Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

scholarly journals Molecular cloning of a cDNA encoding canine factor IX

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 207-212 ◽  
Author(s):  
JP Evans ◽  
HH Watzke ◽  
JL Ware ◽  
DW Stafford ◽  
KA High
Keyword(s):  
Gene Transfer ◽  
Amino Acid Level ◽  
Protein A ◽  
Factor Ix ◽  
Hemophilia B ◽  
Leader Peptide ◽  
Causative Mutation ◽  
Coding Region ◽  
Isolation And Characterization

Abstract Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.

scholarly journals Molecular cloning of a cDNA encoding canine factor IX

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 207-212 ◽  
Author(s):  
JP Evans ◽  
HH Watzke ◽  
JL Ware ◽  
DW Stafford ◽  
KA High
Keyword(s):  
Gene Transfer ◽  
Amino Acid Level ◽  
Protein A ◽  
Factor Ix ◽  
Hemophilia B ◽  
Leader Peptide ◽  
Causative Mutation ◽  
Coding Region ◽  
Isolation And Characterization

Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.

scholarly journals Characterization of a New Alloantigen (SH) on the Human Neutrophil Fcγreceptor IIIb

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
Juergen Bux ◽  
Ernst-Ludwig Stein ◽  
Philippe Bierling ◽  
Patricia Fromont ◽  
Mary Clay ◽  
...  
Keyword(s):  
Nucleotide Sequence Analysis ◽  
Polymorphism Analysis ◽  
Mutational Event ◽  
Coding Region ◽  
Specific Pcr ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Antigen Frequency ◽  
Neonatal Neutropenia

Abstract Polymorphic structures of the neutrophil Fcγreceptor IIIb (FcγRIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on FcγRIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody–specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the FcγRIIIb. Nucleotide sequence analysis of the FcγRIIIb coding region from a SH(+) individual showed a single-base C→A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN I showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the FcγRIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the FcγRIIIb. Our findings show the existence of an additional polymorphism of the FcγRIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.

Identification and characterization of undifferentiated mast cells in mouse bone marrow

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4282-4289 ◽  
Author(s):  
Maria Célia Jamur ◽  
Ana Cristina G. Grodzki ◽  
Elsa H. Berenstein ◽  
Majed M. Hamawy ◽  
Reuben P. Siraganian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Immunoglobulin E ◽  
Mouse Bone Marrow ◽  
Immunomagnetic Isolation ◽  
Polymerase Chain ◽  
Identification And Characterization ◽  
Mast Cell Population

Abstract Sequential immunomagnetic isolation with 2 monoclonal antibodies was used to purify and characterize an undifferentiated mast cell in adult mouse bone marrow that had not been previously recognized. This cell represents 0.02% of the cells in the bone marrow, is CD34+, CD13+, and c-kit+, and does not express FcϵRI. However, by polymerase chain reaction (PCR) the cell contains message for the α and β subunits of FcϵRI, mast cell–specific proteases, and carboxypeptidase A. Morphologically, this cell has a large nucleus, little cytoplasm, few cytoplasmic organelles, and no cytoplasmic granules. In vitro, in the presence of interleukin-3 (IL-3) and stem cell factor (SCF) these cells differentiate only into a granulated mast cell that now expresses CD13, c-kit, mast cell–specific gangliosides, FcϵRI, and binds immunoglobulin E (IgE). When injected into lethally irradiated mice, these cells are able to reconstitute the mast cell population in the spleen.

scholarly journals Identification and functional characterization of a missense mutation in resistin in two patients with severe obesity and insulin resistance

2011 ◽  
Vol 164 (6) ◽  
pp. 927-936 ◽  
Author(s):  
Sigri Beckers ◽  
Fenna de Freitas ◽  
Doreen Zegers ◽  
An Verrijken ◽  
Armand V Peeters ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Functional Characterization ◽  
Mutant Protein ◽  
Morbidly Obese ◽  
Male Body ◽  
Coding Region ◽  
Obese Adults ◽  
Obese Mother

ObjectiveIn this study, we hypothesized that mutations in the resistin encoding gene, RETN, may cause a monogenic form of obesity.Design/methodsWe screened the coding region of RETN in 81 morbidly obese adults, 263 overweight and obese children/adolescents, and 116 healthy lean subjects. In vitro experiments include qPCR, ELISA, and western blot for WT and mutant resistin transfected into 3T3-L1 adipocytes.ResultsMutation analysis identified five sequence variants in our patient populations: 3′-UTR +87 G/A, 3′-UTR +100 A/G, T73T, IV3-61 C/A, and C78S. In our control population, we only found the 3′-UTR +87 G/A variant. We started functional experiments for the C78S mutation that was found in a 20-year-old obese male (body mass index (BMI)=39.7 kg/m2) and his obese mother (BMI=31.9 kg/m2). In vitro testing demonstrated that the mutation does not impair mRNA expression. We identified a 100-fold lower extracellular protein concentration for mutant resistin compared with WT levels using a resistin ELISA on cell culture medium (P=4.87×10−6). We also detected a decreased intracellular concentration for the mutant protein (tenfold lower relative levels, P=0.007). The plasma resistin levels of the proband and his mother, however, did not differ significantly from lean control individuals.ConclusionsIn conclusion, we identified the first missense mutation in resistin in a morbidly obese proband and his obese mother. Functional testing of the mutant protein suggests that the C78S mutant protein is degraded, possibly resulting in a decreased extracellular concentration, which may predispose to obesity.

scholarly journals Construction, Rescue, and Characterization of Vectors Derived from Ovine Atadenovirus

2003 ◽  
Vol 77 (22) ◽  
pp. 11941-11951 ◽  
Author(s):  
Peter Löser ◽  
Christian Hofmann ◽  
Gerald W. Both ◽  
Wolfgang Uckert ◽  
Moritz Hillgenberg
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Human Factor ◽  
Insertion Site ◽  
Factor Ix ◽  
Vector System ◽  
Virus Rescue ◽  
Human Factor Ix

ABSTRACT Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

Six Novel and Three Recurrent Mutations in Nine Austrian Patients with Hemophilia B

1994 ◽  
Vol 72 (01) ◽  
pp. 074-077 ◽  
Author(s):  
J Walter ◽  
I Pabinger-Fasching ◽  
H H Watzke
Keyword(s):  
Direct Sequencing ◽  
Point Mutations ◽  
Factor Ix ◽  
Hemophilia B ◽  
Causative Mutation ◽  
Genetic Defects ◽  
Missense Mutations ◽  
Enzymatic Amplification ◽  
Recurrent Mutations ◽  
Polymerase Chain

SummaryIn this report we describe the molecular basis of the factor IX (FIX) deficiency in nine patients with severe (n = 6), moderate (n = 1) or mild (n = 2) hemophilia B. The following genetic defects were identified by enzymatic amplification with the polymerase chain reaction (PCR) and subsequent direct sequencing of all exons and exon-intron-junctions: patient B.B. (FIX “Vienna I”): deletion of nucleotides 6343 to 6362; patient M.H. and W. J. (FIX “Vienna II”): nucleotide 17704 (C to G), Gin 97 to Glu; patient L. K. (FIX “Vienna III”): nucleotide 17761 (C to T), Arg 116 to stop; patient U. A. (FIX “Vienna IV”): nucleotide 10415 (C to G), Pro 55 to Ala; patient H.G. (FIX “Vienna V”): nucleotide 6488 (C to T), Thr 38 to lie; patient H. M. (FIX “Vienna VI”): nucleotide 31276 (G to C), Trp 385 to Cys; patient L. C. (FIX “Vienna VII”): deletion of nucleotide 6700; patient S.F. (FIX “Vienna VIII”): nucleotide 10392 (A to T), Asp 47 to Val. The causative mutation was detected in the FIX gene in each of the nine patients with hemophilia B. There was one small deletion, one point deletion and seven point mutations. The latter include six missense mutations and one nonsense mutation. The mutations in Vienna III, IV and V have already been described in previous studies. The two deletions, Vienna I and Vienna VII have not been reported previously. The genetic defects observed in Vienna II, VI and VIII are novel missense mutations which result in amino acid changes at residues 97,47 and 385, respectively.

In vitro characterization of K5A and K5R variants of Factor IX

2021 ◽  
Author(s):  
S Knoll Machado ◽  
T Kraushaar ◽  
J Röder ◽  
P Claar ◽  
C Hardy ◽  
...  
Keyword(s):  
Factor Ix ◽  
In Vitro Characterization

Truncation of Glycoprotein (GP) IIIa (▵ 616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4712-4720 ◽  
Author(s):  
Milagros Ferrer ◽  
Jianming Tao ◽  
Gema Iruı́n ◽  
Matilde Sánchez-Ayuso ◽  
José González-Rodrı́guez ◽  
...  
Keyword(s):  
Complex Formation ◽  
Novel Mutation ◽  
Molecular Genetic ◽  
Coding Region ◽  
Chain Reaction ◽  
Molecular Genetic Study ◽  
Nonsense Mutations ◽  
Glanzmann Thrombasthenia ◽  
Polymerase Chain ◽  
Exon 11

This work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846→T transversion in exon 11 of GPIIIa that changes Glu616→Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846→T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-▵GPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from in-frame skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon.

scholarly journals The Malmo polymorphism of factor IX: establishing the genotypes by rapid analysis of DNA

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2104-2107 ◽  
Author(s):  
JB Graham ◽  
GR Kunkel ◽  
GS Tennyson ◽  
ST Lord ◽  
DM Fowlkes
Keyword(s):  
Population Studies ◽  
Coagulation Factor ◽  
Rapid Analysis ◽  
Factor Ix ◽  
Small Samples ◽  
Coding Region ◽  
Chain Reaction ◽  
X Ray ◽  
Polymerase Chain ◽  
Enzyme Recognition

Abstract A DNA polymorphism in the coding region of coagulation factor IX-- potentially valuable for carrier detection, prenatal diagnosis, and population studies--was described in 1985. It had been discovered with monoclonal antibodies that distinguish between threonine and alanine as the 148th residue of the peptide. Its use as a diagnostic tool has been limited because threonine-containing factor IX (Malmo A) is dominant to alanine-containing factor IX (Malmo B) in immunoassays of plasma; therefore, detection of Malmo heterozygotes is not possible in all instances. A DNA method for recognizing all heterozygotes has been developed, but it also has limitations. We report the development of another DNA procedure based on amplification of the relevant DNA with the polymerase chain reaction (PCR). This method is quick, avoids the use of isotopes and x-ray film, and specifically identifies all the Malmo genotypes: hemizygotes, homozygotes, and heterozygotes. The procedure can be performed satisfactorily on small samples of blood (less than 1 mL) as suggested by Kogan et al (N Engl J Med 317:985, 1987). The method described is applicable to any genetic polymorphism that overlaps a restriction enzyme recognition site.

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