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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3457-3466
Compound-Heterozygous Mutations in the Plasminogen Gene Predispose
to the Development of Ligneous Conjunctivitis
By
Volker Schuster,
Silvia Seidenspinner,
Petra Zeitler,
Cornelia Escher,
Uwe Pleyer,
Wolfgang Bernauer,
E. Richard Stiehm,
Sherwin Isenberg,
Stefan Seregard,
Thomas Olsson,
Anne-Marie Mingers,
Christian Schambeck, and
Hans Wolfgang Kreth
From the Children's Hospital and the Central Laboratory, University
of Würzburg, Würzburg, Germany; the Department of
Ophthalmology, Charité, Humboldt University, Berlin, Germany; the
Department of Ophthalmology, University of Zürich, Zürich,
Switzerland; the Jules Stein Eye Institute, Departments of
Ophthalmology and Pediatrics, UCLA School of Medicine, Los Angeles, CA;
the St Erik's Eye Hospital, Stockholm, Sweden; and the Department of
Ophthalmology, Visby Hospital, Visby, Sweden.
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ABSTRACT |
Homozygous type I plasminogen deficiency has been identified as a
cause of ligneous conjunctivitis. In this study, 5 additional patients
with ligneous conjunctivitis are examined. Three unrelated patients (1 boy, 1 elderly woman, and 1 man) had plasminogen antigen levels of less
than 0.4, less than 0.4, and 2.4 mg/dL, respectively, but had
plasminogen functional residual activity of 17%, 18%, and 17%,
respectively. These subjects were compound-heterozygotes for different
missense mutations in the plasminogen gene: Lys19  Glu/Arg513
His, Lys19 Glu/Arg216 His, and
Lys19 Glu/Leu128 Pro,
respectively. The other 2 patients, a 14-year-old boy and his
19-year-old sister, who both presented with a severe course of the
disease, exhibited plasminogen antigen and functional activity levels
below the detection limit (<0.4 mg/dL and <5%, respectively). These subjects were compound-heterozygotes for a deletion mutation (del
Lys212) and a splice site mutation in intron Q (Ex17 + 1del-g) in the plasminogen gene. These findings show that certain
compound-heterozygous mutations in the plasminogen gene may be
associated with ligneous conjunctivitis. Our findings also suggest that
the severity of clinical symptoms of ligneous conjunctivitis and its
associated complications may depend on the amount of plasminogen
functional residual activity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LIGNEOUS CONJUNCTIVITIS is an uncommon
form of chronic conjunctivitis that may initially present with
unspecific symptoms such as tearing and conjunctival hyperemia. At the
beginning, pseudomembranes develop on the palpebral surfaces that may
later form thick nodular masses with subsequent loss of the normal
mucosa. In 1847, the first report of ligneous conjunctivitis describes a 46-year-old man with bilateral pseudomembraneous
conjunctivitis.1 Because the pseudomembranes may have a
wood-like consistency, the disease was later termed "ligneous"
conjunctivitis.2 In addition to the ocular lesions, many
patients show similar pseudomembranes in the mucosa of the mouth, the
tongue, the nasopharynx, the tracheobronchial tree, and the female
genital tract.3-8 Histological examination of
pseudomembranes from affected eyes or mucosal tissue from the genital
tract exhibits a disrupted epithelium replaced by a massive deposition
of fibrin and amorphous hyalin-like eosinophilic material, accompanied
by an inflammatory cellular infiltration.6,9-14 Ligneous
conjunctivitis has recently been linked to severe type I plasminogen
deficiency by Mingers et al.8,15 In 3 patients with
ligneous conjunctivitis distinct homozygous mutations in the
plasminogen gene were identified.16,17 Similar fibrin-rich conjunctival lesions that are indistinguishable from human ligneous conjunctivitis were also described in mice with homozygously disrupted plasminogen genes (Plg / mice) or in mice
homozygously deficient for both the urokinase-type plasminogen
activator (u-PA) and the tissue-type plasminogen activator (t-PA) genes
(u-PA//t-PA/
mice).18 Ligneous conjunctivitis has been also reported
after use of the antifibrinolytic agent tranexamic acid during
treatment of menorrhagia.19 Tranexamic acid blocks the
lysine binding sites on the kringle domains of plasminogen and leads to
dissociation of the fibrin(ogen)-plasminogen complex. These findings
point to the central role of functional plasmin(ogen) deficiency in the
pathogenesis of ligneous conjunctivitis.
We report here distinct compound-heterozygous mutations in the
plasminogen gene in 5 patients with ligneous conjunctivitis.
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MATERIALS AND METHODS |
Patients
Patient no. 1.
This 5-year-old boy is the first child of healthy nonconsanguinous
German parents. Episodes of venous thrombosis were not observed in any
member of the family.
At 13 months of age, he developed chronic bilateral tarsal ligneous
conjunctivitis (Table 1) after an infection
with streptococci group A. Repeated surgical excision of conjunctival
pseudomembranes and treatment with topical antibiotics and chymotrypsin
resulted in complete recovery of the left eye, but only transient and
short-term improvement of the right eye. Histological examination of
the pseudomembrane exhibited massive exsudation of fibrin with an inflammatory cellular infiltration (mainly CD4+ T cells and
CD19+ B cells), a disrupted and infolded epithelium,
granulation tissue, and an amorphous, eosinophilic, hyaline material.
At 3.5 years of age, a large pedunculated fibrin-rich pseudomembranous
lesion of the right upper tarsal conjunctiva
(Fig 1A) developed that was completely
excised, followed by intensive topical administration of steroids and
long-term topical heparin (5,000 U/mL) according to the recommendations
of De Cock et al.11 Within 6 months, the conjunctival
pseudomembrane had almost completely disappeared and has not recurred.
Patient no. 2.
This previously healthy 71-year-old woman first developed unilateral
ligneous conjunctivitis at the age of 69 years (Fig 1B and Table 1). At
that time, a pseudomembrane developed over the palpebral conjunctiva of
the right upper eye lid. It recurred rapidly after surgical removal.
Treatment with topical heparin eye drops (1,000 to 5,000 U/mL), topical
cyclosporine A (2%), and corticosteroids did not prevent recurrences
of the pseudomembranes and surgical excision was performed three times.
During this time period, visual acuity decreased from 20/30 to 20/70.
Other mucous membranes were unaffected. Histopathology of excised
pseudomembrane showed a disrupted conjunctival epithelium and massive
fibrinous tissue with infiltration of inflammatory cells, mainly
lymphocytes, granulocytes, and some macrophages. The family history is
unremarkable, without eye disease or venous thrombosis.
Patients no. 3 and 4.
This 19-year-old white female (patient no. 3) first developed
conjunctivitis at 3 weeks of age (Table 1) and was treated with
multiple eye drops, including corticosteroids. At 3 years of age, she
developed bilateral conjunctival pseudomembranes that were surgically
removed; a diagnosis of ligneous conjunctivitis was made. These
membranes have recurred repeatedly, necessitating surgical removal on
18 different occasions, the last removal occurring 2 years ago.
However, the rate of conjunctival membrane formation has decreased in
recent years. At 5 years of age, she developed hoarseness and was noted
to have a ligneous membrane on the vocal cords. She also showed
asthma-like symptoms. She continued to have intermittent lung problems
and at 8 years of age she developed a pneumomediastinum. At 8 years of
age, she had her first of 20 bronchoscopies to remove thickened
membranes from her laryngo-tracheobronchial tree. At 16 years of age,
she developed an abscess of the left lung necessitating bronchoscopic
drainage of this area. She also had had gingival membranes and nodular,
calcified masses in the renal collecting system, demonstrable by
ultrasound and pyelography. Treatment with multiple eye drops,
corticosteroids, local heparin, and multiple courses of antibiotics
have been ineffective.
Her 14-year-old brother (patient no. 4) developed conjunctivitis at 9 months of age that became severe at 4 years of age. At 5 years of age,
he had surgical removal of the ligneous conjunctival membranes. These
have recurred frequently necessitating surgery on 15 occasions, the
last time occurring 1 year ago. There is some scarring of the upper
lids with symmetrical ptosis. Since 5 years of age, he has had
membranes on the gums associated with intermittent bleeding, geographic
tongue, and sinusitis. He also had membrane formation in the pharynx
and in the renal collecting system. Furthermore, duodenal ulceration
and an eosinophilic gastric infiltration were observed. He has been
treated in the same way as his sister and, in addition, has received
intravenous Igs and thalidomide without dramatic effects. Papers
concerning the clinical course of patients no. 3 and 4 and the
bronchial lesions of patient no. 3 have already been
published.3,7
Patient no. 5.
This currently 23-year-old man developed unilateral ligneous
conjunctivitus on the right upper eyelid at 9 months of age (Table 1).
After surgical excision, pseudomembranes recurred within 8 months.
During the first 10 years of life the patient required local excision
of recurrent pseudomembranes combined with cryotherapy on 12 occasions.
At 12 years of age, he gradually developed bilateral limbal dots and
itching suggestive of bilateral vernal conjunctivitis. At this time,
the disease could be adequately controlled by a combination of topical
steroids, antihistamines, and levocabastine. After a few years, limbal
disease activity gradually decreased and treatment was discontinued.
There has been no formation of extraocular pseudomembranes during the
entire course of disease and there have been no signs of active
conjunctival disease for the past 5 years.
Coagulation Studies
Quantitative determination of plasminogen antigen in citrated plasma of
patients was performed by 1% agarose
immunoelectrophoresis20 using a polyclonal rabbit
antiplasminogen antiserum (OSCB; Behringwerke AG, Marburg, Germany).
The lower limit of detection of plasminogen antigen in human plasma is
0.4 mg/dL.
Plasminogen functional activity in citrated plasma after activation
with streptokinase was assessed using a chromogenic assay with
p-nitroaniline as substrate (Berichrom Plasminogen; Behringwerke AG).
The lower limit of detection is 5% plasminogen functional activity.
Quantitative determination of t-PA in citrated plasma was performed
using enzyme-linked immunosorbent assay (ELISA; Asserachrom t-PA;
Boehringer Mannheim, Mannheim, Germany). Quantitative determination of
u-PA in citrated plasma was performed by ELISA (IMUBIND uPA Strip-well
ELISA kit; American Diagnostica GmbH, Pfungstadt, Germany). Plasminogen
activator inhibitor type 1 (PAI-1) activity in citrated plasma was
determined by a chromogenic assay (Baxter Diagnostics AG,
Düdingen, Switzerland).
Mutation Analysis
Genomic DNA was prepared from peripheral blood samples of all patients
with ligneous conjunctivitis as well as from healthy family members.
Thereafter, DNA samples were amplified by polymerase chain reaction
(PCR) using a set of primer pairs flanking all 19 exons, including
intron boundaries of the human plasminogen gene.21,22 To
screen for plasminogen gene mutations, we used the method of
single-strand conformation polymorphism (SSCP) analysis, which permits
the detection of point mutations and other sequence alterations
according to a variant migration pattern of the mutant versus the
wild-type DNA fragment in gel electrophoresis, as described previously.16 Amplified and subsequently denatured PCR
segments were electrophoresed in a nondenaturing 5% polyacrylamide gel (Roth, Karlsruhe, Germany) containing 5% glycerol in 0.5× TBE buffer at room temperature for 7 hours at 10 W. The gel was dried and
exposed to Kodak XAR-5 films (Eastman Kodak, Rochester,
NY). Variant bands of single-stranded DNA fragments that
differed in their migration pattern were excised from dried gels,
dissolved in 1× TE buffer, purified with MicroSpin columns
(Pharmacia, Freiburg, Germany), and reamplified. Amplified PCR products
were again purified with MicroSpin columns and directly cycle-sequenced
by the dideoxy-termination method using 35S-dATP and the
Exo( )Pfu Cyclist DNA Sequencing Kit (Stratagene, Heidelberg,
Germany) according to the manufacturer's directions. Samples were
electrophoresed on 6% polyacrylamide/8.3 mol/L urea sequencing gels
(Roth), dried, and exposed to Kodak XAR-5 films.
Direct Analysis of Plasminogen Mutations by Amplification
Mutagenesis and by Restriction Fragment Length Polymorphism (RFLP)
Analysis
For the direct detection of the plasminogen gene mutation
Arg216 His in exon 7 (patient no. 2), genomic
DNA was amplified by PCR with the following primer pair flanking the
mutation in exon 7: 5'-AACCTGAAGAAGAATTACTGTG-3' (forward)
and 5'-CGGGATCCTGGAATCTGGGTGTGCATCATAC-3' (reverse). The first primer has a single sequence mismatch at position
779 (C G) that results in the introduction of an artificial BstE II restriction site into only the wild-type exon 7 allele. Amplified PCR products were digested with BstE II (GIBCO,
Eggenstein, Germany), electrophoresed in a 2.5% agarose gel, and
visualized by ethidium bromide staining as described
recently.16 The wild-type BstE II-digested PCR
fragment has a size of 119 bp; the mutant (uncut) PCR fragment has a
size of 141 bp.
The missense mutation Arg513 His (G A
at position 1671) in exon 13 of plasminogen gene (patient no. 1)
disrupts the restriction site for the restriction enzyme Mae
III. To detect this mutation directly, amplified PCR products of
plasminogen exon 13 were digested with Mae III (Boehringer
Mannheim), electrophoresed in a 2% agarose gel, and visualized by
ethidium bromide staining. The (uncut) mutant PCR fragment has a size
of 184 bp, the wild-type Mae III-digested PCR fragment has a
size of 139 bp.
The deletion mutation of a guanosine at position +1 in intron Q of the
plasminogen gene (mutation name, Ex17 + 1del-g) in patients no. 3 and 4 disrupts the restriction site for the restriction enzyme Sty I. To detect this mutation directly, amplified PCR products of plasminogen
exon 17/intron Q were digested with Sty I (Boehringer
Mannheim), electrophoresed in a 2% agarose gel, and visualized by
ethidium bromide staining. The (uncut) mutant PCR fragment has a size
of 203 bp; the wild-type Sty I-digested PCR fragment has a size
of 143 bp.
The missense mutation Leu128 Pro (T C
at position 516) in exon 5 of the plasminogen gene (patient no. 5)
produces a restriction site for the restriction enzyme Msp I. To detect this mutation directly, amplified PCR products of plasminogen
exon 5 were digested with Msp I (Boehringer Mannheim),
electrophoresed in a 2% agarose gel, and visualized by ethidium
bromide staining. The (uncut) wild-type PCR fragment has a size of 221 bp, whereas the mutant Msp I-digested PCR fragment has a size
of 144 bp.
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RESULTS |
Patients No. 1 Through 5 With Ligneous Conjunctivitis
Coagulation studies in patient no. 1 and his family.
The coagulation system of patient no. 1 at the age of 4 years and 4 months showed a plasminogen functional activity of 17% (normal range,
80% to 120%) and a plasminogen immunoreactive antigen level of less
than 0.4 mg/dL (normal range, 6 to 25 mg/dL; Table 2).
Fibrinogen, t-PA (Table 2), u-PA (Table 2), and PAI-1 of the patient
and all healthy family members tested were within the normal range
(data not shown). Plasminogen functional activities in the mother,
father, and the brother (1 year and 8 months old) were 54%, 88%, and
121%, respectively. Plasminogen antigen concentrations were 7.0, 9.0, and 15.0 mg/dL, respectively (Table 2).
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Table 2.
Plasminogen Values in Plasma and Molecular Genetic
Findings in Five Patients With Ligneous Conjunctivitis and One Healthy
Female With Heterozygous Type I Plasminogen Deficiency
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Molecular genetic findings in patient no. 1 and his family.
An altered SSCP band pattern was found in this family when primers
specific for plasminogen exon 2 (Fig 2), exon 7 (data
not shown), and exon 13 (data not shown) were used. Direct sequencing of abnormal single strands of plasminogen exon 2 from patient no. 1 (Fig 2, lane 4; band c and c') demonstrated an A G
point mutation at position 188 leading to an amino acid exchange at position 19 (Lys19 Glu; not shown). The father
was shown to be heterozygous (Table 2). This mutation is located in the
proactivation peptide (NH2-terminal acidic domain of
plasminogen) 59 amino acids upstream of the
Lys77-Lys78 cleavage site.21
Examination of the younger brother and the mother and 50 healthy
controls showed the wild-type pattern of plasminogen exon 2 (data not
shown).
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| Fig 2.
SSCP analysis of plasminogen gene exon 2 in patient no.
1, his healthy parents, and his healthy brother and in healthy subject
H. Radiolabeled single-stranded PCR products of plasminogen exon 2 were
electrophoresed in a nondenaturing 5% polyacrylamide gel as described
in Materials and Methods. Lane 1, healthy control (C); lane 2, father
of patient no. 1; lane 3, mother of patient no. 1; lane 4, patient no.
1 with ligneous conjunctivitis; lane 5, brother of patient no. 2;
lane 6, healthy subject H; lane 7, healthy control. dsDNA,
double-stranded DNA.
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Direct sequencing of abnormal single strands of plasminogen exon 13 from patient no. 1 demonstrated a G A point mutation at
position 1671 leading to an amino acid exchange at position 513 (Arg513 His; data not shown). The mother was
also heterozygous for this Arg513 His mutation.
The Arg513 His mutation could be easily
differentiated from the wild-type allele by RFLP analysis (Fig 3).
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| Fig 3.
Direct detection of missense mutation Arg513
His in plasminogen exon 13 by RFLP analysis. Amplified PCR
products of plasminogen exon 13 were digested with Mae III,
electrophoresed in a 2% agarose gel, and visualized by ethidium
bromide staining. The (uncut) mutant type PCR fragment has a size of
184 bp; the wild-type Mae III-digested PCR fragment has a size
of 139 bp. , no Mae III added to PCR product; +,
Mae III added to PCR product. Lane M, 100-bp ladder; lane 1, father of patient no. 1; lane 2, mother of patient no. 1; lane 3, brother of patient no. 1; lane 4, patient no. 1; lanes 5 through 8, healthy controls. The larger (mutant type) allele is found after
Mae III-digestion in patient no. 1 and his mother (lanes 2+
and 4+, heterozygous), but not in the father and the brother of
patient no. 1 (lanes 1+ and 3+) and not in 4 healthy controls
(lanes 5+, 6+, 7+, and 8+).
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This mutation is located in the kringle 5 domain of plasminogen,
downstream from the intrakringle disulfide bridge
Cys512-Cys536.21 Examination of the
younger brother and the father and 50 healthy controls showed the
wild-type pattern of plasminogen exon 13 (data not shown).
In this family, two different plasminogen gene mutations were
independently transmitted from the father (Lys19 Glu) and the mother (Arg513 His) to patient no.
1, who therefore is a compound-heterozygote for these mutations. The
combination of these mutations leads to undetectable plasminogen
antigen (<0.4 mg/dL) and markedly decreased functional activity
(17%). However, due to the limited sensitivity of the assays, we
cannot exclude traces of plasminogen antigen in this subject (Table 2).
In the mother, who is heterozygous for the Arg513
His mutation, plasminogen values (antigen concentration, 7.0 mg/dL; functional activity, 54%) are reduced by
approximately 50% (heterozygous range).
In the father, who is heterozygous for the Lys19 Glu mutation, plasminogen values (antigen concentration, 9.0 mg/dL;
functional activity, 88%) are in the low normal range (antigen
concentration, 6 to 25 mg/dL; activity, 80% to 120%).
Coagulation studies in patient no. 2.
Coagulation studies performed at the age of 71 years showed plasminogen
functional activity of 18% (normal range, 80% to 120%) and
plasminogen antigen of less than 0.4 mg/dL (normal range, 6 to 25 mg/dL). Levels of fibrinogen, t-PA (Table 2), u-PA (Table 2), and PAI-1
were normal (data not shown).
Molecular genetic findings in patient no. 2.
Altered SSCP band patterns in genomic DNA of patient no. 2 were found
by SSCP analysis when primers specific for plasminogen exon 2 and exon
7 were used (data not shown). Direct sequencing of the altered exon 7 single strands from patient no. 2 exhibited a G A
point mutation at position 780 leading to an amino acid substitution at
position 216 (Arg216 His; data not shown). The Arg216 His mutation could be easily
differentiated from the wild-type allele by amplification mutagenesis
(Fig 4). Examination of 50 healthy controls by this
method showed only the wild-type allele (data not shown).
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| Fig 4.
Direct detection of plasminogen exon 7 mutation in
patient no. 2 by amplification mutagenesis. A part of plasminogen exon
7 containing the mutation (G A at position 780) was
amplified by PCR as described in Materials and Methods. The first
primer has a single sequence mismatch that results in the introduction
of an artificial BstE II restriction site into the wild-type
allele only. Amplified PCR products were digested with BstE II,
electrophoresed in a 2.5% agarose gel, and visualized by ethidium
bromide staining. The wild-type BstE II-digested PCR fragment
has a size of 119 bp; the mutant (uncut) PCR fragment has a size of 141 bp. , no BstE II added to PCR product; +, BstE II
added to PCR product. Lane M, 100-bp ladder; lane 1, healthy control;
lane 2, homozygous Turkish girl with ligneous
conjunctivitis16; lane 3, patient no. 2. The larger
(mutant) allele is found after BstE II-digestion in an
unrelated girl with ligneous conjunctivitis (lane 2+,
homozygous)16 and in patient no. 2 (lane 3+,
heterozygous), but not in the healthy control (lane 1+).
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This Arg216 His mutation is located in the
kringle 2 domain of plasminogen, downstream from the intrakringle
disulfide bridge Cys215-Cys238.21
It has been shown in another family with type I plasminogen deficiency
that heterozygous Arg216 His mutation in the
plasminogen gene leads to reduction of plasminogen functional activity
and plasminogen antigen by approximately 50%.16
Direct sequencing of the altered exon 2 single strands from patient no.
2 exhibited a A G point mutation at position 188 leading to
an amino acid exchange at position 19 (Lys19 Glu). Examination of 50 healthy controls by SSCP-PCR analysis showed only the wild-type pattern of plasminogen exon 2 (data not shown).
Coagulation studies in patients no. 3 and 4 and their family.
When the coagulation system of patients no. 3 and 4 was examined at 14 and 19 years of age, respectively, no plasminogen functional activity
was found (<5%; normal range, 80% to 120%). Plasminogen antigen
levels were below the detection limit of the assay used (<0.4 mg/dL;
normal range, 6 to 25 mg/dL; Table 2). Plasminogen functional
activities in both the healthy mother and the healthy father were 52%,
whereas plasminogen antigen concentrations were 6.0 and 12.0 mg/dL,
respectively. Fibrinogen, t-PA (Table 2), u-PA (Table 2), and PAI-1
were within the normal range in all family members (data not shown).
Molecular genetic findings in patients no. 3 and 4 and other members
of family 3.
An altered SSCP band pattern was found in patients no. 3 and 4 when
primers specific for plasminogen exon 7 (Fig 5) and exon 17, including intron boundaries (data not shown), were used. Direct sequencing of abnormal single strands of plasminogen exon 7 from patients no. 3 and 4 (Fig 5, lanes 4 and 5; band b) demonstrated a
(heterozygous) 3-bp deletion (AAG) at position 767-769 leading to
deletion of a lysine residue at position 212 (del Lys212;
data not shown). The mother is heterozygous for this
Lys212-deletion (Table 2). Examination of 50 healthy
controls showed only the wild-type pattern of plasminogen exon 7 (data
not shown).
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| Fig 5.
SSCP analysis of plasminogen gene exon 7 in patients no.
3 and 4 and her parents. Radiolabeled single-stranded PCR products of
plasminogen exon 7 were electrophoresed in a nondenaturing 5%
polyacrylamide gel as described in Materials and Methods. Lane 1, healthy control (C); lane 2, mother; lane 3, father; lane 4, patient
no. 4 with ligneous conjunctivitis; lane 5, patient no. 3. dsDNA,
double-stranded DNA. The double-stranded DNA of patients no. 3 and 4 (lanes 4 and 5) and of the mother (lane 2) shows two bands. The shorter
band is the mutant allele with a 3-bp deletion (AAG) at position
767-769. Note that alleles a and d of the mother (lane 2) are slightly
faster migrating when compared with bands a and d of a healthy control
with the wild-type sequence (lane 1). This is due to two polymorphisms,
a silent C T base exchange at position 847 in plasminogen
exon 7 (Cys238) and a T G base exchange at
position 15 in plasminogen intron F.
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Direct sequencing of plasminogen exon 17/intron Q from patients no. 3 and 4 and their father demonstrated a (heterozygous) 1-bp deletion of a
guanosine at position +1 in intron Q (name of mutation: Ex17+1del-g),
probably resulting in an aberrant splicing (data not shown). This
mutation could be easily differentiated from the wild-type allele by
RFLP analysis after digestion of PCR fragments with the restriction
enzyme Sty I (Fig 6).
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| Fig 6.
Direct detection of deletion mutation (Ex17 + 1del-g)
in plasminogen intron Q by RFLP analysis. Amplified PCR products of
plasminogen exon 17/intron Q were digested with Sty I,
electrophoresed in a 2% agarose gel, and visualized by ethidium
bromide staining. The (uncut) mutant type PCR fragment has a size of
203 bp; the wild-type Sty I-digested PCR fragment has a size of
143 bp. , no Sty I added to PCR product; +, Sty I
added to PCR product. Lane M, 100-bp ladder; lane 1, healthy control;
lane 2, mother of patients no. 3 and 4; lane 3, father of patients no.
3 and 4; lane 4, patient no. 4; lane 5, patient no. 3. The larger
(mutant) allele is found after Sty I-digestion in patients no.
3 and 4 (lanes 4+ and 5+, heterozygous) and their father (lane
3+, heterozygous), but not in the healthy control and not in the
mother of patients no. 3 and 4 (lanes 1+ and 2+).
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Coagulation studies in patient no. 5 and his family.
The coagulation system of patient no. 5 at the age of 23 years showed a
plasminogen functional activity of 17% (normal range, 80% to 120%)
and a plasminogen immunoreactive antigen level of 2.4 mg/dL (normal
range, 6 to 25 mg/dL; Table 2). Fibrinogen, t-PA (Table 2), u-PA (Table
2), and PAI-1 were within the normal range in all family members (data
not shown). Plasminogen functional activities in the mother and the
father were 57% and 70%, respectively. Plasminogen antigen
concentrations were 7.0 and 14.0 mg/dL, respectively (Table 2).
Molecular genetic findings in patient no. 5 and his family.
An altered SSCP band pattern was found in this family when primers
specific for plasminogen exon 2 (data not shown) and exon 5 (Fig 7) were used. Direct sequencing of abnormal single
strands of plasminogen exon 2 from patient no. 5 demonstrated an A
G point mutation at position 188 leading to an amino acid
exchange at position 19 (Lys19 Glu; data not
shown). The father is heterozygous for this mutation (Table 2).
Examination of the mother and 50 healthy controls showed the wild-type
pattern of plasminogen exon 2 (data not shown).
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| Fig 7.
SSCP analysis of plasminogen gene exon 5 in patient no. 5 and his healthy parents. Radiolabeled single-stranded PCR products of
plasminogen exon 5 were electrophoresed in a nondenaturing 5%
polyacrylamide gel as described in Materials and Methods. Lane 1, healthy control (C); lane 2, father of patient no. 5; lane 3, mother of
patient no. 5; lane 4, patient no. 5. dsDNA, double-stranded DNA.
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Direct sequencing of the abnormal single strands of plasminogen exon 5 from patient no. 5 (Fig 7, lane 4; band c) demonstrated a T C point mutation at position 516 leading to an amino acid exchange at position 128 (Leu128 Pro; data not
shown). The mother (Fig 7, lane 3) was also heterozygous for this
mutation. The Leu128 Pro mutation could be
easily differentiated from the wild-type allele by RFLP analysis
(Fig 8). Examination of the the father and 50 healthy
controls showed the wild-type pattern of plasminogen exon 5.
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| Fig 8.
Direct detection of missense mutation Leu128
Pro in plasminogen exon 5 of patient no. 5 by RFLP analysis.
Amplified PCR products of plasminogen exon 5 were digested with
Msp I, electrophoresed in a 2% agarose gel, and visualized by
ethidium bromide staining. The (uncut) wild-type PCR fragment has a
size of 221 bp; the mutant Msp I-digested PCR fragment has a
size of 144 bp. , no Msp I added to PCR product; +,
Msp I added to PCR product. Lane M, 100-bp ladder; lane 1, healthy control; lane 2, father of patient no. 5; lane 3, mother of
patient no. 5; lane 4, patient no. 5. The shorter (mutant type) allele
is found after Msp I-digestion in patient no. 5 and his mother
(lanes 3+ and 4+, heterozygous), but not in his father (lane 2+)
and not in a healthy control (lane 1+).
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Leu128 is located in the kringle 1 domain of plasminogen 5 amino acids upstream of the intrakringle disulfide bridge
Cys133-Cys157.21
In this family, two different plasminogen gene mutations were
independendly transmitted from the father (Lys19 Glu) and the mother (Leu128 Pro) to patient no.
5, who therefore is a compound-heterozygote for these mutations. The
combination of these mutations leads to markedly decreased plasminogen
antigen (2.4 mg/dL) and functional activity (17%; Table 2).
Polymorphisms in the plasminogen gene.
A silent C T base exchange at position 847 in plasminogen
exon 7 (Cys238)21,23 in combination with a T
G base exchange at position 15 in plasminogen intron
F was found in a homozygous pattern in patient no. 1 and the father of
patient no. 2; it was also found in a heterozygous pattern in patient
no. 2, his healthy brother, patients no. 3 and 4, and their mother (not
shown). The second allele of the mother of patients no. 3 and 4 exhibits the Cys238 polymorphism without the intron F
polymorphism. A silent C T base exchange at
position 1018 (Phe295) and a silent A G base exchange at position 1159 (Gln342) was found in
a heterozygous pattern in patients no. 3 and 4.21,23 The
mother was homozygous for the former and heterozygous for the second
polymorphism (not shown).
Molecular genetic findings in a healthy subject with heterozygous
type I plasminogen deficiency (subject H).
In this healthy woman, the plasminogen functional activity was 57%
(normal range, 80% to 120%) and the plasminogen antigen level was 7.5 mg/dL (normal range, 6 to 25 mg/dL; Table 1). Molecular genetic studies
showed a heterozygous C A point mutation in plasminogen exon
2 at position 159 leading to an amino acid exchange (Thr9
Asn; Fig 2, lane 6, SSCP pattern; sequencing data not shown;
Table 2). This novel missense mutation lies in the
NH2-terminal fragment of plasminogen, the proactivation
peptide, which is released during the conversion of Glu-plasminogen to
Lys-plasminogen by cleavage between
Lys77-Lys78.21
| |
DISCUSSION |
We have shown that certain compound-heterozygous mutations in the
plasminogen gene (Lys19 Glu/Arg216
His, Lys19 Glu/Arg513 His, Lys19 Glu/Leu128
Pro, and del Lys212/Ex17 + 1del-g) are
associated with ligneous conjunctivitis. It is possible that, as a
consequence of these mutations, mRNA transcription and/or the half-life
of mRNA of the mutant plasminogen allele may be markedly decreased. It
is also conceivable that the liver may synthesize a truncated
plasminogen molecule that is rapidly cleared from the circulation. Some
of the missense mutations in the plasminogen gene, described here,
affect a lysine (Lys19 Glu, del
Lys212) or an arginine residue (Arg216
His, Arg513 His). It has been suggested that lysine and arginine side chains of plasminogen interact
with the lysine binding sites of the kringles of the plasminogen
molecule and stabilize specific protein conformations.24 These mutations may therefore change the folding and conformation of
the mutant plasminogen molecule, leading to impaired secretion from the
liver into the blood stream. This mechanism has been recently
demonstrated in vitro by genetic engineering techniques for type I
plasminogen deficiency caused by a Ser572 Pro
mutation.25 Further in vitro studies are needed to show
whether the plasminogen variants, described here, exhibit an altered
binding capacity to cells and fibrinogen and whether the activation by
t-PA, u-PA, or other plasminogen activators is altered.
The plasminogen gene is well conserved in humans and different animal
species. The plasminogen gene mutations in patients with type I
plasminogen deficiency described in this and other studies16,17 ( Table 3) have not been
identified in different animals studied so far, except for
His216, which has been found in mice.26 Amino
acid residues Thr9, Leu128, Arg216,
Arg513, Ser572, and Trp597 are
conserved in all animal species studied so far: Rhesus
monkey,27 Macropus eugenii (GENBANK Accession No.
AF012297), pig,28-30 ox,31-33
horse,34,35 mouse (except for
His216),26 and hedgehog.36,37
Partial coding sequence of the N-terminal end of the plasminogen gene
shows the conservation of Thr9 also in sheep, goat, rabbit,
and dog.26,34 In the cat, Thr9 is
deleted.26 Lys19 is conserved in Rhesus
monkey,27 Macropus eugenii (GENBANK accession
AF012297), rabbit, canine, hedgehog, and mouse, but is replaced by an
arginine in pig, horse, ox, sheep, and goat.26,29,31,34
Inherited plasminogen deficiency has been classified into two types,
true deficiency (type I) and dysplasminogenemia (type II). In the
former, the level of immunoreactive plasminogen is reduced in parallel
with its functional activity. Six distinct plasminogen gene mutations
leading to type I plasminogen deficiency have been previously
described: Ser572 Pro, Ala675
Thr, Glu460 Stop,
Arg216 His, Trp597 Stop,
and Trp597 Cys (Table
3).16,17,22,38 We have described here six new mutations in
the plasminogen gene (Thr9 Asn,
Lys19 Glu, Leu128 Pro,
del Lys212, Arg513 His, and Ex17 + 1del-g) that lead to type I plasminogen deficiency (Table 3). The
prevalence of type I plasminogen deficiency has been recently
calculated as 0.26% (25 of 9,611 subjects) in a large epidemiological
study in United Kingdom.39 The theoretically predicted
prevalence of compound-heterozygotes (including homozygotes) would
therefore be approximately 7 per 1,000,000. Most subjects with
heterozygous type I plasminogen deficiency are clinically healthy. Two
recent epidemiological studies suggest that heterozygous type I
plasminogen deficiency by itself is not a risk factor for thrombosis.39,40 Furthermore, none of the 8 reported
patients with ligneous conjunctivitis and homozygous or
compound-heterozygous type I plasminogen deficiency developed
thrombosis (this report and Schuster et al16 and Schott
et al17).
In type II plasminogen deficiency, or dysplasminogenemia, the level of
immunoreactive plasminogen is normal (or only slightly reduced),
whereas the functional activity is markedly reduced (Table
3).41 Four distinct plasminogen gene mutations
(Ala601 Thr, Val355 Phe,
Asp676 Asn, and Gly732 Arg) have been identified in patients with dysplasminogenemia (Table
3).23,42-46,54 The prevalence of dysplasminogenemia has
been calculated as 0.03% (3 of 9,611 subjects) in an epidemiologic
study in United Kingdom.39 In Japan and China, the
prevalence rate is greater than 2%.46,47 Only a small
fraction of subjects with type II plasminogen deficiency will develop
thrombosis, suggesting that this plasminogen variant by itself is not a
risk factor for vascular occlusions.
Our previous15-17 and current results show that homozygous
and compound-heterozygous type I plasminogen deficiency may be
associated with ligneous conjunctivitis. In contrast, ligneous
conjunctivitis has never been described in subjects with type II
plasminogen deficiency.23 The severity of clinical symptoms
in patients with homozygous or compound-heterozygous type I plasminogen
deficiency seems to depend mainly on the amount of plasminogen
functional residual activity. Five patients with ligneous
conjunctivitis and severe type I plasminogen deficiency studied so far
who showed undetectable plasminogen functional
activity15-17 (patients no. 3 and 4 of the present report)
exhibited severe multisystem disease. In contrast, 3 patients of this
study who had a plasminogen functional residual activity of 17% and
18%, presented with a much milder course of disease. In these cases,
formation of pseudomembranes was confined to the conjunctivae.
To our knowledge, 2 of the patients described in this report are the
first cases in which the upper gastrointestinal tract (duodenal ulcer
and eosinophilic gastric infiltration in patient no. 4) and the kidneys
(patients no. 3 and 4) are also affected. Involvement of the kidneys
has been documented so far only rarely in dogs with ligneous
conjunctivitis48 and in plasminogen-deficient mice
(Plg/ mice)49 and occasionally in
plasminogen activator-deficient mice (t-PA/
mice and
u-PA//t-PA/
mice).50 In contrast, gastric and duodenal ulcers are
common in Plg/ and
u-PA//t-PA/
mice.49-53
Therapeutic options in the treatment of multisystem complications in
patients with severe type I plasminogen deficiency are limited. In one
boy with homozygous type I plasminogen deficiency and bilateral
ligneous conjunctivitis treated so far, repeated intravenous infusions
of Lys-plasminogen resulted in complete resolution of conjunctival
pseudomembranes.17 For different reasons (short half-life
of Lys-plasminogen and high costs), this procedure will not be possible
in most cases. In patients with recurrent life-threatening
complications (ie, severe bronchopneumonias, upper airway obstruction,
imminent blindness), liver transplantation may be another therapeutic
approach. In patients in whom formation of pseudomembranes is
restricted to the conjunctivae, topical treatment with heparin,
steroids, and  -chymotrypsin immediately after excision of
pseudomembranse may lead to acceptable results.11 Further
in vitro and in vivo studies may eventually help to develop plasminogen
variants with higher specific activity and a longer half-life for
therapeutic replacement therapy in patients with severe type I
plasminogen deficiency.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted January 19, 1999.
Supported by the Deutsche Forschungsgemeinschaft (Grant Schu 560/4-1 to
V.S.).
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. section
1734 solely to indicate this fact.
Presented in part on the XIVth International Congress on Fibrinolysis
and Thrombolysis, Ljubljana, June 22-26, 1998; Fibrinolysis & Proteolysis 12:20, 1998 (abstr 54, suppl 1)
Address reprint requests to Volker Schuster, MD, Children's Hospital,
University of Würzburg, Josef-Schneider-Stra e 2, D-97080
Würzburg, Germany; e-mail: Schuster{at}mail.uni-wuerzburg.de.
| |
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ABSTRACT
INTRODUCTION