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Cardiovascular risk assessment of dyslipidemic children: analysis of biomarkers to identify monogenic dyslipidemia

2014; Elsevier BV; Volume: 55; Issue: 5 Linguagem: Inglês

10.1194/jlr.p043182

1539-7262

Ana Margarida Medeiros, Ana Catarina Alves, Pedro Aguiar, Mafalda Bourbon,

Lipid metabolism and disorders

The distinction between a monogenic dyslipidemia and a polygenic/environmental dyslipidemia is important for the cardiovascular risk assessment, counseling, and treatment of these patients. The present work aims to perform the cardiovascular risk assessment of dyslipidemic children to identify useful biomarkers for clinical criteria improvement in clinical settings. Main cardiovascular risk factors were analyzed in a cohort of 237 unrelated children with clinical diagnosis of familial hypercholesterolemia (FH). About 40% carried at least two cardiovascular risk factors and 37.6% had FH, presenting mutations in LDLR and APOB. FH children showed significant elevated atherogenic markers and lower concentration of antiatherogenic particles. Children without a molecular diagnosis of FH had higher levels of TGs, apoC2, apoC3, and higher frequency of BMI and overweight/obesity, suggesting that environmental factors can be the underlying cause of their hypercholesterolem≥ia. An apoB/apoA1 ratio ≥0.68 was identified as the best biomarker (area under the curve = 0.835) to differentiate FH from other dyslipidemias. The inclusion in clinical criteria of a higher cut-off point for LDL cholesterol or an apoB/apoA1 ratio ≥0.68 optimized the criteria sensitivity and specificity. The correct identification, at an early age, of all children at-risk is of great importance so that specific interventions can be implemented. apoB/apoA1 can improve the identification of FH patients. The distinction between a monogenic dyslipidemia and a polygenic/environmental dyslipidemia is important for the cardiovascular risk assessment, counseling, and treatment of these patients. The present work aims to perform the cardiovascular risk assessment of dyslipidemic children to identify useful biomarkers for clinical criteria improvement in clinical settings. Main cardiovascular risk factors were analyzed in a cohort of 237 unrelated children with clinical diagnosis of familial hypercholesterolemia (FH). About 40% carried at least two cardiovascular risk factors and 37.6% had FH, presenting mutations in LDLR and APOB. FH children showed significant elevated atherogenic markers and lower concentration of antiatherogenic particles. Children without a molecular diagnosis of FH had higher levels of TGs, apoC2, apoC3, and higher frequency of BMI and overweight/obesity, suggesting that environmental factors can be the underlying cause of their hypercholesterolem≥ia. An apoB/apoA1 ratio ≥0.68 was identified as the best biomarker (area under the curve = 0.835) to differentiate FH from other dyslipidemias. The inclusion in clinical criteria of a higher cut-off point for LDL cholesterol or an apoB/apoA1 ratio ≥0.68 optimized the criteria sensitivity and specificity. The correct identification, at an early age, of all children at-risk is of great importance so that specific interventions can be implemented. apoB/apoA1 can improve the identification of FH patients. Dyslipidemia is one of the major cardiovascular risk factors. It can be due to a monogenic condition, but can also be secondary to specific conditions such as obesity, diabetes mellitus, hypothyroidism (1Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents; National Heart, Lung, and Blood Institute. 2011. Expert panel on integrated guidelines for cardiovascular health and risk reduction in children and adolescents: summary report. Pediatrics., 128(Suppl 5): S213–S256.Google Scholar), or even to polygenic or environmental causes (2Yusuf S. Hawken S. Ounpuu S. Dans T. Avezum A. Lanas F. McQueen M. Budaj A. Pais P. Varigos J. et al.Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study.Lancet. 2004; 364: 937-952Abstract Full Text Full Text PDF PubMed Scopus (8412) Google Scholar). Because lipids, and other cardiovascular risk factors, track into adulthood, and the aggregation of classical risk factors such as lipid levels, blood pressure, BMI, diabetes mellitus, and tobacco use is associated with an even higher cardiovascular risk (3Berenson G.S. Srinivasan S.R. Bao W. Newman III, W.P. Tracy R.E. Wattigney W.A. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. The Bogalusa Heart Study.N. Engl. J. Med. 1998; 338: 1650-1656Crossref PubMed Scopus (3113) Google Scholar, 4Strong J.P. Malcom G.T. McMahan C.A. Tracy R.E. Newman III, W.P. Herderick E.E. Cornhill J.F. Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention from the Pathobiological Determinants of Atherosclerosis in Youth Study.JAMA. 1999; 281: 727-735Crossref PubMed Scopus (732) Google Scholar), it is important to identify at-risk children at a young age so therapeutic measures and/or lifestyle modifications can be implemented early in life to decrease their cardiovascular risk (1Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents; National Heart, Lung, and Blood Institute. 2011. Expert panel on integrated guidelines for cardiovascular health and risk reduction in children and adolescents: summary report. Pediatrics., 128(Suppl 5): S213–S256.Google Scholar, 5Daniels S.R. Greer F.R. Lipid screening and cardiovascular health in childhood.Pediatrics. 2008; 122: 198-208Crossref PubMed Scopus (969) Google Scholar). Familial hypercholesterolemia (FH) is the most frequently diagnosed inherited lipid disorder in children and adolescents (6Daniels S.R. Gidding S.S. de Ferranti S.D. Pediatric aspects of familial hypercholesterolemias: recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S30-S37Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). FH is an autosomal dominant condition resulting in severely elevated LDL cholesterol (LDL-C) concentrations in plasma from birth and has a frequency of about 1:400/500 in most populations (7Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolaemia.in: Scriver C.R. Beaudet A.L. In Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill, New York1995: 1981-2030Google Scholar). FH is mainly due to loss-of-function mutations in the LDL receptor gene (LDLR) (7Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolaemia.in: Scriver C.R. Beaudet A.L. In Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill, New York1995: 1981-2030Google Scholar) or the apolipoprotein B gene (APOB) (8Soria L.F. Ludwig E.H. Clarke H.R. Vega G.L. Grundy S.M. McCarthy B.J. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B-100.Proc. Natl. Acad. Sci. USA. 1989; 86: 587-591Crossref PubMed Scopus (467) Google Scholar). Gain-of-function mutations in proprotein convertase subtilisin kexin type 9 gene (PCSK9) (9Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2168) Google Scholar), or even in other genes yet to be described, are a rare cause of FH (10Hopkins P.N. Toth P.P. Ballantyne C.M. Rader D.J. Familial hypercholesterolemias: prevalence, genetics, diagnosis and screening recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S9-S17Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 11Soutar A.K. Naoumova R.P. Mechanisms of disease: genetic causes of familial hypercholesterolemia.Nat. Clin. Pract. Cardiovasc. Med. 2007; 4: 214-225Crossref PubMed Scopus (456) Google Scholar). FH confers lifelong risk of atherosclerosis beginning in childhood and is associated with premature CVD (pCVD), so early screening is justified (6Daniels S.R. Gidding S.S. de Ferranti S.D. Pediatric aspects of familial hypercholesterolemias: recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S30-S37Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12DeMott K. Nherera L. Shaw E.J. Minhas R. Humphries S.E. Kathoria M. Ritchie G. Nunes V. Davies D. Lee P. et al.Clinical Guidelines and Evidence Review for Familial Hypercholesterolaemia.The Identification and Management of Adults and Children with Familial Hypercholesterolaemia. National Collaborating Centre for Primary Care and Royal College of General Practitioners. 2008; (London)Google Scholar, 13Catapano A.L. Reiner Z. De Backer G. Graham I. Taskinen M.R. Wiklund O. Agewall S. Alegria E. Chapman M. Durrington P. et al.ESC/EAS guidelines for the management of dyslipidaemias. The task force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS).Atherosclerosis. 2011; 217: 3-46Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar). Evidence from studies with treated and untreated FH children indicates that early identification and treatment is associated with reduced subclinical evidence of atherosclerosis (6Daniels S.R. Gidding S.S. de Ferranti S.D. Pediatric aspects of familial hypercholesterolemias: recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S30-S37Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14Rodenburg J. Vissers M.N. Wiegman A. van Trotsenburg A.S. van der Graaf A. de Groot E. Wijburg F.A. Kastelein J.J. Hutten B.A. Statin treatment in children with familial hypercholesterolemia: the younger, the better.Circulation. 2007; 116: 664-668Crossref PubMed Scopus (238) Google Scholar). However, in most of the cohorts described, a mutation causing disease can be found in varying percentages, ranging from 20 to 90% of the cases (15Futema M. Plagnol V. Whittall R.A. Neil H.A. Humphries S.E. Use of targeted exome sequencing as a diagnostic tool for familial hypercholesterolaemia.J. Med. Genet. 2012; 49: 644-649Crossref PubMed Scopus (63) Google Scholar, 16Motazacker M.M. Pirruccello J. Huijgen R. Do R. Gabriel S. Peter J. 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The identification of specific biomarkers that can help to distinguish between monogenic and polygenic/environmental dyslipidemia is important so both groups of children receive the appropriate treatment and/or counseling to reduce their cardiovascular risk. Previous studies in adults have selected plasma levels of apoB and apoA1 as good predictors of cardiovascular risk and the apoB/apoA1 ratio has been considered the best marker of the balance between atherogenic and antiatherogenic particles (2Yusuf S. Hawken S. Ounpuu S. Dans T. Avezum A. Lanas F. McQueen M. Budaj A. Pais P. Varigos J. et al.Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study.Lancet. 2004; 364: 937-952Abstract Full Text Full Text PDF PubMed Scopus (8412) Google Scholar, 20Walldius G. Jungner I. Holme I. Aastveit A.H. Kolar W. Steiner E. 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Here we present the cardiovascular risk assessment of a cohort of children with a clinical diagnosis of FH in order to identify useful biomarkers for clinical criteria improvement to distinguish between monogenic and polygenic/environmental dyslipidemia in clinical settings. A total of 237 unrelated children (2–17 years old) were referred as index patients to the Portuguese FH Study (PFHS) (24Bourbon M. Rato Q. Portuguese Familial Hypercholesterolemia Study: presentation of the study and preliminary results.Rev. Port. Cardiol. 2006; 25: 999-1013PubMed Google Scholar, 25Bourbon M. Alves A.C. Medeiros A.M. Silva S. Soutar A.K. Familial hypercholesterolaemia in Portugal.Atherosclerosis. 2008; 196: 633-642Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Medeiros A.M. Alves A.C. Francisco V. Bourbon M. Update of the Portuguese Familial Hypercholesterolaemia Study.Atherosclerosis. 2010; 212: 553-558Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27Alves A.C. Medeiros A.M. Francisco V. Gaspar I.M. Rato Q. Bourbon M. Molecular diagnosis of familial hypercholesterolemia: an important tool for cardiovascular risk stratification.Rev. Port. Cardiol. 2010; 29: 907-921PubMed Google Scholar) during 1999–2012, mainly by pediatricians, cardiologists, and clinical geneticists countrywide. Only children with two independent altered fasting lipid profiles were recruited for this study. The recruitment criteria applied was having a clinical diagnosis of FH according to an adaptation of the Simon Broome criteria (24Bourbon M. Rato Q. Portuguese Familial Hypercholesterolemia Study: presentation of the study and preliminary results.Rev. Port. Cardiol. 2006; 25: 999-1013PubMed Google Scholar): children were admitted when presenting total cholesterol (TC) >260 mg/dl or LDL-C >155 mg/dl, and a family history of hypercholesterolemia or pCVD. In a few cases, children under the age of 10 were admitted to the study with lower cut-off points for cholesterol values (TC >200 mg/dl or LDL-C >120 mg/dl) when a severe dyslipidemia was present in one of the parents (TC >300 mg/dl or LDL-C >200 mg/dl). Family history of hypercholesterolemia was defined if hypercholesterolemia (TC >290 mg/dl) was present in at least one of the parents and additionally in other members of the family (siblings, grandparents, and/or uncles). According to Simon Broome criteria, pCVD was defined if any of the following events: angina, myocardial infarction, percutaneous transluminal coronary angioplasty, or coronary artery bypass grafting, occurred for the first time before 55 years of age in males and 65 years of age in females. Additionally 138 children (1–17 years old) were referred to the PFHS cascade screening program as being related to a previously identified FH patient (children, grandchildren, nephews, cousins, or siblings). Exclusion criteria were thyroid dysfunction and diabetes. Parents and other relatives with and without a clinical diagnosis of FH were also recruited. Written informed consent was obtained from all participants before their inclusion in the study. The study protocol and database were previously approved by the National Institute of Health (INSA) Ethical Committee and the National Data Protection Commission. TC, LDL-C, and TGs greater than the 95th percentile, for age and sex, were defined according to the reference values for the Spanish population (28Gómez-Gerique J.A. Gutiérrez-Fuentes J.A. Montoya M.T. Porres A. Rueda A. Avellaneda A. Rubio M.A. Lipid profile of the Spanish population: the DRECE (diet and risk of cardiovascular disease in Spain) study. DRECE study group.Med. Clin. (Barc.). 1999; 113: 730-735PubMed Google Scholar), due to the absence of percentile distributions for fasting serum lipids in the Portuguese population. Because values did not differ greatly between sexes and ages, the following mean values were assumed for the 95th percentile: TC >225 mg/dl, LDL-C >135 mg/dl, and TGs >125 mg/dl. BMI percentiles were calculated for age and gender according to the Centre for Disease Control growth charts (29Kuczmarski R.J. Ogden C.L. Guo S.S. Grummer-Strawn L.M. Flegal K.M. Mei Z. Wei R. Curtin L.R. Roche A.F. Johnson C.L. 2000 CDC Growth Charts for the United States: methods and development.Vital Health Stat. 11. 2002; 246: 1-190PubMed Google Scholar) as recommended by the Portuguese Directorate-General for Health. Three BMI percentile cut-offs were defined in this work: 75th, 85th, and 95th. Overweight and obesity were defined as a BMI greater than the 85th and 95th percentile, respectively (29Kuczmarski R.J. Ogden C.L. Guo S.S. Grummer-Strawn L.M. Flegal K.M. Mei Z. Wei R. Curtin L.R. Roche A.F. Johnson C.L. 2000 CDC Growth Charts for the United States: methods and development.Vital Health Stat. 11. 2002; 246: 1-190PubMed Google Scholar). Stage 1 and stage 2 hypertension were defined as systolic or diastolic blood pressure greater than the 95th and 99th percentile, respectively, for age and gender according to the "The Fourth Report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents" tables (30National High Blood Pressure Education Working Group on High Blood Pressure in Children and Adolescents. 2004. The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics., 114: 555–576.Google Scholar), as recommended by the Portuguese Directorate-General for Health. Demographic and clinical data on cardiovascular risk factors, such as blood pressure, weight and height, physical activity, personal medical history, lipid profile, and family history of pCVD, as well as lipid-lowering measures, were obtained by the assistant clinician for all index patients in a form adapted from the Simon Broome Registry at the time of referral to the study. For relatives, a simpler form was fulfilled with only demographic data, personal medical history of pCVD, lipid profile, and lipid-lowering measures. Fasting blood samples were collected from individuals at the time of referral to the study. TC, direct LDL-C, HDL cholesterol (HDL-C), TGs, apoA1, apoB, and lipoprotein (a) [Lp(a)] were determined for all individuals in a Cobas Integra 400 plus system (Roche) by enzymatic colorimetric and immunoturbidimetric methods. Additionally, in all children included as index patients and received after 2010, serum levels of apoA2, apoC2, apoC3, apoE, and sdLDL (sLDL-EX "SEIKEN" kit) were measured by direct quantification in an RX Daytona analyzer (Randox Laboratories), mostly by enzymatic colorimetric and immunoturbidimetric methods. The genetic diagnosis of FH was performed by the molecular analysis of APOB (two fragments of exons 26 and 29), LDLR (including the study of splice regions and large rearrangements), and PCSK9 genes as reported previously (26Medeiros A.M. Alves A.C. Francisco V. Bourbon M. Update of the Portuguese Familial Hypercholesterolaemia Study.Atherosclerosis. 2010; 212: 553-558Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Mutations were considered to be pathogenic if cosegregation of the mutation with the phenotype was observed and if mutations were previously described in other populations. Pathogenicity of novel variants was assessed according to Cotton and Scriver criteria (31Cotton R.G. Scriver C.R. Proof of "disease causing" mutation.Hum. Mutat. 1998; 12: 1-3Crossref PubMed Scopus (160) Google Scholar): cosegregation analysis (in at least 80% of the relatives), absence in a panel of a minimum of 50 normolipidemic individuals, amino acid nature and conservation in different species, and, when possible, by functional assays as reported before (32Bourbon M. Duarte M.A. Alves A.C. Medeiros A.M. Marques L. Soutar A.K. Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.J. Med. Genet. 2009; 46: 352-357Crossref PubMed Scopus (37) Google Scholar, 33Silva S. Alves A.C. Patel D. Malho R. Soutar A.K. Bourbon M. In vitro functional characterization of missense mutations in the LDLR gene.Atherosclerosis. 2012; 225: 128-134Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Statistical analysis was performed using SPSS software (version 17.0 for Windows; SPSS, Chicago, IL). Comparison of frequencies between qualitative variables was carried out using the chi-squared test. Mean values of quantitative variables were compared with the Student's t-test or ANOVA for independent data while median values were compared with the nonparametric Mann-Whitney or Kruskal Wallis median tests. Pearson correlation was conducted to determine associations between variables. P < 0.05 was considered to be statistically significant. Biomarker cut-off values were determined from receiver operating characteristic (ROC) curves with the area under the curve (AUC) >0.7 using pretreatment values for FH and non-FH children in order to find biomarkers to distinguish these two groups. For biomarker selection, criteria were sensitivity and specificity values above 50% and sensitivity higher than specificity. The value that maximized the sum of sensitivity and specificity was selected as the optimal cut-off point for each biomarker. Different criteria for the clinical diagnosis of FH were established using novel cut-off points and were compared with the genetic diagnosis using cross-tables. Sensitivity, specificity, and kappa statistic were calculated to evaluate the inter-diagnostic agreement. Kappa statistic ranges between negative values and 1, indicating no agreement and perfect agreement, respectively, among raters. A total of 237 unrelated children (131 girls and 106 boys) were referred to PFHS. Mean age at inclusion was 10.0 ± 3.6 years (2–17 years). All children were reported to be nonsmokers. Physical symptoms such as xanthoma were absent and, therefore, all children were classified as "possible FH" according to Simon Broome criteria. Clinical and biochemical characteristics are shown in Table 1.TABLE 1.Clinical and biochemical characteristics of all the children included in the studyDemographic and Clinical DataaAt the time of referral to PFHS.Cardiovascular Risk FactorsaAt the time of referral to PFHS.Lipids (mg/dl)DemographicBMI (kg/m2) (n = 218)aAt the time of referral to PFHS.19.8 ± 4.2Basic lipid profilebHighest values reported without treatment.n = 237Index patients237 95th percentile26.1%TG97.5 ± 57.31st or 2nd degree relative with CVD24.5%Physical activityn = 207Advanced lipid profilecAdvanced lipid profile performed at INSA recorded only for children without treatment.n = 155Physical symptoms≤2 h/week66.7%Lp(a)55.9 ± 61.1Xanthomas0.0%3-4 h/week24.2%apoA1149.1 ± 28.1Therapeutic measures5-6 h/week7.7%apoB100.0 ± 31.9On diet49.4%≥7 h/week1.4%Specific lipid profiledSpecific lipid profile performed at INSA (only available after 2010) recorded only for children without treatment.n = 90On medication32.3%Blood pressuren = 150apoA228.8 ± 5.4Normal64.7%apoC23.7 ± 1.5Prehypertension19.3%apoC38.4 ± 2.2Stage 1 and 2 hypertension16.0%apoE3.1 ± 0.8Smoking habits0.0%sdLDL29.9 ± 14.9Data are expressed as mean ± SD unless otherwise noted.a At the time of referral to PFHS.b Highest values reported without treatment.c Advanced lipid profile performed at INSA recorded only for children without treatment.d Specific lipid profile performed at INSA (only available after 2010) recorded only for children without treatment. Open table in a new tab Data are expressed as mean ± SD unless otherwise noted. Besides the dyslipidemia present in all the children referred to PFHS as index patients, other cardiovascular risk factors such as obesity/overweight, hypertension, physical inactivity, and family history of pCVD (1st/2nd degree relative) were also evaluated (Table 1). TC and/or LDL-C above the 95th percentile were the most frequent cardiovascular risk factors in the study population (89.5%), followed by overweight/obesity (41.7%), family history of pCVD (24.5%), TGs >95th percentile (16%), and hypertension (16%). About 40% carried at least two cardiovascular risk factors. A molecular defect was identified in 89 children referred as index patients. A total of 85 children had a LDLR mutation and 4 had an APOB mutation (37.6%). In the APOB, two different mutations were found that have been reported before as a cause of FH (supplementary Table I). In the LDLR, 63 different mutations were found and considered pathogenic: 42 were null alleles or had been proven to be pathogenic by functional studies; 14 did not have functional studies but had been reported in FH patients from other populations and in general fulfilled Cotton and Scriver criteria (31Cotton R.G. Scriver C.R. Proof of "disease causing" mutation.Hum. Mutat. 1998; 12: 1-3Crossref PubMed Scopus (160) Google Scholar) for a mutation causing disease, with exception for 4 mutations that showed low penetrance in children (2 functional mutations and 2 without functional studies), and another 3 mutations that showed weak cosegregation but had been described before as mutations causing disease. The remaining seven mutations were novel and predicted to be pathogenic according to Cotton and Scriver criteria (31Cotton R.G. Scriver C.R. Proof of "disease causing" mutation.Hum. Mutat. 1998; 12: 1-3Crossref PubMed Scopus (160) Google Scholar) (supplementary Table I). Homozygous or compound heterozygous were not identified in the present cohort. No mutations were found in the PCSK9 gene. In the remaining 148 children (62.4%), no mutations considered pathogenic were identified using the current molecular biology techniques previously published (26Medeiros A.M. Alves A.C. Francisco V. Bourbon M. Update of the Portuguese Familial Hypercholesterolaemia Study.Atherosclerosis. 2010; 212: 553-558Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27Alves A.C. Medeiros A.M. Francisco V. Gaspar I.M. Rato Q. Bourbon M. Molecular diagnosis of familial hypercholesterolemia: an important tool for cardiovascular risk stratification.Rev. Port. Cardiol. 2010; 29: 907-921PubMed Google Scholar). The cascade screening program performe