Six Different Point Mutations in Seven Danish Families with Symptomatic Protein C Deficiency

1995 ◽  
Vol 73 (02) ◽  
pp. 186-193 ◽  
Author(s):  
Bent Lind ◽  
Marianne Schwartz ◽  
Sixtus Thorsen
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Family Members ◽  
Point Mutations ◽  
Patient Specific ◽  
Missense Mutations ◽  
Coding Region ◽  
Protein C Deficiency ◽  
Epidermal Growth Factor Domain ◽  
Increased Risk

SummarySix different point mutations of the protein C gene are described in seven Danish families with protein C deficiency associated with an increased risk of venous thromboembolism. All affected family members are heterozygotes for the mutated protein C genotype. One mutation is a G2992→A transition at position +5 in the 5’ splice site of intron D. The other five mutations affect the protein coding region. One is a Cl432→T transition in exon III converting the highly conserved Arg15 to Trp in the Gla-domain. Another mutation is a G3157→C transversion in exon V converting the non-conserved Gly72 to Arg in the epidermal growth factor domain. The remaining three mutations are located in non-conserved amino acid positions in exon IX and affect the serine proteinase domain. The first is a G8559→C transversion converting Gly282 to Arg. The second is a C8571→T transition (present in two families) converting Arg286 to Cys. The third is a C8695→T transition converting Pro327 to Leu. In each family the protein C deficiency cosegregates or probably cosegregates (one family, G8559→C) with the mutation. All affected family members exhibit a reduction of both the antigen and the functional plasma concentration of protein C to approximately 50% of normal indicating that the mutated protein C is not present (type 1 deficiency) or only present in low amounts in plasma. Agarose gel electrophoresis followed by Western blotting shows that the Arg15→Trp substitution is associated with a normal as well as an abnormal migrating plasma protein C band. This provides positive evidence for that both the normal and mutated alleles are expressed (type 2 deficiency). The five other mutations are associated with only one band with the mobility of normal protein C. In one family a novel G1390→A transition converting the normal Ala1 to Thr was demonstrated. This mutation is not linked to the patient specific G8559→C transversion. In conclusion one splice site mutation and five different missense mutations of the protein C gene are described.

scholarly journals Splice site mutation in the human protein C gene associated with venous thrombosis: demonstration of exon skipping by ectopic transcript analysis

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2423-2432
Author(s):  
B Lind ◽  
WW van Solinge ◽  
M Schwartz ◽  
S Thorsen
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Family Members ◽  
Exon Skipping ◽  
Splice Site Mutation ◽  
Peripheral Blood Lymphocytes ◽  
Nucleotide Sequencing ◽  
Liver Protein ◽  
Site Mutation ◽  
Protein C Deficiency

Heterozygosity for a G-->C mutation converting the highly conserved Gln184 (CAG) to His (CAC) was identified at the last nucleotide of exon 7 of the protein C gene in two family members with deep vein thrombosis. As the nucleotide is a part of the 5 splice site of intron G, it was examined how the mutation affected splicing of protein C pre- mRNA. Relevant protein C cDNA fragments were amplified with polymerase chain reaction after reverse transcription of ectopic mRNA from peripheral blood lymphocytes. Southern blot analysis and nucleotide sequencing of these fragments showed a fragment (A) corresponding to correctly spliced mRNA originating from the normal allele and a fragment (B) corresponding to a truncated mRNA lacking exon 7, originating from the mutant allele. A third fragment (C) lacking exons 7 and 8 was identified in both affected and unaffected family members, as well as in normal controls. Analysis of human liver protein C mRNA indicated that the ectopic lymphocyte mRNA was qualitatively representative for the tissue-specific mRNA. In conclusion, evidence is provided showing that the mutation abolishes formation of correctly spliced mRNA. This agrees with the observation that the mutation results in a type 1 protein C deficiency.

scholarly journals Splice site mutation in the human protein C gene associated with venous thrombosis: demonstration of exon skipping by ectopic transcript analysis

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2423-2432 ◽  
Author(s):  
B Lind ◽  
WW van Solinge ◽  
M Schwartz ◽  
S Thorsen
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Family Members ◽  
Exon Skipping ◽  
Splice Site Mutation ◽  
Peripheral Blood Lymphocytes ◽  
Nucleotide Sequencing ◽  
Liver Protein ◽  
Site Mutation ◽  
Protein C Deficiency

Abstract Heterozygosity for a G-->C mutation converting the highly conserved Gln184 (CAG) to His (CAC) was identified at the last nucleotide of exon 7 of the protein C gene in two family members with deep vein thrombosis. As the nucleotide is a part of the 5 splice site of intron G, it was examined how the mutation affected splicing of protein C pre- mRNA. Relevant protein C cDNA fragments were amplified with polymerase chain reaction after reverse transcription of ectopic mRNA from peripheral blood lymphocytes. Southern blot analysis and nucleotide sequencing of these fragments showed a fragment (A) corresponding to correctly spliced mRNA originating from the normal allele and a fragment (B) corresponding to a truncated mRNA lacking exon 7, originating from the mutant allele. A third fragment (C) lacking exons 7 and 8 was identified in both affected and unaffected family members, as well as in normal controls. Analysis of human liver protein C mRNA indicated that the ectopic lymphocyte mRNA was qualitatively representative for the tissue-specific mRNA. In conclusion, evidence is provided showing that the mutation abolishes formation of correctly spliced mRNA. This agrees with the observation that the mutation results in a type 1 protein C deficiency.

Severe Arterial Cerebral Thrombosis in a Patient with Protein S Deficiency (Moderately Reduced Total and Markedly Reduced Free Protein S): A Family Study

1989 ◽  
Vol 61 (01) ◽  
pp. 144-147 ◽  
Author(s):  
A Girolami ◽  
P Simioni ◽  
A R Lazzaro ◽  
I Cordiano
Keyword(s):  
Arterial Thrombosis ◽  
Protein C ◽  
Protein S ◽  
Protein S Deficiency ◽  
Protein C Deficiency ◽  
Free Protein ◽  
Her Family ◽  
S Deficiency ◽  
Increased Risk ◽  
Patient Reported

SummaryDeficiency of protein S has been associated with an increased risk of thrombotic disease as already shown for protein C deficiency. Deficiencies of any of these two proteins predispose to venous thrombosis but have been only rarely associated with arterial thrombosis.In this study we describe a case of severe cerebral arterial thrombosis in a 44-year old woman with protein S deficiency. The defect was characterized by moderately reduced levels of total and markedly reduced levels of free protein S. C4b-bp level was normal. Protein C, AT III and routine coagulation tests were within the normal limits.In her family two other members showed the same defect. All the affected members had venous thrombotic manifestations, two of them at a relatively young age. No other risk factors for thrombotic episodes were present in the family members. The patient reported was treated with ASA and dipyridamole and so far there were no relapses.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

Severe Homozygous Protein C Deficiency: Identification of a Splice Site Missense Mutation (184, Q → H) in Exon 7 of the Protein C Gene

1994 ◽  
Vol 72 (01) ◽  
pp. 065-069 ◽  
Author(s):  
J M Soria ◽  
D Brito ◽  
J Barceló ◽  
J Fontcuberta ◽  
L Botero ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Gene Product ◽  
Splice Site ◽  
Protein C ◽  
Purpura Fulminans ◽  
Single Strand Conformation Polymorphism ◽  
Donor Splice Site ◽  
Protein C Deficiency ◽  
Donor Splice ◽  
Newborn Child

SummarySingle strand conformation polymorphism (SSCP) analysis of exon 7 of the protein C gene has identified a novel splice site missense mutation (184, Q → H), in a newborn child with purpura fulminans and undetectable protein C levels. The mutation, seen in the homozygous state in the child and in the heterozygous state in her mother, was characterized and found to be a G to C nucleotide substitution at the -1 position of the donor splice site of intron 7 of the protein C gene, which changes histidine 184 for glutamine (184, Q → H). According to analysis of the normal and mutated sequences, this mutation should also abolish the function of the donor splice site of intron 7 of the protein C gene. Since such a mutation is compatible with the absence of gene product in plasma and since DNA sequencing of all protein C gene exons in this patient did not reveal any other mutation, we postulate that mutation 184, Q → H results in the absence of protein C gene product in plasma, which could be the cause of the severe phenotype observed in this patient.

scholarly journals Structural Studies of TREM-2 Mutants Linked to Neurodegenerative Diseases

2014 ◽  
Vol 70 (a1) ◽  
pp. C248-C248
Author(s):  
Daniel Kober ◽  
Tom Brett
Keyword(s):  
Neurodegenerative Diseases ◽  
Neurodegenerative Disease ◽  
Structural Changes ◽  
Expression System ◽  
Point Mutations ◽  
Myeloid Cells ◽  
Surface Protein ◽  
Patient Specific ◽  
Structural Studies ◽  
Increased Risk

Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) is an extracellular surface protein expressed on myeloid cells of the monocyte/macrophage lineage including dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have revealed point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. This represents the first molecular link between inflammatory processes and neurodegenerative disease. TREM-2 modulates the innate immune inflammatory response; however, the biological ligand for TREM-2 remains elusive. As a first step towards understanding the role of TREM-2 in neurodegenerative disease, we have undertaken structural and biophysical studies of wild-type and mutant TREM-2 proteins. We developed a mammalian-cell based expression system for the successful production of TREM-2 in quantities suitable for structural studies. We have crystallized the TREM-2 Ig domain and determined the structure at 3.3 Å resolution. Analysis of this structure reveals the location of disease-linked mutations and produces hypotheses about their involvement in structural stability and ligand binding. In addition, we are studying the affect these mutations have on the stability of the protein using biochemical stability and surface expression assays. We are also pursuing structural studies of the point mutants to elucidate any structural changes caused by mutation. These studies are crucial to understanding the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.

scholarly journals Deficiency of the natural anticoagulant proteins in women with pregnancy related venous thromboembolism

2009 ◽  
Vol 62 (1-2) ◽  
pp. 53-62 ◽  
Author(s):  
Gorana Mitic ◽  
Ljubica Povazan ◽  
Radmila Lazic ◽  
Dragan Spasic ◽  
Milana Maticki-Sekulic
Keyword(s):  
Venous Thromboembolism ◽  
Protein C ◽  
Protein S ◽  
Single Woman ◽  
Control Group ◽  
Protein S Deficiency ◽  
Clotting Factors ◽  
Protein C Deficiency ◽  
S Deficiency ◽  
Increased Risk

Inherited thrombophilia can be defined as a predisposition to thrombosis caused by heritable defects, such as mutations in genes encoding the natural anticoagulants or clotting factors. Pregnancy related risk of VTE is sixfold increased comparing to non pregnant age matched women. Pregnancy is an independent risk factor for the development of venous thromboembolism and this risk is further increased by the presence of thrombophilia. Aim of the study: The aim of the study was to evaluate the association between deficiency of natural anticoagulants: antithrombin, protein C and protein S and pregnancy related thromboembolism. We have determined the activities of antithrombin, proten C and protein S in 74 women with pregnancy related thrombosis and in 45 healthy women who had at least two uncomplicated pregnancies. Among the women with the history of venous thromboembolism antithrombin deficiency was found in 4 (5.4%), protein C deficiency in 2 (2.7%) and protein S deficiency in 5 (6.76%). The total of 11 (14.6%) women was found to be deficient. Not a single woman in the control group was found to be deficient in natural anticoagulants. Deficiencies of coagulation inhibitors are associated with an increased risk of venous thrombosis during pregnancy and puerperium (p= 0.006). Antithrombin, protein C and protein S deficient women are at higher risk of developing venous thromboembolism during antepartal period (p= 0.0097). Prophylactic treatment with heparin should be recommended from the very beginning of the following pregnancy in women with antithrombin, protein C or protein S deficiency.

scholarly journals Identification of mutations in 90 of 121 consecutive symptomatic French patients with a type I protein C deficiency. The French INSERM Network on Molecular Abnormalities Responsible for Protein C and Protein S deficiencies

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2598-2605 ◽  
Author(s):  
S Gandrille ◽  
M Aiach
Keyword(s):  
Protein C ◽  
Polypeptide Chain ◽  
Large Population ◽  
Protein S ◽  
Type I ◽  
Missense Mutations ◽  
Protein C Deficiency ◽  
Molecular Abnormalities ◽  
Recurrent Mutations ◽  
Sporadic Cases

By studying the protein C gene of 121 consecutive patients with symptomatic type I protein C deficiency, we detected 55 different candidate mutations in 90 cases. The mutations, 76% of which were missense changes, were distributed throughout the gene. More than half the missense mutations involved Cys, Phe, Pro, or Gly, amino acids known to affect the structure of the polypeptide chain by various mechanisms. Thus, 40% of protein C deficiencies may be caused by polypeptide chain instability rather than a lack of expression of the mutated allele; this may also account for phenotypic heterogeneity. Seventeen of the 55 different mutations were found in apparently unrelated families. Half the French families we studied bore one of these 17 mutations. The wide variety of mutations suggests that both sporadic cases and a founder effect contribute to the spectrum of protein C mutations in a given population. The differences in both unique and recurrent mutations in French and Dutch populations-the only large population samples so far studied-support this hypothesis.

Compound Heterozygous Protein C Deficiency Caused by Two Mutations, Arg-178 to Gin and Cys-331 to Arg, Leading to Impaired Secretion of Mutant Protein C

1994 ◽  
Vol 72 (06) ◽  
pp. 814-818 ◽  
Author(s):  
Yuichi Sugahara ◽  
Osamu Miura ◽  
Shinsaku Hirosawa ◽  
Nobuo Aoki
Keyword(s):  
Disulfide Bond ◽  
Protein C ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Expression Vectors ◽  
Compound Heterozygous ◽  
Protein C Deficiency ◽  
Peptide Cleavage ◽  
Mutant Proteins ◽  
Heterozygous Protein C Deficiency

SummaryThe protein C gene in a patient apparently homozygous for protein C deficiency was analyzed. Two different point mutations, each located in a different allele, were detected to reveal that the patient is a compound heterozygote. Mutation of Arg-178 (CGG) to Gin (CAG) [mutation I] was detected in exon VII, in the vicinity of activation peptide cleavage site by thrombin. Mutation of Cys-331 (TGC) to Arg (CGC) [mutation II] was found in exon IX, at one of the sites involved in disulfide bond formation in the catalytic domain of the heavy chain. The alteration of Cys-331 to Arg disables the formation of the disulfide bond and would alter the protein conformation. Transient expression assays using COS-7 cells transfected with protein C expression vectors containing each one of these two mutations suggested that each of the two mutations would lead to the protein C deficiency by an impairment of secretion of the respective mutant proteins.

Detection of Ten New Mutations by Screening the Gene Encoding Factor IX of Danish Hemophilia B Patients

1995 ◽  
Vol 73 (05) ◽  
pp. 774-778 ◽  
Author(s):  
Marianne Schwartz ◽  
Jørgen Ingerslev ◽  
Elma Scheibel ◽  
Lise Rud Nielsen
Keyword(s):  
Point Mutations ◽  
Splice Site Mutation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Missense Mutations ◽  
Coding Region ◽  
Site Mutation ◽  
Gene Encoding ◽  
Base Pair Deletion ◽  
Wide Range

SummaryHemophilia B is caused by a wide range of mutations. In order to characterize the mutations among patients in Denmark, we have systematically screened the entire coding region, the promoter region and exon flanking sequences of the gene encoding factor IX using single strand conformation and heteroduplex analyses. Patients from 32 different families were examined, and point mutations (23 different) were found in all of them. Ten of the mutations have not been reported by others; they include a splice site mutation, a single base pair deletion, and missense mutations. Notably, the study contains a female patient and a previously described Leyden mutation. In ten families with sporadic cases of hemophilia B, all 10 mothers were found to be carriers. The origin of two of these mutations was established.

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