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Familial hypercholesterolemia. The determination of phenotype
Jansen, A.C.M.
Publication date
2003
Link to publication
Citation for published version (APA):
Jansen, A. C. M. (2003). Familial hypercholesterolemia. The determination of phenotype.
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Susceptibilityy genes for all-cause
mortalityy from familial
hypercholesterolemia: :
aa large parent-offspring study
Angeliquee CM Jansen
1, John JP Kastelein
1, Eric JG Sijbrands*
Vascularr Medicine', Academic Medical Center, University of Amsterdam, Amsterdam,, Department of Metabolic and Vascular diseases2, Erasmus Medical Center,
Abstract t
Background d
Thee risk of cardiovascular disease and mortality among patients w i t h familial hypercholesterolemiaa varies to a large extent. We recently studied the contribution of genetic variantss in candidate genes for atherosclerosis and identified six polymorphisms associated withh cardiovascular risk. It is unknown whether such susceptibility genes also contribute to thee mortality from this disorder.
Methodss and results
Inn the present study, we analyzed the relationship between 65 polymorphisms in 36 candidate geness and all-cause mortality in the parents of 1473 FH index patients. Among 2946 parents, 19700 deaths took place in 110.725 person years. The overall standardized mortality ratio wass 1.44(95% CI: 1.37 to 1.51; p<0.001). Multivariate Cox survival analysis revealed that threee variants were indeed associated with increased mortality; C(-482)T in the apolipoprotein C33 gene, G!n27Glu in the p2-adrenergic receptor gene, and Thr71 lie in the apolipoprotein
BB gene. Conversely, the Thr26Asn substitution in the tumor necrosis factor beta gene and thee C825T variant in the G-protein B3 subunit were related to decreased mortality.
Conclusions s
Wee identified five polymorphisms in candidate genes for atherosclerotic vascular disease thatt were significantly associated with mortality in FH patients. Our findings support the generallyy presumed multifactorial model in which additional, heritable susceptibility genes influencee intermediate traits and life expectancy in FH. Moreover, it provides evidence that thee mortality risk of patients with familial hypercholesterolemia is certainly also based on additionall genetic risk factors for cardiovascular disease.
Introduction n
Cardiovascularr disease (CVD) is the number one cause of death in the western world. Carrierss of mutations in the low-density lipoprotein (LDL) receptor gene are exposed to severelyy increased serum cholesterol levels from birth onwards. This condition is thought to acceleratee the ageing of the arterial vessel wall and causes premature onset of cardiovascular diseasee resulting in excess mortality.1 Obviously, this paradigm of the relationship between LDL-cholesteroll and atherogenesis begins as a monogenic disorder but during life additional riskk factors determine trie uuiuen uf disease. Such a model \s supported by the known largee variation in the risk of cardiovascular disease2 and mortality3'4 among patients with familiall hypercholesterolemia. Their cardiovascular disease risk has been related to the type off LDL receptor mutation2, as well as to classical CVD risk factors such as male gender", smoking79,, lipoprotein(a)10 and low plasma high-density lipoprotein (HDL)-cholesterol levels.55111 Recently, we have identified additional genetic risk factors for cardiovascular diseasee in a large cohort with familial hypercholesterolemia.12 We do not know, however, whetherr such additional susceptibility genes also contribute to the mortality from the disorder, butt knowledge of this relationship could improve the identification of patients who are at particularlyy high risk and guide the development of strategies aimed at the individual to preventt premature death. The purpose of our study was to investigate whether susceptibility geness for cardiovascular disease also influence overall mortality. In the present study, we analyzedd the relationship between 65 polymorphisms in 36 candidate genes and mortality fromm familial hypercholesterolemia in the parents of 1473 index cases.
Methods s
Wee randomly selected a cohort of 4000 out of 9300 apparently unrelated, severely hypercholesterolemicc patients collected from 27 different hospitals throughout the Netherlands.. Their DNA was stored at a central DNA and Biobank at the Academic Medical Centree of the University of Amsterdam.
Familiall hypercholesterolemia was diagnosed according to the following criteria: (i) the presencee of a documented LDL receptor mutation, or (ii) an LDL-cholesterol level above the 95thh percentile for gender and age, in combination with (a) the presence of tendon xanthomas inn the patient or in a first-degree relative, or (b) an LDL-cholesterol level above the 95th percentilee for age and gender in a first-degree relative, or (c) proven coronary artery disease inn the patient or in a first-degree relative under the age of 60 years.
Extensivee clinical and biochemical information was acquired by reviewing the index patients' medicall records and with questionnaires. In these questionnaires, patients were asked to
providee the dates of birth and death of their parents. Additional information was sought fromm general practitioners and hospitals that patients had visited formerly. The Ethics Institutionall Review Board of each participating hospital approved the protocol.
Molecularr analyses
Wee extracted genomic DNA from peripheral blood leukocytes by standard procedures. Genotypingg was performed by Roche Molecular Systems, CA, USA. The laboratory procedures usedd have been described earlier by Cheng et aU3 We chose 65 polymorphisms in 36 candidatee genes that had previously been characterized and associated with cardiovascular diseasee by their contribution to lipid metabolism, blood pressure regulation, coagulation andd hemostasis, homocysteine metabolism, endothelial function, cell adhesion, inflammation orr plaque stability.
Statisticall analysis
Thee mortality of all parents of the index patients was compared with the mortality of the Dutchh population standardized for age, gender and calendar period - standardized mortality ratioo (SMR) = 1.0 for the Dutch population - as described previously.3" Inclusion of the parentall years before the birth of the index case would result in selection on years without deathh and consequently to underestimation of the mortality risk. Therefore, we excluded thesee years from the analysis. Each parent had 0.5 probability of being affected. Hence, the observedd SMRs exhibit 50% of the excess mortality from familial hypercholesterolemia. Thee cumulative survival of the parents according to genetic polymorphisms was analyzed withh multivariate Cox proportional hazard regression analysis and the Kaplan-Meier method. Parentss that were still alive at the moment of the study were censored at the date the questionnairee was filled out. First, each polymorphism was assessed individually with the usee of an additive or dominant model. The genetic model with the maximum log-likelihood wass selected as "best fitting" inheritance model and entered into a multivariate model. As covariates,, we added gender and the year of birth of the parent, since mortality in familial hypercholesterolemiaa is known to vary with calendar period.4 Statistical analyses were performedd using SPSS software (version 10.0, Chicago, Illinois). Hazard ratios with a p-valuee of less than 0.05 were considered statistically significant.
Results s
Inn 1473 FH index patients, complete genotypes for all 65 polymorphisms and information onn the parents for the mortality analyses were available. The remaining 927 patients could nott be analyzed because DNA data were not complete and/or information about their
parentss was unavailable.
Thee cohort of 1473 index patients consisted of 690 males and 783 females, with a mean agee of 50.5 12.6 years. A total of 459 patients (31.2 %) had a cardiovascular event (27.9 %% coronary artery disease, 2.9 % cerebrovascular disease and 4.3 % peripheral arterial disease).. Classical risk factors, such as diabetes mellitus and hypertension occurred in 5.4 % andd 8.5 % of patients, whereas 69.3 % of patients had ever smoked. As expected, mean totall cholesterol and LDL-cholesterol levels were increased (9.61 2.01 mmol/L and 7.48 1.977 mmol/L, respectively). Mean HDL-cholesterol (1.23 0.35 mmol/L), and median trinlvrpriHee levels 11.54; interouartile ran^e: 1.09 - 2 . 1 3 mmol^L^ were in the norma! ranne off our general population.
Amongg all 2946 parents, 1970 deaths took place in 110.725 person years. The overall SMR off the parents was 1.44 (95% CI: 1.37 to 1.51; p<0.001). Mortality reached a maximum beforee 1975 and fell thereafter (data not shown). The cumulative survival in the parents accordingg to gender is shown in Figure 1. The SMR of men was 1.53 (95% CI: 1.43 to 1.63; p<0.001)) and of women 1.33 (1.24 to 1.43; p<0.001). The mortality risk of men relative to womenn was 2.27 (95% CI: 2.07 to 2.48; p<0.001). When the index patients had cardiovascularr disease, excess mortality was observed in his/her parents (HR 1.21; 95% CI
1.100 - 1.32; p<0.001).
Figuree 1. Cumulative survival in the 2946 parents
accordingg to gender
00 20 40 60 80 100 Agee (years)
Ann overview of the 65 polymorphisms in the 36 genes involved in the study is presented in Tablee 1. Table 2 shows the polymorphisms that had significant effects on the mortality risk off the parents. In this table, we also show their relation with effects on the cardiovascular diseasee risk in the 1473 index cases. Three polymorphisms were associated with increased mortality;; C(-482)T in the apolipoprotein (apo) C3 gene, Gln27Glu in the (3,-adrenergic receptorr gene, and Thr71 lie in the apoB gene. Conversely, Thr26Asn in the tumor necrosis factorr beta gene and C825T in the G-protein B3 subunit were significantly associated with decreasedd mortality. None of these 5 polymorphisms were in linkage disequilibrium and all
weree in Hardy-Weinberg equilibrium. Although not significant, the point estimates of cardiovascularr disease risk in the index cases were in line with the findings on mortality risk inn the parents.
Tablee 1. The 65 polymorphisms examined in the Gene e
Lipidd metabolism
Lipoprotein(a) )
AA pol ipo protein A4
Apolipoproteinn B Apolipoproteinn C3 Apolipoproteinn E {^-adrenergicc receptor Cholesteryll ester transferr protein Hepaticc lipase Loww density lipoprotein receptor r
Lipoproteinn lipase
Paraoxonase e
Paraoxonase-2 2 Peroxisomee proliferator-activatedd receptor gamma2
Polymorphism m C93T T G121A A Thr347Ser r Gln360His s Thr71lle e C(-6411 )A C(482)T T T(455)C C C1100T T C3175G(Sstl) ) T3206G G Cys112Arg g ArglBSCys s Trp64Arg g C(-631)A A C(-629)A A He405Val l Asp442Gly y G(+1)A A (+3)TT ins in intron 14 TaqlB B C(480)T T Ncol+AA in exon 18 T(-93)G G Asp9Asn n Asn291Ser r Ser447Term m Met55Leu u Gln192Arg g Ser311Cys s Pro12Ala a
Bloodd pressure regulation (part 1)
Dipeptidyl l carboxypeptidasee 1 (=Angiotensin-conn verting enzyme) )
Angiotensinogen n
Angiotensinn II receptor type 1 Alpha-adducin n
Atriall natriuretic peptide precursorr A Aluu element l/D inn intron 16 Met235Thr r A1166C C Gly460Trp p G664AA (Val7Met) T2238CC (Sea I) atudy y Gene e Polymorphism m
Bloodd pressure regulation (part II)
B2-adrenergicc receptor
Epitheliall sodium channel
G-proteinn 83 subunit
Coagulationn and hemostasis
Coagulationn factor II (Prothrombin) ) Coagulationn factor V Coagulationn factor VII
B-fibrinogen n Glycoproteinn la Glycoproteinn Ilia Plasminogen-activatorr inhibitor typee 1 Homocysteinee metabolism
Cystathionee beta synthase
Methylenetetrahydrofolate e reductase e
Endotheliall function
Nitricc oxide synthase type 3
Arg16Gly y G!n27Glu u Trp493Arg g Ala663Thr r C825TT (splice variant) G20210A A Arg506Gln n Arg353Gln n (-323)) 10-bp del/ins G(455)A A G873A A Leu33Pro o (-675)) 5G/4G G11053T T Combination n He278Thrr and 68-bp insertion n C677T T A(-922)G G C(-690)T T G894TT (Glu298Asp)
Celll adhesion, inflammation and plaque stability
E-selectinn (=endothelial leukocyte adhesionn molecule-1; ELAM) Intercellularr adhesion mo!ecu!e-1 Tumorr necrosis factor alfa
Tumorr necrosis factor betaa (lymphotoxin alpha) Metalloproteinasee 3 (Stromelysin-I) ) Ser128Arg g Leu554Phe e Gly214Arg g G(-376)A A G(-308)A A G(-244)A A G(-238)A A Thr26Asn n (-1171)5A/6A A
Discussion n
Wee analyzed the relationship between genetic variants in candidate genes for atherosclerosis andd alkause mortality in familial hypercholesterolemia. Polymorphisms in three genes, namely thosee encoding apoCIII, the (^-adrenergic receptor and apoB, were associated with increased mortality,, while polymorphisms in the genes encoding tumor necrosis factor beta and G-proteinn B3 subunit were related to a better life expectancy.
Wee recently studied the relationship between the same set of polymorphisms and the occurrencee of cardiovascular disease in another cohort of FH patients and identified five susceptibilityy genes.12 Remarkably, only genetic variants in the apoC3 gene yielded significant hazardd ratios in both analyses. Taken together, our findings support the generally presumed multifactoriall model in which additional susceptibility genes are inherited and influence intermediatee traits and life expectancy. Moreover, it provides evidence that the mortality riskk of patients with familial hypercholesterolemia is certainty also partly based on additional geneticc risk factors for cardiovascular disease.
Interestingly,, the apoC3 gene was related to both mortality as well as to cardiovascular disease inn our earlier analysis. This gene is part of the complex apolipoprotein gene cluster apoA1-C3-A4-A5.. Apolipoproteins play a central role in lipid metabolism and this cluster of apolipoprotein geness has been identified as a locus with significant effects on triglyceride levels and HDL-cholesterol.14155 These lipoprotein particles have been described to play an important role in thee occurrence of cardiovascular disease in familial hypercholesterolemia.51116 Remarkably, twoo different polymorphisms in the apoC3 gene were related to CVD and mortality. However, manyy polymorphisms in this complex gene cluster are in linkage disequilibrium and these twoo polymorphisms may be linked to the same unidentified functional defect in or nearby thee apoA1-C3-A4-A5 gene cluster.
Beta2-adrenergicc receptors are cell-surface receptors that mediate vasodilation in response too adrenergic agonists, and in healthy myocardium they mediate chronotropic and inotropic responsess to adrenergic agents.'7 Common polymorphisms have been associated with an alteredd vascular response to sympathetic stimulation.18 So far, little is known about their associationn with major cardiovascular endpoints; we found an increase in all-cause mortality inn carriers of a common polymorphism in this gene.
Thee Thr71 Me polymorphism in the apolipoprotein B gene was significantly associated with increasedd mortality in our study. ApoB is a structural protein for the LDL-cholesterol particle andd the ligand for the LDL receptor. The Thr71 Me mutation in the apoB gene has been relatedd with elevated levels of apoB-containing lipid particles.19-20 A recent meta-analysis suggestedd a relationship between several polymorphisms in the apoB gene and the risk of coronaryy artery disease.21
Tablee 2. Polymorphisms associated with cumulative survival in the parents and their relationship with CVD
inn the index cases
Gene e
AA pol i po protein C3 B2-adrenergicc receptor Apolipoproteinn B
Tumorr necrosis factor beta (lymphotoxinn alpha) G-protetnn B3 subunit Polymorphism m C(-482)T T Gln27Glu u Thr71lle e Thr26Asn n C825T T HRR indicates hazard ratio; CVD, cardiovascular
Bestt fitting geneticc model Dominant t Dominant t Additive e Additive e Dominant t disease e HR R 1.29 9 1.14 4 1.10 0 0.87 7 0.89 9 Totall mortality 95%% CI 1.099 - 1.52 1.011 - 1.28 1.02-- 1.19 0.79-0.96 6 0.811 -0.99 P P 0.003 3 0.03 3 0.02 2 0.005 5 0.03 3
Tumorr necrosis factor beta {or lymphotoxin alpha) acts as a pro-inflammatory factor. Genetic variantss in this gene might contribute to an increased risk for myocardial infarction. Indeed recently,, a large-scale, association study using 92,788 SNP's identified variants in this gene ass strongg risk factors for myocardial infarction.22
Lastly,, the C825T variant in the G-protein B3 subunit gene, a candidate gene for hypertension andd obesity, lead to a longer survival in our FH cohort study. Few studies have addressed thee role of this gene in the occurrence of CVD. Recently, the T-allele was found to be associatedd with the presence and severity of coronary artery disease in angiographically examinedd patients.23
All-causee mortality is by definition very heterogeneous, although in case of FH it is very likely thatt the majority of deaths is caused by cardiovascular disease. It is nevertheless remarkable thatt we found effects of five polymorphisms in candidate genes for atherosclerosis. In fact, wee analyzed both parents who had a 0.5 probability of having familial hypercholesterolemia andd we still were able to observe the burden of FH. Nonetheless, we preferred to analyze all parents,, because analyses restricted to certainly-affected parents could lead to an underestimationn of the mortality risk as a result of selection on living parents.
Thee strength of the present study lies in the fact that we avoided such a selection. In addition,, by excluding index cases, this mortality method also reduces selection on cardiovascularr disease. This was illustrated by the fact that the observed SMR was comparable too our earlier findings in a large pedigree without selection on cardiovascular disease/
Onee could argue that a number of our findings might be a random result of multiple testing.. This is, however, highly unlikely, because we analyzed functional polymorphisms in
CVD D HR R 1.05 5 1.13 3 1.03 3 0.93 3 1.03 3 95%% CI 0.82-- 1.34 0.93-- 1.37 0.92-- 1.16 0.80-- 1.09 0.88-- 1.21 P P 0.7 7 0.2 2 0.6 6 0.4 4 0.7 7
aa large population and the observed risk estimates were all pointing in the expected directions.. In addition, they were in line with the estimates of cardiovascular risk in the indexx cases. Moreover, some associations were unexpectedly strong considering that we analyzedd all-cause mortality in a model that certainly has a small sensitivity to detect effects: wee analyzed mean effects in parents with a probability of carrying a polymorphism. We are convincedd that knowledge on additional genetic risk from future genome-wide analyses willl improve the identification of patients who are at particularly high risk.
Wee found a discrepancy between the susceptibility genes for cardiovascular disease12 and all-causee mortality. This is however not unexpected since pathophysiological mechanisms involvedd in cardiovascular disease may be different from those underlying fatal coronary arteryy events. In addition, all-cause mortality is very heterogeneous and, furthermore, the discrepancyy between the findings is probably also based on the larger period of exposure inn the parents when compared to the index patients.
Finally,, considering the limitations of association studies, we suggest our results should be replicatedd before conclusions can be drawn about familial hypercholesterolemia in general andd before these results can be extrapolated to cohorts without hypercholesterolemia. However,, we conclude, from the results obtained in our study, that polymorphisms in the apoC33 gene, the P2-adrenergic receptor gene and the apoB gene contribute substantially
too excess mortality, while polymorphisms in the tumor necrosis factor-p gene and the G-proteinn B3 subunit gene are related to better life expectancy among patients with familial hypercholesterolemia.. Moreover, our findings suggest that these susceptibility genes for all-causee mortality do constitute an inherited risk of cardiovascular disease.
Acknowledgments s
Thiss study was supported by a grant of the Netherlands Heart Foundation (98/165). John JPP Kastelein is an established investigator of the Netherlands Heart Foundation (grant 2000D039)) We are indebted to Suzanne Cheng and Jia Li of Roche Molecular Systems, Alameda,, California, USA, for performing the genotyping and generously providing research reagents.. We thank all the patients and the specialists of the participating Lipid Clinics.
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