Lipoprotein and apolipoprotein abnormalities in familial combined hyperlipidemia: a 20-year prospective study

Atherosclerosis 159 (2001) 471– 481 www.elsevier.com/locate/atherosclerosis

Lipoprotein and apolipoprotein abnormalities in familial combined hyperlipidemia: a 20-year prospective study Marguerite J. McNeely a,*, Karen L. Edwards c, Santica M. Marcovina a, John D. Brunzell a, Arno G. Motulsky a,b, Melissa A. Austin c a

Department of Medicine, School of Medicine, Uni6ersity of Washington, Box 356429, Seattle, WA 98195, USA b Department of Genetics, Uni6ersity of Washington, Seattle, WA 98195, USA c Department of Epidemiology, Uni6ersity of Washington, Seattle, WA 98195, USA Received 5 October 1999; received in revised form 3 April 2001; accepted 6 April 2001

Abstract In order to characterize the lipoprotein abnormalities in familial combined hyperlipidemia (FCHL) and to describe factors associated with the stability of the FCHL phenotype during 20-year follow-up, 287 individuals from 48 families with FCHL originally identified in the early 1970s (baseline) were studied. Hyperlipidemia was defined as lipid-lowering medication use, or Eage- and sex-specific 90th percentile for triglycerides or cholesterol. Triglyceride, cholesterol and medical history data were obtained at baseline and 20-year follow-up. Additional follow-up measures included HDL-C, LDL-C, LDL particle size, lipoprotein(a), apolipoprotein (apo) A-I, apoB, and apoE polymorphism. Longitudinally, two-thirds of relatives were consistently normolipidemic or hyperlipidemic, and one third were discordant for hyperlipidemic status at baseline and 20-year follow-up. Individuals with hyperlipidemia at baseline and/or follow-up had higher apoB levels than those with consistently normal lipids (PB 0.05), whereas small LDL size was associated with concurrent hyperlipidemia. Among individuals who were normolipidemic at baseline, the following variables were independently associated with development of hyperlipidemia over 20 years: older age at baseline, male sex, greater increase in BMI during follow-up, and apoE alleles epsilon 2 or epsilon 4. In conclusion, apoB is associated with hyperlipidemia and apoE polymorphism is associated with later onset of hyperlipidemia in FCHL. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Familial combined hyperlipidemia; Cholesterol; Triglycerides; Lipoproteins; Genetics; Adult; Child

1. Introduction Familial combined hyperlipidemia (FCHL) was first described over 20 years ago as a dominantly inherited lipoprotein disorder with variable phenotypic expression [1,2]. At that time, routine lipoprotein measurements were limited to triglycerides and total cholesterol. Thus, FCHL was characterized as a disorder in which affected individuals from a given family have elevations in triglyceride, cholesterol, or both of these lipids [2,3]. Abbre6iations: Apo, apolipoprotein; FCHL, familial combined hyperlipidemia; HDL-C, high density lipoprotein cholesterol; Lp(a), lipoprotein(a); LDL-C, low density lipoprotein cholesterol. * Corresponding author. Tel.: +1-206-2215273; fax: +1-2066165365. E-mail address: [email protected] (M.J. McNeely).

Despite advances in measurement of various lipoproteins and apolipoproteins, the definition of this disorder remains unchanged, largely because the genetic basis of the disorder remains elusive [4]. FCHL is a relatively common condition. It is identified in about 14–19% of individuals less than age 60 with documented coronary disease [5], depending on whether or not FCHL is considered to encompass hyperapobetalipoproteinemia [6]. FCHL is often associated with hyperinsulinemia, impaired glucose tolerance, obesity, and increased risk of coronary artery disease, suggesting shared features with the insulin resistance syndrome [6–10]. Several studies have shown that low density lipoprotein (LDL) subclass phenotype B (small, dense LDL) [11,12] and elevated levels of apolipoprotein B-100 (apoB) [13 –16] may be common features of FCHL. Cardiovascular disease risk is associated with

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both LDL subclass phenotype B and elevated apoB [17 – 20], and may account for the increased risk of cardiovascular disease reported with FCHL. ApoE polymorphism is also associated with cardiovascular disease risk and lipid levels in FCHL. The three common alleles for apoE are epsilon 2, 3, and 4. In most populations, the majority of individuals are homozygous for epsilon 3 (E3/E3). Several studies have shown that individuals with at least one epsilon 2 allele have higher triglycerides, higher HDL-C, lower LDL-C, and lower cardiovascular disease risk than individuals with E3/E3. In contrast, individuals with at least one epsilon 4 allele also have higher triglycerides, but lower HDL-C, higher LDL-C and apoB, and higher cardiovascular disease risk compared to individuals with E3/ E3 [21–28]. Among affected relatives from FCHL families, apoE polymorphism has been shown to influence lipid phenotypic expression: individuals with E4/ E4 had higher LDL-C and lower triglycerides than those with E3/E2 [29]. The purpose of this study is to better characterize the frequency of lipoprotein and apolipoprotein abnormalities among relatives in families with FCHL based on a unique sample of families originally ascertained in the early 1970s and subsequently resampled between 1994 and 1997. These prospective data also were used to evaluate stability of lipoprotein characteristics over time, providing an opportunity to examine variables associated with the hyperlipidemic FCHL phenotype.

2. Methods

2.1. Family ascertainment and study subjects This analysis is based on a prospective family study derived from two baseline family studies conducted in the early 1970s at the University of Washington. Baseline study 1 identified 47 probands who were myocardial infarction survivors and whose families met criteria for FCHL [2]. Baseline study 2 identified 24 probands with hypertriglyceridemia (without clinically evident coronary disease) whose families met criteria for FCHL [3]. Of these 71 FCHL families, 48 families had at least one relative who participated at baseline and 20-year follow-up. In order to minimize potential bias associated with the differing recruitment strategies used in the baseline studies, probands were excluded. A total of 287 firstand second-degree relatives of the probands participated at follow-up. All relatives in these families were of European descent (Caucasian). Written, informed consent was obtained for all participants both at baseline and follow-up. The University of Washington Institutional Review Board approved this study.

2.2. Non-participants There were 684 first- and second-degree relatives who had cholesterol and triglyceride values measured at baseline, 397 of whom did not participate at follow-up. Of these 397 non-participants, 114 (29%) were deceased at follow-up. Other reasons for non-participation included: inability to contact (n= 245, 62%), refusal (n = 28, 7%), and residence outside the country (n= 10, 3%). Thus, half (283/570) of eligible relatives presumed to be alive did not participate at follow-up. The mean lipid values for the 397 non-participants at baseline were 227.4961.5 for cholesterol and 168.29 239.7 for triglycerides (mean9 SD). These were not significantly different than the values for participants, which are shown in Table 1 (P= 0.721 for cholesterol, P= 0.622 for ln triglycerides, adjusted for age and sex). A similar proportion of participants and non-participants had cholesterol or triglyceride values E age- and sex-specific 90th percentiles [30] at baseline (48.4% vs. 45.3%, chi-square P= 0.428).

2.3. Measurements Participants completed a medical history questionnaire at baseline and follow-up. This information included birth date, sex, height, weight, medications, and smoking history. Participants were also asked if a physician had ever diagnosed them with diabetes, hypertension or a myocardial infarction. Body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Smoking history was missing for three participants at baseline. Self-reported height or weight data was missing for seven individuals at baseline and four individuals at follow-up. Change in variables over the follow-up period was calculated as the follow-up value minus the baseline value (e.g. DBMI = BMI at follow-up-BMI at baseline). Baseline fasting lipids were measured using an AutoAnalyzer (AA) II. Total cholesterol was measured using the N-24a method [2], and triglyceride using a semi-automated method modified from the procedure of Carlson [31,32]. These methods have been compared to those developed for the Lipid Research Clinics. No systematic difference was noted for cholesterol, but a linear relationship was noted between the old and new triglyceride methods (r= 0.96; Brunzell, unpublished data). Therefore, baseline triglyceride values were adjusted using a linear regression equation. Thus, baseline triglyceride levels reported are comparable to those using current methods. At the follow-up visit, fasting lipids, lipoproteins, and apolipoproteins were measured at the Northwest Lipid Research Laboratory. LDL cholesterol (LDL-C) levels were derived using the Friedewald algorithm (LDLC) = (total cholesterol) -(triglyceride/5) if the triglyce-

Table 1 Characteristics of relatives in 48 FCHL families at baseline Baseline study 1 (n= 3D 30 families)

Total (n=3D 48 families)

1st degree relatives (n= 88)

2nd degree relatives (n=78)

Total (n =166)b

1st degree relatives (n = 48)

2nd degree relatives (n =73)

Total (n= 121)b

(n=287)

32.5 (16.3) 48.9% (43) 157.1 (155.6) 228.2 (57.3)

19.4 (8.2) 46.2% (36) 80.6 (64.3) 192.0 (33.7)

26.4 (14.7) 47.6% (79) 121.1 (127.2) 211.2 (50.9)

31.3 (14.3) 50.0% (24) 172.2 (229.1) 217.5 (51.7)

24.3 (10.8) 48.0% (35) 109.3 (90.1) 194.7 (47.5)

27.0 (12.7) 48.8% (59) 134.2 (162.4) 203.7 (50.2)

26.7 (13.9) 48.1% (138) 126.7 (143.0) 208.0 (50.7)

59.1% (52) 23.1 (4.3) 29.5% (26) 2.3% (2) 4.5% (4)

38.5% (30) 21.4 (3.7) 15.6% (12) 1.3% (1) 5.1% (4)

49.4% (82) 22.4 (4.1) 23.0% (38) 1.8% (3) 4.8% (8)

47.9% (23) 24.0 (3.6) 31.9% (15) 6.3% (3) 4.2% (2)

34.3% (25) 22.5 (3.9) 27.8% (20) 1.4% (1) 9.6% (7)

39.7% (48) 23.1 (3.8) 29.4% (35) 3.3% (4) 7.4% (9)

45.3% (130) 22.7 (4.0) 25.7% (73) 2.4% (7) 5.9% (17)

3.4% (3) (0)

1.3% (1) (0)

2.4% (4) (0)

1.1% (1) (0) 6.8% (6) 1.1% (1)

(0) 2.6% (2) 3.8% (3) (0)

0.6% 1.2% 5.4% 0.6%

(1) (2) (9) (1)

(0) (0)

(0) (0)

(0) (0)

2.1% (1) 4.2% (2) 4.2% (2) (0)

(0) 1.4% (1) 4.1% (3) (0)

0.8% (1) 2.5% (3) 4.1% (5) (0)

1.4% (4) (0) 0.7% 1.7% 4.9% 0.3%

(2) (5) (14) (1)

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Age (years) Male TG (mg/dl)a Cholesterol (mg/dl) Hyperlipidemiac BMI (kg/m2)d Current smokere Estrogen use Oral contraceptives Diuretic use Beta blocker use M.If Diabetes Hypertension Lipid medications

Baseline study 2 (n = 3D 18 families)

Data are mean (SD) or column% (n) a Triglycerides. b There are no significant differences between baseline study 1 and 2. c Hyperlipidemia defined as lipid-lowering medication use or Eage- and sex-specific 90th percentile for triglycerides or cholesterol based on Lipid Research Clinic data [30]. d Self-reported height or weight data missing for seven participants (five from study 1, six from study 2). e Smoking history missing for three participants (one from study 1, two from study 2). f Myocardial Infarction.

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ride value was below 400 mg/dl. If the triglyceride value was 400 mg/dl or over, LDL-C was measured using the Lipid Research Clinic beta quantification method. Lipoprotein(a) (Lp(a)) was measured using enzyme immunoassay (monoclonal antibody MAb a-40), as previously described [33]. ApoA-I and apoB were assayed nephelometrically using reference materials from the International Federation of Clinical Chemistry standardization project [34,35]. The major LDL subclass diameter was determined in duplicate using 2– 14% polyacrylamide gel electrophoresis as previously described [36]. In addition, the LDL subclass phenotype was determined for each participant [36]. LDL subclass phenotype A is defined as a predominance of large LDL and LDL subclass phenotype B defined as a predominance of small LDL [17]. Thirty relatives were classified with an intermediate phenotype [36], and were considered a distinct LDL subclass phenotype (non-phenotype B) for this analysis. ApoE phenotype was determined by a combination of isoelectric focusing and western blotting techniques, using a modification of a previously published method [37]. Ten milliliter of plasma was reduced by incubation with buffer containing 0.77% DDT in 0.25% Tween-20, loaded onto a polyacrylamide/urea gel (pH range 4–8), and allowed to reach isoelectric focus points for 90 min. Protein fractions were transferred to a nitrocellulose filter by capillary diffusion for 2 h, and then soaked in a 2% non-fat milk solution to block non-specific protein binding sites. The filter was then incubated with an anti-apoE specific antibody, and after several washings, allowed to react with an anti-goat-IgG HRP conjugate. An ECL detection kit (Amersham Life Sciences, Inc.) was used to visualize apoE specific immunoreactivity, allowing the six apoE phenotypes to be distinguished by their typical banding patterns.

2.4. Definition of hyperlipidemia and familial combined hyperlipidemia Hyperlipidemia at baseline and follow-up was defined as E90th percentile of cholesterol and/or triglyceride using age- and sex-specific Lipid Research Clinics reference values [30]. Values at or above the 90th percentile were used to define hyperlipidemia based on the work of Porkka and colleagues, showing that at this level about 45% of offspring with one parent affected with FCHL are hyperlipidemic [38]. Individuals taking lipid-lowering medication were considered to be hyperlipidemic regardless of lipid levels. A family was considered to meet criteria for FCHL if E 2 first-degree relatives of the proband were hyperlipidemic at baseline, at least one of whom was hypercholesterolemic and at least one of whom was hypertriglyceridemic, excluding relatives under age 18. All 48 families included in this study met these criteria

based on lipid data from all first-degree relatives plus the 48 probands who participated at baseline.

2.5. Definition of abnormal lipoproteins and apolipoproteins Lipid Research Clinic age- and sex-specific reference values were used to determine abnormalities in HDL-C and LDL-C at follow-up [30,39]. Elevated levels of apoB at follow-up were defined using age- and sexspecific reference values from the National Health and Nutrition Examination Survey III (NHANES III), which are referable to the International Federation of Clinical Chemistry reference materials [40].

2.6. Statistical analysis All statistical tests were performed using Stata software version 5.0 (Stata Corporation, College Station, TX). Triglyceride and Lp(a) data were skewed; therefore statistical tests used natural logarithmic transformation of these data. For cross-sectional analyses at baseline, logistic regression was used to determine if various independent variables differed by baseline study (dependent variable), adjusting for age and sex. For longitudinal analyses, linear regression was used for analysis of continuous dependent variables measured at follow-up, using hyperlipidemic status at baseline as an independent variable, and adjusting for covariates. Logistic regression models were used to examine the association between various dichotomous dependent variables measured at follow-up (LDL subclass phenotype, presence/absence of elevated LDL-C, or elevated apo B) and hyperlipidemic status at baseline, adjusting for covariates. Covariates included: age at follow-up, sex, baseline study, and medication use at follow-up. Medications included estrogens (oral contraceptives or post-menopausal hormone replacement), diuretics and beta-blockers. Individuals taking lipid-lowering medications at follow-up (n= 32) were excluded from all analyses of lipid, lipoprotein, or apolipoprotein concentrations measured at follow-up. Dichotomous lipoprotein and apolipoprotein variables (above or below a given percentile) based on age- and sex-specific reference values were not further adjusted for age or sex. Models that included the change in value of a variable during the follow-up period were further adjusted for the baseline value of that variable (e.g. any model of Dcholesterol included baseline cholesterol as a covariate). Associations between various lipoprotein and apolipoprotein measures and stability of the hyperlipidemic FCHL phenotype were tested using linear or logistic regression models, and the following categories of hyperlipidemia: (1) normolipidemic at baseline and follow-up (reference group); (2) normolipidemic at

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baseline, hyperlipidemic at follow-up; (3) hyperlipidemic at baseline, normolipidemic at follow-up; and (4) hyperlipidemic at baseline and follow-up. A multivariable logistic regression model was used to test which variables were independently associated with the development of hyperlipidemia over 20 years among FCHL relatives who were normolipidemic at baseline. The independent variables used in this model were: age at baseline; sex (men vs. women); BMI at baseline; change in BMI; apoE phenotype (E3/E3 – reference group, E3/E2, and E3/E4 or E4/E4); baseline study (baseline study 1 vs. baseline study 2); diabetes at follow-up; and medication use at follow-up (estrogens, diuretics, or beta-blockers). Because these comparisons may violate assumptions of independence due to familial relationships, all regression models utilized a robust variance estimator (or sandwich estimator), with kindred as the clustering variable [41]. This procedure uses a robust estimate of variance and relaxes the assumption of independence for individuals from the same kindred.

475

3.3. Characteristics of relati6es at follow-up

3. Results

Thirty-two individuals were taking lipid-lowering medication at follow-up (nine were normolipidemic at baseline and 23 were hyperlipidemic at baseline). These individuals were excluded from subsequent analyses of lipid, lipoprotein, or apolipoprotein data obtained at follow-up. Sixty-one individuals were taking medications that can affect lipid and lipoprotein levels at follow-up, including oral contraceptives, postmenopausal estrogens, diuretics and beta-blockers. The proportion of individuals taking these medications at follow-up was 17.8% (28/157) among those with normal lipids at baseline and 25.4% (33/130) among those with hyperlipidemia at baseline (chi square P = 0.120). Subsequent analyses were adjusted for use of these medications at follow-up. Sixteen individuals reported diabetes at follow-up. The proportion with diabetes at follow-up was 3.8% (6/157) among those with normal lipids at baseline, and 7.7% (10/130) among those with hyperlipidemia at baseline (chi-square test P= 0.150). Only 9 individuals with diabetes were not taking lipid-lowering medication at follow-up (four were normolipidemic and five were hyperlipidemic at baseline (chi-square test P= 0.392).

3.1. Characteristics of relati6es at baseline and by baseline study

3.4. Comparisons of follow-up measurements by hyperlipidemic status at baseline

General characteristics of the 287 relatives at baseline are shown in Table 1. About half (136/287, 47%) were first-degree relatives of the probands. First-degree relatives tended to be older and have a higher prevalence of age-related conditions, such as history of myocardial infarction, diabetes and hypertension, compared with second-degree relatives. There were no significant differences in baseline characteristics by baseline study. Subsequent analyses combine first- and second- degree relatives, and participants from both baseline studies, but are adjusted for age, sex and baseline study. Fiftyfive percent (75/136) of first-degree relatives and 36% (55/151) of second-degree relatives had hyperlipidemia defined by values at or above the 90th percentile.

Lipid, lipoprotein, and apolipoprotein results by hyperlipidemic status at baseline are presented in Table 2. By definition, mean triglyceride and cholesterol levels at baseline were higher among relatives with hyperlipidemia compared to those with normal lipids at baseline. At follow-up, triglyceride, cholesterol, LDL-C, and apoB concentrations were higher among those with hyperlipidemia at baseline (all PB0.001). There was no association between change in triglyceride values during 20-year follow-up and hyperlipidemic status at baseline. On average, cholesterol values increased during 20-year follow-up among individuals who were normolipidemic at baseline, whereas they decreased slightly among hyperlipidemic relatives (PB0.001, adjusted for age, sex, baseline cholesterol, medication use at follow-up, and baseline study), suggesting regression to the mean. Hyperlipidemic individuals tended to have smaller LDL size, but the results did not reach statistical significance (adjusted P=0.099). There were no significant differences in baseline BMI, BMI change, HDL-C, apoA-I, or Lp(a) by hyperlipidemic status at baseline. Lipoproteins and apolipoproteins were further characterized, using various age- and sex-specific percentiles to define abnormal levels (data not shown in table). The most common abnormalities at follow-up among relatives with hyperlipidemia at baseline were LDL subclass phenotype B (30%), elevated apoB (28% were E90th

3.2. Cross-sectional analysis of factors associated with hyperlipidemia at baseline In univariate analyses, none of the following variables were associated with hyperlipidemia at baseline: age at baseline, BMI at baseline, sex, smoking, diabetes at baseline, medication use at baseline (estrogens or diuretics), baseline study, or apoE polymorphism. Likewise, when these variables were included in a multivariable logistic regression model, none were significantly associated with hyperlipidemia at baseline.

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percentile), and elevated LDL-C (19% were E 90th percentile). Regardless of whether the 90th or 95th percentile was used to define hyperlipidemia at baseline, the prevalence of apoB and LDL-C above the 75th, 90th or 95th percentile at follow-up was higher in the hyperlipidemic group than in the normolipidemic group (P0 0.002, adjusted for age, sex, baseline study and medication use at follow-up, excluding 32 individuals taking lipid lowering medication at follow-up). The prevalence of LDL subclass phenotype B was not significantly different between those with and without hyperlipidemia at baseline (29.9% vs. 22.3% using the 90th percentile definition of hyperlipidemia, adjusted P = 0.109; 32.4% vs. 22.8% for the 95th percentile definition, adjusted P=0.137). The prevalence of abnormal apoB levels, LDL-C levels, and LDL subclass phenotype B was also examined by type of hyperlipidemia at baseline (90th percentile definition, data not shown in table). This analysis included 107 relatives who were hyperlipidemic at baseline (23 individuals taking lipid-lowering medication at follow-up were excluded). Seventeen percent (18/107)

had hypertriglyceridemia with normal cholesterol, 60% (64/107) had hypercholesterolemia with normal triglycerides, and 23% (25/107) had both hypertriglyceridemia and hypercholesterolemia at baseline. The frequency of LDL subclass phenotype B at follow-up was 61% (11/ 18) in the hypertriglyceridemic group, 17% (11/64) in the hypercholesterolemic group, and 40% (10/25) in the group with both hypercholesterolemia and hypercholesterolemia (P B 0.001, adjusted for age, sex, baseline study, and medication use; hypertriglyceridemia vs. hypercholesterolemia groups). The frequency of elevated LDL-C or apoB at follow-up (75th, 90th or 95th percentile) was not significantly different across categories of hyperlipidemia at baseline.

3.5. Factors associated with stability of hyperlipidemic status at baseline and follow-up Of the 287 participants, 110 (38.3%) were normolipidemic at baseline and follow-up, 47 (16.4%) were normolipidemic at baseline and hyperlipidemic at follow-up, 49 (17.1%) were hyperlipidemic at baseline

Table 2 Comparison of lipids, lipoproteins, and apolipoproteins at follow-up by hyperlipidemic status at baseline Normolipidemic relatives at baseline

Hyperlipidemica relatives at baseline

Baseline N Triglycerides (mg/dl) Cholesterol (mg/dl)

157 82.79 44.0 180.8 9 29.7

130 179.8 9194.4 240.8 9 51.4

Follow-up n BMI (kg/m2) DBMI (kg/m2) c Triglycerides (mg/dl) DTriglycerides (mg/dl) Cholesterol (mg/dl) DCholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) LDL size (A, ) apoA-I (mg/dl) apoB (mg/dl)d Lp(a) (nmol/L)

148 26.995.6 4.69 4.8 157.19 103.7 77.5 997.0 197.89 30.6 19.8 9 33.4 47.09 14.0 118.99 28.9 265.799.1 137.6926.8 102.69 20.6 9.7 911.0

107 27.2 95.5 4.895.4 207.4 9 163.0 42.69197.2 233.6 9 42.2 −1.39 60.6 48.9 9 19.8 142.8 9 38.1 264.1 99.4 143.19 34.7 123.79 26.5 8.3 911.9

P valueb, adjusted for age and sex

– –

0.684 0.813 0.003 0.785 B0.001 B0.001 0.443 B0.001 0.200 0.139 B0.001 0.099

P valueb, adjusted for age, sex, medications, and baseline study

– –

0.483 0.660 B0.001 0.975 B0.001 B0.001 0.732 B0.001 0.099 0.264 B0.001 0.179

Data are means 9S.D. a Hyperlipidemia defined asEage- and sex-specific 90th percentile for triglycerides or cholesterol based on Lipid Research Clinic data at baseline [30]. Excludes 32 individuals on lipid lowering medication at follow-up (nine were normolipidemic and 23 were hyperlipidemic at baseline). b P values based on natural logarithm of triglycerides, Dtriglycerides, and Lp(a) values. P values for DBMI were further adjusted for baseline BMI; P values for Dtriglycerides were further adjusted for baseline triglycerides, and P values for Dcholesterol were further adjusted for baseline cholesterol. c For DBMI, data missing for 11 individuals (seven missing baseline BMI, four missing follow-up BMI). d For apoB, data missing for two individuals.

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Fig. 1. Association between LDL size at follow-up and stability of hyperlipidemia among relatives in FCHL families. Data are mean 9 SEM. Comparisons are to group with consistently normal lipids at baseline and follow-up; **PB 0.001, adjusted for age, sex, medication use at follow-up (estrogens, diuretics, and beta-blockers), and baseline study.

and normolipidemic at follow-up, and 81 (28.2%) were hyperlipidemic at baseline and follow-up. Thus, twothirds (191/287, 66.6%) were concordant and one-third were discordant for hyperlipidemic status at baseline and follow-up. Because small LDL size and elevated apoB were the most common abnormalities among those with hyperlipidemia at baseline, these measurements were compared among relatives with concordant and discordant lipid status at baseline and follow-up. LDL particle diameter at follow-up was associated with concurrent hyperlipidemia at follow-up but not baseline lipid status (P B0.001; Fig. 1). Findings were similar for LDL subclass phenotype B (P B0.001; Table 3). In contrast, apoB levels at follow-up were lowest among individuals with consistently normal lipids, highest among those with consistent hyperlipidemia, and intermediate among individuals who were hyperlipidemic at only one time point (Table 3 and Fig. 2). Among the 157 individuals who were normolipidemic at baseline, the development of hyperlipidemia over 20-year follow-up was independently associated with older age at baseline, male sex, larger increase in BMI during follow-up, and apoE epsilon 2 or 4 relative to

E3/E3 (Table 4). These findings were also independent of BMI at baseline, baseline study, diabetes at followup, and medication use at follow-up (estrogens, diuretics, or beta-blockers).

4. Discussion This is a descriptive study of lipoproteins, apolipoproteins, and LDL size in FCHL using both baseline and follow-up data at a 20-year interval. Our findings support previous reports that : 50% of those with a first-degree affected relative in FCHL families have cholesterol or triglyceride levels above the 90th percentile [38], although not all first-degree relatives were sampled in our study. This finding would be consistent with autosomal dominant inheritance of FCHL [6], although it does not exclude the possibility of an oligogenic etiology. These results are also consistent with prior reports that FCHL is associated with small LDL particle size and elevated apoB [11–16,38]. In this study, one third of participants were discordant for hyperlipidemic status at baseline and 20 year follow-up (16.4% changed from normolipidemic to hy-

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perlipidemic status and 17.1% changed from hyperlipidemic to normolipidemic status). Similar or higher degrees of discordance have been reported in individuals without known FCHL. In a study of 564 US veterans, 75% (40/53) of those with hypercholesterolemia (\ 90th percentile) had a short-term follow-up cholesterol level below the 90th percentile without treatment [42]. In 169 healthy postmenopausal Danish women followed for 7–12 years, 16% (15/93) initially classified as hypercholesterolemic (E 240 mg/dl) had normal longterm cholesterol levels, and 20% (15/76) of those with initially normal cholesterol had elevated long-term cholesterol levels [43]. In 646 pediatric patients aged 3 – 19 years, 59% (62/105) changed from hypercholesterolemic (E200 mg/dl) to normocholesterolemic status, and 7% (38/541) changed from normocholesterolemic to hypercholesterolemic status on two consecutive measurements obtained over 3 years [44]. Regression to the mean [42,45], and secular trends toward decreasing cholesterol levels in the general population [46] are other factors that may account for reduction in cholesterol levels over 20-year follow-up. While we are not aware of comparable longitudinal Table 3 Association between LDL subclass phenotype and elevated plasma apo B levels at follow-up and stability of hyperlipidemia among relatives in FCHL families Baseline

Normal lipids Sample size, (n) LDL subclass phenotype B, % (n) ApoBE75th percentileb, % (n) ApoBE90th percentileb, % (n) Hyperlipidemia a Sample size, (n) LDL subclass phenotype B, % (n) ApoBE75th percentileb, % (n) ApoBE90th percentileb, % (n)

Follow-up Normal lipids

Hyperlipidemiaa

110 8.2% (9)

38 63.2% (24)c

7.3% (8)

36.8% (14)c

0.9% (1)

21.1% (8)c

49 12.2% (6)

58 44.8% (26)c

26.5% (13)c

78.6% (44)c

4.1% (2)

48.2% (27)c

Hyperlipidemia defined as lipid-lowering medication use, or E age- and sex-specific 90th percentile for triglyceride or cholesterol. Individuals taking lipid-lowering medications at follow-up were excluded (n = 32; all were hyperlipidemic at follow-up, 9/32 were normolipidemic at baseline and 23/32 were hyperlipidemic at baseline). b ApoB data missing for 2 individuals who were hyperlipidemic at baseline and follow-up. Percentiles are age- and sex- specific, so further adjustment for these variables not done. c P00.001, compared to group with normal lipids at baseline and follow-up, adjusted for age, sex, medication use at follow-up (estrogens, diuretics or beta-blockers), and baseline study. a

reports of discordant hypertriglyceridemic status, an even larger proportion of individuals are likely to cross above or below a given threshold since the day to day intra-individual variability has been reported to be higher for triglycerides (20%) than for cholesterol (5%) [47]. The relationship between small LDL size and hypertriglyceridemia is well documented [19,36]. In our study, small LDL size was only associated with baseline hypertriglyceridemia and concurrent hyperlipidemia rather than a diagnosis of FCHL over a 20-year time span. LDL size among relatives who were hyperlipidemic at baseline but normolipidemic at the time of LDL size determination during follow-up was similar to that of relatives who were consistently normolipidemic. In contrast to the findings for LDL size, apoB levels were highest among individuals with consistent hyperlipidemia, intermediate among those with transient hyperlipidemia, and lowest among consistently normolipidemic individuals over the follow-up period. This suggests that apoB may be an important measure of the FCHL phenotype. These findings are consistent with other studies showing a close relationship between apoB and FCHL [48,49]. Bredie and colleagues suggested a single gene effect on apoB by segregation analysis among 40 FCHL kindreds [49]. Jarvik and colleagues also noted a mendelian locus with large effects on apoB levels defined by segregation analysis [48]. Yet apoB levels alone do not completely explain the FCHL phenotype. For example, FCHL may be the result of two (or more) common gene effects which segregate independently within the same family. ApoB levels and LDL subclass may be two such gene effects [11,48,51,52], although the genes themselves remain unknown. FCHL may therefore be an oligogenic disorder [50]. We also identified several factors associated with later development of hyperlipidemia in relatives from FCHL families. These included older age, male sex, increase in BMI, and apoE allele epsilon 2 or 4. Interestingly, results of the cross-sectional analysis at baseline demonstrated that age at baseline, sex, BMI at baseline, and apoE polymorphism were not associated with hyperlipidemic status. Thus, it remains unclear whether these factors are associated with the development of hyperlipidemia via mechanisms distinct from FCHL, or whether these represent risk factors for delayed phenotypic expression of underlying FCHL. This issue will likely remain unresolved until the genetic basis for FCHL is understood. In the interim however, this information may be useful for designing genetic studies of FCHL. There are several limitations to this study. In the absence of genetic markers for FCHL, the definition of hyperlipidemia remains somewhat arbitrary. Neverthe-

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Fig. 2. Association between plasma apoB at follow-up and stability of hyperlipidemia among relatives in FCHL families. Data are mean 9 SEM. Comparisons are to group with consistently normal lipids at baseline and follow-up; *P =0.019, **PB0.001, adjusted for age, sex, medication use at follow-up (estrogens, diuretics and beta-blockers), and baseline study.

less, our main conclusions about the relationship between apoB, LDL size, and the hyperlipidemic phenotype of FCHL were consistent regardless of whether hyperlipidemia was defined using 90th or 95th percentile cut-offs. The prevalence of elevated apoB and LDLC in FCHL may be underestimated in this study due to the exclusion of lipid-lowering medication users, especially since clinical guidelines for the use of these medications emphasize LDL-C reduction [53]. Even so, only 17.7% (23/130) of individuals who were hyperlipidemic at baseline were taking lipid-lowering medication at follow-up. Another factor that may have lowered mean apoB and LDL-C levels in this study is survival bias. Since apoB and LDL-C are strongly associated with cardiovascular disease, individuals with high apoB and LDL-C levels may have been more likely to die during the 20-year follow-up period than individuals with a more favorable lipid profile. While apoB and LDL-C were not measured at baseline, total cholesterol values of non-participants were not significantly different from those of participants. Therefore, the potential effect of survival bias is probably small. Finally, this study included only Caucasians, and it remains to be seen if FCHL phenotypes vary with ethnicity.

In summary, elevated apoB is common among hyperlipidemic individuals from families with FCHL. LDL subclass phenotype B (which is not phenotypically related to apoB levels) is also common in FCHL, but was Table 4 Factors independently associated with the development of hyperlipidemia at 20-year follow-up among 145 FCHL relatives who were normolipidemic at baselinea Variables in the logistic regression model

Odds ratio (95% P value CI)

Age at baseline, years Sex (men vs. women) BMI at baseline (kg/m2) b Change in BMI (kg/m2) b apoE phenotype E3/E2c

1.08 (1.01, 4.10 (1.85, 0.93 (0.78, 1.17 (1.06, 4.35 (1.66, 11.34) 3.70 (1.52, 1.09 (0.50, 0.60 (0.12, 1.50 (0.54,

apoE phenotype E4/E3 or E4/E4c Baseline study (study 1 vs. study 2) Diabetes at follow-up Estrogen, diuretic or beta-blocker medication use at follow-up

1.15) 0.017 9.06) B0.001 1.10) 0.408 1.30) 0.002 0.003 9.01) 2.38) 2.86) 4.16)

0.004 0.833 0.519 0.437

Hyperlipidemia defined as lipid-lowering medication use, or E age- and sex-specific 90th percentile for triglyceride or cholesterol. b BMI data missing for 2 individuals. c Relative to apoE phenotype E3/E3. Ten individuals with E2/E4 were excluded. a

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only associated with baseline hypertriglyceridemia and concurrent hyperlipidemia. Although elevated apoB levels cannot be recommended as a substitute for traditional criteria for FCHL at this time, it is likely to be a useful adjunct phenotypic measure in studies designed to elucidate the genetic basis of FCHL. ApoE polymorphism was also associated with development of hyperlipidemia during 20-year follow-up, but not with hyperlipidemia in cross-sectional analysis at baseline. Further study is needed to determine how well apoB and apoE polymorphism distinguish relatives with variable expression of FCHL from unaffected relatives with transient hyperlipidemia.

[8]

[9]

[10]

[11]

[12]

Acknowledgements [13]

This research was supported by NIH grants HL49513 and HL-30086, and was performed during Dr. Austin’s tenure as an Established Investigator of the American Heart Association. A portion of the studies were performed on the General Clinical Research Center of the University of Washington (NIH RR 37). The authors would like to extend a special thanks to all the family members who participated in this study. We also thank Kody Wallace, Katrina Van Halen, Francine Romero, Aruna Kamenini, Pam Lovy, Barbara Scheffler, and Margaret Poole for their perseverance in re-contacting baseline study participants and identifying additional eligible family members. Database management and programming was the work of Jon Diemer, Drew Levy, Tom Frasier and Andy Louie. We are grateful for the support of Dr. Joseph Goldstein in the development of this study.

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