biochim Biophys Acta. 1990;1043:1-6. [Medline] [Order article via Infotrieve]
  72. Rall SC Jr, Weisgraber KH, Mahley RW, Ogawa Y, Fielding CJ, Utermann G, Haas J, Steinmetz A, Menzel H-J, Assmann G. Abnormal lecithin:cholesterol acyltransferase activation by a human apolipoprotein A-I variant in which a single lysine residue is deleted. J Biol Chem. 1984;259:10063-10070. [Abstract/Free Full Text]
  73. Deeb SS, Cheung MC, Peng R, Wolf AC, Stern R, Albers JJ, Knopp RH. A mutation in the human apolipoprotein A-I gene: dominant effect on the level and characteristics of plasma high density lipoproteins. J Biol Chem. 1991;266:13651-13660.
  74. Matsunaga T, Hiasa Y, Yanagi H, Maeda T, Hattori N, Yamakawa K, Yamanouchi Y, Obara T, Hamaguchi H. Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene. Proc Natl Acad Sci U S A. 1991;88:2793-2797. [Abstract/Free Full Text]
  75. Gualandri V, Franceschini G, Sirtori CR, Gianfranceschi G, Orsini GB, Cerrone A, Menotti A. AIMilano apolipoprotein identification of the complete kindred and evidence of a dominant genetic transmission. Am J Hum Genet. 1985;37:1083-1097. [Medline] [Order article via Infotrieve]
  76. Franceschini G, Sirtori CR, Bosisio E, Gualandri V, Orsini GB, Mogavero AM, Capurso A. Relationship of the phenotypic expression of the A-IMilano apoprotein with plasma lipid and lipoprotein patterns. Atherosclerosis. 1985;58:159-174. [Medline] [Order article via Infotrieve]
  77. Nichols WC, Dwulet FE, Liepnieks J, Benson MD. Variant apolipoprotein AI as a major constituent of a human hereditar