Clinical Chemistry 48: 2066-2070, 2002;
(Clinical Chemistry. 2002;48:2066-2070.)
© 2002 American Association for Clinical Chemistry, Inc.
Lack of Association between Increased Carotid Intima-Media Thickening and Decreased HDL-Cholesterol in a Family with a Novel ABCA1 Variant, G2265T
Seung Ho Hong1,1,
Ward Riley2,
Jeffrey Rhyne1,
Gina Friel1 and
Michael Miller1a
1 Department of Medicine, University of Maryland and Veterans Administration Medical Center, Baltimore, MD 21201.
2 Department of Neurology, Wake Forest University School of Medicine, Winston-Salem, NC 27104.
aAddress correspondence to this author at: Department of Medicine, University of Maryland, 22 S. Greene St., S3B06, Baltimore, MD 21201. Fax 410-328-4382; e-mail mmiller{at}heart.umaryland.edu.
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Abstract
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Low HDL-cholesterol (HDL-C) concentrations are inversely correlated with cardiovascular disease, and previous studies have demonstrated that variants in the ATP-binding cassette transporter, ABCA1, are responsible for a proportion of HDL-C deficiency states. We identified a novel variant in ABCA1 in a kindred with decreased HDL-C. This variant was not identified in >200 chromosomes of healthy individuals. The proband, a heterozygote for G2265T, developed premature coronary artery disease. In addition to low HDL-C, six biological family members heterozygous for the ABCA1 variant exhibited low HDL-C concentrations compared with unaffected family members (0.83 ± 0.32 vs 1.33 ± 0.36 mmol/L; P = 0.009). Despite the decreased HDL-C, carotid artery B-mode ultrasound studies failed to reveal increased intima-media thickening in affected individuals compared with age- and sex-matched controls. Although these data extend previous observations that a single defective ABCA1 allele may lead to decreased HDL-C, associated evidence of early atherosclerosis was not confirmed.
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Introduction
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HDL particles play a pivotal role in reverse cholesterol transport (RCT)2
by transporting free cholesterol from peripheral tissues to the liver. Inherently, molecular abnormalities in any of the proteins regulating HDL metabolism may therefore potentially impair RCT or advance atherothrombosis. Observational studies have identified an inverse relationship between coronary artery disease (CAD) and the HDL-cholesterol (HDL-C) concentration, even when total cholesterol concentrations are desirable (1). The recent identification of the ATP-binding cassette transporter, ABCA1, as an important regulator of HDL metabolism and RCT has demonstrated that variations in this gene appear to be a noteworthy contributor to HDL deficiency states.
Despite the recent characterization of ABCA1 mutations as the cause of Tangier disease (TD) and familial hypoalphalipoproteinemia (2)(3)(4)(5), there have been little data evaluating clinical measures of atherosclerosis in individuals with identified defects in ABCA1. This is an important issue to resolve because low-HDL syndromes are heterogeneous and may not necessarily be linked to premature vascular disease (6). For example, CAD has been reported in individuals with TD before age 60 years, although premature, symptomatic CAD at <40 years has not been reported (7). As such, identifying individuals at potentially high risk of CAD would seemingly have important clinical implications.
In view of the burgeoning evidence invoking ABCA1 in HDL metabolism and RCT, we identified a novel ABCA1 mutation in a kindred with moderately low HDL-C but without a history of TD. In addition, a noninvasive surrogate of atherosclerosis was used to evaluate the impact of low HDL-C concentrations on premature vascular disease.
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Materials and Methods
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study participants
The proband (II-3), a Caucasian resident of Jacksonville, FL, had decreased HDL-C (0.34 mmol/L) and apolipoprotein AI (apoA-I; 0.49 g/L) concentrations (Fig. 1
, arrow). When he was 44 years of age, he was diagnosed with CAD and underwent coronary artery bypass grafting. Before the development of symptomatic CAD, the proband had smoked heavily (40 cigarettes daily) for 25 years. The proband’s father (I-1) evidenced no history of premature CAD and died of a brain tumor at the age of 74 years. The proband’s paternal uncle (I-3) had no history of symptomatic CAD and died in 1999 of an alcohol-related illness. At present, there is no history of symptomatic CAD in other biological family members, whose risk factors include hypertension (II-2) and cigarette smoking (III-5). A total of 17 biological family members were evaluated in this kindred, and all participants gave their informed consent before participation.
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Figure 1. Pedigree of a kindred showing HDL-C segregation of W590L mutation.
Shaded symbols indicate heterozygotes. The corresponding value under each symbol represents the HDL-C concentration in mmol/L. ? indicates that the value is not known.
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pcr amplification and single-strand conformation polymorphism analysis
Genomic DNA was isolated from the peripheral whole blood of study participants. Templates for single-strand conformation polymorphism (SSCP) analysis were enzymatically amplified from genomic DNA of the participants by PCR, using each pair of primers and reaction conditions described previously (3)(8)(9)(10)(11). The primers were made to amplify all coding regions and splice site junctions. PCR products were mixed with 6x loading dye (950 mL/L formamide, 20 mmol/L EDTA, 0.5 g/L bromphenol blue, 0.5 g/L xylene cyanol FF), denatured for 10 min at 96 °C, and placed on ice. SSCP analysis was performed by electrophoresis using 6% or 8% nondenatured polyacrylamide gels at 5–10 W for 
24 h at room temperature.
sequencing of pcr-amplified dna
PCR products showing SSCP shifts were purified by use of a PCR purification reagent set from Qiagen and sequenced manually with a thermo sequenase 33P-labeled terminator cycle sequencing reagent set (Amersham). To identify variants of other primary HDL candidate genes as well as ABCA1 in the proband, all exon regions and splice site junctions of the apoA-I (APOAI), lecithin cholesterol acyl transferase (LCAT), lipoprotein lipase (LPL), phospholipid transfer protein (PLTP), and scavenger receptor class B type I (SR-BI) genes were amplified and sequenced (12)(13)(14)(15). Evaluation for PLTP mutations was conducted in the laboratory of Dr. X-C. Jiang.
determination of concentrations of plasma lipids and lipoprotein subclasses
Blood samples were collected from all participants after an overnight fast. The concentrations of plasma total cholesterol and triglycerides were measured by enzymatic methods. HDL-C was determined by the heparin–manganese precipitation method. The concentrations of apoA-I and apoB were measured by use of single radial immunodiffusion plates. LDL-cholesterol was calculated by the formula of Friedewald et al. (16).
measurement of carotid artery intima-media thickness
A standardized B-mode ultrasound examination (17) was performed to identify the mean intima-media thickness (IMT) of the common carotid artery in predefined 10-mm segments of the near and far walls located between 10 and 20 mm proximal to the flow divider tip of the carotid bifurcation, on the left and right sides of the neck. Sonographers used a high-resolution 5- to 10-MHz Acuson Aspen ultrasound system to obtain B-mode images on S-VHS videotapes. All images were read at the Wake Forest School of Medicine, and carotid IMT measurement were recorded by readers blinded to HDL variant status. Each value represents the mean of up to 20 mean IMT measurements obtained from multiple interrogation angles.
statistical analysis
The lipid concentrations between two groups were compared by the Student t-test. The data are expressed as mean ± SD. Statistical significance was defined as P <0.05.
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Results
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SSCP analysis of the intron–exon boundaries led to the identification of a novel ABCA1 mutation. The affected individuals in the kindred were heterozygous for a G2265T substitution (Fig. 2
, arrow) with predicted conversion of Trp to Leu (W590L). Although this mutation did not alter a restriction enzyme site, it was detected by SSCP analysis and confirmed by direct sequencing of the affected individuals. The point mutation was not detected during the screening of >200 chromosomes of healthy Caucasians.
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Figure 2. DNA sequence analysis of ABCA1 gene in the proband and a control.
Arrow indicates the site of the G T transition at position 2265.
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Lipid, lipoprotein, and apolipoprotein concentrations of the proband (shown in bold) and family members of the kindred are shown in Table 1
. Mean lipid concentrations were compared between carriers (+) and noncarriers (-) of the ABCA1 variant. Significant differences between (+) and (-) individuals for the W590L mutation were noted for HDL-C (P = 0.009) and apoA-I (P = 0.036). Affected family members with the ABCA1 mutant exhibited decreased HDL-C and apoA-I. HDL-C and apoA-I concentrations were lowest in the proband (II-3), his son (III-3), and the proband’s youngest sibling (II-8), whereas the other heterozygotes for G2265T (I-3, II-5, and III-4), evidenced only mildly decreased HDL-C without decreased apoA-I. Finally, four individuals (III-1, III-6, III-8, and III-10) had low HDL-C (<1.03 mmol/L, or < 40 mg/dL) in the absence of the G2265T variant.
B-Mode ultrasound studies were performed in 30 participants, including 5 of the 6 participants with the ABCA1 variant (I-3 died before testing), unaffected biological family members (n = 10), and age- and sex-matched controls (n = 15; Table 2
). The mean carotid IMT in affected individuals was 0.6192 cm compared with 0.689 cm (P, not significant) for age- and sex-matched controls. Biological family members without the mutation evidenced slightly lower carotid IMT compared with individuals with the ABCA1 variant, G2265T (0.588 cm), but these measurements were not significantly different compared with age- and sex-matched controls (0.576 cm). We were also unable to identify a significant correlation between HDL-C and carotid IMT in the small subset of individuals tested.
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Table 2. B-Mode ultrasound assessment of carotid IMT in individuals with the ABCA1 variant, G2265T, unaffected biological family members, and age- and sex-matched controls.
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Discussion
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We identified a novel ABCA1 variant, G2265T, associated with decreased HDL-C but not with increased carotid IMT thickness. Although the proband developed premature CAD, he also had a long-standing history of cigarette smoking, which may have played an important contributory role. In addition to the proband, the genetic variant was also present in five additional family members. Although three of these family members are relatively young (<50 years of age), neither of the two oldest affected family members, sibling II-5 (57 years of age) and paternal uncle I-3 (who died at age 81 years), had manifested symptomatic CAD, emphasizing the complexity inherent in interpreting the clinical relevance of low HDL-C. That is, although some low-HDL-C syndromes coincide with premature CAD, others do not (6)(7)(18)(19). In fact, individuals with apoA-IMilano, a disorder characterized by very low HDL-C but without premature CAD, have also been shown to have decreased carotid IMT (20). Although the proband in the present study evidenced premature CAD, there was no evidence of increased carotid IMT among the five individuals studied with the ABCA1 variant G2265T, suggesting that this ABCA1 variant may be insufficient for promoting early atherosclerosis unless environmental triggers (e.g., cigarette smoking) intervene. In contrast, increased carotid IMT and premature CAD have been reported in association with defective ABCA1 alleles (21)(22), although the extent and severity of concomitant CAD risk factors were not identified (23).
The W590L mutation site in ABCA1 resides in a highly conserved hydrophobic region within the extracellular segment of the NH2 terminus (24)(25) and is identical to amino acid 605 of ABCR. Two mutations in amino acids 602 and 608 of ABCR have been reported in association with Stargardt disease (26), a recessive childhood retinal degeneration syndrome. Other mutations in ABCA1 associated with TD and in close proximity to W590S have been identified in amino acids 587 and 597 (3)(27). Finally, Bodzioch et al. (4) reported a mutation in an individual with TD at the same nucleotide (2265) that led to a predicted conversion of Trp to Ser (W590S). Thus, the mutation reported here at amino acid 590 in ABCA1 identifies an important region with predicted physiologic relevance.
The mean HDL-C concentrations were significantly different between affected and unaffected biological family members with ABCA1 variant G2265T. However, only four of the six affected individuals displayed low HDL-C (
1.03 mmol/L). To rule out the possibility of additional variants causing HDL-C deficiency in the proband, all coding regions and splice site junctions for the following HDL candidate genes were sequenced: APOAI, LCAT, LPL, PLTP, and SR-BI. However, no other mutations were identified, indicating that a single defective ABCA1 allele may lead to familial HDL deficiency, as has been demonstrated previously (3)(8)(28). Alternatively, fluctuation in HDL-C among family members may have resulted, in part, from linkage disequilibrium between G2265T and other common variants in exons or promoter regulatory factors of ABCA1.
In conclusion, affected individuals with G2265T evidenced decreased HDL-C. However, despite the resulting low HDL-C and history of premature CAD in the proband, there was no evidence of increased carotid IMT in the proband or affected individuals. The lack of an association between the mutation and carotid IMT may have reflected the small number of individuals studied. Alternatively, despite reduced activity from defective ABCA1, other regulators of cholesterol efflux (29) may have compensated sufficiently to preserve RCT and limit early plaque deposition. Regardless, these data do not implicate the ABCA1 variant G2265T as promoting early atherosclerosis despite its association with isolated low HDL-C.
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Acknowledgments
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This study was supported by an American Heart Association Grant-In-Aid (Mid Atlantic Region), Veterans Affair Merit Award, and NIH award (HL-61369). The carotid ultrasound examinations were performed by Teresa Crotts, Lois Hoots, and Mitzie Spainhour from the Wake Forest University School of Medicine. We acknowledge Dr. X-C Jiang (SUNY-Downstate Medical Center, Brooklyn, NY) for evaluation of genetic variants in PLTP.
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Footnotes
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1 Current address: Department of Science Education, Jeju National University, Jeju 690-061, Korea. 
2 Nonstandard abbreviations: RCT, reverse cholesterol transport; CAD, coronary artery disease; HDL-C, HDL-cholesterol; TD, Tangier disease; apoA-I, apolipoprotein AI; SSCP, single-strand conformation polymorphism; and IMT, intima-media thickness.
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Abstract
Introduction
