Cardiovascular disease in familial hypercholesterolaemia: Influence of low-density lipoprotein receptor mutation type and classic risk factors

Atherosclerosis 200 (2008) 315–321

Cardiovascular disease in familial hypercholesterolaemia: Influence of low-density lipoprotein receptor mutation type and classic risk factors R. Alonso a,∗ , N. Mata b , S. Castillo c , F. Fuentes d , P. Saenz e , O. Mu˜niz f , J. Galiana g , R. Figueras h , J.L. Diaz i , P. Gomez-Enterr´ıa j , M. Mauri k , M. Piedecausa l , L. Irigoyen m , R. Aguado n , P. Mata a , on behalf of the Spanish Familial Hypercholesterolaemia Group1 a

Lipid Clinic, Fundaci´on Jim´enez D´ıaz, Madrid, Spain b Public Health Institute, Madrid, Spain c Diagnostic Genetic Unit, L´ acer S.A., Barcelona, Spain d Hospital Reina Sof´ıa, C´ ordoba, Spain e Hospital de M´ erida, Badajoz, Spain f Hospital Virgen del Roc´ıo, Sevilla, Spain g Hospital Ciudad Real, Ciudad Real, Spain h Hospital Bellvitge, Barcelona, Spain i Hospital Juan Canalejo, Coru˜ na, Spain j Hospital Central, Asturias, Spain k Hospital de Terrassa, Barcelona, Spain l Hospital General Universitario de Elche, Alicante, Spain m Hospital Santiago Apostol, Vitoria, Spain n Hospital General de Leon, Spain Received 7 August 2007; received in revised form 30 November 2007; accepted 18 December 2007 Available online 20 February 2008

Abstract Aim: To determine the effect of the type of mutation in low-density lipoprotein receptor gene and the risk factors associated with the development of premature cardiovascular disease (PCVD) in a large cohort of heterozygous familial hypercholesterolemia (hFH) subjects with genetic diagnosis in Spain. Methods and results: A cross-sectional study was conducted on 811 non-related FH patients (mean age 47.1 ± 14 years, 383 males and 428 females) with a molecular defect in the low-density lipoprotein receptor (LDLR) gene from the Spanish National FH Register. Prevalence of PCVD was 21.9% (30.2% in males and 14.5% in women, P < 0.001). Mean age of onset of cardiovascular event was 42.1 years in males and 50.8 years in females. Of those patients with PCVD, 59.5% of males and 27% of females suffered a second cardiovascular (CV) event. In multivariate analysis male gender, age, tobacco consumption (ever), and total cholesterol/HDL-cholesterol (TC/HDL-C) ratio were significantly associated with PCVD. Two hundred and twenty different mutations were found with a large heterogeneity. Patients carrying null-mutations had significantly higher frequency of PCVD and recurrence of CV events. No relationship with Lp(a) levels and genotype of Apo E were found. Conclusions: This study confirms the importance of identifying some classic risk factors such as smoking and TC/HDL-C ratio, and also the type of mutation in LDLR gene in order to implement early detection and intensive treatment for the prevention of cardiovascular disease in FH patients. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Familial hypercholesterolaemia; Cardiovascular disease; Cardiovascular risk factors; LDL-receptor mutations

∗ 1

Corresponding author. Tel.: +34 915 504910; fax: +34 915 435001. E-mail address: [email protected] (R. Alonso). For Spanish Familial Hypercholesterolaemia Group see: www.colesterolfamiliar.com.

0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.12.024

316

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321

1. Introduction Familial hypercholesterolaemia (FH) is the most common monogenic disorder associated with the development of severe and premature cardiovascular disease, affecting one in 400–500 people in the general population [1]. FH is a very heterogeneous disease and it is caused by mutations in the gene encoding the low-density lipoprotein receptor (LDLR); worldwide, over 1000 mutations have been identified to date. In the Spanish FH Register more than 200 different mutations have been detected from all over the country [2]. Several studies have shown that at least 50% of males and 20% of females with FH who do not receive effective treatment will suffer a coronary event by the age of 50 years [3,4]. Also, an increase in the relative risk for fatal coronary events has been observed in FH patients below 40 years [4]. Although FH is a monogenic disorder, the incidence and characteristics of cardiovascular disease of affected subjects vary considerably in different cohorts and countries, even in cases that share the same mutation [5,6]. This suggests that other metabolic, environmental and genetic factors could explain the differences in the susceptibility to coronary artery disease in these patients [6,7]. Previous studies have shown conflicting results related to some of the classic and emerging cardiovascular risk factors (CVRF), as well as the type of mutation and their impact in the development of PCVD in FH patients [8–15]. This is probably related to the sample size of the studies and the criteria used for the diagnosis of this disorder. Therefore, the aim of this study was to determine the effect of the type of LDLR gene mutation and classic cardiovascular risk factors in the development of premature cardiovascular disease in a large cohort of Spanish patients with molecular diagnosis of FH.

2. Methods

Demographic data, medical history, lipid-lowering therapy, physical examination, pre-treatment lipid profile and familial history of cardiovascular disease were obtained from all patients using a standardised report form at the inclusion in the register. Classic CVRF were evaluated according to International Guidelines [18]. Cardiovascular disease was defined if any of the following criteria were found: (1) myocardial infarction proved by at least two of the following criteria: (a) classical symptoms (chest pain >15 min), (b) serial ECG specific changes, (c) >2 upper limit normal of cardiac enzymes; (2) angina pectoris, diagnosed as classic symptoms in combination with at least one positive result of the following: (a) exercise test, (b) nuclear scintigram, (c) dobutamine stress ultrasound, (d) >70% stenosis on coronary angiography; (3) percutaneous coronary intervention (ACTP) or coronary artery bypass grafting; (4) ischaemic stroke, demonstrated by CT or MRI scan or documented transitory ischaemic attack; (5) intermittent claudication defined as classic symptoms and at least one positive result of the following: (a) ankle/arm index <0.9, (b) stenosis >50% determined by angiography or doppler ultrasound; (6) abdominal aortic aneurism; (7) peripheral arterial bypass graft or percutaneous transluminal angioplasty. Cardiovascular disease was considered premature if the first event occurred before 55 years old in males and 65 years old in females. 2.2. Laboratory methods Blood samples were obtained after 12 h fasting. Total cholesterol (TC), triglycerides (TG), and HDL-C were measured by standardized enzymatic methods in a central laboratory. When TG levels were lower than 350 mg/dL, LDL cholesterol (LDL-C) was calculated using the Friedew¨ald formula. Lipoprotein (a) (Lp(a)) was measured using kinetics nefelometry with polyclonal antibodies (Beckman, USA) [19]. Apolipoprotein E genotypes were determined by the method of Hixson and Vernier as previously described [20].

2.1. Subjects 2.3. Molecular genetic analysis Subjects were selected from the Spanish FH Register, supported by the “Fundaci´on Espa˜nola Hipercolesterolemia Familiar”. The main characteristics of the register have been previously reported [2,16]. In summary, a total of 77 lipid clinics throughout Spain recruited FH patients with the same clinical criteria. Physicians attended at least 3 different meetings in order to homogenize diagnosis criteria. The genetic test was performed to subjects with clinical diagnosis of FH (score ≥ 6 using an adapted version of the Dutch scoring system) [17]. All non-related patients ≥18 years old with genetic diagnosis of heterozygous FH were included in the study. In order to homogenize the sample, patients with familial defective apolipoprotein B disorder were excluded from the analysis. A written informed consent was obtained from all participants before their inclusion in the register and the protocol was approved by the local ethics committee.

Genomic DNA was isolated from whole blood samples using standard methods. All the patients were heterozygous carriers for any mutation in the LDLR gene associated with hypercholesterolaemia. The genetic diagnosis was made using molecular biology techniques as described [21]. Briefly, samples were analyzed using a DNA-microarray (Progenika, Bilbao, Spain) and capillary sequencing was conducted by using multiplex PCR conditions and sequence reactions previously described [21,22]. Negative samples for the DNA-array or sequencing were also analyzed for large deletions or insertions using an adapted quantitative fluorescent multiplex PCR methodology [23]. Mutations were classified as receptor-negative or receptordefective depending on their functional class as reported previously in the literature. Those mutations with non-

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321

reported functional class in the literature were classified as “unknown”. Receptor-negative mutations included: (1) point mutations that cause a premature stop codon; (2) missense mutations affecting the fifth cystein rich repeat in the ligand binding domain of the LDLR gene (class 2A mutation); (3) small deletions or insertions causing a frame shift and a premature stop codon and (4) large rearrangements. Receptor-defective mutations were the rest of inframe point mutations and inframe small deletions and insertions 2.4. Statistical analysis All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS v13.0, Chicago, IL, USA). An initial descriptive analysis was carried out using number of cases and percentages for qualitative variables and mean and standard deviation for quantitative variables with a normal distribution. For those quantitative variables with an abnormal distribution, median and interquartilic range was estimated. Furthermore, comparisons of frequencies between qualitative variables were carried out using the Chi-squared test. Mean values of quantitative variables were compared with the Student’s t-test for independent data while median values were compared with the non-parametric median test. A logistic regression analysis (stepwise) was conducted to determine the variables associated with PCVD including those variables that were significantly predictive for PCVD in bivariate analysis. The relationship between variables was considered statistically significant if the Pvalue <0.05. The magnitude of the association was estimated

317

using the odds ratio (OR) with a confidence interval (CI) of 95%. The Kaplan–Meier method was performed to determine PCVD-free survival time regarding the type of mutation using the age limits of 55 years in men and 65 years in women. Survival curves were compared by the Breslow test. The independent contribution of each significant variable to the length of PCVD-free survival was evaluated by multivariate Cox regression. For multivariate analysis and for Kaplan–Meier curves only null and defective mutations were considered. Pre-treatment TC, HDL-C, TG, LDL-C and TC/HDL-C ratio were used for statistical analysis.

3. Results A total of 811 (428 females and 383 males) non-related FH patients were included in the analysis. Clinical and biochemical characteristics are shown in Table 1. Mean age at inclusion was 45.5 years for males and 48.4 years for females (range from 18 to 83 years). Significant differences were found in tobacco consumption (P < 0.001), hypertension (P < 0.01), waist diameter (P < 0.001), TC, HDL-C (P < 0.001), triglycerides (P < 0.001) and TC/HDL-C ratio (P < 0.001) between males and females. Mean LDL-C levels at inclusion in the register was 220 ± 67.2 mg/dL, and there were no differences between gender (data not shown). More than 80% of the subjects were receiving lipidlowering therapy at their inclusion (668 cases from 774; data was missing in 37 cases). Statins were the most frequent

Table 1 Baseline characteristics of FH subjects included in the study (N = 811) All 811

Males 383 (47.2)

Females 428 (52.8)

P

Age (year) CV disease (%)a

47.1 ± 14 21.9

45.5 ± 13.3 30.2

48.4 ± 14.4 14.5

0.004 0.000

Tobacco consumption (%) Current Ex-smokers Never

20.3 26.1 53.6

20.0 42.4 37.6

20.5 11.4 68.1

0.000

Hypertension (%) Diabetes mellitus (%) BMI (kg/m2 )b Waist diameter (cm) Xanthomas (%) TC (mg/dL)b TG (mg/dL)c HDL-C (mg/dL)b LDL-C (mg/dL)b TC/HDL-C ratiob Lp(a) (mg/dL)c Type of LDL-r gene mutation (null/defective/unknown) Apo E genotype (E2/E3/E4)

15.3 3.5 26.4 ± 4.5 87.5 ± 12.9 28.5 417.2 ± 84.3 106.0 (80.0–145.0) 52 ± 14.6 335.9 ± 79.8 8.6 ± 3.3 29.5 (11.7–67.7) (30.8/56.1/13) (5.0/75.4/19.6)

11.6 3.5 26.6 ± 3.7 93.4 ± 10.2 30.3 409.3 ± 74.7 113.5 (88.7–153.2) 46.5 ± 12.6 330.9 ± 71.3 9.4 ± 3.5 28.6 (13.0–62.2) (32.7/54.7/12.6) (3.4/78.5/18.0)

18.7 3.4 26.1 ± 5.1 82.6 ± 13 26.8 424.3 ± 91.6 100.0 (72.0–139.0) 57 ± 14.5 340.5 ± 86.5 7.9 ± 3.0 30.9 (9.3–73.8) (29.1/57.5/13.5) (6.3/72.7 /21.0)

0.008 0.938 0.122 0.000 0.275 0.014 0.000 0.000 0.126 0.000 0.350 0.537 0.204

CV: cardiovascular; BMI: body mass index; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol. Values of lipid profile correspond to highest levels without lipid-lowering therapy. a Data from 776 subjects. b Data are expressed as mean ± standard deviation (S.D.). c Data are expressed as median and interquartilic range.

318

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321

lipid-lowering agents used and less than 30% of patients were receiving the highest dose (80 mg/day) of simvastatin or atorvastatin in monotherapy or in combination with resins. 3.1. Cardiovascular disease and classic risk factors Data of cardiovascular disease was not available in 35 subjects. Overall prevalence of PCVD was 21.9% and was significantly higher in males (30.2% in men vs. 14.5% in women; P < 0.001). The first CV event occurred 8 years earlier in males than in females (42.1 years vs. 50.8 years, respectively; P < 0.001). Coronary artery disease (CAD) represented more than 80% of the first vascular event. Myocardial infarction was the most frequent first event in men (57.7%), and angina pectoris was in women (50.9%). Approximately 60% of males and 27% of females had a second CV event (P < 0.05), 1–3 years after the first event. Family history of PCVD in first degree relatives was present in 44.4%, and the mean age of onset was 47.4 ± 9.6 years. Multiple regression analysis showed that the most important risk factors associated to PCVD in this population were male gender (OR = 1.98; CI 95%: 1.09–3.56), tobacco consumption (OR = 1.80; CI 95%: 1.03–3.16), higher TC/HDL-C ratio (OR = 1.26; CI 95%: 1.01–1.58) and age (OR = 1.05; CI 95%: 1.02–1.07). In men, the best predictors were tobacco consumption (OR = 2.93; CI 95%: 1.34–6.37), the presence of a null-mutation (OR = 2.09; CI 95%: 1.04–4.21) and age (OR = 1.04; CI 95%: 1.01–1.07), and in women, the best predictors were TC/HDL-C ratio (OR = 1.67; CI 95%: 1.08–2.60), HDL-C levels (OR = 1.07; CI 95%: 1.01–1.13) and age (OR = 1.06; CI 95%: 1.02–1.10) (Table 2). 3.2. Molecular analysis and effect of type of mutation in CVD A total of 204 different point mutations and 16 different large rearrangements have been detected along LDLR gene; 13 of them are novel mutations. The 10 most frequent mutations represent 31.5% confirming the high genetic heterogeneity of Spanish FH population (Table 3). According to the known residual binding activity of the mutated LDL

Table 2 Predictors of cardiovascular disease in familial hypercholesterolemia

All subjects Gender (male) Tobacco (ever smokers) TC/HDL-C ratio Age

Adjusted OR

P

CI 95%

1.98 1.80 1.26 1.05

0.023 0.039 0.041 0.000

1.09 1.03 1.01 1.02

3.56 3.16 1.58 1.07

Males Tobacco (ever/never) Type of mutation (null/defective allele) Age

2.93 2.09

0.007 0.040

1.34 1.04

6.37 4.21

1.04

0.005

1.01

1.07

Females TC/HDL-C ratio HDL-C Age

1.67 1.07 1.06

0.022 0.023 0.003

1.08 1.01 1.02

2.60 1.13 1.10

Multiple regression analysis. Variables included in the model: age, gender, tobacco (ever vs. never smokers), hypertension, BMI, family history of PCVD, Lp(a), apo E genotype, type of mutation, pre-treatment lipid profile (LDL-C, HDL-C, triglycerides, TC/HDL-C ratio).

receptor, 248 patients had mutations classified as null, 451 as defective and in the remaining 112 subjects the activity was unknown. Patients carrying null-mutations had significantly higher TC/HDL-C ratio (P < 0.05), frequency of PCVD (OR = 1.68; CI 95%: 1.10–2.40, P < 0.01), recurrence of CV events (P < 0.05) and family history of PCVD in first degree relatives (OR = 1.67; CI 95%: 1.20–2.30, P < 0.01) compared with patients carrying defective-mutations. There were no differences in xanthomas, LDL-C and HDL-C levels between both groups (Table 4). Kaplan–Meier curves were performed to determine the PCVD-free survival time depending on type of mutation (null vs. defective mutations). The Breslow test showed significant differences in men (P < 0.01) but not in women (P = 0.8) (Fig. 1). In men, the mean PCVD-free survival time was 51 years for carriers of null-mutations and 53 years for carriers of defective mutations (P < 0.01). Multivariate Cox regression showed that predictors related to PCVD-free survival time in men were the presence of null-mutation (OR = 1.84; CI 95%: 1.18–2.89), age (OR = 1.02; CI 95%: 1.004–1.03) and tobacco consumption (OR = 2.28; CI 95%: 1.33–3.92).

Table 3 Most frequent mutations of the Spanish FH Registry Exon

Nucleotide change

Estimated effect

N (%)

3 9 I12 4 17 + 11 Prom. 9 I16 6+1 I9

c.313 + 1G > C + c.274C > G c.1342C > T c.1845 + 1G > C Q133X c.2393 2401del9 + N543H c.(−135) C > G c.1197 1205del9 c.2389 + 4G > A c.829G > A + c.12G > A c.1358 + 1G > A

Splicing defect + p.Q71E LBD Stop codon p.Q427X Splicing defect Stop codon p.Q133X In frame p.L778 F780del + p.N543H EGFPHD Modified transcriptional activity In frame p.Y379 F381del Unknown p.E256K LBD + Stop codon p.W(-18)X Splicing defect

44 (5.4) 32 (3.9) 31 (3.8) 29 (3.6) 25 (3.1) 23 (2.8) 18 (2.2) 18 (2.2) 18 (2.2) 18 (2.2)

Prom: promoter; LBD: ligand binding domain; EGFPHD: EGF precursor homology domain; MSD: membrane spanning domain.

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321

319

Table 4 Characteristics of FH patients according to the type of mutation N (%)a

Null-mutation 248 (35.5%)

Defective-mutation 451 (64.5%)

P

Males Females Age (year)b BMI (kg/m2 )b Xanthomas Cardiovascular disease (%) Age of onset CVDb % Recurrence of CVD PCAD (%) in first degree relatives TC (mg/dL)b HDLc (mg/dL)b TG (mg/dL)c LDL-C (mg/dL)b TC/HDL-C ratiob

50.8% 49.2% 47.4 ± 14.2 26.2 ± 4.5 28.4% 27.2% 44.9 ± 9.1 53.1% 48.3% 419.9 ± 77.4 50.3 ± 13.4 110.0 (84.0–144.5) 339.6 ± 69.8 8.9 ± 3.3

46.7% 53.3% 46.9 ± 13.9 26.6 ± 4.8 27.8% 18.2% 45.2 ± 9.5 48.1% 35.9% 411.2 ± 78 52.7 ± 14.8 100.0 (79.0–138.0) 330.0 ± 72.3 8.3 ± 2.9

0.294 0.670 0.356 0.866 0.006 0.318 0.041 0.002 0.167 0.066 0.065 0.129 0.046

a N = 699 with known residual binding activity in the mutated protein. Other cases with functional mutation but with unknown activity were excluded from the analysis. b Data are expressed as mean ± standard deviation (S.D.). c Data are expressed as median and interquartilic range.

Fig. 1. Kaplan–Meier curves for PCVD-free survival in FH men (left box) and women (right box) with null (solid line) and defective (dotted line) mutations in LDLR gene in men (left box).

In women, the predictors were age (OR = 1.046; CI 95%: 1.01–1.07), HDL-C (OR = 1.04; CI 95%: 1.00–1.08) and TC/HDL-C ratio (OR = 1.40; CI 95%: 1.08–1.81).

4. Discussion This is a large cross-sectional study in a non-related population with definitive molecular diagnosis of FH that were recruited from all over the country. Therefore, there was no possibility of including patients with other types of severe hypercholesterolaemia, and also the selection bias toward genetically isolated communities was minimized. In this cohort, the overall prevalence of PCVD was 21.9%, confirming that the CAD risk in FH patients is extremely high compared to the general population in Spain estimated in 2.6% for the same age [24]. However, the prevalence of PCVD in this population is lower compared to other FH series from western countries, ranging from 30 to 39%

[8,9,11,12,14], but comparison between our cohort and other studies is difficult to make because there are important differences in the diagnosis criteria of FH (most of the previous studies included patients with clinical diagnosis), the CVD rates and prevalence of CVRF in the general population in each country and the genetic background of FH in each population. Besides these methodological differences, some environmental factors could also explain in part, the different prevalence of CVD between countries. A possible modulating factor could be the Mediterranean diet, which is enriched in monounsaturated fat (olive oil). This diet has been shown to have beneficial effects on cardiovascular risk factors and also it has other antiatherogenic properties beyond the effect on lipid levels [25]. These effects could contribute to reduce the incidence of cardiovascular events in the general population even in Spanish patients suffering FH. Although there are some differences in the mean age of onset of cardiovascular disease, the results of this study are in agreement with others showing an earlier and more severe

320

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321

development of PCVD in men than in women [9], probably related to differences in the lipoprotein profile (men had lower HDL-C and a higher TC/HDL-C ratio) and in the higher rate of smokers in men. In the multivariate analysis, male gender, tobacco consumption, TC/HDL-C ratio and age were the most important predictors for the development of cardiovascular disease in hFH subjects. These findings are consistent with the evidence from other cross-sectional studies showing that classic risk factors are important predictors for CAD in this patients [9,11]. In our study, HDL-C was a good predictor in women but not in men, and TC/HDL-C ratio was it in both sexes, suggesting a gender-specific lipoprotein influence on coronary heart disease as demonstrated previously by others [8,12,26,27]. However, due to the small number of PCVD events occurring in females, the statistic power to identify predictors in this subgroup is probably lower than in males. Other studies have shown that low HDL-C is a powerful risk factor for CAD in men, in women and in both sexes [11,27]. In Spain, a study carried out in a small FH group with genetic diagnosis from a specific region of the country showed that HDL-C and TC/HDL-C ratio were the most important coronary risk factors, independently of the type of mutation [26]. Recently, conclusive evidence on the protective effects of raised HDLC levels against carotid atherosclerosis progression in FH patients has been published [28], suggesting that even in FH, a disorder with very high levels of LDL-C, the HDL-C levels have the same importance as in general population. In respect to other CV factors, the majority of the studies have not shown any relationship between potential risk factors, such as homocysteine levels, C-reactive protein, Lp(a) and PCVD in FH patients [10,11]. In our study, Lp(a) levels and ApoE genotype were not associated with the development of CAD, even in younger FH patients. The effects of the type of mutation in lipoprotein levels and in the risk of suffering cardiovascular disease have been analyzed previously with ambiguous results, [8,14,15,22]. In our study, although there is an important molecular heterogeneity, the prevalence of PCVD and recurrence of events in patients carrying a null-mutation was 1.7 folds higher than those with defective-mutations. These results are in line with other studies that have shown a very high risk of CAD in adult patients carrying severe mutations [14] and also in children with FH [29]. The difference in cardiovascular risk related to the type of mutation found in our study, could not be explained by differences in lipoprotein levels, except by a discrete but significant higher TC/HDL-C ratio in null-mutation carriers. On the other hand, patients carrying a null-mutation have a higher risk of CVD in their first degree relatives suggesting that additional familial risk factors could contribute to the CVD burden of FH. Due to the molecular heterogeneity found in Spain and in order to establish an accurate analysis of the impact of the mutation type in LDLR gene in CVD risk, it is necessary to carry out a cohort study over a long period of time including a representative sample of patients sharing the same mutation.

4.1. Practical utility of the results Our results confirm a failure in the prevention of cardiovascular disease in Spanish FH patients. This might be explained in part by a late diagnosis of the disorder, the undertreatment with lipid-lowering drugs and the inadequate management of other risk factors such as smoking. In this study, most patients were receiving statin therapy at inclusion; however, the LDLC levels were far from the therapeutic goals proposed for FH [30]. Therefore, a more aggressive lipid-lowering therapy in order to reduce LDL-C levels over 45% is mandatory [31]. On the other hand, in order to provide an early diagnosis, the development of a case finding strategy and then a cascade screening program in their relatives by using a DNA-based diagnosis [22,32,33] is a feasible and cost effective method into clinical practice [34,35]. The diagnosis of these patients will permit a multifactorial approach specially related to changes in dietary habits, quit smoking and increase physical activity in order to prevent the development of CVD. In conclusion, in this large group of molecularly defined FH subjects, classic risk factors such as smoking and TC/HDL-C ratio account for a sizeable part of the risk of PCVD development. Moreover, patients carrying a nullmutation have a higher risk of PCVD. These findings point out that the early genetic detection of FH subjects is essential in order to implement appropriate early-intervention strategies based on therapeutic lifestyle changes, and on initiating an effective lipid-lowering therapy that will prevent the development of premature CVD.

Acknowledgements This study was supported by grants from the Spanish Ministry of Health (SAF2001-2466-C05-02; RT/G03-181) and Fundaci´on Espa˜nola Hipercolesterolemia Familiar.

References [1] Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGrawHill; 2001. p. 2863–913. [2] Pocovi M, Civeira F, Alonso R, Mata P. Familial hypercholesterolamia in Spain: case-finding program, clinical and genetic aspects. Semin Vasc Med 2004;4:67–74. [3] Slack J. Risks of ischaemic heart-disease in familial hyperlipoproteinaemic states. Lancet 1969;2:1380–2. [4] Scientific Steering Committee on behalf of the Simon Broome Register Group. The risk of fatal coronary heart disease in familial hypercholesterolaemia. BMJ 1991;303:893–6. [5] Thompson GR, Seed M, Niththyananthan S, et al. Genotypic and phenotypic variation in familial hypercholesterolaemia. Arteriosclerosis 1989;9:175–80. [6] Sijbrands E, Westendorp R, Defesche J, de Meier PH, Smelt A, Kastelein J. Mortality over two centuries in large pedigree with familial hypercholesterolaemia: family tree mortality study. BMJ 2001;322:1019–23.

R. Alonso et al. / Atherosclerosis 200 (2008) 315–321 [7] Jansen A, van Aalst-Cohen E, Tanck M, et al. Genetic determinants of cardiovascular disease risk in familial hypercholesterolaemia. Arterioscler Thromb Vasc Biol 2005;25:1475–81. [8] Ferrieres J, Lambert J, Lussier-Cacan S, Davignon J. Coronary artery disease in heterozygous familial hypercholesterolaemia patients with the same LDL receptor gene mutation. Circulation 1995;92: 290–5. [9] Jansen AC, van Aalst-Cohen ES, Tanck MW, et al. The contribution of classical risk factors to cardiovascular disease in familial hypercholesterolaemia: data in 2400 patients. J Intern Med 2004;256:482– 90. [10] Mozas P, Castillo S, Reyes G, et al. Apo E genotype is not associated with cardiovascular disease in heterozygous subjects with Familial Hypercholesterolaemia. Am Heart J 2003;145:999–1005. [11] Neil HA, Seagroatt V, Betteridge D, et al. Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia. Heart 2004;90:1431–7. [12] Simon Broome Familial Hyperlipidaemia Register Group and Scientific Committee. Genetic causes of familial hypercholesterolaemia in UK patients: relation to plasma lipid levels and coronary heart disease risk. J Med Genet 2006;43:943–9. [13] Gaudet D, Vohl MC, Couture P, et al. Contribution of receptor negative versus receptor defective mutations in the LDL-receptor gene to angiographically assessed coronary artery disease among young (25–49 years) versus middle-aged (50–64 years) men. Atherosclerosis 1999;143:153–61. [14] Bertolini S, Cantafora A, Averna M, et al. Clinical expression of familial hypercholesterolaemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype. Arterioscler Thromb Vasc Biol 2000;20:41–52. [15] Umans-Eckenhausen MA, Sijbrands EJ, Kastelein JJ, Defesche JC. Low-density lipoprotein receptor gene mutations and cardiovascular risk in a large genetic cascade screening population. Circulation 2002;106:3031–6. [16] Mata P, Alonso R, Castillo S, Pocovi M. MEDPED and the Spanish familial hypercholesterolaemia foundation. Atherosclerosis (Suppl) 2002;2(3):9–11. [17] Defesche J. Familial hypercholesterolaemia. In: Betteridge DJ, editor. Lipids and Vascular Disease. London, UK: Martin Dunitz Ltd.; 2000. P65–76. [18] National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. (Adult Treatment Panel III): JAMA 2001; 285: 2486–97. [19] Marcovina SM, Albers JJ, Scanu AM, et al. Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory medicine to evaluate analytical methods for the determination of plasma lipoprotein (a). Clin Chem 2000;46:1956–67. [20] Hixson J, Vernier D. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990;31:545–8.

321

[21] Mozas P, Castillo S, Tejedor D, et al. Molecular characterization of familial hypercholesterolaemia in Spain: identification of 39 novel and 77 recurrent mutations in LDLR. Hum Mutat 2004;24:187. [22] Tejedor D, Castillo S, Mozas P, et al. Reliable low-density DNA array based on allele-specific probes for detection of 118 mutations causing familial hypercholesterolaemia. Clin Chem 2005;51:1137–44. [23] Heath KE, Day IN, Humphries SE. Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low-density lipoprotein receptor gene. J Med Genet 2000;37:272–80. [24] Baena JM, del Val Garcia JL, Tomas J, et al. Cardiovascular disease epidemiology and risk factors in primary care. Rev Esp Cardiol 2005;58:367–73. [25] Lopez-Miranda J, Badimon L, Bonanome A, et al. Monounsaturated fat and cardiovascular risk. Nutr Rev 2006;64:S2–12. [26] Real JT, Chaves FJ, Martinez-Uso I, Garcia-Garcia AB, Ascaso JF, Carmena R. Importance of HDL cholesterol levels and the total/HDL cholesterol ratio as a risk factor for coronary heart disease in molecularly defined heterozygous familial hypercholesterolaemia. Eur Heart J 2001;22:465–71. [27] Hill JS, Hayden M, Frohlich J, Pritchard P. Genetic and environmental factors affecting the incidence of coronary artery disease in heterozygous familial hypercholesterolaemia. Arterioscler Thromb 1991;11:290–7. [28] Junyent M, Cofan M, Nu˜nez I, Gilabert R, Zambon D, Ros E. Influence of HDL cholesterol on preclinical carotid atherosclerosis in familial hypercholesterolaemia. Arterioscler Thromb Vasc Biol 2006;26:1107–13. [29] Koeijvoets K, Rodenburg J, Hutten B, Wiegman A, Kastelein J, Sijbrands E. Low-density lipoprotein receptor genotype and response to pravastatin in children with familial hypercholesterolaemia: substudy of an intima-media thickness trial. Circulation 2005;112:3168–73. [30] International Panel on Management of Familial Hypercholesterolaemia. Guidelines for the diagnosis and management of heterozygous familial hypercholesterolaemia. Atherosclerosis 2004;173:55–68. [31] Smilde TJ, Wissen S, Wollersheim H, Trip MD, Kastelein JJ, Stalenhoef AF. Effect of aggressive versus conventional lipid lowering on atherosclerosis progression in familial hypercholesterolaemia (ASAP): a prospective, randomised, double-blind trial. Lancet 2001;357:577–81. [32] Umans-Eckenhausen MA, Defesche JC, Sijbrands EJ, Scheerder RL, Kastelein JJ. Review of first 5 years of screening for familial hypercholesterolaemia in the Netherlands. Lancet 2001;357:165–8. [33] van Aalst-Cohen E, Jansen A, Tanck M, et al. Diagnosing familial hypercholesterolaemia: the relevance of genetic testing. Eur Heart J 2006;27:2240–6. [34] Wonderling D, Umans-Eckenhausen MA, Marks D, Defesche JC, Kastelein JJ, Thorogood M. Cost-effectiveness analysis of the genetic screening program for familial hypercholesterolaemia in The Netherlands. Semin Vasc Med 2004;4:97–104. [35] Leren TP. Cascade genetic screening for familial hypercholesterolaemia. Clin Genet 2004;66:483–7.