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Coagulation and Transfusion Medicine / Protein S Deficiency

Genotype and Laboratory and Clinical Phenotypes of Protein S Deficiency

Sebastian Duebgen, MD, 1 Teresa Kauke, MD, 1 Christoph Marschall, PhD, 2 Andreas Giebl, MD, 1 Susanne Lison, MD, 1 Christina Hart, MD, 3 Andrea Dick, PhD, 1 and Michael Spannagl, MD 1

Key Words: Protein S deficiency; PROS1; Genotype; Phenotype; Laboratory assessment

DOI: 10.1309/AJCP40UXNBTXGKUX

Upon completion of this activity you will be able to:

• distinguish conditions of acquired protein S deficiency from situations in which hereditary deficiency seems probable.

• decide under which circumstances mutational analysis of the PROS1 gene is recommendable and whether normal findings in sequence analysis should be followed by deletion diagnostics (multiplex ligation- dependent probe amplification).

• provide advice about the significance of a PROS1 mutation to asymptomatic affected family members.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 316. Exam is located at www.ascp.org/ajcpcme.

Abstract

The diagnosis of thrombophilia caused by protein

S deficiency remains difficult. From 2005 to 2010, we

documented 135 patients with suspected hereditary protein S deficiency for whom mutational analysis of the PROS1 gene had been performed by direct double-stranded sequencing of the amplified 15 exons including splice sites. Multiplex ligation-dependent probe amplification was performed on 12 of 15 exons in cases with no mutation found but a large deletion in the PROS1 gene was suspected. Mutations were identified in 49 patients, 9 by familial screening. Altogether, 17 new and 11 previously described mutations of PROS1 were identified among the 49 patients. After the exclusion of acquired protein S deficiency due to

pregnancy or hormonal contraceptives, there remained only 1 case with protein S activity levels less than 40% that could not be explained by sequence variations or deletions in the examined regions of the PROS1 gene. After the exclusion of conditions associated with acquired protein S deficiency, persistently low protein

S activity levels are highly indicative of a genetic

alteration in PROS1. We observed a clear correlation

between the laboratory phenotype and the type of mutation.

Protein S (gene symbol, PROS1; GeneID, 5627; MIM No. 176880) was first described in 1979 by DiScipio and Davie 1 as a glycoprotein containing γ-carboxylated glutamic acid residues similar to other proteins involved in coagulation processes. In contrast with other vitamin K–dependent clot- ting factors, protein S, which was named after the place of its first isolation, Seattle, WA, lacks serine protease activity. Further studies demonstrated that it has cofactor function for activated protein C. Under normal conditions, more than 60% of protein S is bound to the C4b binding protein, and unbound protein S is available to form a complex with activated pro- tein C in the presence of phospholipids and calcium ions. This complex irreversibly inactivates factors Va and VIIIa by proteolysis, thus interfering with the formation of the pro- thrombinase and tenase complexes on procoagulant surfaces. Thrombin inactivates protein S by proteolytic cleavage. Two highly homologous genes are located near the cen- tromeric region of chromosome 3, at 3p11.1-3q11.2: one is the active, encoding gene PROS1, and the other, PROS2 or PROSP, is, in all likelihood, a pseudogene with no open read- ing frame. PROS2 lacks exon 1, which contains the transla- tional start site and encodes for a signal peptide. Furthermore, there are several destructive mutations contained in PROS2. 2 Owing to the cofactor function of protein S, the laboratory phenotype is difficult to assess. Typically, measurement is per- formed using a latex ligand immunoassay for the determina- tion of free antigen and using clotting assays that detect protein S–mediated enhancement of the anticoagulant function of acti- vated protein C. In addition to congenital deficiencies, there are numerous physiologic and pathologic conditions that lead to decreased protein S levels. Inflammation leads to increased

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C4b binding protein levels and concomitantly decreases the levels of unbound and functional protein S. Pregnancy and

hepatic disorders, such as hepatitis and cirrhosis, and treat- ment with hormonal contraceptives 3 and the lingering effect

of previous anticoagulation similarly lead to decreased levels.

Materials and Methods

Patients From 2005 to 2010, 5,851 patients with thrombophilia

or the suspicion of an inherited thrombophilia attended the hemostasis outpatient clinics of the University of Munich, Munich, Germany, for diagnostic studies. The suspicion of familial protein S deficiency was raised in 170 cases, and the patients were registered into a database. Of these, 135 patients consented to molecular analysis of the PROS1 gene. All patients came from a Caucasian background. In addition

Coagulation and Transfusion Medicine / Original Article

PROS1 Gene Analysis

Following the protocol proposed by Ten Kate et al, 6 all 15 exons of the PROS1 gene were screened for PROS1 sequence

variants by direct sequencing. In addition, multiplex ligation- dependent probe amplification (MLPA) 7 was performed to detect target sequence copy number changes in cases in which no mutation was detected by direct sequencing and repeatedly low protein S values suggested a genetically determined defi- ciency. For MLPA, we used the SALSA KIT P112 PROS1 from MRC-Holland, Amsterdam, the Netherlands. 8 In contrast with DNA sequencing, MLPA focuses on copy number changes. Before exponential amplification by polymerase chain reaction (PCR), the MLPA probes hybrid- ize with adjacent regions of the DNA and are ligated using

a thermostable ligase. Subsequently, PCR is used to amplify

the original probes, and copy number changes are evaluated by electrophoresis by comparing the relative amounts of the products. The probe mix used for the detection of deletions in the PROS1 gene contained probes for 12 of 15 PROS1 exons.

to

assessments of the concentration of the free antigen, protein

Deletions in exons 3, 8, and 14 could not be detected with this

S

activity, and prothrombin time, several other patient char-

MLPA system. Two probes were present for exon 1, and one

acteristics were collected, including medical history, family history, other explaining risk factors (factor V Leiden and pro- thrombin G20210A mutant, antiphospholipid antibodies), and other causes of acquired protein S deficiencies (pregnancy, hormonal contraception, chronic systemic inflammation).

probe each for a short distance 5' of the PROS1 promoter and for the PROS pseudogene were included. In addition, 16 ref- erence probes that detect several different autosomal chromo- somal locations were included in the probe mix. Heterozygous deletions of recognition sequences were expected to produce

Protein S Assays

Stago, Asnières, France). 5 This clotting assay detects protein

35% to 50% reduced relative peak area for the amplification product of that probe.

a

The measurement of protein S was performed by the determination of free antigen and by measuring protein S

Results

activity. Free protein S was quantified using the HemosIL Free Protein S latex ligand immunoassay 4 (Instrumentation Laboratory SpA, Milan, Italy). Protein S activity was mea- sured using the STA Protein S Clotting assay (Diagnostica

PROS1 gene analysis was performed in 135 patients with suspected protein S deficiency Table 1 . There were 85 patients with venous thromboembolism (VTE). In this group, 46 patients were determined to have plasma protein S activ-

S

activity as the enhancement of protein C activity in a system

ity levels lower than 60%, and the corresponding mutations

enriched for factor Va, resulting in a prolonged clotting time.

were found in 28 of them (17 unrelated persons, 4 pairs of

Table 1Overview of the Examined Collective Sorted by Clinical and Laboratory Phenotype *

Laboratory Phenotype

Clinical Phenotype

Pathologic Protein S Activity < 60%

Unachievable Protein S Activity Due to Intake of VKA but Suspicion of Protein S Deficiency Owing to (Family) History

Normal Protein S Activity but Reported Former Deficiency or Family History of Protein S Deficiency

Total

Own VTE VTE in family history Stroke/TIA Abortion Healthy, no family history of VTE Total

28/46 (61)

7/27 (26)

1/12 (8)

36/85 (42)

10/23 (43)

0/0 (0)

0/8 (0)

10/31 (32)

1/5 (20)

0/4 (0)

0/0 (0)

1/9 (11)

0/0 (0)

0/0 (0)

0/1 (0)

0/1 (0)

1/8 (13)

0/0 (0)

1/1 (100)

2/9 (22)

40/82 (49)

7/31 (23)

2/22 (9)

49/135 (36.3)

TIA, transient ischemic attack; VKA, vitamin K antagonist; VTE, venous thromboembolism. * Data are given as number of PROS1 mutations found/number of patients (percentage).

© American Society for Clinical Pathology

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Protein S–Free Antigen (%)

Protein S Activity (%)

Duebgen et al / Protein S Deficiency

120

 
120   100 80     60     40 20     0

100

100
100
120   100 80     60     40 20     0

80

80  
80  
 
 
 

60

60  
 
 

40

40

20

   

0

120

 

100

100
100

80

 
 
 
 

60

60

40

40
40

20

20

0

 

No

Missense

Nonsense

Large

No

Missense

Nonsense

Large

Mutation

Mutation

Mutation

Deletion

Mutation

Mutation

Mutation

Deletion

Type of Mutation

 

Type of Mutation

Figure 1Protein S activity denoted as percentage of the standard according to the different types of mutation, with each dot representing a single case. The corresponding popu- lation means and standard deviations are shown in Table 2.

relatives, and 1 family with 3 members). The remaining 39 patients with VTE were receiving oral anticoagulation (n = 27) or had borderline values but had family or medical histo- ries that suggested protein S deficiency (n = 12). In 8 of these cases, mutations in the PROS1 gene were identified. Because of pathologic protein S levels or familial protein S deficiency, 31 patients who themselves had no pathology but did have VTE in their family histories were genetically examined. A total of 10 patients (2 unrelated persons and 4 couples of rela- tives) had mutations in the PROS1 gene. A separate group of 9 patients had a history of cerebrovascular infarction or repeated transient ischemic attacks. One patient with low protein S activity was revealed to have a corresponding mutation in the PROS1 gene. One patient had recurrent abortion and border- line values for protein S, but no mutation could be verified. Finally, 2 cases from a group of 9 patients who had no clinical manifestations in their own or their family histories and who were referred to us because of low protein S levels showed mutations in the PROS1 gene.

Laboratory Phenotype of Protein S Deficiency The following phenotypic analysis excluded patients receiving vitamin K antagonist treatment, patients who were pregnant or receiving hormonal contraceptives, and patients with other causes of acquired protein S deficiency. Figure

1 shows the protein S activity levels of the remaining 82 patients, grouped according to results of genetic analysis.

Figure 2 Protein S free antigen denoted as percentage of

the standard according to the different types of mutation, with each dot representing a single case. The corresponding popu- lation means and standard deviations are shown in Table 3.

There were significant differences in the levels of resid- ual activity when grouped by the type of mutation. Patients without mutations detectable by the test system typically showed protein S activity levels more than 40%. One case in which mutations were not detectable showed persistently low levels. Whether a deletion existed in exons 3, 8, and 14 in this case could not be examined. Nonsense mutations and large deletions were linked to the strongest reduction of the level of activity. The 2 missense mutations that did not show patho- logic activities were the previously unknown polymorphisms c.1016T>A and c.1138A>C. The mean values and SDs are shown in Table 2 . Free antigen levels of protein S are depicted in Figure

2 according to the type of mutation. The corresponding mean values and SDs are shown in Table 3 . As shown in

Table 2Population Means and SDs of Protein S Activities According to Type of Mutation *

Protein S Activity (%)

Type of Mutation

No. of Patients

μ ± σ

No mutation

44

58 ± 16 45 ± 17 24 ± 6.3 26 ± 10

Missense

24

Nonsense

8

Large deletion

6

* Patients taking a vitamin K antagonist or hormonal contraceptives or who were pregnant were excluded.

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Table 3Population Means and SDs of Protein S Free Antigen According to Type of Mutation *

Protein S Free Antigen (%)

Type of Mutation

No. of Patients

μ ± σ

No mutation

42

63 ± 19 47 ± 24 29 ± 23 19 ± 6.6

Missense

23

Nonsense

8

Large deletion

6

* Patients taking a vitamin K antagonist or hormonal contraceptives or who were pregnannt were excluded.

Table 4 , 8-17 normal and pathologic findings for free antigen levels in different patients with the same mutation are not unusual. Similar to measurement of activity nonsense muta- tions and large deletions were linked to the strongest reduction of free antigen concentration.

Genetic Background of Protein S Deficiency By using sequence analysis and multiplex ligation- dependent probe amplification, we identified mutations in

Coagulation and Transfusion Medicine / Original Article

49 patients from 35 families. We were able to genetically characterize 7 new and 8 previously described missense muta- tions with amino acid exchanges, 2 unknown and 1 previously described base pair exchanges resulting in a stop codon, 3 unknown frame shift mutations, and 4 new and 1 previously known splice site mutations. Large deletions were identified by MLPA in 4 families. In 1 family, we characterized a previ- ously unknown deletion of exon 9 and the bordering intron regions (Table 4).

Discussion

The purpose of our study was to assess genotypes and laboratory and clinical phenotypes in patients with hereditary protein S deficiency. During 5 years, in our collection of 5,851 single cases that were referred to our hemostasis out- patient clinics because of their own or familial thrombophilia, suspicion of protein S deficiency arose in 170 patients. Of these, 135 were examined genetically, and mutations were identified in 49. Although the database included laboratory and clinical parameters, the total significance of protein S

Table 4Laboratory, Clinical, and Genetic Data for Patients With Mutations in PROS1

 

Protein S

 

Family

Already

Case

Activity

Free Antigen

Clinical

History

Type of

Nucleotide

Amino Acid

Listed

No.

Patients *

(%)

(%)

Manifestations

of VTE

Mutation

Exon

Exchange

Exchange

in HGMD

1

1

46

56

Asymptomatic

Yes

Missense

2

c.121C>T

p.Arg41Cys

No

2

2

30-52

54-108

DVT

Yes

Missense

2

c.122G>A

p.Arg41His

Yes

9

3

1

31

30

DVT

No

Missense

2

c.200A>C

p.Glu67Ala

Yes

9

4

3 + 1

38-53

44-77

DVT/PE

Yes

Missense

2

c.233C>T

p.Thr78Met

Yes

9

5

1

+ 1

39

62

DVT

No

Missense

6

c.556T>C

p.Cys186Arg

No

6

1

39

33

DVT

Yes

Missense

6

c.557G>A

p.Cys186Tyr

Yes

10

7

1

36

56

DVT/PE

No

Missense

7

c.701A>G

p.Tyr234Cys

Yes

11

8

1

38

38

DVT

Yes

Missense

7

c.727G>C

p.Asp243His

Yes

12

9

1

79

76

DVT

Yes

Missense

10

c.1016T>A

p.Leu339Gln

No

10 2

41

18

DVT

Yes

Missense

10

c.1085A>G

p.Gln362Arg

No

11 1

107

Asymptomatic

No

Missense

10

c.1138A>C

p.Asn380His

No

12 1

55

57

Asymptomatic

No

Missense

11

c.1252A>T

p.Asn418Tyr

No

13 1

1 +

34-59

35-52

Asymptomatic

Yes

Missense

13

c.1501T>C

p.Ser501Pro

Yes

13

14 3 + 3

29-39

15-30

DVT/PE/CVT

Yes

Missense

13

c.1543C>T

p.Arg515Cys

Yes

14

15 2

46-49

29-76

DVT

Yes

Missense

14

c.1676T>C

p.Ile559Thr

No

16 1

28

71

DVT

No

Nonsense

2

c.100C>T

p.Gln34X

No

17 1

VKA

VKA

DVT/PE/CVT

No

Nonsense

2

c.150-152delA

p.Lys50AsnfsX77

No

18 1

VKA

VKA

DVT

No

Nonsense

Intron 9

c.965+1delG

p.IVS9+1delG

No

19 1

VKA

VKA

DVT/PE

Yes

Nonsense

Intron 9

c.965+4A>G

p.IVS9+4A>G

No

20 1

25

23

DVT

Yes

Nonsense

Intron 10

c.1155+1G>A

p.IVS10+1G>A

No

21 1

18

21

DVT

No

Nonsense

Intron 10

c.1155+5G>A

p.IVS10+5G>A

Yes 15

22 1

29

16

DVT

Yes

Nonsense

11

c.1168G>T

p.Glu390X

No

23 1

VKA

VKA

PE

Yes

Nonsense

11

c.1244dupA

p.Pro416AlafsX22 No

 

24 2 + 1

13-25

11-16

DVT/PE

Yes

Nonsense

12

c.1351C>T

p.Arg451X

Yes 16

25 1

32

17

DVT/PE

Yes

Nonsense

Intron 13

c.1645-1G>A

p.IVS13-1G>A

No

26 3

27

60

DVT

Yes

Nonsense

14

c.1570delC

p.Leu524fsX525

No

1 +

27 1 + 2

13-27

10-26

DVT/PE

Yes

Large deletion

Entire gene

delPROS1

delPROS1

Yes 8,17

28 2

33-41

17-25

DVT/PE

Yes

Large deletion

9

c.891-?_

ex9del

No

965+?del

CVT, cerebral venous sinus thrombosis; DVT, deep vein thrombosis; HGMD, Human Gene Mutation Database; PE, pulmonary embolism; VKA, intake of vitamin K antagonist.

*

Patients from different families are separated by +. Nomenclature according to the Human Genome Variation Society.

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Duebgen et al / Protein S Deficiency

deficiency in our collection can only be estimated owing to

vitamin K antagonist intake. Owing to the prevalence of oral anticoagulants, protein S deficiency as a cause for throm- bophilia is presumably underdiagnosed. The prevalence of familial protein S deficiency was estimated to be between 0.03% and 0.13% by Dykes et al 18 in a Scottish study that examined 3,788 healthy volunteers. Others have presumed higher frequencies of approximately 2% in the general popu- lation 19 and 1% to 13% in patients with thrombosis. 20 The clinical phenotype of symptomatic protein S defi- ciency is recurrent deep vein thrombosis and pulmonary embolism. Case reports have suggested an association with warfarin necrosis. 21 There is no clear evidence for protein

S deficiency being a risk factor for arterial infarction, eg,

cerebral insult, even in the case of patent foramen ovale. 22-24 In approximately half of the cases with VTE, the event is unprovoked and is not preceded by a risk situation, such as air travel, hormonal contraception or replacement therapy, preg- nancy or puerperium, immobilization, surgery, or trauma. 25 However, in a prospective cohort study by Sanson et al 26 with 70 asymptomatic carriers of a protein S deficiency, the annual incidence of an initiating thromboembolic event seemed to

be 0.4% per year. In comparison, antithrombin and protein C

deficiency showed yearly incidence rates of 1.6% and 1.0%, respectively, in the same study. Because venous thromboembolic events in the patient or

family history are typically the features that lead to referral to our hemostasis outpatient clinics, a selection bias should exist

in our collection. Therefore, a comparison between different

groups within this collection would be uninformative. Our results show, however, that in patients with borderline results for coagulation testing, anamnesis for VTE is crucial for the assessment of pretest probability of mutational analysis. Following the analysis of the data for 135 patients for whom the suspicion of protein S deficiency had been raised and who consented to genetic testing, there remained only 1 case with protein S activity values less than 40% that could not be explained by mutation or deletion in the PROS1 gene using this test system. Whether in this case with known severe familial protein S deficiency a single deletion in the unscreened exons 3, 8, and 14 exists or whether in rare cases other genetic defects can lead to that phenotype is unclear. Thus, our findings support the observation by Mulder et al 27

that low cutoff values increase the diagnostic performance of protein S assays. For a long time, the observation of patients with a consistent history of familial protein S deficiency and no pathologic finding in PROS1 sequence analysis has puzzled

the experts. However, gene linkage analysis by Lanke et al 28 revealed an association with the locus of PROS1. Johansson

et al 17 detected large deletions in the PROS1 gene in 3 of 8

families studied by using quantitative PCR. The introduction

of MLPA simplified the detection of large deletions within the

genome. By this method, Pintao et al 8 found large deletions of the PROS1 gene in 6 of 18 patients with protein S deficiency who were negative for sequence variants. In our patient col- lection, large deletions were found in 6 patients from 4 differ- ent families. As mentioned, there remained only 1 case with activity levels lower than 40% that could not be explained by sequence variation or deletion in the PROS1 gene. Therefore, we affirm the statement that large deletions are a common finding in patients who test negatively for PROS1 sequence variations and who have consistently low levels of protein S. The phenotypic expression of protein S deficiency has conventionally been described as 1 of 3 types. A lack of free protein and activity is classified as type I. Patients with type II deficiency have normal levels of free antigen but diminished activity, and this type is usually attributed to missense muta-

tions. 29 Type III has been described as normal total protein levels but reduced free protein and, hence, reduced activity. However, it could not be shown that type III deficiency has

a causative effect on thrombosis. 30 As changes from one type

to another are observable not only in different persons in the

same family but also in the same person at different times, this classification is questionable. One reason for these changes might be that testing in hemostasis is prone to comparatively high interassay, batch-to-batch, and day-to-day variability such that these fluctuations may obscure existing differences between selected groups. Furthermore, the heterogeneity of PROS1 mutations contradicts the notion of accurately defined states of deficiency. In our opinion, the laboratory phenotype

of protein S deficiency is a continual decrease in activity and

free antigen, and this decrease can show great differences not only between patients with the same mutation but also at dif- ferent times in the same person. However, from our results, we can assert a clear decrease in protein and activity levels

in the genetically defined categories ranging from borderline

values in patients in whom no genetic changes were observ- able to missense and nonsense mutations to repeatedly low values in patients with large deletions. With regard to the many difficulties the diagnosis of protein S deficiency implies, Marlar and Gausman 31 recently

proposed a diagnostic algorithm for the assessment of protein

S abnormalities. We agree with the points that the evalua-

tion of the pretest probability is the most important item and that, ideally, tests with pathologic results should be repeated after 4 to 6 weeks. However, we challenge the point that the measurement of free antigen concentrations should precede activity testing as a criterion for inclusion in further testing. There are not enough data to show that antigen binding is more sensitive than coagulation tests for protein S deficiency.

As protein S activity reflects the function of the protein, we consider its measurement also necessary for exclusion of pro- tein S deficiency.

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Conclusion

Protein S deficiency is a known risk factor for VTE,

although its real impact and relevance in our thrombosis col- lection is unclear owing to the widespread intake of vitamin

K antagonists. In terms of protein S deficiency as a risk factor

for arterial thrombosis, there is scant evidence in the form of case reports. The diagnosis of familial protein S deficiency remains difficult because numerous conditions lead to acquired defi- ciency status, and the assessment of the laboratory phenotype

is awkward, with wide variations owing to batch specificities

and unstable reagents. The deterioration of the PROS1 gene

is the leading cause of familial protein S deficiency. Since the

introduction of screening methods for large deletions, there remain few cases in which no detectable sequence variation or deletion in the PROS1 gene can explain the hereditary deficiency. The classical categorization of types I through III by antigen and activity levels overestimates the performance of the available test systems and is, therefore, questionable. As

the deficiency of free antigen and activity correlates with the type of mutation in the PROS1 gene, a corresponding grading

of protein S deficiency would be more meaningful.

From the 1 Department of Hemostasis and Transfusion Medicine, University of Munich, Munich, Germany; 2 Center for Human Genetics and Laboratory Medicine, Dr. Klein & Dr. Rost, Martinsried, Germany; and 3 Department of Haematology/ Oncology, University of Regensburg, Regensburg, Germany.

Address reprint requests to Dr Duebgen: Hemostasis Outpatient Clinics, Munich University Hospital, Ziemssenstr 1, D-80336 Munich, Germany.

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