BIOCHEMICAL
Vol. 168, No. 3, 1990 May 16, 1990
IDENTIFICATION
OF THE MUTATION RESPONSIBLE
APOLIPOPROTEIN Carmine
*
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1118-1127
CII
Crecchio*,
DEFICIENCY
Antonio
FOR A CASE OF PLASMATIC
(APO CII-BARI)l
Capurso'and
Centro SMME-CNR and Dipartimento di Biochimica ICattedra di Geriatria e Gerontologia, Istituto Universitl di Bari, Italy
Received
March
16,
Pepe *,2
Gabriella
e Biologia Molecolare, di Medicina Clinica,
1990
We studied a case of familial Apolipoprotein CII deficiency. By Southern hybridization, amplification and sequence analysis, the genetic defect was identified. It consists in a point mutation C->G in the third exon of the gene causing a premature stop codon. Truncated at the aa. 36 of the mature form, the protein loses its functional domains, becomes inefficient and cannot be detected in the plasma, because of its high instability. The mutation destroys an RsaI site, present in the normal gene sequence. This point mutation is useful in the diagnosis of this Apolipoprotein CII deficiency. 01990 Academic Press, Inc.
Apolipoprotein
CII
lipoprotein role
(VLDL)
and high
in human lipid
lipase
(LPL)
increased Two major which
the
types
a genetic
approximately Toronto; of Apo CII
results
normal
level,
Apo CII-St.Michael which
can only
deficient
0006291x/90 Copyright All rights
(HDL),
of protein but
is
low
density
a fundamental of lipoprotein
(1). xantomas
and
atherosclerosis. patients
have been recessive
which unable
is
The other
be detected
in
$1.50 1118
described, tract
in the
the plasma
in
(2).
the
One
plasma
at
LPL (Apo CII-
has a markedly
Theriological be addressed Universita'
present
to activate
(3,4).
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
plays
activator
as an autosomal
1Reported at the 5th International Roma, 1989. 2To whom correspondence should chimica e Biologia Molecolare, 70126 Bari, Italy.
of the very
in hypertriglyceridemia, and early
inherited mutant
lipoprotein
hydrolysis
of Apo CII is
component
as physiological
of pancreatitis
defect
possesses
density
triglyceride
deficiency
risk
the main
metabolism,
in the
The Apo CII
(Apo CII),
by using
reduced very
level sensitive
Congress,
at Dipartimento di Biodi Bari, Via Amendola 165/A,
Vol.
BIOCHEMICAL
168, No. 3, 1990
techniques cases
(Apo
the
CII-Padova;
circulating
Apo CII-Paris
been
Apo CII-Hamburg
Apo CIl
(5-7).
was completely
RESEARCH COMMUNICATIONS In particular,
undetectable
in two
(Apo
CII-Nijmegen;
(8-9).
The structural have
AND BIOPHYSICAL
organization
studied
The molecular few patients
and the sequence
by two research basis
groups
of the Apo CII
(4,7,8,9,12,13).
of the
normal
Apo CII
gene.
recently
defined
case
of Apo CII
(10.11).
defect
has been
We characterized
another
in a
deficiency. The study abnormally Apo CII
concerns high
an italian
level
(14,15).
any circulating
as regard
this
defect.
on intestinal
reactivity protein
is
and a total
in
either
of the that,
proband,
an of plasmatic
were
considered
in the
clearly
not
able
homozygote
experiments,
two probands,
at least
deficiency methods
immunofluorescence
cells
having
carried showed
tissue
examined,
genetic
origin
of
responsible
for
positive Apo CII
synthesized.
This
evidence
caused
In this
study
we describe
in
the young
deficiency
However,
indicating
two siblings
and immunoblotting
Apo CII
mucosa
(16),
with
of triglycerides
Electrophoresis
to reveal
out
family
us to research
the
the mutation girl
P.I.
(Apo
MATERIALS
the
the defect. Apo CII
CII-Bari).
AND METHODS
The DNAs were extracted from peripheral standard method, with some modificati.on
blood (Guanti.,
cells according unpublished).
to the
The DNAs were digested according the suppliers' instructions,transferred to nitrocellulose membrane and hybridized with a full length cDNA ofnormal human Apo CII (kindly supplied by Dr. Sidoli).Sequences which separately contained each of four Apo CII exons and the flanking parts of the introns were amplified using, as primers, 20-22 base long oligonucleotides (Applied Biosystem). The primers were synthesized with internal restriction site, in order to digest and clone the amplified products. PCR procedure was carried out by using the Gene Amp Kit in the DNA-thermal cycler(PerkinElmer Cetus), with some modifications to the manifacturers' instructions. Samples were subjected to 25-30 cycles of polymerization, each consisting of denaturation 1 min at 94"C, annealing 1 min 30 set at 55-60°C (depending on the primer composition), extension 2 min 30 set at 72'C. In the last cycle, the extension was carried out for 10 min to ensure the completeness the amplified DNA was digested and of the reaction. After control on gel, cloned in pUC18 vector. Several positive clones of each amplification product were sequenced on both strands with the dideoxy method, according to Sanger (17) using universal direct and reverse primers. The strategies of amplification and sequencing are shown in Fig.3.
1119
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS First,
we checked
the Apo CII defect.
gene of P.I.
This
shown
blot
EcoRI or with
using
and Fig.2.
BamHI,
for
by digestions
analysis,
in Fig.1
existence
and we looked
was performed
and Southern are
the possible
for
showed
of a large
an RFLP associated with
the
several
Apo CII
In Fig.1 an hybridization
pattern
B
with
in the
restriction
cDNA, as probe.
the DNA of P.I.,
A
rearrangement
enzymes The results
digested
with
in agreement
with
C
a
4.8 Kb
3.8 3.5
P,
n
4 El 500
E2
E3
;
t
E4
bp
Fig.l. a) Hybridizations of P.I. DNA with Apo CII cDNA probe. A- digestion with BanHI; B= digestion with TaqI; C= digestion with EcoRI. 10,ug DNA /lane were run on 0.72 agarose gel in 4OeM Tris-2OmM Naac-Z&f EDTA, pH 7.6, at 7OMa. 4 hrs. The filters were prehybridised in 10% Destran Sulphate-4xSSC-0.1% SDS-0.2% NaPPi-SX Denhart's solution-lOOpg/ml calf tymus DNA, at 6S°C, for 6 hrs. Then the hybridization was carried out in a fresh solution of the same composition , at the same temperature for at least 16 hrs, in presence of the nick-translated probe (s.a.=2xlO%pm/ pg). The filters were washed to O.SxSSC and autoradiographed with an intensifying screen. These experiments performed with the DNA of the parents and a normal subject gave the aame results. b) Organization and restriction map of normal Apo CII gene.
q = EcoRI;A= polymorphic
site
BamRI;O= absent
PstI;O=TaqI. in our proband.
The asterisk as explained
1120
indicates in the
the
text.
TaqI
Vol.
168,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
A
RESEARCH
COMMUNICATIONS
B
bp
1500 1200
670
Fig.2. Blot hybridizations of EcoRIxPstI digested DNAs. A= normal DNA; B= P.I. DNA. The DNAs were double digested as described in the text and hybridized at the same conditions of the experiments of Fig.1. The different intensity of the radioactive bands is in agreement with the very different content in exonic sequences.
the
physical
map and the
DNA digested
with
TaqI
sequence gave the
pattern,
depending
on the loss
60% of
the Caucasian
population
deficiency
(18).
exons
in
(exon
II)
Fig-la.
By the
a larger
again
unique
and a smaller
with
double
in
digestion
a pattern
I),
kb (exons this
that
TaqI
site,
be associated
of 1.5 kb (exon one of 0.7
gene
(10,ll).
kb band of hybridization:
3.8
and cannot
DNA, digested
hybridized
human Apo CII
of one polymorphic
EcoRIxPstI
fragment
The P.I.
of the normal
is
with
common to Apo CII
we separated
another III
this
the four
fragment
and IV),
way and probed
of 1.2 kb
as shown
with
the
in
Apo CII
perfectly
coincided
with
that
existence
of an RFLP for
the
cDNA,
of normal
DNA (Fig.2). All
these
enzymes P.I.
and clearly
cannot
point identify
experiments
mutation this
excluded suggested
be attributed in
was amplified
"in
shown
3. Four
in Fig.
that
the molecular
to a large
the coding
mutation,
the
part
a portion
vitro",
cloned pairs
of
basis
rearrangement, the
of P.I.
gene.
1121
In order
genome,
and sequenced,
of oligonucleotide
but
of the
defect
most
probably
in to a
to precisely
containing following
primers
tested
the the
were
four
exons,
strategy
used
to amplify
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
EcoRl
-215bp . . . CGGAGGCGAATTCTCAGAGTGAGGGT....... . ..GCCTCCGCTTAAGAGTCTCACTCCCA....... CGGAGGCCAATTCTCAGAGT --> 51
5,
C
Not-dstectabl.
111
TAG
c--x
low
Table
level
59
l-Continued
CONSEQUENCE
ALTERED
12 Toronto
c-->c
PROTEIN LENGTE
SITE
Shift-pramatop
--
74 AA
Shift-extension
n.d.
96 a~
Defect.splicinq Shift-ptem.stop
+ DdeI - EphI
74 AA
Shift-pram.stop
- BphI
17 aa
- RsaI
36 ~a
--
71 aa
- R#AI
36 ~a
4
S.Michml 7 Hambur9 s Ni jmsgen 13
Premature
Padovs
9
Parim
Loa*
Bari
the
point
III
= splice
cases
mutation
also
consider
are
genetically
type
may affect
the
Bari
of
case
two cases
different the
a transversion so that cases.
the
consequent
The difference
be explained
by Li
premature in detection rate
the
Padova
respectively) stop
aa.
protein
of degradation 1125
theory,
However,
et al.
(*)
syndrome,
case which
at the
of the
in
any if
the
we exon
the mutation.
characterizing
and C->A,
by a different
for
to the
and,
in any position.
reported
target
to be similar (C->G
each other
gene
recently
the heterogeneity
appears
*top
junction.
seems to be a preferential In spite
fnit.aita
Premature
S.J.
All
stop
(13).
Both
affects 36 is in
after
the Apo CII-
the
springs
same codon,
common to the the
plasma
secretion,
from
two
could depending
Vol.
BIOCHEMICAL
168, No. 3, 1990
on the differences patients
(of
course,
concentration What, of the the
in sex,
cannot
in
our
age and general
technical
is
same population
differences
really
say,
of Apo CII
deficiency
establish
the
of significance
that
the
at themolecular origin
of
larger
number
identification It
would
individuals, position phenotypical
event
occurs
level
is
the Apo CII
studied
just
useful
about
of the
two
the Apo CII
be interesting
so far,
the
these
it
is
from
whether
also
in the
the exon
but
to
it
into
area
ethnic
seems
the
genetic of a
could
origin
sequencesfrom
can occur III,
the
difficult
availability
a restricted
some mutations
in
new case studied
the
more gene
in mind
very
observation,
between
individuals
same nucleotide
more insight
association
of
in
Bearing
In particular,
particularly
manifestation
hit
of hotspot.
to gaining
to analyse
to discover
that
by chance.Therefore,any
patients
of a possible
is
of this
deficiency.
of data
of an exon,
conditions
determining
mutations
in a sort
few cases
unlike
in
interesting
two different
we could
degree
health
be excluded).
opinion,
same codon,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
without
aid
the
and mutation. "normal"
in a silent being
syndrome.
ACKNOWLEDGMENTS We thank Prof. Saccone for her suggestions and advice. Grateful thanks are due to Prof. Guanti whoseexperience in the field has been a source of unfailing help and support. We are indebited to Dr. La Rosa for the clinical assessment of the family and its active collaboration. Thanks are due to the student M.A. Di Stefano for her help in some of the reported experiments and to Mr. 3. Blackwood for the revision of the English text. Work financially supported by the grant REGIONE PlJGLIA:RICERCA SANITARIA FINALIZZATA and partially by a grant of Italian Ministry of Education.
REFERENCES 1) La Rosa, JC., Levy, RI., Herbert, R., Lux, SE., Fredrickson, DS.(1970) Biochem. Biophys. Res. Commun. 41, 57-61. 2) Cox, DW., Breckenridge, WC., Little, JA.(19?8) N. Engl. J. Med. 299, 1421-2424. 3) Connelly, PW., Maguire, GF., Hofmann, T., Little, JA. (1978) Proc.Natl. Acad. Sci. USA 84, 270-273. 4) Connelly, PW., Maguire, GF., Little, JA. (1989) in Human Apolipoprotein Mutant 2. (Sirtori, CR., Franceschini, G., Brewer, HB. Jr, Hassmann, G., eds) Vol. 167, pp. 121-126, Plenum Press, New York and London. 5) Baggio, G., Manzato, E., Gabelli, C., Fellin, R., Martini, S., Baldo, Enzi G., Verlato, F., Baiocchi, MR., Sprecher, DL., Kashyap, ML., Brewer, HB.Jr. and Crepaldi, G..(1986) J. Clin. Invest. 77, 520-527.
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AND
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6) Fojo, SS., Baggio, G., Gabelli, C., Higuchi, K., Bojanovski, M., Gregg, RE., Brewer, HB. Jr (1988) Biochem. Biophys. Res. Corn. 154. 73-79. 7) Fojo, SS., Beisiegel, U., Beil, U., Higuchi, K., Bojanovski, M., Gregg, RE., Greten, H. and Brewer, BB.Jr. (1988) J. Clin. Invest. 82, 1489-1494. 8) Fojo, SS., Stalenhoef, AFH., Marr, K., Gregg, RE, Ross, RS., Brewer, HB. Jr (1988) J. Biol. Chem. 263, 17913-17916. 9) Fojo, SS., de Gennes, J.L., Chapman, J., Parrot, C., Lohse, P.,Kwan,SS., Truffert, J. and Brewer, H.B.Jr.(1989) J. Biol. Chem.264, 20839-20842. 10) Wei, CF., Tsao, YK., Robberson, DL., Gotto, AM., Brown, K., Chan,L. (1985) J. Biol. Chem. 260, 15211-15221. 11) Fojo, SS., Law., Brewer, HB. Jr (1987) FEBS Lett. 231, 221-226. 12) Cox, DW., Wills, DE., Quan, F., Ray, P. (1988) J. Med. Gen. 25,649-652. 13) Fojo, SS., Lohse, P., Parrott, C., Baggio, G., Gabelli, C., Thomas, F., Hoffmann, J. and Brewer, HB. Jr. (1989) J. Clin. Invest. 84, 1215-1219. 14) Capurso, A., Pace, L., Bonomo, L., Catapano, AL., Schilirh, C., La Ro sa, M., Assmann, G. (1980) Lancet 1, 268. 15) Catapano, AL., Mills, GL., Roma, P., La Rosa, M., Capurso, A. (1983) Clin. Chim. Acta 130, 317-327. 16) Capurso, A., Mogavero, AM., Resta. F., Di Tommaso, M., Taverniti,P., Turturro, F., La Rosa, M., Marcovina, S. and Catapano, AL. (1988) J. Lipid Res. 29, 703-711. 17) Sanger, F., Nicklen, S., Coulson, AR. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 18) Humphries, SE., Williams, L., Myklebost, O., Stalenhoef. AFH., Demacker, PNM., Baggio, G., Crepaldi, G., Galton, DJ. and Williamson, R. (1984) Hum. Genet. 67, 151-155. 19) Jackson, RL., baker, HN., Gilliam, EB.. Gotto, AM (1977) Proc.Natl. Acad. Sci. USA 74, 1942-1945. 20) Hospattankar, AV., Fairwell, T., Ronan, R., Brewer, HB. Jr. (1984) J. Biol. Chem. 259, 318-322. 21) Kinnunen, PKJ., Jackson, XL., Smith, LC., Gotto, AM., Sparrow, JT. (1977) Proc. Natl. Acad. Sci. USA 74, 4848-4851.
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