pihusch et al-2001-american journal of reproductive immunology

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AJRI 2001; 46:124–131Printed in Ireland - all rights resered .

Thrombophilic Gene Mutations and RecurrentSpontaneous Abortion: Prothrombin Mutation

Increases the Risk in the First TrimesterRUDOLF PIHUSCH, TINA BUCHHOLZ, PETER LOHSE, HEIKE RÜBSAMEN, NINA ROGENHOFER, UWE

HASBARGEN, ERHARD HILLER, AND CHRISTIAN J. THALER

Pihusch R, Buchholz T , Lohse P, Rübsamen H , Rogenhofer N , Hasbargen U , Hiller E ,

Thaler CJ . Thrombophilic gene mutations and recurrent spontaneous abortion:

Prothrombin mutation increases the risk in the first trimester. AJRI 2001; 46:124–131

© Munksgaard , 2001

PROBLEM: Thrombophilic predisposition may be one of the underlying causes of recurrent spontaneous abortions (RSA). We studied the prevalence of five throm-

bophilic gene mutations in patients with RSA.

METHOD OF STUDY: 102 patients with two or more consecutive abortions and 128

women without miscarriage were analyzed for factor V Leiden mutation (FVL),

prothrombin G20210A mutation (PTM), C677T mutation in the 5,10-methylenete-

trahydrofolate reductase (MTHFR) gene, glycoprotein IIIa (GPIIIa) C1565T poly-

morphism, and -fibrinogen G-455A polymorphism by polymerase chain reaction

(PCR) techniques.

RESULTS: No differences in the prevalence of FVL, MTHFR T/T, GPIIIa and

-fibrinogen polymorphism were detected. Heterozygous PTM occurred more often in

patients with RSA. This effect was significant in a subgroup with abortions exclusively

in the first trimester (6.7% vs. 0.8%, P=0.027, OR 8.5).

CONCLUSIONS: In contrast to the other mutations and polymorphisms, het-

erozygous PTM is more common in patients with abortions in the first trimester. Thismight reflect an influence of PTM on pathogenesis of early pregnancy loss.

Key words:

-Fibrinogen, factor V Leiden,

genetic thrombophilia, GPIIIa,

habitual abortion, MTHFR

RUDOLF PIHUSCHERHARD HILLER

Haemostaseology Research

Laboratory, Department of

Haematology and Oncology,

Klinikum der Universität

München-Großhadern, Munich,

Germany

TINA BUCHHOLZ

NINA ROGENHOFER

UWE HASBARGEN

CHRISTIAN J. THALER

Department of Obstetrics and

Gynecology, Klinikum der

UniversitätMünchen-Großhadern, Munich,

Germany

PETER LOHSE

HEIKE RU BSAMEN

Molecular Biology Laboratory,

Department of Clinical

Chemistry, Klinikum der

Universität

München-Großhadern, Munich,

Germany

Address reprint requests to

Christian J. Thaler, Department

of Obstetrics and Gynecology,Klinikum der Universität

München-Großhadern, 81377

Munich, Germany.

E-mail:

[email protected]

Submitted September 25, 2000;

revised December 5, 2000;

accepted December 6, 2000.

© MUNKSGAARD, 2001


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GENETIC THROMBOPHILIA AND RECURRENT SPONTANEOUS ABORTIONS / 125

INTRODUCTION

Recurrent spontaneous abortions (RSA) are a major

concern in gynecology, affecting about 1 – 5% of cou-

ples1,2 and frequently accompanied by maternal mor-

bidity as well as a considerable psychological burden.

Despite intense anatomic, endocrinologic, and im-

munologic screening efforts, up to 30 – 50% of RSA

remain unexplained.3,4

Common findings in placentae from recurrent

aborti are fibrin deposition and thrombi in intervillous

spaces and fetal stem vessels, associated with fetal

hypoperfusion, hypoxia, and fetal demise.5 The under-

lying pathophysiological mechanisms remain unclear,

although it has been speculated that increased coagu-

lation or decreased fibrinolytic activities may be po-

tential causes of RSA. The precedence of this disorder

is the antiphospholid syndrome, where activation of

coagulation generates arterial and venous clot forma-

tion and secondary placental insuf ficiency.6

During the last years, several genetic risk factors of

thrombophilia have been identified.7 – 14 The known

thrombophilic mutations, which were considered in

this study, are recapitulated in Table I. Besides caus-

ing hereditary thrombophilia, several studies demon-

strated that factor V Leiden mutation (FVL),

prothrombin mutation (PTM), and 5,10-methylenete-trahydrofolate reductase (MTHFR) mutations may

also increase a woman’s risk of recurrent pregnancy

loss in the second and third trimester,15 – 25 possibly by

affecting the maternal – fetal circulation.

We studied these mutations in our collective con-

sisting of abortions mainly in the first trimenon and

furthermore included polymorphisms in the glyco-

protein IIIa (GPIIIa) and -fibrinogen gene loci that

have so far not been investigated in this collective,

TABLE I. Genetic Risk Factors for Thrombosis7–14,26–30

Name Mutation Pathomechanism Prevalence (Caucasians) Clinical effect

FVL 1691 G A substitution HeterozygosityBlocked inactivation Heterozygosity increases

of factor Va byin the gene of approximately 5–10% the risk for thrombosis

coagulation factor V by approximatelyactivated protein C,

seven-fold,resulting in reduced

homozygosityclearance of factor

increases it by Va

approximately 80-fold

Heterozygosity increasesElevated prothrombin20210 G A substitutionPTM Heterozygosity

risk for thrombosis bylevels in plasma approximately 1–2%in the 3-untranslated

region of the approximately two-fold;

prothrombin gene increased risk of

myocardial infarction

and cerebral vein

thrombosis

TL-MTHFR Thermolabile variant of Possible increased riskHomozygosity for the TL-MTHFR

for venous thrombosisapproximately 10%677 CT substitution the enzyme with

and arterial infarctionin the MTHFR gene reduced catalytic

activity and elevated

plasma levels of

homocysteine

GPIIIa PI A1/ A2 PI A2 allelePlatelet GPIIIa is PI A2 allele potentially1565 CT substitution

in the gene for plateletpolymorphism essential for more often in subjectsapproximately 15%

with unstable anginaGPIIIa (PI A2

allele) aggregation andand myocardialthrombus formation

infarction

A / A genotype causes A allele−455 G A substitution Possibly associated with-Fibrinogen

polymorphism arterial complicationsapproximately 20%higher plasmain the promotor of the

fibrinogen levelsgene for the -chain

FVL, factor V Leiden mutation; PTM , prothrombin mutation; TL-MTHFR, thermolabile 5 ,10 -methylenetetrahydrofolate reductase, GPIIIa,

glycoprotein IIIa.

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 46, 2001


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/ PIHUSCH ET AL.126

although they may be of importance in the pathogen-

esis of arterial occlusions.14,26 – 30

MATERIALS AND METHODS

RSA Patients and ControlsWe analyzed 102 Caucasian women with two or more

unexplained consecutive abortions at 25 weeks of

gestation. The routine work up of these patients in-cluded vaginal ultrasound, hormonal evaluation (folli-

cle-stimulating hormone, luteinizing hormone,

prolactin, dehydroepiandosterone [DHEA], testos-

terone, thyroid-stimulating hormone), determination of

auto-antibodies (antinuclear antibodies [ANA], anti-

mitochondrial antibodies [AMA], IgG and IgM anti-

cardiolipin antibodies), dilute Russell viper venom time

(DRVVT) and lupus-sensitive activated partial throm-

boplastin time (aPTT).

If anticardiolipin antibodies (ACA) IgG was greater

than 20 IU/mL or ACA IgM was greater than 12

IU/mL in two consecutive measurements (more than 4

weeks apart) or if the DRVVT test and the lupus-sen-

sitive aPTT were positive, patients were considered as

having an antiphospholipid syndrome and were ex-

cluded from the study. All functional tests were per-

formed at least 2 months after the last pregnancy.

Patients with chromosomal aberrations were also ex-

cluded. None of the patients had deficiencies of an-

tithrombin, protein C, or protein S as determined by

functional assays.

One hundred and twenty-eight women with at least

one healthy term delivery and no history of abortions

or pregnancy-associated complications served as con-

trols. They were recruited from a series of consecutivepregnant women that delivered healthy term infants at

our institution between November 1998 and February

1999. Patients after artificial reproduction techniques

were excluded.

Each RSA patient and each control patient gave a

written informed consent allowing polymerase chain

reaction (PCR) testing for the mutations and polymor-

phisms mentioned in this paper.

Genetic StudiesThe FVL, PTM, MTHFR, GPIIIa, and -fibrinogen

genotype analyses were performed in all patients and

controls. Genomic DNA was extracted from white

blood cells using the QIAmp DNA blood mini kit

(QIAGEN, Hilden, Germany) and amplified by the

PCR with gene-specific primer pairs. Each 50-L reac-

tion contained 10 mM Tris – HCl, pH 8.3, 50 mM KCl,

1.5 mM MgCl2, 0,01% gelatin, 200 M of each dNTP,

20 M of each forward and reverse primer, approxi-

mately 200 ng of high molecular weight DNA, and 1.25

units Taq DNA Polymerase (Sigma-Aldrich, Deisen-

hofen, Germany). After denaturation at 95°C for 3 min,

DNA was amplified for 40 cycles at 95°C for 30 s, 59°C

(GPIIIa), 60°C (FVL and PTM), 62°C (-fibrinogen),

65°C (MTHFR), and finally annealed at 72°C for 2 min.

The positive assay control was DNA from a normal

(FVL and -fibrinogen) or homozygous mutant subject

(GPIIIa, MTHFR, and PTM), while a water blank

served as negative control for each analysis.Following PCR, the newly generated products were

digested with the appropriate restriction enzyme (New

England BioLabs) and electrophoresed in 2 – 3% low

melting point agarose gels (Gibco BRL Life Technolo-

gies, Karlsruhe, Germany). Mnl I digestion of the 288-

bp PCR product of the factor V gene generated

fragments of 158, 93, and 37 bp for the normal allele.

Digestion products of the mutant allele were 158 and

130 bp in size. The prothrombin fragment of 230 bp was

cleaved by HindIII in case the GA mutation was

present and yielded two smaller ones of 190 and 40 bp.

The 219-bp PCR product of the normal MTHFR allele

was also not digested by HinfI, whereas the mutantallele was characterized by two fragments, 176 and 43

bp in length.

For the PIA1/A2 polymorphism, the resultant PCR

product was digested with Msp I, generating fragments

of 275, 69, and 6 bp in case of the A1 allele and of 173,

102, 69, and 6 bp in case of the A2 allele. The 5-flanking

region and part of the first exon of the -fibrinogen gene

was enzymatically amplified by PCR as described by

Thomas et al.,14 using slightly modified oligonucle-

otides.

Statistical AnalysisResults of the two groups were compared with theMann – Whitney U-test, Fisher’s exact test, and Pear-

son’s chi-square test for categorial variables. Odds ratio

(OR) and its 95% confidence intervals (CI) were calcu-

lated. Statistical analysis was performed with the Statis-

tical Package for the Social Sciences (SPSS for

Windows 9.0, SPSS Inc., Chicago, IL).

RESULTS

Patient Demographics and Pregnancy DataThe demographic characteristics and pregnancy data of

the RSA patients and the controls are shown in Table

II. Women with recurrent abortions were significantly

older (P0.001), had a significant higher number of

pregnancies (P0.001), and less healthy children (P

0.001).

We documented 351 abortions in the RSA group. Of

the RSA patients, 65.7% had two or three abortions and

34.3% had four or more abortions. The median per

© MUNKSGAARD, 2001


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TABLE II. Demographic and Pregnancy Data of the Study Populations

All patients P value First-trimester patients P value Controls

102 75Number 128

Age 35 (22–48) 0.001 35 (22–48) 0.001 32 (18–44)

4 (2–9)Pregnancies 0.001 4 (2–9) 0.001 1 (1–4)

3 (2–9) 0.001 3 (2–8)Pregnancy Losses 0.001 0 (0–0)

First trimester 3 (0–8) 3 (2–8)

Second trimester 0 (0–3) –

61.8% (63) 61.3%(46)Primary

38.2% (39) 38.7%(29)Secondary

0 (0–4) 0.001 0 (0–4) 0.001 1 (1–4)Deliveries

All alues are medians ( range ) and percentage ( numbers ) , respecti ely.

P alues were determined by the Mann – Whitney U -test.

RSA patient was 3 (range 2 – 9), whereas, by definition,

none of the controls had abortions. The abortions wereclassified as primary in 63 patients (61.8%) and as

secondary in 39 patients (38.2%). This amounts to a

total of 217 primary and 134 secondary abortions. The

patients with secondary abortions had significantly

more pregnancies (median 5, range 3 – 9 vs. median 4,

range 2 – 9; P0.001) and, by definition, more normal

deliveries with healthy infants (median 1, range 1 – 4 vs.

median 0, range 0 – 2) than the primary aborters.

Seventy-five (73.5%) of the women had abortions

exclusively in the first trimester (at 12 weeks of

gestation), the remaining 27 (26.5%) had additional

abortions in the second trimester (gestation week 13 –

24). The first-trimester RSA patients had a total of 244abortions, 46 (61.3%) were classified as having primary

abortions, 29 (38.7%) as having secondary abortions.

Prealence of Thrombophilic MutationsEight women with RSA and 11 women of the control

group (7.9 vs. 8.6%, NS) were heterozygous for FVL,

while homozygous carriers were not detected (Table

III). The frequency of the 1691 A allele was 4.0 vs.

4.3% (NS). The PTM occurred only as the het-

erozygous form in five RSA patients and in one control

(4.9 vs. 0.8%, NS). The frequency of the 20210 A allele

was 2.5 vs. 0.4% (P=0.0265). The OR among het-

erozygous carriers for the PTM was 6.27 (95% CI

0.75 – 52.8). Heterozygosity for the MTHFR 677C/T

mutation was found in 47 RSA patients and in 61

controls (46.1 vs. 47.7%, NS), homozygosity (thermo-

labile [TL]-MTHFR) in 14 RSA patients and in 12

controls (13.7 vs. 9.4%, NS). The frequency of the

677T allele was 36.8 vs. 33.2% (NS). The 1565 C/T

(PIA1/A2) genotype of platelet GPIIIa was found in 21

RSA patients and in 31 controls (20.6 vs. 24.4%, NS),

while the 1565 T/T (PIA2/A2

) genotype was present inone RSA patient and in two controls (1.0 vs. 1.6%,

NS). The frequency of the 1565 T (PIA2) allele was 11.3

vs. 13.8% (NS). The −455 G/A genotype in the

promoter region of the -fibrinogen gene was detected

in 33 RSA patients and in 48 controls (32.4 vs. 37.5%,

NS), the −455 A/A genotype in eight RSA patients

and in 11 controls (7.8 vs. 8.6%, NS). Accordingly, the

frequency of the −455 A allele was 24.0 vs. 27.3%

(NS).

Subgroup analysis of those 75 RSA patients with

abortions occurring exclusively in the first trimester

demonstrated no significant difference in the preva-

lence of the FVL and 677C/T MTHFR mutations orof the GPIIIa and -fibrinogen polymorphisms. How-

ever, heterozygosity for the PTM occurred significantly

more often in the group of first-trimester RSA when

compared to all RSA patients as well as to the controls

(6.7 vs. 0.8%, P=0.027, Table IV). The OR was 8.53

(95% CI 1.02 – 71.7) for heterozygous carriers of the

G20210A PTM. Correspondingly, in the controls, the

prothrombin G/G genotype (wildtype) was signifi-

cantly increased (99.2 vs. 93.3%, P=0.027) and the

prevalence of the 20210A allele 3.3 vs. 0.4% (P=

0.009). Because there was from literature an a priori

basis for evaluating PTM, the P0.009 value does not

require a Benferroni correction for multiple compari-sons. No statistically significant difference existed be-

tween patients with primary and secondary abortions.

DISCUSSION

In this case – control study, the prevalence of FVL and

the TL-MTHFR was not increased in women who



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/ PIHUSCH ET AL.128

had RSA while otherwise healthy. This is in contrast

to previously published data: Ridker et al.15 found an

OR of 2.3 for FVL in 113 women with three or more

recurrent abortions before the third trimenon com-

pared to 437 postmenopausal multipara without abor-

tions. Meinardi et al.16 also reported an increased risk

of fetal loss (even single) and stillbirth in 228 carriers

of FVL compared to 121 healthy relatives. The OR

were 2.08 (95% CI 1.33 – 3.25) for loss before 20 weeksof gestation and 1.60 (95% CI 0.58 – 4.43) for stillbirth,

with homozygous carriers having an even higher risk.

In contrast to these two studies, however, our

cohort included mainly women with abortions in the

first trimester. It has been proposed that the patho-

genesis of first-trimester abortions is different from

loss of pregnancies occurring in the second and third

trimester, as profound structural and genetic abnor-

malities are common at this early stage of embryonal

development.31 This might dilute the influence of FVL

on pathogenesis of abortion in our study. Indeed, the

study of Younis et al.,25 who concentrated only onpregnancy losses after sonographic detection of a

fetal pulse, thereby excluding very early abortions

and many pregnancy losses due to failure of early

TABLE III. Prevalence of the Genotypes in all Recurrent Spontaneous Abortion Patients and Controls

P value OR (95% CI)ControlsPatients

128102Total

FVL

92.1% (93) 91.4% (117) NS1691 G/G

NS8.6% (11)7.9% (8)1691 A /G1691 A / A 0.0% (0) 0.0% (0) NS

Frequency of 1691 G 96.0% (194) 95.7% (245) NS

NSFrequency of 1691 A 4.3% (11)4.0% (8)

MTHFR mutation

40.2% (41) 43.0% (55) NS677 C/C

677 C/T 46.1% (47) 47.7% (61) NS

677 T/T 13.7% (14) 9.4% (12) NS

Frequency of 677C 63.2% (129) 66.8% (171) NS

Frequency of 677T NS33.2% (85)36.8% (75)

GPIIIa mutation

74.0% (94)78.4% (80)1565 C/C (PI A1/ A1 ) NS

1565 C/T (PI A1/ A2 ) 20.6% (21) 24.4% (31) NS

1565 T/T (PI A2/ A2 ) 1.0% (1) 1.6% (2) NS

Frequency of 1565 C 88.7% (181) NS86.2% (219)

NS13.8% (35)11.3% (23)Frequency of 1565 T

-fibrinogen mutation

−455 G/G NS59.8% (61) 53.9% (69)

−455 G/ A 32.4% (33) 37.5% (48) NS

8.6% (11)7.8% (8)−455 A / A NS

NS72.7% (186)76.0% (155)Frequency of −455 G

Frequency of −455 A 24.0% (49) 27.3% (70) NS

PTM

20210 G/G 95.1% (97) 99.2% (127) 0.0626.27 (0.75–52.8)0.0620.8% (1)4.9% (5)20210 A /G

0.0% (0)20210 A / A 0.0% (0) NS

Frequency of 20210 G 97.5% (199) 99.6% (255) 0.027

0.4% (1)2.5% (5)Frequency of 20210 A 0.027

OR, odds ratio; CI , con fidence interal ; FVL, factor V Leiden mutation; MTHFR, 5 ,10 -methylenetetrahydrofolate reductase; GPIIIa,

glycoprotein IIIa; PTM , prothrombin mutation; NS , not signi ficant.

Percentage ( number ) of patients and controls with the respecti e genotype.

© MUNKSGAARD, 2001


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TABLE IV. Prevalence of Prothrombin Genotype in Patients with First-trimester Recurrent Spontaneous Abortion

and Controls

Patients Controls P value OR (95% CI)

75 128Total

PTM

93.3% (70) 99.2% (127) 0.02720210 G/G

6.7% (5) 0.8% (1)20210 A /G 0.027 8.53 (1.02–71.7)

20210 A / A 0.0% (0) 0.0% (0) NS

96.7% (145) 99.6% (255) 0.009Frequency of 20210 G

3.3% (5) 0.4% (1) 0.009Frequency of 20210 A

OR, odds ratio; CI , con fidence interal ; PTM , prothrombin mutation.

Percentage ( number ) of patients and controls with the respecti e genotype.

NS , not signi ficant.

embryonic differentiation, was able to show an in-

creased prevalence of FVL in women who recurrently

aborted within the first trimester. However, as these

data reflect ethnic specificities and analyze a relatively

small number of highly selected patients (n=37), theymight overestimate the true role of FVL.

Perhaps more relevant, impaired placental perfusion

might not be critical for embryonic development dur-

ing very early gestation, due to very favorable placen-

tal/embryonic ratio at this stage. In agreement with

this explanation, several cohort studies17 – 23 found an

increased prevalence of FVL and TL-MTHFR only in

patients loosing their pregnancies in the second and

third trimester. Even more persuasive is the prospec-

tive study of Preston et al.24 In a large European

cohort, FVL and other coagulation inhibitor deficien-

cies (antithrombin, protein C, or protein S) increased

the risk of fetal death only after 28 weeks of gestation.The fact, that 2 consecutive abortions were used as

inclusion criterion in our study, does not seem to

explain our findings, as even those reports, which

concentrate on patients with 3 abortions within the

first trimester,15,17,18 failed to show an increased risk

with FVL or TL-MTHFR. In summary, we conclude

that, in unselected populations, FVL and TL-

MTHFR do not appear to play a relevant role for

first-trimester abortions.

In our study, we were unable to detect an in fluence

of the GPIIIa PIA2 allele on RSA. The GPIIIa PIA2/A2

has been associated with unstable angina, myocardial

infarction,26 and stent occlusion27; however, these data

have been contradicted by other studies.28,29 At

present, our data do not suggest an unfavorable influ-

ence of GPIIIa PIA2/A2 polymorphism on pregnancies.

Furthermore, our study did not reveal any effects of

the −455 G/A polymorphism in the promotor of the

-fibrinogen gene on recurrent fetal loss in the first or

second trimester. This gene polymorphism has been

associated with elevated fibrinogen levels30 and con-

secutive risk of ischemic heart disease. However, our

data do not indicate an increased risk of placental

perfusion or functional problems caused by the dis-

crete rise in fibrinogen concentrations that has beenshown for the -fibrinogen −455 G/A polymorphism.

Our study demonstrates that the prevalence of the

prothrombin 20210GA genotype is significantly in-

creased in patients with first-trimester abortions with

an OR of 8.4 and, therefore, has to be considered as

an important risk factor for RSA. In earlier studies,

Kutteh et al.,17 Gris et al.,21 and Brenner et al.22 have

identified this genotype as a risk factor for second-

and third-trimester abortions. Our significant findings

for the first trimester might be due to the composition

of the study group, as previous studies had relatively

small numbers of patients with early abortions that, in

contrast, represents 73% of the patients in our collec-tive. This may also explain the observed differences in

risk: our study revealed a much higher OR in the first

trimester than that reported for second- and third-

trimester abortions. Our findings implicate that PCR

testing for PTM might be valuable in all patients with

a history of abortions, as clinical clues for the pres-

ence of the mutation (history of thrombosis) are not

reliable and screening tests (PTM, aPTT) are normal

in these patients.

As PTM causes only a moderately thrombophilic

state,9,10 it is presently not known how it might cause

failure of first-trimester pregnancies. In contrast to

FVL, however, the PTM is associated with problems

in both venous9 and arterial system.10 In this respect,

PTM might resemble the antiphospholipid syndrome

that has been well documented to cause abortions6 as

well as arterial and venous thromboses. In addition,

increased prothrombin levels in heterozygous mothers

might not only affect plasmatic coagulation. Besides

fibrin generation, thrombin also activates platelets,



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/ PIHUSCH ET AL.130

smooth muscle cells, fibroblasts, mesangial cells, and

macrophages,32 all of which are represented within

placental tissues. Thrombin is able to induce cellular

responses, such as proliferation, chemotaxis, and inhi-

bition of neuronal outgrowth. Thus, increased

prothrombin levels might affect placental function by

influencing pivotal mechanisms, such as cell adhesion,

smooth muscle cell proliferation, and vasculogenesis.33

It might be rewarding to study these aspects inmiscarriages of mothers carrying the heterocygous

prothrombin mutation.

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