WO2000034515A1

WO2000034515A1 - Use of factor x polymorphism in the diagnosis and treatment of factor x and/or factor xa mediated diseases

Info

Publication number: WO2000034515A1
Application number: PCT/GB1999/003973
Authority: WO
Inventors: Rakesh Anand; John Edward Norris Morten; John Craig Smith
Original assignee: AstraZeneca UK Ltd; AstraZeneca AB
Current assignee: AstraZeneca UK Ltd; AstraZeneca AB
Priority date: 1998-12-05
Filing date: 1999-11-30
Publication date: 2000-06-15
Anticipated expiration: 2001-06-05
Also published as: EP1135534A1; JP2002531144A; AU1396700A; GB9826747D0

Abstract

This invention relates to polymorphisms in the human Factor X gene, in particular to the discovery of two single nucleotide polymorphisms in the coding sequence of the human Factor X gene. The invention also relates to methods and materials for analysing allelic variation in the Factor X gene, and to the use of Factor X polymorphism in the diagnosis and treatment of Factor X and/or Factor Xa-mediated diseases, such as thrombotic diseases.

Description

USE OF FACTOR X POLYMORPHISM IN THE DIAGNOSIS AND TREATMENT OF FACTOR X AND/OR FACTOR XA MEDIATED DISEASES

This invention relates to polymorphisms in the Factor X gene. The invention also relates to methods and materials for analysing allelic variation in the Factor X gene, and to the use of Factor X polymorphism in the diagnosis and treatment of Factor X and/or Factor Xa-mediated diseases, such as thrombotic diseases.

Factor Xa is one of a cascade of proteases involved in the complex process of blood coagulation. The protease known as thrombin is the final protease in the cascade and Factor Xa is the preceding protease which cleaves prothrombin to generate thrombin. Factor Xa is produced by cleavage ofthe zymogen precursor Factor X, by activated factor VII. For a review ofthe process of blood coagulation see Rock and Wells (1997) Crit Rev Clin Lab Sci 34, 475-501 and for a review of the Biochemistry of Factor X see Hertzberg (1994) Blood Reviews 8, 56-62.

Certain compounds are known to possess Factor Xa inhibitory properties and the 5 field has been reviewed by R.B. Wallis, Current Opinion in Therapeutic Patents. 1993,

1173- 1179 and Yamazaki (1995) Drugs of the Future 20, 911-918. Thus it is known that two proteins, one known as antistatin and the other known as tick anticoagulant protein (TAP), are specific Factor Xa inhibitors which possess antithrombotic properties in various animal models of thrombotic disease. 0 It is also known that certain non-peptidic compounds possess Factor Xa inhibitory properties. Ofthe low molecular weight inhibitors mentioned in the review by R.B. Wallis, all possessed a strongly basic group such as an amidinophenyl or amidinonaphthyl group.

The sequence of Factor X was published by Leytus et al (1986) Biochemistry 25, 5098-5102. The sequence was submitted to the EMBL database as separate exons: Exon 1 5 (EMBL Nccession Number -L00390), Exon 2 (EMBL Nccession Number - L00391), Exon 3 (EMBL Nccession Number - L00392), Exon 4 (EMBL Nccession Number - L00393), Exon 5 (EMBL Nccession Number - L00394), Exon 6 ((EMBL Nccession Number - L00395), Exon 7 (EMBL Nccession Number - L00396), and Exon 8 (EMBL Nccession Number - L29433). All positions herein relate to the position in the appropriate EMBL Nccession number unless 0 stated otherwise or apparent from the context. Mutations in the Factor X gene which lead to Factor X deficiency and a clinical phenotype are well documented (For a review of Factor X mutations and Factor X deficiency see Cooper et al (1997) Thrombosis and Haemostasis 78, 161-172).

Other variation in DNN sequence (polymoφhisms) may not lead to Factor X deficiency but may increase the probability of pathological conditions or affect drug response or may be genetically linked to other polymoφhisms which do so.

One approach is to use knowledge of polymoφhisms to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed "pharmacogenetics") . Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymoφhisms are used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymoφhism detection: Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16, 33.

Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous. Thus there is a need for improved approaches to pharmaceutical agent design and therapy.

The present invention is based on the discovery of two single nucleotide polymoφhisms (SNPs) in the coding sequence ofthe human Factor X gene.

According to one aspect ofthe present invention there is provided a method for the diagnosis of a single nucleotide polymoφhism in a Factor X gene in a human, which method comprises determining the sequence ofthe nucleic acid ofthe human at position 41 in exon 5 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00396 and determining the status ofthe human by reference to polymoφhism in the Factor X gene.

According to another aspect ofthe present invention there is provided a method for the diagnosis of a single nucleotide polymoφhism in a Factor X gene in a human, which method comprises determining the sequence ofthe nucleic acid ofthe human at position 41 in exon 5 ofthe Factor X gene as defined by the position in EMBL

ACCESSION NO. L00394, and/or at position 57 in exon 7 ofthe Factor X gene as defined by the position in EMBL

ACCESSION NO. L00396 and determining the status ofthe human by reference to polymoφhism in the Factor X gene.

The term human includes both a human having or suspected of having a Factor X- mediated disease and an asymptomatic human who may be tested for predisposition or susceptibility to such disease. At each position the human may be homozygous for an allele or the human may be a heterozygote. In one embodiment ofthe invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at exon 5 position 41 is presence of C and or T.

In another embodiment ofthe invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at exon 7 position 57 is presence of C and/or T.

Subsequently to the present invention, Cargill et al have confirmed the presence of a single nucleotide polymoφhism in human Factor X at exon 5 position 41 and/or at exon 7 position 57 (Cargill et al., Nature Genetics, 22, 231-239, 1999).

The method for diagnosis is preferably one in which the sequence is determined by a method selected from amplification refractory mutation system and restriction fragment length polymoφhism.

In another aspect ofthe invention we provide a method for the diagnosis of Factor X- and/or Factor Xa-mediated disease, which method comprises: i) obtaining sample nucleic acid from an individual, ii) detecting the presence or absence of a variant nucleotide at position 41 in exon 5 ofthe

Factor X gene as defined by the position in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 ofthe Factor X gene as defined by the position in EMBL

ACCESSION NO. L00396, iii) determining the status ofthe individual by reference to polymoφhism in the Factor X gene. Allelic variation at exon 5 position 41 consists of a single base substitution from C (the published base), preferably to T. Allelic variation at exon 7 position 57 consists of a single base substitution from C (the published base), preferably to T.

The status ofthe individual may be determined by reference to allelic variation at any one or both positions optionally in combination with any other polymoφhism that is or becomes known.

The test sample of nucleic acid is conveniently a sample of blood, bronchoalveolar lavage fluid, sputum, or other body fluid or tissue obtained from an individual. It will be appreciated that the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part ofthe region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic variation.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of variant nucleotides at one or more polymoφhic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Table 1 lists a number of mutation detection techniques, some based on the PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in Table 2. Further amplification techniques are listed in Table 3. Many current methods for the detection of allelic variation are reviewed by Nollau et al., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997. Abbreviations:

Table 1 - Mutation Detection Techniques

General: DNA sequencing, Sequencing by hybridisation Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage

* Note: not useful for detection of promoter polymoφhisms.

Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Ohgonucleotide arrays (DNA Chips)

Solution phase hybridisation: Taqman™ - US-5210015 & US-5487972 (Hoffmann-La

Roche), Molecular Beacons - Tyagi et al (1996), Nature Biotechnology, 14, 303; WO

95/13399 (Public Health Inst, New York)

Extension Based: ARMS™, ALEX™ - European Patent No. EP 332435 Bl (Zeneca Limited), COPS - Gibbs et al (1989), Nucleic Acids Research, 17, 2347.

Incorporation Based: Mini-sequencing, APEX Restriction Enzyme Based: RFLP, Restriction site generating PCR Ligation Based: OLA Other: Invader assay

Table 2 - Signal Generation or Detection Systems

Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)

Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry

Table 3 - Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

Preferred mutation detection techniques include ARMS™, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.

Particularly preferred methods include ARMS™ and RFLP based methods. ARMS™ is an especially preferred method.

In a further aspect, the diagnostic methods ofthe invention are used to assess the efficacy of therapeutic compounds in the treatment of Factor X and/or Factor Xa-mediated diseases, such as thrombotic diseases.

Assays, for example reporter-based assays, may be devised to detect whether one or more ofthe above polymoφhisms affect transcription levels and/or message stability.

Individuals who carry particular allelic variants ofthe Factor X gene may therefore exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react to different diseases. In addition, differences in protein regulation arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy. The diagnostic methods ofthe invention may be useful both to predict the clinical response to such agents and to determine therapeutic dose. In a further aspect, the diagnostic methods ofthe invention, are used to assess the predisposition of an individual to diseases mediated by Factor X and/or Factor Xa. This may be particularly relevant in the development of thrombotic disease and other diseases which are modulated by Factor X and/or Factor Xa. The present invention may be used to recognise individuals who are particularly at risk from developing these conditions.

Low frequency polymoφhisms may be particularly useful for haplotyping as described below. A haplotype is a set of alleles found at linked polymoφhic sites (such as within a gene) on a single (paternal or maternal) chromosome. If recombination within the gene is random, there may be as many as 2" haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs. One approach to identifying mutations or polymoφhisms which are correlated with clinical response is to carry out an association study using all the haplotypes that can be identified in the population of interest. The frequency of each haplotype is limited by the frequency of its rarest allele, so that SNPs with low frequency alleles are particularly useful as markers of low frequency haplotypes. As particular mutations or polymoφhisms associated with certain clinical features, such as adverse or abnormal events, are likely to be of low frequency within the population, low frequency SNPs may be particularly useful in identifying these mutations (for examples see: Linkage disequilibrium at the cystathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma levels of homocysteine. Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse JF, London J, Stansbie D, Humphries SE, and Gudnason V; and Variation at the von willebrand factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of three novel single nucleotide polymoφhisms in the vWF gene promoter. Blood (1999) 93:4277-83, Keightley AM, Lam YM, Brady JN, Cameron CL, Lillicrap D).

In a further aspect, the diagnostic methods ofthe invention are used in the development of new drug therapies which selectively target one or more allelic variants ofthe Factor X gene. Identification of a link between a particular allelic variant and predisposition to disease development or response to drag therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity of variants implicated in the disease process whilst minimising effects on other variants.

In a further diagnostic aspect ofthe invention the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes. In the accompanying Example 2 we provide details of convenient engineered restriction enzyme sites that are lost or gained as a result of a polymoφhism ofthe invention. According to another aspect ofthe present invention there is provided a nucleic acid comprising any one ofthe following polymoφhisms: the nucleic acid of EMBL ACCESSION No. L00394 with T at position 41 as defined by the position in EMBL ACCESSION No. L00394; 5 the nucleic acid of EMBL ACCESSION No. L00396 with T at position 57 as defined by the position in EMBL ACCESSION No. L00396; or a complementary strand thereof or an antisense sequence thereto or a fragment thereof of at least 20 bases comprising at least one polymoφhism.

Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least

10 30 bases.

Novel sequence disclosed herein, may be used in another embodiment ofthe invention to regulate expression ofthe gene in cells by the use of antisense constructs. To enable methods of down-regulating expression ofthe gene ofthe present invention in mammalian cells, an example antisense expression construct can be readily constructed for instance using the

15 pREPIO vector (Invitrogen Coφoration). Transcripts are expected to inhibit translation of the gene in cells transfected with this type construct. Antisense transcripts are effective for inhibiting translation ofthe native gene transcript, and capable of inducing the effects (e.g., regulation of tissue physiology) herein described. Oligonucleotides which are complementary to and hybridizable with any portion of novel gene mRNA disclosed herein

20 are contemplated for therapeutic use. U.S. Patent No. 5,639,595, Identification of Novel Drugs and Reagents, issued Jun. 17, 1997, wherein methods of identifying ohgonucleotide sequences that display in vivo activity are thoroughly described, is herein incoφorated by reference. Expression vectors containing random ohgonucleotide sequences derived from previously known polynucleotides are transformed into cells. The cells are then assayed for a

25 phenotype resulting from the desired activity ofthe ohgonucleotide. Once cells with the desired phenotype have been identified, the sequence ofthe ohgonucleotide having the desired activity can be identified. Identification may be accomplished by recovering the vector or by polymerase chain reaction (PCR) amplification and sequencing the region containing the inserted nucleic acid material.nucleotide molecules can be synthesized for

30 antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O- alkylRNA, or other ohgonucleotide mimetics. U.S. Patent No. 5,652,355, Hybrid Oligonucleotide Phosphorothioates, issued July 29, 1997, and U.S. Patent No. 5,652,356, Inverted Chimeric and Hybrid Oligonucleotides, issued July 29, 1997, which describe the synthesis and effect of physiologically-stable antisense molecules, are incoφorated by reference. Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.

The invention further provides nucleotide primers which can detect the polymoφhisms of the invention.

According to another aspect ofthe present invention there is provided an allele specific primer capable of detecting a Factor X gene polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL ACCESSION NO. L00396.

An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e.g. as used for ARMS™ assays. The allele specific primer is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.

An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof are also contemplated wherein about 6-8 ofthe nucleotides at the 3' terminus correspond with the allele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or 1 ofthe remaining nucleotides may be varied without significantly affecting the properties ofthe primer.

Primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example "Protocols for

Oligonucleotides and Analogues; Synthesis and Properties," Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1^st Edition. If required the primer(s) may be labelled to facilitate detection.

According to another aspect ofthe present invention there is provided an allele-specific ohgonucleotide probe capable of detecting a Factor X gene polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL

ACCESSION NO. L00396.

The allele-specific ohgonucleotide probe is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides. The design of such probes will be apparent to the molecular biologist of ordinary skill.

Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length. In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene. However, if required one or more mismatches may be introduced, provided that the discriminatory power ofthe ohgonucleotide probe is not unduly affected. The probes ofthe invention may carry one or more labels to facilitate detection.

According to another aspect ofthe present invention there is provided a diagnostic kit comprising an allele specific ohgonucleotide probe ofthe invention and/or an allele-specific primer of the invention.

The diagnostic kits may comprise appropriate packaging and instructions for use in the methods ofthe invention. Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase.

In another aspect ofthe invention, the single nucleotide polymoφhisms of this invention may be used as genetic markers in linkage studies. This particularly applies to the polymoφhism at exon 7 position 57 because of its informative frequency (see below). The

Factor X gene has been mapped to chromosome 13q34 (Bowcock et al, Genomics 16, 486-

496, 1993).

According to another aspect ofthe present invention there is provided a method of treating a human in need of treatment with a Factor Xa ligand antagonist drag in which the method comprises: i) diagnosis of a single nucleotide polymoφhism in Factor X gene in the human, which diagnosis comprises determining the sequence ofthe nucleic acid at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL

ACCESSION NO. L00396. and determining the status ofthe human by reference to polymoφhism in the Factor X gene; and ii) administering an effective amount of a Factor Xa ligand antagonist drag.

The term " Factor Xa ligand antagonist drag" includes drags acting at Factor Xa and/or Factor X but the former is preferred.

Factor Xa ligand antagonist drags possess activity in the treatment or prevention of a variety of medical disorders where anticoagulant therapy is indicated, for example in the treatment or prevention of thrombotic conditions such as coronary artery and cerebro-vascular disease. Further examples of such medical disorders include various cardiovascular and cerebrovascular conditions such as myocardial infarction, the formation of atherosclerotic plaques, venous or arterial thrombosis, coagulation syndromes, vascular injury including reocclusion and restenosis following angioplasty and coronary artery bypass surgery, thrombus formation after the application of blood vessel operative techniques or after general surgery such as hip replacement surgery, the introduction of artificial heart valves or on the recirculation of blood, cerebral infarction, cerebral thrombosis, stroke, cerebral embolism, pulmonary embolism, ischaemia and angina (including unstable angina).

Preferably determination ofthe status ofthe human is clinically useful. Examples of clinical usefulness include deciding which antagonist drug or drags to administer and/or in deciding on the effective amount ofthe drag or drags. Inhibitors of Factor Xa have been disclosed in the following publications: European patent application EP 540051 A, Daiichi; WO98/21188, Zeneca Ltd and WO96/10022, Zeneca Ltd. According to another aspect ofthe present invention there is provided use of a Factor Xa ligand antagonist drug in preparation of a medicament for treating a Factor Xa and/or Factor

X-mediated disease in a human diagnosed as having a single nucleotide polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL

ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL

ACCESSION NO. L00396.

According to another aspect ofthe present invention there is provided a pharmaceutical pack comprising a Factor Xa- ligand antagonist drag and instructions for administration ofthe drag to humans diagnostically tested for a single nucleotide polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL ACCESSION NO. L00396. According to another aspect of the present invention there is provided a computer readable medium comprising at least one novel polynucleotide sequence ofthe invention stored on the medium. The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis or any other bioinformatic analysis. The reader is referred to Bioinformatics, A practical guide to the analysis of genes and proteins, Edited by A D Baxevanis & B F F Ouellette, John Wiley & Sons, 1988. Any computer readable medium may be used, for example, compact disk, tape, floppy disk, hard drive or computer chips.

The polynucleotide sequences ofthe invention, or parts thereof, particularly those relating to and identifying the single nucleotide polymoφhisms identified herein represent a valuable information source, for example, to characterise individuals in terms of haplotype and other sub-groupings, such as investigation of susceptibility to treatment with particular drags. These approaches are most easily facilitated by storing the sequence information in a computer readable medium and then using the information in standard bioinformatics programs or to search sequence databases using state ofthe art searching tools such as "GCC". Thus, the polynucleotide sequences ofthe invention are particularly useful as components in databases useful for sequence identity and other search analyses. As used herein, storage ofthe sequence information in a computer readable medium and use in sequence databases in relation to 'polynucleotide or polynucleotide sequence ofthe invention' covers any detectable chemical or physical characteristic of a polynucleotide ofthe invention that may be reduced to, converted into or stored in a tangible medium, such as a computer disk, preferably in a computer readable form. For example, chromatographic scan data or peak data, photographic scan or peak data, mass spectrographic data, sequence gel (or other) data.

The invention provides a computer readable medium having stored thereon one or a more polynucleotide sequences ofthe invention. For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of a polynucleotide ofthe invention, a polynucleotide consisting of a polynucleotide ofthe invention, a polynucleotide which comprises part of a polynucleotide ofthe invention, which part includes at least one ofthe polymoφhisms ofthe invention, a set of polynucleotide sequences wherein the set includes at least one polynucleotide sequence ofthe invention, a data set comprising or consisting of a polynucleotide sequence ofthe invention or a part thereof comprising at least one ofthe polymoφhisms identified herein.

A computer based method is also provided for performing sequence identification, said method comprising the steps of providing a polynucleotide sequence comprising a polymoφhism ofthe invention in a computer readable medium; and comparing said polymoφhism containing polynucleotide sequence to at least one other polynucleotide or polypeptide sequence to identify identity (homology), i.e. screen for the presence of a polymoφhism.

The invention will now be illustrated but not limited by reference to the following

Examples. All temperatures are in degrees Celsius. In the Examples below, unless otherwise stated, the following methodology and materials have been applied.

AMPLITAQ™, available from Perkin-Elmer Cetus, is used as the source of thermostable

DNA polymerase.

General molecular biology procedures can be followed from any ofthe methods described in "Molecular Cloning - A Laboratory Manual" Second Edition, Sambrook, Fritsch and

Maniatis (Cold Spring Harbor Laboratory, 1989).

Electropherograms were obtained in a standard manner: data was collected by ABI377 data collection software and the wave form generated by ABI Prism sequencing analysis

(2.1.2).

Example 1

Identification of Polymorphisms

1. Methods

DNA Preparation DNA was prepared from frozen blood samples collected in EDTA following protocol I

(Molecular Cloning: A Laboratory Manual, p392, Sambrook, Fritsch and Maniatis, 2^nd

Edition, Cold Spring Harbor Press, 1989) with the following modifications. The thawed blood was diluted in an equal volume of standard saline citrate instead of phosphate buffered saline to remove lysed red blood cells. Samples were extracted with phenol, then phenol/chloroform and then chloroform rather than with three phenol extractions. The DNA was dissolved in deionised water.

Template Preparation

Exons 5 and 7 were amplified from genomic DNA by PCR. Templates were prepared using the ohgonucleotide primers described below.

Exon 5 was amplified in a two step PCR reaction with an annealing temperature of 68° and denaturation temperature of 94°. Exon 7 was amplified in a three step PCR reaction with an annealing temperature of 64°, extension temperature of 72° and denaturation temperature of 94°. Each step was 1 minute. Both reactions were carried out in l.OmM MgCl₂ buffer.

For analysis generally 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR.

Forward oligos were modified by the addition of Ml 3 forward sequence to the 5' end for use in dye-primer sequencing.

Dye Primer Sequencing

Dye-primer sequencing using Ml 3 forward primer was as described in the ABI protocol

P/N 402114 for the ABI Prism™ dye primer cycle sequencing core kit with "AmpliTaq FS"™

DNA polymerase, modified in that the annealing temperature was 45° and DMSO was added to the cycle sequencing mix to a final concentration of 5 %. The extension reactions for each base were pooled, ethanol/sodium acetate precipitated, washed and resuspended in formamide loading buffer. 4.25 % Acrylamide gels were run on an automated sequencer (ABI 377, Applied Bio systems).

2. Results 5 Novel Polymorphisms

Frequency is the allele frequency ofthe variant allele in control subjects, "eng"' = engineered RFLP

10 Example 2

Engineered restriction site primers for detection of polymorphisms

Standard methodology can be used to detect the polymoφhism at position 41 (as defined by the position in EMBL ACCESSION NO L00394) and the polymoφhism at position 57 (as defined by the position in EMBL ACCESSION NO. L00396) based on the materials set out 15 below using a cDNA template.

Primer Sequence 5 '-3'

17-40Nco I ACGGAAGCTCTGCAGCCTGGACCA SEQ ID NO.5 20 58-81 Spe I TAGGATGTAGAACTCGCTCAGACT SEQIDNO.6

T at position 41 generates an engineered Nco I site in the diagnostic fragment 17-156 described above. T at 57 generates an engineered Spe I site in the diagnostic fragment 1-81 as described above. Sequence Listing Free Text

SEQ ID NO.1 <223>Description of Artificial Sequence: exon 5 forward primer

SEQ ID NO.2 <223>Description of Artificial Sequence: exon 5 reverse primer SEQ ID NO.3 <223>Description of Artificial Sequence: exon 7 forward primer

SEQ ID NO.4 <223>Description of Artificial Sequence: exon 7 reverse primer

SEQ ID NO.5 <223>Description of Artificial Sequence: 17-40 Nco I primer

SEQ ID NO.6 <223>Description of Artificial Sequence: 58-81 Spe I primer

Claims

1. A method for the diagnosis of a single nucleotide polymoφhism in a Factor X gene in a human, which method comprises determining the sequence ofthe nucleic acid ofthe human at position 41 in exon 5 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00396, and determining the status ofthe human by reference to polymoφhism in the Factor X gene.

2. A method for diagnosis according to claim 1 in which the single nucleotide polymoφhism is further defined as: the single nucleotide polymoφhism at exon 5 position 41 is presence of C and/or T; the single nucleotide polymoφhism at exon 7 position 57 is presence of C and/or T.

3. A method for diagnosis according to claim 1 or 2 in which the sequence is determined by a method selected from amplification refractory mutation system and restriction fragment length polymoφhism.

4. Use of a method according to any of claims 1 - 3 for predicting the clinical response to a therapeutic compound, or for determining the therapeutic dose of a compound, in the treatment of Factor X- and/or Factor Xa- mediated disease.

5. Use of a method according to any of claims 1 - 3 for assessing the predisposition of an individual to diseases mediated by Factor X and/or Factor Xa.

6. A nucleic acid comprising any one ofthe following polymoφhisms: the nucleic acid of EMBL ACCESSION NO. L00394 with T at position 41 as defined by the position in EMBL ACCESSION NO. L00394; and/or the nucleic acid of EMBL ACCESSION NO. L00396 with T at position 57 as defined by the position in EMBL ACCESSION NO. L00396; or a complementary strand thereof or an antisense sequence thereto or a fragment thereof of at least 20 bases comprising at least one polymoφhism.

7. An allele-specific primer capable of detecting a Factor X gene polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00394 and/or at position 57 in exon 7 in the Factor X gene as defined by the position in EMBL ACCESSION NO. L00396.

5

8. An allele-specific ohgonucleotide probe capable of detecting a Factor X gene polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the position in EMBL ACCESSION NO. L00394 and/or at position 57 in exon 7 in the Factor X gene as defined by the position in EMBL ACCESSION NO. L00396.

10

9. A diagnostic kit comprising an allele-specific primer as defined in claim 7 or an allele- specific ohgonucleotide probe as defined in claim 8.

10. A method of treating a human in need of treatment with a Factor Xa ligand antagonist 15 drag in which the method comprises:

(i) diagnosis of a single nucleotide polymoφhism in the Factor X gene in the human, which diagnosis comprises determining the sequence ofthe nucleic acid at position 41 in exon 5 of the Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL ACCESSION 20 NO. L00396, and determining the status of the human by reference to polymoφhism in the Factor X gene; and (ii) administering an effective amount of a Factor Xa ligand antagonist drug.

25 11. Use of a Factor Xa ligand antagonist drug in the preparation of a medicament for treating a Factor Xa and/or Factor X mediated disease in a human diagnosed as having a single nucleotide polymoφhism at position 41 in exon 5 ofthe Factor X gene as defined by the positions in EMBL ACCESSION NO. L00394, and/or at position 57 in exon 7 in the Factor X gene as defined by the positions in EMBL ACCESSION NO. L00396.

30

12. A computer readable medium comprising at least one nucleic acid sequence as defined in claim 6 stored on the medium.