Common and Rare ABCA1 Variants Affecting Plasma HDL Cholesterol
Abstract—Mutations in ABCA1, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of ABCA1 can affect plasma HDL cholesterol in the general population. To address this issue, we developed a screening strategy to find common ABCA1 variants. This strategy involved long-range amplification of genomic DNA by using coding sequences only, followed by deep sequencing into the introns. This method helped us to characterize a new set of amplification primers, which permitted amplification of virtually all of the coding sequence of ABCA1 and its intron-exon boundaries with a single DNA amplification program. With these new sequencing primers, we found 3 novel ABCA1 mutations: a frameshift mutation (4570insA, A1484S→X1492), a missense mutation (A986D) in a TD family, and a missense mutation (R170C) in aboriginal subjects with FHA. We also used these sequencing primers to characterize 4 novel common amino acid variants in ABCA1, in addition to 5 novel common silent variants. We tested for association of the ABCA1 I/M823 variant with plasma HDL cholesterol in Canadian Inuit and found that M823/M823 homozygotes had significantly higher plasma HDL cholesterol compared with subjects with the other genotypes. The results provide proof of principle of the effectiveness of this approach to identify both rare and common ABCA1 genomic variants and also suggest that common amino acid variation in ABCA1 is a determinant of plasma HDL cholesterol in the general population.
- Received April 20, 2000.
- Accepted May 30, 2000.
Mutations in the gene encoding ATP-binding cassette transporter 1 (ABCA1) have been reported in patients with Tangier disease (TD),1 2 3 4 5 an autosomal recessive disorder characterized by extremely depressed plasma concentrations of HDL cholesterol, abnormal deposition of cholesteryl esters in the reticuloendothelial system, and disordered cellular cholesterol efflux. ABCA1 has been shown to regulate the apolipoprotein- (apo) mediated lipid removal pathway from cells,6 and both TD patients and mice with a disrupted Abca1 gene have defective transport of lipids from the Golgi to the plasma membrane.7 Furthermore, mutations in ABCA1 have been found in some patients with familial hypoalphalipoproteinemia (FHA),8 a relatively more common disorder than TD. Cellular cholesterol efflux appears to be abnormal in some patients with FHA due to mutant ABCA1, but there is no obvious tissue deposition of cholesterol esters, and the low plasma HDL cholesterol concentrations demonstrate an apparent autosomal codominant pattern of expression in ABCA1 mutation carriers.8 Therefore, ABCA1 is an attractive candidate gene for the determination of plasma HDL cholesterol concentration in the general population.
To this point, common variants and polymorphisms in ABCA1 and their potential association with plasma concentrations of HDL cholesterol have not been reported. To identify common genomic variants in ABCA1 for association studies, we applied a genomic screening strategy under a single set of amplification primers, which would amplify almost all of the ABCA1 coding sequence by using the same amplification conditions. Using these primers to sequence genomic DNA, we discovered 2 new TD mutations, 1 new FHA mutation, and many common variants in ABCA1, 1 of which was associated with variation in plasma HDL cholesterol.
The proband in the TD family (the Figure⇓; subject II-4) was previously reported to have severe aortic stenosis, associated with deposition of cholesteryl ester in the aortic valve, and early atherosclerosis.9 The kindred has been subsequently extended and a total of 8 members have been studied, including both parents (subjects I-1 and I-2); 2 siblings with TD (II-1 and II-3); a sibling with low HDL cholesterol, but no other findings, thus being clinically compatible with heterozygous FHA (II-5); and a clinically normal brother and his son (II-2 and III-1, respectively). The biochemical data from these 8 family members are shown in the Figure⇓ .
We selected from our dyslipidemia archival sample repository 6 subjects who conformed to stringent biochemical criteria for heterozygous FHA, which were plasma HDL cholesterol and/or apo AI concentration below the second percentile for age and sex, with plasma triglyceride concentration below the 50th percentile for age and sex. None of these subjects had any physical manifestations compatible with TD. This screening sample with FHA included 3 aboriginal Canadian subjects, 2 taken from our well-characterized sample of Oji-Cree10 and 1 taken from our well-characterized sample of Inuit.11
Samples from normolipidemic subjects taken from 6 ethnic groups (38 Europeans, 44 Oji-Cree, 30 Inuit, 36 Africans, 37 Chinese, and 38 South Asians) were studied to determine allele and genotype frequencies when a new polymorphism was identified. Association analyses with plasma HDL cholesterol concentration were carried out in normolipidemic samples by using amino acid polymorphisms that were shown to have sufficient heterozygosity or information content, thus ensuring sufficient numbers of subjects with each genotype for parametric statistical analysis.
Determining the Intron-Exon Boundaries of ABCA1
Genomic DNA was isolated from the whole blood of all study subjects by using an established method (Puregene, Gentra Systems). Long-distance polymerase chain reaction (LPCR) amplification was performed to identify intron-exon boundaries of the ABCA1 gene. Human ABCA1 cDNA sequence (Genome Data Base AJ012376) and murine ABCA1 cDNA sequence (Genome Data Base X75926) were aligned and used to infer the approximate position of each exon for the human ABCA1 gene. LPCR primers were designed with the intention of annealing within the deduced interior regions of exons, although there could be no a priori guarantee of avoiding annealing to intron-exon boundaries, which would result in a failure to amplify. In such an instance, different primers would be designed and the procedure would be repeated. In addition, primers were chosen to share an optimal annealing temperature of 62°C, which would allow for many reactions to proceed simultaneously within a single amplification device using an identical cycling program, thus increasing the throughput of the entire strategy.
Expand 20-kb Plus enzyme mix (Roche) was used for LPCR according to the manufacturer’s instructions. LPCRs were performed in a Perkin-Elmer gene amplification 9600 apparatus. Genomic DNA from a normal subject was used as the amplification template for all of these experiments. The cycle conditions for the LPCR were as follows: denaturing at 92°C for 2 minutes, followed by 10 cycles of a denaturation step at 92°C for 10 seconds, an annealing step at 62°C for 30 seconds, and an extension step at 68°C for 18 minutes. An autoextension feature was used to add 10 seconds to the extension step in each cycle for 20 cycles and a final 7-minute extension step at 68°C. The products of successful amplification reactions from genomic DNA ranged in size from 4 to 15 kb. Each successful LPCR was subsequently found to have amplified between 4 and 6 exons of ABCA1.
LPCR amplification products were electrophoresed in 0.6% agarose gels and purified by using the QIAEX II gel extraction kit (QIAGEN). Each pair of LPCR primers was used to define the 3′ boundary of the first exon by using the 5′-most primer and the 5′ boundary of the last exon by using the 3′-most primer of each LPCR. For the exons contained within each LPCR product, internal primers were then designed to sequence in forward and reverse directions, beginning within the deduced central regions of the exons. When successful, these tracings would begin with a known exon sequence but would either end before the intron-exon boundary, in which case a new primer would be designed and the sequencing strategy would continue in a “walking” fashion, or continue through the intron-exon junction and into a previously unknown intron sequence. The position of an intron-exon boundary within the LPCR product was obvious after alignment with the published cDNA sequences by using Sequence Navigator (Applied Biosystems). When additional intron sequence information was required, a sequencing primer could be designed by using the newly derived intron sequence information, and the sequencing could continue, in theory, throughout the whole intron.
Once the intron-exon boundaries were determined (Table⇓ I; please see http://www.ahajournals.org), the ABCA1 gene–coding region in study samples could be sequenced with different PCR primers, which were designed to amplify the genomic region containing each individual exon, the intron-exon boundaries, and a long intronic stretch based on the genomic sequence obtained from the LPCR mapping strategy described above. In addition, the primers were chosen to share an optimum annealing temperature of 56°C (Table⇓ II; please see http://www.ahajournals.org), which allowed for optimal use of a single amplification apparatus.
ABCA1 Genomic Mutations and Polymorphisms
Samples from 7 subjects were analyzed extensively: TD family subject II-4 and the 6 subjects with FHA, as described above. Direct genomic DNA sequencing of the individual exons was carried out by using the primers in Table⇑ II (please see http://www.ahajournals.org). All amplification reactions were carried out in a 50-μL mixture containing 32 pmol of each primer; 0.2 mmol/L each of dATP, dCTP, dGTP, and dTTP; 1.5 mmol/L MgCl2; 50 mmol/L KCl; 20 mmol/L Tris HCl (pH 8.4); and 2.5 U Taq Platinum DNA polymerase (GIBCO BRL, Life Technologies). Each amplification cycle consisted of a denaturation step at 94°C, an annealing step at 56°C, and an extension step at 72°C for 30 seconds each, followed by a final extension step for 10 minutes at 72°C and cooling to 4°C. The amplification products were then run on 1.5% agarose gels and purified with the QIAEX II gel extraction kit (QIAGEN). Purified amplification products were directly sequenced by the ABI 377 Prism automated DNA sequencer. DNA sequences were aligned both with published ABCA1 exon sequences3 4 and with each other by using ABI Sequence Navigator software.
Once a sequence variant was identified in a sample, its presence was confirmed by fresh reamplification and resequencing of the culprit exon from the patient sample on another day. When the DNA change, which was usually a single nucleotide polymorphism (SNP), affected a restriction endonuclease recognition site, restriction fragment length polymorphism analysis would be used as the basis of a rapid method to genotype the specific SNP for the purposes of determining allele and genotype frequencies in various ethnic groups and for studies of association with plasma HDL cholesterol. In contrast, when the DNA change or SNP did not affect a restriction endonuclease recognition site, genotypes would be inferred from electrophoretogram traces from direct genomic DNA sequencing to determine allele and genotype frequencies in whites only.
The significance of deviations of the observed genotype frequencies from those predicted by the Hardy-Weinberg equation were evaluated with χ2 tests. sas (version 6.12) was used for statistical comparisons involving HDL cholesterol.12 To test whether a common variation in ABCA1 was associated with variation in plasma HDL cholesterol, we genotyped the ABCA1 I/M823 variant in a previously reported normolipidemic Inuit sample.11 We assumed a recessive model for M823, in which the genotype variable was set at 0 for subjects with I823/I823 and M823/I823 genotypes and 1 for subjects with the M823/M823 genotype. Covariates in the ANOVA were age, sex, body mass index (BMI), and current cigarette smoking.11 The proportion of variation in a plasma HDL cholesterol concentration from each covariate was estimated from partial regression coefficients.
Intron-Exon Boundaries of ABCA1 and Primers for Genomic Sequencing
The strategy outlined in the Methods section allowed for determination of >96% of the intron-exon boundaries within a period of 6 weeks (Table⇑ I; please see http://www.ahajournals.org). Owing to insufficient human cDNA sequence information, we could not determine the 2 boundaries of exon 1 and the 5′ boundary of exon 2. For as-yet-undetermined reasons, the 5′ boundary of exon 5 and the 3′ boundaries of exon 4 were not accessible through this strategy. The availability of heretofore unpublished intron sequences allowed for the development of amplification primers for individual exons (Table⇑ II; please see http://www. ahajournals.org). These primers could amplify at a constant annealing temperature, allowing for all amplification of all exons from a single genomic source simultaneously within a single amplification apparatus.
Rare Mutations in ABCA1 in TD and FHA
The sequencing strategy outlined above resulted in the identification of 2 novel ABCA1 mutations in the TD family. These were a frameshift mutation in exon 33, namely 4570insA, A1484S→X1492, and a missense mutation in exon 21, namely A986D (the Figure⇑ and Table⇑ I). Although the frameshift mutation within codon 1484 did not alter a restriction site, it was easily genotyped because of the characteristic appearance of the electrophoretogram tracings of genomic DNA sequences of exon 33 of ABCA1 in heterozygotes for the frameshift (the Figure⇑ ). Digestion of an amplified fragment containing exon 21 with restriction endonuclease Tth111I revealed a 279-bp fragment for alleles containing A986 and smaller 144- and 135-bp fragments for alleles containing D986 (the Figure⇑ ).
The sequencing strategy also resulted in the identification of a rare ABCA1 variant, namely R170C, in 1 of the subjects with FHA who was part of the screening experiment. This subject was Oji-Cree, and subsequent screening of 80 Oji-Cree samples by using mismatch amplification primers and digestion with SfuI revealed 2 more Oji-Cree heterozygotes for ABCA1 C170/R170. Both of these subjects were found to have plasma HDL cholesterol concentrations below the 5th percentile for age and sex. However, the mutation was completely absent from 230 subjects from other ethnic groups, suggesting that it is probably a population-specific variant that determines FHA in Oji-Cree. Its presence in such a small number of Oji-Cree suggests that it arose relatively recently in the history of these people.
Common Amino Acid Variants in ABCA1
The sequencing strategy outlined above resulted in the identification of 4 common amino acid variants in ABCA1, namely R/K159 in exon 6, V/I765 in exon 16, I/M823 in exon 17, and R/K1527 in exon 34 (Table 1⇑) in the subjects who had been selected for the screening experiments. Restriction analysis by mismatch priming and digestion with restriction endonucleases BsmI and BstNI for I/M823 and R/K1527, respectively, was carried out to determine the genotype and allele frequencies of these markers in the normolipidemic ethnic samples. Restriction analysis with the use of the naturally occurring DpnII recognition site affected by V/I765 was carried out to determine the genotype and allele frequencies of this marker in the normolipidemic ethnic samples. Genotypes for the R/K159 variant were determined from the characteristic appearance of electrophoretogram tracings of genomic DNA sequences of exon 6 for calculation of the genotype and allele frequencies in whites only. None of the genotype frequencies for any of the variants deviated from the expectations of the Hardy-Weinberg equation in any study sample.
The ABCA1 R/K159, V/I765, I/M823, and R/K1527 variants were each markedly polymorphic in whites (Table 1⇑). The ABCA1 V/I765, I/M823, and R/K1527 variants were each also markedly polymorphic in other ethnic groups, with a wide range of allele frequencies.
Common Silent Variants in ABCA1
The sequencing strategy outlined above also resulted in the identification of 5 common silent variants in ABCA1, namely 876C/T and 888G/A in exon 8, 1980C/A in exon 14, 2820G/A in exon 19, and 4221G/A in exon 30 (Table 1⇑) in white subjects. Although none of these variants altered a naturally occurring restriction endonuclease recognition site, genotypes could be easily determined from the characteristic appearance of electrophoretogram tracings of genomic DNA sequences of the individual exons. This comparison allowed for calculation of the genotype and allele frequencies in whites only. The ABCA1 876C/T, 888G/A, 1980C/A, 2820G/A, and 4221G/A SNPs were each markedly polymorphic in whites (Table 1⇑). None of the genotype frequencies for any of the variants deviated from the expectations of the Hardy-Weinberg equation in any study sample.
In addition, we observed several disparities in the ABCA1 genomic DNA sequence from the screening sample and sequences that had been previously published. Allele frequencies for these disparate genotypes were determined from the characteristic appearance of electrophoretogram tracings of genomic DNA sequences of the individual exons in 28 whites; for each disparity, the DNA sequence was monomorphic in the control sample (Table 2⇓).
Association of ABCA1 I/M823 With Plasma HDL Cholesterol Variation
ANOVA revealed that age, sex, BMI (in kg/m2), smoking, and ABCA1 I/M823 genotype (assuming a recessive model for M823) were each a significant source of variation for plasma HDL cholesterol in Inuit (Tables 3⇓ and 4⇓). Simple mean HDL cholesterol concentration was significantly higher in M823/M823 homozygotes than in subjects with the I823/I823 and M823/I823 genotypes (Table 4⇓). Multivariate regression analysis indicated that age, sex, BMI, smoking, and ABCA1 I/M823 genotype (assuming a recessive model for M823) accounted for 29.4%, 42.0%, 13.5%, 8.2%, and 7.0%, respectively, of attributable variation in the plasma HDL cholesterol concentration (P<0.0001, <0.0001, =0.0034, =0.021, and =0.031, respectively). Post hoc analyses revealed no associations with other lipoprotein variables, such as plasma triglycerides and apo AI (data not shown).
We report herein (1) a strategy to identify intron-exon boundaries of a gene, without any need for cloning; (2) the application of this method to identify intron-exon boundaries of ABCA1; (3) the use of the acquired intron information to derive a set of amplification primers that amplified >96% of the ABCA1 coding sequence at a single annealing temperature, allowing for more effective use of amplification hardware; (4) the use of the amplification primers for genomic DNA sequencing, which resulted in (5) the identification of 3 novel rare ABCA1 mutations in TD and FHA, of which 1 appears to be an Oji-Cree–specific variant; and (6) the identification of 4 novel common amino acid variants and 5 novel common silent variants in ABCA1. We have noted a wide range of allele frequencies of these common markers across ethnic groups. Finally, we found an association of the common ABCA1 I/M823 variant with plasma HDL cholesterol in Canadian Inuit: M823/M823 homozygotes had significantly higher plasma HDL cholesterol compared with subjects with the other genotypes. This finding suggests that common variation in ABCA1 may be a source of variation of plasma HDL cholesterol in the general population.
The aforementioned method to transform a cDNA sequence into genomic amplification primers (designated TCGA) allowed us to rapidly identify 12 novel ABCA1 genomic sequence variants, with no need for cloning. The large quantity of intron sequence provided by TCGA was sufficient to allow the design of genomic amplification primers that fulfilled 3 criteria: (1) the amplified fragment contained the entire exon sequence; (2) the amplified fragment contained both intron-exon boundaries; and (3) the GC content of the primers could be optimized to allow for annealing at a specific temperature. The optimal annealing temperature could be kept constant for all genomic DNA amplification reactions developed this way, thereby permitting many reactions to proceed simultaneously in a single amplification apparatus under a single set of conditions.
In the postgenomic era, expressed sequence tags (ESTs), defined as complete or partial cDNA sequences, can mark genes for further study and analysis. However, the EST database will not necessarily be directly helpful to investigators who seek to identify human mutations from genomic DNA. For this application, reliable primers and conditions are required for amplification from human genomic DNA samples. Because there is no information regarding the intron-exon boundaries for the vast majority of ESTs mapped to date, a robust, general method such as TCGA is required, which might assist the large-scale, rapid conversion of EST data into intron sequence data. The intron sequences can be used, in turn, to create primers for the amplification and sequencing of coding regions from a genomic DNA template. TCGA will not necessarily fully characterize the intron size and complete intron sequence: only enough sequence is obtained to allow for clean amplification of coding regions and margins from a genomic template. However, the “walking” TCGA strategy of sequencing through the intron can be serially used until the next contiguous intron-exon boundary is crossed and the next exon is entered.
There are several reasons to acquire intron sequence for the study of candidate genes in human diseases. First, some diseases can result from mutations directly at or close to the intron-exon boundaries. Thus, sufficient flanking sequence within the intron is required to develop amplification primers to first identify and then to screen for a mutation close to these boundaries. Second, a small subset of some diseases can be due to mutations well inside introns. Again, sufficient flanking sequence within the intron is required to develop amplification primers to identify and then screen for an intron mutation in such patients. Finally, knowledge of intron sequence can permit development of clean and effective amplification strategies that fully capture coding regions, where most mutations would be expected to reside. Furthermore, TCGA allows for the use of genomic DNA from easily accessible whole-blood samples as the template for amplification and sequencing, with no need for cloning or further modifications. Approaches to find disease mutations in the absence of knowledge of the intron-exon boundaries, such as sequencing of the cDNA derived from reverse transcriptase–treated mRNA from tissues in diseased patients, are more cumbersome by comparison. In addition, such methods depend on mRNA being expressed in the diseased tissue, in contrast to the independence of mRNA expression required to detect DNA changes at the genomic level.
We used TCGA to identify rare ABCA1 mutations in TD and FHA subjects. The 2 TD family members who were simple heterozygotes for ABCA1 A986D, namely I-1 and II-5, had depressed though not undetectable plasma concentrations of HDL cholesterol and apo AI (the Figure⇑ ). In contrast, the plasma HDL cholesterol and apo AI concentrations in the simple heterozygote for 4570insA, namely I-2, were within the normal range (the Figure⇑ ). This finding suggests that the A986D mutation, which occurs between the Walker domains A and B of the first ABC in ABCA1,1 had an autosomal codominant influence on HDL cholesterol and apo AI. In contrast, the 4570insA mutation, which creates a premature truncation that results in the deletion of most of the second transmembrane domain and the second ABC of ABCA1,1 had a recessive influence on the TD phenotype. The Oji-Cree FHA R170C mutation, which occurs in the 5′ intracytoplasmic domain of ABCA1,1 appeared to have an autosomal codominant influence on the biochemical phenotype. The mechanism(s) through which ABCA1 mutations can lead to the TD and FHA phenotypes are not understood at present. Presumably, once reliable in vitro tests of function are developed, these 3 rare ABCA1 mutations can be studied to determine their impact on cellular cholesterol trafficking, which might then contribute to an understanding of the phenotype in intact humans.
To this point, common variants and SNPs in ABCA1 and their potential associations with plasma HDL cholesterol have not been reported in the literature. The R/K159 variant occurs within the 5′ intracytoplasmic domain of ABCA1,1 the V/I765 variant occurs within the first group of transmembrane domains of ABCA1,1 the I/M823 variant occurs upstream of the Walker A motif of the first ABC of ABCA1,1 and the R/K1527 variant occurs within the second group of transmembrane domains of ABCA1.1 Interestingly, the allele frequencies vary widely across populations (Table 1⇑), a finding whose significance is uncertain at this time.
That common ABCA1 variants might affect HDL in subgroups of the general population was demonstrated by the finding in normal Inuit that the ABCA1 I/M823 genotype was a significant source of variation in plasma HDL cholesterol concentration (Tables 3⇑ and 4⇑). It should be noted that the sample sizes of the various ethnic groups were relatively small for this screening experiment and that replication in larger samples would be important. Inuit with the M823/M823 genotype had significantly higher mean plasma HDL cholesterol than did Inuit with the other 2 genotypes (1.42±0.39 versus 1.67±0.53 mmol/L). The I/M823 marker accounted for 7% of the variation in HDL cholesterol, an effect size that was comparable to that of cigarette smoking, although it was smaller than those due to age, sex, and BMI. More association studies with these common ABCA1 variants are now required to determine whether such modest associations with HDL cholesterol are more general. The mechanism(s) through which this common ABCA1 variant can affect plasma HDL cholesterol is not clear at present and cannot be resolved by the current study.
In addition to the rare mutations and common variants shown in Table⇑ 1, we observed several disparities between the ABCA1 genomic DNA sequence obtained from the screening sample of whites and ABCA1 sequences that had been previously published (Table⇑ 2). For each disparity, the DNA sequence was monomorphic in our control sample of 28 whites (Table 2⇑), suggesting that small amendments might be required in some of the deposited reference sequences for ABCA1.1 4
In summary, our results provide proof of principle that TCGA is an effective way to develop primers that are part of an amplification strategy to find mutations in human genomic DNA. TCGA permitted rapid assessment of >96% of the ABCA1 coding sequence and resulted in the identification of 12 novel ABCA1 sequence variants. Three of the novel variants were extremely rare and were seen in subjects with either TD or FHA. One of the common amino acid variants, namely ABCA1 I/M823, was significantly associated with variation in plasma HDL cholesterol in Inuit. The results suggest that TCGA can identify both rare and common ABCA1 genomic variants and that common variation in ABCA1 can be a determinant of plasma HDL cholesterol.
This work was supported by grants from the National Institutes of Health (1-R21-DK44597-01), the Ontario Ministry of Health (No. 04307), the MRC (Canada, No. MT13430), the Canadian Genetic Diseases Network, and the Blackburn Group. Dr Hegele is a Career Investigator (No. CI-2979) of the Heart and Stroke Foundation of Ontario. Dr Harris is supported by the Ontario Ministry of Health Career Scientist Program. Dr Young is a Senior Scientist of the MRC (Canada). The authors wish to thank Carol Anderson and Pearl Campbell for their expert assistance. We acknowledge the cooperation and assistance from the chief and council of the community of Sandy Lake, the Sandy Lake community surveyors, the Sandy Lake nurses and the staff of the University of Toronto Sioux Lookout program, and the members of the Health Canada research team.
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