Hum Genet (1992) 90:472-473
human . genet,cs 9 Springer-Verlag 1992
D N A variants
A novel missense mutation in the antithrombin III gene (Ala387 >Val) causing recurrent venous thrombosis Deborah White l, Ginger Abraham 1, Chris Carter 2, Vijay V. Kakkar 1, David N. Cooper 1 Charter Molecular Genetics Laboratory, Thrombosis Research Institute, Manresa Road, London SW3 6LR, UK 2pathology Department, Huddersfield Royal Infirmary, Huddersfield HD3 3EA, UK Received: 12 May 1992 / Revised: 24 July 1992
Abstract. A novel G C T - + G T T transition in the antit h r o m b i n I I I ( A T I I I ) gene, resulting in an Ala387--~Val substitution near the reactive site, was detected in a patient with recurrent venous thrombosis and A T I I I activity/antigen levels consistent with type I A T I I I deficiency.
Introduction A n t i t h r o m b i n I I I ( A T I I I ) is the m a j o r physiological inhibitor of t h r o m b i n and as such fulfils a central role in the regulation of haemostasis (Barrowcliffe and T h o m a s 1987). The exposed reactive site of A T I I I (Arg393-Ser394) presents an ideal substrate to thrombin with which it forms a stable complex. A T I I I also binds heparin strongly, which greatly enhances the rate of formation of the protease/inhibitor complex. A T I I I deficiency is not u n c o m m o n , occurring at a frequency of b e t w e e n 1/5000 and 1/2000 in the general population (Cosgriff et al. 1983; Beresford 1988). A b o u t 3% of patients presenting with venous t h r o m b o t i c disease have an inherited deficiency of A T I I I (Gladson et al. 1988). The h u m a n A T I I I gene has been localized to chromosome lq23-q25 (Bock et al. 19851 and comprises seven exons spaced out over 14 kb g e n o m i c D N A (Prochownik et al. 1985; Bock et al. 19881. W e report here a novel point mutation in exon 6 of the A T I I I gene causing type I A T I I I deficiency and recurrent venous thrombosis.
thrombosis and pulmonary embolism. He has experienced further episodes of varicose veins, superficial thrombophlebitis and venous ulceration and is now on long-term Warfarin therapy. His ATIII activity and antigen measurements were 50% and 60% respectively, consistent with heterozygous type I ATIII deficiency. His mother died from a pulmonary embolism, whereas his asymptomatic brother and his brother's children reportedly exhibit low ATIII levels. Polymerase chain reaction (PCR) amplification of the seven exons of the ATIII gene (Bock et al. 1988) was carried out as described (Grundy et al. 1991). PCR/direct sequencing was performed using the same oligonucleotides as primers.
Results and discussion The propositus was screened for mutation within the coding sequence of his A T I I I genes by P C R / d i r e c t sequencing. This revealed the heterozygous presence of a novel GCT---~GTT transition that lies within exon 6 and is predicted to convert the Ala residue at position 387 of the protein to Val (Fig. 1). No other deviations from the wild-type D N A sequence (Bock et al. 1988) were noted other than previously k n o w n D N A sequence p o l y m o r phisms (Lane et al. 1991). Several lines of evidence are consistent with this lesion being responsible for the disease phenotype. (1) The Ala387 substitution has not been described before in n u m e r o u s sequencing studies (reviewed by C o o p e r 19911
Materials and methods ATIII activity was measured by the two-stage amidolytic method (Scully 19831 in the presence of heparin using the chromogenic substrate Th-1 (Immuno, Sevenoaks, UK). ATIII antigen measurement was performed by radial immunodiffusion assay using commercial plates (Behring Diagnostics, Hounslow, UK) and an ovine/rabbit antiserum to human ATIII (Behring). The propositus, of English origin, first came to clinical attention at the age of 21 after developing a left iliofemoral deep vein
Corre,spondence to: D. N. Cooper
Fig. 1. PCR/direct sequencing of exon 6 (sense strand) of the ATII1 gene around the site of the heterozygous GCT---~GTT transition at Ala387 in the propositus (P) and a healthy control (C)
473 o f b o t h d i s e a s e a n d w i l d - t y p e A T I I I alleles a n d is t h e r e fore u n l i k e l y to b e a n e u t r a l p o l y m o r p h i s m . (2) T h e loc a t i o n o f a m i n o acid r e s i d u e A l a 3 8 7 in the A T I I I p r o t e i n a p p e a r s to b e e i t h e r s t r u c t u r a l l y o r f u n c t i o n a l l y i m p o r tant since this a m i n o acid r e s i d u e is c o n s e r v e d in the hom o l o g o u s h u m a n serpins a l p h a - l - a n t i c h y m o t r y p s i n , C1 i n h i b i t o r a n d p l a s m i n o g e n a c t i v a t o r i n h i b i t o r 1 (Ye et al. 1987). (3) T h e s u b s t i t u t i o n o f a l a n i n e by v a l i n e at t h e P7 r e s i d u e at t h e b a s e of t h e a l p h a helical r e a c t i v e c e n t r e l o o p is likely to e x e r t a m a r k e d effect on the t e r t i a r y structure o f the A T I I I p r o t e i n (Stein et al. 1990). P r e v i o u s l y r e p o r t e d p o i n t m u t a t i o n s in r e s i d u e s A l a 3 8 2 , A l a 3 8 4 , G l y 3 9 2 , A r g 3 9 3 , Ser394 a n d P r o 4 0 7 h a v e all r e s u l t e d in d e f e c t i v e o r a b s e n t t h r o m b i n i n h i b i t i o n ( C o o p e r 1991).
References Barrowcliffe TW, Thomas DP (1987) Antithrombin III and hepafin. In: Bloom AL, Thomas DP (eds) Haemostasis and thrombosis, 2nd edn. Churchill Livingstone, Edinburgh, pp 849-869 Beresford CH (1988) Antithrombin III deficiency. Blood Rev 2: 239-250 Bock SC, Harris JF, Balazs I, Trent JM (1985) Assigment of the human antithrombin III structural gene to chromosome lq2325. Cytogenet Cell Genet 39 : 67-69 Bock SC, Marrinan JA, Radziejewska E (1988) Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site. Biochemistry 27:61716178
Cooper DN (1991) The molecular genetics of familial venous thrombosis. Blood Rev 5 : 55-70 Cosgriff TM, Bishop DT, Hershgold E J, Skolnick MH, Martin BA, Baty BJ, Carlson KS (1983) Familial antithrombin III deficiency: its natural history, genetics, diagnosis and treatment. Medicine 62 : 209-220 Gladson CL, Sharrer I, Hach V, Beck KH, Griffin JH (1988) The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients and venous thrombosis. Thromb Haemost 59 : 18-22 Grundy CB, Thomas F, Millar DS, Krawczak M, Melissari E, Lindo V, Moffat E, Kakkar VV, Cooper DN (1991) Recurrent deletion in the human antithrombin III gene. Blood 78 : 10271032 Lane DA, Ireland H, Olds RJ, Thein SL, Perry D J, Aiach M (1991) Antithrombin III: a database of mutations. Thromb Haemost 66 : 657-661 Prochownik EV, Bock SC, Orkin SH (1985) Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily. J Biol Chem 260 : 9608-9612 Scully MF (1983) The use of an automated analyzer in the evaluation of antithrombin III and heparin. Semin Thromb Haemost 9 : 309-313 Stein PE, Leslie AGW, Finch JT, Turnell WG, McLaughlin PJ, Carrell RW (1990) Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature 347:99-102 Ye RD, Wun T-C, Sadler JE (1987) cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta. J Biol Chem 262 : 3718-3725
human . genet,cs 9 Springer-Verlag 1992
D N A variants
A novel missense mutation in the antithrombin III gene (Ala387 >Val) causing recurrent venous thrombosis Deborah White l, Ginger Abraham 1, Chris Carter 2, Vijay V. Kakkar 1, David N. Cooper 1 Charter Molecular Genetics Laboratory, Thrombosis Research Institute, Manresa Road, London SW3 6LR, UK 2pathology Department, Huddersfield Royal Infirmary, Huddersfield HD3 3EA, UK Received: 12 May 1992 / Revised: 24 July 1992
Abstract. A novel G C T - + G T T transition in the antit h r o m b i n I I I ( A T I I I ) gene, resulting in an Ala387--~Val substitution near the reactive site, was detected in a patient with recurrent venous thrombosis and A T I I I activity/antigen levels consistent with type I A T I I I deficiency.
Introduction A n t i t h r o m b i n I I I ( A T I I I ) is the m a j o r physiological inhibitor of t h r o m b i n and as such fulfils a central role in the regulation of haemostasis (Barrowcliffe and T h o m a s 1987). The exposed reactive site of A T I I I (Arg393-Ser394) presents an ideal substrate to thrombin with which it forms a stable complex. A T I I I also binds heparin strongly, which greatly enhances the rate of formation of the protease/inhibitor complex. A T I I I deficiency is not u n c o m m o n , occurring at a frequency of b e t w e e n 1/5000 and 1/2000 in the general population (Cosgriff et al. 1983; Beresford 1988). A b o u t 3% of patients presenting with venous t h r o m b o t i c disease have an inherited deficiency of A T I I I (Gladson et al. 1988). The h u m a n A T I I I gene has been localized to chromosome lq23-q25 (Bock et al. 19851 and comprises seven exons spaced out over 14 kb g e n o m i c D N A (Prochownik et al. 1985; Bock et al. 19881. W e report here a novel point mutation in exon 6 of the A T I I I gene causing type I A T I I I deficiency and recurrent venous thrombosis.
thrombosis and pulmonary embolism. He has experienced further episodes of varicose veins, superficial thrombophlebitis and venous ulceration and is now on long-term Warfarin therapy. His ATIII activity and antigen measurements were 50% and 60% respectively, consistent with heterozygous type I ATIII deficiency. His mother died from a pulmonary embolism, whereas his asymptomatic brother and his brother's children reportedly exhibit low ATIII levels. Polymerase chain reaction (PCR) amplification of the seven exons of the ATIII gene (Bock et al. 1988) was carried out as described (Grundy et al. 1991). PCR/direct sequencing was performed using the same oligonucleotides as primers.
Results and discussion The propositus was screened for mutation within the coding sequence of his A T I I I genes by P C R / d i r e c t sequencing. This revealed the heterozygous presence of a novel GCT---~GTT transition that lies within exon 6 and is predicted to convert the Ala residue at position 387 of the protein to Val (Fig. 1). No other deviations from the wild-type D N A sequence (Bock et al. 1988) were noted other than previously k n o w n D N A sequence p o l y m o r phisms (Lane et al. 1991). Several lines of evidence are consistent with this lesion being responsible for the disease phenotype. (1) The Ala387 substitution has not been described before in n u m e r o u s sequencing studies (reviewed by C o o p e r 19911
Materials and methods ATIII activity was measured by the two-stage amidolytic method (Scully 19831 in the presence of heparin using the chromogenic substrate Th-1 (Immuno, Sevenoaks, UK). ATIII antigen measurement was performed by radial immunodiffusion assay using commercial plates (Behring Diagnostics, Hounslow, UK) and an ovine/rabbit antiserum to human ATIII (Behring). The propositus, of English origin, first came to clinical attention at the age of 21 after developing a left iliofemoral deep vein
Corre,spondence to: D. N. Cooper
Fig. 1. PCR/direct sequencing of exon 6 (sense strand) of the ATII1 gene around the site of the heterozygous GCT---~GTT transition at Ala387 in the propositus (P) and a healthy control (C)
473 o f b o t h d i s e a s e a n d w i l d - t y p e A T I I I alleles a n d is t h e r e fore u n l i k e l y to b e a n e u t r a l p o l y m o r p h i s m . (2) T h e loc a t i o n o f a m i n o acid r e s i d u e A l a 3 8 7 in the A T I I I p r o t e i n a p p e a r s to b e e i t h e r s t r u c t u r a l l y o r f u n c t i o n a l l y i m p o r tant since this a m i n o acid r e s i d u e is c o n s e r v e d in the hom o l o g o u s h u m a n serpins a l p h a - l - a n t i c h y m o t r y p s i n , C1 i n h i b i t o r a n d p l a s m i n o g e n a c t i v a t o r i n h i b i t o r 1 (Ye et al. 1987). (3) T h e s u b s t i t u t i o n o f a l a n i n e by v a l i n e at t h e P7 r e s i d u e at t h e b a s e of t h e a l p h a helical r e a c t i v e c e n t r e l o o p is likely to e x e r t a m a r k e d effect on the t e r t i a r y structure o f the A T I I I p r o t e i n (Stein et al. 1990). P r e v i o u s l y r e p o r t e d p o i n t m u t a t i o n s in r e s i d u e s A l a 3 8 2 , A l a 3 8 4 , G l y 3 9 2 , A r g 3 9 3 , Ser394 a n d P r o 4 0 7 h a v e all r e s u l t e d in d e f e c t i v e o r a b s e n t t h r o m b i n i n h i b i t i o n ( C o o p e r 1991).
References Barrowcliffe TW, Thomas DP (1987) Antithrombin III and hepafin. In: Bloom AL, Thomas DP (eds) Haemostasis and thrombosis, 2nd edn. Churchill Livingstone, Edinburgh, pp 849-869 Beresford CH (1988) Antithrombin III deficiency. Blood Rev 2: 239-250 Bock SC, Harris JF, Balazs I, Trent JM (1985) Assigment of the human antithrombin III structural gene to chromosome lq2325. Cytogenet Cell Genet 39 : 67-69 Bock SC, Marrinan JA, Radziejewska E (1988) Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site. Biochemistry 27:61716178
Cooper DN (1991) The molecular genetics of familial venous thrombosis. Blood Rev 5 : 55-70 Cosgriff TM, Bishop DT, Hershgold E J, Skolnick MH, Martin BA, Baty BJ, Carlson KS (1983) Familial antithrombin III deficiency: its natural history, genetics, diagnosis and treatment. Medicine 62 : 209-220 Gladson CL, Sharrer I, Hach V, Beck KH, Griffin JH (1988) The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients and venous thrombosis. Thromb Haemost 59 : 18-22 Grundy CB, Thomas F, Millar DS, Krawczak M, Melissari E, Lindo V, Moffat E, Kakkar VV, Cooper DN (1991) Recurrent deletion in the human antithrombin III gene. Blood 78 : 10271032 Lane DA, Ireland H, Olds RJ, Thein SL, Perry D J, Aiach M (1991) Antithrombin III: a database of mutations. Thromb Haemost 66 : 657-661 Prochownik EV, Bock SC, Orkin SH (1985) Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily. J Biol Chem 260 : 9608-9612 Scully MF (1983) The use of an automated analyzer in the evaluation of antithrombin III and heparin. Semin Thromb Haemost 9 : 309-313 Stein PE, Leslie AGW, Finch JT, Turnell WG, McLaughlin PJ, Carrell RW (1990) Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature 347:99-102 Ye RD, Wun T-C, Sadler JE (1987) cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta. J Biol Chem 262 : 3718-3725
