April 2009 | Vol 6 | N.º 4 | CNIC-7 [PDF (766K)]

Prognostic factors of cardiovascular disease mortality and morbidity in a cohort of families with genetic diagnoses of familial hypercholesterolemia

Rodrigo Alonso¹, Nelva Mata², Lina Badimón³, Francisco Pérez-Jiménez⁴ and Pedro Mata¹, on behalf of the Spanish Familial Hypercholesterolaemia Group^*
Correspondence Pedro Mata. Lipid Clinic, Internal Medicine Department. Fundación Jiménez Díaz. Avda. Reyes Católicos 2, Madrid-28040, Spain. Telephone: +34­915504800 (3231). Fax: +34­915042206.
Email pmata@fjd.es

Competing interests
The authors declared no competing interests.

Acknowledgment
We thank the families for their valuable contribution and participation and the Fundación Hipercolesterolemia Familiar, Ciber Fisiopatologia Obesidad y Nutricion (CB06/03) and Instituto Salud Carlos III, Spain.

Abstract | Familial Hypercholesterolemia (FH) is a frequent genetic disorder caused by mutations in the low-density lipoprotein receptor It is associated with a high risk of severe and premature coronary artery disease (CAD). Although FH is a monogenic disorder, a wide variation in phenotypic expression exists. This is especially true in the onset and severity of CAD and is explained partly by the molecular defect and partly by classic cardiovascular risk factors. Additional environmental, metabolic and genetic factors may contribute to the atherosclerotic burden and clinical expression of cardiovascular disease. To gain insight into the prognostic factors and mechanisms that influence the development of CAD and mortality in FH, a long-term prospective follow-up of a molecularly well-defined FH cohort using a multidisciplinary approach is necessary. In 2003, we began the current study with the following aims: 1) determine the absolute and relative mortality of treated FH patients; 2) determine the relative risk of non fatal cardiovascular events in the FH population; 3) analyze the prognostic role of classic risk factors, dietary habits and genetic polymorphisms and their interaction in FH clinical expression; 4) evaluate the role of imaging in risk stratification; and 5) estimate the long-term quality of life of subjects and families with this genetic disorder.

Background
Familial hypercholesterolemia (FH) is the most com­mon genetic cause of severe and premature coronary artery disease (CAD). This monogenic disorder is caused by mutations in the gene that encodes the LDL receptor (LDL-r) and has an autosomal dominant mode of inheritance with a high penetrance that affects one in 400 to 500 individuals in its heterozygous form.¹ Due to these mutations, there is a lower expression of function­al LDL-rs on the liver and an accumulation of LDL cho­lesterol (LDL-c) beginning at birth, causing premature atherosclerosis and cardiovascular disease. To date, more than 800 functional mutations in this gene have been described worldwide, with more than 200 in Spain alone.^2,3

Mortality in FH
The importance of FH lies in its high incidence of fatal and non-fatal premature cardiovascular disease (CVD). Moreover, FH shortens life expectancy by 20 to 30 years.⁴ The first report of a significant increase in FH risk described a 51% chance of fatal or non-fatal coro­nary heart disease by the age of 50 in men and a corre­sponding risk of 12% in women.⁵

The mean age of onset of CVD is between 40 and 45 years in males and 10 years later in females with FH. Moreover, other studies on FH patients in Japan and Finland have shown that CVD (sudden death and myo­cardial infarction) is the principal cause of death in this population.^6,7 In the past 2 decades, additional studies on the prospective follow-up of patients with a clinical diagnosis of FH and the risk of CAD have been published.^8,9 The first report of the Simon Broome Registry, published in 1991 and based on the 9-year (1980-1989) follow-up of 526 patients meeting the clinical criteria of FH, demonstrated an approximate 100-fold increase in FH-related mortality from coro­nary heart disease and a nearly 10-fold increase in total mortality, especially in young adults.8 This excess in mortality was not found in patients over 60 years of age. A second report from the same registry, including 1185 subjects with a clinical diagnosis of FH from lipid clin­ics followed from 1980 to 1995, confirmed the earlier report, showing an increased relative risk of a fatal coro­nary event that declined after 60 years of age in young males (48-fold) and females (125-fold). These values re­mained significantly higher in women, but not in men, between the ages of 60 and 79 years.9 The decline in relative risk of death from CAD after the age of 60 years could be explained in part by selective survival due to earlier death in the most susceptible subjects and reduced risk among survivors. Interestingly, a decline in the relative risk for coronary mortality in patients aged 20-59 years was observed after 1992, suggesting that the introduction of statins as a part of therapy im­proved the prognosis of FH patients.9 Recently, an ex­tension of previous reports studying a cohort of patients with a "definite" or "possible" clinical diagnosis of FH followed for up to 26 years showed a significant reduction (approximately 33%) in coronary mortality since the widespread use of statins was established.¹⁰ In patients without known CAD at registration, all-cause mortality was significantly reduced by about one third, mainly due to a reduction of fatal cancer. This result could be attributed to an increase in surveillance that led to patients listening to advice regarding concerns such as indications in dietary changes and smoking cessation given as part of routine clinical care. This hypothesis could not be addressed in this study, however. In the Simon Broome Register, FH patients were classified by clinical criteria and, as a result, some misclassification of polygenic hypercholesterolemia as FH most likely oc­curred.

Data from a cohort of families with FH in The Netherlands showed that the mortality is particularly high in middle-age male patients and that additional risk fac­tors can modulate the mortality associated with FH.¹¹ A recent publication of an 8.5-year follow-up of more than 2,100 FH patients showed that statin treatment reduced the risk of coronary heart disease by 80%. The risk of myocardial infarction in statin-treated patients older than 55 years was not significantly greater than that observed in an age-matched sample from the Dutch general population.¹²
Risk factors for cardiovascular disease in FH

In Spain, data from the Spanish FH Registry showed that 30.2% of men and 14.5% of women with a genetic diagnosis of FH had some manifestation of premature CAD at the time of inclusion.^13,14 It is therefore possible that some individuals with very high plasma cholesterol levels are more susceptible to developing atherosclerotic disease than others.

Despite being a monogenic disorder, the clinical ex­pression of FH varies considerably, even in cases that share the same mutation, particularly with regard to cholesterol levels and the age of onset and severity of CAD.15 Previous studies have shown that classical cardiovascular risk factors and the type of mutation in the LDL-r gene partially contribute to the development of CAD.^14,16,17 Age, gender and tobacco consumption are well-recognized determinant factors of a high risk of CAD in different cohorts14,16. Conversely, a higher risk of CAD in patients with LDL-r null alleles compared with defective allele mutations is also well documented.^14,17,18

Although the genetic defect is most likely the most important factor in the clinical expression of FH, other genetic, environmental (particularly those relating to diet, tobacco consumption and physical activity) and metabolic factors could play an important role in modulating the atherosclerotic burden in this population. The majority of variation that can be attributed to sus­ceptibility to CAD is caused by variations in genes that may have minor effects on phenotype in the general population but could have more pronounced effects in FH patients based on gene-gene interaction.

In Spain, the 22% of premature CAD in FH patients14 is nearly 10-fold higher than that reported in the general population¹⁹ but lower when compared with other FH cohorts from western countries.^16,20 Th is diff erence in CAD prevalence could be explained by some environmental factors, such as diet. The Mediterranean diet, which is enriched in monounsaturated fat (olive oil), vegetables, fruits, fish and fibre, has been shown to have beneficial effects on cardiovascular risk factors with ad­ditional anti-atherogenic properties beyond the effects on lipid levels.²¹ These effects may contribute to a reduc­tion of the incidence of cardiovascular events in the general population, even in Spanish patients suffering from FH. Patients in Tunisia with a severe mutation in the LDL-r gene do not express early coronary disease most likely a result of their dietary habits or additional environmental factors.²²

Extremely high LDL-c levels are key determinants of CAD in FH patients compared with the general popula­tion; however, they do not completely explain the high risk of cardiovascular disease in these patients. Other lipoproteins can also influence the phenotypic expres­sion of atherosclerosis in FH. Indeed, HDL-cholesterol levels constitute an independent risk factor for FH.²³ Low HDL levels have been associated with an increased risk for CHD; however, the mechanisms underlying the relationship between LDL, risk of atherosclerosis-asso­ciated HDL and atheroprotection in FH patients remain to be elucidated. A key factor in the evolution of sub­clinical atherosclerosis to an ischemic event is the in­creased vulnerability of atheromatous plaques, which precipitates rupture and subsequent thrombosis. Be­yond the role of HDL and LDL in lipid transport, recent studies suggest that these lipoprotein fractions are en­riched in proteins with a role in the molecular processes involved in atherothrombosis.^24,25 Thus, characteriza­tion of HDL and LDL lipidome/proteome patterns in FH may provide relevant insight into the mechanisms underlying their role in the development, progression and complication of atheromatous plaques and in the promotion of premature CAD manifestation.

Imaging and cardiovascular risk in FH
MRI of arteries is ideal for longitudinal studies of the progression of atherosclerosis because it is a non-inva­sive procedure and is superior to other imaging modali­ties in providing information on plaque composition and volume at multiples sites.²⁶ MRI has been used in hypercholesterolemic patients to analyse the effect of statin therapy on aortic and carotid atherosclerotic lesions.^27,28 The evidence in FH, however, is scarce. Only one, small case-control study demonstrated that an in­tensive and long-term lipid-lowering therapy is associ­ated with a decreased lipid content in atherosclerotic plaques in carotid arteries measured using MRI.²⁹ The non-invasive analysis of arteries in vivo from FH patients using MRI therefore provides a unique opportu­nity for longitudinally assessing changes in atheroscle­rotic lesions and, in particular, their tissue composition in this high risk population. These studies could result in a better risk stratification of these patients and also may elucidate the role of other genetic and environmental factors in the development of vascular disease before it becomes clinically evident.

Project
The main objective of this prospective study is to deter­mine the prognostic factors for all-cause mortality and morbidity. This study will be performed in a large, long­term prospective cohort of subjects with a genetic diag­nosis of familial hypercholesterolemia and their rela­tives.

Methodological approach
A nationwide recruitment of patients to the follow-up study of FH families began in 2003 and is ongoing. Patients from the Spanish FH registry (property of the Spanish FH Foundation) with a genetic diagnosis of FH are contacted by telephone from the Spanish FH Foun­dation (Cohort Coordination Centre) and invited to participate in this study, along with their relatives. Th ree categories of subjects are admitted to the study: 1) index cases with a genetic diagnosis of FH, 2) relatives with a positive genetic test for FH, and 3) relatives with a nega­tive test for FH. The study currently includes over 1400 subjects (271 index cases and 1200 relatives) who are followed-up by a standardized protocol. Clinical char­acteristics and mean serum lipid and lipoprotein con­centrations at registration for men and women are shown in Table 1. This study was approved by the local ethics committee and all subjects gave written informed consent before their inclusion in the study.

Registration visit and follow-up
The demographic and clinical characteristics of subjects include date of birth, employment and educational status, details of past medical history (especially cardiovascular disease), cardiovascular risk factors (lipid profile, high blood pressure, type 2 diabetes, obesity, smoking status), physical examination and previous treatment for hypercholesterolemia. The Quality of life Survey (SF-12), food frequency and physical activity question­naire are recorded on a standardized registration form. Physical examination includes weight, height, body mass index, waist diameter and supine blood pressure measured with a standardized OMRON MX3 sphygmo­manometer. The presence of tendon xanthomas, arcus cornealis and signs of peripheral vascular disease is also reported. All data are registered on a computer file specifically designed for this study.

For the detection of fatal and non fatal cardiovascular events, an active epidemiological surveillance has been developed using a mixed system of active search and passive notification. In the case of non fatal cardiovascular events, a summary of the relevant hospital case notes is required from the doctor who included the sub­ject in the study and classified the subject according WHO-MONICA criteria. In the case of death, a copy of the death certificate from the National Statistic Institute is required. Causes of death are coded by one investigator using the International Classification of Diseases (9th revision).

Follow-up visits are carried out every 3 years and a standardized phone call is made every year to all subjects included in the study. Patients and families with FH also receive a card with the telephone numbers they are required to use in the case of event.

Laboratory methods
Venous blood samples are taken after 12 hours of fasting at inclusion and every 3 years. Genomic DNA is isolated from whole blood or saliva samples using standard methods. The genetic diagnosis is made using a DNA microarray (Progenika, Bilbao, Spain) as previously described.³ DNA, serum and plasma samples are distrib­uted in aliquots and preserved at –80 ºC until use. Serum total cholesterol, triglyceride and HDL cholesterol levels are measured in a centralized laboratory using enzy­matic methods. Serum LDL-c concentration is calculated using the Friedewald formula. Candidate genes other than LDL-r that are related to cardiovascular dis­ease will be analysed through SNP association. Diff er­ential HDL and LDL lipid and protein patterns in FH and their association with the onset of CAD will be analysed using state-of-the-art techniques based on lipidomic and proteomic techniques. Characterisation of circulating microparticles will be performed by flow­cytometry and proteomic analysis. Functional effects of circulating microparticles in FH and at the onset of CAD will be investigated in vitro on a cell culture of en­dothelial cells (angiogenesis assays) and with platelet adhesion studies using a flow chamber. Plasma quantification of specific protein candidates will be based on an analysis of antigen levels by western blot, ELISA, and XMap techniques.

MRI Study
A subgroup of patients with a genetic diagnosis of FH and a control group without an LDL-r mutation, matched by age and gender, will be randomly selected from the cohort. A sequential MRI will be performed at inclusion and after 2 years of follow-up using a 1.5T whole-body MRI system (General Electric, Spain).

In summary, FH is a frequent genetic disorder associ­ated with premature atherosclerosis and cardiovascular mortality and disease. Early detection and treatment are mandatory to prevent the increased burden of premature CAD and death. The clinical phenotype is highly modifiable by the presence of environmental and metabolic factors, the type of LDL-r mutation and coin­heritance of other genetic factors. This large, long-term prospective follow-up study is an excellent model of translational research to evaluate and determine the principal prognostic factors related to CAD morbidity and mortality. It will permit analysis using post-genomic research approaches for gene-gene, gene-environment, and protein-protein interactions and their contribution to atherosclerotic plaque burden and early manifestation of CVD.

This long-term follow-up will assess the prognosis, safety and efficacy in older patients who began statin treatment in adulthood.