Identification of Two Novel Point Mutations in the Human Protein S Gene Associated With Familial Protein S Deficiency and Thrombosis

Individuals with thrombosis who were believed to possess associated familial protein S deficiency were analyzed for mutations in the protein S gene by a two-step process. First, the individuals were analyzed for protein S Pro 626 A/G dimorphism in both their genomic DNA and reverse-transcribed (RT) polymerase chain reaction (PCR)–amplified cDNA from peripheral blood cell mRNA. If a heterozygote expressed both alleles at the mRNA level at this site in genomic DNA, a search for point mutations was made by direct cDNA sequencing. RT-PCR amplification of exons 1-6 with mRNA from two twin sisters, each of whom has severe type I protein S deficiency, revealed both larger and smaller fragments in addition to the expected 504–base pair fragment in normal individuals. A donor splice-site mutation at position +4 of the 5′ end of intron A was subsequently identified in both sisters and their mother. This mutation would lead to incorrect precursor mRNA splicing and the observed cDNA products. Translation of the altered mRNAs would result in a truncated protein without biological activity. In a second family, cDNA sequencing revealed a T→G mutation at codon 603 (Ile→Ser) in exon 15 of the protein S gene in an individual with protein S deficiency (mixed type) and a history of thrombosis. The same mutation was also detected in the proband's mother and grandmother, both of whom also exhibit protein S deficiency and thrombotic disease. This mutation occurs within a disulfide loop of protein S that is believed to be responsible for binding to C4b binding protein and may result in greater affinity between protein S and C4b, consequently leading to thrombotic disease.

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A hitherto unknown splice site defect in the protein S gene (PROS1): the mutation results in allelic exclusion and causes type I and type III protein S deficiency

British Journal of Haematology ◽

10.1046/j.1365-2141.1997.3883202.x ◽

1997 ◽

Vol 99 (2) ◽

pp. 298-300 ◽

Cited By ~ 4

Author(s):

Stefan Mustafa ◽

Ingrid Pabinger ◽

Katalin Varadi ◽

Walter-Michael Halbmayer ◽

Klaus Lechner ◽

...

Keyword(s):

Splice Site ◽

Protein S ◽

Allelic Exclusion ◽

Type I ◽

Protein S Deficiency ◽

Type Iii ◽

S Gene ◽

S Deficiency

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A Novel Splice Acceptor Site Mutation which Produces Multiple Splicing Abnormalities Resulting in Protein S Deficiency Type I

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614631 ◽

1999 ◽

Vol 82 (07) ◽

pp. 65-71 ◽

Cited By ~ 14

Author(s):

Hideki Tatewaki ◽

Hiroko Iida ◽

Mutsuko Nakahara ◽

Hiroko Tsuda ◽

Sachiko Kinoshita ◽

...

Keyword(s):

Molecular Mechanisms ◽

Protein S ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Type I ◽

Protein S Deficiency ◽

Site Mutation ◽

Exon 2 ◽

S Gene ◽

S Deficiency

SummaryIn an attempt to explore the molecular mechanisms for protein S deficiency, a patient with such a deficiency was examined at the DNA, RNA and protein levels. Nucleotide analyses revealed that the proband, the mother and the grandmother had a G → C substitution in the invariant AG dinucleotide at the splicing acceptor site of intron A/exon 2. This patient was heterozygous for this substitution and the mutant allele was inherited from the proband’s mother and grandmother. Reverse transcription-polymerase chain reaction analysis demonstrated several kinds of splicing abnormalities such as exon skipping and cryptic splicing, in addition to correct splicing. Semiquantitation of mRNA for the protein S gene revealed that the amount of the proband’s mRNA was reduced to 60% of normal. Thus, this mutation impaired the normal processing of mRNA for the protein S gene, resulting in the subject’s severe protein S deficiency.

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Identification of Eight Point Mutations in Protein S Deficiency Type I – Analysis of 15 Pedigrees

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653863 ◽

1995 ◽

Vol 73 (05) ◽

pp. 750-755 ◽

Cited By ~ 16

Author(s):

P H Reitsma ◽

E Gómez ◽

S R Poort ◽

R M Bertina

Keyword(s):

Direct Sequencing ◽

Molecular Genetic ◽

Point Mutations ◽

Protein S ◽

Type I ◽

Protein S Deficiency ◽

Genetic Abnormality ◽

Genetic Studies ◽

S Deficiency ◽

Deficiency Type

SummaryWe describe molecular genetic studies of 15 patients with protein S deficiency type I (i. e. reduced total protein S antigen). All the exons of the PROS 1 gene were analyzed both by PCR and direct sequencing in all 15 probands. This analysis led to the identification of point mutations affecting eight individuals. One of these mutations (codon -25, insertion of T) has been described previously in a Dutch pedigree. The other mutations are novel and all are located in exons that code for the protein S domain that is homologous to the steroid hormone binding globulins. They include two amino acid replacements (one individual with 340 Gly → Val, and two individuals with 467 → Val Gly), and four frameshift mutations due to either one bp deletions (in codon 261 deletion of T and in codon 267 deletion of G) or insertions (in codon 565 insertion T and after codon 578 insertion of C). Studies performed in six families (totalling 43 subjects) showed cosegregation of the genetic abnormality with reduced plasma protein S levels, and provided genetic evidence for a heterozygous protein S deficiency in 25 of them. The yield of mutations in this study (53%) confirms that the percentage of protein S deficient cases in which a point mutation is found remains low.

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Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency

British Journal of Haematology ◽

10.1111/j.1365-2141.1993.tb08662.x ◽

1993 ◽

Vol 85 (1) ◽

pp. 173-175 ◽

Cited By ~ 6

Author(s):

G. Marchetti ◽

C. Legnani ◽

P. Patracchini ◽

D. Gemmati ◽

M. Ferrati ◽

...

Keyword(s):

Gene Polymorphism ◽

Mrna Level ◽

Protein S ◽

Protein S Deficiency ◽

S Gene ◽

S Deficiency

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The Frequency of Type I Heterozygous Protein S and Protein C Deficiency in 141 Unrelated Young Patients with Venous Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642558 ◽

1988 ◽

Vol 59 (01) ◽

pp. 018-022 ◽

Cited By ~ 139

Author(s):

C L Gladson ◽

I Scharrer ◽

V Hach ◽

K H Beck ◽

J H Griffin

Keyword(s):

Venous Thrombosis ◽

Protein C ◽

Antithrombin Iii ◽

Young Patients ◽

Protein S ◽

Type I ◽

Protein S Deficiency ◽

Protein C Deficiency ◽

Low Protein ◽

S Deficiency

SummaryThe frequency of heterozygous protein C and protein S deficiency, detected by measuring total plasma antigen, in a group (n = 141) of young unrelated patients (<45 years old) with venous thrombotic disease was studied and compared to that of antithrombin III, fibrinogen, and plasminogen deficiencies. Among 91 patients not receiving oral anticoagulants, six had low protein S antigen levels and one had a low protein C antigen level. Among 50 patients receiving oral anticoagulant therapy, abnormally low ratios of protein S or C to other vitamin K-dependent factors were presented by one patient for protein S and five for protein C. Thus, heterozygous Type I protein S deficiency appeared in seven of 141 patients (5%) and heterozygous Type I protein C deficiency in six of 141 patients (4%). Eleven of thirteen deficient patients had recurrent venous thrombosis. In this group of 141 patients, 1% had an identifiable fibrinogen abnormality, 2% a plasminogen abnormality, and 3% an antithrombin III deficiency. Thus, among the known plasma protein deficiencies associated with venous thrombosis, protein S and protein C. deficiencies (9%) emerge as the leading identifiable associated abnormalities.

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A Mutation in the Protein S Pseudogene Is Linked to Protein S Deficiency in a Thrombophilic Family

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651024 ◽

1989 ◽

Vol 62 (03) ◽

pp. 897-901 ◽

Cited By ~ 10

Author(s):

Hans K Ploos van Amstel ◽

Pieter H Reitsma ◽

Karly Hamulyák ◽

Christine E M de Die-Smulders ◽

Pier M Mannucci ◽

...

Keyword(s):

Total Protein ◽

Protein S ◽

Southern Analysis ◽

Type I ◽

Protein S Deficiency ◽

S Deficiency ◽

The Family ◽

Dna Pattern ◽

Deficiency Type ◽

S Genes

SummaryProbands from 15 unrelated families with hereditary protein S deficiency type I, that is having a plasma total protein S concentration fifty percent of normal, were screened for abnormalities in their protein S genes by Southern analysis. Two probands were found to have a deviating DNA pattern with the restriction enzyme Mspl. In the two patients the alteration concerned the disappearance of a Mspl restriction site, CCGG, giving rise to an additional hybridizing Mspl fragment.Analysis of relatives of both probands showed that in one family the mutation does not co-segregate with the phenotype of reduced plasma protein S. In the family of the other proband, however, complete linkage between the mutated gene pattern and the reduced total protein S concentration was found: 12 heterozygous relatives showed the additional Mspl fragment but none of the investigated 26 normal members of the family. The mutation is shown to reside in the PSβ gene, the inactive protein S gene. The cause of type I protein S deficiency, a defect PSα gene has escaped detection by Southern analysis. No recombination has occurred between the PSα gene and the PSβ gene in 23 informative meioses. This suggests that the two protein S genes, located near the centromere of chromosome 3, are within 4 centiMorgan of each other.

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Protein S mRNA in Patients with Protein S Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653862 ◽

1995 ◽

Vol 73 (05) ◽

pp. 746-749 ◽

Cited By ~ 5

Author(s):

E Sacchi ◽

M Pinotti ◽

G Marchetti ◽

G Merati ◽

L Tagliabue ◽

...

Keyword(s):

Gene Polymorphism ◽

Total Protein ◽

Restriction Analysis ◽

Protein S ◽

Type I ◽

Protein S Deficiency ◽

Phenotypic Analysis ◽

S Deficiency ◽

Deficiency Type ◽

S Genes

SummaryA protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) from free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.

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