SAFEHEART is an open, multicenter, long-term prospective cohort study in a well-defined FH population, conducted in nineteen outpatient lipid clinics in Spain. Recruitment of subjects from FH families began in 2004 and is still ongoing. Inclusion criteria are: 1) index cases (IC) with genetic diagnosis of FH, 2) relatives over 15 years old with genetic diagnosis of FH, 3) relatives over 15 years old without a genetic diagnosis of FH (control group). Relatives with high cholesterol due to other causes in the control group were not excluded.

To assure and improve the quality of data, physicians attended to 3 different meetings where the different questionnaires and procedures were carefully explained. This study was approved by the local ethics committees and all eligible subjects gave written informed consent.

A Coordinating Centre was created to organize and to implement the follow-up of cases. A standardized phone call is made every year to the subjects to know any relevant changes in lifestyles, medication, and cardiovascular events. An active epidemiological surveillance system has been developed for the detection of fatal and non fatal cardiovascular events that are classified according to WHO-MONICA criteria [¹³].

Premature CHD is defined if one of the following events occurs in males before 55 years old and in females before 65 years old, 1) Myocardial infarction, proved by at least two of the following: classic symptoms (> 15 minutes); specific electrocardiographic changes; elevated cardiac enzymes (> 2× upper limit of normal). 2) Angina pectoris, diagnosed as classic symptoms in combination with at least one unequivocal result of one of the following: exercise test, nuclear scintigram, dobutamine stress ultrasound scan, > 70% stenosis on a coronary angiogram. 3) Percutaneous coronary intervention or other invasive procedures and coronary artery bypass grafting.

Demographic and clinical characteristics of subjects include: age, educational status, medical history focused on cardiovascular disease (CVD), classic cardiovascular risk factors (hypertension, type 2 diabetes, smoking status), physical examination and current treatment for hypercholesterolemia and other risk factors. History of CVD is obtained from medical charts provided by the subjects at inclusion. A quality of life (SF-12) [¹⁴], food frequency [Vázquez C, Alonso R, Garriga M, de Cos A, de la Cruz JJ, Fuentes-Jiménez F, Mata P. Validation of a food frequency questionnaire in Spanish patients with Familial Hypercholesterolaemia. Nutr Metab Cardiovasc Dis, in press] and physical activity [¹⁵] surveys are recorded on standardized form. Physical examination includes weight (kg), height (cm), body mass index (Kg/m²) and waist circumference (cm). Blood pressure is measured twice in supine position with an Omron MX3 sphyngomanometer.

Venous blood samples are taken after 12 hours of fasting. Serum, plasma and DNA samples are aliquotted and preserved at -80°C in a biobank located at the Cardiovascular Research Center in Barcelona. DNA is isolated from whole blood using standard methods and the genetic diagnosis of FH is made using a DNA-microarray² (Progenika SA, Bilbao, Spain). Serum total cholesterol, triglycerides and HDL-cholesterol levels are measured in a centralized laboratory using enzymatic methods. Serum LDL-c concentration is calculated using the Friedewald formula [¹⁶].

Mutations were classified as receptor-negative or receptor defective depending on their functional class as reported previously[⁷]. Those mutations with non reported functional class in the literature were classified as "unknown".

Maximum statin dose was defined as previously described [¹²]: simvastatin 80 mg/day, pravastatin 40 mg/day, lovastatin 80 mg/day, fluvastatin 80 mg/day, atorvastatin 80 mg/day, rosuvastatin 20-40 mg/day. Maximum statin (MS) dose combined with ezetimibe 10 mg/day was considered as maximum combined therapy (MCT). The following treatment was considered giving at least a 50% reduction in LDL-c baseline-levels: simvastatin 20 or 40 or 80 mg/day in combination with ezetimibe 10 mg/day, pravastatin 40 mg/day in combination with ezetimibe 10 mg/day, fluvastatin 80 mg/day in combination with ezetimibe 10 mg/day, atorvastatin 40 or 80 mg/day, atorvastatin 10 or 20 mg/day in combination with ezetimibe 10 mg/day, rosuvastatin 20 or 40 mg/day and rosuvastatin 10 mg/day in combination with ezetimibe 10 mg/day.

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 a non normal distribution, median and interquartilic-range were estimated. 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. The relationship between variables was considered statistically significant if the p value < 0.05.

A binary logistic regression analysis (forward) was conducted to determine the variables associated with the attainment of an LDL-c < 100 mg/dl, including those variables proved to be statistically significant in bivariate analyses and possible confounders (age, gender, tobacco, diabetes mellitus, high blood pressure, presence of CAD, type of mutation, type of LLT). The magnitude of the association was estimated using the adjusted odds ratio (OR) with a confidence interval (CI) of 95%. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS v 18.0, Chicago, IL, USA).

Most of the IC (88.3%) and FH relatives (73.3%) have been clinically diagnosed by the specialist. However, 25.4% of relatives with molecular diagnosis of FH (mean age 44.8 years, SD 15.9) did not know their clinical condition. Respect to genetic diagnosis, 120 different functional mutations in LDL-r and Apo B genes have been identified in this cohort and 25 mutations represent 65.0% of the cases recruited. Type of mutation was classified as receptor-negative or null mutations in 56.3% of FH subjects, while 35.2% had defective mutations.

Prevalence of current smokers was 28.4% in FH subjects and 34.6% in the control group (P < 0.001). There were no significant differences in the prevalence of type 2 diabetes and hypertension between both groups. HDL-c levels were lower in FH patients compared with controls (P < 0.001) and there were no differences between FH subjects with null and defective LDL-r mutations (49.1 ± 11.8 and 51.2 ± 14.3 mg/dl, respectively, P = 0.208).

History of CVD was present in 14.0% of FH patients, almost 5 times the prevalence found in non-FH subjects (p < 0.001). Coronary heart disease was the most frequent CVD (93.4%) and it was premature in 80.0% of cases. First cardiovascular event occurred earlier in index cases (IC) compared with FH and non-FH relatives (P < 0.001) (Table 1). Prevalence of cardiovascular disease, especially CHD was higher in patients carrying null mutations than those carrying defective mutations in LDL-r gene (14.8% vs. 10.6% respectively, P < 0.05).

At inclusion, 1056 (83.7%) FH cases (97.0% of IC and 78.0% of relatives) were receiving LLT compared to 18.8% of non-FH subjects (p < 0.001). Nearly all cases on LLT were receiving statins (95.4%), either in monotherapy (58.3%) or in combined therapy (37.0%), especially with ezetimibe (31.3%). The most frequent prescribed statins were Atorvastatin (57.3%), Simvastatin (28.9%) and Rosuvastatin (2,9%). Mean doses of statins were: Atorvastatin 44.8 mg (SD: 25.8), Simvastatin 40.7 mg (SD: 23.3) and Rosuvastatin 28.8 mg (SD: 12.7). Only 13.4% of FH subjects were receiving MS dose, and 13.6% were receiving MCT.

LDL-c level was not available in 91 FH cases.

Mean LDL-c level was 220.7 mg/dl (SD: 72.5) in those FH cases that were not receiving LLT (16.3%) compared to 186.5 mg/dl (SD: 65.6) of LDL-c level in FH cases on LLT (83.7%). Of those FH patients receiving LLT, only 33 (3.4%) had an LDL-c level < 100 mg/dl, while 149 (15.5%) had an alternative LDL level < 130 mg/dl. On the other hand, 270 FH patients (29.0%) receiving combined therapy (of whom 47.7% were on MCT) did not reach LDL-c goal. Cases with an LDL-c > 160 mg/dl and > 200 mg/dl in the MCT group were 54 (41.5%) and 21 (16.1%), respectively. LDL-c levels in patients using combined therapy with ezetimibe was slightly higher but not significant in those FH subjects with defective mutation compared with null mutation (171.2 ± 67.6 vs. 161.2 ± 57.2, respectively, P = 0.227).

Characteristics of FH patients according to attainment of LDL-c goal are shown in Table 2. Patients with an LDL-c on target were older, had a higher prevalence of CVD history, type 2 diabetes mellitus, hypertension, and were mostly treated with combined therapy than those subjects not on target (p < 0.05). The percentage of cases receiving LLT expected to reduce LDL-c levels at least 50% was 51.5% and was higher (63.6%) in patients on goal compared to those patients not on target (49.9%), although this difference was not statistically significant. However, LDL-c levels were lower in those patients with more intensive LLT (174.0 ± 62.2 mg/dl vs. 196.5 ± 63.8 mg/dl in patients receiving medication to reduce LDL-C ≥50% or below, respectively). There were no differences in LDL-c goal attainment between cases with null and defective mutations. The attainment of LDL-c goal was slightly higher but not statistically significant in those cases treated by specialists compared to general practitioners (59.3% vs 55.2%, P = 0.677).

Logistic regression analysis showed that combined therapy using statin and ezetimibe was significantly associated with the achievement of LDL-c goal (OR adjusted: 2.22; IC95%:1.10-4.48), after adjustment for age, sex, CVD and cardiovascular risk factors.

Fundación Jiménez Díaz, Madrid: PM, RA. Health Promotion and Epidemiology, Spanish Ministry of Health and Fundación Hipercolesterolemia Familiar, Madrid: NM. Centro de Investigación Cardiovascular CSIC-ICCC, Hospital Sant Pau and IIB-Sant Pau, Barcelona: LB, TP. IMIBIC/Hospital Reina Sofía, Córdoba: FF, FPJ, JLM. Hospital Virgen del Rocío, Sevilla: OM, J. Villar-Ortiz. Hospital Abente y Lago, A Coruña: JLD. Hospital de Lugo, Lugo: JIV, R. Argueso. Hospital San Pedro de Alcántara, Cáceres: JFS. Hospital San Pedro, Logroño: A. Brea, D. Mosquera. Hospital de Vitoria, Vitoria: LI. Hospital Comarcal Vega Baja, Orihuela: JM Cepeda. Hospital General de Albacete, Albacete: MAB. Hospital de Elche, Alicante: MP, AM Hidalgo. Hospital Central de Asturias, Oviedo: P Gómez-Enterría, C Martínez-Faedo. Hospital de Mérida, Badajoz: P Saenz. Hospital Clínico, Barcelona. D. Zambón. Hospital Ramón y Cajal, Madrid: C Vázquez, F. Arrieta. Hospital de Donostia, Donostia: F. Almagro. Hospital Ciudad Real, Ciudad Real: J Galiana. Hospital de Igualada: J. Panisello. Hospital Nuestra Señora de la Candelaria, Tenerife: F Pereyra, M Muros.

FH, Familial Hypercholesterolemia; FHR, Familial Hypercholesterolemia relatives; CVD, Cardiovascular Disease; LLT, Lipid lowering therapy; BMI, Body Mass Index; LDL-C, cholesterol transported on low-density lipoproteins; HDL-C, cholesterol transported on High Density Lipoproteins. * values are expressed as Mean and standard deviation; ** values are expressed as Median and interquartilic range.

FH, Familial hypercholesterolemia; LLT, Lipid-lowering therapy; CVD, Cardiovascular disease; BMI (Body mass index) (A) LDL-ch levels were uknown in 91 cases; (B) percentage calculated with total cases in each group. (C) Combined therapy includes only any statin + ezetimibe 10 mg; (D), Maximum therapy includes any statin at maximum dose + ezetimibe 10 mg; *Mean and Standard Deviation (SD).