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, MD1
Key Words: Protein S deficiency; PROS1; Genotype; Phenotype; Laboratory assessment
DOI: 10.1309/AJCP40UXNBTXGKUX

CME/SAM

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 ligationdependent 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 Davie1 as a glycoprotein containing -carboxylated glutamic acid residues similar to other proteins involved in coagulation processes. In contrast with other vitamin Kdependent clotting 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 protein 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 prothrombinase and tenase complexes on procoagulant surfaces. Thrombin inactivates protein S by proteolytic cleavage. Two highly homologous genes are located near the centromeric 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 reading frame. PROS2 lacks exon 1, which contains the translational 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 performed using a latex ligand immunoassay for the determination of free antigen and using clotting assays that detect protein Smediated enhancement of the anticoagulant function of activated 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 treatment with hormonal contraceptives3 and the lingering effect of previous anticoagulation similarly lead to decreased levels.

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 ligationdependent 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 deficiency. 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 hybridize 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. Deletions in exons 3, 8, and 14 could not be detected with this MLPA system. Two probes were present for exon 1, and one probe each for a short distance 5' of the PROS1 promoter and for the PROS pseudogene were included. In addition, 16 reference probes that detect several different autosomal chromosomal locations were included in the probe mix. Heterozygous deletions of recognition sequences were expected to produce a 35% to 50% reduced relative peak area for the amplification product of that probe.

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 to assessments of the concentration of the free antigen, protein S activity, and prothrombin time, several other patient characteristics were collected, including medical history, family history, other explaining risk factors (factor V Leiden and prothrombin G20210A mutant, antiphospholipid antibodies), and other causes of acquired protein S deficiencies (pregnancy, hormonal contraception, chronic systemic inflammation). Protein S Assays The measurement of protein S was performed by the determination of free antigen and by measuring protein S activity. Free protein S was quantified using the HemosIL Free Protein S latex ligand immunoassay4 (Instrumentation Laboratory SpA, Milan, Italy). Protein S activity was measured using the STA Protein S Clotting assay (Diagnostica Stago, Asnires, France).5 This clotting assay detects protein S activity as the enhancement of protein C activity in a system enriched for factor Va, resulting in a prolonged clotting time.

Results
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 activity levels lower than 60%, and the corresponding mutations were found in 28 of them (17 unrelated persons, 4 pairs of

Table 1 Overview of the Examined Collective Sorted by Clinical and Laboratory Phenotype*
Laboratory Phenotype Unachievable Protein S Activity Due to Intake of VKA but Suspicion of Protein S Deficiency Owing to (Family) History 7/27 (26) 0/0 (0) 0/4 (0) 0/0 (0) 0/0 (0) 7/31 (23) Normal Protein S Activity but Reported Former Deficiency or Family History of Protein S Deficiency Total 1/12 (8) 0/8 (0) 0/0 (0) 0/1 (0) 1/1 (100) 2/22 (9) 36/85 (42) 10/31 (32) 1/9 (11) 0/1 (0) 2/9 (22) 49/135 (36.3)

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

Pathologic Protein S Activity < 60% 28/46 (61) 10/23 (43) 1/5 (20) 0/0 (0) 1/8 (13) 40/82 (49)

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

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120

120

100 Protein SFree Antigen (%) No Missense Nonsense Large Mutation Mutation Mutation Deletion Type of Mutation

100

Protein S Activity (%)

80

80

60

60

40

40

20

20

0 No Missense Nonsense Large Mutation Mutation Mutation Deletion Type of Mutation

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

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 population means and standard deviations are shown in Table 3.

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 histories 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 relatives) 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 borderline 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.
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There were significant differences in the levels of residual 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 pathologic 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 2 Population Means and SDs of Protein S Activities According to Type of Mutation*
Protein S Activity (%) Type of Mutation No mutation Missense Nonsense Large deletion
*

No. of Patients 44 24 8 6

58 16 45 17 24 6.3 26 10

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

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Table 3 Population Means and SDs of Protein S Free Antigen According to Type of Mutation*
Protein S Free Antigen (%) Type of Mutation No mutation Missense Nonsense Large deletion
*

No. of Patients 42 23 8 6

63 19 47 24 29 23 19 6.6

49 patients from 35 families. We were able to genetically characterize 7 new and 8 previously described missense mutations 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 previously unknown deletion of exon 9 and the bordering intron regions (Table 4).

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

Discussion
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 mutations 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 ligationdependent probe amplification, we identified mutations in

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 outpatient 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 4 Laboratory, Clinical, and Genetic Data for Patients With Mutations in PROS1
Protein S Case No. Patients*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 2 1 3+1 1+1 1 1 1 1 2 1 1 1+1 3+3 2 1 1 1 1 1 1 1 1 2+1 1 3 1+1+2 2

Activity (%)
46 30-52 31 38-53 39 39 36 38 79 41 107 55 34-59 29-39 46-49 28 VKA VKA VKA 25 18 29 VKA 13-25 32 27 13-27 33-41

Free Antigen (%)

56 54-108 30 44-77 62 33 56 38 76 18 57 35-52 15-30 29-76 71 VKA VKA VKA 23 21 16 VKA 11-16 17 60 10-26 17-25

Clinical Manifestations
Asymptomatic DVT DVT DVT/PE DVT DVT DVT/PE DVT DVT DVT Asymptomatic Asymptomatic Asymptomatic DVT/PE/CVT DVT DVT DVT/PE/CVT DVT DVT/PE DVT DVT DVT PE DVT/PE DVT/PE DVT DVT/PE DVT/PE

Family History Type of of VTE Mutation

Yes Yes No Yes No Yes No Yes Yes Yes No No Yes Yes Yes No No No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Nonsense Large deletion Large deletion

Exon
2 2 2 2 6 6 7 7 10 10 10 11 13 13 14 2 2 Intron 9 Intron 9 Intron 10 Intron 10 11 11 12 Intron 13 14 Entire gene 9

Nucleotide Exchange
c.121C>T c.122G>A c.200A>C c.233C>T c.556T>C c.557G>A c.701A>G c.727G>C c.1016T>A c.1085A>G c.1138A>C c.1252A>T c.1501T>C c.1543C>T c.1676T>C c.100C>T c.150-152delA c.965+1delG c.965+4A>G c.1155+1G>A c.1155+5G>A c.1168G>T c.1244dupA c.1351C>T c.1645-1G>A c.1570delC delPROS1 c.891-?_ 965+?del

Amino Acid Exchange

p.Arg41Cys p.Arg41His p.Glu67Ala p.Thr78Met p.Cys186Arg p.Cys186Tyr p.Tyr234Cys p.Asp243His p.Leu339Gln p.Gln362Arg p.Asn380His p.Asn418Tyr p.Ser501Pro p.Arg515Cys p.Ile559Thr p.Gln34X p.Lys50AsnfsX77 p.IVS9+1delG p.IVS9+4A>G p.IVS10+1G>A p.IVS10+5G>A p.Glu390X p.Pro416AlafsX22 p.Arg451X p.IVS13-1G>A p.Leu524fsX525 delPROS1 ex9del

Already Listed in HGMD

No Yes9 Yes9 Yes9 No Yes10 Yes11 Yes12 No No No No Yes13 Yes14 No No No No No No Yes15 No No Yes16 No No Yes8,17 No

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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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 thrombophilia is presumably underdiagnosed. The prevalence of familial protein S deficiency was estimated to be between 0.03% and 0.13% by Dykes et al18 in a Scottish study that examined 3,788 healthy volunteers. Others have presumed higher frequencies of approximately 2% in the general population19 and 1% to 13% in patients with thrombosis.20 The clinical phenotype of symptomatic protein S deficiency 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, pregnancy or puerperium, immobilization, surgery, or trauma.25 However, in a prospective cohort study by Sanson et al26 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 al27 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 al28 revealed an association with the locus of PROS1. Johansson et al17 detected large deletions in the PROS1 gene in 3 of 8 families studied by using quantitative PCR. The introduction
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of MLPA simplified the detection of large deletions within the genome. By this method, Pintao et al8 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 collection, large deletions were found in 6 patients from 4 different 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 mutations.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 different 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 observable 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 Gausman31 recently proposed a diagnostic algorithm for the assessment of protein S abnormalities. We agree with the points that the evaluation 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 protein 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 collection 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 deficiency 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 1Department of Hemostasis and Transfusion Medicine, University of Munich, Munich, Germany; 2Center for Human Genetics and Laboratory Medicine, Dr. Klein & Dr. Rost, Martinsried, Germany; and 3Department 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.

References
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25. ten Kate MK, van der Meer J. Protein S deficiency: a clinical perspective. Haemophilia. 2008;14:1222-1228. 26. Sanson BJ, Simioni P, Tormene D, et al. The incidence of venous thromboembolism in asymptomatic carriers of a deficiency of antithrombin, protein C, or protein S: a prospective cohort study. Blood. 1999;94:3702-3706. 27. Mulder R, Ten Kate MK, Kluin-Nelemans HC, et al. Low cut-off values increase diagnostic performance of protein S assays. Thromb Haemost. 2010;104:618-625. 28. Lanke E, Johansson AM, Hillarp A, et al. Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1. J Thromb Haemost. 2004;2:1918-1923.

29. Garca de Frutos P, Fuentes-Prior P, Hurtado B, et al. Molecular basis of protein S deficiency. Thromb Haemost. 2007;98:543-556. 30. Libourel EJ, Bank I, Veeger NJ, et al. Protein S type III deficiency is no risk factor for venous and arterial thromboembolism in 168 thrombophilic families: a retrospective study. Blood Coagul Fibrinolysis. 2005;16:135140. 31. Marlar RA, Gausman JN. Protein S abnormalities: a diagnostic nightmare. Am J Hematol. 2011;86:418-421.

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