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A-IMilano
Apoprotein
DECREASED
HIGH
DENSITY
LIPOPROTEIN
CHOLESTEROL
LEVELS
WITH
SIGNIFICANT
LIPOPROTEIN
MODIFICATIONS
AND
WITHOUT
CLINICAL
ATHEROSCLEROSIS
IN
AN
ITALIAN
FAMILY
GUIDo
FRANCESCHINI
and
CESARE
R.
SIRTORI,
Center
E.
Grossi
Paoletti,
University
of
Milan,
Milan,
Italy
ANTONIO
CAPURSO,
II
Medical
Clinic,
University
of
Bari,
Bari,
Italy
KARL
H.
WEISGRABER
and
ROBERT
W.
MAHLEY,
Gladstone
Foundation
Laboratories
for
Cardiovascular
Disease,
University
of
California,
San
Francisco,
California
94140
A
B
S
T
R
A
C
T
Significant
hypertriglyceridemia
with
a
very
marked
decrease
of
high
density
lipoproteins
(HDL)-cholesterol
levels
(7-14
mg/dl)
was
detected
in
three
members
(father,
son,
and
daughter)
of
an
Italian
family.
The
three
affected
individuals
did
not
show
any
clinical
signs
of
atherosclerosis,
nor
was
the
atherosclerotic
disease
significantly
present
in
the
family.
Lipoprotein
lipase
and
lecithin:cholesterol
acyltransferase
activites
were
normal
or
slightly
re-
duced.
Morphological
and
compositional
studies
of
HDL
in
the
subjects
showed
a
significant
enlargement
of
the
lipoprotein
particles
(-
120
vs.
-94
A
for
control
HDL)
and
a
concomitant
increase
in
the
triglyceride
content.
Analytical
isoelectric
focusing
of
HDL
apo-
proteins
provided
evidence
for
multiple
isoproteins
in
the
apoprotein(apo)-A-I
range,
with
nine
different
bands
being
detected
instead
of
the
usual
four
bands
observed
in
normal
subjects.
Two-dimensional
im-
munoelectrophoresis
against
apo-A
antiserum
indi-
cated
a
clear
reduction
of
apo-A
in
the
alpha
electro-
phoretic region,
with
splitting
of
the
protein
"peak."
The
observation
in
otherwise
clinically
healthy
sub-
jects
of
hypertriglyceridemia,
reduced
HDL-choles-
terol,
and
marked
apoprotein
abnormalities,
without
a
significant
incidence
of
atherosclerotic
disease
in
the
family
suggests
this
is
a
new
disease
entity
in
the
field
of
lipoprotein
pathology,
very
probably
related
to
an
altered
amino
acid
composition
of
the
apo-A-I
protein
(see
Weisgraber
et
al.
1980.
J.
Clin.
Invest.
66:
901-907).
Address
requests
for
reprints
to
R.
W.
Mahley,
M.D.,
Ph.D.,
Gladstone
Foundation
Laboratories,
P.O.
Box
40608,
San
Francisco,
Calif.
94140.
Received
for
publication
22
April
1980
and
in
revised
form
8
July
1980.
892
J.
Clin.
Invest.
C
The
American
Society
for
INTRODUCTION
Plasma
levels
of
high
density
lipoproteins
(HDL)'
and,
in
particular,
of
HDL-cholesterol,
have
received
in-
creasing
attention
in
the
past
few
years
because
of
the
negative
statistical
correlation
between
HDL-choles-
terol
levels
and
the
incidence
of
ischemic
vascular
dis-
eases,
both
of
the
heart
(1,
2)
and
of
the
central
nervous
system
(3).
Inversely,
a
protective
effect
has
been
postulated
for
increased
HDL
levels,
on
the
assump-
tion
that
in
this
condition
an
increased
cholesterol
re-
moval
from
tissues
(4,
5)
and/or
a
decreased
delivery
of
cholesterol
by
the
"low
density
lipoprotein
recep-
tors"
(6)
may
occur.
At
the
two
extremes
of
HDL-cholesterol
distribution,
a
striking
difference
in
the
risk
of
clinical
athero-
sclerosis
is
found;
in
particular,
subjects
with
markedly
elevated
HDL-cholesterol
may
have
a
"longevity
syn-
drome"
(7),
whereas
a
familial
pattern
of
decreased
HDL-cholesterol
may
be
associated
with
a
high
inci-
dence
of
myocardial
infarction,
particularly
when
hypercholesterolemia
is
also
present
(8).
Decreased
HDL-cholesterol
levels
are
frequently
found
in
pa-
tients
with
hypertriglyceridemia
(Fredrickgon
types
I,
IV,
and
V),
where
a
negative
correlation
has
been
sug-
gested
(9)
but
not
generally
confirmed
(10)
between
tri-
glyceride
levels
in
very
low
density
lipoproteins
(VLDL)
and
HDL-cholesterol.
A
decrease
in
the
C-Il
peptide
activation
of
lipoprotein
lipase
is,
in
this
case,
suggested
as
the
mechanism
of
hypertriglyceridemia
(11).
Patients
with
a
complete
or
almost
complete
absence
'
Abbreviations
used
in
this
paper:
HDL,
high
density
lipo-
proteins;
LCAT,
lecithin:cholesterol
acyltransferase;
LDL,
low
density
lipoproteins;
LPL,
lipoprotein
lipase;
TG,
plasma
triglycerides;
VLDL,
very
low
density
lipoproteins.
Clinical
Investigation,
Inc.
*
0021-9738/80/11/0892/09
*1.00
Volume
66
November
1980
892-900
of
HDL
are
diagnosed
as
having
Tangier
disease
(12).
In
this
disease,
the
total
absence
of
HDL
and
HDL-
cholesterol
is
usually
accompanied
by
hypolipidemia,
multiple
lipoprotein
abnormalities
(13,
14),
and
at
times
deficient
activities
of
lipoprotein
lipase
and
lecithin:
cholesterol
acyltransferase
(15).
Characteristic
clinical
symptoms
(enlarged
tonsils,
splenomegaly)
are
sec-
ondary
to
cholesterol
accumulation
in
tissues
(16).
In
Tangier
disease,
a
defective
assembly
of
HDL
is
postu-
lated,
based
on
a
normal
presence
of
apoprotein(apo)-
A-I
(the
major
protein
component
of
HDL)
in
the
mucosal
cells
of
the
intestinal
wall
(17)
and
on
the
de-
tection
of
apo-A-I
in
the
infranatant
proteins
after
ultra-
centrifugation
of
plasma
lipoproteins
(18).
Further-
more,
a
structural
abnormality
of
A-I
has
been
proposed
as
the
defect
in
Tangier
disease
(18).
Contrary
to
expec-
tations,
in
Tangier
disease
clinical
atherosclerosis
is
not
found,
possibly
because
cholesterol
in
the
tissues
removed
by
nascent
HDL
is
not
deposited
in
arterial
smooth
muscle
cells
but
only
in
tissue
macro-
phages
(19).
The
clinical
observation
of
a
family
with
hypertri-
glyceridemia
and
a
very
severe
reduction
of
plasma
HDL,
HDL-cholesterol,
and
apo-A-I
levels,
without
clinical
findings
of
Tangier
disease
or
clinical
athero-
sclerosis,
prompted
more
detailed
clinical
and
bio-
chemical
investigations
of
this
syndrome.
Analysis
of
HDL
apoproteins
provided
evidence
that
this
is
the
first
human
disease
characterized
by
an
abnormal
amino
acid
composition
of
an
apolipoprotein,
as
will
be
described
in
the
accompanying
report
(20).
METHODS
Patients.
The
family,
D.,
consists
of
a
father
and
three
chil-
dren-a
boy
and
two
girls-of
the
respective
ages
in
1980
of
49,
19, 13,
and
12.
The
father,
D.V.,
was
referred
to
the
Lipid
Center
in
Milan
in
1975
because
of
moderate
hypertriglyceri-
demia
(total
plasma
triglyceride
levels
between
300
and
400
mg/dl)
apparently
resistant
to
diet
and
clofibrate
treatment.
The
patient's
private
physician
indicated
that
clofibrate
treatment
was
poorly
tolerated
and
that
it
appeared
to
induce
a
marked
increase
of
plasma
triglyceride
(TG)
levels.
This
finding
could
not
be
confirmed
because
the
patient
refused
to
repeat
this
medication.
At
the
first
clinic
visit
(6
May
1975),
standard
lipid
and
lipoprotein
analysis
indicated
a
moderate
hypertriglyceri-
demia
(244
mg/dl)
with
a
normal
total
plasma
cholesterol
(220
mg/dl).
On
agarose
electrophoresis,
a
wide,
heavily
stained
pre-beta
band
and
an
almost
complete
absence
of
an
alpha
lipoprotein
band
were
observed.
The
HDL-cholesterol
level,
determined
at
this
visit
by
selective
precipitation
of
very
low
and
low
density
lipoproteins
(21),
was
12
mg/dl
(Fig.
1).
Historically,
the
patient
D.V.
complained
only
of
irregular
bowel
habits,
markedly
influenced
by
psychological
condi-
tions.
This
had
been
diagnosed
as
"irritable
colon."
He
had
undergone
several
tonsillectomies
(at
ages
3,
30,
and
32),
apparently
for
repeated
streptococcal
infections.
The
ear-
nose-throat
specialists
had
not
noted
any
unusual
characteris-
tics
of
the
tissue
after
tonsillectomy.
The
patient
reported
numerous
episodes
of
lymphoadenopathies
and
bronchitis.
At
age
36
he
had
undergone
total
gastrectomy
because
of
a
bleeding
duodenal
ulcer.
Subsequently,
bowel
complaints
were
frequent.
The
patient
denied
any
symptoms
indicating
coronary
or
peripheral
artery
disease.
Coronary
disease
was
described
only
in
one
cousin,
and
diabetes
mellitus,
in
a
brother.
The
parents
of
the
subject
are
alive.
The
mother,
age
73,
has
suf-
fered
a
stroke,
but
otherwise
both
parents
are
healthy.
The
father
is
79.
The
physical
examination
of
the
patient
disclosed
a
nor-
mally
developed,
slightly
overweight
(166
cm,
75
kg)
white
male.
No
abdominal
masses
could
be
palpated.
Eye
grounds
and
EKG
were
normal,
as
were
the
standard
biochemical
tests
and
urinalysis.
In
the
years
1975-78,
diet
and
drug
treatment
were
ad-
ministered
to
lower
the
markedly
elevated
TG
levels
of
the
patient.
Polyenyl
phospholipids
(600
mg/d)
(22)
(Lipostabil,
Nattermann,
German
Federal
Republic)
were
given
at
the
suggestion
of
Dr.
H.
Peeters
(Brussels,
Belgium).
Metformin
treatment
(Glucophage,
Spemsa,
Florence,
Italy)
was
also
administered
(23).
Based
on
recent
experimental
and
clinical
evidence
(17,
24)
suggesting
a
role
for
the
intestine
in
apo-
protein
A-I
synthesis,
a
diet
markedly
enriched
in
fat
(-55-
60%
of
calories)
was
also
recently
prescribed.
The
wife
and
children
of
the
subject
were
also
examined
for
lipid
and
lipoprotein
levels
and
for
physical
signs.
The
wife,
of
German
origin,
had
a
normal
lipid
and
lipoprotein
profile.
The
boy,
D.M.,
and
the
younger
girl,
D.A.,
examined
at
ages
15
and
9,
were
also
found
to
have
markedly
decreased
HDL-cholesterol
levels.
In
contrast,
the
other
daughter,
D.E.,
age
10,
had
a
normal
HDL-cholesterol
level
(42
mg/dl)
and
no
other
lipid
abnormalities.
She
was
later
found
to
have
none
of
the
typical
apoprotein
changes
detected
in
the
father
and
in
her
brother
and
sister
(see
below).
Therefore,
she
was
not
studied
further,
except
for
a
detailed
investigation
of
plasma
lipoprotein
composition.
The
two
children
with
decreased
HDL
levels
also
had
slightly
elevated
TG
levels.
In
the
boy,
a
type
IV
electro-
phoretic
profile
was
detected.
Both
children
have
undergone
tonsillectomies;
the
ear-nose-throat
specialist
stated
that
the
removed
tonsils
showed
only
signs
of
infection.
Neither
of
the
two
has
any
significant
physical
signs
and
hepatospleno-
megaly
was
absent.
Biochemical
tests
disclosed
only
an
elevation
of
alkaline
phosphatase
in
the
girl
(403
mU/ml
vs.
normal
values
of
60-170
mU/ml).
This
finding
could
not
be
attributed
to
any
specific
disease
or
syndrome.
No
attempts
were
made
to
lower
TG
in
the
boy,
except
to
suggest
a
mod-
erately
reduced
carbohydrate
diet.
In
the
5
yr
these
two
chil-
dren
have
been
observed
in
our
center,
their
HDL-choles-
terol
levels
have
not
changed
significantly,
and
they
have
shown
a
normal
rate
of
physical
and
intellectual
growth.
Preparation
of
lipoproteins
and
apoproteins.
Ultracentrif-
ugal
isolation
of
lipoproteins
was
carried
out
according
to
the
procedure
described
by
Havel
et
al.
(25).
For
routine
follow-up
of
the
patients,
the
National
Institutes
of
Health
guidelines
(26)
were
applied,
i.e.,
ultracentrifugation
of
plasma
at
d
<
1.006
and
precipitation
of
low
density
lipo-
proteins
(LDL)
in
the
infranate
by
heparin-MnC12
(20).
When
HDL
were
isolated
by
ultracentrifugation,
they
were
sub-
jected
to
only
one
ultracentrifugal
washing,
due
to
the
paucity
of
the
available
material.
Lipoprotein
fractions
were
exhaustively
dialyzed
against
0.15
M
NaCl,
1
mM
EDTA
(pH
7.4).
Samples
were
delipi-
dated
with
ethanol:diethyl
ether
(3:1
vol/vol)
according
to
Brown
et
al.
(27).
Polyacrylamide
gel
electrophoresis.
Polyacrylamide
gel
electrophoresis
of
isolated
apoproteins
was
carried
out
by
two
Clinical
Aspects
of
a
Family
with
a
Variant
of
A-I
Apoprotein
893
600-
500
_
400
mg
/dl
300
200
100
20
-
HDL
CHOL]
lo-
F.
*
I,I
I
I
*
I
I
.
ZI
I
Z
II
I
I
I
1. 1.
I
.
I
."I
I
I
I
*
I
IIH
!
1.
*
H
ep..
MnC
12
:11
1.1.11
I.1
1.
1.
I
1.
.1
.1
r.-,l
111.
I
I
I.
.
.1
1.
I.,
,
I
11-11
.
.11.
n-
n..
IS
LOW
CHO-LOW
CAL
DIETTWh
M
FA
I
IPEPL
METFORIMIN
1~~~~~~~~~~~~~~0
mgi
13 1
550
IDg/d1
1
2/75
6/75
12/75
6/76
12/76
6/77
12/77
6/78
12/78
6/79
DATES
FIGURE
1
Clinical
course
of
patient
DAV.
Plasma
cholesterol
and
TG,
HDL-cholesterol
and
bodcy
weight.
A
relative
stability
of
the
HDL-cholesterol
levels,
in
spite
of
the
drug
and
diet
treatmleints,
is
noted,
particularly
when
the
ultracentrifuge
determinations
(without
asterisks)
are
conisidered.
Polyenyl
phospholipids
(PEPL)
and
metformin
did
not
appear
to
exert
any
significant
effect
on
plasma
lipid
and
lipoproteins.
Notice
the
tendency
of
total
plasma
cholesterol
levels
to
increase
as
body
weight
rises.
techniques,
viz.,
sodium
dodecyl
sulfate
(SDS)
and
urea
gel
electrophoresis.
For
SDS-polyacrylamide
gel
electropho-
resis,
the
procedure
described
by
Weber
and
Osborn
(28)
was
followed
using
10%
acrylamide
gels
containing
0.1%
SDS.
5%
mercaptoethanol
was
added
as
a
reducing
agent
only
in
specified
samples.
Molecular
weights
were
determined
from
recorded
scans
by
comparison
with
added
molecular
weight
markers
and
with
the
location
of
apoproteins
of
known
molecular
weights.
The
procedure
for
the
urea
gel
electro-
phoretic
separatioin
followed
the
methodology
described
by
Davis
(29),
using
7.5
and
10%
acrylamide
gels
containing
8.0
M
urea.
Isoelectric
focusing.
Polyacrylamide
gel
isoelectric
fo-
cusing
was
carried
out
at
different
pH
ranges
after
apoprotein
solubilization
in
100
,l
8.0
M
urea
and
20
,pl
40%/c
sucrose
(wt/vol).
In
specified
samples,
5
,Al
of
mercaptoethanol
was
also
added
to
the
system.
7.5%
polyacrylamide
gels
contaill-
ing
8.0
NI
urea
and
2%
Ampholine
(LKB
Produkter,
Brommlla,
Sweden)
were
used
for
the
pH
ranges
3.5-
10.0
and
4.0-6.0.
The
sample
(50-75
,g
of
proteins)
was
applied
to
the
top
of
each
gel
(0.5
x
9
cmll)
and
overlaid
with
the
upper
electrolyte
(0.02
M
NaOH),
the
upper
chamber
being
filled
with
the
solu-
tion.
The
lower
electrolyte
was
0.01
NI
H3PO4.
The
gels
were
stained
according
to
the
method
of
Malik
anid
Berrie
(30).
A
blank
gel
was
included
for
the
determiinlation
of
the
pH
profiles.
Enizyme
activity
determiniationt.
Lipoproteini
lipase
(LPL)
activitv
was
determinied
in
the
three
subjects
by
intravenous
894
Franceschini,
Sirtori,
Capurso,
Weisgraber,
and
Mahley
THERAPIES
body
80-
wt
kg
70-
12/79
injection
of
100
IU/kg
sodium-heparin
(Liquemin,
Roche
Diagnostics
Div.,
Hoffman-La
Roche
Inc.,
Nutley,
N.
J.).
Plasma
samples
were
drawn
after
5
and
45
min,
and
the
lipolytic
activity
determined
on
Intralipid
(Vitrum
AB,
Stock-
holm,
Sweden)
substrate,
as
suggested
by
Boberg
(31).
This
technique
was
recently
shown
to
assess
the
LPL
activity
ac-
curately
without
being
affected
by
the
hepatic
lipase
activity
(32).
Lecithin:cholesterol
acyltransferase
(LCAT)
was
tested
in
the
three
subjects
by
the
nonisotopic
method
described
by
Patsch
et
al.
(33).
A
decrease
in
plasma
free
cholesterol
concentration
was
measured
by
enzyme
methodology
(Boehringer
Biochima,
German
Federal
Republic),
before
and
after
incubation
at
37°C.
Immunological
methods.
Immunological
analyses
of
plasma
apo-B
and
apo-A-I
were
carried
out
with
monospecific
antisera
raised
in
rabbits.
The
apo-B
content
of
our
samples
was
measured
by
radial
immunodiffusion,
using
commercial
immunoplates
(M-Partigen,
beta
lipoprotein,
Behringwerke
AG,
Marburg-Lahn,
German
Federal
Republic).
As
an
apo-B
reference
standard,
two
preparations
were
used:
a
standard
serum,
purchased
from
Behringwerke,
and
an
LDL
prepara-
tion
(fraction
d
=
1.030-1.050),
previously
tested
against
anti-
apo-B,
apo-A-I,
apo-CII,
apo-CIII,
albumin,
and
total
immuno-
globulins,
to
demonstrate
the
presence
of
protein
con-
taminants
other
than
apo-B.
The
apo-A-I
content
of
native
serum
lipoprotein
fractions
and
of
the
d
>
1.210
bottom
fraction
were
measured
by
elec-
troimmunodiffusion
(34).
Two
preparations
were
used
as
standards,
including
a
freshly
prepared
pool
of
100
normo-
lipemic
subjects
and
a
preparation
of
pure
apo-A-I
obtained
by
column
chromatography
(18).
A
two-dimensional
immunoelectrophoretic
analysis
was
also
carried
out
on
whole
plasma,
which
was
reacted
against
anti-apo-A
(A-I