Genetic Determinants of Cardiovascular Disease Risk inFamilial Hypercholesterolemia
Angelique C.M. Jansen, Emily S. van Aalst-Cohen, Michael W.T. Tanck, Suzanne Cheng,Marcel R. Fontecha, Jia Li, Joep C. Defesche, John J.P. Kastelein
Objective—To investigate the contribution of polymorphisms in multiple candidate genes to cardiovascular disease (CVD)risk in a large cohort of patients with heterozygous familial hypercholesterolemia (FH).
Methods and Results—We genotyped 1940 FH patients for 65 polymorphisms in 36 candidate genes. During 91.451person-years, 643 (33.1%) patients had at least 1 cardiovascular event. Multifactorial Cox survival analysis revealed thatthe G20210A polymorphism in the prothrombin gene was strongly associated with a significantly increased CVD risk(GA versus GG; P�0.001).
Conclusions—In a large cohort of FH patients, we found that the G20210A polymorphism in the prothrombin gene isstrongly associated with CVD risk. Our results constitute a step forward in the unraveling of the hereditary propensitytoward CVD in FH and might lead to better risk stratification and hence to more tailored therapy for CVD prevention.(Arterioscler Thromb Vasc Biol. 2005;25:1475-1481.)
Familial hypercholesterolemia (FH) is a common heredi-tary disease, characterized by elevated levels of plasma
low-density lipoprotein cholesterol (LDL-C) and prematurecardiovascular disease (CVD).1 Characteristically, the meanage of onset of CVD is between 40 and 45 years in male FHpatients and in female FH patients 10 years later.1,2 Never-theless, the phenotypic expression of this disorder, in terms ofonset and severity of atherosclerotic vascular disease, variesconsiderably.3
Unfortunately, a paucity of solid data exists on factors thatcontribute to these phenotypic differences. Previous studieshave mostly focused on classical CVD risk factors and thefunctional variety among LDL receptor mutations.4–6 Al-though both influence the occurrence of CVD, they can onlypartially explain the observed large differences. We recentlystudied the contribution of classical risk factors to CVD in alarge cohort of FH patients and demonstrated that �20% ofthe variation in CVD occurrence could be explained by theserisk factors alone.7 Therefore, other still unknown and possi-bly genetic factors play an undeniable role in the develop-ment of CVD in these patients. Genetic differences affectsusceptibility to disease and whereas premature atherosclero-sis can be linked in rare cases to single-gene disorders, mostindividuals do not carry such DNA defects. The “commondisorder, common variant” theory predicts that the majorityof population-attributable variation in susceptibility to prev-
alent disease is caused by variants that occur in high fre-quency in multiple genes.8
Such genetic variation may also play an important role inthe development of CVD in FH. This is substantiated by thefact that clustering of CVD occurs in FH kindred.9 Unfortu-nately, large-scale association studies involving multiplepolymorphisms are lacking in FH. Our objective, therefore,was to investigate the contribution of polymorphisms inmultiple candidate genes to CVD risk in a large cohort ofpatients with heterozygous FH.
MethodsStudy Design and Study PopulationThe present investigation was a retrospective, multicenter, cohortstudy. The study design and study population have been describedelsewhere.7 Briefly, lipid clinics in the Netherlands submit DNAsamples from clinically suspected FH patients to a central laboratoryfor LDL receptor mutation analysis.10 We randomly selected hyper-cholesterolemic patients from this DNA bank database with the aidof a computer program (Microsoft Excel). These patients had beenreferred from 27 lipid clinics throughout the Netherlands (Figure I,available online at http://atvb.ahajournals.org).
Phenotypic data (including detailed information on CVD) wereacquired by reviewing patient’s medical records by a trained team ofdata collectors.7 Guidelines for data collection from medical recordswere constructed for the purpose of the study and have beenpublished.11 Written informed consent was obtained from all livingpatients. The Ethics Institutional Review Board of each participatinghospital approved the protocol.
Original received November 3, 2004; final version accepted April 21, 2005.From the Departments of Vascular Medicine (A.C.M.J., E.S.v.A.-C., J.C.D., J.J.P.K.) and Clinical Epidemiology and Biostatistics (M.W.T.T.),
Academic Medical Center, University of Amsterdam, the Netherlands; and the Department of Human Genetics (S.C., M.R.F., J.L.), Roche MolecularSystems, Inc, Alameda, Calif.
Correspondence to John J.P. Kastelein, Department of Vascular Medicine, Academic Medical Center, Meibergdreef 9, P.O.Box 22700, room F4-159.2,1100 DE Amsterdam, the Netherlands. E-mail [email protected]
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000168909.44877.a7
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Power CalculationTo calculate CVD risk associated with genetic polymorphisms, atleast 2000 FH patients were needed to reach a power of 80% todetect an odds ratio of 1.9 or more for carriers of the rare allele(please see http://atvb.ahajournals.org for the exact powercalculation).
Selection of Patients and Inclusion CriteriaOn reviewing 4000 medical records, a total of 2400 patients fulfilledthe inclusion and exclusion criteria for participation and wereincluded in the study (Figure I and Table I, available online athttp://atvb.ahajournals.org). The FH diagnostic criteria were basedon internationally established criteria.12–14
Selection of DNA Polymorphisms andGenetic AnalysesWe genotyped 65 polymorphisms in 36 candidate genes that werepreviously implicated in CVD.15 Complete genotypes for all 65polymorphisms could be obtained for 1940 (80.1%) of patients. Inthe remaining 460 patients, complete genotyping was impaired bytechnical difficulties. First, the amount of DNA was insufficient foradequate amplification in 235 patients. Among the remaining sam-ples, complete genotyping for all polymorphisms was not possible.The missing data rate ranged from 0.51% to 5.3%, with an averagerate of 1.7%. These patients did not differ clinically from the 1940patients and were excluded from further analyses. Genomic DNAwas extracted from peripheral blood leukocytes by standard proce-dures. Each sample was amplified using 2 pools of biotinylatedpolymerase chain reaction primers. Each polymerase chain reactionproduct pool was then hybridized to a linear array of sequence-specific oligonucleotide probes and alleles were detected using ahydrogen peroxidase-based chromogenic reaction, essentially asdescribed previously.15 Samples were blinded for genotyping. Theaccuracy of genotyping in 500 randomly selected DNA samples wasassessed by re-analysis of several polymorphisms in 3 genes (CETP,MTHFR, LPL). Less than 0.5% of discordant results were found.
Statistical AnalysisDifferences between subgroups were tested with �2 statistics orindependent sample t test (for triglycerides [skewed distribution] onlog-transformed data). To adjust for the effects of age and sex, weused multiple linear or logistic regression. Allele frequencies were
calculated by genotype counting and for each (biallelic) poly-morphism the deviation from Hardy–Weinberg equilibrium wastested by a �2 test with 1 degree of freedom. A likelihood ratio testwas used to detect pair-wise linkage disequilibrium16 and the extentof disequilibrium was expressed in terms of D��D/Dmax or D/Dmin.17
Cox proportional hazard regression with backward stepwise selec-tion was used to model the association of multiple polymorphismssimultaneously with the occurrence of CVD. Polymorphisms weretreated as categorical variables and patients homozygous for thecommon allele were used as the reference category. Follow-upstarted at birth and ended for each individual at the date of the firstoccurrence of established CVD. Patients without CVD were cen-sored at the date of the last lipid clinic visit or at the date of deathattributable to other causes. Because polymorphisms might expresstheir untoward effects by way of, for example, hypertension, diabetesmellitus, obesity, or dyslipidemia, we did not introduce these factorsas covariates in our models. Instead, we added those covariates thatfunction independently from the polymorphisms: sex and smoking(time-dependent). For smoking, we implemented a linearly decreas-ing risk effect for the 3 years after cessation.18 Statistical analyseswere performed using SPSS software (version 11.5; Chicago, Ill).Polymorphisms with P�0.05 for the likelihood ratio test wereconsidered to be suggestively associated; those with P�0.001 wereconsidered statistically significant.
ResultsClinical characteristics of the 1940 completely genotypedpatients are described in Table 1. During 91.451 person-years, 643 (33.1%) patients had at least 1 cardiovascularevent, including 29 individuals who died from documentedCVD events. Mean age of onset of CVD was 48.2 years.Patients with CVD were older, more often males and smok-ers, and had a higher prevalence of hypertension and diabetesmellitus. More obesity and higher total cholesterol levelswere also observed among CVD patients but were notsignificant after adjustment for age and sex. LDL-C levels didnot differ between CVD and non-CVD patients. High-densitylipoprotein cholesterol levels were lower (1.15�0.32 versus1.24�0.36 mmol/L; P�0.001) and median triglyceride levels
TABLE 1. Clinical Characteristics of 1940 FH Patients With and WithoutCardiovascular Disease
CVD� CVD� PP Adjusted forAge and Sex
Patient N (%) 643 (33.1) 1297 (66.9)
Male, % 60.8 42.0 �0.001 NA
Age at first lipid clinic visit, y 50.3 (�11.3) 42.2 (�12.5) �0.001 NA
Age at last lipid clinic visit, y 56.4 (�11.4) 46.6 (�12.8) �0.001 NA
Smoking, ever, % 82.9 70.4 �0.001 �0.001
Hypertension, % 17.0 6.3 �0.001 �0.001
Diabetes mellitus, % 10.6 3.5 �0.001 �0.001
Body mass index, kg/m2 25.6 (�3.2) 24.8 (�3.6) �0.001 0.2
Total cholesterol, mmol/L 9.63 (�2.24) 9.41 (�1.88) 0.042 0.1
higher (1.76 versus 1.48 mmol/L; P�0.001) in patients withCVD.
The 36 genes and 65 polymorphisms examined in the studyand the frequencies of the least common alleles of thepolymorphisms are presented in Table 2. All but 11 of the 65polymorphisms studied were in Hardy–Weinberg equilib-rium. The same polymorphisms showed deviation fromHardy–Weinberg equilibrium in patients with and withoutcardiovascular disease (data not shown). For 3 polymor-phisms in the CETP gene (Asp442Gly, �1 GA, and�3insT/in14), only homozygous wild-type individuals werefound, which were excluded from further analyses.
Multifactorial Cox survival analysis, which included the 62polymorphisms simultaneously, adjusted for sex and smok-ing, revealed that the G20210A polymorphism in the pro-thrombin gene exhibited the strongest association with anincreased risk of CVD (GA versus GG; P�0.001) (Table 3).Heterozygous carriers of the G20210A polymorphismshowed clearly reduced cardiovascular event-free survivalrates (Figure). Subgroup analyses for gender, total cholester-ol, LDL-C, high-density lipoprotein cholesterol, and triglyc-eride tertiles and year of birth (before 1930, 1930 to 1949,1950 to 1969, or after 1970) were performed. For eachsubgroup, hazard ratios (HR) of 2.0 (not significant) werefound, suggesting that the observed HR is not caused by aspecific subgroup within the present population (data notshown). In addition, 4 other polymorphisms were identifiedto be suggestively associated with the risk of CVD (P�0.05).
TABLE 2. The 65 Polymorphisms in 36 Candidate GenesExamined in the Study
Gene Polymorphism Freq*
Lipid Metabolism
Lipoprotein(a) C93T 0.156
G121A 0.148
Apolipoprotein A4 Thr347Ser 0.189
Gln360His 0.080
Apolipoprotein B Thr71Ile 0.347
Apolipoprotein C3 C(�641)A 0.381
C(�482)T 0.267
T(�455)C 0.373
C1100T 0.260
C3175G (SstI) 0.111
T3206G 0.372
Apolipoprotein E Cys112Arg 0.200
Arg158Cys 0.054
�3-adrenergic receptor Trp64Arg 0.063
Cholesteryl ester transfer protein C(�631)A 0.074
C(�629)A 0.488
Ile405Val 0.327
Asp442Gly 0.000
�1GA 0.000
�3insT/intron 14 0.000
TaqIB 0.437
Hepatic lipase C(�480)T 0.224
Low density lipoprotein receptor NcoI�/- in exon 18 0.264
Heterozygous and homozygous carriers of the Met235Thrvariant in the angiotensinogen gene and homozygous carriersof the Thr347Ser variant in the apolipoprotein (apo) A4 genehad an increased CVD risk (HR Met235Thrhet 1.25 (95% CI,1.05 to 1.48) and HR Met235Thrhom 1.23 (95% CI, 0.98 to1.54), respectively, and HR Thr347Serhom 1.37 (95% CI, 0.97to 1.93). Conversely, homozygous carriers of the Ser311Cysvariant in the paraoxonase-2 gene and homozygous carriersof the C1100T variant in the apoC3 gene had a decreasedCVD risk (HR, Ser311Cyshom 0.69 [95% CI, 0.47 to 1.01] andHR, C1100Thom 0.65 [95% CI, 0.46 to 0.91], respectively).Strong linkage disequilibrium was observed between thepolymorphisms C1100T in apoC3 and Thr347Ser in apoA4(D���0.928). Therefore, multifactorial Cox survival analy-sis was performed using the genotype combination of thepolymorphisms C1100T in apoC3 and Thr347Ser in apoA4(Table 4). Compared with homozygotes for the commonalleles, the SerSer�CC combination showed a significanteffect on risk (HR 1.43 [95% CI, 1.01 to 2.03]), whereasThrThr�TT combination showed a significant protectiveeffect on risk (HR, 0.69 [95% CI, 0.49 to 0.97]).
We investigated and confirmed the associations betweenspecific polymorphisms and hypertension, diabetes mellitus,obesity, and dyslipidemia, which were not introduced ascovariates in our models. However, to enable comparison ofour results with those from earlier studies, we performed thesame Cox regression model while adjusting not only for sexand smoking but also for hypertension (time-dependent),diabetes mellitus (time-dependent), body mass index, totalcholesterol, LDL-C, high-density lipoprotein cholesterol, tri-glycerides, lipoprotein(a), and homocysteine levels. The de-tected HRs were similar (data not shown). The genotypedistributions of polymorphisms associated with cardiovascu-lar disease are depicted in Table 5.
DiscussionWe investigated the contribution of a large number ofpolymorphisms in multiple candidate genes to CVD risk inFH patients. Strikingly, the G20210A polymorphism in the
TABLE 3. Multifactorial Cox Regression of Polymorphisms Associated WithCardiovascular Disease
Chromosome Gene Polymorphism P* HR 95% CI
1p Angiotensinogen Met235Thr 0.033
Met/Thr vs Met/Met 1.25 1.05–1.48
Thr/Thr vs Met/Met 1.23 0.98–1.54
7q Paraoxonase-2 Ser311Cys 0.049
Ser/Cys vs Ser/Ser 1.09 0.93–1.28
Cys/Cys vs Ser/Ser 0.69 0.47–1.01
11p Prothrombin G20210A �0.001
G/A vs G/G 2.44 1.56–3.83
A/A vs G/G No A/A patients
11q Apolipoprotein A4 Thr347Ser 0.049
Thr/Ser vs Thr/Thr 0.89 0.74–1.06
Ser/Ser vs Thr/Thr 1.37 0.97–1.93
11q Apolipoprotein C3 C1100T 0.023
C/T vs C/C 1.01 0.86–1.20
T/T vs C/C 0.65 0.46–0.91
*According to the likelihood ratio test.
Kaplan–Meier curves for cardiovascular event-free survival byG20210A genotype.
prothrombin gene was most strongly related to a significantlyincreased CVD risk. In addition, 4 other polymorphisms weresuggestively associated with CVD (P�0.05). Two wereassociated with increased CVD risk, namely the Met235Thrvariant in the angiotensinogen gene and the Thr347Servariant in the apoA4 gene. In contrast, the Ser311Cyssubstitution in the paraoxonase-2 gene and the C1100Tvariant in the apoC3 gene were associated with decreasedCVD risk. To our knowledge, this is the largest exploratorystudy on the association of genetic variants and CVD risk inFH.
The strengths of the present study lie in several areas. Tobegin with, by recruiting patients from all over the country,and by using our patient registration database, we minimizedselection bias toward large families and genetically isolatedcommunities. Moreover, by choosing a cohort design andderiving our “cases” and “controls” from a common popula-tion, we reduced the chance of population stratification andspurious associations.
Second, the large size of the cohort provided sufficientstatistical power to detect relatively small relative risks orHRs. Our initial power statement was based on calculating anodds ratio. A power statement for HR was difficult toformulate, because other, less easy to predict, assumptionslike median survival in control patients would have had tohave been included in that power analysis. In retrospect, theaverage median survival of homozygous wild-type patientswas 60 years, with a maximum follow-up of 85 years. Thus,in our cohort, HR of 1.6 (or 0.7 protective) could have beendetected with a power of 80% (with a Bonferroni corrected
2-sided P�0.0008) using 1940 patients and assuming that10% of the patients carried at least 1 rare allele. Because thecarrier frequency was even higher than 10% for most poly-morphisms, the actual power to detect a HR of 1.6 or higherexceeded 80%.
Last, we applied more stringent criteria for statisticalsignificance to avoid problems of “repetitive testing,” whichoften occurs in studies of multiple polymorphisms. Further-more, to examine possible false-positive associations causedby linkage disequilibrium between loci, we simultaneouslyassessed the effects of multiple polymorphisms in a multifac-torial Cox model. Importantly, a cohort study design enabledus to determine HRs. Many association studies have hadcase-control designs with odds ratios as the outcome param-eter. Whereas odds ratios mimic relative risks remarkablywell when the outcome of interest is rare, this is not the casefor CVD in FH, in which odds ratios might exaggerate theactual risk.
Several limitations of our study should also be mentioned.First, in this retrospective study, the primary source of datawas the patient’s medical records. Medical records are pri-marily intended for patient care and not for research purposes.Second, no standardized information was available on life-style factors, such as dietary habits and physical activity. Asa result, the interaction between these environmental factorsand genetic variants could not be studied.
Third, our study included patients that were referred to alipid clinic. In theory, patients with the most detrimentalgenetic profiles might have died before referral. Therefore,genetic polymorphisms associated with more severe CVD orearly death could have been missed, leading to an underesti-mation of the risk. Fourth, 11 of the 65 polymorphismsstudied were not in Hardy–Weinberg equilibrium. The exactreason for the deviation is not known and we can onlyspeculate on this. Importantly, the deviation from Hardy–Weinberg equilibrium is not because of mixed ethnic groups,because the Dutch population is known to be a homogenousone. More than 99% of our patients were white and patientswere randomly selected from all over the country. In addition,most deviations were caused by an excess of heterozygotes,which makes genotyping errors unlikely. Furthermore, theaccuracy of genotyping in 500 randomly selected DNAsamples was assessed by re-analysis of several polymor-phisms in three genes, revealing that �0.5% of the resultswere discordant. However, it must be stressed that these 3re-analyzed polymorphisms were not among the polymor-phisms that were not in Hardy–Weinberg equilibrium. Fi-nally, and most importantly, the 11 SNPs showed the samedeviation from Hardy–Weinberg equilibrium in patients withCVD as well as in patients without CVD; therefore, we are ofthe opinion that this has not affected our results significantly.Fifth, it should be stressed that by the nature of this explor-atory study the impact of the polymorphisms on intermediateend points such as coagulation factors (eg, prothrombin),blood pressure levels, angiotensinogen levels, and apoli-poprotein levels, could not be assessed in the present study.Therefore, we could not determine the relationship of theseintermediate end points with CVD, nor the subsequent
TABLE 5. Distributions of Polymorphisms Associated WithCardiovascular Disease
Gene and Polymorphism
CVD� CVD�
N % N %
Angiotensinogen Met235thr
Met/Met 220 34.2 503 38.8
Met/Thr 313 48.7 605 46.6
Thr/Thr 110 17.1 189 14.6
Paraoxonase-2 Ser311Cys
Ser/Ser 375 58.3 755 58.2
Ser/Cys 240 37.3 450 34.7
Cys/Cys 28 4.4 92 7.1
Prothrombin G20210A
G/G 623 96.9 1275 98.3
G/A 20 2.9 22 1.7
A/A 0 0 0 0
Apolipoprotein A4 Thr347Ser
Thr/Thr 426 66.3 875 67.5
Thr/Ser 179 27.8 367 28.3
Ser/Ser 38 5.9 55 4.2
Apolipoprotein C3 C1100T
C/C 360 56.0 718 55.4
C/T 247 38.4 467 36.0
T/T 36 5.6 112 8.6
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pathological mechanisms involved in the development ofCVD.
Our results are in line with earlier findings. The prothrom-bin mutation G20210A leads to increased plasma levels ofprothrombin and an increased risk of venous thrombosis.19
Many studies have focused on the causative role of thismutation in arterial disease, often with negative results.20,21
However, in a comprehensive meta-analysis investigating thepossible link between the G20210A prothrombin gene variantand different forms of premature ischemic heart disease in12 043 patients, the G20210A polymorphism was shown tobe a significant risk factor for myocardial infarction at ayoung age.22 In patients aged 55 years and younger, the oddsratio was 1.77 (95% CI, 1.16 to 3.42); in those aged 45 yearsand younger, the odds ratio was 2.30 (95% CI, 1.27 to 4.59).These odds ratios are similar to the HRs found in our study.In another meta-analysis, the association between theG20210A variant and arterial circulatory events was con-firmed.23 In addition, 2 other large studies showed a pro-nounced effect of this polymorphism on CVD events indyslipidemic patients.24,25 Although the relationship betweenthis prothrombin mutation and CVD remains the subject ofdebate in non-FH populations, these findings support thestrong association between G20210A carriership and prema-ture CVD risk in our FH patients who have severe dyslipid-emia from birth onwards.
Recently, the M235T polymorphism in the angiotensino-gen gene was associated with increased levels of angioten-sinogen and a corresponding increase in the risk of hyperten-sion, as assessed in 45 267 individuals.26 However, arelationship between this variant and CVD risk has neverbeen established in a large-scale study. Again, in our FHpatients, the combined effect of elevated angiotensinogen andLDL-C levels might explain the increased risk of CVD.
We identified 2 common variants in the ApoA1-C3-A4-A5gene cluster to be suggestively associated with CVD risk(Thr347Ser in apoA4 and C1100T in apoC3). Both appear tohave (statistically) independent effects, because they re-mained significant in the multifactorial model and the esti-mated HRs of the univariate and multifactorial model werealmost identical for both variants (data not shown). Apoli-poproteins play a central role in lipid metabolism and thiscluster of apolipoprotein genes has been identified as a locuswith significant effects on triglyceride levels.27–29 Remark-ably, in the first prospective study on genetic variants in thisgene cluster and CVD risk in the general population, exactlythe same 2 polymorphisms were associated with an increaseand decrease in risk, respectively.30 However, in their multi-factorial model, which also included other known risk factorssuch as body mass index and blood pressure, only theThr347Ser variant remained significantly associated withCVD risk. They concluded that the apparent protective effectof C1100T in the apoC3 gene is most likely caused by stronglinkage disequilibrium across the gene cluster. When bodymass index, blood pressure, total cholesterol, and triglycer-ides were also added to our multifactorial model, the effect ofthe Thr347Ser variant was no longer significant (data notshown). However, this apparent discrepancy could be causedby differences in genotype frequencies between the 2 popu-
lations. Furthermore, in both populations the inference offunctionality for these variants was hampered by the almostcomplete absence of patients homozygous for both the347Ser and the 1100T allele.
Many studies support the antiatherogenic role ofparaoxonase-1, an HDL-associated enzyme.31 The protectiveeffect is generally considered to be caused by the ability ofparaoxonase-1 to attenuate oxidative modification of lipopro-tein particles. Paraoxonase-2, a family member ofparaoxonase-1, is reported to have antioxidant properties andantiatherogenic capacities as well, although its exact physio-logical role is still unknown.31 Our results confirm a relation-ship between paraoxonase-2 and CVD, and are in concor-dance with the findings of an earlier study in FH patients, inwhich homozygotes for the Ser311Cys variant ofparaoxonase-2 seem to be protected against CVD.32
Considering the limitations of association studies, wesuggest our results should be replicated in addition to beingperformed in prospective studies of large and well-definedFH populations, in which genetic and environmental modifi-ers should be carefully monitored. The results of thishypothesis-generating study constitute a basis for furtherresearch and form a step forward in the unraveling of theunderlying mechanisms of CVD in FH.
AcknowledgmentsThis study was supported by a grant of the Netherlands HeartFoundation (98/165). Kastelein is an established investigator of theNetherlands Heart Foundation (grant 2000D039). We are indebted toRMS personnel who supported this study. We acknowledge themembers of the independent adjudication committee: R. J. G. Peters,MD, PhD, cardiologist; J. Stam, MD, PhD, neurologist; and D.Legemate, MD, PhD, vascular surgeon. We thank all the patients andthe specialists of the participating lipid clinics.
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Jansen et al CVD Risk and Genetic Variation in FH 1481
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Assuming that 10% of the patients carried at least one rare allele, and that 30% of patients
homozygous for the common allele would experience a CVD event, an odds ratio of 1.9 or
more could be detected with a power of 80% (p-value= 0.0008; Bonferroni correction for 65
polymorphisms to be tested). Based on the results of the pilot study, we assessed that
approximately 50% of the patients selected from the DNA-bank database would fulfil the
inclusion criteria. Therefore, we had to review the medical records of twice as many patients
as needed based on the power calculation. For the final study, we selected 4000 patients from
the database.
Table I and Figure I
Table I. Inclusion and exclusion criteria
Inclusion criteria Males and females
Age 18 years and older
FH diagnostic criteria:
I. Presence of a documented LDL-receptor mutation
Or
II. LDL-cholesterol level above the 95th percentile for sex and age,
In combination with at least one of the following:
(a) the presence of typical tendon xanthomas in the patient or in a first degree relative
(b) an LDL-cholesterol level above the 95th percentile for age and sex in a first degree relative
(c) proven CAD in the patient or in a first degree relative under the age of 60 years
Exclusion criteria Secondary causes of hypercholesterolemia such as renal, liver or thyroid disease
Hypercholesterolemia due to other genetic defects, such as familial defective apolipoprotein B
CAD indicates coronary artery disease
1
CVD risk and genetic variation in FH
Figure I. Selection of study patients
9300 patients – 62 lipid clinics
4000 patients – 27 lipid clinics
Preselection of patients from larger lipid clinics only
9188 patients – 48 lipid clinics
Selected at random (using Microsoft Excel)
DNA samples from patients, stored in central DNA bank database
Excluded after reviewing medical record for inclusion and exclusion criteria
1600Included after reviewing medical record for inclusion and exclusion criteria
2400
2
Marcel R. Fontecha, Jia Li, Joep C. Defesche and John J.P. KasteleinAngelique C.M. Jansen, Emily S. van Aalst-Cohen, Michael W.T. Tanck, Suzanne Cheng,
Genetic Determinants of Cardiovascular Disease Risk in Familial Hypercholesterolemia
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