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2.
Thromb Haemost ; 86(4): 1023-7, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11686319

ABSTRACT

We report the identification of a new mutation resulting in type I antithrombin (AT) deficiency and the mechanism by which the deficiency arose. The single base substitution of G to A at nucleotide 2709 was identified in a proband with a family history of venous thrombosis. The mutation results in a substitution of 82 Ser by Asn, creating a new glycosylation site. Expression studies were then carried out, to confirm Asn-linked glycosylation occurred at this consensus site and that this resulted in the AT deficient phenotype. Cell-free translations using rabbit reticulocyte lysate in the presence of microsomes demonstrated that the 82 Asn variant was post-translationally processed efficiently. The 82 Asn variant protein was of a higher molecular weight than normal AT. consistent with the addition of a fifth glycan chain. Incubation of translation product with endoglycosidase H, confirmed that the higher molecular weight product had resulted from additional carbohydrate. Expression of the 82 Asn variant in COS-7 cells resulted in intracellular accumulation, with a low level of secretion of the protein into culture supernatant, consistent with type I AT deficiency. The addition of an extra carbohydrate side chain to residue 82 of antithrombin may block post-translational folding. trapping the variant intracellulary.


Subject(s)
Amino Acid Substitution , Antithrombin III Deficiency/genetics , Antithrombin III/genetics , Mutation, Missense , Point Mutation , Protein Processing, Post-Translational , Thrombophilia/genetics , Venous Thrombosis/etiology , Adolescent , Adult , Animals , Antithrombin III/biosynthesis , Antithrombin III/metabolism , Antithrombin III Deficiency/classification , COS Cells , Cell-Free System , Chlorocebus aethiops , Exocytosis , Female , Glycosylation , Humans , Male , Molecular Weight , Mutagenesis, Site-Directed , Pedigree , Protein Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Substrate Specificity , Transfection , Venous Thrombosis/genetics
4.
Blood ; 93(12): 4242-7, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10361121

ABSTRACT

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (approximately 70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of beta antithrombin (the very high affinity fraction) greatly increased (approximately 20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of beta antithrombin in regulating coagulation.


Subject(s)
Antithrombin III Deficiency/genetics , Antithrombin III/genetics , Asparagine/genetics , Mutation , Threonine/genetics , Adult , Amino Acid Sequence , Antithrombin III/chemistry , Antithrombins/analysis , Consensus Sequence , Female , Glycosylation , Humans , Isoelectric Focusing , Male , Middle Aged , Sequence Analysis, DNA , Thrombosis/genetics
5.
Blood ; 92(12): 4671-6, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9845533

ABSTRACT

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T-->C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the -10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, -10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue -10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue -10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.


Subject(s)
Antithrombins/deficiency , Antithrombins/genetics , Protein Processing, Post-Translational , Adult , Amino Acid Substitution/genetics , Animals , Antithrombins/chemistry , COS Cells , Cell-Free System , DNA Mutational Analysis , Dogs , Female , Gene Expression , Glycosylation , Humans , Leucine/genetics , Microsomes/metabolism , Pancreas/metabolism , Point Mutation/genetics , Proline/genetics , Protein Sorting Signals/genetics , Protein Sorting Signals/metabolism , Transfection
6.
Aust N Z J Psychiatry ; 32(6): 823-9, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10084347

ABSTRACT

OBJECTIVE: This paper outlines the methodologies used, and preliminary descriptive data collected, on a cohort of familial bipolar disorder (BPD) probands and first-degree relatives taking part in a descriptive and genetic study into familial BPD in New Zealand. METHOD: Fifty-five bipolar probands and 67 first-degree relatives were interviewed using the modified Diagnostic Interview for Genetic Studies (DIGS) and Family Interview for Genetic Studies (FIGS). Data was also collated from other sources. Blood samples were taken for DNA genomic analysis. RESULTS: New Zealand families in which BPD segregates proved willing participants in this familial based genetic research. The methodologies used were acceptable. High rates of comorbidity were found in probands (27.3% met DSM-IV criteria for panic disorder/sub-threshold panic disorder; 12.7% for phobic disorder; 1.8% for obsessive-compulsive disorder; 9.1% for alcohol-related disorders and 7.3% for an eating disorder) and relatives (major depression 34.3%; panic disorder/sub-threshold panic disorder 12.0%; phobias 11.9% and alcohol-related disorders 11.9%). The polarity of index BPD illness was related to age of onset and frequency of comorbidity. Suicidal behaviour was common. CONCLUSIONS: Psychiatric genetic research in New Zealand families is highly feasible. Emerging trends in the familial transmission of BPD include high rates of comorbidity, illness patterns based on polarity of index episode and frequent suicidal behaviour. Such trends will be delineated further as numbers accrue, perhaps enabling identification of more homogenous phenotypic subgroups than currently produced by diagnostic schemes.


Subject(s)
Bipolar Disorder/epidemiology , Bipolar Disorder/genetics , Family , Adolescent , Adult , Age of Onset , Aged , Bipolar Disorder/diagnosis , Cohort Studies , Comorbidity , DNA/genetics , Data Collection , Feeding and Eating Disorders/diagnosis , Feeding and Eating Disorders/epidemiology , Feeding and Eating Disorders/genetics , Female , Humans , Male , Mental Disorders/diagnosis , Mental Disorders/epidemiology , Mental Disorders/genetics , Middle Aged , New Zealand/epidemiology , Panic Disorder/diagnosis , Panic Disorder/epidemiology , Panic Disorder/genetics , Patient Selection , Phenotype , Phobic Disorders/diagnosis , Phobic Disorders/epidemiology , Phobic Disorders/genetics , Suicide, Attempted/psychology , Suicide, Attempted/statistics & numerical data
8.
Pathology ; 29(4): 341-7, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9423212

ABSTRACT

Venous thrombosis represents a manifestation of disordered hemostatic balance. The classical presentation is of pain and swelling of the lower limb, although clinical history and examination are notoriously misleading in reaching a diagnosis. A number of acquired predispositions have been associated with a tendency to thrombosis, such as immobilisation, surgery, malignancy and certain types of oral contraception, but in at least half of the instances no predisposition can be identified. A variety of genetic risk factors have also been identified. Mutations within the genes for antithrombin, protein C and protein S are associated with a venous thromboembolic phenotype. The commonest thrombophilic predisposition however is a variant of coagulation factor V, factor V Leiden, which results from a single amino acid substitution rendering the factor V molecule resistant to activated protein C. Factor V Leiden is present in approximately 5% of individuals of European origin, and is found in up to 40% of those with confirmed venous thrombosis. Increasingly it is recognised that venous thrombosis should be considered a polygenic disorder, with interactions between the various single gene defects which predispose to thrombosis, as well as normal genetic variation between individuals in the levels of both procoagulant and anticoagulant proteins, all determining which individuals will express the phenotype of venous thrombosis.


Subject(s)
Thrombophlebitis/genetics , Thrombophlebitis/physiopathology , Antithrombin III/genetics , Antithrombin III/physiology , Blood Coagulation/physiology , Factor V/genetics , Factor V/physiology , Humans , Mutation , Protein C/genetics , Protein C/physiology , Protein S/genetics , Protein S/physiology , Risk Factors
9.
Diabetologia ; 39(11): 1325-8, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8932999

ABSTRACT

A high proportion of the female patients who are members of maturity onset diabetes of the young (MODY) pedigrees, and whose diabetes mellitus is due to a glucokinase mutation, originally presented with gestational diabetes. To establish whether glucokinase mutations could be a common cause of gestational diabetes, we studied 50 subjects who presented with gestational diabetes and on follow-up had hyperglycaemia (5.5-10.0 mmol/l). Screening for glucokinase mutations using single-stranded conformational polymorphism (SSCP) analysis detected a missense mutation at position 299 (Gly299-->Arg) in three subjects. As two pedigrees in the Oxford area had the same glucokinase mutation, we suspected the role of a founder-effect, and carried out pedigree extension, haplotype construction (using microsatellite markers GCK1 and GCK2) and mutation screening of at-risk subjects from the same geographical area. One of the gestational diabetic subjects was found to be related to one of the previous pedigrees via her paternal grandmother. Subjects with the mutation were found to have the Z + 4/2 (GCK1/ GCK2) haplotype, suggesting that the observed high prevalence of the Gly299-->Arg glucokinase mutation in the Oxford region was due to a founder-effect. Since glucokinase mutations predominantly induce subclinical hyperglycaemia, it is likely that in the locality of other pedigrees there will be undiagnosed subjects with the same glucokinase mutation, which remains undetected unless pregnancy occurs.


Subject(s)
Diabetes, Gestational/epidemiology , Diabetes, Gestational/genetics , Glucokinase/genetics , Mutation/genetics , Adult , Alleles , Diabetes, Gestational/enzymology , Exons/genetics , Female , Genetic Markers/genetics , Glucokinase/classification , Haplotypes/genetics , Humans , Male , Pedigree , Polymorphism, Restriction Fragment Length , Pregnancy , Prevalence , United Kingdom/epidemiology
10.
Pathology ; 28(4): 339-42, 1996 Nov.
Article in English | MEDLINE | ID: mdl-9007953

ABSTRACT

There is a high degree of genetic heterogeneity underlying antithrombin deficiency indicating that a number of genetic mechanisms are responsible for the disorder. We report the identification of a five nucleotide (CAGAA) deletion in exon 2 of the antithrombin gene that results in a shift in the frame of translation of the mRNA and introduces a premature STOP signal in codon 70. The deleted nucleotides represent one repeat of a duplicated sequence within codons 36-39. The deletion may have arisen by slippage and mispairing of the repeated sequences at the replication fork during DNA synthesis.


Subject(s)
Antithrombin III/genetics , Sequence Deletion , Adult , Amino Acid Sequence , Antithrombin III Deficiency , Base Sequence , Electrophoresis, Polyacrylamide Gel , Female , Frameshift Mutation , Humans , Male , Middle Aged , Pedigree , Sequence Analysis, DNA
11.
Blood Rev ; 10(2): 59-74, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8813337

ABSTRACT

Antithrombin is the major proteinase inhibitor of thrombin and other blood coagulation proteinases. Antithrombin has two functional domains, a heparin binding site and a reactive centre (that complexes and inactivates the proteinase). Its deficiency results in an increased risk of venous thromboembolism. Appreciable progress has been made in recent years in understanding the structure and function of this protein, the genetic cause of inherited deficiency and its clinical consequence. The structure of antithrombin is now considered in terms of the models derived from X-ray crystallography, which have provided explanations for the function of its heparin interaction site and of its reactive loop. The structural organization of the antithrombin gene has been defined and numerous mutations have been identified that are responsible for antithrombin deficiency: these may reduce the level of the protein (Type I deficiency), alter the function of the protein (Type II deficiency, altering heparin binding or reactive sites), or even have multiple or 'pleiotropic effects' (Type II deficiency, altering both functional domains and the level of protein).


Subject(s)
Antithrombin III Deficiency , Antithrombin III/chemistry , Antithrombin III/genetics , Crystallography, X-Ray , Humans , Mutation , Polymorphism, Genetic
12.
Br J Haematol ; 89(3): 602-9, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7734360

ABSTRACT

We report the characterization of three variant antithrombins with reduced heparin binding as the primary abnormality. Two of these variants, antithrombin Southport (Leu 99 to Val, 2759 C to G) and antithrombin Vienna (Gln 118 to Pro, 5349 A to C) were novel, whereas the third, Pro 41 to Leu, has been previously described as antithrombin Basel. All three variants exhibited reduced binding for heparin on crossed immunoelectrophoresis and in a quantitative monoclonal antibody-based assay. The mutations were characterized by direct sequence analysis of enzymatically amplified genomic DNA and all affected individuals were heterozygous for the mutations. These three mutations do not occur at the sites of the basic amino acids directly involved in heparin binding nor do they result in a change in charge of the affected residue. It seems probable that they reduce heparin affinity either by perturbing the initial contact site involved in the heparin-binding domain (Arg 47, Arg 129 and possibly Arg 24), or by preventing the subsequent heparin-induced conformational change.


Subject(s)
Antithrombins/genetics , Heparin/metabolism , Point Mutation , Thrombosis/genetics , Adult , Antithrombins/metabolism , Female , Humans , Immunoelectrophoresis, Two-Dimensional , Male , Middle Aged , Models, Molecular , Thrombosis/blood
13.
Blood ; 84(12): 4209-13, 1994 Dec 15.
Article in English | MEDLINE | ID: mdl-7994035

ABSTRACT

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768-69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-->Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7-1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.


Subject(s)
Antithrombin III Deficiency , Antithrombin III/genetics , Base Sequence , Female , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/mortality , Heterozygote , Humans , Male , Molecular Sequence Data , Netherlands/epidemiology , Point Mutation , Sequence Deletion , Terminator Regions, Genetic
15.
Nucleic Acids Res ; 22(17): 3556-9, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7937056

ABSTRACT

Antithrombin III is the most important inhibitor of coagulation proteinases such as thrombin and factor Xa. Inherited deficiency of antithrombin III is a well recognised risk factor for the early development of venous thromboembolism. The gene for antithrombin III is located at chromosome 1q 23-25 and its structural organisation has been described. A database of mutations of the antithrombin III gene has been compiled and a recent update lists 184 entries. These entries are listed according to subtype of deficiency and to nucleotide sequence number. There are 68 reports of type I 'classical' and 116 reports of type II 'variant' deficiencies. This summary considers the entries in terms of the number of unique molecular events, the nature of the genetic defects and the role of CpG dinucleotides in deficiency. Sample listings of type I and II deficiency entries are provided.


Subject(s)
Antithrombin III/genetics , Databases, Factual , Mutation , Antithrombin III Deficiency , Chromosomes, Human, Pair 1 , Humans , Molecular Sequence Data
16.
Thromb Haemost ; 72(2): 198-202, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7831651

ABSTRACT

Inherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.


Subject(s)
Antithrombins/deficiency , Point Mutation , Thrombosis/genetics , Antithrombins/genetics , Codon/genetics , Female , Homozygote , Humans , Infant , Infant, Newborn , Male
18.
Hum Mutat ; 4(1): 31-41, 1994.
Article in English | MEDLINE | ID: mdl-7951256

ABSTRACT

Two (ATT) trinucleotide repeat polymorphisms have been identified in the tails of Alu repeat elements in intron 5 of the antithrombin gene. The frequency and distribution of allele sizes for the Alu 5 and Alu 8 tail polymorphisms have been defined in a sample Caucasian population. The Alu 5 polymorphism has two alleles while that of Alu 8 has 10 alleles with a heterozygosity of 0.83. These polymorphisms have been used in combination with four previously described polymorphisms within the antithrombin gene to construct antithrombin gene haplotypes in the sample Caucasian population. Twenty-two different haplotypes were observed, with the Alu 8 polymorphism being particularly useful in subdividing the core haplotype based on the previously identified polymorphisms. The haplotype data were used to investigate the origin of repeat mutations within the antithrombin locus. We compared the haplotypes associated the mutant antithrombin genes in five families with the mutation 2759C-->T (L99F) and five families with the mutation 5381C-->T (R129Stop). The mutation 2759C-->T (L99F), which occurs within a non-CpG dinucleotide, was carried on a gene associated with an identical haplotype in each of the five families. The mutation 5381C-->T (R129Stop), a single base substitution within a CpG dinucleotide, was associated with at least two different haplotypes. The findings suggest a founder effect in the five families sharing the 2759C-->T (L99F) and at least two independent origins for the CpG dinucleotide mutation 5381C-->T (R129Stop).


Subject(s)
Antithrombins/genetics , Mutation , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA Primers/genetics , Gene Frequency , Haplotypes , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Polymorphism, Genetic , Terminology as Topic , White People/genetics
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