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Journal of the American College of Cardiology Vol. 44, No. 7, 2004© 2004 by the American College of Cardiology Foundation ISSN 0735-1097/04/$30.00Published by Elsevier Inc. doi:10.1016/j.jacc.2004.06.070

HDL and Coronary Artery Disease

Novel ApoA-I Mutation (L178P) Leads tondothelial Dysfunction, Increased Arterial Wallhickness, and Premature Coronary Artery Disease

. Kees Hovingh, MD,* Alison Brownlie, PHD,† Radjesh J. Bisoendial, MD,* Marie Pierre Dube, PHD,†ohannes H. M. Levels, PHD,* Wilma Petersen, BSC,* Robin P. F. Dullaart, MD, PHD,‡rik S. G. Stroes, MD, PHD,* Aeilko H. Zwinderman, PHD,§ Eric de Groot, MD, PHD,*ichael R. Hayden, MD, PHD, CHB,†� Jan Albert Kuivenhoven, PHD,* John J. P. Kastelein, MD, PHD*

msterdam and Groningen, The Netherlands; and Vancouver, British Columbia, Canada

OBJECTIVES We investigated the consequences of an apolipoprotein A-I (apoA-I) gene defect with regardto lipid metabolism, endothelial function, arterial wall thickness, and coronary artery disease(CAD) risk.

BACKGROUND Due to limited numbers of carriers of the apoA-I defects, data on the consequences of suchdefects have remained inconclusive.

METHODS Lipids and lipoproteins were measured in 54 apoA-I (L178P) carriers and 147 nonaffectedsiblings. Flow-mediated dilation (FMD) was assessed in 29 carriers and 45 noncarriers, andcarotid intima-media thickness (IMT) could be determined in 33 heterozygotes and 40controls. Moreover, CAD risk was evaluated for all apoA-I mutation carriers.

RESULTS Heterozygotes exhibited lower plasma levels of apoA-I (�50%; p � 0.0001) and high-densitylipoprotein cholesterol (�63%; p � 0.0001). In addition, carriers had impaired FMD (p �0.012) and increased carotid IMT (p � 0.001), whereas multivariate analysis revealed thatheterozygotes had a striking 24-fold increase in CAD risk (p � 0.003).

CONCLUSIONS Heterozygosity for a novel apoA-I mutation underlies a detrimental lipoprotein profile thatis associated with endothelial dysfunction, accelerated carotid arterial wall thickening, andseverely enhanced CAD risk. Importantly, the extent of atherosclerosis in these subjects wassimilar to the burden of premature arterial wall abnormalities seen in patients with familialhypercholesterolemia. These data illustrate the pivotal role in humans of apoA-I in theprotection against CAD. (J Am Coll Cardiol 2004;44:1429–35) © 2004 by the AmericanCollege of Cardiology Foundation

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rospective epidemiologic studies have shown that high-ensity lipoprotein (HDL) cholesterol plasma levels are in-ersely related to coronary artery disease (CAD) risk (1). This

See page 1436

grees with the finding that low plasma levels of HDLholesterol are a common form of dyslipidemia in patientsuffering from premature CAD (2). Moreover, the importancef increasing HDL cholesterol levels is illustrated by a 2% to% reduction of major cardiovascular events associated with amg/dl (0.026 mmol/l) increase of HDL cholesterol on five

From the *Department of Vascular Medicine, Academic Medical Center,msterdam, The Netherlands; †Xenon Genetics Inc., Vancouver, British Columbia,anada; ‡Department of Internal Medicine, Academic Hospital, Groningen, Theetherlands; §Department of Clinical Epidemiology and Biostatistics, Academicedical Center, Amsterdam, The Netherlands; �Center for Molecular Medicine and

herapeutics, Children and Women’s Hospital, University of British Columbia,ancouver, British Columbia, Canada. Dr. Kastelein is an established investigatorf the Netherlands Heart Foundation (2000D039). Drs. Hovingh and Albertuivenhoven are supported by the Netherlands Heart Foundation (2000.115 and998T011, respectively). Dr. Hayden is holder of a Canada Research Chair in Humanenetics. This work was supported by grants from the Canadian Institutes of Healthesearch to Dr. Hayden and from Xenon Genetics to Dr. Hayden.Manuscript received January 20, 2004; revised manuscript received June 2, 2004,

bccepted June 22, 2004.

ears of fenofibrate treatment (3). The atheroprotective role ofDL is partly ascribed to its role in reverse cholesterol

ransport, by which HDL transports cholesterol from periph-ral cells to the liver and steroidogenic organs (4). In addition,DL displays significant anti-oxidant and anti-inflammatory

roperties (5).Apolipoprotein A-I (apoA-I), the major protein constituent

f the HDL particle, is critical to HDL metabolism. Itrovides HDL with structural integrity and is required forormal HDL function. Human apoA-I is expressed in the

iver and small intestine, and secretion into plasma results in deovo HDL production. In this process, apoA-I is lipidatedhrough adenosine triphosphate (ATP)-binding cassette AIransporter-mediated cholesterol efflux, which results in theormation of disc-shaped pre-beta1 HDL particles (6). Leci-hin/cholesterol acyltransferase, an enzyme that uses apoA-I ascofactor, subsequently esterifies free cholesterol on the nas-

ent HDL particle, which leads to the formation of larger andpherical HDL. Finally, apoA-I has been shown to play anmportant role in one of the last steps of the reverse cholesterolransport process. As a ligand of the scavenger receptor B-I, itacilitates the specific uptake of cholesteryl esters from HDL

y the liver, albeit in mice (7). It is not known whether, in

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1430 Hovingh et al. JACC Vol. 44, No. 7, 2004Atherosclerosis in ApoA-I (L178P) Carriers October 6, 2004:1429–35

umans, apoA-I also binds to the recently described novelepatic receptor (ectopic beta chain of ATP synthase) to enableolo-HDL particle uptake (8).

To date, over 40 apoA-I gene defects have been de-cribed, which may have severe consequences for HDLetabolism (9). The risk of CAD associated with these

poA-I gene defects, however, has not been elucidated. Thiss primarily due to limited numbers of carriers of apoA-Iefects, and, as a consequence, inconclusive data are pro-ided by the literature. Some apoA-I mutations supposedlynderlie CAD (L159P) (10), whereas others show no effectL144R) (11) or even protection against CAD (R173C)12).

We identified a novel apoA-I mutation (L178P) inutch families suffering from familial hypo-alpha-

ipoproteinemia. We report on the consequences of thispoA-I mutation for lipid metabolism in a large group ofeterozygotes compared with family controls. As surrogatend points for CAD, we subsequently measured endothelialunction by flow-mediated dilation (FMD) and carotidntima-media thickness (IMT). Finally, cardiovascular endoints in the affected and control individuals were scorednd assessed by multivariate analyses.

ETHODS

tudy population. The subjects were recruited from autch population-based study to identify genes that controlDL cholesterol levels (performed in collaboration with theenter for Molecular Medicine and Therapeutics at theniversity of British Columbia, Canada; and Xenon Ge-etics Inc., British Columbia, Canada). The populationomprised 94 probands who met the following inclusionriteria: 1) HDL cholesterol plasma level below the fifthercentile for age and gender; 2) absence of other primary orecondary lipid disorders (i.e., diabetes, alcohol abuse); and) high likelihood of inherited low HDL cholesterol (de-ned as an HDL cholesterol below the fifth percentile forge and gender and absence of other primary or secondaryipid disorders in at least one first-degree family member).nformed consent was obtained from all subjects for plasmaampling, storage, genetic analysis, and vascular tests, underprotocol approved by the ethics committee of the Aca-

emic Medical Center in Amsterdam. Past medical history,resence of cardiovascular risk factors, use of medication,nd information on geographic origin of the probands were

Abbreviations and AcronymsapoA-I � apolipoprotein A-ICAD � coronary artery diseaseFMD � flow-mediated dilationHDL � high-density lipoproteinIMT � intima-media thicknessLDL � low-density lipoprotein

ssessed by a questionnaire. m

NA analysis and mutation detection. DNA was ex-racted from peripheral blood leukocytes, as described pre-iously (13). Genotyping, linkage analysis, haplotyping, andequencing were used in an attempt to localize the molecularefects in families segregating low HDL. In one of theamilies (Family #11), this exercise resulted in the identifi-ation of a novel C/T point mutation at nucleotide 643 inxon 4 of the ApoA-I gene (using Genbank entryI4557320 as reference sequence), predicting the exchange

f a leucine for a proline residue at position 178 (L178P) inhe mature ApoA-I protein. The leucine residue at position78 is highly conserved with the exception of the pig, wherehis residue is instead a phenylalanine (14). The variant wasound on the affected haplotype and fully co-segregatedith hypo-alpha-lipoproteinemia in this family. In supportf the causal nature of this mutation, the sequence variantas not detected in 374 control chromosomes (data not

hown).Using polymerase chain reaction-restriction fragment

ength polymorphism analysis, we identified five additionaleterozygous carriers in the remaining 93 low HDL pro-ands (Family #8, #12, #28, #61, and #94). It is of note thatll six probands originate from the same geographic regionn the Netherlands, suggesting common ancestry. A Markovhain Monte Carlo with a bayesian integration approach

15) (described under Statistical Analysis) was used for thestimation of age of the mutation.aboratory analysis. Blood samples were drawn after anvernight fast. Total plasma cholesterol and triglyceridesere determined by an enzymatic colorimetric procedure

CHOD-PAP, Boehringer, Mannheim, Germany); HDLholesterol was measured as cholesterol remaining afterrecipitation of apoB-containing lipoproteins by MnCl2.ow-density lipoprotein (LDL) cholesterol was calculatedsing the Friedewald formula. Plasma apoA-I and apoBoncentrations were determined by immunonephelometry.mmunoelectrophoresis was used to quantify LpAI (Sebia,sy-les Moulineaux, France), and LpAI:AII was calculateds the difference between total apoA-I and LpAI concen-rations (16).low-mediated dilation. Flow-mediated vasodilation waseasured as previously described (17). In short, FMD was

ssessed after 10 h of fasting. A blood pressure cuff waslaced below the elbow of the right arm. After a 10-minest, the diameter of the brachial artery was visualized in thentecubital fossa using a 7.5-Mhz transducer. Reactiveyperemia was induced by inflating the blood pressure cuffo 220 mm Hg for 4 min. After release of the cuff, reactiveasodilation was monitored at 30-s intervals during 5 min.

all track measurements were performed and digitallytored by the same sonographer, who was blinded as to theenetic status of the participants. Baseline vessel luminaliameter was calculated as the average of three baselineeasurements. The FMD results were calculated using the

ollowing equation: (maximal lumen after hyperemia �

ean diameter at baseline)/diameter at baseline � 100%.

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1431JACC Vol. 44, No. 7, 2004 Hovingh et al.October 6, 2004:1429–35 Atherosclerosis in ApoA-I (L178P) Carriers

ntima-media thickness. The IMT measurements wereerformed as earlier described (18). In short, high-esolution B-mode ultrasound images were acquired us-ng an Acuson 128XP/10v (Acuson Corp., Mountain-iew, California), with a 7.0-MHz linear array trans-ucer. Segments of 10 mm of the following predefinedall segments were scanned: the common carotid artery,

he carotid bulb, and the internal carotid artery. Thecquired images were saved as JPEG image files. Onemage reader, blinded as to the genetic status of theatient, measured the IMT of the far wall of thoseegments. The mean combined IMT of these threeegments was used to compare carriers and noncarriers.tatistical analysis. A Markov Chain Monte Carlo with

bayesian integration approach was applied usingMLE�2.14 (15) to estimate the age of the ApoA-I

L178P) mutation. The six pedigree disease haplotypes and6 independent control haplotypes taken from the pedi-rees, genotyped at 10 microsatellite markers spanning 12M, were used for the estimation. Program parameters wereet to 1,000,000 replications; average population growth wasalculated to 0.18 per generation for the last 300 years; andhe haplotype fraction sampled in the Northern region ofhe Netherlands (Friesland) was set to 0.012.

Continuous outcomes were compared between affectednd unaffected subjects using the general linear mixed modelith pedigree number as a random factor to account for the

ssociation between subjects from the same pedigree. Forichotomous outcomes, a similar generalized linear mixedodel was applied with pedigree as random factor. The Cox

roportional hazard model extended with a frailty-arameter per pedigree was used to determine event-freeurvival rates for affected and unaffected subjects. Theroportional hazard conditions were met. Analyses wereerformed using the Statistical Program for the Socialciences (Version 10.0, SPSS Inc., Chicago, Illinois) and-plus for the Cox regression with frailty parameter. Theignificance level was set at p �0.05 (two-tailed).

ESULTS

emographic and genetic characteristics of study co-ort. Active recruitment in six kindreds yielded 54 het-rozygotes for the apoA-I defect and 147 family controls.he number of cases and controls in the different pedigreesere distributed as follows—Family #11: 12 carriers and 28

ontrols; Family #8: 7 carriers and 4 controls; Family #12:3 carriers and 49 controls; Family #28: 3 carriers and 11ontrols; Family #61: 6 carriers and 5 controls; and Family94: 13 carriers and 50 controls.All individuals were Caucasian of Dutch descent and

ived in an isolated part in the Northern region of theetherlands. The demographic data of carriers and noncar-

iers are given in Table 1. Mean age, age range, male/female t

atios, and the prevalence of hypertension (defined by these of antihypertensive medication) and smoking wereimilar in both groups.

Univariate linear regression revealed that the effects of thepoA-I mutation described in subsequent sections were notifferent among the six families, suggesting homogeneity ofhe total population. Genotyping and haplotyping of theseamilies with genetic markers in close proximity to thepoA-I gene demonstrated that they shared a 2.67-cMaplotype segment, suggestive of common ancestry. Twoindreds even shared a 7.62-cM haplotype. The age of thepoAI L178P mutation was estimated at 20 generations,ith the 95% confidence interval (CI) ranging from 12 to 35enerations.ipids, lipoproteins, and apolipoproteins. Comparedith control subjects, heterozygotes for the apoA-I defectresented with a 52% mean decrease of apoA-I levels (0.70s. 1.47 g/l, p � 0.0001) (Table 2) and a 62% decrease ofDL cholesterol levels (0.44 vs. 1.22 mmol/l, p � 0.0001).hese reductions of HDL cholesterol were reflected by a

ignificant decrease of HDL containing only apoA-ILpA-I: 0.31 vs. 0.60 g/l, p � 0.0001) and HDL containingoth apoA-I and apoA-II (LpA-I:A-II: 0.39 vs. 0.87 g/l, p

0.0004). In the absence of effects on triglycerides andDL cholesterol, the reduction of HDL cholesterol was

able 1. Demographic Characteristics of Heterozygotes for thepoA-I (L178P) Defect and Unaffected Family Controls

ApoAI (L178P)Heterozygotes

(n � 54)Noncarriers

(n � 147)

ean age (yrs) 37.6 � 19.2 37.9 � 16.8ge range 3–81 0.8–90ales/females 32/22 68/79

moker (%) 9 (17.6) 48 (30.6)ypertension (%) 4 (7.4) 9 (6.1)MI (kg/m2) 24.9 � 5.0 25.0 � 5.3

ll data are expressed as the mean value � SD or number (%) of subjects. Differencesetween carriers and noncarriers were not significant.

BMI � body mass index.

able 2. Lipids, Lipoproteins, and Apolipoproteins of Carriersnd Noncarriers of ApoA-I Mutation (L178P)

ApoAI (L178P)Carriers(n � 54)

Noncarriers(n � 147) p Value

poA-I (g/l) 0.70 � 0.31 1.47 � 0.33 �0.0001DL cholesterol (mmol/l) 0.44 � 0.22 1.22 � 0.36 �0.0001LpA-I (g/l) 0.31 � 0.10 0.60 � 0.13 �0.0001LpA-I:A-II (g/l) 0.39 0.87 0.0004

C (mmol/l) 4.20 � 1.06 4.72 � 0.99 0.001G (mmol/l) 1.31 � 0.88 1.19 � 1.28 0.09DL cholesterol (mmol/l) 3.16 � 0.92 2.98 � 0.91 0.23ipoprotein(a) (mg/l) 182.78 � 209.0 213 � 165.9 0.45poB (g/l) 0.99 � 0.26 0.89 � 0.20 0.21

ll data are given as the mean value � SD.ApoA-I � apolipoprotein A-I; apoB � apolipoprotein B; HDL � high-density

ipoprotein; LpA-I � HDL containing apoA-I; LpA-I:A-II � HDL containingpoA-I and apoA-II; LDL � low-density lipoprotein; TC � total cholesterol; TG �

riglycerides.

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1432 Hovingh et al. JACC Vol. 44, No. 7, 2004Atherosclerosis in ApoA-I (L178P) Carriers October 6, 2004:1429–35

irrored by a decrease of total cholesterol levels (4.20 vs..72 mmol/l, p � 0.001) in apoA-I (L178P) heterozygotes.ipoprotein(a) levels were not different in affected andnaffected subjects.low-mediated vasodilation. Flow-mediated dilationould be assessed in 29 carriers (18 men and 11 women) and

igure 1. Log-transformed (logFMD) results are significantly lower inarriers of the apoA-I (L178P) mutation compared with family controls.nverted triangles � apoA-I (L178P) carriers; circles � family controls.

igure 2. Mean combined carotid intima media thickness (IMT) increasehe rate of progression of carotid wall thickening over time is similar in L17

ApoA-I (L178P) carriers; open diamonds � patients with FH; � � family

5 family controls (24 men and 21 women). Controls wereignificantly younger than carriers (29.2 vs. 40.7 years, p �.01). We found no association between age and FMD inhis cohort (p � 0.18). In addition, smoking habits and therevalence of medically treated hypertension did not differmong both groups. The median FMD was significantlyeduced in apoA-I (L178P) carriers compared with familyontrols (3.5% vs. 5.5%, p � 0.012) (Fig. 1). This differenceemained significant after exclusion of heterozygotes (n � 5)ith from CAD.

MT. The IMT measurements were performed in 33eterozygotes (12 women and 21 men) and 40 familyontrols (18 women and 22 men). The mean carotid IMTf mutation carriers was significantly increased comparedith that of family controls (0.79 vs. 0.56 mm, p � 0.0001).he cross-sectional data were subsequently plotted against

ge, which suggested that the progression of carotid wallhickening over time differed significantly between carriersnd controls. Our data show a 0.0047- or 0.0082-mmcombined) carotid IMT increase per year of age in controlsnd heterozygotes, respectively (p � 0.001) (Fig. 2). Thempact of the apoA-I (L178P) mutation is clear whenonsidering similar rates of carotid wall thickening in aohort of 215 patients with familial hypercholesterolemiaFig. 2) (19).

ear of age is significantly higher in heterozygotes compared with controls.terozygotes and familial hypercholesterolemia (FH) patients. Solid circles

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1433JACC Vol. 44, No. 7, 2004 Hovingh et al.October 6, 2004:1429–35 Atherosclerosis in ApoA-I (L178P) Carriers

ardiovascular events. Among the 54 L178P heterozy-otes, eight subjects (15%) had CAD (i.e., acute myocardialnfarction, coronary artery bypass graft operation, or coro-ary angioplasty), of which six occurred at a premature agebefore the age of 55 and 60 years in men and women,espectively). In contrast, only two of 147 control subjects1.4%) suffered from an event, of which none occurredrematurely. Using univariate analysis, this difference wasighly statistically significant (p � 0.0001; data not shown).n multivariate analysis, controlling for age, gender, and

edigree, the apoA-I (L178P) carrier status was found toonvey a 24-fold increase in the risk of developing CADTable 3). The odds ratio to develop CAD when carryinghe apoA-I gene mutation was 18.9 (CI 2.3 to 153.8, p �.003) after excluding the six probands. The difference invent-free survival between carriers and controls, as shownn Figure 3, further illustrates the severe consequences ofhis mutation (p � 0.008).

ISCUSSION

his report shows the detrimental consequences of a novelpoA-I (L178P) mutation on HDL metabolism, endothe-ial function, carotid arterial wall IMT and CAD risk in aopulation comprising 54 heterozygous carriers. The novelefect was identified in six families, all originating from theortheastern part of the Netherlands. Evidence of a

ounder effect for L178P was obtained from haplotype

able 3. Multivariate Analysis of Risk of Coronary Arteryisease Attributable to the L178P Mutation Carrier Status

Controlled for Age, Gender,and Pedigree n OR (95% CI)

pValue

ncluding pedigree probandsCAD risk (all ages) 201 24.1 (3.2–179.1) 0.0009CAD risk (age 20 yrs and over) 167 23.7 (3.2–174.6) 0.0009

xcluding pedigree probandsCAD risk (all ages) 195 18.9 (2.3–153.8) 0.0030CAD risk (age 20 yrs and over) 161 18.5 (2.3–148.0) 0.0030

AD � coronary artery disease; CI � confidence interval; OR � odds ratio.

igure 3. Event-free survival is significantly lower in apoA-I (L178P)arriers than in controls. Bottom broken line � apoA-I (L178P) carriers;

pop line � family controls. p � 0.008.

nalysis, which demonstrated that all carriers share a.67-cM haplotype surrounding the gene.poA-I (L178P): effects on apoA-I levels, HDL choles-

erol, and triglycerides. Although we observed a 50%ecrease of apoA-I levels in apoA-I (L178P) carriers,eterozygotes for other apoA-I defects have been associatedith even greater apoA-I reductions (up to 80%) (10,20).his has been attributed to dominant negative effects of

ome mutations, and, possibly, L178P does not exhibit suchroperties. In this respect, it is of interest to note that weere not able to detect apoA-I (L178P) in plasma by meansf mass spectrometry of the apoA-I protein moiety ofsolated HDL (data not shown). This suggests that the178P variant is not properly processed in the cell, resulting

n absent or greatly diminished secretion into the circula-ion. Alternatively, the mutant protein may be hypercatabo-ized on secretion (thereby rendering plasma levels too lowor detection), an effect that has been described for otherpoA-I variants (21,22). These data imply that the effectsbserved can be solely attributed to half normal levels ofild-type apoA-I.Heterozygous carriers of the apoA-I (L178P) defect

resented with 62% reductions in HDL-C. Similar reduc-ions have been described for other apoA-I mutations23,24) although some reports describe HDL-C reductionsp to 80% of normal (24). Finally, we noted no effects onG levels, which is in contrast with a study that revealedigher TG levels in men suffering from an other apoA-Idelta K107) defect (25).poA-I (L178P): cardiovascular disease. Although mu-

ine studies have unequivocally shown that apoA-I protectsgainst atherosclerosis (26,27), and epidemiologic data inumans clearly indicate that apoA-I levels are a strongredictor of CAD (28), susceptibility for CAD has beenhown to vary significantly between carriers of apoA-Iefects. Ten of 22 apoA-I–deficient subjects (13 differentamilies) have been described to suffer from CAD to variousegrees (29–33).It is of interest to note that two publications describedutations at the same amino acid position. Two heterozy-

ous carriers for the Leu178His defect were shown to sufferrom low HDL cholesterol and amyloidosis, but no signs ofAD were reported (34). Interestingly, no amyloidosis wasbserved in our L178P mutation carriers. The majority ofpoA-I mutations associated with amyloidosis change the

soelectric point of the protein (14). This might underlie theifference in clinical phenotype between L178H and178P. The L178H introduces a positively charged residue,hereas L178P does not. Also, it should be noted that the178H protein was identified in the plasma of its carriers,hile this was not the case in heterozygous carriers of the178P defect. During the preparation of this manuscript,

kewaki et al. (35) reported on a 51-year-old female ho-ozygote for a premature stop codon at residue 178

nderlying HDL cholesterol deficiency (0.08 mmol/l) and

remature heart failure. In this case, the number of carriers

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1434 Hovingh et al. JACC Vol. 44, No. 7, 2004Atherosclerosis in ApoA-I (L178P) Carriers October 6, 2004:1429–35

n � 3) left no opportunity to investigate the directorrelation between the mutation and the risk for CAD.aken together, the relatively small number and young agef individuals with apoA-I gene defects have until nowompromised detailed studies into CAD risk.

Some of these difficulties can be overcome by usingurrogate markers to assess atherosclerotic burden, such asMD and carotid artery IMT (17,36,37). Both FMD andMT correlate with established risk factors and have beenhown to have predictive value for future vascular events17). It is clear from our data that heterozygotes for apoA-IL178P) suffer from endothelial dysfunction as comparedith family controls, which is likely related to the low levelsf HDL cholesterol in these patients. This hypothesis isupported by experiments in which infusion of reconstitutedDL acutely improved endothelial function in patientsith isolated low HDL cholesterol levels, due to ABCA1efects (38). It is well recognized that impaired endothelium-ependent vasomotor response, caused by diminished nitricxide bioavailability, precedes structural arterial wall alter-tions. This, in turn agrees with our finding of an increasedarotid IMT in affected subjects compared with familyontrols. However, we needed to account for the differencen age between the two groups. We therefore calculated theate of progression of arterial wall thickening by plotting theean carotid artery wall thickness against age. The rate of

rogression in affected subjects was found to be significantlyncreased.

Finally, the size of our cohort allowed us to not onlyorrelate apoA-I (L178P) with low HDL cholesterol, re-uced FMD, and increased IMT progression, but also withdefinite increased risk for cardiovascular complications.ultivariate analysis revealed a dramatic 24-fold increase inAD, as defined by myocardial infarction, coronary arteryypass graft surgery, and percutaneous transluminal coro-ary angioplasty.Together, our data illustrate that the L178P defect is

eleterious. This brings us to address IMT measurements inarriers of another interesting mutation in apoA-I denoteds apoA-I-Milano (R173C). In contrast to all other apoA-Iefects, apoA-I-Milano has been described to be beneficialespite the fact that carriers present with a 60% reduction inDL cholesterol (39). It has been described that this

ariant has increased potential to promote cellular choles-erol efflux (40). Others have recently shown that infusionsf ApoA-I-Milano/phospholipid complexes in CAD pa-ients result in a significant regression of coronary plaque41). Regarding IMT, 21 heterozygous carriers of thepoA-I-Milano (R173C) mutation were found to haveimilar average thicknesses of carotid segments comparedith control subjects (n � 42), further underlining theistinct properties of this peculiar Milano variant (39).onclusions. We unambiguously show that isolated lowDL cholesterol due to heterozygosity for apoA-I (L178P)

s marked by endothelial dysfunction, accelerated carotid

rterial wall thickening, and an increased incidence of

remature vascular events compared with their family con-rols. In this cohort, genetically defined low HDL choles-erol appears equally detrimental as hereditary high LDLholesterol with respect to IMT progression. The datanderscore the pivotal importance of normal apoA-I andDL cholesterol levels as a defense against all stages of

ardiovascular disease and furthermore indicate that thera-eutic intervention to raise HDL cholesterol may be equallyffective to reduce CAD risk as drugs that lower LDLholesterol.

cknowledgmentshe authors thank A. Smit, J. Gort, and P. Rol for their

ssistance in the vascular studies, as well as A. H. E. M.lerkx for laboratory support.

eprint requests and correspondence: Dr. John J. P. Kastelein,epartment of Vascular Medicine, F4-159.2, Academic Medicalenter, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.-mail: e.vandongen@amc.uva.nl.

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