Abstract
Activation of the complement system seems an important link between inflammation and atherogenesis. The Y402H polymorphism of complement factor H (CFH) has been associated with cardiovascular events, but results are conflicting and possibly modified by age of onset of cardiovascular disease (CVD).
We determined whether or not the Y402H polymorphism influenced CVD risk in a multicentre cohort study involving 2016 unrelated patients with familial hypercholesterolaemia (FH), who have an extremely increased susceptibility to premature CVD. We identified 261 individuals who were homozygous for the polymorphism (CC genotype; 12.9%), 929 individuals who were heterozygous (TC genotype; 46.1%), and 826 individuals carried the wild-type (TT genotype; 41.0%). During 95 115 person years, 644 patients had a cardiovascular event. Carriers of the CC genotype had a decreased risk of CVD (hazard ratio 0.67, 95% confidence interval 0.51–0.87; P = 0.003) relative to the other genotype groups. This association was unaltered after adjustment for clinically relevant cardiovascular risk factors or age effects.
Among patients with severely increased risk of early onset CVD, the Y402H CFH variant was inversely associated with susceptibility to CVD. This suggests that CFH is a modifier gene of CVD.
Introduction
Atherosclerosis is a chronic inflammatory disorder and its initiation and progression are thought to be influenced by activation of the complement system.1–3 Previous studies have shown that, although complement activation is nearly absent in normal arteries, it is extensively activated in atherosclerotic lesions.4,5 During complement activation, host cells are protected from being damaged as innocent bystanders by regulatory proteins, including complement factor H (CFH).6 Immunohistochemical studies have detected CFH in early human coronary artery lesions.7 Interestingly, CFH was exclusively located in the superficial layer of the arterial intima, suggesting that it may protect the arterial wall from damage by excess complement activation.7
Several variants in the CFH gene have been identified, but the Y402H polymorphism (rs 1061170) is of particular interest because it is located within the binding site for heparin and C-reactive protein.6 It is proposed that the replacement of tyrosine for histidine at position 402 in exon 9 may affect the binding properties of CFH on host surfaces, potentially influencing complement activation, immune responses, and inflammation.8 Three independent genomic studies identified the Y402H polymorphism of CFH that may account for almost half of all age-related macular degeneration (AMD) cases.9–11 Age-related macular degeneration is a progressive late-onset disease affecting central vision and is the leading cause of irreversible blindness in the elderly western population.12 Several replication studies established the association of this CFH variant with increased risk of AMD in different ethnic populations and different clinical subtypes of the disease.13 Epidemiological, genetic, and pathological evidence suggest that similar pathways may be involved in the aetiologies of AMD and atherosclerosis.14
To date, seven studies have been conducted on potential associations between the CFH Y402H polymorphism and cardiovascular disease (CVD).15–21 In contrast with the strong and consistent findings in AMD, however, they have yielded conflicting results. Recently, it was postulated that this inconsistency may be due to heterogeneity in age of onset of CVD.21 This hypothesis could explain the association of the CFH Y402H polymorphism with increased risk of CVD in studies regarding elderly subjects and the inverse association with risk of early CVD in two other populations.17,20,21 However, previous studies had limited power to detect stratum-specific effects and the proposed inverse relationship between the CFH Y402H and risk of earlier onset CVD should be confirmed in a population at increased risk of premature CVD.
Patients with heterozygous familial hypercholesterolaemia (FH) have severely elevated low-density lipoprotein (LDL) cholesterol levels and as a result they belong to those at highest risk of premature CVD.22 The atherosclerotic burden of FH shows, however, large variation.23 The disorder is considered to be an exemplary model to analyse secondary (or modifier) genes involved in CVD.24 In the present study, we assessed the relationship between the Y402H polymorphism in CFH and susceptibility to CVD in a large cohort of patients with heterozygous FH.
Methods
Study design and patients
The study population and data collection of the FH cohort have been described in detail elsewhere.25 In brief, lipid clinics throughout The Netherlands submitted DNA samples of clinically suspected patients with FH to a central laboratory for LDL receptor mutation analysis. From this database, we randomly selected a cohort of 4000 out of 9300 patients, who were collected between 1989 and 2002. We excluded subjects with secondary causes of hypercholesterolaemia and those with hypercholesterolaemia caused by other genetic defects, such as familial defective apolipoprotein B. A total of 2400 unrelated patients, aged 18 years and older, fulfilled the diagnostic criteria for FH as they were previously published.25 DNA was available of 2016 patients with heterozygous FH for the present analyses. Of these patients, over 99% were Caucasian and 1055 (52.3%) individuals had a documented LDL receptor mutation. The Institutional Review Board of each participating hospital approved the study protocol and informed consent was obtained from all patients.
We assessed cardiovascular risk factors using the following definitions. Smoking was defined as ever smoking. Hypertension was defined as systolic blood pressure >140 mmHg or diastolic blood pressure >90 mmHg at three consecutive office visits. Patients on anti-hypertensive medication were only added to the hypertension group, when a documented diagnosis was available. Diabetes mellitus was defined as patients using anti-diabetic medication or fasting plasma glucose >6.9 mmol/L. Lipid levels were determined in fasting patients not using lipid-lowering medication for at least 6 weeks. Total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured by standard methods. Low-density lipoprotein cholesterol was calculated with the Friedewald equation.
Cardiovascular disease
The primary clinical endpoint of the present genetic association study was CVD, defined as fatal or non-fatal CVD using internationally accepted criteria as published before.25 Secondary endpoints of the study were coronary heart disease, cerebral artery disease, and peripheral artery disease. Coronary heart disease was defined as: (i) myocardial infarction, (ii) percutaneous coronary intervention or other invasive procedures, (iii) coronary artery bypass grafting, and (iv) angina pectoris. Cerebral artery disease was defined as: (i) ischaemic stroke and (ii) documented transient ischaemic attack. Peripheral artery disease was defined as: (i) peripheral arterial bypass grafting, (ii) peripheral percutaneous transluminal angioplasty or other percutaneous invasive intervention, and (iii) intermittent claudication.
Molecular analysis
Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols.26 Considering the concordance of previous association studies for CFH Y402H in AMD populations, we investigated specifically this polymorphism. The Y402H polymorphism in CFH was detected with molecular analysis of the CFH gene with the standard protocol of the 7900HT Taqman (Applied Biosystems, Foster City, USA). Two oligonucleotide primers were used to identify the replacement of thymine (T) by cytosine (C) on position 1277.9
Statistical analyses
All data were analysed using SPSS for Windows software package version 15.0 (SPSS Inc., Chicago, IL, USA). Deviations of the genotype distribution from that expected for a population in Hardy–Weinberg equilibrium were tested using χ2 statistics with one degree of freedom. Genotype and allele frequencies among patients with and without history of CVD were compared with the χ2 test. Differences between CFH genotypes were tested with χ2 statistics for dichotomous variables and independent sample t-test for continuous variables. If dependent variables were not normally distributed, logarithmic transformations were applied. To adjust statistical tests for age and gender, we used multiple linear regression analysis.
Cox proportional hazard regression analysis was used to determine the association between the CFH variant and the occurrence of CVD. The proportional hazards assumption was tested by drawing log minus log plots of the survival function and was met for all Cox proportional hazard models. Follow-up started at birth and ended at the first occurrence of established fatal or non-fatal CVD. Patients without CVD were censored at the date of the last lipid clinic visit or at the date of death attributable to other causes. Complement factor H genotype was analysed according to an additive, dominant, or recessive genetic model, respectively. The recessive genetic model (homozygous carriers relative to heterozygous and wild-type carriers) had the highest log-likelihood (data not shown) and was used in the present analyses. The cumulative CVD-free survival, without adjustment for co-variables, was illustrated with the Kaplan–Meier method. Initially, we adjusted in the multiple Cox regression analysis for variables that are independent of the polymorphism: year of birth, gender, and smoking. For smoking, we implemented a linearly decreasing risk effect for the 6 years after cessation.27 Additionally, adjustment was made for all other variables that were significant risk factors of CVD in Cox regression analysis. The presence of the homozygous CFH variant in young patients, who did not yet express their high cardiovascular risk, could potentially have led to lower risk estimates. Therefore, we tested the effect of age by separate analyses of age tertiles and by adjusting for age tertiles in the Cox regression analysis. All reported P-values are based on two-sided tests of significance. Statistical significance was assessed at the 5% level.
Results
Genotype and allele frequencies
We observed homozygosity for the Y402H polymorphism in 261 (CC genotype; 12.9%) patients, heterozygosity in 929 (TC genotype; 46.1%) patients, and 826 (TT genotype; 41.0%) patients carried the wild-type. Genotype distributions in the total cohort and in the patients with and without history of CVD were in Hardy–Weinberg equilibrium (P > 0.18).
To compare our results with previous case–control studies, we reported all genotype and allele frequencies in Table 1. The distribution of CFH genotypes was significantly different between patients with and without history of CVD (P = 0.003) as a result of higher frequency of the CC genotype in patients without history of CVD (P = 0.001).
Genotype and allele frequencies for the T→C variation in CFH gene coding for Y402H variant
| Genotype or allele | Patients with CVD | Patients without CVD |
|---|---|---|
| n (frequency) | n (frequency) | |
| TT | 270 (0.419) | 556 (0.405) |
| TC | 314 (0.488) | 615 (0.448) |
| CC | 60 (0.093) | 201 (0.147) |
| T allele | 854 (0.663) | 1727 (0.629) |
| C allele | 434 (0.337) | 1017 (0.371) |
| Genotype or allele | Patients with CVD | Patients without CVD |
|---|---|---|
| n (frequency) | n (frequency) | |
| TT | 270 (0.419) | 556 (0.405) |
| TC | 314 (0.488) | 615 (0.448) |
| CC | 60 (0.093) | 201 (0.147) |
| T allele | 854 (0.663) | 1727 (0.629) |
| C allele | 434 (0.337) | 1017 (0.371) |
CFH, complement factor H; CVD, cardiovascular disease.
Characteristics of patients
Comparisons of general characteristics among the genotypes are presented in Table 2. Patients with the CC genotype had significant lower plasma triglyceride levels [difference (±standard deviation), 0.14 ± 0.07 mmol/L; P = 0.01]. We found no significant differences in demographics, the presence of smokers, hypertension, and diabetes mellitus, body mass index (BMI), and other plasma lipid concentrations between the CC genotype and other genotypes.
Characteristics of 2016 patients with familial hypercholesterolaemia according to the genotypes of the CFH Y402H polymorphism
| Characteristic | TT genotype | TC genotype | CC genotype | P-valuea |
|---|---|---|---|---|
| (n = 826) | (n = 929) | (n = 261) | ||
| Age at first lipid clinic visit (years) | 45.2 (±12.4) | 44.7 (±12.7) | 43.9 (±13.2) | 0.2 |
| Age at last lipid clinic visit (years) | 50.3 (±12.8) | 49.8 (±13.3) | 48.9 (±13.9) | 0.2 |
| Males (%) | 47.3 | 48.5 | 44.8 | 0.3 |
| Smoking, ever (%) | 73.5 | 74.1 | 67.5 | 0.05b |
| Hypertension (%) | 9.9 | 8.4 | 7.7 | 0.7b |
| Diabetes (%) | 5.6 | 6.2 | 5.4 | 0.9b |
| Body mass index (kg/m2) | 25.1 (±3.3) | 25.1 (±3.6) | 24.9 (±3.5) | 0.5b |
| Total cholesterol (mmol/L) | 9.44 (±2.02) | 9.60 (±1.99) | 9.47 (±1.92) | 0.8b |
| Low-density lipoprotein cholesterol (mmol/L) | 7.30 (±1.96) | 7.40 (±1.91) | 7.42 (±1.85) | 0.6b |
| High-density lipoprotein cholesterol (mmol/L) | 1.22 (±0.36) | 1.23 (±0.37) | 1.19 (±0.34) | 0.07b |
| Triglycerides (mmol/L) | 1.80 (±1.04) | 1.81 (±0.99) | 1.66 (±1.01) | 0.02b,c |
| Characteristic | TT genotype | TC genotype | CC genotype | P-valuea |
|---|---|---|---|---|
| (n = 826) | (n = 929) | (n = 261) | ||
| Age at first lipid clinic visit (years) | 45.2 (±12.4) | 44.7 (±12.7) | 43.9 (±13.2) | 0.2 |
| Age at last lipid clinic visit (years) | 50.3 (±12.8) | 49.8 (±13.3) | 48.9 (±13.9) | 0.2 |
| Males (%) | 47.3 | 48.5 | 44.8 | 0.3 |
| Smoking, ever (%) | 73.5 | 74.1 | 67.5 | 0.05b |
| Hypertension (%) | 9.9 | 8.4 | 7.7 | 0.7b |
| Diabetes (%) | 5.6 | 6.2 | 5.4 | 0.9b |
| Body mass index (kg/m2) | 25.1 (±3.3) | 25.1 (±3.6) | 24.9 (±3.5) | 0.5b |
| Total cholesterol (mmol/L) | 9.44 (±2.02) | 9.60 (±1.99) | 9.47 (±1.92) | 0.8b |
| Low-density lipoprotein cholesterol (mmol/L) | 7.30 (±1.96) | 7.40 (±1.91) | 7.42 (±1.85) | 0.6b |
| High-density lipoprotein cholesterol (mmol/L) | 1.22 (±0.36) | 1.23 (±0.37) | 1.19 (±0.34) | 0.07b |
| Triglycerides (mmol/L) | 1.80 (±1.04) | 1.81 (±0.99) | 1.66 (±1.01) | 0.02b,c |
CFH, complement factor H.
Values are given as means ± standard deviation.
aComparison between the CC genotype and other genotypes.
bAdjusted for age and gender.
cStatistical testing after logarithmic transformation.
Complement factor H genotype and cardiovascular disease
During 95 115 person years, 644 patients had their first cardiovascular event. Mean age of onset of CVD (±SD) was 48.5 ± 10.7 years. In Figure 1, the Kaplan–Meier curves show the CVD-free survival of patients with the CC genotype and TT/TC genotype. Without adjustment for co-variables, carriers of the CC genotype had a decreased risk of CVD compared with carriers of other genotypes (HR 0.67, 95% CI 0.51–0.87; P = 0.003). As shown in Table 3, year of birth, male sex, smoking, the presence of diabetes, higher BMI, lower levels of HDL cholesterol, and higher triglyceride levels were significantly associated with an increased cardiovascular risk in univariate analyses. In Cox regression analysis with adjustment for year of birth, gender, and smoking, the CC genotype was significantly associated with 0.66 decreased risk of CVD (P = 0.003; Table 3). Additional adjustment for other risk factors did not change the effect of CC genotype on the decreased susceptibility to CVD in our population (P = 0.003). Furthermore, we found similar results among men and women (data not shown).
Kaplan–Meier curves for cardiovascular disease-free survival among familial hypercholesterolaemia patients with the CC genotype and TT/TC genotypes of complement factor H gene. Carriers of the CC genotype had a decreased risk of CVD compared with carriers of other genotypes (RR 0.67, 95% CI 0.51–0.87; P = 0.003).
Kaplan–Meier curves for cardiovascular disease-free survival among familial hypercholesterolaemia patients with the CC genotype and TT/TC genotypes of complement factor H gene. Carriers of the CC genotype had a decreased risk of CVD compared with carriers of other genotypes (RR 0.67, 95% CI 0.51–0.87; P = 0.003).
Hazard ratio of cardiovascular disease estimated with Cox regression analyses in patients with familial hypercholesterolaemia
| Variables | Hazard ratio (95% CI) | P-value | |
|---|---|---|---|
| Analyses of single variables | |||
| Year of birth | 1.07 | (1.06–1.08) | <0.001 |
| Males | 2.96 | (2.51–3.48) | <0.001 |
| Smoking | 1.90 | (1.54–2.34) | <0.001 |
| Diabetes mellitus | 1.31 | (1.03–1.66) | 0.03 |
| Hypertension | 1.13 | (0.91–1.40) | 0.3 |
| Body mass index | 1.03 | (1.00–1.06) | 0.03 |
| Low-density lipoprotein cholesterol | 0.97 | (0.92–1.01) | 0.1 |
| High-density lipoprotein cholesterol | 0.37 | (0.27–0.49) | <0.001 |
| Triglycerides | 1.23 | (1.08–1.39) | 0.001 |
| CC genotype of CFH gene | 0.67 | (0.51–0.87) | 0.003 |
| Analyses of CC genotype of CFH gene adjusted for | |||
| Year of birth, gender, and smoking | 0.66 | (0.50–0.87) | 0.003 |
| Year of birth, gender, smoking, diabetes, body mass index, high-density lipoprotein cholesterol, triglycerides | 0.55 | (0.37–0.81) | 0.003 |
| Analyses of CC genotype of CFH gene and age tertilesa | |||
| 19–43 years | 1.26 | (0.64–2.46) | 0.5 |
| 44–55 years | 0.52 | (0.30–0.90) | 0.02 |
| 56–86 years | 0.68 | (0.47–0.98) | 0.04 |
| Variables | Hazard ratio (95% CI) | P-value | |
|---|---|---|---|
| Analyses of single variables | |||
| Year of birth | 1.07 | (1.06–1.08) | <0.001 |
| Males | 2.96 | (2.51–3.48) | <0.001 |
| Smoking | 1.90 | (1.54–2.34) | <0.001 |
| Diabetes mellitus | 1.31 | (1.03–1.66) | 0.03 |
| Hypertension | 1.13 | (0.91–1.40) | 0.3 |
| Body mass index | 1.03 | (1.00–1.06) | 0.03 |
| Low-density lipoprotein cholesterol | 0.97 | (0.92–1.01) | 0.1 |
| High-density lipoprotein cholesterol | 0.37 | (0.27–0.49) | <0.001 |
| Triglycerides | 1.23 | (1.08–1.39) | 0.001 |
| CC genotype of CFH gene | 0.67 | (0.51–0.87) | 0.003 |
| Analyses of CC genotype of CFH gene adjusted for | |||
| Year of birth, gender, and smoking | 0.66 | (0.50–0.87) | 0.003 |
| Year of birth, gender, smoking, diabetes, body mass index, high-density lipoprotein cholesterol, triglycerides | 0.55 | (0.37–0.81) | 0.003 |
| Analyses of CC genotype of CFH gene and age tertilesa | |||
| 19–43 years | 1.26 | (0.64–2.46) | 0.5 |
| 44–55 years | 0.52 | (0.30–0.90) | 0.02 |
| 56–86 years | 0.68 | (0.47–0.98) | 0.04 |
CFH, complement factor H.
aMultiple Cox regression analyses of CC genotype of CFH gene adjusted for year of birth, gender, and smoking according to the age tertiles in the cohort.
Age effects
The average (±SD) age at end of follow-up of patients without CVD was 46.6 ± 12.8 years and this was significantly lower than the mean age (48.5 ± 10.7 years) at onset of CVD in the other group. The presence of the homozygous CFH variant in young patients, who did not yet express their high cardiovascular risk, could potentially have led to lower risk estimates. To test for effects of the CC genotype on CVD risk with age, we stratified the cohort by age tertiles and performed separate Cox regression analyses in these three age strata. The results of these analyses are shown in Table 3. In the lower age tertile, the CC genotype was not associated with CVD. The point-estimates of the hazard rates in the two upper age tertiles were similar to the findings in the total cohort. Actually, we may have underestimated the cardioprotective effect of the CC genotype in our total cohort as a result of the presence of young patients. Therefore, we performed an analyses restricted to the upper two age tertiles adjusting for year of birth, gender, and smoking and found a hazard ratio of 0.61 (95% CI, 0.45–0.82; P = 0.001). Finally, we performed a Cox regression analysis with adjustment for year of birth, gender, smoking, and age tertiles: patients with the CC genotype had 0.66 (95% CI, 0.50–0.88; P = 0.004) times less frequent CVD relative to patients with the other genotypes.
Complement factor H genotype and secondary outcomes
As expected, coronary heart disease was the most prevalent cardiovascular event in the population: in 549 (85.2%) of the FH patients with a history of CVD the first event was coronary heart disease, in 39 (6.1%) patients the first event was cerebral artery disease, and peripheral artery disease was the first event in 56 (8.7%) patients. The relationship between the CC genotype of the CFH gene and these three major cardiovascular events adjusted for year of birth, gender, and smoking has been shown in Table 4. The CC genotype was significantly associated with a 0.67 decreased risk of coronary heart disease (P = 0.009). Although we found no significant association between the CC genotype and other major cardiovascular events, probably as a result of lower prevalence in our cohort, the hazard ratio of carriers with the CC genotype was 0.39 for cerebral artery disease (P = 0.07) and 0.71 for peripheral artery disease (P = 0.3).
Hazard ratio of cardiovascular events estimated with Cox regression analyses in patients with familial hypercholesterolaemia
| Cardiovascular event | n | Event rate (%) | Hazard Ratio (95% CI) | P-value | |
|---|---|---|---|---|---|
| Coronary artery disease | 573 | 28.5 | 0.67 | (0.50–0.90) | 0.009 |
| Cerebral artery disease | 74 | 3.7 | 0.39 | (0.14–1.08) | 0.07 |
| Peripheral artery disease | 90 | 4.5 | 0.71 | (0.34–1.46) | 0.3 |
| Cardiovascular event | n | Event rate (%) | Hazard Ratio (95% CI) | P-value | |
|---|---|---|---|---|---|
| Coronary artery disease | 573 | 28.5 | 0.67 | (0.50–0.90) | 0.009 |
| Cerebral artery disease | 74 | 3.7 | 0.39 | (0.14–1.08) | 0.07 |
| Peripheral artery disease | 90 | 4.5 | 0.71 | (0.34–1.46) | 0.3 |
Discussion
In this large, multicentre cohort study of patients at increased susceptibility to premature CVD, we demonstrated that the CC genotype of the CFH gene, present in ∼13% of the population, was significantly associated with a two-fold decreased risk of CVD. Adjustment for clinically relevant cardiovascular risk factors and age effects did not influence the effect. We also analysed the risk of coronary, cerebral, and peripheral events separately and found similar decreased hazard ratios in carriers of the CC genotype.
Atherosclerosis is characterized by a strong inflammatory component.6 Data are accumulating that activation of the complement system is an important link between inflammation and atherogenesis.2 Complement activation can arise through the classical, alternative, or lectin pathway. All three pathways initiate activation of a C3 convertase enzyme which leads to the production of C3a and C3b and then to the terminal C5b-9 membrane attack complex, causing cell lysis. Complement activation is nearly absent in normal arteries, but is considerably active in atherosclerotic lesions as demonstrated by C5b-9 depositions in atherosclerotic plaques.3 In addition, the extent of deposition was correlated with the severity of the lesion.17 Furthermore, the expression of messenger RNA for complement proteins was upregulated in vascular cells of atherosclerotic lesions,9 and elevated levels of activated complement products were found in plasma and atherosclerotic plaques of patients with myocardial infarction.28
During complement activation, host cells are protected from being damaged by CFH. Complement factor H modulates the complement cascade initiated by the alternative pathway: it inhibits the activation of C3 to C3a and C3b and inactivates existing C3b, thereby preventing uncontrolled complement activation.6 CFH was also present in human coronary artery lesions but was exclusively located in the superficial layer of the arterial intima, suggesting that CFH may protect the arterial wall from damage by excess complement activation.7
Complement factor H is encoded by a member of the Regulator of Complement Activation gene cluster, a group of closely linked genes located on chromosome 1q32 coding for several of the complement regulatory proteins. A large number of polymorphisms have been identified in CFH, but their potential influence on the level of expression or function of CFH are unknown.7 Interestingly, the Y402H polymorphism is located within the binding site for heparin and C-reactive protein. Recently, it has been demonstrated that the wild-type and the 402H variant differentially recognize heparin in vitro.29 This functional alteration may affect binding of CFH to the proteoglycan layer on the arterial intima, potentially influencing complement activation and inflammation in the atherosclerotic plaque.
Previous studies assessed the relationship between the CFH Y402H and susceptibility to CVD, but they have yielded conflicting results.15–21 Four case–control studies reported no significant association between this CFH variant and cardiovascular events.15,16,18,19 Two prospective population-based cohort studies in the elderly found that carriers of the CC genotype had an increased risk for myocardial infarction and cardiovascular mortality, respectively.17,20 In contrast, the Nurses’ Health Study and the Health Professionals Follow-up Study observed no overall association of the Y402H variant with risk of coronary heart disease among men, but an inverse association among women.20 This inverse association became consistently stronger among both men and women after stratifying by earlier age of coronary heart disease onset. However, post-hoc analyses in sub-populations should always be interpreted with caution and confirmed in other populations.
Patients with heterozygous FH have a severely increased predisposition to premature cardiovascular events.22 However, the risk shows considerable variation among patients with FH: 40% of untreated patients reach a normal life span whereas excess mortality caused by CVD occurs in the remaining 60%.23 Clearly secondary genetic factors and life style determine the burden from this monogenic disorder.30 The monogenic background and the large variation of CVD risk offer a unique opportunity to analyse secondary genes involved in CVD.24
In the present study, we confirmed the inverse relationship of the Y402H polymorphism and CVD in a population with high risk of premature CVD. The mean age of onset of CVD in our FH subjects was 48.5 years and is considerably earlier than in all other cardiovascular association studies of the CFH Y402H variant. In the youngest age tertile, however, we found no relationship between CVD susceptibility and the CFH polymorphism, probably due to the low prevalence of cardiovascular events in this age category. Obviously, most cardiovascular events occurred in the intermediate age tertile and, consequently, the inverse association with CVD was strongest in these individuals. The reason for the age effects observed in the relationship between the CFH Y402H polymorphism and CVD is yet unknown and warrants further investigation to establish the functional consequences of the genetic variant and its impact on complement activation and complex diseases such as AMD and atherosclerosis.
A limitation of our study is that the influence of the Y402H polymorphism on potential intermediate traits such as C-reactive protein levels and other inflammation markers could not be determined. Furthermore, association studies would ideally be performed using haplotype-based analysis instead of focusing on single nucleotide polymorphism. The CFH Y402H polymorphism, however, has been consistently associated with AMD and therefore we restricted our analysis to this polymorphism. An advantage of this single nucleotide polymorphism-based approach is that multiple testing is circumvented. Of course we tested several additional characteristics such as the influence of possible confounders in the relationship between Y402H and CVD and the type of cardiovascular event. In the classical approach for correcting P-values, the nominal P-value is multiplied by the number of hypotheses tested (Bonferroni correction). In the present study, Bonferroni correction did not change the hazard ratios (P < 0.05). However, this correction is probably too conservative, because all outcomes are highly correlated with each other.
In conclusion, in this large cohort study of hypercholesterolaemia patients at increased susceptibility of early onset CVD, we found a significant two-fold decreased risk of CVD in patients homozygous for the CC genotype of CFH gene. The association was not explained by known cardiovascular risk factors such as age, gender, smoking, diabetes, and cholesterol levels, suggesting that CFH is a modifier gene of CVD.
Conflict of interest: none declared.
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