A Novel Splice Acceptor Site Mutation of Protein S Gene in Affected Individuals with Type I Protein S Deficiency: Allelic Exclusion of the Mutant Gene

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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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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Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy

Blood ◽

10.1182/blood.v86.7.2632.2632 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2632-2641 ◽

Cited By ~ 16

Author(s):

CJ Formstone ◽

AI Wacey ◽

LP Berg ◽

S Rahman ◽

D Bevan ◽

...

Keyword(s):

Splice Site ◽

Mutation Screening ◽

Protein S ◽

Allelic Exclusion ◽

Type I ◽

Protein S Deficiency ◽

Rt Pcr ◽

S Deficiency ◽

Polymerase Chain ◽

Reverse Transcript

Abstract The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele (“allelic exclusion”). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position-1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

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Protein S deficiency type I: identification of point mutations in 9 of 10 families

Blood ◽

10.1182/blood.v86.9.3444.bloodjournal8693444 ◽

1995 ◽

Vol 86 (9) ◽

pp. 3444-3451 ◽

Cited By ~ 25

Author(s):

S Mustafa ◽

I Pabinger ◽

C Mannhalter

Keyword(s):

Stop Codon ◽

Direct Sequencing ◽

Protein S ◽

Type I ◽

Missense Mutations ◽

Protein S Deficiency ◽

Site Mutation ◽

Frame Shift ◽

S Deficiency ◽

Deficiency Type

We identified potentially causative mutations in the active protein S gene (PROS 1) by direct sequencing of PROS 1-specific polymerase chain reaction (PRC) products of all 15 exons, including exon-intron boundaries in 10 families with hereditary protein S deficiency type I. Seven different mutations were found in 9 of 10 families, including one frame shift mutation, a previously published splice site mutation (both occurring in two unrelated families), four missense mutations, and a stop codon at the beginning of exon 12. In family studies, cosegregation of the mutation with the disease could be demonstrated for five mutations; for two missense mutations, this was not possible due to limited family data. All seven mutations were the only abnormalities identified in the respective index patients and were absent in 44 to 62 normal individuals. Therefore, they most likely represent the causal gene defects. For five mutations, analysis of ectopic RNA could be performed. Mutant transcripts were present in the case of the frame shift and three of the missense mutations, while no mutant RNA could be detected in the case of the stop codon.

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Poor Relationship between Phenotypes of Protein S Deficiency and Mutations in the Protein S Alpha Gene

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614891 ◽

1999 ◽

Vol 82 (12) ◽

pp. 1634-1638 ◽

Cited By ~ 13

Author(s):

José Hermida ◽

Pier Mannuccio Mannucci ◽

Elena Faioni

Keyword(s):

Splice Site ◽

Genetic Defect ◽

Splice Site Mutation ◽

Protein S ◽

Nucleotide Sequencing ◽

Type I ◽

Protein S Deficiency ◽

Site Mutation ◽

Single Strand Conformational Polymorphism ◽

S Deficiency

SummaryBy single strand conformational polymorphism, nucleotide sequencing and enzyme restriction, we analyzed the protein S α gene in 17 protein S-deficient probands and in their available family members. The relationship between genotype and phenotype was also evaluated. Twelve different sequence variations were identified in 17 probands. Ten were putative causal mutations distributed in 16 probands: 4 were nonsense, 5 missense and one a splice site mutation. In most families in which a mutation was identified, more than one phenotype of PS deficiency was present. The same splice site mutation (intron j G-A, exon 10+5) was associated with type I deficiency in one family and with type I/III in another unrelated family. A phenotypic discrepancy was also observed for the Arg474Pro, Gly597Asp and Arg410stop mutations. Glu26Ala, previously reported in kindreds with type I deficiencies, was found in association with I, II and III phenotypes in four unrelated kindreds. Phenotypic analysis of protein S deficiency is poorly related to the underlying genetic defect.

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Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy

Blood ◽

10.1182/blood.v86.7.2632.bloodjournal8672632 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2632-2641 ◽

Cited By ~ 1

Author(s):

CJ Formstone ◽

AI Wacey ◽

LP Berg ◽

S Rahman ◽

D Bevan ◽

...

Keyword(s):

Splice Site ◽

Mutation Screening ◽

Protein S ◽

Allelic Exclusion ◽

Type I ◽

Protein S Deficiency ◽

Rt Pcr ◽

S Deficiency ◽

Polymerase Chain ◽

Reverse Transcript

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele (“allelic exclusion”). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position-1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

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A Novel Mutation in Intron K of the PROS1 Gene Causes Aberrant RNA Splicing and Is a Common Cause of Protein S Deficiency in a UK Thrombophilia Cohort

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615020 ◽

1998 ◽

Vol 79 (06) ◽

pp. 1086-1091 ◽

Cited By ~ 7

Author(s):

M. E. Daly ◽

M. Makris ◽

F. E. Preston ◽

I. R. Peake ◽

N. J. Beauchamp

Keyword(s):

Rna Splicing ◽

Novel Mutation ◽

Thrombotic Event ◽

Protein S ◽

Acceptor Site ◽

Type I ◽

Protein S Deficiency ◽

S Deficiency ◽

A Minor ◽

Aberrant Rna

SummaryIn the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.

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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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