Original Contributions

Compound Heterozygosity for an Apolipoprotein A1 Gene Promoter Mutation and a Structural Nonsense Mutation With Apolipoprotein A1 Deficiency

Akira Matsunaga, Jun Sasaki, Hua Han, Wei Huang, Mari Kugi, Takafumi Koga, Sadanori Ichiki, Tomoko Shinkawa, Kikuo Arakawa
https://doi.org/10.1161/01.ATV.19.2.348
Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:348-355
Originally published February 1, 1999

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Abstract

Abstract—Apolipoprotein (apo) A1 plays a central role in the metabolism of HDL. We describe a novel genetic variant of the apoA1 gene identified in a patient with low concentrations of plasma HDL cholesterol. The proband, a 12-year-old Japanese boy, exhibited markedly low levels of both plasma apoA1 and HDL cholesterol. Genomic DNA sequencing of apoA1 genes of the patient showed a compound heterozygosity for an A to C substitution at 27 bp upstream of the transcription start site of 1 apoA1 allele, and a C to T substitution in another allele at residue 84 resulting in aberrant termination. The point mutation at nucleotide position –27 changed ATAAATA of the putative TATA box signal sequence to ATACATA. In addition to this mutation, the patient was heterozygous for a G to A substitution at position –75. Immunoblotting of an isoelectric focusing electrophoresis gel of the proband’s plasma showed a trace amount of normal apoA1. No measurable plasma apoA1 and HDL cholesterol in a patient with homozygosity for nonsense mutation at residue 84 has been reported previously. To determine the effects of substitution either at position –27 or –75, plasmids containing the 5′-flanking region of the human apoA1 promoter fused to the CAT reporter gene were constructed and transfected in HepG2 cells. A construct with the A to C substitution at position –27 showed 41.8±4.2%, and G to A substitution at position –75 showed 72.8±15.2% (means±SD, n=3) of CAT activities, compared with the wild-type promoter sequence. A construct with the double substitutions at positions –27 and –75 showed only 22.8±1.3% (mean±SD, n=3) activity relative to the wild type. Our patient is the first case with a TATA box mutation etiologically related to lipoprotein disorders.

  • Received May 7, 1998.
  • Accepted July 10, 1998.

Several epidemiological and clinical studies have demonstrated that the concentration of plasma HDL correlates inversely with the risk of coronary artery disease (CAD) and that HDL is a powerful predictor of CAD.1 Studies of transgenic mice and rabbits have shown that overexpression of apoA1 protects them from the development of vascular lesions.2 3 The evidence in transgenic animals indicates that apoA1 is the major factor responsible for the antiatherogenic effects of HDL. ApoA1 is known to play a central role in regulation of the efflux and transport of cholesterol from peripheral tissues to the liver, and as a cofactor for lecithin:cholesterol acyltransferase (LCAT), which is responsible for the formation of most cholesteryl esters in plasma.4 Recently, scavenger receptor BI (SR-BI) was identified as a HDL receptor.5 The apoA1 gene is adjacent to the genes encoding apoC3 and A4 on chromosome 11q23.6 Mature apoA1 is a 243–amino acid single polypeptide chain synthesized by the liver and intestine. ApoA1 gene mRNA translates the 267–amino acid prepropeptide, which undergoes intracellular cleavage leading to a 249–amino acid propeptide. The propeptide is secreted and undergoes extracellular proteolysis giving rise to mature apoA1.

To date, >10 pedigrees of patients with the apoA1 mutation and extremely low concentrations of HDL cholesterol have been described.7 8 9 10 11 12 13 14 Eight different mutations of the apoA1 gene have been identified in homozygous forms which cause almost a complete absence of plasma HDL cholesterol and apoA1. One such mutation was shown to be caused by a deletion of the entire apoA1/C3/A4 gene complex,7 the second one was the result of a rearrangement involving the apoA1 and C-III genes,8 and others were caused by the synthesis of truncated apoA1, presumably because of premature termination of translation.10 11 12 14 15 16 Recently, we reported apoA1 Sasebo, which showed extremely low concentrations of HDL cholesterol caused by truncated apoA1.13 On the other hand, Miccoli et al17 reported that compound heterozygosity for an apoA1 (Leu141Arg)Pisa, as well as a frameshift mutation in exon 3 of apoA1 gene, contributed to the absence of HDL cholesterol, corneal opacities, and CAD. Heterozygous deletion mutation of amino acid 146 to 160 of apoA1 Seattle with extremely low concentrations of plasma apoA1 and HDL cholesterol has also been reported.9 A common G to A transition at –75 bp from the transcription start site of the apoA1 gene is associated with altered levels of HDL cholesterol, although these finding have not been consistent. Apart from the –75 G/A mutation, no mutation in proximal 5′-flanking region of apoA1 gene related to plasma apoA1 or HDL cholesterol levels has been reported. In this report, we describe a compound heterozygous TATA box and nonsense mutation of apoA1 gene in a patient with a markedly low concentration of plasma HDL.

Methods

Subjects

A 12-year-old Japanese boy developed hematuria, fatty liver, and obesity 4 years before presentation. Hematuria disappeared 1 year after the point out. At presentation, he was 145 cm tall and weighed 59 kg (body mass index, 28 kg/m2). Physical examination showed no xanthoma, tonsillar hypertrophy, or corneal opacity. Laboratory tests showed plasma aspartate aminotransferase and alanine aminotransferase of 133 and 76 IU/L, respectively. The concentrations of total plasma cholesterol, triglyceride, and HDL cholesterol were 3.93, 1.08, and 0.20 mmol/L, respectively. The paternal uncle had sudden death when he was 54 years old, but the other family history was negative for premature cardiovascular disease. The proband’s mother died at the age of 34 years because of ovarian cancer. The father did not have symptoms suggestive of coronary or vascular insufficiency. Blood samples were available from the father, paternal aunt, and paternal grandmother of the proband (Figure 1⇓). Our request for maternal family blood samples was declined. The proband and his paternal family members gave their informed consent before participation in this study.

Figure 1.
Figure 1.

Pedigree of the proband’s family. Arrow indicates the proband; Symbols, apoA1 genotype and gender of other family members; squares, males; circles, females. Numbers appearing next to individuals refer to those listed in Tables 2⇓⇓ and 3⇓.

Lipoprotein Analysis

Plasma lipoproteins were separated by ultracentrifugation,18 and the concentrations of cholesterol and triglyceride were determined by an enzymatic method.19 The concentrations of apolipoproteins were determined by the single immunodiffusion method.20 Isoelectric focusing gel electrophoresis (IEF) was performed by the 1-step method described previously by Takada et al.21 In brief, 1 μL of plasma was incubated for 1 hour at room temperature with 25 μL of 10 mmol/L Tris-HCl (pH=8.2) containing 1% sodium decyl sulfate, 2% ampholyte (pH=4 to 6), and 2 μL of mercaptoethanol. The mini-slab gel system (Biometra-Multigel G-44, Biometra, Gottingen, Germany) with 7.2% polyacrylamide gel containing 5 M urea and 2% ampholyte, pH 4 to 6, was used for IEF. ApoA1 bands were detected by immunoblot with polyclonal rabbit anti-apoA1 antiserum (Daiichi Pure Chemicals Co, Tokyo). Plasma samples from gender- and age-matched normolipidemic healthy volunteers were used as normal controls.

DNA Amplification and Single-Strand Conformation Polymorphism

DNA was extracted from peripheral blood leukocytes as described previously.22 Polymerase chain reaction (PCR) primers listed in Table 1⇓ were used for single-strand conformation polymorphism (SSCP). The nucleotide numbers are based on the published sequence for apoA1 DNA.23 PCR was performed for 35 cycles of 96°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. To screen for a possible apoA1 gene mutation, we used SSCP analysis as described by Orita et al24 with minor modifications. For this purpose, 0.5 μL of purified PCR products were diluted with 12.5 μL of 50% formamide solution, heated at 95°C for 5 minutes, and applied (8 μL/lane) to 7.2% polyacrylamide gel. During electrophoresis at 45 W, the temperature of the gel was kept at 24°C or 4°C with a built-in water jacket connected to an internal thermostat-regulated water circulator. After electrophoresis, the bands were detected by silver staining (Bio-Rad Laboratory).

View this table:
Table 1.

Oligonucleotide Primers Used in PCR-SSCP and Sequence of apoA1 Gene

DNA Sequencing

DNA segments carrying all 4 exons of apoA1 were amplified by PCR.25 PCR primers listed in Table 1⇑ were used for sequencing. The PCR-amplified DNA was isolated by electrophoresis on a 0.8% agarose gel and ligated into pT7Blue-T vector (Novagen). Twelve clones from each allele were sequenced using T7 DNA polymerase (Sequenase; United States Biochemical Co) according to the dideoxynucleotide chain termination method or using an ABI 373A DNA sequencer.26

PCR-Mediated Site-Directed Mutagenesis and Restriction Fragment Length Polymorphism

Because 3 different transcriptional start sites for human apoA1 gene have been reported,27 28 the A to C substitution of 5′-franking promoter can be shown by 3 different numbers from each transcriptional start site as –27, –28, or –30. In this study, we used number –27 from the transcriptional start site in the liver.28 To identify point mutations, a G to A substitution at position –75, an A to C substitution at –27, a G to A substitution at codon 37, and a C to T substitution at codon 84, PCR-mediated site-directed mutagenesis was performed.25 Leukocyte DNA was amplified by PCR using primer A (5′-AGGACCAGTGAGCAGCAACATGGCC-3′) and B (5′-GGCAAGGCCTGAACCTTGAGCT-3′) for nucleotide position –75 polymorphism,26 primer C (5′-GCTTGCTGTTTGCCCACTCT-3′) and D (5′-CCAGCTCTTGCAGGGCCAAT-3′) for nucleotide position –27 mutation, primer E (5′-AGAGACTATGTGTCCCAGTTTGAAGGCTCG-3′) and F (5′-GATATTAGGTGAGGACTCGGCC-3′) for the G to A substitution at codon 37,21 and primer G (5′-AAAGGAGACAGAGGGCCTGAGC-3′) and H (5′-CGCGCATCTCCTCGCCCAGAGG-3′) for the C to T substitution at codon 84. The regions of primer pairs, A and B, C and D, E and F, and G and H, were amplified by 35 cycles of PCR at 96°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute and digested with MscI, TspEI, TaqI, and AfaI, respectively. After electrophoresis using 5% polyacrylamide gel, these gels were visualized with ethidium bromide.

Construction of CAT Fusion Genes

The wild-type and mutant sequences of the apoA1 promoter were obtained by PCR amplification of the genomic DNAs from normal control, the proband or homozygous carrier of mutation at position –75, followed by the Kunkel method of site-directed mutagenesis.29 Amplification of the apoA1 promoter region was accomplished by PCR with primers I (5′-CCTAAGCTTCTCTGCCAACACA-ATGGAC-3′) and J (5′-GAAGTCTAGAGAGCGGGAGAAGAC-CTCAGG-3′) (+119 to –333 bp) that contain HindIII and XbaI sites at 5′ and 3′ terminals, respectively. The 452 bp fragments obtained were subcloned into pT7Blue-T vector, and the normal and mutant alleles were confirmed by DNA sequencing with an ABI 373A DNA sequencer. The genomic subclone was digested with HindIII/XbaI and cloned into pCAT vector (Promega Biotec).

Transient Expression of Recombinant Reporter Constructs

Plasmid DNAs (10 μg/dish) from various constructs were prepared and transfected into cultured cells by the lipofectin method (Gibco). All cultured cells were maintained in DMEM (Gibco) and supplemented with 10% FCS, penicillin, and streptomycin at 37°C in 5% CO2. Human hepatoma (HepG2) cells were seeded (1.5×106 cells/100-mm dish) for 40 hours before transfection. To correct for variation in DNA uptake by cells, 3 mg of the plasmid pRSV-β-gal30 were cotransfected with each test construct. After transfection, the cells were incubated for 24 hours, then gently scraped into prechilled extraction buffer. Protein extracts from transfected cells were prepared by 3 cycles of freeze-thaw, and the CAT and β-galactosidase enzyme activities of the cell extracts were assayed. In each experiment, the CAT enzymatic activity was normalized relative to that of the β-galactosidase activity. The CAT assay mixture contained 0.1 μCi of [14C]chloramphenicol, 0.25 mmol/L Tris-HCl (pH=7.8), 0.8 mmol/L acetyl-CoA, and 80 μL of extract in a final volume of 160 μL. The reaction mixture was incubated at 37°C for 1 hour and extracted with 0.5 mL of ethyl acetate. Ethyl acetate was evaporated and the pellets redissolved in 20 μL of ethyl acetate and analyzed by thin-layer chromatography on aluminum sheet Silica Gel 60 (20×20 cm, E. Merck Darmstadt, Germany) in chloroform/methanol (95:5) mixture for 45 minutes. CAT activity was evaluated by visualizing the extent of conversion of [14C]chloramphenicol to its acetylated form, and quantitated by cutting out the respective spots followed by counting.

Statistical Analysis

Differences between groups in the concentrations of lipids, lipoproteins, and apolipoproteins were evaluated for statistical significance with a 2-tailed unpaired test. A P value <0.05 was considered statistically significant. Data are expressed as mean±SD.

Results

Plasma Lipids and Apolipoproteins

The concentrations of plasma lipids and apolipoproteins of the proband and other family members are presented in Table 2⇓⇓. The concentrations of plasma HDL cholesterol and apoA1 were extremely low in the proband, and the levels of apoA2, apoC2, and apoC3 were low. However, plasma LDL cholesterol and apo B were normal. The father (II-2) had low levels of plasma HDL cholesterol and apoA1 (45% to 55% of normal controls). The proband’s grandmother (I-1) who was 78 years old and had moderate hypertriglyceridemia, showed slightly low concentrations of plasma HDL cholesterol and apoA1. The father (II-2) had moderate hypertriglyceridemia and aunt (II-1) had moderate hypercholesterolemia. High concentrations of plasma VLDL cholesterol were observed in the father (II-2) and the grandmother (I-1) who had the E2 allele. Agarose gel electrophoresis of the proband’s plasma showed a very weak α-migrating band (data not shown). IEF and immunoblotting of the proband’s plasma also showed a trace of apoA14 isoform, in agreement with normal apoA1 (Figure 2⇓).

Figure 2.
Figure 2.

Isoelectric focusing gel electrophoresis and immunoblotting. Plasma (1 μL) was incubated for 1 hour at room temperature with 25 μL of 10 mmol/L Tris-HCl (pH=8.2) containing 1% sodium decyl sulfate, 2% ampholyte (pH=4 to 6), and 2 μL of mercaptoethanol. Used for IEF was 7.2% polyacrylamide gel containing 5 M urea and 2% ampholyte, pH 4 to 6. Lane 1 indicates normal control; lane 2, proband.

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Table 2.

Concentrations of Plasma Lipoprotein and Apolipoprotein in Different Members of Family

View this table:
Table 2A.

Continued

SSCP Analysis

Figure 3⇓ shows SSCP results for exon 4 of the apoA1 gene in the proband and control. Denatured fragments of exon 4 from the proband were separated into 4 distinct bands of different mobility (Figure 3⇓, lane 2). This mutation was detected the codon 84 mutation by DNA sequence.

Figure 3.
Figure 3.

SSCP analysis for DNA fragments of exon 4 of apoA1 gene from the proband and control. Purified PCR products (0.5 μL) with primers 7 and 10 in Table 1⇑ were diluted with 12.5 μL of 50% formamide solution, heated at 95°C for 5 minutes, and applied (8 μL/lane) to 7.2% polyacrylamide gel. Single strands of DNA fragments from the proband with a G to A substitution at residue 84 shows abnormal migration. Visualization was achieved by silver staining. Lane 1 indicates normal control; lane 2, proband; arrows, abnormal bands.

DNA Analysis

To determine the molecular basis of the apoA1 variants, we amplified genomic DNA by PCR and sequenced all 4 exons and the promoter region of apoA1 gene in the proband. Sequencing of 12 separate clones of exon 4 showed a C to T substitution at codon 84 in 5 clones that led to a Gln to stop codon (Figure 4A⇓). This substitution was identical with the mutation detected by SSCP analysis of exon 4. The second substitution was found in exon 3, 5 of 12 separate clones showed a G to A transition at codon 37 that leads to an Ala to Thr substitution (data not shown). The third substitution was found in 27 bp upstream from the transcriptional start site of apoA1 gene; 8 of 12 separate clones showed an A to C substitution (Figure 4B⇓). The transversion at position –27 led to a ATAAATA to ATACATA substitution in the TATA box sequence. A heterozygous common G to A substitution at position –75 of apoA1 gene was also found in the same allele that showed the substitution at position –27, and substitutions at codon 37 and codon 84 were in another allele (data not shown). Polyacrylamide gel electrophoresis was used to identify variants at position –27 and codon 84 after digestion with restriction enzymes, TspEI and AfaI. Restriction fragment length polymorphism (RFLP) was used in other members of the family to identify additional carriers of the mutation (Figure 5A⇓ and 5B⇓). The proband and his father (Figure 5A⇓, lanes 3 and 4, respectively) were confirmed as heterozygotes of the substitution at position –27 by the loss of the TspEI site. PCR-amplified DNA of exon 4 of the apoA1 gene from the proband (Figure 5B⇓, lane 1) was subjected to AfaI digestion to confirm the nonsense mutation characterized by the creation of an AfaI site.

Figure 4.
Figure 4.

Sequence analysis of PCR-amplified DNA of apoA1 gene from the proband. The PCR-amplified DNA was sequenced after subcloning into T7Blue-T vectors. The sequence shown is the sense strand. A, Graphic representation generated by ABI 373 analysis software (version 1.2.0) shows a C to T substitution at codon 84. B, Autoradiography of sequencing gels of DNA from the proband shows an A to C substitution at position –27. The substituted nucleotide is marked with an asterisk.

Figure 5.
Figure 5.

Restriction fragment length analysis of apoA1 gene. A, analysis for the A to C substitution at position –27. PCR products generated with use of primers C and D as described under Methods and digested with TspEI were electrophoresed on 5% polyacrylamide. Lane M, marker from a digest of φX174; lane 1, grandmother (I-1); lane 2, paternal aunt (II-1); lane 3, proband (III-1); lane 4, father (II-2); lane 5, nondigested control. PCR-amplified DNAs of the promoter region from the proband and his father (lanes 3 and 4) subjected to TspEI showed 170 bp and 150 bp bands, indicating loss of a TspEI site, and DNAs from the other family members (lanes 1 and 2) showed 150 bp band resulting from complete digestion of TspEI. B, analysis for the C to T substitution at codon 84. PCR products generated with use of primers G and H were digested with AfaI. Lane M, marker from a digest of φX174; lane 1, proband (III-1); lane 2, father (II-2); lane 3, normal control; lane 4, nondigested control. The proband (lane 1) DNA subjected to AfaI showed 173 bp and 156 bp bands, indicating heterozygosity for a nonsense mutation at codon 84.

DNA sequencing and RFLP analysis showed that only the proband had a compound heterozygous mutation, whereas the father had a heterozygous A to C substitution at position –27, and other members of the family did not have these mutations (Figure 1⇑ and Table 3⇓). All examined family members were heterozygous or homozygous carriers of apoA1 (Ala37Thr). It was reported that the frequency of apoA1 (Ala37Thr) was about 8% in healthy Japanese persons.25

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Table 3.

Haplotypes of Different Family Members

Transcription Efficiency by Mutation of ApoA1 Promoter

To determine whether an A to C substitution at position –27 of the apoA1 gene is involved in the regulation of its expression, we constructed plasmids containing fusions between the bacterial CAT gene and DNA fragments spanning +119 to –333 of the apoA1 5′-flanking region (Figure 6⇓). We compared the promoter regions of the apoA1 gene, including a substitution at the nucleotide position –27 mutation (–75G/–27C), substitutions at the nucleotide position –75 and –27 (–75A/–27C), a substitution at position –75 (–75A/–27A), and wild type (–75G/–27A). The results of assay for CAT enzymatic activity showed that the wild-type sequence (–75G/–27A) in the –333/+119AI-CAT construct expressed substantial levels of CAT activity in HepG2 (Figure 7⇓). The mutant sequences, (–75G/–27C) and (–75A/–27C), in the CAT constructs expressed 41.8±4.2% and 22.8±1.3% (n=3) of CAT activity relative to the wild-type sequence, respectively. On the other hand, the substitution at nucleotide position –75 (–75A/–27A) had 72.8±15.2% (n=3) of CAT activity relative to the wild type. These results suggest that nucleotide –27 of the apoA1 gene is important and sufficient for expression in HepG2 cells.

Figure 6.
Figure 6.

Structures of the apoA1 promoter–CAT fusion genes. The transcriptional start site of apoA1 gene is indicated by +1. The coordinates of the constructs are –333 to +119 from the transcriptional start site.

Figure 7.
Figure 7.

Effect of nucleotide substitutions of the apoA1 promoter on the transcription of the CAT gene. A, CAT assays in HepG2 cells. Analysis was performed as described under Methods. The designations, –75G/–27C, –75A/–27C, and –75A/–27A, indicate the mutated promoter sequences shown in Figure 6⇑. Negative control is the pCAT vector. B, schematic representation of CAT assays. The results shown are averages of 3 independent transfections.

Discussion

We identified apoA1 mutations in a 12-year-old Japanese boy with apoA1 and HDL deficiencies. DNA sequence analysis of the apoA1 gene in our patient showed the substitution Gln to stop codon at residue 84 in 1 allele as previously reported.11 The other allele of our patient was affected by an A to C substitution at position –27 which was involved in the TATA box and a common polymorphism of G to A substitution at position –75 in the apoA1 promoter region. The results of DNA sequence analysis were confirmed by digestions with restrictive enzymes. The proband had an additional mutation in the apoA1 gene, apoA1 (Ala37Thr), which was not related to plasma concentrations of HDL cholesterol and apoA1.11 25 The proband must have inherited the apoA1 (Ala37Thr) from his mother, not his father (II-2), because the grandmother (I-1) was a homozygous carrier of this mutation and did not have the codon 84 mutation. Therefore, apoA1 (Ala37Thr) was not related to low plasma HDL cholesterol and apoA1 in this case.

In the patient described here, determination of lipoprotein showed reduced plasma concentrations of apoA1 and HDL cholesterol. The low level of HDL cholesterol reflected reductions in both HDL2 and HDL3 fractions (Table 2⇑⇑), although a more marked reduction was present in the HDL2 fraction. IEF and immunoblotting of the proband’s plasma showed a trace of normal apoA14 band (Figure 2⇑). These results suggest that the proband secreted a low amount of normal apoA1 in the plasma.

The heterozygous carrier of the substitution at position –27 of apoA1 gene showed about 45% to 55% of plasma apoA1 and HDL cholesterol, whereas in the compound heterozygous carrier the levels were about 10% of the wild type. In addition, no measurable levels of plasma apoA1 and HDL cholesterol were reported previously in a homozygosity for the same nonsense point mutation at codon 84 of apoA1.11 Heterozygosities for other nonsense and frameshift mutations in apoA1 were reported to have about 50% of apoA1 and HDL cholesterol levels.17 31 Thus, the mutation of TATA box region of apoA1 gene may exert a negative effect on HDL cholesterol levels similar to that of the nonsense mutation at codon 84, as well as affect the synthesis of apoA1.

The genes coding for apoA1 and 2 other apolipoproteins, apoC3 and apoA4, are closely linked and tandemly organized within a 15-kb DNA segment in the long arm of human chromosome 11.6 Certain regions of the chicken, rabbit, rat, and human apoA1 5′-flanking sequences are highly conserved.32 One of these regions corresponds to the typical eukaryotic gene TATA box. The TATA box is important for the correct initiation of gene transcription. In this regard, 2 different human genes with TATA box mutations were reported previously.33 34 Several different TATA box point mutations of human β-globin gene caused β-thalassemia,33 35 and 1 point mutation in the TATA box of 17β-hydroxysteroid dehydrogenase type 1 was reported in a patient with hereditary breast cancer.34 It has also been reported that these TATA box mutations in the β-globin gene showed 20% to 55% production of β-globin mRNA in transient expression compared with normal β-gene.36 37 The apoA1 gene was the third human gene that showed a point mutation in the TATA box.

Previous studies of the expression of human apoA1 gene in several different human cell lines by RNA-blotting analysis indicated that the gene is expressed only in certain cell lines of hepatic (HepG2, Hep3B, and PLC/PRF/5) and intestinal (Caco-2) origin.38 Detailed analysis of the nucleotide sequences involved in the apoA1 gene transcription has been performed, and some of the transcription factors have been identified.28 39 40 41 Taylor et al42 reported that a minimal segment of the rat apoA1 gene was nucleotides –46 to –7 including the TATA box. Sastry et al27 reported that sequences within the –256 to –41 nucleotide and –2052 to –192 nucleotide, located 5′ but not 3′ to the TATA box of the human apoA1 gene, were necessary and sufficient for expression in HepG2 and Caco-2 cells, respectively. Recently, Harnish et al39 reported that apoA1 gene expression in liver cells was activated synergistically by hepatocyte nuclear factors 3 and 4 bound to 3 cis-acting elements (–214 to –192; –169 to –146; and –134 to –119). In the present study, the constructs of apoA1 gene promoter including these cis-acting elements was used for expression of the CAT reporter gene. We demonstrated that an A to C substitution at position –27 within the TATA box and a G to A substitution at position –75 of the human apoA1 gene were associated with reduced CAT activities.

A common polymorphism caused by a G to A transition at position –75 of apoA1 gene has been studied previously.43 44 Studies in Caucasians have shown that individuals with the A allele at position –75 have higher levels of HDL cholesterol or apoA1 than do individuals homozygous for G allele, the common allele.43 44 However, other studies including our previous report found no effect of A allele on the concentrations of HDL cholesterol.26 45 46 In vitro analysis of the effects of –75 polymorphism on transcription has yielded conflicting results. For example, Smith et al47 reported that G to A substitution at position –75 in the apoA1 promoter showed 68% of CAT activities. Tuteja et al48 reported that the G to A substitution in a promoter construct including –330 to +1 showed about 50% of CAT activities, but CAT activities of the same substitution in a construct spanning the region –1469 to +397 were similar to those of the wild-type construct. On the other hand, Angotti et al49 reported that the G to A substitution increased transcription by about 5- to 7-fold in a construct spanning the region from –256 to +29. In the present study, the G to A substitution at position –75 showed 72.8% of CAT activity, a finding similar to that of Smith et al.47 Furthermore, the construct with the mutant (–75A/–27C) showed only 22.8% activities of the wild type. These data suggest that the –75 and –27 nucleotide substitutions had the additive effect for reduced CAT activities. This is the first case of TATA box mutation in human apolipoproteins.

Despite a nearly complete absence of HDL, a highly variable susceptibility to premature CAD is present in patients with apoA1 deficiencies. In 4 different families including the nonsense mutation at codon 84, homozygous patients for different apoA1 null alleles suffered from CAD.7 8 11 16 In contrast, CAD was absent in 5 apoA1-deficient patients.10 12 13 14 15 Possible reasons for such variability include age of the carriers, the small sample size of each kindred, presence of other coronary risk factors, and presence of abnormal circulating apoA1 variants. Because the proband described here was still 12 years old, we could not obtain any assessment of susceptibility to CAD despite the very low concentrations of plasma HDL cholesterol. The patient was obese and had a fatty liver, but no relationship is expected between apoA1 variants and these abnormalities. The number of family members in this study was too small to allow establishment of a firm genotype/phenotype association with coronary atherosclerosis.

In conclusion, the compound heterozygous mutant of an A to C substitution at position –27 in addition to common G to A substitution at position –75 together with nonsense mutation at residue 84 of apoA1 gene may together contribute to lowering plasma apoA1 and HDL cholesterol levels.

Acknowledgments

This work was supported partly by grants from the Japanese Ministry of Education, Science, Sports and Culture (No. 08671195 to J.S., No. 08671196 to A.M.). The authors wish to thank Dr. Yoshio Misumi and Dr. Fumiko Arakawa (Department of Biochemistry, Fukuoka University) for their technical advice and Akiyo Hamachi for her expert technical assistance. We also thank Prof. Takashi Imamura (National Institute of Genetics) for discussions and comments.

References

  1. ↵
    Gordon D, Rifkind BM. High density lipoproteins: the clinical implications of recent studies. N Engl J Med. 1989;321:1311–1315.
    OpenUrlCrossRefPubMed
  2. ↵
    Rubin EM, Krauss RM, Spangier EA, Verstuyft JG, Gift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature. 1991;353:265–267.
    OpenUrlCrossRefPubMed
  3. ↵
    Duverger N, Kruth H, Emmanuel F, Caillaud J-M, Viglietta C, Castro G, Tailleux A, Fievet C, Fruchart JC, Houdebine LM, Denefle P. Inhibition of atherosclerosis development in cholesterol-fed human apolipoprotein A-I-transgenic rabbits. Circulation. 1996;94:713–717.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995;36:211–228.
    OpenUrlAbstract
  5. ↵
    Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996;271:518–520.
    OpenUrlAbstract
  6. ↵
    Karathanasis KS. Apolipoprotein multigene family: tandem organization of human apolipoprotein A-I-C-III and AIV genes. Proc Natl Acad Sci U S A. 1985;82:6374–6378.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Ordovas JM, Cassidy DK, Civeira K, 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;264:16339–16345.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Karathanasis SK, Ferris E, Haddad IA. DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis. Proc Natl Acad Sci U S A. 1987;87:7198–7203.
  9. ↵
    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.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    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.
  11. ↵
    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 U S A. 1991;88:2793–2797.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Römling R, von Eckardstein A, Funke H, Motti C, Fragiacomo GC, Noseda G, Assmann G. A nonsense mutation in the apolipoprotein A-I gene in associated with high-density lipoprotein deficiency and periorbital xanthelasmas. Arterioscler Thromb. 1994;14:1915–1922.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Moriyama K, Sasaki J, Takada Y, Matsunaga A, Fukui J, Albers JJ, Arakawa K. A cysteine containing truncated apolipoprotein A-I associated with high density lipoprotein deficiency. Arterioscler Thromb Vasc Biol. 1996;16:1416–1423.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Takata K, Saku K, Ohta T, Takata M, Bai H, Jimi S, Liu R, Sato H, Kajiyama G, Arakawa K. A new case of apoA-I deficiency showing codon 8 nonsense mutation of the apoA-I gene without evidence of coronary heart disease. Arterioscler Thromb Vasc Biol. 1995;15:1866–1874.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Lacker KJ, Dieplinger H, Novicka 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.
  16. ↵
    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.
  17. ↵
    Miccoli R, Bertolotto A, Navalesi R, Odoguardi L. Compound heterozygosity for a structural apolipoprotein A-I variant, apoA1(L141R)pisa, and an apolipoprotein A-I null allele in patients with absence in HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation. 1996;94:1622–1628.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Schumaker VN, Puppione DL. Sequential flotation ultracentrifugation. Methods Enzymol. 1986;128:155–170.
    OpenUrlCrossRefPubMed
  19. ↵
    Sasaki J, Arakawa K. Effect of nifedipine on serum lipids, lipoproteins, and apolipoproteins in patients with essential hypertension. Curr Ther Res. 1987;41:845–851.
    OpenUrl
  20. ↵
    Goto Y, Akanuma Y, Harano Y. Determination by SRID method of normal values of serum apolipoproteins (A-I, A-II, B, C-II, C-III and E) in normolipidemic healthy Japanese subjects. J Clin Biochem Nutr. 1986;1:73–88.
  21. ↵
    Takada Y, Sasaki J, Ogata S, Nakanishi T, Ikehara Y, Arakawa K. Isolation and characterization of apolipoprotein A-I Fukuoka (110Glu→Lys): a novel apolipoprotein valiant. Biochim Biophys Acta. 1990;1043:169–176.
    OpenUrlPubMed
  22. ↵
    Takada Y, Sasaki J, Seki M, Ogata S, Teranishi Y, Arakawa K. Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA. J Lipid Res. 1991;32:1275–1280.
    OpenUrlAbstract
  23. ↵
    Shoulders CC, Kornblihtt AR, Munro BS, Baralle FE. Gene structure of human apolipoprotein A-I. Nucleic Acids Res. 1983;11:2827–2837.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    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.
    OpenUrlCrossRefPubMed
  25. ↵
    Araki K, Sasaki J, Matsunaga A, Takada Y, Moriyama K, Hidaka K, Arakawa K. Characterization of two new human apolipoprotein A-I variants: Apolipoprotein A-I Tsushima (Trp-108→Arg) and A-I Hita (Ala-95→Asp). Biochim Biophys Acta. 1994;1214:272–278.
    OpenUrlPubMed
  26. ↵
    Matsunaga A, Sasaki J, Mori T, Moriyama K, Nishi K, Hidaka K, Arakawa K. Apolipoprotein A-I gene promoter polymorphism in patients with coronary artery disease and healthy controls. Nutr Metab Cardiovasc Dis. 1995;5:269–275.
  27. ↵
    Sastry KN, Seedorf U, Karathanasis SK. Different cis-acting DNA elements control expression of human apolipoprotein AI gene in different cell types. Mol Cell Biol. 1988;8:605–614.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Higuchi KS, Law SW, Hoeg JM, Schumacher UK, Meglin N, Brewer JHB. Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5′-flanking region of the human ApoA-I gene. J Biol Chem. 1988;263:18530–18536.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985;82:488–492.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Hall CV, Jacobs PE, Ringold GM, Lee F. Expression and regulation of Escherichia coli lacZ fusions in mammalian cells. J Mol Appl Genet. 1983;2:101–109.
    OpenUrlPubMed
  31. ↵
    Nakata K, Kobayashi K, Yanagi H, Shimakura Y, Tsuchiya S, Arinmi T, Hamaguchi H. Autosomal dominant hypoalphalipoproteinemia due to a completely defective apolipoprotein A-I gene. Biochem Biophys Res Commun. 1993;196:950–955.
    OpenUrlCrossRefPubMed
  32. ↵
    Lamon-Fava S, Sastry R, Ferrari S, Rajavashisth TB, Lusis AJ, Karathanasis SK. Evolutionary distinct mechanisms regulate apolipoprotein A-I gene expression: differences between avian and mammalian apoA-I gene transcription control regions. J Lipid Res. 1992;33:831–842.
    OpenUrlAbstract
  33. ↵
    Poncz M, Ballantine M, Solowiejczyk D, Barak I, Schwartz E, Surrey S. β-thalassemia in a Kurdish Jew: single base changes in the T-A-T-A box. J Biol Chem. 1982;257:5994–5996.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Peltoketo H, Piao Y, Mannermaa A, Ponder BA, Isomaa V, Poutanen M, Winqvist R, Vihko R. A point mutation in the putative TATA box, detected in nondiseased individuals and patients with hereditary breast cancer, decreases promoter activity of the 17 beta-hydroxysteroid dehydrogenase type 1 gene 2 (EDH17B2) in vitro. Genomics. 1994;23:250–252.
    OpenUrlCrossRefPubMed
  35. ↵
    Baysal E, Carver MFH. The β- and γ-thalassemia repository. Hemoglobin. 1995;19:213–236.
    OpenUrlPubMed
  36. ↵
    Orkin SH, Sexton JP, Cheng T-C, Goff SC, Giardina JV, Lee JI, Kazazian HHJ. ATA box transcription mutation in β-thalassemia. Nucleic Acids Res. 1983;11:4727–4734.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Takihara Y, Nakamura T, Yamada H, Takagi Y, Fukumaki Y. A novel mutation in the TATA box in a Japanese patient with beta +-thalassemia. Blood. 1986;67:547–550.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Eggerman TL, Hoeg JM, Meng MS, Tombragel A, Bojanovski D, Brewer HBJ. Differential tissue-specific expression of human apoA-I and apoA-II. J Lipid Res. 1991;32:821–828.
    OpenUrlAbstract
  39. ↵
    Harnish DC, Malik S, Kilbourne E, Costa R, Karathanasis SK. Control of apolipoprotein AI gene expression through synergistic interactions between hepatocyte nuclear factors 3 and 4. J Biol Chem. 1996;271:13621–13628.
    OpenUrlCrossRefPubMed
  40. ↵
    Ginsburg GS, Ozer J, Karathanasis SK. Intestinal apolipoprotein AI gene transcription is regulated by multiple distinct DNA elements and is synergistically activated by the orphan nuclear receptor, hepatocyte nuclear factor 4. J Clin Invest. 1995;96:528–538.
  41. ↵
    Papazafiri P, Ogami K, Ramji DP, Nicosia A, Monaci P, Cladaras C, Zannis VI. Promoter elements and factors involved in hepatic transcription of the human ApoA-I gene positive and negative regulators bind to overlapping sites. J Biol Chem. 1991;266:5790–5797.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Taylor AH, Wishart P, Lawless DE, Raymond J, Wong NCW. Identification of functional positive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI promoter. Biochemistry. 1996;35:8281–8288.
    OpenUrlCrossRefPubMed
  43. ↵
    Pagani F, Sidoli A, Giudici GA, Barenghi L, Vergani C, Baralle EF. Human apolipoprotein A-I gene promoter polymorphism: association with hyperalphalipoproteinemia. J Lipid Res. 1990;31:1371–1377.
    OpenUrlAbstract
  44. ↵
    Sigurdsson GJ, Gudnason V, Sigurdsson G, Humphries SE. Interaction between a polymorphism of the apoA1 promoter region and smoking determines plasma levels of HDL and apoA1. Arterioscler Thromb. 1992;12:1017–1022.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Barre DE, Guerra R, Verstraete R, Wang Z, Grundy SM, Cohen JC. Genetic analysis of a polymorphism in the human apolipoprotein A-I gene promoter: effect on plasma HDL-cholesterol levels. J Lipid Res. 1994;35:1292–1296.
    OpenUrlAbstract
  46. ↵
    Akita H, Chiba H, Tsuji M, Hui SP, Takahashi Y, Matsuno K, Kobayashi K. Evaluation of G-to-A substitution in the apolipoprotein A-I gene promoter as a determinant of high-density lipoprotein cholesterol level in subjects with and without cholesteryl ester transfer protein deficiency. Hum Genet. 1995;96:521–526.
    OpenUrlPubMed
  47. ↵
    Smith JD, Brinton EA, Breslow JL. Polymorphism in the human apolipoprotein A-I gene promoter region: association of the minor allele with decreased production rate in vivo and promoter activity in vitro. J Clin Invest. 1992;89:1796–1800.
  48. ↵
    Tuteja R, Tuteja N, Melo C, Casari G, Baralle FE. Transcription efficiency of human apolipoprotein A-I promoter varies with naturally occurring A to G transition. FEBS. 1992;304:98–101.
    OpenUrlCrossRefPubMed
  49. ↵
    Angotti E, Mele E, Costanzo F, Avvedimento EV. A polymorphism (G→A transition) in the -78 position of the apolipoprotein A-I promoter increases transcription efficiency. J Biol Chem. 1994;269:17371–17374.
    OpenUrlAbstract/FREE Full Text
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  • Compound Heterozygosity for an Apolipoprotein A1 Gene Promoter Mutation and a Structural Nonsense Mutation With Apolipoprotein A1 Deficiency
    Akira Matsunaga, Jun Sasaki, Hua Han, Wei Huang, Mari Kugi, Takafumi Koga, Sadanori Ichiki, Tomoko Shinkawa and Kikuo Arakawa
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:348-355, originally published February 1, 1999
    https://doi.org/10.1161/01.ATV.19.2.348
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