Hum Genet (1992) 90:472-473

D N A variants

human . genet,cs

�9 Springer-Verlag 1992

A novel missense mutation in the antithrombin III gene (Ala387 causing recurrent venous thrombosis

> Val)

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 transit ion in the anti- th rombin I I I ( A T I I I ) gene, resulting in an Ala387--~Val substitution near the reactive site, was detected in a pa- tient with recurrent venous thrombosis and A T I I I activ- i ty/antigen levels consistent with type I A T I I I deficiency.

Introduction

Ant i th rombin I I I ( A T I I I ) is the major physiological in- hibitor of thrombin and as such fulfils a central role in the regulation of haemostasis (Barrowcliffe and Thomas 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 hepar in strongly, which greatly enhances the rate of format ion of the pro- tease/ inhibi tor complex.

A T I I I deficiency is not u n c o m m o n , occurr ing at a fre- quency of be tween 1/5000 and 1/2000 in the general pop- ulation (Cosgriff et al. 1983; Beresford 1988). A b o u t 3% of patients present ing with venous thrombot ic disease have an inheri ted deficiency of A T I I I (Gladson et al. 1988). The human A T I I I gene has been localized to chro- mosome lq23-q25 (Bock et al. 19851 and comprises seven exons spaced out over 14 kb genomic D N A (Prochownik et al. 1985; Bock et al. 19881. We repor t here a novel point mutat ion 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 ven- ous ulceration and is now on long-term Warfarin therapy. His ATIII activity and antigen measurements were 50% and 60% re- spectively, consistent with heterozygous type I ATIII deficiency. His mother died from a pulmonary embolism, whereas his asympto- matic 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 de- scribed (Grundy et al. 1991). PCR/direct sequencing was perform- ed using the same oligonucleotides as primers.

Results and discussion

The propositus was screened for muta t ion within the coding sequence of his A T I I I genes by PCR/d i rec t se- quencing. This revealed the he terozygous presence of a novel GCT---~GTT transit ion that lies within exon 6 and is predicted to convert the Ala residue at posit ion 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 known D N A sequence polymor- phisms (Lane et al. 1991).

Several lines of evidence are consistent with this le- sion being responsible for the disease phenotype. (1) The Ala387 substitution has not been described before in numerous sequencing studies (reviewed by Cooper 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 mea- surement 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 atten- tion 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 transi- tion at Ala387 in the propositus (P) and a healthy control (C)

473

of bo th d isease and wi ld - type A T I I I al le les and is there- fore un l ike ly to be a neu t ra l p o l y m o r p h i s m . (2) The lo- ca t ion of amino acid res idue Ala387 in the A T I I I p ro t e in a ppea r s to be e i the r s t ruc tura l ly or func t iona l ly impor - tant s ince this amino acid res idue is conse rved in the ho- 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 , C1 inh ib i to r and p l a s m i n o g e n ac t iva tor inh ib i to r 1 (Ye et al. 1987). (3) The subs t i tu t ion of a lan ine by va l ine at the P7 res idue at the base of the a lpha hel ical reac t ive cen t re loop is l ikely to exer t a m a r k e d effect on the te r t i a ry struc- ture of the A T I I I p ro t e in (Ste in et al. 1990). Prev ious ly r e p o r t e d po in t m u t a t i o n s in res idues Ala382 , Ala384 , Gly392, Arg393 , Ser394 and Pro407 have all r esu l ted in defec t ive or absen t t h r o m b i n inh ib i t ion ( C o o p e r 1991).

References

Barrowcliffe TW, Thomas DP (1987) Antithrombin III and hepa- fin. In: Bloom AL, Thomas DP (eds) Haemostasis and throm- bosis, 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 lq23- 25. 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 re- gion near the inhibitor reactive site. Biochemistry 27:6171- 6178

Cooper DN (1991) The molecular genetics of familial venous throm- bosis. Blood Rev 5 : 55-70

Cosgriff TM, Bishop DT, Hershgold E J, Skolnick MH, Martin BA, Baty BJ, Carlson KS (1983) Familial antithrombin III deficien- cy: its natural history, genetics, diagnosis and treatment. Medi- cine 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 defi- ciency 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 : 1027- 1032

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 evalua- tion 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