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Lipid levels and their genetic regulation in patients with familial hypercholesterolemia and familial defective apolipoprotein B-100: the MEDPED Slovakia Project
Atherosclerosis Supplements 4 (2003) 3–5
Lipid levels and their genetic regulation in patients with familial hypercholesterolemia and familial defective apolipoprotein B-100: the MEDPED Slovakia Project Branislav Vohnout a,j,∗ , Katar´ına Rašlová a , Juraj Gašparovi˘c a , Jana Franeková b , Lubom´ıra Fábryová c , Martina Belošovi˘cová d , Gustáv Ková˘c e , Claudia Šebová e , Eva Rajecová f , Jozef Stavný g , Miron Babjak h , Maria B. Donati i , Licia Iacoviello j a
j
Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovak Republic b OKB NsP Žilina, Bratislava, Slovak Republic c NsP Milosrdn´ı bratia, Bratislava, Slovak Republic d DFNsP Bratislava, Slovak Republic e FNsP aDerera Bratislava, Slovak Republic f UTaRCH Bratislava, Slovak Republic g OKB NsP Poprad, Slovak Republic h OKB NsP Humenne, Slovak Republic i Center for High Technology Research and Education in Biomedical Sciences, Catholic University, 86100 Campobasso, Italy Angela Valenti Laboratory of Genetic and Environmental Risk Factors for Thrombotic Disease, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy
Abstract We examined, from a cohort of 165 families, 529 individuals for familial hypercholesterolemia (FH). Utilising clinical criteria for diagnosis, we identified 122 patients (n = 41 families) as having FH. With PCR testing, 31 individuals (n = 12 families) were found to have familial defective Apo B-100 (FDB). From the cohort, 102 normolipidemic (NL) individuals served as a control group. Patients with FH had the highest levels of total cholesterol (TC), LDL-cholesterol (LDL-C) and apolipoprotein B (Apo B), followed by FDB patients and the normolipidemic relatives had the lowest levels (P < 0.0001 for all parameters). We did not find any effect of Apo E genotypes on lipid levels in the NL or FH group. Therefore, other genetic and/or environmental factors may be responsible for the diversity in the clinical expression in these populations. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Familial hypercholesterolemia; Familial defective Apo B-100; Apo E; Polymorphism
1. Introduction Familial hypercholesterolemia (FH) is an autosomal co-dominant genetic disease characterised by markedly elevated LDL-cholesterol (LDL-C) levels, presence of tendon xanthomas and premature atherosclerosis [1]. Familial defective apolipoprotein B-100 (FDB) is another monogenic disorder associated with primary hypercholesterolemia. The classical form of FDB is due to R3500Q mutation [1].
∗
Corresponding author. E-mail address: [email protected] (B. Vohnout).
In some FDB heterozygotes, the clinical picture is indistinguishable from those with heterozygous FH, although the former tends to have less severe hypercholesterolemia [2]. It is expected that such a phenotype is due to a compensatory mechanisms through elevated very low density lipoprotein (VLDL) remnant uptake by apolipoprotein E (Apo E) mediated LDL receptor pathway [3]. Apo E serves as a ligand for the Apo B/E receptor and Apo E polymorphism is an important factor determining the variability of plasma lipid levels. Polymorphism of the Apo E gene may play an important role in determining diversity in the clinical expression of FH, although, no clear pattern of Apo E alleles has been established in FH patients [4].
1567-5688/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1567-5688(03)00023-0
4
B. Vohnout et al. / Atherosclerosis Supplements 4 (2003) 3–5
2. Patients and methods Five hundred and twenty-nine subjects from 165 families with familial occurrence of hypercholesterolemia were screened for the presence of FH or FDB. Subjects were recruited from Lipid Clinics working with the MEDPED project in Slovakia. Venous blood samples were collected in EDTA tubes after overnight fasting. Plasma levels of lipids, lipoproteins and apolipoproteins were measured by standard automated enzymatic methods. LDL-C was calculated using the Friedewald formula. Clinical diagnosis of FH was based on widely accepted criteria [1,5]. In each family, a previously identified patient was classified as the proband. Subsequently, first-degree and relevant second-degree relatives of the proband were invited to the Lipid Clinic for evaluation. The Apo B R3500Q mutation was detected by allele specific polymerase chain reaction (PCR) [6] in all of the probands. Positive finding of the mutation resulted in relatives of the proband also being evaluated for the mutation. Genotypes for Apo E were determined by PCR gene amplification and cleavage with HhaI [7]. Multivariate analysis was used to identify correlates among the different groups (FH, FDB or NL). Apo E genotype, sex, age and body mass index (BMI) were factors examined to predict changes in lipid and apolipoprotein levels between the groups and in each group separately. The frequencies of the Apo E alleles and genotypes were determined by genotype count and compared with the values predicted on the basis of the assumption of Hardy–Weinberg equilibrium performing the exact test of Hardy–Weinberg proportion for multiple alleles. P < 0.05 was considered as statistically significant.
3. Results Of the 165 families (n = 529 individuals), we identified 122 affected persons with a clinical diagnosis of FH (from 41 families, mean: 2.98 FH patients per family, range: 1–8 patients per family). Twelve families were identified as having FDB by PCR, totalling 31 affected patients (mean: 2.6 FDB patients per family, range: 1–5 patients per family). The control group consisted of 102 normolipidemic (NL) relatives from the cohort. Main characteristics of the patients with FH, FDB and NL are listed in Table 1. There was a difference in age and BMI distribution between the groups (P < 0.05 and 0.01, respectively); the main determinant of the difference was older age and higher BMI in FH patients as compared to NL group (P < 0.005 for both). As expected, there was a statistically significant difference in total cholesterol (TC), LDL-C and Apo B levels among the groups. Patients with FH had the highest levels of total cholesterol, LDL-C and Apo B, followed by FDB patients and the normolipidemic relatives had the lowest levels (P < 0.0001 for all parameters). The same trend was found with triglyceride (TG) levels (P < 0.001). FH patients had lower
Table 1 Characteristics of individuals with FH, FDB and normolipidemic (NL) controls
Number of families Number of patients Age (years) BMI (kg/m2 )a Females (%) TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) TG (mmol/l) Apo AI (g/l)b Apo B (g/l)c
FH
FDB
41 122 35.7 24.0 59.2 9.1 7.0 1.3 1.5 1.2 1.5
12 31 32.6 23.7 54.8 7.4 5.5 1.3 1.3 1.2 1.1
± 18.3& ± 4.4# ± ± ± ± ± ±
0.14∗ 0.13∗ 0.02& 0.08∗∗ 0.03‡ 0.03∗
NL
± 15.8 ± 5.3 ± ± ± ± ± ±
0.27 0.25 0.05 0.15 0.05 0.06
102 28.8 22.1 55.9 4.9 3.0 1.4 1.0 1.3 0.7
± 16.0 ± 4.4 ± ± ± ± ± ±
0.15 0.14 0.03 0.09 0.03 0.03
Values are mean ± S.E., except for age and BMI where mean ± S.D.; TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglycerides; Apo, apolipoprotein. a Data on BMI were missing for 11 FH patients, 7 NL controls and 1 FDB patient. b Data on Apo AI were missing for 55 FH patients, 32 NL controls and 12 FDB patients. c Data on Apo B were missing for 23 FH patients, 11 NL controls and 6 FDB patients. ∗ P < 0.0001. ∗∗ P < 0.001. & P < 0.05. ‡ P < 0.005. # P < 0.01.
high density lipoprotein cholesterol (HDL-C) and Apo AI levels compare to normolipidemic controls (P < 0.05 and <0.005, respectively). All comparisons were adjusted for age, sex and BMI. Apo E polymorphism was evaluated only in unrelated persons (41 FH, 12 FDB and 49 NL group). Genotype distribution was in Hardy–Weinberg equilibrium for all groups. The frequencies of Apo E genotypes were 12.2, 2.4, 70.7 and 14.7% for genotypes 23, 24, 33 and 34 in FH and 0, 0, 91.7 and 8.3% in FDB group. NL group Apo E genotypes were 10.2, 0, 69.4 and 20.4%. There was no difference in the frequency of Apo E genotypes between FH, FDB and normolipidemic subjects (P = 0.67). The impact of Apo E genotypes on lipid levels was analysed using a general linear regression model with age, sex and BMI as covariates. An association between Apo E polymorphism and lipid levels could not be demonstrated in FH and normolipidemic subjects (data not shown). Because of insufficient number of subjects, the FDB group was not analysed.
4. Discussion Although clinical and biochemical phenotypes of FDB bear some similarity to those of FH, mean levels of plasma LDL-C in FDB are only mildly elevated compared with FH [1]. In our study, patients with FH had significantly higher levels of TC, LDL-C and Apo B then patients with FDB. This finding has been explained by an Apo E mediated
B. Vohnout et al. / Atherosclerosis Supplements 4 (2003) 3–5
clearance of VLDL and intermediate density lipoprotein (IDL) particles in FDB [3]. In our study, FDB was diagnosed using the PCR method and FH clinically, based on strict lipid criteria. Therefore, we cannot exclude that this underestimates the real prevalence of FH patients in our cohort and explain to a certain degree, differences in lipid levels between FDB and FH patients. Apo E polymorphism can be a factor that determines diversity in the clinical expression of FH. Previous studies have examined the effect of Apo E alleles on the phenotypic expression of FH with ambiguous results [4]. We did not find any effect of the Apo E genotype on lipid levels in the FH or NL group. Similarly, in a previous study, we did not find any significant effect of Apo E genotype on lipid levels in Slovak premature myocardial infarction (MI) men versus their spouses and control couples who did not have a personal or family history of MI [8]. Therefore, we conclude that Apo E polymorphisms are not responsible for the variability in lipid levels in either the normal or diseased Slovak population. Other genetic and/or environmental factors may be responsible for the diversity in the clinical expression of FH. Acknowledgements The authors thank Natália Arvayová, Adriana Uher˘ciková and Zuzana Obernauerová for excellent assistance in the examination of the patients, Dr. Monika Ivani˘cová for her help in patients’ examination and evaluation, Dr. Augusto Di Castelnuovo for his helpful suggestions in statistical evalu-
5
ation and Dr. Susan Stephenson for her useful suggestions. Branislav Vohnout, M.D. was a recipient of aMarie Curie Individual Fellowship (HPMF-CT-1999-00099)
References [1] WHO Familial Hypercholesterolemia. Human Genetics Program. Division of Noncommunicable Diseases. Geneva, WHO/HGN/FH/ CONC/98.7. p. 1–45. [2] Hansen PS, Defesche JC, Kastelein JJP, et al. Phenotypic variation in patients heterozygous for familial defective apolipoprotein B (FDB) in three European countries. Arterioscler Thromb Vasc Biol 1997;17:741–7. [3] Miserez AR, Keller W. Differences in the phenotypic characteristics of subjects with familial defective apolipoprotein B-100 and familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15:1719– 29. [4] Hill JS, Hayden MR, Frohlich J, Pritchard PH. Genetic and environmental factors affecting the incidence of coronary artery disease in heterozygous Familial Hypercholesterolemia. Arterioscler Thromb 1991;11:290–7. [5] Williams RR, Hunt SC, Schumacher C, et al. Diagnosing heterozygous Familial Hypercholesterolemia using new practical criteria validated by molecular genetics. Am J Cardiol 1993;72:171–6. [6] Schuster H, Rauh G, Muller S, et al. Polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100. Anal Biochem 1992;204:22–5. [7] Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990;31:545–8. [8] Raslova K, Smolkova B, Vohnout B, et al. Risk factors for atherosclerosis in survivors of myocardial infarction and their spouses: comparison to controls without personal and family history of atherosclerosis. Metabolism 2001;50:24–9.
Lipid levels and their genetic regulation in patients with familial hypercholesterolemia and familial defective apolipoprotein B-100: the MEDPED Slovakia Project Branislav Vohnout a,j,∗ , Katar´ına Rašlová a , Juraj Gašparovi˘c a , Jana Franeková b , Lubom´ıra Fábryová c , Martina Belošovi˘cová d , Gustáv Ková˘c e , Claudia Šebová e , Eva Rajecová f , Jozef Stavný g , Miron Babjak h , Maria B. Donati i , Licia Iacoviello j a
j
Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovak Republic b OKB NsP Žilina, Bratislava, Slovak Republic c NsP Milosrdn´ı bratia, Bratislava, Slovak Republic d DFNsP Bratislava, Slovak Republic e FNsP aDerera Bratislava, Slovak Republic f UTaRCH Bratislava, Slovak Republic g OKB NsP Poprad, Slovak Republic h OKB NsP Humenne, Slovak Republic i Center for High Technology Research and Education in Biomedical Sciences, Catholic University, 86100 Campobasso, Italy Angela Valenti Laboratory of Genetic and Environmental Risk Factors for Thrombotic Disease, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy
Abstract We examined, from a cohort of 165 families, 529 individuals for familial hypercholesterolemia (FH). Utilising clinical criteria for diagnosis, we identified 122 patients (n = 41 families) as having FH. With PCR testing, 31 individuals (n = 12 families) were found to have familial defective Apo B-100 (FDB). From the cohort, 102 normolipidemic (NL) individuals served as a control group. Patients with FH had the highest levels of total cholesterol (TC), LDL-cholesterol (LDL-C) and apolipoprotein B (Apo B), followed by FDB patients and the normolipidemic relatives had the lowest levels (P < 0.0001 for all parameters). We did not find any effect of Apo E genotypes on lipid levels in the NL or FH group. Therefore, other genetic and/or environmental factors may be responsible for the diversity in the clinical expression in these populations. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Familial hypercholesterolemia; Familial defective Apo B-100; Apo E; Polymorphism
1. Introduction Familial hypercholesterolemia (FH) is an autosomal co-dominant genetic disease characterised by markedly elevated LDL-cholesterol (LDL-C) levels, presence of tendon xanthomas and premature atherosclerosis [1]. Familial defective apolipoprotein B-100 (FDB) is another monogenic disorder associated with primary hypercholesterolemia. The classical form of FDB is due to R3500Q mutation [1].
∗
Corresponding author. E-mail address: [email protected] (B. Vohnout).
In some FDB heterozygotes, the clinical picture is indistinguishable from those with heterozygous FH, although the former tends to have less severe hypercholesterolemia [2]. It is expected that such a phenotype is due to a compensatory mechanisms through elevated very low density lipoprotein (VLDL) remnant uptake by apolipoprotein E (Apo E) mediated LDL receptor pathway [3]. Apo E serves as a ligand for the Apo B/E receptor and Apo E polymorphism is an important factor determining the variability of plasma lipid levels. Polymorphism of the Apo E gene may play an important role in determining diversity in the clinical expression of FH, although, no clear pattern of Apo E alleles has been established in FH patients [4].
1567-5688/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1567-5688(03)00023-0
4
B. Vohnout et al. / Atherosclerosis Supplements 4 (2003) 3–5
2. Patients and methods Five hundred and twenty-nine subjects from 165 families with familial occurrence of hypercholesterolemia were screened for the presence of FH or FDB. Subjects were recruited from Lipid Clinics working with the MEDPED project in Slovakia. Venous blood samples were collected in EDTA tubes after overnight fasting. Plasma levels of lipids, lipoproteins and apolipoproteins were measured by standard automated enzymatic methods. LDL-C was calculated using the Friedewald formula. Clinical diagnosis of FH was based on widely accepted criteria [1,5]. In each family, a previously identified patient was classified as the proband. Subsequently, first-degree and relevant second-degree relatives of the proband were invited to the Lipid Clinic for evaluation. The Apo B R3500Q mutation was detected by allele specific polymerase chain reaction (PCR) [6] in all of the probands. Positive finding of the mutation resulted in relatives of the proband also being evaluated for the mutation. Genotypes for Apo E were determined by PCR gene amplification and cleavage with HhaI [7]. Multivariate analysis was used to identify correlates among the different groups (FH, FDB or NL). Apo E genotype, sex, age and body mass index (BMI) were factors examined to predict changes in lipid and apolipoprotein levels between the groups and in each group separately. The frequencies of the Apo E alleles and genotypes were determined by genotype count and compared with the values predicted on the basis of the assumption of Hardy–Weinberg equilibrium performing the exact test of Hardy–Weinberg proportion for multiple alleles. P < 0.05 was considered as statistically significant.
3. Results Of the 165 families (n = 529 individuals), we identified 122 affected persons with a clinical diagnosis of FH (from 41 families, mean: 2.98 FH patients per family, range: 1–8 patients per family). Twelve families were identified as having FDB by PCR, totalling 31 affected patients (mean: 2.6 FDB patients per family, range: 1–5 patients per family). The control group consisted of 102 normolipidemic (NL) relatives from the cohort. Main characteristics of the patients with FH, FDB and NL are listed in Table 1. There was a difference in age and BMI distribution between the groups (P < 0.05 and 0.01, respectively); the main determinant of the difference was older age and higher BMI in FH patients as compared to NL group (P < 0.005 for both). As expected, there was a statistically significant difference in total cholesterol (TC), LDL-C and Apo B levels among the groups. Patients with FH had the highest levels of total cholesterol, LDL-C and Apo B, followed by FDB patients and the normolipidemic relatives had the lowest levels (P < 0.0001 for all parameters). The same trend was found with triglyceride (TG) levels (P < 0.001). FH patients had lower
Table 1 Characteristics of individuals with FH, FDB and normolipidemic (NL) controls
Number of families Number of patients Age (years) BMI (kg/m2 )a Females (%) TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) TG (mmol/l) Apo AI (g/l)b Apo B (g/l)c
FH
FDB
41 122 35.7 24.0 59.2 9.1 7.0 1.3 1.5 1.2 1.5
12 31 32.6 23.7 54.8 7.4 5.5 1.3 1.3 1.2 1.1
± 18.3& ± 4.4# ± ± ± ± ± ±
0.14∗ 0.13∗ 0.02& 0.08∗∗ 0.03‡ 0.03∗
NL
± 15.8 ± 5.3 ± ± ± ± ± ±
0.27 0.25 0.05 0.15 0.05 0.06
102 28.8 22.1 55.9 4.9 3.0 1.4 1.0 1.3 0.7
± 16.0 ± 4.4 ± ± ± ± ± ±
0.15 0.14 0.03 0.09 0.03 0.03
Values are mean ± S.E., except for age and BMI where mean ± S.D.; TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglycerides; Apo, apolipoprotein. a Data on BMI were missing for 11 FH patients, 7 NL controls and 1 FDB patient. b Data on Apo AI were missing for 55 FH patients, 32 NL controls and 12 FDB patients. c Data on Apo B were missing for 23 FH patients, 11 NL controls and 6 FDB patients. ∗ P < 0.0001. ∗∗ P < 0.001. & P < 0.05. ‡ P < 0.005. # P < 0.01.
high density lipoprotein cholesterol (HDL-C) and Apo AI levels compare to normolipidemic controls (P < 0.05 and <0.005, respectively). All comparisons were adjusted for age, sex and BMI. Apo E polymorphism was evaluated only in unrelated persons (41 FH, 12 FDB and 49 NL group). Genotype distribution was in Hardy–Weinberg equilibrium for all groups. The frequencies of Apo E genotypes were 12.2, 2.4, 70.7 and 14.7% for genotypes 23, 24, 33 and 34 in FH and 0, 0, 91.7 and 8.3% in FDB group. NL group Apo E genotypes were 10.2, 0, 69.4 and 20.4%. There was no difference in the frequency of Apo E genotypes between FH, FDB and normolipidemic subjects (P = 0.67). The impact of Apo E genotypes on lipid levels was analysed using a general linear regression model with age, sex and BMI as covariates. An association between Apo E polymorphism and lipid levels could not be demonstrated in FH and normolipidemic subjects (data not shown). Because of insufficient number of subjects, the FDB group was not analysed.
4. Discussion Although clinical and biochemical phenotypes of FDB bear some similarity to those of FH, mean levels of plasma LDL-C in FDB are only mildly elevated compared with FH [1]. In our study, patients with FH had significantly higher levels of TC, LDL-C and Apo B then patients with FDB. This finding has been explained by an Apo E mediated
B. Vohnout et al. / Atherosclerosis Supplements 4 (2003) 3–5
clearance of VLDL and intermediate density lipoprotein (IDL) particles in FDB [3]. In our study, FDB was diagnosed using the PCR method and FH clinically, based on strict lipid criteria. Therefore, we cannot exclude that this underestimates the real prevalence of FH patients in our cohort and explain to a certain degree, differences in lipid levels between FDB and FH patients. Apo E polymorphism can be a factor that determines diversity in the clinical expression of FH. Previous studies have examined the effect of Apo E alleles on the phenotypic expression of FH with ambiguous results [4]. We did not find any effect of the Apo E genotype on lipid levels in the FH or NL group. Similarly, in a previous study, we did not find any significant effect of Apo E genotype on lipid levels in Slovak premature myocardial infarction (MI) men versus their spouses and control couples who did not have a personal or family history of MI [8]. Therefore, we conclude that Apo E polymorphisms are not responsible for the variability in lipid levels in either the normal or diseased Slovak population. Other genetic and/or environmental factors may be responsible for the diversity in the clinical expression of FH. Acknowledgements The authors thank Natália Arvayová, Adriana Uher˘ciková and Zuzana Obernauerová for excellent assistance in the examination of the patients, Dr. Monika Ivani˘cová for her help in patients’ examination and evaluation, Dr. Augusto Di Castelnuovo for his helpful suggestions in statistical evalu-
5
ation and Dr. Susan Stephenson for her useful suggestions. Branislav Vohnout, M.D. was a recipient of aMarie Curie Individual Fellowship (HPMF-CT-1999-00099)
References [1] WHO Familial Hypercholesterolemia. Human Genetics Program. Division of Noncommunicable Diseases. Geneva, WHO/HGN/FH/ CONC/98.7. p. 1–45. [2] Hansen PS, Defesche JC, Kastelein JJP, et al. Phenotypic variation in patients heterozygous for familial defective apolipoprotein B (FDB) in three European countries. Arterioscler Thromb Vasc Biol 1997;17:741–7. [3] Miserez AR, Keller W. Differences in the phenotypic characteristics of subjects with familial defective apolipoprotein B-100 and familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15:1719– 29. [4] Hill JS, Hayden MR, Frohlich J, Pritchard PH. Genetic and environmental factors affecting the incidence of coronary artery disease in heterozygous Familial Hypercholesterolemia. Arterioscler Thromb 1991;11:290–7. [5] Williams RR, Hunt SC, Schumacher C, et al. Diagnosing heterozygous Familial Hypercholesterolemia using new practical criteria validated by molecular genetics. Am J Cardiol 1993;72:171–6. [6] Schuster H, Rauh G, Muller S, et al. Polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100. Anal Biochem 1992;204:22–5. [7] Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990;31:545–8. [8] Raslova K, Smolkova B, Vohnout B, et al. Risk factors for atherosclerosis in survivors of myocardial infarction and their spouses: comparison to controls without personal and family history of atherosclerosis. Metabolism 2001;50:24–9.