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. 1997;17:83-90

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 Miettinen, H. E.
Right arrow Articles by Kontula, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miettinen, H. E.
Right arrow Articles by Kontula, K.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:83-90.)
© 1997 American Heart Association, Inc.

Articles

Apolipoprotein A-IFin

Dominantly Inherited Hypoalphalipoproteinemia Due to a Single Base Substitution in the Apolipoprotein A-I Gene

Helena E. Miettinen; Helena Gylling; Tatu A. Miettinen; Jorma Viikari; Lars Paulin; Kimmo Kontula

the Institute of Biotechnology (H.E.M., L.P., K.K.) and the Department of Medicine (H.E.M.H.G., T.A.M., K.K.), University of Helsinki, and the Department of Medicine, University of Turku (J.V.), Finland.

Correspondence to Helena E. Miettinen, MD, Department of Medicine, University of Helsinki, Haartmaninkatu 4, 00290 Helsinki, Finland. E-mail helena.miettinen@hyks.mailnet.fi.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-IFin). The T-to-G substitution destroys an Fsp I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-IFin mutation. Isoelectric focusing revealed the presence of the mutant apo A-IFin protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-IFin, causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.


Key Words: high-density lipoprotein • catabolism • mutation • reverse cholesterol transport • gene expression


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Epidemiological studies have shown that low serum HDL-C and apo A-I concentrations are strongly associated with increased risk for CAD.1 The exact pathophysiology of this association, however, is still a matter of controversy. The concept of reverse cholesterol transport2 is the widely accepted model for explaining the inverse correlation between HDL concentration and CAD risk. Apo A-I is the major protein component of HDL and is considered to play an important role in reverse cholesterol transport. Accordingly, apo A-I is the most potent activator of LCAT3 and has a capacity to promote cholesterol efflux from peripheral cells.4 5 Genetic factors influencing structural or functional properties of apo A-I and HDL may impair the process of reverse cholesterol transport and thus modify risk of atherosclerosis. There are, however, many genetic disorders, including apo A-I and LCAT deficiencies, in which serum HDL-C and apo A-I levels are severely diminished or even undetectable, yet they only seldom seem to be associated with premature CAD (for review see References 6 and 7).

The gene coding for apo A-I is located in the vicinity of the apo C-III and A-IV genes in chromosome 11.8 The apo A-I gene encodes a 267–amino acid preproapo A-I, which is cotranslationally modified to a 251–amino acid proapo A-I. The cleavage of the prosegment produces the mature apo A-I protein, consisting of 243 amino acids. Several mutations causing HDL deficiency have been identified in the apo A-I gene.9 10 11 Typically affected subjects are homozygous for the mutation, and heterozygous family members have about half of normal or normal HDL-C and apo A-I concentrations; thus, the phenotype is inherited in a codominant fashion. In addition, population screening studies have revealed several natural variants of apo A-I that do not associate with serum HDL-C concentrations.

There are three kindreds,12 13 14 however, in which heterozygosity for an apo A-I mutation was found to result in a phenotype with greatly altered serum HDL-C level. One of these mutations, apo A-IMilano, has been characterized extensively.14 15 Carriers of the apo A-IMilano are heterozygous for Arg173->Cys substitution16 and have reduced serum HDL-C and apo A-I levels (33% and 60% of normal, respectively) and slight hypertriglyceridemia.17 Cysteine residue, normally not present in apo A-I, provides the mutant protein with capability to form dimers. Despite their reduced serum HDL-C levels, subjects heterozygous for apo A-IMilano do not seem to be at risk for premature CAD. It has even been proposed that the mutant A-IMilano protein could exert a protective effect against development of CAD.18

In this report we describe a kindred with severely diminished serum HDL-C and apo A-I levels yet without an apparently increased liability to develop CAD. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Our study was undertaken to examine the genetic basis of the dominantly inherited hypoalphalipoproteinemia in the kindred and the impact of the underlying DNA alterations in the affected family members on lipoprotein metabolism.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Patients
The study group consisted of a kindred with severe hypoalphalipoproteinemia (Fig 1Down). The proband, a 65-year-old woman, was a Finnish citizen living in Turku, which is located in southwestern Finland, but both of her parents were probably of Russian origin. In a routine medical checkup, the proband was found to have extremely low serum HDL-C and apo A-I concentrations, while the apo A-II concentration was about half of that of the nonaffected family members. HDL-C, apo A-I, and apo A-II levels were diminished to approximately the same extent in all affected family members. This study was approved by the Ethical Review Committee of the Department of Medicine, University of Helsinki.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Diagram of the pedigree under study. Question marks indicate subjects not available for studies.

Measurement of Serum Lipids and Lipoproteins and Assay of Cholesterol Esterification
Serum total cholesterol, triglycerides, apo A-I, and apo A-II were analyzed with commercial kits (Boehringer Diagnostica and Orion Diagnostica). Serum lipoproteins were separated by ultracentrifugation into density classes.19 The percentages of cholesterol esters in serum and HDL fraction were determined by measuring their free cholesterol content separately (commercial kit from Boehringer). In vitro cholesterol esterification from serum was measured by incubating 1.5 mL serum with [3H]cholesterol for 24 hours at 37°C followed by separation of 3H-labeled cholesterol esters by thin-layer chromatography.20

Southern Blot Analysis
DNA was isolated from 20 mL of venous blood.21 DNA (10 µg) was digested with the restriction enzymes Sac I, Msp I, Xmn I, and Pst I (New England Biolabs), fractionated by gel electrophoresis on a 0.6% agarose gel, and transferred to nylon filters (Hybond-N, Amersham International) by standard techniques.22 The filters were hybridized with a 32P-labeled 2.2-kb human genomic apo A-I probe (a gift from Dr Jan L. Breslow, The Rockefeller University, New York).

DNA Amplification and Sequencing
Genomic DNA (150 ng) was amplified by PCR in a 50-µL mixture containing 50 pmol of each primer (Table 1Down), 0.2 mmol/L each of dATP, dCTP, dGTP, and dTTP, and 0.6 IU of Dynazyme DNA polymerase (Finnzymes) in the reaction buffer supplied by the manufacturer. Thirty cycles consisting of denaturation at 95°C, annealing at 53°C to 59°C, and extension at 72°C for 1 minute each were run in a programmable thermal cycler. The nucleotide numbering of the apo A-I gene follows that presented by Shoulders et al.23


View this table:
[in this window]
[in a new window]
  		Table 1. Sequences and Positions of the Primers Used in PCR, DNA Sequencing, and Mutagenesis

PCR-amplified DNA fragments were directly sequenced with a commercial sequencing kit (Sequenase 2.0, United States Biochemical Corp) by a solid-phase sequencing method24 using streptavidin-coated magnetic beads (Dynabeads M 280 streptavidin, DYNAL AS) to generate single-stranded DNA. Strong DNA secondary structures in the G-C–rich area of exon 4 and compressions in the sequencing gel were resolved by using 7-deaza-dGTP nucleotides (United States Biochemical Corp) in sequencing reactions and by running the reactions on an 8% polyacrylamide gel containing 30% formamide.

Assay of the Apo A-IFin Mutation by PCR and Restriction Enzyme Analysis
Genotyping of the kindred was done by PCR combined with restriction analysis. PCR was performed using primers F and K for 30 cycles at 95°C, 58°C, and 72°C for 1 minute each. The PCR product, 487 bp in size, was digested with the restriction enzyme Fsp I (New England Biolabs) and run on a 2.5% agarose gel. The T-to-G base substitution destroys an existing Fsp I cutting site, resulting in an amplification product 487 bp in size, while the normal allele produces two fragments (294 and 193 bp) on digestion with Fsp I.

In Vitro Mutagenesis of Apo A-I cDNA
A full-length human apo A-I cDNA (a gift from Dr S. Metzger, The Rockefeller University, New York) in vector pUC was subcloned into BamHI–Sma I site in vector pGEM4Z. The T-to-G transversion at position 2024 of the apo A-I gene was introduced to apo A-I pGEM4Z using the principle of an overlap extension in site-directed mutagenesis.25 Two overlapping PCR fragments were amplified, in separate reactions, with the primer E and the reverse mutagenic primer L and with the mutagenic primer M and a reverse primer K. PCR was performed for 30 cycles at 95°C, 55°C, and 72°C for 1 minute each under the conditions described above. These two amplification reactions yielded two mutated PCR fragments (387 bp and 308 bp in size, respectively) containing an overlap stretch of 20 bp. The fragments were purified on a 2% agarose gel using a commercial kit (Qiagen gel extraction kit, Qiagen). In the final overlap PCR reaction, 10 µL each of the two purified PCR products was used as templates, together with the primers E and K. The PCR mixture was first denatured at 100°C for 5 minutes and then amplified for 40 cycles at 95°C, 58°C, and 72°C for 1 minute each. PCR products were run on a 2.5% agarose gel, and the extension fragment (695 bp) was purified (Qiagen gel extraction kit, Qiagen). The mutated extension fragment was digested with the restriction enzymes Bsu36I and Dra III (New England Biolabs) and ligated to apo A-I pGEM4Z after its digestion with Bsu36I and Dra III. DNA sequences of the mutated apo A-I pGEM4Z constructs were confirmed by dideoxynucleotide sequencing.

Transient Transfection of COS Cells
Wild-type and mutated apo A-I pGEM4Z constructs were digested with the restriction enzymes Sma I and BamHI, and the apo A-I cDNAs released were subcloned to an expression vector pSVL SV40 (Pharmacia LKB Biotechnology Inc) carrying the SV40 late promoter. Transfection of the COS-7 (monkey kidney) cells was performed as described previously.26 In brief, subconfluent COS-7 cells were transfected with the wild-type and the mutated apo A-I cDNAs in pSVL SV40 and with the transfection vector pSVL SV40 alone using the liposome (lipofectamine reagent, GIBCO-BRL) transfection method. All transfections were performed in triplicate under serum-free conditions (OPTI-MEM, GIBCO-BRL). Transfections were carried out using 5 µg DNA and following the manufacturer's transfection protocol. Supernatants were harvested after 72 hours. Cells were collected by centrifugation, resuspended in 0.25 mol/L Tris, pH 7.5, and lysed by repeated (three times) freezing in carbon ice/ethanol and melting in a 37°C water bath. Intracellular extracts were collected after centrifugation and stored at 4°C until DNA slot-blot hybridization analysis was performed.

Transfection efficacy was controlled by slot blotting intracellular DNA from each cell culture dish, along with a control standard DNA, to nylon filters.27 After hybridization of the filters with a digoxigenin-labeled pSVL SV40, the amount of plasmid DNA present in the cell extract was estimated by reading the intensities of the slots.

Immunochemical Studies
To examine the properties of the mutant apo A-I expressed in vitro, COS cells 48 hours posttransfection were starved for 1 hour in a medium without methionine (MEM-methionine). [35S]Methionine (Amersham) was then added to the cell medium (100 µCi/mL), and cells were grown for an additional 5 hours. After collection of the supernatants, protease inhibitors (1 mmol/L PMSF, 5 µg/mL aprotinin, and 5 µg/mL leupeptin) were added. Aliquots (1 mL) of the labeled supernatants were immunoprecipitated by using a polyclonal swine apo A-I antibody (Orion Corp) and protein A–Sepharose (Pharmacia). Immunoprecipitated proteins were run on SDS/12% acrylamide gels under both reducing and nonreducing conditions.

To compare the physicochemical properties of the circulating normal and mutant apo A-I molecules, serum and HDL samples (2 µL) were electrophoresed under reducing and nonreducing conditions on SDS/12% acrylamide gel and transferred onto nitrocellulose filter (Hybond-C, Amersham) using standard techniques. Immunoblotting was performed with a polyclonal swine apo A-I primary antibody (Orion Corp) and an alkaline phosphatase–conjugated anti-swine IgG secondary antibody. Proteins were visualized by using colorimetric detection (DIG DNA labeling and detection kit, Boehringer Mannheim GmbH) according to the protocols provided by the manufacturer.

Isoelectric focusing was performed using serum, HDL, and HDL infranatant (ultracentrifuge d>1.21 g/L) samples (2 µL). Samples were delipidated and dissolved in a buffer (30 µL) containing 0.01 mol/L Tris HCl (pH 8.2), 1% decylsulfate, 1% ß-mercaptoethanol, and 2.5% carrier ampholytes.28 The separating gradient covered the range of pH 4 to 6.5. Immunoblotting was carried out as described above.

Northern Blot Analysis
Poly(A)-containing RNA was isolated 72 hours posttransfection using Dynabeads oligo dT magnetic beads (DYNAL) according to the instructions provided by the manufacturer. RNA samples (1 µg) were electrophoresed in a formaldehyde/agarose gel mixture and transferred to a nylon filter (Hybond-N, Amersham) by standard techniques. A full-length apo A-I cDNA probe was labeled with digoxigenin using a commercial kit (DIG DNA labeling kit, Boehringer Mannheim). Filters were prehybridized in 0.25 mol/L Na2HPO4, 1 mmol/L EDTA, 20% SDS, and 0.5% blocking reagent (Boehringer Mannheim) at 68°C for 60 minutes and hybridized with digoxigenin-labeled apo A-I cDNA probe at 68°C for 12 hours. Filters were washed at 68°C with 0.1x SSC/1% SDS three times for 20 minutes each followed by detection of the hybridized DNA using a commercial technique (DIG luminescent detection kit for nucleic acids, Boehringer Mannheim).

Apo A-I Kinetics In Vivo
Fasted EDTA plasma (50 mL) was used for separation of autologous HDL by serial preparative ultracentrifugations. Autologous apo A-I was isolated from HDL with guanidine hydrochloride incubation29 and iodinated with 125I by a modification of the iodine-monochloride method.30 31 About 99% of the radioactivity of apo A-I was precipitable by 15% trichloroacetic acid.29

Potassium iodide was given perorally daily to each subject, starting 3 days before injection and extending up to 14 days postinjection. The total amount of radioactivity per injection did not exceed 20 µCi. After the injection, blood samples were collected and counted for 14 days. The die-away curves for the tracer were constructed, and the FCR was determined from these data using a two-pool model.32 The TR was determined by multiplying the FCR by the respective pool size of the tracer. Plasma volume was calculated as 4.5% of body weight. To examine whether the normal and mutant tracer apo A-I species reassociate equally with HDL in vivo, we counted the radioactivities of the HDL and HDL infranatant (d>1.21 g/L) fractions from three postinjection samples of both an affected and nonaffected subject. In both subjects, 95% of radioactivity was recovered from the HDL and 5% from the infranatant fractions.

Statistical Methods
Mean levels of serum lipids and lipoproteins were compared using Student's t test. Before comparison, serum triglyceride levels were transformed logarithmically.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Serum Lipids and Clinical Data
Serum lipid and lipoprotein analysis indicated that seven members of the family had greatly reduced serum HDL-C concentrations, in the range of 0.15 to 0.42 mmol/L (Table 2Down). Pedigree analysis suggested an autosomal-dominant inheritance of this lipoprotein abnormality (Fig 1Up). Affected family members had serum HDL-C concentrations about 20% and apo A-I concentrations about 25% of those of the nonaffected members. Serum apo A-II concentration was reduced to about 50% of normal, and serum triglyceride concentrations were elevated (3.58±0.75 versus 1.94±0.91 mmol/L, affected versus nonaffected). Esterification percentages of serum total and HDL cholesterol were lower in affected family members than in nonaffected ones (Table 2Down). Cholesterol esterification in vitro, as measured by percentage of 3H-labeled cholesterol esterified during 24 hours, was also lower in affected family members than in nonaffected ones (26.6±1.74%, n=5, versus 39.4±1.8%, n=6, respectively).


View this table:
[in this window]
[in a new window]
  		Table 2. Lipids and Genotypes of the Kindred

Clinical examination did not reveal corneal clouding, lipid arcus, or skin or tendon xanthomas, nor was there any clinical evidence of CAD (eg, exercise-induced angina, past history of myocardial infarction, electrocardiographic abnormalities suggestive of ischemia, or a history of coronary bypass operation) in any of the family members.

Immunoblotting and Isoelectric Focusing
Immunoblotting of serum and HDL of affected and nonaffected subjects gave a single protein band of {approx}28 kD in molecular size (data not shown). Isoelectric focusing of serum and HDL of the affected subjects revealed the presence of mutant proapo and apo A-I proteins with 1+ charge difference compared with the normal proapo and apo A-I proteins (Fig 2Down). For reasons unknown, a weaker and less well-demarcated band was also present at position +1 in the control lanes. The normal apo A-I focusing pattern was also present in the serum and HDL of the affected subjects (Fig 2Down). We investigated the possible dissociation of the mutant proteins from HDL by carrying out isoelectric focusing analysis of the apo A-I species in ultracentrifuge d>1.21 g/L. This infranatant fraction of the affected family members consisted of the mutant proapo and apo A-I proteins in addition to the normal apo A-I protein (Fig 2Down). The intensities of the mutant and normal apo A-I bands were much weaker than those of serum and HDL but apparently similar to those of the HDL infranatant of a normal subject.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. Isoelectric focusing of apo A-I present in serum (left), HDL (middle), and HDL infranatant fractions (ultracentrifuge d>1.21 g/L; right). The major isoform of apo A-I is designated as 0, while proapo A-I migrates at the position +2. Proapo and apo A-IFin proteins migrate at the positions +3 and +1, respectively. The upper arrow on the right indicates the proapo A-IFin and the lower arrow apo A-IFin proteins. P1 and P2 indicate affected family members 9 and 11, respectively (cf Fig 1Up), and N1 and N2, normal subjects.

Identification of the Apo A-I Gene Mutation
Southern blot analyses did not show any major rearrangements in the apo A-I gene of the proband (data not shown). All four exons and all exon-intron boundaries of her apo A-I gene were then sequenced. Comparison of the sequencing data to the wild-type apo A-I gene sequence23 revealed heterozygosity for a single T-to-G transversion in exon 4 at position 2024 substituting arginine for leucine at position 159 of the mature apo A-I protein (Fig 3Down). The T-to-G mutation, here designated as apo A-IFin, produces a stretch of 10 nucleotides containing C and G only that was found to form strong DNA secondary structures requiring the use of 7-deaza-dGTP in sequencing reactions in addition to the use of formamide in sequencing gel to prevent problems related to compressions in the sequencing gel.



View larger version (59K):
[in this window]
[in a new window]
  		Figure 3. DNA sequence analysis of exon 4 of the proband and a control subject. The T-to-G substitution at position 2024 is indicated by an arrow.

Sequencing data were confirmed by restriction typing (Fig 4Down). The T-to-G mutation at position 2024 abolishes an Fsp I cutting site in the mutant allele. Digestion of the 487-bp PCR products of exon 4 of the proband with Fsp I thus produces an undigested 487-bp fragment in addition to two fragments 294 bp and 193 bp in size, derived from the normal allele, demonstrating that the patient is heterozygous for apo A-IFin mutation. Fsp I restriction analysis was subsequently used for genotyping of the whole kindred. All seven affected family members (HDL-C<0.5 mmol/L) were shown to be heterozygous for the apo A-IFin mutation, whereas none of the eight nonaffected family members were carriers of this mutation.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. PCR assay for the apo A-IFin mutation. Primers F and K (Table 1Up) were used to amplify a 487-bp DNA fragment, which was digested with the restriction enzyme Fsp I and size-fractionated on a 2% agarose gel. The A-IFin mutation destroys an existing Fsp I cutting site, resulting in a fragment of 487 bp instead of the normal cleavage products of 294 and 193 bp in size. The sizes (in bp) of the fragments are shown on the left and the fragment carrying the A-IFin mutation is indicated on the right. Lane 1: DNA 100-bp ladder; lanes 2, 4, 9, and 10: control subjects; and lanes 3 and 5 through 8: affected subjects with the apo A-IFin mutation.

Expression of the Mutant Apo A-I cDNA in COS Cells
Wild-type and apo A-IFin–mutated apo A-I cDNAs were expressed in COS cells using the expression vector pSVL SV40. Northern blot hybridization analysis of RNA extracted from the COS cells, transfected with either the wild-type or mutant apo A-I cDNA, showed roughly similar levels of mature apo A-I mRNA, whereas no detectable apo A-I mRNA was present in the cells transfected with the expression vector pSVL SV40 alone (Fig 5Down). After labeling with [35S]methionine and immunoprecipitation with an apo A-I antibody, protein products encoded by the two different cDNAs were analyzed by electrophoresis. COS cells transfected with the mutant or wild-type, or with both the wild-type and mutant, apo A-I cDNAs secreted an immunoprecipitable 28-kD protein, compatible with the size of the mature apo A-I, into the cell media (Fig 6Down).



View larger version (94K):
[in this window]
[in a new window]
  		Figure 5. Northern blot hybridization analysis of poly(A)-containing RNA from the transfected COS cells. Lane 1: RNA of COS cells transfected with the expression vector pSVL SV40 alone; lane 2: RNA of the COS cells transfected with the wild-type apo A-I cDNA in pSVL SV40; and lane 3: RNA of the COS cells transfected with the A-IFinFin mutated apo A-I cDNA in pSVL SV40. Equal amounts of sample (1 µg RNA) were loaded in each lane. Positions of 28S and 18S rRNA are indicated on the left.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 6. Immunoprecipitation of apo A-I from the media of transfected COS cells. COS cells were transfected with wild-type or A-IFin–mutated apo A-I cDNA and the resulting protein products labeled with [35S]methionine, immunoprecipitated with apo A-I antiserum, and analyzed by electrophoresis. Lane 1: immunoprecipitation of the media of COS cells transfected with the expression vector pSVL SV40 alone; lanes 2, 3, and 8: immunoprecipitation of the media of COS cells transfected with the wild-type apo A-I cDNA; lanes 4 and 5: immunoprecipitation of the media of COS cells transfected with the A-IFin–mutated apo A-I cDNA; lanes 6 and 7: immunoprecipitation of the media of COS cells transfected with both the wild-type and A-IFin–mutated apo A-I cDNAs; and lane 9: molecular weight marker. The sizes (kD) of the protein standards are indicated on the right.

In Vivo Studies of Apo A-I Kinetics
Results from the in vivo studies of the apo A-I kinetics in affected subjects 9 and 11 and the nonaffected subject 8, as well as a group of nonrelated healthy control subjects, are summarized in Table 3Down and Fig 7Down. The affected family members had higher apo A-I FCRs in comparison with the FCR of the nonaffected family member and the upper value of the 95% CI of the control subjects (Table 3Down). The TR of apo A-I was within the normal range in the affected subject 11 and subnormal (below the 95% CI of the control subjects) in the affected subject 9. Activities of the HDL infranatant (d>1.21) counted from three postinjection samples were similar in both the nonaffected and affected subjects (5% versus 7% of the total activity of HDL).


View this table:
[in this window]
[in a new window]
  		Table 3. Apo A-I Kinetics in 2 Affected and 1 Nonaffected Family Members and in 13 Control Subjects



View larger version (12K):
[in this window]
[in a new window]
  		Figure 7. Decay curves of the apo A-I kinetic studies. {blacktriangleup}, Control subjects (mean±95% CI, n=13); {bullet}, affected subject 9; {circ}, affected subject 11.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We have identified a novel point mutation of the apo A-I gene, designated as apo A-IFin, causing dominantly inherited hypoalphalipoproteinemia in 7 individuals of a 15-member kindred with severely diminished serum HDL-C and apo A-I concentrations. The mutation, a single T-to-G substitution in exon 4 at position 2024, substitutes arginine for leucine at residue 159 of the mature apo A-I protein and has a profound effect on serum HDL-C and apo A-I levels. On genotyping of the family members by a PCR-based restriction typing analysis, all affected subjects were found to be heterozygous for the apo A-IFin mutation, while the nonaffected members had the wild-type sequence.

Isoelectric focusing of serum and HDL samples of affected subjects revealed that the mutant protein is expressed and secreted in vivo, together with the normal apo A-I, and is present in both serum and HDL of mutation carriers (Fig 2Up). Immunoblotting studies of samples of the affected subjects showed that the intensities of the mutant and normal apo A-I bands in their HDL infranatant were much weaker than those of their serum and HDL but apparently similar to those of the HDL infranatant of a normal subject. These data suggest that most mutant apo A-I protein is present in HDL itself and not abnormally dissociated from HDL to serum fractions of higher density.

Previous reports of a number of apo A-I gene mutations causing hypoalphalipoproteinemia9 10 11 have shown that the respective phenotype is usually inherited in a codominant fashion. Accordingly, homozygous subjects have reportedly low or undetectable serum HDL-C and apo A-I levels, whereas heterozygous carriers have about half of normal or normal HDL-C concentrations. In our kindred, all affected subjects were genotyped as heterozygotes for the apo A-IFin mutation, thus having one normally functioning apo A-I allele and one mutant allele producing apo A-IFin protein, as was demonstrated by isoelectric focusing of serum and HDL. It is therefore intriguing that the affected subjects were found to have serum HDL-C concentration only 20% and apo A-I concentration only 25% of those of their nonaffected relatives.

The mechanism for such a negative dominant effect is obscure. The mutant protein could somehow interact with its normal counterpart, eg, suppressing its secretion or enhancing its catabolism. The expression and secretion of the mutant and normal apo A-I proteins and their possible interactions were examined in detail by expressing the normal and mutated apo A-I cDNAs, separately or together, in COS cells. Northern blot analysis of the transfected COS cells showed comparable levels of apo A-I mRNA, indicating that mutation does not affect transcription or stability of mRNA to a significant extent. Immunoprecipitation of the culture media of the same COS cells also showed that the mutant and wild-type apo A-I cDNAs directed the synthesis of comparable amounts of a similar-sized 28-kD apo A-I protein. Transfection of the COS cells with the normal and mutant apo A-I cDNAs together suggested that the mutant apo A-I does not interfere with the secretion of the normal protein (Fig 6Up).

Substitution of arginine for leucine leads to the placement of two consecutive arginine residues at positions 159 and 160 of the mature apo A-I protein, which could make the mutant protein more susceptible to proteolytic cleavage.33 It is of interest that two different mutations in the apo A-I gene creating two consecutive arginines in the mature apo A-I protein have been described previously, both in association with amyloidosis. In apo A-I Iowa (Gly26->Arg)13 and in apo A-I Leu60->Arg,34 the N-terminal proportions of apo A-I act as amyloid proteins being deposited into several organs. In both of the previous cases, however, nothing favored the idea that the Arg-Arg sequence had a role in proteolysis of the apo A-I protein or in the actual process of amyloidogenesis. The affected family members in our kindred had no clinical findings suggestive of amyloidosis. Expression studies in COS cells did not show any abnormal proteolytic cleavage products of the mutant protein nor did immunoblotting of serum and HDL reveal any subfragments of the apo A-I protein.

Two affected subjects and one nonaffected member of the family volunteered for in vivo kinetic studies of apo A-I based on the use of autologous 125I-labeled apo A-I. In both the affected and nonaffected subjects, 95% of the tracer was recovered in the HDL and only 5% in HDL infranatant (d>1.21 g/L) fractions of the postinjection samples, suggesting that mutant apo A-I is reassociated with circulating HDL particles to approximately the same extent as normal apo A-I, thus permitting the use of the two-pool method in kinetic analysis. FCRs of the affected subjects were higher than those of the nonaffected subject and control subjects, indicating that increased catabolism of apo A-I protein could provide an explanation for profoundly diminished HDL-C and apo A-I concentrations. A low TR in one of the affected subjects may have contributed to the low values. Hypercatabolism of apo A-I has been previously observed in a number of conditions, including apo A-I Milano35 and apo A-IIowa mutations,36 Tangier disease,37 apo A-I deficiency without a detectable apo A-I gene mutation,38 and LCAT deficiency.39 However, otherwise normal subjects with low HDL-C, with or without hypertriglyceridemia, have also been shown to have increased apo A-I FCR.40 41 In hypertriglyceridemia, CETP-mediated exchange of HDL cholesterol esters to VLDL triglycerides may accelerate, thus making HDL particles triglyceride rich and susceptible to lipolysis. Lipolysis of HDL core triglycerides could loosen the binding of apo A-I to HDL particles and thus facilitate apo A-I clearance by the kidneys.42 In hypertriglyceridemic transgenic mice overexpressing the apo C-III gene, serum HDL level was modestly decreased.43 However, in mice overexpressing both the apo C-III and CETP genes, HDL concentration was diminished by 50% and in mice overexpressing each of the apo C-III, CETP, and apo A-I genes, HDL level was reduced by 60%.44 Affected subjects in our kindred presented with moderate hypertriglyceridemia, which might have slight impact on apo A-I FCR and serum HDL-C concentrations.

The exact conformation of apo A-I is not known at present but, on the basis of computer simulation, apo A-I protein has been postulated to consist of several homologous amphipathic alpha helices of 22 amino acids and two helices of 11 amino acids,45 46 each terminated by a ß-turn. These helices are thought to mediate two functions, LCAT activation and lipid binding, of apo A-I.47 While studies using site-directed mutagenesis have indicated that several apo A-I domains are important for LCAT activation, it is especially the deletion of domain 143-164 or 165-186 that almost abolishes LCAT activation by apo A-I.48 49 Apo A-ISeattle, a de novo mutation deleting amino acids from Glu146 to Arg160, diminished HDL-C apo A-I concentrations to levels below 15% of normal when present in a heterozygous form.12 However, point mutations at residues 143 (Pro->Arg)50 and 165 (Pro->Arg)51 52 have been shown to affect serum lipoprotein levels and LCAT cofactor activity only slightly. In contrast, Apo A-IFin, a single point mutation substituting arginine for leucine at residue 159, has a profound effect on apo A-I concentration, thus further underscoring the importance of this domain of the apo A-I protein. The observation that cholesterol esterification percentages in serum and HDL were lower in affected subjects than nonaffected ones favors the idea that LCAT is not fully activated. Further support for this assumption comes from studies showing that the in vitro cholesterol esterification percentages within 24 hours were lower in affected subjects than in nonaffected ones. The clarification of the impact of the apo A-IFin on activation of LCAT requires comparative experiments with artificial liposomes by using biosynthetic normal apo A-I and apo A-IFin protein preparations as cofactors of LCAT.

Approximately 30 mutations of the apo A-I gene, many of which do not affect lipoprotein levels, have been detected so far.9 10 11 34 Mutations, most commonly the C->T transition, are nonrandomly distributed throughout the A-I gene53 : substitutions are overrepresented in the 10 N-terminal amino acids and in residues 103-177. Four different mutations have been detected in a hot spot for mutations present at codons 3 through 5 consisting of a run of seven consecutive C's.51 54 55 56 CpG dinucleotides dominate the structure in codons 120 through 208 and may explain the hypervariability of this region. Apo A-IFin is situated in a CpG-rich stretch of the apo A-I gene but unexpectedly changes thymidine to cytosine, thus creating a 10-nucleotide run containing C and G only. This stretch may even alter the secondary structure of DNA, which was manifested as technical difficulties in DNA sequencing. Thus, a partial stop of the sequenase enzyme was encountered at the site of the mutation, with continuation of the reaction some nucleotides further but with lower intensity, a phenomenon described previously with DNA sequences possessing unexceptionally strong secondary structures.57

Association between apo A-I deficiency and premature CAD is still a matter of debate, and further investigation of the exact role of apo A-I in reverse cholesterol transport is needed. Evaluation of the risk between apo A-I deficiency states and CAD is hampered by the fact that apo A-I defects are genetically heterogeneous and very rare in the population. An unequivocal propensity for CAD has been demonstrated in specific conditions, such as homozygosity for the apo A-I Gln 84->Stop or Q(-2)X mutations9 58 or major rearrangements of the apo A-I/C-III/A-IV gene complex.59 60 In most kindreds with hypoalphalipoproteinemia due to either apo A-I or LCAT deficiency, no clear association of the genetic defect to CAD has been observed. It is possible that even trace amounts of normally functioning apo A-I protein could be sufficient for the process of reverse cholesterol transport or that factors other than apo A-I, such as apo E–containing plasma lipoprotein {gamma}-LpE,61 could also trigger cholesterol efflux from cells. These assumptions are further supported by experiments in transgenic mice: mice lacking the apo A-I gene do not seem to be at increased risk for CAD,62 but mice overexpressing A-I are protected from atherosclerosis.63 It has even been postulated that a mutant apo A-I protein, such as apo A-IMilano, could act as an antiatherogenic agent and exert protection against premature CAD.18 Carriers of the apo A-IFin mutation did not present any clinical signs of CAD despite their very low serum HDL-C levels. The CAD risk cannot, however, be ruled out owing to the relatively small sample size and young age of four affected family members. Thus, many important questions, such as the prevalence of the apo A-IFin mutation in different populations and the exact functional role of the apo A-IFin protein in reverse cholesterol transport and its possible influence on development of CAD, remain to be answered.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CAD = coronary artery disease
CETP = cholesterol ester transfer protein
FCR = fractional catabolic rate
HDL-C = HDL cholesterol
LCAT = lecithin:cholesterol acyltransferase
PCR = polymerase chain reaction
TR = transport rate
CI = confidence interval


*    Acknowledgments
 
This study was supported by grants from the Medical Council of the Finnish Academy, The Sigrid Juselius Foundation, Orion Research Foundation, Finnish Medical Society Duodecim, and the University of Helsinki. The 2.2-kb human genomic apo A-I probe was a gift from Dr Jan L. Breslow, and full-length human apo A-I cDNA was a gift from Dr S. Metzger, both of The Rockefeller University, New York. Kaija Kettunen, Eeva Gustafsson, Leena Kaipiainen, and Leena Saikko provided excellent technical assistance.

Received November 28, 1995; revision received May 22, 1996;
*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical implications of recent studies. N Engl J Med. 1989;321:1311-1316.[Medline] [Order article via Infotrieve]
  2. Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968;9:155-167.[Abstract]
  3. Fielding CJ, Shore VG, Fielding PE. A protein cofactor of lecithin:cholesterol acyltransferase. Biochem Biophys Res Commun. 1972;46:1493-1498.[Medline] [Order article via Infotrieve]
  4. Bielicki JK, Johnson WJ, Weinberg RB, Blick JM, Rothblat GH. Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol. J Lipid Res. 1992;33:1699-1709.[Abstract]
  5. Hara H, Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein–like lipoproteins and reduction of cellular cholesterol. J Biol Chem. 1991;266:3080-3086.[Abstract/Free Full Text]
  6. Breslow JL. Familial disorders of high density lipoprotein metabolism. In: Scriver DR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Book Co; 1989:1251-1266.
  7. Schaefer EJ. Clinical, biochemical, and genetic features in familial disorders of high density lipoprotein deficiency. Arteriosclerosis. 1984;4:303-322.[Abstract/Free Full Text]
  8. Karathanasis SK. Apolipoprotein multigene family: tandem organization of apolipoprotein A-I, C-III and A-IV genes. Proc Natl Acad Sci U S A. 1985;82:6374-6378.[Abstract/Free Full Text]
  9. Ng DS, Leiter LA, Vezina C, Connelly PW, Hegele RA. Apolipoprotein A-I Q(-2)X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. J Clin Invest. 1994;93:223-229.
  10. Romling R, von Eckardstein A, Funke H, Motti C, Fragiacomo GC, Noseda G, Assmann G. A nonsense mutation in the apolipoprotein A-I gene is associated with high-density lipoprotein deficiency and periorbital xanthelasmas. Arterioscler Thromb. 1994;14:1915-1922.[Abstract/Free Full Text]
  11. Assmann G, von Eckardstein A, Funke H. High density lipoproteins, reverse transport of cholesterol, and coronary artery disease: insights from mutations. Circulation. 1993;87(suppl III):III-28-III-34.
  12. 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;266:13654-13660.[Abstract/Free Full Text]
  13. Nichols WC, Gregg RE, Brewer HB Jr, Benson MD. A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy. Genomics. 1990;8:318-323.[Medline] [Order article via Infotrieve]
  14. Franceschini G, Sirtori CR, Capurso A, Weisgraber KH, Mahley RW. A-I Milano apoprotein. J Clin Invest. 1980;66:892-900.
  15. Weisgraber KH, Bersot TP, Mahley RW. A-I Milano apoprotein isolation and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins. J Clin Invest. 1980;66:901-907.
  16. Weisgraber KH, Rall SC, Bersot TP, Mahley RW, Franceschini G, Sirtori CR. Apolipoprotein A-IMilano: detection of normal A-I in affected subjects and evidence for a cysteine for arginine substitution in the variant A-I. J Biol Chem. 1983;258:2508-2513.[Free Full Text]
  17. Franceschini G, Sirtori CR, Bosisio E, Gualandri V, Orsini GB, Mobavero AM, Capurso A. Relationship of the phenotypic expression of the A-I Milano apoprotein in plasma lipid and lipoprotein patterns. Atherosclerosis. 1985;58:159-174.[Medline] [Order article via Infotrieve]
  18. Ameli S, Hultgardh-Nilsson A, Cercek B, Shah PK, Forrester JS, Ageland H, Nilsson J. Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury in hypercholesterolemic rabbits. Circulation. 1994;90:1935-1941.[Abstract/Free Full Text]
  19. Lipid and lipoprotein analysis. In: Manuals of Laboratory Operations. Washington, DC: Lipid Research Clinic Program; 1974:63-70. US Dept of Health, Education, and Welfare publication NIH175-628.
  20. Dobiasova M, Schutzova M. Cold labeled substrate and estimation of cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay. Physiol Bohemoslov. 1986;35:319-327.
  21. Bell GI, Karam JH, Rutter WJ. Polymorphic DNA region adjacent to the 5' end of the human insulin gene. Proc Natl Acad Sci U S A. 1981;78:5759-5783.[Abstract/Free Full Text]
  22. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98:503-517.[Medline] [Order article via Infotrieve]
  23. Shoulders CC, Kornblihtt AR, Munro BS, Baralle FE. Gene structure of human apolipoprotein A-I. Nucleic Acids Res. Gene.. 1983;11:2827-2838.
  24. Hultman T, Bergh S, Moks T, Uhlen M. Bidirectional solid-phase sequencing of in vitro amplified plasmid DNA. Biotechniques. 1991;10:84-93.[Medline] [Order article via Infotrieve]
  25. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Nucleic Acids Res. Gene. 1989;77:51-59.
  26. Miettinen H, Gylling H, Ulmanen I, Miettinen TA, Kontula K. Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase (LCAT) deficiency. Arterioscler Thromb Vasc Biol. 1995;15:1-8.
  27. Abken H, Reifenrath B. A procedure to standardize CAT reporter gene assay. Nucleic Acids Res. 1992;20:3527.[Free Full Text]
  28. Menzel H-J, Kladetzky R-G, Assmann G. One-step screening method for the polymorphism of apolipoproteins A-I, A-II, and A-IV. J Lipid Res. 1982;23:915-922.[Abstract]
  29. Vega GL, Gylling H, Nichols AV, Grundy SM. Evaluation of a method for study of kinetics of autologous apolipoprotein A-I. J Lipid Res. 1991;32:867-875.[Abstract]
  30. Billheimer DW, Eisenber S, Levy RI. The metabolism of very low density lipoproteins. I. Preliminary in vitro and in vivo observations. Biochim Biophys Acta. 1972;260:212-221.[Medline] [Order article via Infotrieve]
  31. McFarlane AS. Efficient trace-labeling of proteins with iodine. Nature. 1958;182:53.[Medline] [Order article via Infotrieve]
  32. Matthews CME. The theory of tracer elements with 131I-labelled plasma proteins. Phys Med Biol. 1957;2:36-53.[Medline] [Order article via Infotrieve]
  33. Seidah NG, Chretien M. Proprotein and prohormone convertases of the subtilisin family. Trends Endocrinol Metab. 1992;3:133-140.[Medline] [Order article via Infotrieve]
  34. Soutar AK, Hawkins PN, Vigushin DM, Tennent GA, Booth SE, Hutton T, Nguyen O, Totty NF, Feest TG, Hsuan JJ, Pepys MB. Apolipoprotein A-I mutation Arg-60 causes autosomal dominant amyloidosis. Proc Natl Acad Sci U S A. 1992;89:7389-7393.[Abstract/Free Full Text]
  35. Roma P, Gregg RE, Meng MS, Ronan R, Zech L, Franceschini G, Sirtori CR, Brewer HB Jr. In vivo metabolism of a mutant form of apolipoprotein A-I, apo A-I Milano, associated with familial hypoalphalipoproteinemia. J Clin Invest. 1993;91:1445-1452.
  36. Rader DJ, Gregg RE, Meng MS, Schaefer JR, 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]
  37. Schaefer EJ, Blum CB, Levy RI, Jenkins LL, Alaupovic P, Foster DM, Brewer HB Jr. Metabolism of high density apolipoproteins in Tangier disease. N Engl J Med. 1978;299:905-910.[Abstract]
  38. Emmerich J, Berges B, Tauveron I, Rader D, Santamarina-Fojo S, Schaefer J, Ayrault-Jarrier M, Thieblot P, Brewer HB Jr. Familial HDL deficiency due to marked hypercatabolism of normal apo A-I. Arterioscler Thromb. 1993;13:1299-1306.[Abstract/Free Full Text]
  39. Gylling H, Miettinen TA. Non-cholesterol sterols, absorption and synthesis of cholesterol and apolipoprotein A-I kinetics in a Finnish lecithin-cholesterol acyltransferase deficient family. Atherosclerosis. 1992;95:25-33.[Medline] [Order article via Infotrieve]
  40. Gylling H, Vega GL, Grundy SM. Physiologic mechanisms for reduced apolipoprotein A-I concentrations associated with low levels of high density lipoprotein cholesterol in patients with normal plasma lipids. J Lipid Res. 1992;33:1527-1539.[Abstract]
  41. Brinton EA, Eisenberg S, Breslow JL. Increased apo A-I and apo A-II fractional catabolic rate in patients with or without hypertriglyceridemia. J Clin Invest. 1991;87:536-544.
  42. 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.
  43. Agellon LB, Walsh A, Hayek T, Moulin P, Jiang XC, Shelanski A, Breslow JL, Tall AR. Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice. J Biol Chem. 1991;266:10796-10801.[Abstract/Free Full Text]
  44. Hayek T, Azrolan N, Verdery RB, Walsh A, Chajek-Shaul T, Agellon LB, Tall AR, Breslow JL. Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipoprotein levels, particle sizes, and metabolism. J Clin Invest. 1993;92:1143-1152.
  45. Fitch WM. Phylogenies constrained by the crossover process as illustrated by human hemoglobins and a thirteen-cycle, eleven-amino-acid repeat in human apolipoprotein A-I. Genetics. 1977;86:623-644.[Abstract/Free Full Text]
  46. McLachlan AD. Repeated helical pattern in apolipoprotein A-I. Nature. 1977;267:465-466.[Medline] [Order article via Infotrieve]
  47. Segrest JP. A molecular theory of lipid-protein interactions in the plasma lipoproteins. FEBS Lett. 1974;38:247-253.[Medline] [Order article via Infotrieve]
  48. Minnich A, Collet X, Roghani A, Cladaras C, Hamilton RL, Fielding CJ, Zannis VI. Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I relation between lecithin-cholesterol acyltransferase activation and lipid binding. J Biol Chem. 1992;267:16553-16560.[Abstract/Free Full Text]
  49. Sorci-Thomas M, Kearns MW, Lee JP. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. J Biol Chem. 1993;268:21403-21409.[Abstract/Free Full Text]
  50. Utermann G, Haas J, Steinmetz A, Paetzold R, Rall SCJ, Weisgraber KH. Apolipoprotein A-I Giessen (Pro143->Arg): a mutant that is defective in activating lecithin:cholesterol acyltransferase. Eur J Biochem. 1984;144:325-331.[Medline] [Order article via Infotrieve]
  51. von Eckardstein A, Funke H, Henke A, Altland K, Benninghoven A, Assmann G. Apolipoprotein A-I variants: naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I. J Clin Invest. 1989;84:1722-1730.
  52. Jonas A, von Eckardstein A, Kezdy KE, Steinmetz A, Assmann G. Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants. J Lipid Res. 1991;32:97-106.[Abstract]
  53. von Eckardstein A, Funke H, Walter M, Altland K, Benninghoven A, Assmann G. Structural analysis of human apolipoprotein A-I variants: amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure. J Biol Chem. 1990;265:8610-8617.[Abstract/Free Full Text]
  54. Lackner KJ, Dieplinger H, Nowicka G, Schmitz G. High density lipoprotein deficiency with xanthomas: a defect in reverse cholesterol transport caused by a point mutation in the apolipoprotein A-I gene. J Clin Invest. 1993;92:2262-2273.
  55. Menzel HJ, Assmann G, Rall SC, Weisgraber KH, Mahley RW. Human apolipoprotein A-I polymorphism: identification of amino acid substitutions in three electrophoretic variants of the Munster-3-type. J Biol Chem. 1984;259:3070-3076.[Abstract/Free Full Text]
  56. Nakata K, Kobayashi K, Yanagi H, Shimakura Y, Shigeru T, Arinami T, Hamaguchi H. Autosomal dominant hypoalphalipoproteinemia due to a completely defective apolipoprotein A-I gene. Biochem Biophys Res Commun. 1993;196:950-955.[Medline] [Order article via Infotrieve]
  57. Ranu RS. Relief of DNA polymerase stop(s) due to severity of secondary structure of single-stranded DNA template during DNA sequencing. Anal Biochem. 1994;217:158-161.[Medline] [Order article via Infotrieve]
  58. Matsunaga T, Hiasa Y, Yanagi H, Maeda T, Hattori N, Yamakawa K, Yamanouchi Y, Tanaka I, 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 USA. 1991;88:2793-2797.[Abstract/Free Full Text]
  59. Karathanasis SK, Ferris E, Haddad IA. DNA inversion within the apolipoproteins A-I/C-III/A-IV–encoding gene cluster of certain patients with premature atherosclerosis. Proc Natl Acad Sci U S A. 1987;84:7198-7202.[Abstract/Free Full Text]
  60. Ordovas JM, Cassidy DK, Civeira F, Bisgaier CL, Schaefer EJ. Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11. J Biol Chem. 1989;264:16339-16342.[Abstract/Free Full Text]
  61. Huang Y, von Eckardstein A, Wu S, Maeda N, Assmann G. A plasma lipoprotein containing only apolipoprotein E and with g mobility on electrophoresis releases cholesterol from cells. Proc Natl Acad Sci U S A. 1994;91:1834-1838.[Abstract/Free Full Text]
  62. Li H, Reddick RL, Maeda N. Lack of apo A-I is not associated with increased susceptibility to atherosclerosis in mice. Arterioscler Thromb. 1993;13:1814-1821.[Abstract/Free Full Text]
  63. Rubin EM, Krauss R, Spangler E, Verstuyft S, Clift S. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein A-I. Nature. 1991;353:265-267.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
J. S. Owen, M. S. Bharadwaj, M. J. Thomas, S. Bhat, M. P. Samuel, and M. G. Sorci-Thomas
Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin
J. Lipid Res., January 1, 2007; 48(1): 226 - 234.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
G. K. Hovingh, A. Brownlie, R. J. Bisoendial, M. P. Dube, J. H.M. Levels, W. Petersen, R. P.F. Dullaart, E. S.G. Stroes, A. H. Zwinderman, E. de Groot, et al.
A novel apoA-I mutation (L178P) leads to endothelial dysfunction, increased arterial wall thickness, and premature coronary artery disease
J. Am. Coll. Cardiol., October 6, 2004; 44(7): 1429 - 1435.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
S. Bhat, M. Zabalawi, M. C. Willingham, G. S. Shelness, M. J. Thomas, and M. G. Sorci-Thomas
Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions
J. Lipid Res., July 1, 2004; 45(7): 1207 - 1220.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
J. M. Martin-Campos, J. Julve, J. C. Escola, J. Ordonez-Llanos, J. Gomez, J. Binimelis, F. Gonzalez-Sastre, and F. Blanco-Vaca
ApoA-IMALLORCA impairs LCAT activation and induces dominant familial hypoalphalipoproteinemia
J. Lipid Res., January 1, 2002; 43(1): 115 - 123.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
P. G. Frank and Y. L. Marcel
Apolipoprotein A-I: structure;-function relationships
J. Lipid Res., June 1, 2000; 41(6): 853 - 872.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
M. G. Sorci-Thomas, M. Thomas, L. Curtiss, and M. Landrum
Single Repeat Deletion in ApoA-I Blocks Cholesterol Esterification and Results in Rapid Catabolism of Delta 6 and Wild-type ApoA-I in Transgenic Mice
J. Biol. Chem., April 14, 2000; 275(16): 12156 - 12163.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
H. Han, J. Sasaki, A. Matsunaga, H. Hakamata, W. Huang, M. Ageta, T. Taguchi, T. Koga, M. Kugi, S. Horiuchi, et al.
A Novel Mutant, ApoA-I Nichinan (Glu235->0), Is Associated With Low HDL Cholesterol Levels and Decreased Cholesterol Efflux From Cells
Arterioscler. Thromb. Vasc. Biol., June 1, 1999; 19(6): 1447 - 1455.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.