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

BIOPHYSICAL

RESEARCH

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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.

1127

Identification of the mutation responsible for a case of plasmatic apolipoprotein CII deficiency (Apo CII-Bari).

We studied a case of familial Apolipoprotein CII deficiency. By Southern hybridization, amplification and sequence analysis, the genetic defect was id...
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