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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4608-4615
By
From INSERM U.428, Paris, France; and the Laboratoire
d'Hémostase, Hôpital Henri Mondor, Créteil, France.
The genomic analysis of a 70-year-old man with recurrent deep venous
thrombosis having a protein S (PS)-deficient phenotype corresponding to
both type III and type II evidenced two different mutations: a +5
g
PROTEIN S (PS), A VITAMIN K-dependent
glycoprotein, is a cofactor of the protein C system, an important
natural anticoagulant mechanism. In the presence of PS, activated
protein C (APC) inactivates factor Va (FVa) and factor VIIIa (FVIIIa)
and thereby reduces thrombin generation (for review, see
Esmon1). By binding to FVa2 or
FXa,3 PS can also directly inhibit prothrombin activation as well as the formation of prothrombinase complexes on phospholipid surfaces.4 In human plasma, approximately 60% of PS is
bound to the complement component C4b-binding protein (C4b-BP), and only the free form has APC-cofactor activity. PS deficiency is transmitted as an autosomal dominant trait, and heterozygous subjects belonging to families with the disorder are at risk of recurrent thromboembolic disease in adulthood.5 Severe and early
thrombotic complications can occur in patients with very low PS levels.
However, only one homozygous PS deficiency associated with severe
purpura fulminans in the neonate period has been characterized
genotypically.6 According to the International Society on
Thrombosis and Hemostasis (ISTH) standardization
subcommittee, three types of PS deficiency can be defined on the basis
of total PS levels, free PS levels, and APC cofactor activity. Type I
deficiency is characterized by low total and free PS antigen levels.
Type II deficiency is characterized by normal total and free PS antigen
levels and low APC cofactor activity. Type III is defined by
selectively reduced levels of free PS antigen and activity. However,
two recent reports suggested that type I and type III are two
phenotypic expressions of the same genetic defect.5,7
Two homologous genes for PS map to chromosome 3.8 The
active gene, PROS1, spans over 80 kb and comprises 15 exons. The second
gene, PROS2, has no open reading frame; shows multiple base changes,
stop codons, and frameshifts; and is probably a pseudogene.9-11 The 5 Ninety mutations, including 2 large deletions, have so far been
identified in the PROS1 gene as causal defects.12 Most are point mutations generating nonsense or missense changes in coding or
splice site sequences. Microinsertions/deletions have also been
reported to result in frameshifts and premature stop codons. Most of
these genetic defects cosegregate with type I PS deficiency. So far,
few mutations have been shown to give rise to type III PS
deficiency.13-16 The single point 460 TCC PS type II deficiency is fairly infrequent, and only 5 mutations have
been identified in patients with normal PS concentrations and low APC
cofactor activity. Interestingly, all 5 mutations giving rise to a type
II phenotype are located in the amino-terminal part of PS, which is
homologous to that of other vitamin K-dependent proteins and encodes
the domains interacting with APC.18 Among the 5 mutations
leading to normal expression of nonfunctional PS, 2 are located in the
propeptide at positions We report the first case of type II PS deficiency due to a splice site
mutation resulting in two alternative splice transcripts lacking either
exon 5 or both exons 5 and 6. The variant PS, characterized by plasma
immunoblot and monoclonal antibody (MoAb)-based assays, is probably a
truncated protein lacking EGF1.
Blood Sampling
Assays for PS
Molecular Biology Techniques DNA analysis.
Genomic DNA was isolated from peripheral blood leukocytes by using the
procedure described by Bell et al.24 The PS genes of
patients I1 and II1 were analyzed according to Gandrille et al.19 The 15 exons and intron/exon junctions of PROS1 were
selectively amplified in a polymerase chain reaction (PCR) by applying
the principle of the amplification-refractory mutation system (ARMS). The sequences of the primers (Genset, Paris, France) used in this study
are presented in Table 1. The amplified
fragments were submitted to denaturing gradient gel electrophoresis
(DGGE). Fragments with abnormal behavior were asymmetrically amplified
and sequenced according to the method of Sanger et al,25
except for exon 13, which was analyzed by restriction enzyme analysis.
PS gene exon 13 was amplified using nucleotides specific for PROS 1. The upstream primer sequence 5
RNA extraction.
Platelet-rich plasma (PRP) was prepared from 60 mL of whole blood
collected in EDTA by centrifugation at 125g for 10 minutes. Platelets were collected by centrifugation at 1,800g for 10 minutes, and after elimination of the supernatant, RNA was extracted by using a monophasic solution containing phenol and guanidinium isothiocyanate (Trizol; GIBCO BRL, Cergy-Pontoise, France) according to
the manufacturer's instructions and kept at Reverse transcription (RT) and cDNA amplification. Platelet or liver mRNA (500 to 1,000 ng) was added to 750 ng of hexamers (dp N6; Pharmacia Fine Chemicals, Uppsala, Sweden) and distilled water to a final volume of 20 µL. The mixture was heated to 75°C for 3 minutes and then kept at +4°C. Four microliters of 5× RT buffer (250 mmol/L Tris HCl, pH 8.3, 375 mmol/L KCl, 15 mmol/L MgCl2), 20 U of ribonuclease inhibitor (Promega, Madison, WI), and 1 µL of dATP, dCTP, dGTP, and dTTP (10 mmol/L) from Pharmacia were added and incubated at 42°C for 10 minutes before the addition of 5 U of reverse transcriptase (Moloney murine leukemia virus [M-MLV] Reverse Superscript; GIBCO BRL). After 50 minutes at 42°C, the enzyme was denatured at 94°C for 5 minutes. Then, 20 µL of the RNA-DNA hybrid solution was added to 80 µL of the PCR mixture containing 50 pmol of upstream and downstream primers, 1× PCR buffer (10 mmol/L Tris HCl, pH 8.3, 150 mmol/L KCl, 0.01% [wt/vol] gelatin, and 1.5 mmol/L MgCl2), and 2.5 U of Taq polymerase (Perkin Elmer Cetus Instruments, Norwalk, CT), and the mixture was subjected to PCR. PCR products were analyzed on 6% polyacrylamide gel stained with ethidium bromide solution. Amplified fragments with abnormal molecular weights were excised from the gel and heated at 85°C for 10 minutes in PCR 1× buffer. The genetic material was sequenced as described by Sanger et al.25 Plasma Studies Polyacrylamide gel electrophoresis and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 4% to 12% gradient gels according to Laemmli.26 After electrophoresis in 0.02 mol/L Tris, 0.2 mol/L glycine, 0.5% SDS, proteins were transferred to 0.45-µm nitrocellulose membranes (Biorad, Hercules, CA) using the transblot electrophoretic transfer cell (Biorad) at 150 V for 2 hours. The membranes were then incubated overnight with 5% (wt/vol) nonfat dry milk and 0.5% (vol/vol) Tween 20 in 100 mmol/L Tris, 150 mmol/L NaCl, pH 7.5 (TBS). PS was detected by using polyclonal rabbit anti-PS serum (Assera PS; Stago) and shown with biotinylated antirabbit IgG (Vector Laboratories, Burlingame, CA) diluted according to the manufacturer's instructions. The Vectastain ABC Elit kit (Vector Laboratories) was used to increase the sensitivity of the method. Assay of PS using the MoAb S-12. We used an ELISA method described by Duchemin et al,13 with slight modifications. Control and patient plasma was tested at two dilutions (1:2,000 and 1:4,000). The standard curve was established by using serial dilutions of 1:1,000 to 1:32,000 of normal pooled plasma. Assay of PS using the MoAb HPS 54.
Plates were coated overnight at room temperature with 100 µL of 1:40
rabbit polyclonal anti-PS antibody (Assera PS; Stago) in 50 mmol/L
Na2CO3, pH 9. After this step and all the
following steps, extensive washing in 20 mmol/L Tris, 150 mmol/L NaCl,
pH 7.5, 1% (vol/vol) Tween 20 (washing buffer) was performed.
TBS-bovine serum albumin (BSA; 5%, 200 µL) was added to each well
for 2 hours at room temperature, and then 100 µL of samples diluted
in 0.1% TBS-BSA (1:400 and 1:800) was allowed to react for 18 hours at room temperature. A pool of normal plasma diluted 1:200 to 1:6,400 was
used for the calibration curve. Bound PS was allowed to react for 4 hours with 100 µL of HPS 54 MoAb (50 µg/ml) and then 100 µL of
goat antimouse IgG antibody conjugated to peroxidase (Biorad; 1:5,000)
was added for 1 hour, followed by 100 µL of 3,3
Case Report The proband (I1), a 70-year-old man with a history of deep vein thrombosis, pulmonary embolism, and myocardial infarction since the age of 23, had been on oral anticoagulants for the previous 10 years. He agreed to switch to low molecular weight (LMW) heparin for 1 month to allow us to measure plasma PS concentrations in the absence of vitamin K antagonist. In the two different blood samples obtained during this time, the total PS concentration was borderline (70%), the free PS concentration was moderately decreased (50%), and PS activity was unambiguously below normal (27%). One of his sons (II1), who had had an episode of deep vein thrombosis at the age of 35, was available for laboratory explorations. He repeatedly had a typical type II phenotype with a total PS concentration of 100% and a free PS concentration of 97%, whereas the functional activity was 30% of normal. Neither subject bore the factor V Arg 506 to Gln mutation that might interfere with PS activity measurement.27Identification of Two Different Mutations in Genomic DNA DNA coding regions and exon/intron junction sequences from the propositus (I1) and his son (II1) were screened by DGGE. In patient I1, abnormal migration was observed with exon 5 and exon 13 of the PROS1 gene. Direct sequencing of exon 5 and its flanking regions showed a g a transition in the fifth basepair of the donor splice site
of intron e (ivs e, +5 g a; Fig
1). The DGGE profile of exon 13 was that observed in subjects bearing
the Ser 460 Pro mutation in previous studies (data not shown).
Cleavage of the amplified fragment by the restriction endonuclease
Rsa I13 confirmed the T C transition at
the first nucleotide of codon 460, leading to the replacement of Ser by
Pro in the protein sequence (data not shown).
Identification of Abnormal cDNA Transcripts To determine whether the ivs e, +5 g a substitution was
responsible for the phenotypic expression of a plasma variant, we searched for mutated transcripts in platelet PS mRNAs from patient II1,
who bore only this mutation and had a type II PS phenotype.
Identification of Abnormal PS in Plasma
DNA analysis of the 15 exons and intron/exon junctions of the
PROS1 gene of a PS-deficient patient showed two different genetic abnormalities located on different alleles. The first mutation was a g
to a substitution on the fifth nucleotide of the donor splice site of
intron e (ivs e, +5, g Submitted October 8, 1997;
accepted February 12, 1998.
The authors are grateful to Prof B. Dahlbäck for providing the
HPS 54 MoAb and to Prof R. Bertina for providing the S12
MoAb.
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