Molecular mechanisms of antithrombin deficiency in two Chinese families

2005 ◽  
Vol 94 (12) ◽  
pp. 1172-1176 ◽  
Author(s):  
Rong-Fu Zhou ◽  
Qi-Hua Fu ◽  
Wen-Bin Wang ◽  
Shuang Xie ◽  
Jin Dai ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Direct Sequencing ◽  
Type I ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Chinese Families ◽  
Real Time Quantitative Pcr ◽  
Intracellular Degradation ◽  
Phenotype Analysis ◽  
At Deficiency

SummaryWe investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C>T in proband 1 and 13328G>A in proband 2, predicting a heterozygous Thr98Ile (T98I) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factorV Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-I98 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T98I and A404T mutation for the two families, respectively.

The Molecular Basis of Antithrombin Deficiency in Belgian and Dutch Families

1998 ◽  
Vol 80 (09) ◽  
pp. 376-381 ◽  
Author(s):  
W. Lissens ◽  
S. Seneca ◽  
P. Capel ◽  
B. Chatelain ◽  
P. Meeus ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Gene Deletion ◽  
Direct Sequencing ◽  
Genetic Defect ◽  
Polymorphism Analysis ◽  
Type I ◽  
Type Ii ◽  
Antithrombin Deficiency ◽  
Venous Thromboembolic Events ◽  
At Deficiency

SummaryThe molecular basis of hereditary antithrombin (AT) deficiency has been investigated in ten Belgian and three Dutch unrelated kindreds. Eleven of these families had a quantitative or type I AT deficiency, with a history of major venous thromboembolic events in different affected members. In the other two families a qualitative or type II AT deficiency was occasionally diagnosed.DNA studies of the AT gene were performed, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and intronexon junction regions. Six novel point mutations were identified: four missense, one nonsense mutation and a single nucleotide deletion near the reactive site, causing a frameshift with premature translation termination. In two kindreds the underlying genetic defect was caused by a whole gene deletion, known as a rare cause of AT deficiency. In these cases, Southern blot and polymorphism analysis of different parts of the AT gene proved useful for diagnosis. In another kindred a partial gene deletion spanning 698 basepairs could precisely be determined to a part of intron 3B and exon 4. In two type I and in both type II AT deficient families a previously reported mutation was identified. In all cases, the affected individuals were heterozygous for the genetic defect.

Impaired Cotranslational Processing as a Mechanism for Type I Antithrombin Deficiency

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4671-4676
Author(s):  
Alison C. Fitches ◽  
Ruth Appleby ◽  
David A. Lane ◽  
Valerio De Stefano ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Mammalian Cells ◽  
Novel Mutation ◽  
Secretory Proteins ◽  
Type I ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Hydrophobic Domain ◽  
At Deficiency ◽  
Hydrophobic Nature

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.

Impaired Cotranslational Processing as a Mechanism for Type I Antithrombin Deficiency

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4671-4676 ◽  
Author(s):  
Alison C. Fitches ◽  
Ruth Appleby ◽  
David A. Lane ◽  
Valerio De Stefano ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Mammalian Cells ◽  
Novel Mutation ◽  
Secretory Proteins ◽  
Type I ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Hydrophobic Domain ◽  
At Deficiency ◽  
Hydrophobic Nature

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.

Molecular analysis and genotype-phenotype correlation in patients with antithrombin deficiency from Southern Italy

2012 ◽  
Vol 107 (04) ◽  
pp. 673-680 ◽  
Author(s):  
Giuseppe Castaldo ◽  
Anna Cerbone ◽  
Anna Guida ◽  
Igor Tandurella ◽  
Rosaria Ingino ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Southern Italy ◽  
Stop Codon ◽  
Type I ◽  
Missense Mutations ◽  
Type Ii ◽  
Clinical Expression ◽  
Antithrombin Deficiency ◽  
At Deficiency ◽  
Molecular Bases

SummaryWe sequenced the SERPINC1 gene in 26 patients (11 males) with antithrombin (AT) deficiency (22 type I, 4 type II), belonging to 18 unrelated families from Southern Italy. Heterozygous mutations were identified in 15/18 (83.3%) families. Of them, eight were novel mutations, each being identified in one family. Seven clearly cause impaired protein synthesis (four frameshift, one non-stop, one splicing and one 21bp deletion). One, present in a single patient, is a missense mutation thought to be causative because: a) it is absent in 100 chromosomes from controls; b) it involves a highly conserved amino acid, whose change is predicted to impair AT activity; c) no other mutation is present in the propositus. Severe mutations (i.e. nonsense, frameshift, deletions) were invariably identified in type I patients. In type II patients, 3/4 were missense mutations; the fourth leads to a 19 nucleotides shift in the stop codon. In addition to the type of mutation, the co-existence of other predisposing factors in most patients helps explain the severity of the present type I cases (age at first event, recurrence during prophylaxis). In the five families in which there was more than one member affected, the same genotype and a concordant clinical expression of the disease were found. We conclude that the molecular bases of AT deficiency in Southern Italy are different as compared to other geographic areas, and that molecular analysis and the study of the effect of the mutation may help predict the clinical expression of the disease.

Creation of an Additional Glycosylation Site as a Mechanism for Type I Antithrombin Deficiency

2001 ◽  
Vol 86 (10) ◽  
pp. 1023-1027 ◽  
Author(s):  
Krzysztof Lewandowski ◽  
Robin Olds ◽  
Alison Fitches
Keyword(s):  
Molecular Weight ◽  
Glycosylation Site ◽  
Base Substitution ◽  
Type I ◽  
Antithrombin Deficiency ◽  
Expression Studies ◽  
Glycan Chain ◽  
Reticulocyte Lysate ◽  
History Of ◽  
At Deficiency

SummaryWe 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.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈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 β antithrombin (the very high affinity fraction) greatly increased (≈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 β antithrombin in regulating coagulation.

Molecular Basis of Antithrombin Type I Deficiency: The First Large In-frame Deletion and Two Novel Mutations in Exon 6

1994 ◽  
Vol 72 (04) ◽  
pp. 534-539 ◽  
Author(s):  
J Emmerich ◽  
G Chadeuf ◽  
M Alhenc-Gelas ◽  
M Gouault-Heilman ◽  
P Toulon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Repeat Sequence ◽  
Type I ◽  
Novel Mutations ◽  
Flanking Sequences ◽  
At Deficiency ◽  
Exon 4

SummaryWe report three novel mutations accounting for cases of inherited type I antithrombin (AT) deficiency. Using the polymerase chain reaction (PCR) and direct sequencing of the coding sequences of the AT gene, we found one mutation in exon 4 and two in exon 6. A deletion of 105 bp causing an in-frame deletion of 35 amino acids between Tyr 240 and Gly 276 was found in exon 4. In a second kindred, deletion of two adenines in codon 412-413 introduced a frameshift and a stop codon at position 431. The last mutation was an insertion of ACCG in codon 387, generating a frameshift with a stop codon located at the normal position.The finding of a sequence repeat of nine residues located at the 5’and 3’ ends of the deleted fragment might explain the 105 bp deletion by slippage and mispairing at the replication fork during DNA synthesis. The second mutation is the fourth described within a region of six amino acids (between Phe 408 and Arg 413), which seems to be a cluster of mutations. In this case, the presence of a double repeat sequence - TTCCT and AACA - flanking this region could be particularly favorable for slipped mispairing.These results confirm that human gene mutations are not random events but are strongly influenced by DNA flanking sequences.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247 ◽  
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

Abstract We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈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 β antithrombin (the very high affinity fraction) greatly increased (≈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 β antithrombin in regulating coagulation.

Molecular Mechanism of Type I Congenital Heparin Cofactor (HC) II Deficiency Caused by a Missense Mutation at Reactive P2 Site: HC II Tokushima

2001 ◽  
Vol 85 (01) ◽  
pp. 101-107 ◽  
Author(s):  
Yasuhiko Kanagawa ◽  
Toshio Shigekiyo ◽  
Ken-ichi Aihara ◽  
Masashi Akaike ◽  
Toshio Matsumoto ◽  
...  
Keyword(s):  
Molecular Pathogenesis ◽  
Nucleotide Position ◽  
Type I ◽  
Metabolic Labeling ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Intracellular Degradation ◽  
Atherosclerotic Lesions ◽  
Old Japanese ◽  
Digestion Pattern

SummaryWe found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bbv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC II in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC II mRNA was transcribed at a similar level as that of wild-type.Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.

Impact of the type of SERPINC1 mutation and subtype of antithrombin deficiency on the thrombotic phenotype in hereditary antithrombin deficiency

2014 ◽  
Vol 111 (02) ◽  
pp. 249-257 ◽  
Author(s):  
Anna Pavlova ◽  
Christof Geisen ◽  
Michael Spannagl ◽  
Frauke Bergmann ◽  
Manuela Krause ◽  
...  
Keyword(s):  
Lower Risk ◽  
Type I ◽  
Missense Mutations ◽  
Type Ii ◽  
Antithrombin Deficiency ◽  
Vein Thrombosis ◽  
At Deficiency ◽  
Deficiency Type ◽  
Deep Vein ◽  
The Impact

SummaryMutations in the antithrombin (AT) gene can impair the capacity of AT to bind heparin (AT deficiency type IIHBS), its target proteases such as thrombin (type IIRS), or both (type IIPE). Type II AT deficiencies are almost exclusively caused by missense mutations, whereas type I AT deficiency can originate from missense or null mutations. In a retrospective cohort study, we investigated the impact of the type of mutation and type of AT deficiency on the manifestation of thromboembolic events in 377 patients with hereditary AT deficiencies (133 from our own cohort, 244 reported in the literature). Carriers of missense mutations showed a lower risk of venous thromboembolism (VTE) than those of null mutations (adjusted hazard ratio [HR] 0.39, 95% confidence interval [CI] 0.27–0.58, p<0.001), and the risk of VTE was significantly decreased among patients with type IIHBS AT deficiency compared to patients with other types of AT deficiency (HR 0.23, 95%CI 0.13–0.41, p<0.001). The risk of pulmonary embolism complicating deep-vein thrombosis was lower in all type II AT deficiencies compared to type I AT deficiency (relative risk 0.69, 95%CI 0.56–0.84). By contrast, the risk of arterial thromboembolism tended to be higher in carriers of missense mutations than in those with null mutations (HR 6.08-fold, 95%CI 0.74–49.81, p=0.093) and was 5.9-fold increased (95%CI 1.22–28.62, p=0.028) in type IIHBS versus other types of AT deficiency. Our data indicate that the type of inherited AT defect modulates not only the risk of thromboembolism but also the localisation and encourage further studies to unravel this phenomenon.

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