Hum Genet (1992) 88:707-708

© Springer-Verlag1992

A novel missense mutation in the antithrombin III gene (Ser349 >Pro) causing recurrent venous thrombosis Catherine B. Grundy 1, Steven Holding 2, David S. Millar 1, Vijay V. Kakkar 1, and David N. Cooper 1 1Charter Molecular Genetics Laboratory, Thrombosis Research Institute, Manresa Road, London SW3 6LR, UK 2Haematology Department, Kingston General Hospital, Hull HU3 1UR, UK Received August 14, 1991 / Revised September 10, 1991

Summary. A novel T C C - + C C C transition in the antithrombin III (ATIII) gene resulting in a Ser349-->Pro substitution was detected in three members of a family with recurrent venous thrombosis and A T I I I activity/ antigen levels consistent with type II ATIII defciency.

Introduction Antithrombin III (ATIII) is a single-chain glycoprotein that serves as an inhibitor of thrombin and other serine proteases, including factors IXa and Xa (Barrowcliffe and Thomas 1987). In so doing, it fulfils a central role in the regulation of haemostasis. The exposed reactive site of A T I I I presents an ideal substrate to the target protease with which it forms a stable complex. ATIII also binds heparin strongly and this greatly enhances the rate of formation of the protease/inhibitor complex. A T I I I deficiency is not uncommon, occurring at a frequency of between 1/5000 and 1/2000 in the general population (Cosgriff et al. 1983; Beresford 1988). About 3% of patients presenting with venous thrombotic disease have an inherited deficiency of ATIII (Gladson et al. 1988). The human A T I I I gene has been localized to chromosome lq23-q25 (Bock et al. 1985) and comprises seven exons spaced out over 14 kb genomic D N A (Prochownik et al. 1985; Bock et al. 1988). We report here a novel point mutation in exon 4 of the A T I I I gene causing type II A T I I I deficiency (ATIII antigen > activity) and recurrent venous thrombosis.

Materials and methods ATIII activity was measured by the two-stage amidolytic method (Sculty 1983) in the presence of heparin using the chromogenic substrate Th-1 (Immuno Ltd, 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). Offprint requests to: D. N. Cooper

The English family, whose pedigree is presented in Fig. la, was referred after three members suffered recurrent thrombotic disease. Individual III.2. suffered a deep vein thrombosis (DVT) and multiple pulmonary emboli (PE) at the age of 16. His ATIII activity and antigen measurements were 40% and 60%, respectively, consistent with heterozygous type II ATIII deficiency. His father (II.2) experienced a DVT following appendicectomy at the age of 37 followed by recurrent PE and thrombophlebitis (43% ATIII activity, 64% ATIII antigen). Individual II.3 first experienced multiple PE and DVT at the age of 50 (40% ATIII activity, 60% ATIII antigen). All three patients are on long-term Warfarin therapy and no recurrence of thrombotic episodes has been reported. ATIII activity measurements from three family members with no clinical history of thrombosis (II.1, 139%; II.4, 101%; III.3, 107%) are consistent with their not having inherited the disease allele. Polymerase chain reaction (PCR) amplification of the seven exons of the ATIII gene (Bock et al. 1988) was carried out as described by Grundy et al. (1991). PCR/direct sequencing was performed using the same oligonucleotides as primers.

Results and discussion Individual III.2 was screened for mutation within the coding sequence of his ATIII genes by PCR/direct sequencing. This revealed the heterozygous presence of a novel T C C - + C C C transition within exon 4, a mutation that is predicted to convert the Ser residue at position 349 of the protein to Pro (Fig. lb). No other deviations from the wild-type D N A sequence (Bock et al. 1988) were noted, other than previously known D N A sequence polymorphisms. Both clinically affected relatives of the propositus (II.2 and II.3) possessed this lesion in the heterozygous form, whereas unaffected relatives (II.1, II.4 and III.3) did not. This lesion therefore co-segregates with the clinical phenotype in the family described. Several other lines of evidence are also consistent with this lesion being responsible for the disease phenot)~e (1) A missense mutation (as opposed to a nonsense mutation or gene deletion) was predicted by the observed excess of plasma A T I I I antigen over activity. (2) The Ser 349 substitution has not been described before in numerous sequencing studies (reviewed by Cooper 1991) of either

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r e s i d u e Ser349 in t h e 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 t a n t since t h e c o r r e s p o n d i n g a m i n o acid r e s i d u e is h i g h l y c o n s e r v e d in t h e h o m 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 , alpha-l-antitrypsin and plasminogen activator inhibitors 1 a n d 2 ( Y e et al. 1987). H o w e v e r , t h e c o n s e r v e d r e s i d u e in t h e s e p r o t e i n s is A l a suggesting t h a t t h e c h a n g e to S e r (which is u n i q u e to A T I I I ) is a s s o c i a t e d with p r o p e r t i e s specific to t h e A T I I I p r o t e i n .

References IV

a

P

C 5'

5' Ser/Pro

b

349

T/C " ~ C C

C

Ser 349

C 3'

C"

A G

C T

A

G

Fig.1. a Pedigree of the affected family from northern England. Half-shaded symbols denote individuals with recurrent venous thrombosis and ATIII activity/antigen levels consistent with type II ATIII deficiency, b PCR/direct sequencing of exon 4 (sense strand) around the site of the heterozygous TCC -~ CCC transition at Ser 349 in individual III.2 (P) and a healthy control (C)

disease or wild-type ATIII alleles, and is therefore unlikely to be a neutral polymorphism. (3) The introduction of a proline residue some 10 residues carboxy terminal to an alpha helical region (Ye et al. 1987) probably e x e r t s a m a r k e d effect o n t h e 3 - d i m e n s i o n a l s t r u c t u r e o f t h e A T I I I p r o t e i n . M o r e o v e r , the l o c a t i o n of a m i n o acid

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