Plasma High-Density Lipoproteins and Ischemic Heart Disease Studies in a Large Kindred with Familial Hypercholesterolemia DAN STREJA, M.D.; GEORGE STEINER, M.D.; and PETER 0 . KWITEROVICH, Toronto, Canada; and Baltimore,

Jr., M.D.;

Maryland

The expression of ischemic heart disease was studied in a large kindred with familial hypercholesterolemia. Tendon xanthomas, multiple generation transmission, and the appearance of bimodality in the distributions of total and lowdensity lipoprotein cholesterol were found. The segregation ratio was 0.9 in females and 0.43 in males, a difference first apparent during adolescence. The upper quartile of total and low-density lipoprotein cholesterol contained all but two cases of ischemic disease, whereas the lower quartile of highdensity lipoprotein cholesterol contained one half of the cases. The ratio of high- to low-density lipoprotein cholesterol (range, 0.06 to 1.6) was < 0.20 in each patient with ischemic disease. The association of a low level of high-density lipoprotein cholesterol with ischemic disease persisted after adjustment for differences in other lipids and lipoproteins. A low level of high-density lipoprotein cholesterol, as well as a high level of low-density lipoprotein cholesterol, may influence the development of ischemic heart disease in this disorder.

P R I M A R Y HYPERBETALIPOPROTEINEMIA (type II hyper-

lipoproteinemia) is a common disorder associated with premature ischemic heart disease (1). One genetic form of hyperbetalipoproteinemia is familial hypercholesterolemia, a disorder of plasma cholesterol and low-density (LD) (beta) lipoprotein metabolism (1). Familial hypercholesterolemia is inherited as an autosomal dominant trait that is completely expressed at birth and in early childhood (2-4). Heterozygous patients usually have a twofold to threefold elevation of total plasma and L D lipoprotein cholesterol levels commonly accompanied by tendon and tuberous xanthomas, early corneal arcus, and a marked predilection to premature ischemic heart disease (1-5). The estimated frequency of heterozygotes is one per 500 subjects (5). Homozygous familial hypercholesterolemia is characterized by a fourfold to sixfold increase in total plasma and L D lipoprotein cholesterol levels, atherosclerosis before age 20, and planar and tendon xanthomas in the first decade of life (1, 2). Goldstein and

co-workers (6) have found, in cultured fibroblasts from homozygotes, a deficiency of cell surface receptors for LD lipoproteins (receptor negative) or, in some patients, defective L D lipoprotein receptors (receptor defective). This defect in binding of L D lipoprotein is associated with the faulty regulation of the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl CoA reductase, decreased esterification of cellular cholesterol, and deficient proteolysis of LD lipoproteins (7). Recent evidence from population surveys suggests that a decrease in high-density (HD) (alpha) lipoprotein cholesterol is associated with an increased risk of ischemic heart disease but there may be a beneficial effect from increased plasma levels of H D lipoprotein cholesterol (811). Although the association of increased total plasma and L D lipoprotein cholesterol levels with premature ischemic heart disease has been found in a number of single large kindreds or a collection of smaller kindreds (15), little information is available on whether the plasma concentration of H D lipoprotein cholesterol has any separate effect on the development of ischemic heart disease in patients with familial hypercholesterolemia. The H D lipoprotein cholesterol levels in familial hypercholesterolemia are significantly lower than normal at birth and in early childhood and persist through adult life (1, 3, 4). The possibility therefore exists that the coronary atherosclerosis in familial hypercholesterolemia is exacerbated by low H D lipoprotein levels. We have investigated the signs of familial hypercholesterolemia in a large Newfoundland kindred, emphasizing the interrelationship(s) of plasma H D lipoprotein cholesterol levels and other plasma lipids and lipoproteins and the presence of ischemic heart disease. The data presented here suggest that the low plasma H D lipoprotein cholesterol level in patients with familial hypercholesterolemia makes an individual contribution to the increased risk for ischemic heart disease, which is separate (but not necessarily independent) of the effect of increased total plasma and L D lipoprotein cholesterol concentrations. Methods PATIENT POPULATION

• From the Department of Medicine, University of Toronto, Toronto, Canada; and the Departments of Pediatrics and Medicine, Lipid Research Clinic, Johns Hopkins University School of Medicine, Baltimore, Maryland. Annals of Internal Medicine 8 9 : 8 7 1 - 8 8 0 , 1 9 7 8

Index Case: The proband (111-34, Figure 1) was born in Birchy Bay, Newfoundland. At 5 years of age she was seen by © 1 9 7 8 American College of Physicians

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her doctor because of xanthomas and xanthelasma. She had a serum cholesterol level of 700 mg/dl. Since the age of 6 she has had recurrent episodes of monoarticular arthritis of the large joints. At age 12, a harsh systolic murmur was heard at the base of her heart that radiated to both carotid arteries. She married at age 19 and moved to Toronto. When she was 21 years old, she had a serum cholesterol level of 622 mg/dl. Multiple, large planar xanthomas were present as were xanthomas in the extensor tendons of her hands and her Achilles tendons. An ileal bypass was recommended and done. After surgery the serum cholesterol ranged from 600 to 900 mg/dl. She was then treated with colestipol, niacin, and clofibrate, and the serum cholesterol fell to 487 to 502 mg/dl. The patient could not tolerate cholestyramine or Choloxin®. A year later, she developed angina pectoris. A coronary angiogram showed severe left and circumflex coronary artery disease with a normal right coronary artery. The systolic gradient across the aortic valve was 15 to 20 mm Hg, compatible with mild aortic stenosis. Kindred: We examined 175 members of this kindred (Figure 1) and analyzed a blood sample in our laboratory. (The age, sex, clinical findings [xanthoma, cardiovascular disease], medication, and the levels of the plasma lipid and lipoproteins in the members of this pedigree have been deposited with the National Auxiliary Publication Service.) This included 25 spouses, 94 maternal and 39 paternal relatives of the proband, and 17 subjects (including the proband) who were related to both the maternal and paternal sides of the family. Eighty percent of the living first, second, third, and fourth degree relatives of the proband were studied. The subjects were studied at home or close to home; 91 % of the members of the kindred were living in a limited geographical area in northern Newfoundland. Blood Drawing: All participants in the study were instructed not to change their diet for 1 week and to fast for 12 h before blood drawing. Four subjects (11-006, 11-018, 11-021, III-025) were on lipid lowering medication. Venous blood was collected in tubes that contained either 0.1 ml of 15% EDTA, NaF, or no anticoagulant. Plasma and serum were separated from the erythrocytes and transported on wet ice to Toronto. The plasma samples for lipoprotein analyses were kept at 4 °C for 2 to 4 days before analysis. Plasma and serum used for other tests (see below) were frozen at —20 °C until analyzed. LABORATORY DETERMINATIONS

An isopropanol extract of the plasma was prepared and treated with zeolite (12). Plasma cholesterol and triglycerides in the extract were measured in a Technicon Auto Analyzer II (Technicon Instruments Corp., Tarry town, New York) using modified Lieberman Burchard and fluorometric techniques, respectively (12). In all samples in which the total cholesterol concentration exceeded 200 mg/dl, the plasma was subjected to preparative ultracentrifugation for 16 h at 100 000 X g. The tubes were sliced and the top fraction, which contained very low-density (VLD) (pre-beta) lipoproteins, was analyzed for its cholesterol and triglyceride content (12) and also subjected to electrophoresis on cellulose acetate (13). The VLD lipoproteins from these samples did not have an abnormal ratio of cholesterol to triglycerides or any beta migrating lipoproteins; none of these patients was therefore considered to have type III hyperlipoproteinemia (14). High-density lipoprotein cholesterol was measured on the supernatant after heparin-manganese precipitation of the other plasma lipoproteins (12). Low-density lipoprotein cholesterol was estimated using the formula described by Friedewald, Levy, and Fredrickson (15). Plasma glucose, blood urea nitrogen, serum bilirubin, serum glutamic oxalacetic transaminase and thyroxine were measured on the frozen samples in all subjects with a plasma cholesterol greater than 200 mg/dl and a plasma triglyceride greater than

140 mg/dl. None of the patients had secondary causes of hyperlipidemia. Three subjects (11-011, 11-013, 11-030) had mild hyperglycemia (range, 121 to 140 mg/dl). Each subject was examined for corneal arcus and cutaneous and tendon xanthomas. A cardiovascular history was obtained to evaluate the presence of angina pectoris, intermittent claudication, or myocardial infarction. Each subject was studied without our knowledge of whether he or she was affected. A patient was considered to have ischemic heart disease if he or she had angina pectoris or a documented myocardial infarction. The criterion for angina pectoris was the presence of pressing substernal pain, induced by exercise and relieved by rest. A myocardial infarction was judged to have occurred if a patient had been hospitalized for 2 to 3 weeks during which time he was told he had suffered a heart attack. GENETIC STUDIES

Distributions of Plasma Lipids and Lipoproteins: The lipid and lipoprotein data were plotted with age and the plots inspected for the appearance of bimodality. The standard methods of maximum likelihood (3, 16), probability plots (5, 17), or frequency distributions (18), previously used to test for the presence of bimodality, were not used because there are theoretical problems with their application here. The maximum likelihood method was not developed for use in a single large kindred where familial influences (both genetic and environmental) may influence the independence of the observations. Further, all the methods require an age-correction, which assumes that the correction for affected and nonaffected subjects is the same. Methods are currently being developed to address these problems (Dr. Edmund A. Murphy, personal communication).

Logarithmic transformation of the data was done using natural logarithms to reduce the positive skewness in the distributions. Segregation Ratio Analyses: To determine the ratio of affected to nonaffected subjects in this kindred, we used arbitrarily chosen but experimentally derived cutpoints from an unrelated Canadian population (19). The cutpoints for plasma cholesterol and triglyceride were the upper 5%, adjusted for age and sex. STATISTICS

The means, medians, standard deviations, and standard errors of the means were computed by standard methods using packaged computer programs (20). Tests of significance ("Student's" t test and chi square), regression analyses, and analysis of covariance were done using programs based on standard formulas and tables (20-22). Results DISTRIBUTIONS OF PLASMA LIPIDS A N D LIPOPROTEINS

W h e n the natural logarithms of L D lipoprotein cholesterol were plotted against age, clustering into t w o populations w a s present to inspection in this kindred. A similar graph of the natural logarithms of total plasma cholesterol s h o w e d less clearcut separation into t w o groups. D i s tributions o f the total plasma and L D lipoprotein cholesterol levels were divided into upper (affected) and lower (unaffected) groups o n the assumption that the lowest value in the upper cluster of points belonged with the affected population, while the highest value in the lower cluster of points belonged with the unaffected group. This assignment does not allow for misclassification, w h i c h m a y be as high as 2 7 % and 1 7 % for total plasma and L D lipoprotein cholesterol levels, respectively (3). T h e lowest values in the affected population for total plasma and L D lipoprotein cholesterol levels were 233 and 187 m g / d l ,

Figure 1 . Pedigree of the Newfoundland family. One hundred seventy-five subjects were studied. They were classified as having hypercholesterolemia, hypertriglyceridemia, or both on the basis of age- and sex-adjusted cutpoints, defined as the upper 5 % in an unrelated Canadian population ( 1 9 ) . The propositus (111-34) was judged to have homozygous familial hypercholesterolemia on the basis of a total cholesterol of 7 0 0 m g / d l , xanthomas before the age of 5 years, and the presence of tendon xanthomas and hypercholesterolemia in both the maternal and paternal sides of the family. The paternal and maternal sides of the family were ascertained through the father (IV-28) and the mother (IV-4) of the propositus, respectively. The parents of the proband are therefore considered to be secondary propositi. Streja et al. • HD Lipoprotein and Ischemic Heart Disease

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Table 1. Segregation Ratio Analyses by Kind of Mating*

Kind of Mating /XT

1

Offspring

\

(Number)

Affected

One parent affected with hypercholesterolemia HC+ X N (6) HC-HTG X N (2) HC X HTG (1) HC X NS (5) HC-HTG X NS (2) IHDJ XN(1) IHDJ XNS(2) Total (her N X N (3) N X NS (3) N X HTG (3) N X HC-HTG§ (1) NS X HC§ (1) HC X HC-HTG§ (1) HC X HC§ (1) Total

Nonaffected

Ratio of Affected: Nonaffected

N

HTG

11 1 1 5 3 0 3 24

HCHTG 1 0 1 4 0 0 1 7

14 1 6 15 1 4 0 41

2 3 2 1 1 0 0 9

12:16 1:4 2:8 9:16 3:2 0:4 4:0 31:50

0 0 0 2 2 0 0 4

0 0 1 0 0 2 0 3

14 2 7 1 0 1 2 27

0 1 0 0 0 0 0 1

0:14 0:3 1:7 2:1 2:0 2:1 0:2 7:28

HC

* In these analyses and those found in Table 2, hypercholesterolemia (HC), hypertriglyceridemia ( H T G ) , or both (HC-HTG) were defined as values of plasma lipids above age- and sex-specific cutpoints, which were the upper 95% in an unrelated Canadian population (19). N = normal plasma lipids; NS = not sampled. The following subjects are not included in these analyses: offspring and descendants of both the maternal and paternal sides of the family (matings II-l X 11-27 and II-4 X 11-28); the secondary propositi (II-4 and 11-28); matings in which insufficient information was available on either the parents or the children to categorize the kind of mating (1-2 X 1-8, 111-36 X 111-37, 11-15 X 11-16, III-113 X III-114, III-117X III-118, III-127 X H-128, and III-129X130), and one mating in the fourth generation (111-82 X 111-83). t Xanthomas were present in each of the parents affected with hypercholesterolemia. t Parent had died from premature ischemic heart disease (IHD) (before the age of 40 years) and was judged as "probably" having familial hypercholesterolemia on this basis. § Spouse had primary hypercholesterolemia.

respectively, first two decades; and 262 and 240 mg/dl, third and later decades. The highest values in the unaffected population for total plasma and LD lipoprotein cholesterol concentrations were 217 and 151 mg/dl, respectively, first two decades; and 245 and 189 mg/dl, ^hird and later decades. The geometric means (antilog of the means of the natural logarithms) for total plasma and LD lipoprotein cholesterol levels were 308 and 257 m g / dl, respectively, affected (upper) group; and 174 and 111 mg/dl, nonaffected (lower) group. Low-density lipoprotein cholesterol levels therefore provided a clearer separation of the two populations than did total plasma cholesterol concentrations. The appearance of two populations suggests, but does not prove, that the expression of increased total plasma and LD lipoprotein cholesterol levels in this kindred is influenced by a single major gene (monogenic). When the natural logarithms of H D lipoprotein cholesterol and total plasma triglycerides were plotted against age, the distributions appeared unimodal.

terol level was above a value that represented the upper 5% of an unrelated Canadian population (19). The subjects were also classified as hypertriglyceridemic using similarly derived cutpoints. The segregation ratio analyses were done first for progeny according to the kind of mating (Table 1). A ratio of 31:50 (affectedrnonaffected) was observed in the offspring of matings in which one parent was considered to be affected with hypercholesterolemia (Table 1). This ratio was lower than 1.0 (40.5:40.5) expected by a Mendelian dominant hypothesis (x 2 = 4.44,0.05 >P > 0.02, 1 df). Segregation ratio analyses were next done according to sex and decade to determine if the low ratio of affected to nonaffected could be related to any influence of age or sex (Table 2). The effect of age was most pronounced in the second decade, when the observed ratio (9:21) was significantly lower than the predicted ratio of 15:15 (x 2 = 4 -49, 0.05 > P > 0.02, 1 df). The ratios in the first (x 2 = 0.42, P > 0.50) and third and later decades (x 2 = 0.52, P > 0.40, 1 df) were not significantly different than predicted. The ratios in the female patients were not significantly different than predicted in the first (x 2 = 0.80, P > 0.40), second (x 2 = 0.04, P > 0.80), third and later (X2 _ o.52, P > 0.40), or for all decades combined (X2 = 0.10, P > 0.70). The ratios in male patients were not significantly different than expected in the first decade (x 2 = 0) but were significantly different in the second decade (x 2 = 5.00, 0.05 > P > 0.02) and for all decades combined (x 2 = 6.72, P < 0.02). The segregation ratio in the third and later decades for male subjects was of borderline significance (x 2 = 3.76, 0.10 > P > 0.05). From these analyses, we conclude that the lower ratios of affected to nonaffected subjects in this kindred were due to the effect of presently unknown factors on the expression of the familial hypercholesterolemia gene in males, which is most marked in the second decade but also present later in life.

SEGREGATION RATIO

A segregation ratio analysis (ratio of affected to nonaffected subjects) was done to determine further if the effect of a major gene could be operating in this family. A subject was considered affected or nonaffected with hypercholestero:emia according to whether the plasma choles874

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• Annals of Internal Medicine • Volume 89 • Number

Segregation ratio analyses were also done according to sibship size to determine if, in general, the sibship contained both unaffected and normal children (expected by a Mendelian hypothesis) rather than sibships comprised of all normal or affected children. Within the 19 sibships in which one parent had hypercholesterolemia (Table 1), the distribution of sibship size (number of sibships) was one (six), two (zero), three (three), four (three), five (zero), six (two), seven (one), eight (two), nine (one), and 10 (one). The ratio of affected to normal children observed (expected) in these sibships was one, 3:3 (3:3); three, 7:2 (4.5:4.5); four, 3:9 (6:6); six, 4:8 (6:6); seven, 1:6 (3.5:3.5); eight, 6:10 (8:8); nine, 5:4 (4.5:4.5); and 10, 2:8 (5:5). These analyses show that a few large sibships with many affected children did not contribute an excessive number of hypercholesterolemic subjects to the total segregation ratio. Indeed the larger sibships, in general, had an excess of normal children. This may be due to chance alone because only a relatively small number of sibships were available for study. Segregation ratio analyses were next determined according to the sex of the parent. In 14 of 19 matings (Table 1) the affected parent was female; the ratio of their affected to normal children was 19:30 6

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Table 2. Segregation Ratio Analyses by Sex and Decade

First Decade Male Hypercholesterolemia HC* HC-HTGf Normocholesterolemia N{ HTG§ Segregation ratios Affected :nonaffected

Female

Third and Later Decade

Second Decade Both Sexes

Male

Female

Both Sexes

Male

Female

Both Sexes

i

Male

k\\ Decades Female

5 0

4 0

4 1

2 2

3 0

7 3

12 1

13 5

3 2

5 2

15 0

5 1

10 0

3 4

28 2

13 7

5:5

4:7

13:30

18:20

9:12

5:15

4:6

9:21

3:10

10:7

13:17

Both Sexes

31:50

* HC = hypercholesterolemia. t HC-HTG = hypercholesterolemia and hypertriglyceridemia. % N = normal plasma lipids. § HTG = hypertriglyceridemia:

(0.65). This segregation ratio was very close to that of 12:20 (0.60) observed in the children born to the five affected males. CLINICAL FINDINGS

Ischemic Heart Disease: Ischemic heart disease was present in 15 members of this kindred (including the propositus). None of the spouses had ischemic heart disease. In three subjects with familial hypercholesterolemia (III8, III-11, 111-39), ischemic heart disease was diagnosed between the ages of 30 and 35 years. One person (11-13) had intermittent claudication as well as angina pectoris. Another (1-12) had multiple cerebrovascular accidents and angina pectoris. In two children, aged 14 (IV-7) and 16 (IV-19) years, exercise was limited because of chest pain. In one of these children (IV-19), a loud systolic ejection murmur at the base of the heart was heard. A history of sudden death or clinical ischemic heart disease was also revealed for five deceased members of the kindred. Subject 111-33, sister of the proband (111-34), died at age 13; the autopsy showed extensive atherosclerosis in all three main coronary arteries, myocardial infarction, and cholesterol deposits in the aortic valve. This patient was probably a homozygote for familial hypercholesterolemia. Xanthomas and Corneal Arcus: Xanthomas, early corneal arcus, or both were noted in 15 persons examined and were present in both paternal and maternal relatives of the index case. The xanthomas usually appeared in the third decade. In a 14-year-old boy (IV-10), a corneal arcus was noted. None of the spouses had xanthomas or corneal arcus. LIPOPROTEINS, LIPIDS, A N D ISCHEMIC H E A R T DISEASE

The relation between concentrations of total plasma cholesterol, LD lipoprotein cholesterol, H D lipoprotein cholesterol and triglycerides, and ischemic heart disease was first investigated by computing the prevalence of ischemic heart disease within each quartile of a given lipid or lipoprotein. If there is no relation between the plasma concentrations of the lipids and lipoproteins, then one would expect the cases of ischemic heart disease (N = 1 4 , excluding the propositus) to be spread equally among the quartiles (AT=3.5 per quartile). The distribu-

tion of ischemic heart disease within the four quartiles of both total and LD lipoprotein cholesterol was identified, and each differed significantly from that expected by the null hypothesis (x 2 = 31.33, P < 4 X 10-5, 3 df) (Table 3). There was a marked increase in cases of ischemic heart disease in the upper quartile of total and LD lipoprotein cholesterol compared with the lower three quartiles (x 2 = 30.41, P < 1 X 10-5, i df) (Table 3). N o cases of ischemic heart disease were found in the two lower quartiles. The distribution of ischemic heart disease within the four quartiles of H D lipoprotein cholesterol was also significantly different than expected (x 2 = 7.92, P < 0.05, 3 df) (Table 3). However, in distinct contrast to the distribution of ischemic heart disease in the LD lipoprotein quartile, the lower quartile of H D lipoprotein cholesterol contained one half of the patients with ischemic heart disease (x 2 = 5.14, 0.05 > P > 0.02, 1 df); no cases were present in the upper H D lipoprotein quartile. In the three lower quartiles for H D lipoprotein cholesterol, there was a stepwise decrease in cases of ischemic heart disease as the H D lipoprotein cholesterol levels increased. The cases of ischemic heart disease within the quartiles for total plasma triglycerides were skewed toward the upper quartiles (x 2 = 6.61, 0.10 > P > 0.05, 3 df) (Table 3). The upper quartile of triglyceride was the largest in cases of ischemic heart disease (x 2 = 5.16, 0.05 > P > 0.02, 1 df) but to a lesser extent than the upper quartile for LD lipoprotein and total cholesterol (Table 3). There was a stepwise decrease in cases of ischemic heart disease as glyceride levels fell, and there were more cases in each of the lower three quartiles for triglycerides than in the same quartile for total plasma and LD lipoprotein cholesterol (Table 3). Since these lipid and lipoprotein values were not corrected for age, possibly the distribution of cases of ischemic heart disease within these quartiles might be related to the concomitant increase of ischemic heart disease and lipid and lipoproteins with age. Although the number of subjects was not large enough to definitely examine this question, very similar distributions of ischemic heart disease within the four quartiles of lipids and lipoproteins were obtained when the subjects were divided into those above and below age 45. The interrelationship between lipoproteins and ischemStreja etal.

• HD Lipoprotein and Ischemic Heart Disease

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Table 3. Distribution of Ischemic Heart Disease Within the Four Quartiles of Plasma Lipids and Lipoproteins*

Quartile

Total cholesterol Low-density lipoprotein cholesterol High-density lipoprotein cholesterol Total triglycerides

Lowest

Second

Third

Highest

Of (105-160) t 0 (51-96)

0 (160-183) 0 (97-123)

2 (184-260) 2 (123-208)

12 (262-447) 12 (215-376)

7 (18-35)

4 (35-41)

3 (42-48)

0 (48-87)

1 (18-56)

2 (58-83)

4 (83-109)

7 (111-321)

* The prevalence of ischemic heart disease was estimated (see Methods) according to the quartiles of the plasma lipids and lipoproteins in 148 relatives of the propositus. The proband and her parents (secondary propositi) were not included in the analyses. t Number of subjects with ischemic heart disease. t Range of values in mg/dl.

ic heart disease was next investigated by dividing the subjects over 30 years of age into three groups: Group A, relatives of propositus with ischemic heart disease (12 subjects—six males, six females); group B, relatives of propositus free of ischemic heart disease (20 subjects—11 males, nine females); and group C, unrelated spouses (20 subjects—10 males, 10 females). The mean ages and the plasma lipids and lipoproteins for these groups are presented in Table 4. There was no significant difference between the groups for age. Group A had significantly higher mean levels of total plasma cholesterol (r = 4.25, P < 0.0005), LD lipoprotein cholesterol (r = 4.31, P < 0.0005), and triglycerides (r = 2.61, P < 0.02) than group B. The mean plasma concentration of H D lipoprotein cholesterol, however, was significantly lower in group A than in group B (r = 2.89, P < 0.007). These mean differences were even greater when group A was compared to group C, except for total plasma triglycerides, which were not significantly different. Group B differed from group C only in the mean levels of LD lipoprotein cholesterol (r = 2.09, P < 0.05). Group A had higher mean total plasma and LD lipoprotein cholesterol and triglycerides as well as lower H D lipoprotein cholesterol levels than groups B and C. The question therefore remained as to whether the signs of ischemic heart disease in group A, but not in groups B and C, were also related to differences in the lipids and lipoproteins other than H D lipoprotein cholesterol. An analysis of covariance was done. This technique combines the features of analysis of variance and regression and allows a test of the hypothesis that differences in H D lipoprotein cholesterol between groups A and B or C persist after adjusting for the effect of total and LD lipoprotein cholesterol and triglycerides on H D lipoprotein cholesterol. The values for H D lipoprotein cholesterol in each group were first plotted against the corresponding values for another lipid or lipoprotein and a test (F ratio) done to determine if the slopes of the regression lines were significantly different. None of the F ratios for slopes was significantly different (Table 5). Since the relationships between H D lipoprotein cholesterol and the 876

other lipids and lipoproteins in the three groups were not different, the second part of the analysis was done. A significant difference in the F ratio for elevation indicates that the mean differences in H D lipoprotein cholesterol concentrations between the groups studied persist after adjusting for the effect of a given lipid or lipoprotein on H D lipoprotein cholesterol levels. We found that after adjustment for total plasma cholesterol, mean H D lipoprotein cholesterol remained significantly different when groups A, B, and C were studied together or when group A was compared to either group B or C. After adjusting for either plasma triglycerides or LD lipoprotein cholesterol, the difference in H D lipoprotein cholesterol persisted only between groups A and C. However, the F ratio for H D lipoprotein cholesterol on total triglycerides and LD lipoprotein cholesterol in the group A, B, and C analysis approached the level of statistical significance (Table 5). These data suggest that H D lipoprotein cholesterol is a separate risk factor for ischemic heart disease even after adjusting for differences in plasma total and LD lipoprotein cholesterol and total triglycerides. Since the difference between groups A and B included significant changes for both LD lipoprotein and H D lipoprotein, we also investigated the distribution of the ratio of H D to LD lipoprotein cholesterol in patients with and without ischemic heart disease in this kindred. The H D to LD lipoprotein cholesterol ratios ranged from 0.059 to 1.159. In subjects with ischemic heart disease, the H D to LD lipoprotein cholesterol ratios ranged from 0.006 to 0.20. In the entire population, there were 43 subjects with a ratio 0.20 had no signs or symptoms of ischemic heart disease. Thirty-four of these subjects were over 30 years of age. This suggests that an H D lipoprotein to LD lipoprotein cholesterol ratio 3.20 ( P < 0 . 0 5 ) and 5.10 ( P < 0 . 0 1 ) . t In the analyses of covariance for groups A and B or A and C, there were one and 28 degrees of freedom; significance levels were achieved at an F ratio > 4.20 (P < 0.05) and 7.64 (P < 0.01).

5). The data further suggested that the association of a low level of H D lipoprotein cholesterol with ischemic heart disease persisted after adjustment for total plasma and LD lipoprotein cholesterol levels. Moreover, even though the numbers are small, the only statistically significant difference between hypercholesterolemic adults, aged 30 or older, with xanthomas and ischemic heart disease and similarly affected adults without ischemic heart disease was in the mean H D lipoprotein cholesterol levels. These observations were made in a retrospective study of a large kindred and are not directly comparable with longitudinal studies made in general populations. In addition, the data base does not allow for corrections for confounding "risk" factor variables such as cigarette smoking and hypertension. With these caveats, our results are compatible with those made in epidemiologic, long-term studies where low H D lipoprotein cholesterol levels were found to be an independent "risk" factor for ischemic heart disease (9, 11, 24). Finally, there are always difficulties in expressing lipid and lipoprotein metabolism in statistical terms. For example, in the prospective Livermore study, Gofman, Young, and Tandy (25) were not able to conclude whether the significantly lower levels of H D lipoproteins in cases of de novo ischemic heart disease were in excess of those anticipated by the inverse correlation of H D lipoproteins and VLD and LD lipoproteins (25). This study also suffers from the need to employ a "soft" endpoint, angina pectoris, to define ischemic heart disease in many of the patients. We did use standard criteria, however, and the decision as to the presence or absence of angina pectoris was made without knowledge of the plasma lipid and lipoprotein levels. We were able to obtain additional clinical information from the physicians of 10 of the 12 adults, aged 30 or older, who were Streja eta/.

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considered to have cardiovascular disease at the time they were studied. Six of the eight subjects with angina pectoris had electrocardiographic evidence of myocardial ischemia or infarction, and all but one was being medically treated for angina pectoris. Of the two subjects with a history of myocardial infarction, one had the infarction confirmed by an electrocardiogram while the other had a normal electrocardiogram but was being treated with Isordil and propranolol. Finally, when we compared the relatives who had ischemic heart disease with the diseasefree spouses, we assumed that the two groups had similar "years of risk for heart disease'' because there was no significant difference in the mean ages of the two groups. This assumption is based on the hypothesis that risk for heart disease begins early in life, probably in childhood (26). The average years of risk for heart disease, starting at age 30, were higher in the relatives with ischemic heart disease (19.4 years) than in the spouses (12.7 years). The mean difference of 6.7 years of risk between the two groups can be reduced to 3.4 years if the octogenarian in the diseased group is excluded from the calculations. The findings in this kindred may not be applicable to all families with familial hypercholesterolemia. Low levels of H D lipoprotein cholesterol, however, have been previously described in this syndrome (1, 3, 4), and it is likely that a similar association may be found in many of these families as well. Several tentative inferences can be made on the relevance of high LD but low H D lipoprotein cholesterol levels in familial hypercholesterolemia. First, the pathogenesis of atherosclerosis in familial hypercholesterolemia may share some common mechanism^) with "garden variety" atherosclerosis, since H D and LD lipoprotein cholesterol levels have separate effects on the development of ischemic heart disease in the general population (9, 11, 24). Second, the quantitative change in H D lipoprotein cholesterol concentrations was small compared with that of L D lipoprotein cholesterol concentrations; and the apparent significant contribution of low H D lipoprotein levels to atherosclerosis in relation to this small change is surprising (Table 4). Third, the ratio of H D to L D lipoprotein cholesterol may be a more sensitive predictor of risk for ischemic heart disease than either lipoprotein level alone. Indeed, a ratio of H D to LD lipoprotein cholesterol of less than 0.20 was present in each subject with ischemic heart disease in this family. As the Framingham group has recently pointed out, however, a person may have the same H D / L D lipoprotein ratio at low levels of H D and LD lipoprotein cholesterols or at high levels, and these may have different medical significance (24). The definitive test of the prognostic value of an H D / L D lipoprotein ratio for ischemic heart disease awaits further study. The biochemical mechanism(s) of the "opposite" effects of H D and LD lipoprotein cholesterol levels on the pathogenesis of atherosclerosis is not definitely known. There are several hypotheses. High-density lipoproteins may act as a "scavenger" of cholesterol esters in the vessel wall and carry esterified cholesterol from the arterial smooth muscle cells back to the liver for catabolism (8). Further support for this hypothesis is derived from stud378

ies in patients with Tangier disease, a genetically determined defect in which the H D lipoprotein levels are markedly reduced (27). Patients with Tangier disease have deposits of cholesterol esters in tonsils, bone marrow, liver, spleen, peripheral nerves, and the arterial wall (27). Another hypothesis has been put forward by Carew and colleagues (28), who surmised from their studies of cultured arterial smooth muscle cells that H D lipoproteins inhibit or interfere with the binding of LD lipoproteins to smooth muscle cells, thereby preventing the LD lipoprotein-mediated production of atherogenic cholesterol esters in the arterial cells. The cause of the low H D lipoprotein cholesterol concentration in familial hypercholesterolemia has not been ascertained, but the decreased level may be part of the phenotypic expression of the disorder, particularly since the changes are present from birth (4). One primary genetic defect in familial hypercholesterolemia exists in a functionless receptor for LD lipoprotein on the cell surface (7). Although the precise role of this receptor in the in-vivo metabolism of LD lipoproteins is not completely understood, patients with familial hypercholesterolemia have decreased catabolism of LD lipoproteins in vivo (29). The increased plasma level of LD lipoproteins in familial hypercholesterolemia, therefore, appears to be a manifestation of this primary defect. The relevance, if any, of the deficiency of LD lipoprotein receptors to low H D lipoprotein cholesterol levels is not apparent, since, in cultured human fibroblasts, H D lipoproteins do not appear to bind specifically to the LD lipoprotein receptor (30). However, in cultured human arterial cells (28, 31), porcine, or rat hepatocytes (32, 33) or rat adrenal cells (34), there appears to be some competition of H D with LD lipoproteins for "LD lipoprotein" binding sites. If H D and LD lipoproteins compete for similar binding sites in vivo, one might expect an elevated rather than a depressed level of plasma H D lipoprotein levels. Other possibilities include decreased synthesis or increased catabolism of H D lipoproteins in familial hypercholesterolemia. These observations may have some importance in planning therapy for patients with familial hypercholesterolemia. Currently, a diet low in cholesterol and high in polyunsaturated fats is employed (1). This produces, on average, a 10% to 15% drop in plasma total and LD lipoprotein cholesterol levels (1). Addition of a bile sequestrant can cause a further 25% to 40% drop in LD lipoprotein cholesterol levels (35). In view of the data presented here, consideration should be given to supplementing the above hypocholesterolemic therapy by recommending measures that may increase H D lipoprotein cholesterol concentrations. These include physical exercise, weight reduction, moderate alcohol intake, and cessation of cigarette smoking (9, 11, 24, 36). This study led to several other interesting observations on the expression of familial hypercholesterolemia. The proportion of affected to nonaffected children was less than expected and appears to have been affected by unknown biological factors that operated primarily in male subjects and first became apparent in adolescence. De-

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creased signs of familial hypercholesterolemia in the second decade have been previously observed in a large collection of families (3). In normal adolescent males there is a decrease in L D lipoprotein and total cholesterol levels and an increase in triglycerides and VLD lipoprotein (37). Whether similar factors are operating in adolescent males with familial hypercholesterolemia or are modified by the familial hypercholesterolemia gene is not clear. Alternatively, the discrepant segregation ratios may be related to the arbitrary definition of hypercholesterolemia that we used. This cutpoint may not have minimized misclassification, particularly in the second decade. The deficiency of affected males with familial hypercholesterolemia late in life may be related to differential mortality, more pronounced in the male compared to the female. The failure to confirm a previous observation (3), namely, a lower segregation ratio in children born to affected males than females, may be related to the small number of affected male parents studied here, or the factors responsible for the previous findings may not be operating in this kindred. Finally, a surprising number of subjects in this kindred had triglyceride levels above the 95th percentile of the control population. The absence of bimodality, however, suggests that the mild hypertriglyceridemia in members of this kindred is probably not part of the phenotypic presentation of familial hypercholesterolemia. Rather, it may be related to inherent differences between the Newfoundland and Toronto populations. Our study suggests that levels of H D lipoprotein cholesterol, as well as total plasma and L D lipoprotein cholesterol, may have some importance in the development of ischemic heart disease and atherosclerosis in familial hypercholesterolemia. The data from this kindred provide a useful base for comparing similar information obtained from other families affected with familial hypercholesterolemia and other inherited hyperlipidemic states. ACKNOWLEDGMENTS: The authors thank Dr. J. Ramsay Farah for assistance in the field studies; Drs. Gary Chase and Edmund Murphy for assistance in the statistical analyses; Dr. Carl Breckenridge for the use of his laboratory; Susan Evans for technical assistance; Hazel Smith for constructing the pedigree; and the members of this family and their physicians, Drs. M. A. McVicker, D. P. Black, and J. Sheldon, for their cooperation. Grant support: by the Ontario Heart Foundation and Contract NOl-HV12158-L from the National Heart, Lung, and Blood Institute. Dr. Streja is a former Fellow of the Ontario Heart Foundation. • Requests for reprints should be addressed to Peter O. Kwiterovich, Jr., M.D.; Johns Hopkins Hospital, 600 N. Wolfe St.; Baltimore, MD 21205. Received 5 July 1978; revision accepted 3 August 1978.

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Plasma high-density lipoproteins and ischemic heart disease: studies in a large kindred with familial hypercholesterolemia.

Plasma High-Density Lipoproteins and Ischemic Heart Disease Studies in a Large Kindred with Familial Hypercholesterolemia DAN STREJA, M.D.; GEORGE STE...
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