Vol. 168, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
May 16, 1990 Pages 1118-1127
IDENTIFICATION OF THE MUTATION RESPONSIBLE FOR A CASE OF PLASMATIC
APOLIPOPROTEIN CII DEFICIENCY (APO CII-BARI)l
Carmine Crecchio*, Antonio Capurso'and Gabriella Pepe *,2
* Centro SMME-CNR and Dipartimento di Biochimica e Biologia Molecolare, ICattedra di Geriatria e Gerontologia, Istituto di Medicina Clinica,
Universitl di Bari, Italy
Received March 16, 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 (Apo CII), the main component of the very low density
lipoprotein (VLDL) and high density lipoprotein (HDL), plays a fundamental
role in human lipid metabolism, as physiological activator of lipoprotein
lipase (LPL) in the triglyceride hydrolysis (1).
The Apo CII deficiency results in hypertriglyceridemia, xantomas and
increased risk of pancreatitis and early atherosclerosis.
Two major types of Apo CII deficient patients have been described, in
which the defect is inherited as an autosomal recessive tract (2). One
possesses a genetic mutant of protein which is present in the plasma at
approximately normal level, but is unable to activate the LPL (Apo CII-
Toronto; Apo CII-St.Michael (3,4). The other has a markedly reduced level
of Apo CII which can only be detected in the plasma by using very sensitive
1Reported at the 5th International Theriological Congress, Roma, 1989.
2To whom correspondence should be addressed at Dipartimento di Bio- chimica e Biologia Molecolare, Universita' di Bari, Via Amendola 165/A, 70126 Bari, Italy.
0006291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 1118
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techniques (Apo CII-Padova; Apo CII-Hamburg (5-7). In particular, in two
cases the circulating Apo CIl was completely undetectable (Apo CII-Nijmegen;
Apo CII-Paris (8-9).
The structural organization and the sequence of the normal Apo CII gene.
have been studied by two research groups (10.11).
The molecular basis of the Apo CII defect has been recently defined in a
few patients (4,7,8,9,12,13). We characterized another case of Apo CII
deficiency.
The study concerns an italian family with two siblings having an
abnormally high level of triglycerides and a total deficiency of plasmatic
Apo CII (14,15). Electrophoresis and immunoblotting methods were not able
to reveal any circulating Apo CII in either proband, considered homozygote
as regard this defect. However, immunofluorescence experiments, carried
out on intestinal mucosa cells of the two probands, clearly showed positive
reactivity (16), indicating that, at least in the tissue examined, Apo CII
protein is synthesized.
This evidence caused us to research the genetic origin of the defect.
In this study we describe the mutation responsible for the Apo CII
deficiency in the young girl P.I. (Apo CII-Bari).
MATERIALS AND METHODS
The DNAs were extracted from peripheral blood cells according to the standard method, with some modificati.on (Guanti., unpublished).
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(Perkin- Elmer 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 of the reaction. After control on gel, the amplified DNA was digested and 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.
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RESULTS
First, we checked for the possible existence of a large rearrangement in
the Apo CII gene of P.I. and we looked for an RFLP associated with the
defect. This was performed by digestions with several restriction enzymes
and Southern blot analysis, using the Apo CII cDNA, as probe. The results
are shown in Fig.1 and Fig.2. In Fig.1 the DNA of P.I., digested with
EcoRI or with BamHI, showed an hybridization pattern in agreement with
A B C
a
4.8 Kb
3.8
3.5
P, 4 El E2 E3 E4
500 bp
n ; t
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= BamRI;O= PstI;O=TaqI. The asterisk indicates the TaqI polymorphic site absent in our proband. as explained in the text.
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A B
1500
1200
bp
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 sequence of the normal human Apo CII gene (10,ll).
DNA digested with TaqI gave the unique 3.8 kb band of hybridization: this
pattern, depending on the loss of one polymorphic TaqI site, is common to
60% of the Caucasian population and cannot be associated with Apo CII
deficiency (18). By the EcoRIxPstI double digestion we separated the four
exons in a larger fragment of 1.5 kb (exon I), another fragment of 1.2 kb
(exon II) and a smaller one of 0.7 kb (exons III and IV), as shown in
Fig-la. The P.I. DNA, digested in this way and probed with the Apo CII cDNA,
again hybridized with a pattern that perfectly coincided with that of normal
DNA (Fig.2).
All these experiments excluded the existence of an RFLP for the tested
enzymes and clearly suggested that the molecular basis of the defect in
P.I. cannot be attributed to a large rearrangement, but most probably to a
point mutation in the coding part of the gene. In order to precisely
identify this mutation, a portion of P.I. genome, containing the four exons,
was amplified "in vitro", cloned and sequenced, following the strategy
shown in Fig. 3. Four pairs of oligonucleotide primers were used to amplify
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Vol. 168, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EcoRl 5,
-215bp <--TCTAGCTATTCGTCCTTAA&T . . . CGGAGGCGAATTCTCAGAGTGAGGGT....... . . . . . . . . ..GCCTCCGCTTAAGAGTCTCACTCCCA....... EXON 1 . . . . .
AAAGATCGTTTA. . . ..TTTCTAGCTATTCGTCCTTAAAAT...
CGGAGGCCAATTCTCAGAGT - - > 51 -
+75bp EC0 RI
Hind,,, 5,
-126bp <--TCCCCTTACTCGAGG~GA . GAAACTTGACT- cc-CA.......
::.CTTTGAACTGACCCTTTGGCTCGTGT....... EXON 11 . . . f . . . GAGGGGAATGRGCTCCAAG~TCT. . , . . . . . ..CTCCCCTTACTCGAGGTTCGTAGA...
GGGAAACCGAG--> +56bp
EarnHI 58
-116bp <--GTGCTGACGTAGGTmTC .CCI&GCATCTTCCCAGCCCAGGCCCT.......
: : . GGTTCGT . . . . . . . ACCACGACTGCATCCAGGACCCAG...
AGAAGGGTCGGGTCCGGGA....... EXON 11' . . . . . ..TGGTGCTGACGTAGGTCCTGGGTC... C$$AGCtT,CTTCCCAGCCCAGGCC--> +64bp 5’ Hind 111
Sam”, 5’
-257bp <--GTWiCGFAAAGACT~TT .CTGCATCCAGGACCCACMGTTCAGG....... ..GCCACTGCTTTTCTGAGGATTCAA...
::.GACGTAGGTCCTGGGTCTTCAAGTCC....... EXON IV :::::. .CGGTGACGAMAGACTCCTAAGTT... C~GgATCCAGGACCCAGAAGTT--> +36bF 5’ Barn iin
Fig.3. Strategy of PCR amplification. The numeration refers to the left (-) and right (+) ends of the four exons. Within the sequences of the four oligoprimers the restriction sites used for cloning are indicated. The lower-case letters represent the mismatch. introduced during the synthesis.
four target sequences, each containing one complete exon. The amplified
products were cloned for sequence analysis. Sequencing of more than one
clone of the same amplified segment excluded errors caused by PCR or cloning
artefacts. We sequenced in total about 1.5 kb which included the four exons
with their flanking sequences. A C->G transversionwas found in the third
position of the codon 59, within exon III(Pig.4). More&over, as shown in
Fig. 5, this point mutation destroys an RsaI site, which is present in the
normal sequence. The analysis of several clones confirmed the homozygote
condition of the proband.
DISCUSSION
The main plasmatic form of Apo CII includes a 79 aa. polypeptide plus a
22 aa. signal peptide of which the sequence is known (19.20). The residues
56-79 are directly involved in the activation of LPL and this function
seems to be enhanced by the binding of phospholipids to the immediately
upstream residues (21).
In the Apo CII-Bari case, the biochemical characterization of the
protein was impossible, because of the absence of the circulating form.
Furthermore, we could not extend our study to tW transcriptional products
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A) B)
E4 E3 E2 El
CIIIO
CII CIIIl
CIII2
Cl
NORMAL
P.I.
CTG
CTG
A c G T
/ A G
G(C) A
\
T
G T C
TAC GAG I
TAG GAG stop
10 20 30 1 40 TQQPQQDFHP SPTFLTQVKE SLSSYWESAK TAAQNLYEKT
50 60 70 79 YLPAVDEKLR DLYSKSTAAM STYTGIFTDQ VLSVLKGEE
Fig.4. Biochemical and genie evidence of the Apo CII deficiency of the patient P.I. A) Isoelectrofocusing of delipidated Apo-VLDL. performed on 7.5% PAGE with 2% ampholines pH 4-6. In the proband the Apo CII fraction Is totally absent. 8) Sequencing gel of P.I. DNA. The part of the sequence containing the mutated base(G instead of C) is written alongside the autoradiography. C) The consequence of the transversion in the patient respect to the normal Apo CII gene is pointed out. Within the amino acid sequence of the mature form, the arrow indicates the point where the translation is stopped.
since suitable bioptic material was no longer available. On the other
hand, the previous immunofluorescence experiments on the intestinal mucosa
cells showed the presence of the protein to a degree not inferior to the
normal values (16). We therefore concluded that, in our proband, the Apo
CII gene is more or less normally expressed.
Here we demonstrate that the Apo CII-Bari case depends on a mutation in
the coding region of the gene with considerably alters the protein. The
mutation identified the polymorphism RsaI with is strictly associated to
the defect. Either RFLP analysis or PCR followed by AS0 hybridization
are suitable methods for the screening of this type of Apo CII deficiency.
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C D E
842 bp
Fi.R.5. Digestions of cloned PCR products. A= pBR322xHinfI. as molecular weight marker. B-C-D= three different clones of the amplified genomic region containing the exon III digested with RsaI. The only site for this enzyme present in the normal fragment here is missing because of the mutation. E- pUC18xRsaI. as control of digestion conditions.
Table I summarizes all the so far identified Apo CII gene mutations and
their consequences on the product. The mutations always affect the protein
translation, and in one case the mRNA splicing as well (Apo CII-Hamburg,
(711, resulting in a frame shift and/or premature stop. The mutated
protein is inefficient in the activation of LPL. Mostly the mutation
occurs in the first half of the gene, changing a large part of the amino
acid sequence: thus, not only the functional domains, but the structural
domains of the protein are damaged. In all cases (except the Apo CII-Paris)
the portion of the gene coding for the signal peptide is normal: the
secretion of the protein should be possible (unless the modification of
the structure impairs the membrane transport). Therefore, we think that
the stability of the molecule in the plasma is strongly reduced, as a
result of its inability to form a normal complex with the phospholipid
fraction. This explains the total failure in the detection of plasmatic
Apo CII in some patients, including ours.
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TABLE I. CASES OF APO CII DEFICIENCY DEFINED AT THE GENIC LEVEL
NAHE ETBNIC ORIGIN
PLASMATIC PROTEIN
MDTATION EXON CODDN TYPE
12 Toronto Btitiab + norIM level IV 90 Ae T deletion
4 S .NfCbAd AIlglOSaXOlI + normA level IV 91-92 CaA n.d.insartfon
7 rLmbur9 Turkish very low level II s.). g-7 c-->c
8 Nijme9an Dutch Not-detoctabla III 40 ETG C deletion
13 Padova Italian very low level III 59 TAG C-->A
9 PAri# Prench Not-deteotable II 1 A'JG A-->C
Bari Italian Not-dstectabl. 111 59 TAG c--x
Table l-Continued
CONSEQUENCE ALTERED SITE PROTEIN LENGTE
12 Toronto Shift-pramatop -- 74 AA
4 S.Michml Shift-extension n.d. 96 a~
7 Hambur9
s
Defect.splicinq Shift-ptem.stop
+ DdeI - EphI
74 AA
Ni jmsgen Shift-pram.stop - BphI 17 aa
13 Padovs Premature stop - RsaI 36 ~a
9 Parim Loa* fnit.aita -- 71 aa
Bari Premature *top - R#AI 36 ~a
S.J. = splice junction.
All the cases are genetically different each other and, in theory, any
point mutation type may affect the gene in any position. However, if we
also consider the two cases recently reported by Li et al. (*) the exon
III seems to be a preferential target for the mutation.
In spite of the heterogeneity characterizing the syndrome, the Apo CII-
Bari case appears to be similar to the Padova case (13). Both springs from
a transversion (C->G and C->A, respectively) which affects the same codon,
so that the consequent premature stop at the aa. 36 is common to the two
cases. The difference in detection of the protein in the plasma could
be explained by a different rate of degradation after secretion, depending
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on the differences in sex, age and general health conditions of the two
patients (of course, technical differences in determining the Apo CII
concentration cannot be excluded).
What, in our opinion, is really interesting is that in these individuals
of the same population two different mutations hit the same nucleotide in
the same codon, we could say, in a sort of hotspot. Bearing in mind the
few cases of Apo CII deficiency studied so far, it is very difficult to
establish the degree of significance of this observation, but it seems
unlike that the event occurs just by chance.Therefore,any new case studied
at themolecular level is useful to gaining more insight into the genetic
origin of the Apo CII deficiency. In particular, the availability of a
larger number of data about patients from a restricted area could aid the
identification of a possible association between ethnic origin and mutation.
It would be interesting to analyse also more gene sequencesfrom "normal"
individuals, to discover whether some mutations can occur in a silent
position of an exon, particularly in the exon III, without being
phenotypical manifestation of the 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.
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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. Li- pid 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., Dema- cker, 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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