Familial B-100: Enhanced MB47to - pnas.org

Proc. Nati. Acad. Sci. USA
Vol. 85, pp. 9758-9762, December 1988
Medical Sciences

Familial defective apolipoprotein B-100: Enhanced binding of
monoclonal antibody MB47 to abnormal low density lipoproteins

(hypercholesterolemia/genetic disease/atherosclerosis/apolipoprotein B-100 polymorphism)

KARL H. WEISGRABER*t, THOMAS L. INNERARITY*, YVONNE M. NEWHOUSE*, STEPHEN G. YOUNG*,
KAY S. ARNOLD*, RONALD M. KRAuSSt, GLORIA L. VEGA§, SCOTT M. GRUNDY§,

AND ROBERT W. MAHLEY*

*Gladstone SFaonunFdraatnicoisncoL,abCorAat9o4r1i4e0s-0f6o0r§C;artdDioonvnaesrcuLlaabrorDaitseoarsye,, Cardiovascular Research Institute, Departments of Pathology and Medicine, University of
California, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; and §Center

for Human Nutrition, Departments of Internal Medicine, Biochemistry, and Clinical Nutrition, University of Texas Health Science Center, Dallas, TX 75235

Communicated by Joseph L. Goldstein, September 2, 1988 (received for review July 20, 1988)

ABSTRACT Familial defective apolipoprotein (apo) B-100 However, familial hypercholesterolemia heterozygosity
is a recently described genetic disorder that appears to result accounts for only a fraction of the high plasma cholesterol
from a mutation in the apoB-100 gene. This disorder is levels in the population (10). Most individuals with elevated
characterized by hypercholesteroledia resulting from elevated LDL appear to have receptors with a normal structure or with
plasma conce ntrations of low density lipoprotein LDL. The no detectable dysfunction (11-13). Therefore, other factors
disorder was fist detected in three members of one family. The must contribute to increased plasma levels of LDL in most
LDL from affected subjects binds defectively (:30% of nor- hypercholesterolemic individuals (13, 14). To determine
mal) to LDL receptors, retarding the clearance of LDL from whether other genetic abnormalities of LDL catabolism
result in hypercholesterolemia, attention has recently been
plasma. In the present study, two otflbr members of the affected directed to'moderately hypercholesterolemic subjects who
do not have classic familial hypercholesterolemia heterozy-
family were found to possess abnormal LDL. In addition, gosity. Vega and Grundy (15) have shown that in some
abnormal LDL with a similar binding defect were found in a hypercholesterolemic subjects the catabolic rate for autolo-
second, unrelated family. In both families, the defect is gous LDL is significantly slower than the rate for normal
transmitted over three generations as an autosomal codomi- heterologous LDL. These results suggested that LDL interact
nant trait and all affected members are heterozygotes. Since
there is only one apoB-100 molecule per LDL particle, the abnormally with the LDL receptor, a possibility confimed in
abnormal LDL in heterozygous subjects is made up of two
plpulations of particles: one that has normal binding activity one of these subjects, G.R., by using an in vitro receptor
to receptors and one that binds defectively. To localize the binding assay (16). The LDL from'G.R. and two of his three
mutation in apoB-100, the binding of five apoB-100-specific brothers bound to receptors with -30% the activity of normal
monoclonal antibodies to abnormal LDL was assesd in a LDL. Since the interaction of LDL with the LDL receptor is
solid-phase RIA. Only antibody MB47, whose epitipe is
between residues 3350 and 3506, distinguished abnormal LDL mediated through apoB-100 (2) and the defect appears to be
from normal LDL isolated from control subjects with normal
lipid levels; MB47 bound with a higher affinity (by =60%) to associated with the apoB-100 molecule, this disorder has been
abnormal LDL. In every individual with abnormal LDL, the designated familial defective apoB-100 (16). Because there is
MB47 antibody bound with a higher affinity. The convenience only one molecule of apoB-100 per LDL particle (17); the
of this assay will facilitate screening of large populations to abnormal LDL in heterozygous subjects would be a mixture,
determine the frequency of this disorder. containing a population of particles with normal binding
activity to receptors and a population with defective binding.
In humans, most plasma cholesterol -is contained in the low
density lipoprotein (LDL) density fraction. Plasma concentra- The current study was undertaken to localize the region of
tions of LDL are regulated by the LDL receptor pathway (1), apoB-100 responsible for the defect. Our approach was to test
and the interaction of LDL with the receptor is mediated by the binding of selected apoB-specific monoclonal antibodies to
apolipoprotein (apo) B-100 (2), the predominant apolipoprotein abnormal and normal LDL to determine whether any bound
component of LDL. The structure of the Mr 550,000 apoB-100 with altered affinity to abnormal LDL from familial defective
has recently been determined (3-6). Epidemiological studies apoB-100 subjects. Four of the five antibodies selected were
have established an association between increased plasma known to inhibit the binding of LDL to LDL receptors. Only
concentrations of LDL and an increased incidence of coronary the MB47 antibody described by Young et al. (18) bound to
heart disease (7, 8). An extreme example ofthis association, and abnormal LDL with a higher apparent affinity than it bound to
one that underscores the importance of the LDL receptor in normal LDL. The high-affinity binding of the MB47 antibody
regulating plasma LDL concentrations, is the genetic disorder cosegregates with defective-binding LDL in all R. family
familial hypercholesterolemia (for review, see ref. 9). Subjects members examined thus far. Familial defective apoB-100 has
heterozygous for familial hypercholesterolemia have elevated also been identified in a second, unrelated subject (W.S.) and
plasma LDL and develop premature atherosclerosis. The dis- four family members. The high-affinity binding of the MB47
order results from several different mutations at the gene locus antibody is also linked to abnormal LDL in this second family.
of the LDL receptor that lead to expression of nonfunctional or
dysfunctional receptors (1). MATERIALS AND METHODS

The publication costs of this article were defrayed in part by page charge Subject Material. The probands (G.R. and W.S.) were
payment. This article must therefore be hereby marked "advertisement" recruited from a population of subjects being treated for
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: apo, apolipoprotein; LDL, low density lipopro-
tein(s).

tTo whom reprint requests should be addressed at: Gladstone

Foundation Laboratories for Cardiovascular Disease, P.O. Box
40608, San Francisco, CA 94140-0608.

9758

Medical Sciences: Weisgraber et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9759

primary hypercholesterolemia at the University of Texas RESULTS
Center for Human Nutrition. Subject G.R. was previously
described (16), and subject W.S. was identified in a receptor We previously described familial defective apoB-100 (16) in
binding assay. Normal, control subjects were volunteers with the R. family. By using in vitro receptor binding assays, we
normal plasma lipid levels. Test subjects for verification of determined that LDL from two of the proband's (G.R.)
the antibody RIA were recruited from a church in Walnut brothers bound defectively to LDL receptors, whereas LDL
Creek, CA.
from a third brother and his son bound normally (16). By
Plasma (from blood treated with EDTA, 1 mg/ml) obtained using a competitive fibroblast binding assay, we have ex-
from subjects who had fasted overnight was used for lipo- tended the study to include four additional first-degree
protein analysis or LDL preparation. Total plasma choles- relatives of one of G.R.'s affected brothers (Fig. 1). Two of
these subjects were found to have an LDL binding deficiency
terol and LDL cholesterol concentrations were determined similar to that of the other affected family members: =30% of
with a spectrophotometric assay kit (Boehringer Mannheim); normal [an average of 7.4 pug of LDL protein per ml was
LDL cholesterol concentrations were measured using the required for 50% inhibition of 1251-labeled LDL binding to
Gilford 400E analyzer after precipitation of plasma with normal fibroblasts compared with 2.6 ,ug/ml for normal LDL
heparin and manganese chloride (19, 20).
(Table 1)]. In agreement with previous results (16), the
General Methods. LDL (p = 1.02-1.05 g/ml) were isolated additional subjects with abnormal LDL had a higher plasma
from plasma by sequential ultracentrifugation (40C) at 59,000 cholesterol concentration than age- and sex-matched con-
rpm in a Beckman 60 Ti rotor and were recentrifuged at p = trols, and this hypercholesterolemia is associated with in-
1.05 g/ml. The protein content of LDL was determined creased LDL cholesterol (Table 1).
according to the method of Lowry et al. (21) using bovine
serum albumin as a standard. The binding of unlabeled LDL Familial defective apoB-100 has also been identified in
samples to LDL receptors on cultured human fibroblasts was three generations in a second, unrelated family by using the
determined in competitive receptor binding assays as de- competitive receptor binding assay. Testing showed that 4 of
scribed (22, 23). The concentration of unlabeled test LDL 15 relatives of the proband W.S. have defective-binding LDL
required to displace 50% of control 125I-labeled LDL was (Fig. 2, Table 2). As in the R. family, the defective LDL of
calculated by linear regression analysis of the logarithm of S. family members has =30% of normal binding activity.
concentration (,ug/ml) vs. probits. Probits were obtained Hypercholesterolemia and elevated LDL are also associated
from a probit transformation table (24). with the presence of abnormal LDL in this family, except for
Le.S. (Table 2). Although Le.S. had hypercholesterolemia
Antibodies. Four of the five apoB-100-specific monoclonal
antibodies used block the binding of LDL to LDL receptors: (280 mg/dl in 1985), his present total cholesterol and LDL
4G3, at residues 3029-3132 (5, 25); 3F5, at 3029-3132 (5);
5E11, at 3249-3636 (5, 25); and MB47, at 3350-3506 (5). cholesterol levels are in the normal range. However, he has
Antibody MB3, which does not block LDL binding and
whose epitope is located in the amino-terminal thrombolytic recently developed diabetes and is presently hypertriglycer-
fragment (T4, residues 1-1297), wqs used as a control antibody idemic. Several unaffected S. family members also have
(26, 27). hypercholesterolemia, but its basis is not yet clear.

RIAs. The ability of individual LDL preparations to bind to Because the molecular defect associated with familial
the various apoB-100 monoclonal antibodies and the concen- defective apoB-100 appears to reside in apoB-100 (16), we
trations of apoB-100 in plasma were determined in solid- sought to localize the region of apoB-100 that contained the
phase competition RIAs according to methods prev pusly mutation(s). Because the mutation(s) affects receptor bind-
described (26). Removawell plates (Dynatech, Cq tilly,
VA) were coated at roorq temperature for 2 hr with tffd of ing, we examined the binding to LDL of four apoB-
100-specific monoclonal antibodies (4G3, 3F5, SEll, and
a phosphate-buffered saline buffer (0.15 M Naq:l cont ining MB47), all of which block LDL binding to the LDL receptor.
A fifth antibody, MB3, which does not block receptor binding
0.022 M Na2HPO4 and 0.015 M Na2HPO4,9fI 7.4) containing of LDL and whose epitope is in the amino-terminal region of
apoB, was used as a control. As shown in Fig. 3 A and B,
10 ,ug of control LDL per ml. The plates were wvshed five antibofjes MB3 and 4G3 bound equally well to all LDL
samples. Similar results were obtained with antibodies 5E11
times with washing buffer (phosphpte-buffered saline con- and :F5 (data not shown). However, with antibody MB47
(Fig. 3C), the binding curves of abnormal LDL were dis-
taining 0.3 mM EDTA, 0.02% NaN3, 0.05% Tween 20,0.04% placed to the left compared with those from a normal subject
aprotinin, and 0.1% bovine serum albumin). Wells were or from unaffected family members. This displacement to
blocked by incubation with 200,ul ofblocking buffer (washing
buffer containing 4% bovine serum albumin) for 1 hr at room lowgr LDL concentrations was reproducible in three sepa-
temperature and then were washed five times with washing
buffer. A standard curve for the LDL binding assays was rate assays and probably reflects a higher binding affinity of
constructed by diluting control LDL in dilution buffer abnopmal LDL for antibody MB47 than normal LDL display.
(washing buffer containing 31% bovine serum albumin) to
concentrations ranging from 0.4 to 100 jig of LDL protein per The fPL from affected subjects P.B. and E.S. were also
ml. A range of 0.16-50 ,ug/ml was used for determining the
apoB concentrations in plasma. Twenty-five microliters of Lo ¼LL
standard LDL, unknown LDL samples, or plasma dilutions
(1:100, 1:200, and 1:300) was loaded into each LDL-coated G.R. Sta.R. W.R. C.R. M.R.
well, followed by 25 pA of a fixed concentration of antibody
(either ascites fluid or IgG that was prepared by protein A Ste.R. J.P. M.P. PB.
purification) in dilution buffer. Optimal antibody concentra-
tion was determined in a separate assay. The plates were then E.S.
incubated overnight at 4°C and washed five times with
washing buffer, and the amount of antibody bound was FIG. 1. Pedigree of the R. family. Subjects whose LDL have
determined after a 4-hr incubation at 40C with I251-labeled been examined for receptor binding activity are indicated by initials
sheep anti-mouse IgG (Amersham). In the plasma assay, use below the symbols; the arrow indicates the proband. Half-filled
of the Pro/Pette System (Perkin-Elmer) improved pipetting symbols indicate subjects whose LDL bound defectively to apoB,E
precision compared with manual pipetting. (LDL) receptors, and unfilled symbols indicate subjects whose LDL
bound normally. Crosses indicate that the subject is deceased.

9760 Medical Sciences: Weisgraber et al. Proc. Natl. Acad. Sci. USA 85 (1988)

Table 1. Characterization of R. family members Table 2. Characterization of S. family members

Cholesterol, Cholesterol,

Family Age, mg/dl LDL IC50,* Family Age, mg/dl LDL IC5o,*
Ag/ml
member Sex yr Plasma LDL Ag/ml member Sex yr Plasma LDL

Defective LDL binding Defective LDL binding 9.9
W.S. d 66 231 183 9.4
G.R. d 68 311 202 9.3 J.L. 9 42 247 171 7.4
6.9 K.L. 9 19 253 190 5.4
W.R. d 72 279 206 7.1 M.S. d 52 257 168 6.6
5.6 Le.S. d 68 197 122
C.R. d 70 336 256 8.0

P.B. 9 41 324 265

E.S. 9 22 250 195

7.4 ± 1.4t 7.7 ± 1.9t

Normal LDL binding Normal LDL binding 2.2
2.7
Sta.R. d 64 181 ND 2.9 S.St. 9 38 182 105 2.1

Ste.R. 34 236 158 3.0 T.N. 9 21 141 78 2.4

J.P. 9 42 138 73 2.3 B.S. d 37 243 178 3.2
2.8
M.P. 9 40 149 68 2.4 R.W. 23 219 147 2.4
1.8
2.6 ± 0.4t K.J. 9 21 174 115 2.3
2.8
S.J. 9 18 154 86 1.9

ND, not determined. inhibit 50% of I251-labeled LDL J.J. d 19 171 98 2.4 0.4t
*Concentration of LDL required to
binding to normal cultured human fibroblasts. L.S. d 64 172 102

tx± SD. R.S. d 42 213 146

C.L. 9 39 210 136

consistently -50% more effective in binding to antibody J.M. 9 40 188 112
MB47 than were LDL from unaffected subjects J.P. and M.P.
*As defined in Table 1.
(data not shown) Qr from normal, control subjects. tx± SD.
The foregoing data demonstrate that immunoassays using
proband W.S. were 1.56, 1.95, 1.82, 1.60, and 1.91 (x ± SD -
antibody MB47 can distinguish abnormal LDL from normal
LDL. Because this assay requires prior isolation of LDL by 1.77 ± 0.18).
ultracentrifugation, a more direct assay was developed,
based on a method used to study the polymorphism detected To test the usefulness of this assay as a screening tool and

by antibody MB19 (26). The "apparent" plasma concentra- to rule out the possibility that the high MB47 value was
tion of apoB was determined by using antibody MB47 in a simply an artifact associated with hypercholesterolemia, we
tested 110 subjects. The assay was done in duplicate (in our
competitive RIA. The value is referred to as apparent experience, duplicate analyses are sufficient to distinguish
because the higher affinity of abnormal LDL for the antibody subjects with familial defective apoB-100 from unaffected
would be expected to result in an artificially high plasma subjects), and subject W.S. and another affected proband,
value in subjects with abnormal LDL. The "true" concen- identified in a receptor assay screen, were included as
tration of apoB was determined in a parallel assay using positive controls. The MB47/MB3 ratio was determined for
antibody MB3, and the results from both assays were each subject, was normalized to 1.00 for subjects with a
expressed as a ratio. The MB47/MB3 ratio from normal plasma cholesterol of <240 mg/dl, and was compared with
control subjects was normalized to 1.00 because the absolute the value in subjects with levels >240 mg/dl. There was no
value of this ratio varies slightly from assay to assay. The
ratios for unaffected and affected family members were then significant difference between the ratios of the two groups
expressed relative to this normalized value. As shown in (<240 mg/dl, x ± SD = 1.00 ± 0.11, n = 42; >240 mg/dl,
Table 3 for the R. family, the ratios for normal controls and x + SD = 1.07 ± 0.10, n = 67). The MB47/MB3 ratio vs.
unaffected family members were essentially identical, where-
as the ratio for family members with abnormal LDL was plasma cholesterol concentration is presented in Fig. 4.
significantly higher (-60%). This higher ratio reflects the Fig. 4 also demonstrates that the MB47/MB3 values for
higher binding affinity of antibody MB47 for abnormal LDL. the two positive controls (m, A) clearly lie outside the range
In a similar assay, the 4G3/MB3 ratios for plasma from of values for the test subjects. In addition, one test subject
with a cholesterol level of 355 mg/dl was found to have a
control, unaffected, and affected subjects were not siglifi- MB47/MB3 ratio of 2.37, indicating that this subject was
affected with familial defective apoB-100. This was con-
cantly different (Table 3). In the S. family also, the MB47/MB3 firmed in a receptor binding assay that showed that this
ratio was significantly higher in the 5 affected subjects than in subject's LDL bound defectively to LDL receptors (34% of
the 11 unaffected family members (1.68 vs. 1.01, Table 3). The normal).
ratio is quite reproducible for a given individual. For example,
the normalized values determined on 5 separate days for the DISCUSSION

Familial defective apoB-100 represents a recently described
genetic disorder that is characterized by hypercholesterol-
emia and by abnormal LDL that bind poorly to LDL
receptors (16). The functional defect in the LDL appears to
be caused by a mutation(s) in apoB-100 (16). This results in
increased concentrations of LDL cholesterol in these sub-
jects because of a retarded clearance rate for plasma LDL
(15). We have examined two kindreds (R. and S.) with this
K.L. disorder and have demonstrated transmittance through three
R.W. K.J. S.J. J.J. T.N.
generations (Figs. 1 and 2). The pattern of inheritance, in
FIG. 2. Pedigree of the S. family (legend as in Fig. 1, ).which the affected subjects are heterozygotes, is consistent

Medical Sciences: Weisgraber et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9761

B/B.

0.4 - Ia I
0.3 - I_ S.a \
0.2 -
102 103 10 102 103 10 102 lo,
I~0.1 |- ngLDL/Well

10

FIG. 3. Ability of LDL from selected subjects to compete with control LDL for binding to apoB-100 monoclonal antibodies as determined
in a solid-phase RIA using antibody MB3 (A), antibody 4G3 (B), or antibody MB47 (C). Filled symbols indicate LDL from affected R. family

members; open symbols indicate LDL from unaffected R. family members; X indicates LDL from a normal control subject. B/Bo, ratio of the
amount of antibody bound in the presence of competitor LDL to the amount bound in the absence of competitor LDL.

with the defect being an autosomal codominant trait. Because binding (25). Only antibody MB47 bound with a higher

there is only one molecule of apoB-100 per LDL particle (17), apparent affinity to LDL from affected R. and S. family

the abnormal LDL in these heterozygous subjects is a members than to normal LDL. Antibody MB47 also identi-
mixture of LDL particles: one kind binds normally to
fied the three additional probands whom we have recently
receptors, and one binds defectively. The present study
demonstrates that the hypercholesterolemia associated with found. One of these subjects was identified in the plasma
familial defective apoB-100 is due to increased LDL concen-
screen presented in Fig. 4. These subjects have hypercho-
trations; all but one of the affected subjects had elevated lesterolemia, and their LDL bind defectively to LDL recep-
LDL. The affected members of the R. family were above the tors (==30% of normal activity) (T.L.I., K.H.W., S.G.Y.,
95th percentile for age- and sex-matched controls (28), G.L.V., S.M.G., R.M.K., and R.W.M., unpublished data).

whereas four of the five affected S. family members were The absolute association of high binding affinity of anti-
above the 75th percentile. body MB47 for abnormal LDL with defective receptor
binding of abnormal LDL indicated that a region of apoB-100
In an effort to locate the mutation(s) responsible for the at or near the MB47 epitope was a probable site of mutation(s)
in the apoB-100 in these families. A study carried out at the
apoB-100 defect in familial defective apoB-100, four mono- DNA level in the R. family identified a single-nucleotide
clonal antibodies (4G3, 3F5, 5Ell, and MB47) that inhibit substitution in the apoB gene that results in a glutamine-
LDL binding to the LDL receptor were examined to deter- for-arginine substitution at residue 3500 in apoB-100. All
mine whether any were capable of distinguishing abnormal affected R. and S. family members, as well as the three new
affected probands mentioned above, are heterozygous, each
LDL from normal LDL. The epitopes of these inhibiting
antibodies, as well as others, are clustered near the junction possessing one allele specifying glutamine at 3500, whereas
of thrombolytic fragments T3 and T2 (residue 3249), indicat- all of the unaffected individuals in the R. and S. families are
ing the importance of this region of apoB-100 in receptor

Table 3. Ratio of plasma apoB concentrations determined with
various monoclonal antibodies

No. of 0
Family subjects MB47/MB3
4G3/MB3 E 330[ A

R. family 0r- S U
Defective LDL
binding 300[._.
Normal LDL
binding 5 1.66 ± 0.29* (21) 1.07 ± 0.12t (12) 0C0 270 * 3*s
Normal 4 1.12 ± 0.29t (12) 0.98 ± 0.12t (9)
controls 4 1.00 ± 0.24§ (24) 1.00 ± 0.15§ (10) e- 0

S. family 5 0 S:
Defective LDL 11
binding 4 - 0.
Normal LDL
binding S 240
Normal
controls 0

Si 2101

1.68 ± 0.31* (15) ND E 180
1.01 ± 0.17t (31) ND
1.00 ± 0.09§ (12) ND 1.50 2.00
MB47/MB3
Values are mean ± SD; the number of multiple determinations for 1.00
each subject in each category is given in parentheses. ND, not
determined. FIG. 4. MB47/MB3 ratios vs. plasma cholesterol concentrations

*Significantly different, P < 0.001, from value in family members in a test population. Assays were performed in duplicate on plasma
with normal LDL binding or in normal controls. samples. ., Individual values for each of the test subjects; *, subject
tNot significantly different from value in family members with W.S.; A, new proband identified in a receptor binding assay screen;
normal LDL binding or in normal controls. *, new proband from the test population identified in this assay.
tNot significantly different from value in normal controls.
§Value normalized to 1.00. Error bar indicates the mean + SD of the MB47/MB3 ratio from the
five affected members of the S. family presented in Table 3.

9762 Medical Sciences: Weisgraber et al. Proc. Natl. Acad. Sci. USA 85 (1988)

homozygous, with both alleles specifying arginine at 3500 8. Heiss, G. & Tyroler, H. A. (1982) Proceedings ofthe Workshop
(29). Thus, to date there is a perfect correlation among on Apolipoprotein Quantification (U.S. Dept. of Health and
defective receptor binding activity, increased MB47 affinity, Human Services, National Institutes of Health, Bethesda,
and a glutamine-for-arginine substitution at residue 3500 in all MD), NIH Publ. No. 83-1266, pp. 7-24.
subjects with familial defective apoB-100. Therefore, it
seems likely that the glutamine substitution at residue 3500 is 9. Goldstein, J. L. & Brown, M. S. (1983) in The Metabolic Basis
responsible for the enhanced binding of antibody MB47 by ofInherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B.,
abnormal LDL. Residue 3500 is within the MB47 epitope (5) Fredrickson, D. S., Goldstein, J. L. & Brown, M. S. (Mc-
and, compared with published sequences of apoB-100 (3-6), Graw-Hill, New York), 5th Ed., pp. 672-712.
this substitution is the only difference in this region of the
apoB gene from affected subject G.R. (29). 10. Expert Panel, National Cholesterol Education Program (1988)

Determining the frequency of familial defective apoB-100 Arch. Intern. Med. 148, 36-69.
will require extensive screening studies. Our recent identifi- 11. Wilson, P. W., Garrison, R. J., Castelli, W. P., Feinleib, M.,
cation of three additional probands with familial defective
apo-B100 indicates that this mutation may be common and McNamara, P. M. & Kannel, W. B. (1980) Am. J. Cardiol. 46,
therefore may contribute significantly to primary hypercho-
lesterolemia in the general population. The contribution of 649-654.
this disorder to increasing the risk of atherosclerosis in 12. Lipid Research Clinics Program (1984) J. Am. Med. Assoc. 251,
affected subjects is another important question that can only
be answered by identifying a large group of subjects for 365-374.
further study. The convenience of the parallel immunoassays 13. Brown, M. S. & Goldstein, J. L. (1987) in Harrison's Principles
on plasma using antibodies MB47 and MB3 should allow the
efficient screening of large populations. The identification of of Internal Medicine, eds. Braunwald, E., Isselbacher, K. J.,
an additional proband with familial defective apoB-100 in our Petersdorf, R. G., Wilson, J. D., Martin, J. B. & Fauci, A. S.
test population demonstrates the effectiveness of this assay (McGraw-Hill, New York), 11th Ed., Vol. 2, pp. 1650-1661.
as a screening tool. 14. Grundy, S. M. & Vega, G. L. (1985) J. Lipid Res. 26, 1464-
1475.
We thank Maureen Balestra and Martha Kuehneman for excellent 15. Vega, G. L. & Grundy, S. M. (1986) J. Clin. Invest. 78, 1410-
technical assistance. We also thank Kerry Humphrey and Sylvia 1414.
Richmond for manuscript preparation, James X. Warger for graphic 16. Innerarity, T. L., Weisgraber, K. H., Arnold, K. S., Mahley,
art, and Al Averbach and Sally Gullatt Seehafer for editorial R. W., Krauss, R. M., Vega, G. L. & Grundy, S. M. (1987)
assistance. We are grateful to Drs. Joseph Witztum and Linda Proc. Natl. Acad. Sci. USA 84, 6919-6923.
Curtiss for generously supplying antibody MB47 and to Drs. Ross 17. Elovson, J., Jacobs, J. C., Schumaker, V. N. & Puppione,
Milne and Yves Marcel for providing antibodies 4G3, 3F5, and SE11. D. L. (1985) Biochemistry 24, 1569-1578.
Special thanks are extended to members of the R. and S. families, 18. Young, S. G., Witztum, J. L., Casal, D. C., Curtiss, L. K. &
who graciously participated in these studies. This work was sup- Bernstein, S. (1986) Arteriosclerosis 6, 178-188.
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