Proc. Nail. Acad. Sci. USA
Vol. 84, pp. 8548-8552, December 1987
Genetics

Plasminogen activator inhibitor type 1 gene is located at region
q21.3-q22 of chromosome 7 and genetically linked with
cystic fibrosis

(protease inhibitor/serpin/gene mapping)

K. W. KLINGER*t, R. WINQVISTt§, A. Riccio¶, P. A. ANDREASENII, R. SARTORIO, L. S. NIELSEN11,
N. STUART*, P. STANISLOVITIS*, P. WATKINS*, R. DOUGLAS*, K.-H. GRZESCHIK**, K. ALITALO§,
F. BLASI~tt, AND K. DANO**
*Integrated Genetics, Inc., 31 New York Avenue, Framingham, MA 01701; tDepartment of Clinical Genetics, Oulu University Central Hospital, Kajaanintie
50, SF-90220, Oulu, Finland; §Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland; International Institute of
Genetics and Biophysics, Consiglio Nazionale delle Richerche, 10 Via Marconi, 80123 Naples, Italy; IlFinsen Laboratory, Rigshospitalet, 49
Strandboulevarden, 2100 Copenhagen O., Denmark; **Institut fur Humangenetik der Universitat, Vesaliusweg 12-14, D-4400 Munster, Federal
Republic of Germany; and ttMikrobiologisk Institut, University of Copenhagen, 2A Oster Farimagsgade, 1353 Copenhagen K., Denmark

Communicated by Lester 0. Krampitz, August 13, 1987 (received for review February 19, 1987)

ABSTRACT The regional chromosomal location of the
human gene for plasminogen activator inhibitor type 1 (PAII)
was determined by three independent methods of gene map-
ping. PAII was localized first to 7cen-q32 and then to
7q21.3-q22 by Southern blot hybridization analysis of a panel
of human and mouse somatic cell hybrids with a PAIl cDNA
probe and in situ hybridization, respectively. We identified a
frequent HindIII restriction fragment length polymorphism
(RFLP) of the PAIl gene with an information content of 0.369.
In family studies using this polymorphism, genetic linkage was
found between PAII and the loci for erythropoietin (EPO),
paraoxonase (PON), the met protooncogene (MET), and cystic
fibrosis (CF), all previously assigned to the middle part of the
long arm of chromosome 7. The linkage with EPO was closest
with an estimated genetic distance of 3 centimorgans, whereas
that to CF was 20 centimorgans. A three-point genetic linkage
analysis and data from previous studies showed that the most
likely order of these loci is EPO, PAIl, PON, (MET, CF), with
PAII being located centromeric to CF. The PAII RFLP may
prove to be valuable in ordering genetic markers in the
CF-linkage group and may also be valuable in genetic analysis
of plasminogen activation-related diseases, such as certain
thromboembolic disorders and cancer.

Proteolysis caused by plasminogen activation is involved in
many diverse biological processes (1-5). The two types of
mammalian plasminogen activators, urokinase-type (u-PA)
and tissue-type (t-PA), are both serine proteases with similar
catalytic specificities but are products of different genes,
located on chromosomes 10 and 8, respectively (6-10).
Plasminogen activation is regulated at several levels-e.g.,

by hormonal regulation of the synthesis of the activators and
by modulators of their enzyme activity. One such modulator
is plasminogen activator inhibitor type 1 (PA1l), an -50-kDa
glycoprotein produced by endothelial cells and several
neoplastic cell lines and found in thrombocytes and blood
plasma (11-16). PAIl inhibits u-PA and t-PA and belongs to
the group of serpins, being an argserpin with an arginine
residue at the reactive center (17-20).
We have previously described the derivation of monoclo-

nal antibodies to PAIl and their use for purification of the
inhibitor (21). These antibodies and amino acid sequence data
have allowed the identification of PAIl cDNA clones from
two different sources (17, 18). The PAII gene was assigned

to human chromosome 7 by hybridization analysis of chro-
mosomes sorted onto filters (18).

In the present study the results of cell hybrid and in situ
hybridization analysis placed PAII in the area 7q21-q22,
close to the chromosomal location predicted for cystic
fibrosis (CF) on the basis of genetic linkage between CF and
a number of polymorphic loci (22-28). CF is a common
autosomal recessive disease with a carrier frequency of 1:20
in Caucasian populations. The basic biochemical defect in CF
is as yet unknown and further genetic linkage studies are
needed to identify the responsible genetic locus. We there-
fore identified a restriction fragment length polymorphism
(RFLP) for PAIl and analyzed genetic linkage between the
PAIl gene and CF as well as other genes located in this
region.

MATERIALS AND METHODS
DNA Probes. The construction and characterization of the

PAIR cDNA clone have been described elsewhere (17). A
2.0-kilobase (kb) BamHI insert of pPAI1-A1 plasmid con-
taining the entire coding sequence for PAIl was used for
polymorphism screening and linkage studies. 32P-labeled
probes were synthesized by using the procedure of Feinberg
and Vogelstein (29, 30).
Other DNA polymorphisms used for linkage analysis have

been described already: metD and metH (24, 25), Epo (28).
The markers at the MET locus were analyzed as a haplotype,
assuming no recombination between the Taq I sites and
linkage equilibrium (24).

Somatic Cell Hybrids. The somatic cell hybrids used in this
study were produced by the fusion of a mouse cell line (RAG
or A9) with human cells, including cells containing translo-
cation derivatives of chromosome 7. Determination of the
chromosomal complement of the hybrids was performed by
analysis of G-banded chromosomes combined with testing of
known biochemical markers on chromosome 7 (31), essen-
tially as reported (32). Human GM 3098 and A431 and mouse
RAG cell lines were used as controls.
DNA Analysis. DNA from human, mouse, and mouse-hu-

man cells was prepared using standard techniques. Gene
mapping using DNA panels of hybrid cell lines and RFLP
analyses were carried out by using a modified form of

Abbreviations: u-PA, urokinase-type plasminogen activator; t-PA,
tissue-type plasminogen activator; PAI1, plasminogen activator
inhibitor type 1; RFLP, restriction fragment length polymorphism;
CF, cystic fibrosis; PON, paraoxonase; lod, logarithm ofodds; EPO,
erythropoietin; cM, centimorgans.
tTo whom reprint requests should be addressed.

8548

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 84 (1987) 8549

Southern blotting (33) and nylon membranes [Genetrans
(Plasco) or Hybond-N (Amersham)]. The membranes were
hybridized to 32P-labeled pPAI1-A1 DNA. After hybridiza-
tion the filters were washed in successive changes of de-
creasing concentrations of NaCl/sodium citrate to a final
wash in 15 mM NaCI/1.5 mM sodium citrate/0.1%
NaDodSO4 at 650C. Autoradiography was at -700C.
Paraoxonase (PON) Analysis. Six families were tested for

the serum arylesterase PON, as described (34).
Chromosomal in Situ Hybridization. Hybridization in situ

was performed on metaphase chromosomes essentially as
described by Harper and Saunders (35).

Pedigrees and Families. Probands in inbred populations
were identified, and the pedigrees were constructed as
described (36, 37). Two large extended kindreds with CF
were studied, one Amish Mennonite family containing 21
affected individuals and a Hutterite family containing 9
affected individuals. Seventeen two-generation nuclear fam-
ilies each with two (11 families) or three (6 families) affected
children were also used in the linkage analysis. The estab-
lishment in these families of linkage between the loci and
markers COLIA2, EPO (erythropoietin), PON, D7S8, MET,
and CF has been reported (28, 37, 38).
Linkage Analysis. Two-point analyses and multipoint anal-

yses were performed using the MLINK and LINKMAP
programs, respectively, of the computer package LINKAGE
(39). Logarithm of odds (lod) scores z were calculated by
assuming no sex difference in recombination. Maximal like-
lihood estimates of the lod score (i) and recombination
fraction (0) were calculated by using the computer program
VACO3 (40). The confidence interval for the maximal like-
lihood estimate ofthe recombination fraction was determined
by graphic interpolation (41). The large Amish Mennonite
pedigree was divided into three sections to include one
inbreeding loop in each section, thus simplifying the analysis
and preserving to a large extent the complex interrelation-
ships and common ancestry of the members of this pedigree.

RESULTS
Chromosomal Localization of the PAII Gene. The chromo-

somal localization of the human gene for PAII was identified

C17,

l8kbp- - +- Ho

., ia~~co., Cb.

FIG. 2. Sublocalization of the human PAIl gene on chromosome
7 by using mouse-human somatic cell hybrids. Human (GM 3098,
A431), mouse (RAG, A9), and hybrid (as indicated in Fig. 1) cell
DNA was cleaved with HindIlI restriction endonuclease, resolved
by electrophoresis, and blotted onto membranes hybridized with
32P-labeled plasmid containing a 2.0-kbp insert of human PAtI
cDNA, and specific hybridization was detected by autoradiography.
From the autoradiogram it can be seern that the probe detects
fragments of 22 kbp and/or 18 kbp in the human GM 3098 and A431
cells and in the hybrids 5387-3-cilO, RAG Ru 6-19, and A9 IT 2-21-14.
A faint hybridization signal of 18 kbp was also detected for hybrid
RAG GM 194 6-13 on a longer exposure. This reflects that PAIl was
present in -.30% of the cells in this hybrid line. As shown in Fig. 1,
the only human chromosomal region these cells have in common is
that located between the centromere and band 32 of the long arm of
chromosome 7 (7cen-q32).

by DNA hybridization in mouse-human hybrid cell lines
containing different human chromosomes. Hybridization
with the PAIl cDNA probe to Southern blots of HindIII-
digested DNA revealed two DNA fragments of 22 kilobase
pairs (kbp) and 18 kbp (see below and Fig. 2). The presence
of one or both of these fragments segregated in a variety of
cell lines specifically with the human chromosome 7, whereas
discordance was found with all other human chromosomes
(data not shown). To study the sublocalization of the PAIl
gene, 7 cell lines that contained specific regions of human
chromosome 7 (Fig. 1) were investigated. As it appears from
Fig. 2, a positive signal was only observed in those cell lines

A9 X-28-13

(6 21)

IT A92-21-14
(5 6 12 15 16

17 18 21 X)

GM 194 RAG 6-13

(3 4 12 13 14 15 X)

MH RAG 8-7

(1 3 4 8 12 13 14 15 18 X)

Ru RAG 4-13
(13 5 6 10 11 14

19 20 21 22 X Y)

Ru RAG 6-19
(x)

5387-3-CIIO

(none)

7

FIG. 1. Portion of human chromosome 7 retained in mouse-human hybrid cell lines used for sublocalization of the PAIl gene by Southern
blotting analysis. In parentheses are indicated other human chromosomes (or derivatives) present in the respective cell lines. As illustrated in
Fig. 2, PAII is found in cell lines IT A9 2-21-14, GM 194 RAG 6-13, Ru RAG 6-19, and 5387-3-cl10.

22

21

1531
1 4

13

12

1 12
1

1 11
1 121
1 122

1 123

2l 11

212
213

22

311
312

313

32

33
34
35

36

IT

i

Genetics: Klinger et al.

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Proc. Natl. Acad. Sci. USA 84 (1987)

that had retained the region of the long arm of chromosome
7 located between the centromere and band 32 (7cen-q32).
As an alternative approach, the localization of the PAIl

gene was studied by in situ hybridization on metaphase
preparations ot human lymphocytes with 3H-labeled PAIl
cDNA. As seen from Fig. 3 Left, chromosome 7 was the only
chromosome that showed a grain number (36%) above
background. The majority of the grains on chromosome 7
were associated with bands q2l.3 and q22 of the long arm
(29% of all grains observed, Fig. 3 Right), verifying and
making more precise the localization obtained by the cell
hybrid analysis.
RFLP. DNA from four unrelated individuals was digested

with a variety of different restriction endonucleases (Apa I,
Bgl II, Msp I, EcoRV, Pvu II, Xmn I, Sac I, BstEII, Bcl I, Taq
I, EcoRI, BamHI, Pst I, HindIII, and Nci I) and analyzed by
Southern blotting with the PAIl cDNA probe. Digestion with
HindIII revealed allelic fragments of 22 kbp (Al) and 18 kbp
(A2). A representative autoradiograph of the segregation of
the RFLP in a family is shown in Fig. 4. The segregation
pattern obtained in all of the nuclear families, and both
pedigrees, was consistent with Mendelian inheritance. The
frequencies for Al and A2 were 0.58 + 0.05 and 0.42 ± 0.05,
respectively, in a sample ofDNA preparations accounting for
86 unrelated chromosomes representative of a general North
American population. Because of the nearly optimal ratios of
the major and minor allele, the polymorphism has a high
polymorphism information content (0.369). An additional but
much less frequent RFLP was found after Xmn I digestion.
No polymorphisms were detected for the remaining restric-
tion enzymes that were tested.

Linkage Analysis. The HindIII polymorphism detected by
the PAIl probe was used to test for linkage between PAII and
the CF locus and other markers previously mapped to this
region of the long arm of chromosome 7. The segregation
pattern of the HindIll RFLP was analyzed by Southern blot
analysis using DNA prepared from each individual of the
families described in Materials and Methods. Lod score

22

20[
1 0

CO
z

C:

L1L
0
cc

z:
z

50

40

30

20

10

21

15 3
15 2
1s

IL

13

12

11 2

11 21
1 1 22

1 1 23

2i 1

21 2
21 3

22

31 2

31 3

p q jpI q IIIqpjq Ip q

NsM *I A N

r
q P q TVL q lp qIpjq pqlq

6 7 8 9 10 1 112

113114 15 1617 181 21 X MY

CHROMOSOMES

UO

0

32 * -
33

.1

35
36

7

i2 2

2 2

Ai *
A2 V1. VW

ePAi HindIll

FIG. 4. Inheritance of HindIII RFLP. DNA samples were di-
gested with HindIII and analyzed by Southern blotting with a 2.0-kbp
fragment of the PAIU cDNA probe. The family pedigree is shown
above. The RFLP assignment of each individual is indicated below
the pedigree. ePAi, pPAI1-A1 probe.

analysis of the cosegregation pattern of the CF disease
phenotype and the RFLP, or the RFLP and additional
markers assigned to chromosome 7q, was performed by using
the LINKAGE program (see Materials and Methods).
The results of the two-point linkage analysis are shown in

Table 1. Evidence was obtained for loose linkage between
PAIl and the CF locus. The maximal likelihood estimate of
the recombination distance (6) between PAIR and CF was
0.20 [95% confidence interval, 0.10-0.36; with a maximal lod
score (z) of 1.77 (odds ratio, 59:1)]. This suggested that the
PAIl locus was located within the established CF linkage
group on the long arm of chromosome 7, albeit somewhat
distant from the CF locus. We therefore examined the linkage
relationship between PAII and other markers within this
group. These included the loci for the c-met protooncogene
(MET) (25), the erythropoietin gene (EPO) (28), and the gene
for the serum protein paraoxonase (PON) (34). The results of
these analyses (Table 1) demonstrated that PAII is most

9

9@0

*0

0 0 0 090 0*0

FIG. 3. Localization of human
PAII gene by in situ hybridization
analysis. (Left) Histogram show-
ing the grain distribution in 60
metaphases. Abscissa: chromo-
somes in their relative size propor-
tions. Ordinate: number of silver
grains. Overall, 203 grains were
observed, of which 73 (36%) were
on chromosome 7. Background
grains were found distributed
evenly on all chromosomes.
(Right) Sublocalization of PAIl to
7q21.3-q22 (58 grains = 29% of all
grains observed).

8550 Genetics: Klinger et al.

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Proc. Nati. Acad. Sci. USA 84 (1987) 8551

Table 1. Linkage analysis of gene loci for PAIR vs. CF, EPO, the c-met protooncogene (MET), and paraoxonase (PON)

Number of
informative Lod (z) score at recombinant fraction (6)

Loci families 0.01 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

O = 0.20; 2 = 1.77; 95% confidence interval = 0.10-0.36
PAII vs. CF 13 -0.18 0.68 1.57 1.77 1.61 1.27 0.86 0.47

O = 0.03; z = 10.08; 95% confidence interval = 0.001-0.11
PAII vs. EPO 9 9.98 9.19 8.13 6.95 5.71 4.43 3.14 1.91

O = 0.18; t = 2.23; 95% confidence interval = 0.10-0.33
PARI vs. MET 18 -0.55 1.50 2.16 2.21 1.94 1.49 0.95 0.45

O = 0.28; i = 0.80; 95% confidence interval = 0.10-0.45
PAIl vs. PON 4 -1.38 -0.18 0.38 0.66 0.78 0.79 0.71 0.54

closely linked to EPO, with a maximal likelihood estimate for
O of 0.03 and a maximal lod score of 10.08 (odds ratio, 1.2 x
1010: 1). For linkage ofPAIl with PONand MET, the maximal
likelihood estimates for 0 and z were 0.28 and 0.80 vs. 0.18
and 2.23, respectively.
A three-locus test was performed to establish the position

of PAIl relative to EPO and PON. Of the families that were
informative for each marker tested for linkage to PAII (Table
1), two families were jointly informative for PAII, PON, and
EPO. Results of the three-locus analysis using data obtained
from these families are shown in Fig. 5. In this analysis the
distance between the EPO and PON loci was held constant,
and the relative probability of the PAII locus was calculated
at the indicated intervals. The data are consistent with the
order PON, PAIl, EPO with odds of 40:8:1 over the alter-
native orders PON, EPO, PAII or PAII, PON, EPO,
respectively.

DISCUSSION
We assigned the gene for PAII to 7cen-7q32 based on the
pattern obtained when a human PAIl cDNA was hybridized
to a panel of DNAs isolated from mouse-human somatic cell
hybrids, sublocalized to bands q21.3-q22 by in situ hybrid-
ization.

Analysis ofthe segregation ofthe HindIII RFLP in families
previously typed for the loci CF, MET, PON, COLJA2, and
EPO showed that the PAHl gene is part of this linkage group
on the long arm of chromosome 7. The PAII gene is closely

40
103 o/O 0/00

0

o °'D I 8

30 20 10 0 10 20 30 40 50
PON EPO

Genetic Location (cM)

FIG. 5. Likelihood of the localization of the PAII gene locus in
relation to PON and EPO. Abscissa: genetic distance from PON.
Ordinate: odds ratio for localization ofPAII at the distance indicated
vs. localization of PAII at infinite distance from PON (i.e., no
linkage). The distance between PON and EPO is assumed to be 10
centimorgans (cM). The relative odds for the orders PON, PAII,
EPO: PON, EPO, PAIl: PAIl, PON, EPO are 40:8:1.

linked to the EPO locus (estimated genetic distance, 3 cM),
which recently was found to be tightly linked with the
collagen locus COLJA2, with no evidence of recombination
(0= 0, z = 4.81) (P.W., N.S., P.S., and K.W.K., unpublished
data). On this basis, the 20 (10-36tt)-cM recombination
distance of PAIl from CF found in this study is in good
agreement with those of 11 (3-27*) and 16 (10-26*) cM found
for EPO and COLIA2, respectively (P.W., N.S., P.S., and
K.W.K., unpublished data; ref. 28). Previous multipoint
linkage analysis has established the order (EPO, COLIA2),
PON, (MET, CF) (P.W., N.S., P.S., and K.W.K., unpub-
lished data), with EPO, COL, and PON centromeric to CF.
According to the results of the three-point analysis in the
present study, the most probable location of PAII is between
EPO and PON-i.e., (EPO, COLIA2), PAII, PON, (MET,
CF), with PAII centromeric to CF (see above) and telomeric
to EPO.
PAIl may prove to be an important genetic marker. The

polymorphism information content of 0.369 is high for a
diallelic system; thus, the majority of families will be infor-
mative for gene mapping by linkage analysis.
The PAII RLFP may be valuable in genetic analysis of

plasminogen activation-related diseases, such as cancer and
certain thromboembolic disorders. In preliminary studies, no
rearrangements of the PAIl sequences were found by South-
ern blotting either in 18 carcinoma samples or in 18 additional
cell lines (R.W. and K.A., unpublished data). These included
12 colon carcinomas, 6 breast carcinomas, and tumor cell
lines with previously demonstrated oncogene rearrange-
ments and amplifications (42). However, these results must
be interpreted with caution since small changes would have
been missed due to the scale of the Southern blotting method
and since we do not yet know the genomic organization of the
PAIl sequences. In relation to the role of plasminogen
activation in the breakdown of matrix proteins in cancer, the
close linkage between PAII and the collagen locus COLIA2
is particularly noteworthy because plasmin is an activator of
latent collagenases (43, 44), and PAIl may therefore serve to
protect collagen.
Some thrombotic disorders, including myocardial infarc-

tion in young patients and recurrent deep vein thrombosis,
are in some cases associated with increased plasma levels of
PAIl (45-47), and familial occurrence of venous thrombosis
with high level of PA1l has been reported (48, 49). The
availability of the cloned gene sequence will allow molecular
studies of the pathophysiology of these disorders, and the
segregation of the RFLP in families with thrombosis will
allow us to determine whether there is a genetic component
involving the PAIl locus.

The excellent technical assistance of Helle Abildgaard, Kirsten
Lund Jakobsen, Vivi Kielberg, Anna Margrethe Poulsen, Aita
Haataja, and Irma Kurvinen is gratefully acknowledged. Determi-
nations of PON serum activity were generously provided by Dr. B.

#95% confidence interval.

Genetics: Klinger et al.

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Proc. Natl. Acad. Sci. USA 84 (1987)

LaDu (University of Michigan). We thank Drs. Robert Schwartz and
Richard Doherty (Rochester, NY) for their role in identification and
collection of members of the inbred pedigrees. This work was
supported financially by the Danish Canter Society, the Danish
Medical Research Council, PF Ingegneria Genetica e Basi Molecolari
delle Malattie Ereditarie and PF Oncologia, Consiglio Nazionale
delle Ricerche, Italy, Finnish Cancer Foundation, and National
Institutes of Health Grants HL31916 and AM36921.

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8552 Genetics: Klinger et al.

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