GB2336431A - Method of analysis of chronic ulcers - Google Patents

Abstract

The present invention relates to methods of determining susceptibility of a patient to developing a chronic ulcers such as dermal ulcers, in particular chronic venous ulcers, arterial ulcers, diabetic ulcers and decubitus ulcers (pressure sores). The methods comprise the determination of the polymorphism type of the patient in genes that encode inflammatory cytokines. These methods may also be used to predict the severity of ulcers and the efficacy of the healing response generated by the body. Polymorphism type may be detected by RFLP analysis or hydridisation of probes to or sequencing of PCR products. The inflammatory cytokines may be IL1, IL6, IL8 or TNF-α.

Description

1 Method of analysis of chronic wounds - 2336431 The present invention

relates to methods of diagnosing susceptibility to chronic ulcers such as dermal ulcers. in particular chronic venous ulcers. arterial ulcers. diabetic ulcers and decubitus ulcers (pressure sores). These methods may also be used to predict the severitv of ulcers and the efficacy of the healing response generated by the body.

The pathogenesis of chronic ulcers at present remains unknown. although many of the physiological mechanisms that initiate and cause persistence of ulcers have been studied closely. For example, reduced oxygen extraction. perivascular fibrin cuffing and trapping of cytokines are all observable features of venous ulcers. However. the link between these physiological disturbances and the pathoQenesis of the condition remains elusive.

Venous ulceration alone costs the United Kingdom Health Service about ú 150 to ú600 in'11'on each year and affects around 150,000 patients in the United Kin=gdom. I'vluch of this cost is spent on care in the community, with up to 30% of community nursinQ time spent on treating leg ulcers. Some chronic ulcers respond rapidly to treatment, whilst others do not; indeed. many fall to heal over periods of several years. Furthermore. an ulcer may increase in size rapidly, or may remain static in terms of its size.

The choice of treatment should ideally be related to the prognosis. For example. if the prognosis is very good, then conservative treatment (such as on an out-patient basis) 1 may be indicated. If the prognosis is poor, then a more inten'entional approach may be 1-5 appropriate. involving surgery and skin grafting.

7 At present. there is no objective prognostic test for the severity of chronic ulcers. neither is there any way to estimate the likely time to healing. It is well known that many. factors may influence the course of the disease, and at present it is upon these factors that clinicians and nurses subjectively judge the prognosis. These factors include the nature of the causative disease (for example diabetes. venous insufficiency arterial insufficiency. ischaemia), patient age, nutritional status. ulcer duration. patient compliance with treatment. the nature of the treatment. and- other inexact criteria (Rijswijk. 1993. Skene et al.. 1992).

In many chronic inflammatory diseases. the up-regulation ancl/or dysregulation of 5 cytokine production in inflamed tissue and wound fluid is thought to contribute both directly and/or indirectly to the pathology of the disease. Cytokines are peptide, protein immunomodulators that are produced by activated immune cells including thymusderived T lymphocytes, B lymphocytes and monocyte/macrophages and may also be stored (e.g. in platelets) and synthesised by non-immune cells. The cytokines include interleukins. c o lo ny- stimulating factors for ranulocytes and/or macrophages. tumour 9 necrosis factors. and interferons.

Wound fluid (the extidate from wounds) contains a mixture of serum and tissue-deri,,-ed proteins. including mariv cytokines. Its composition Is thought to reflect the microenvironment of the wound site. This environment may be different within healing w and non-healing chronic wounds; it has been postulated that chronic leg ulcers do not heal because there is a deficit of arowth promoting cytokines (Schultz el al., 1991). Conversely. a net excess of -rowth inhibitine, cvtokines may also be present: wound fluid from lea ulcers has been reported by several groups to inhibit fibroblast and keratinocyte proliferation (Bucalo et al 1989-, Harris et al. 1991 -., Shakespeare et al 1991).

While transient inflammation is a key integral stimulatory process in the healing of acute wounds, excessive and prolonged inflammation can lead to tissue breakdown and can cause wound chronicitv. In cutaneous animal models of inflammation. the response of the dermis to an intradermal injection of endotoxin (LPS) has been described. Neutrophil recruitment in LPS-Induced injury was found to be associated with raised levels of T-NWct. IL-1 and IL-8 (Silber et al., 1994).

A recent report indicates that retention fluid from blisters of partial skin thickness bums. which contain relatively larae amounts of c';tokines and ---ro"ih factors. have a surprisingly high level of IL-8 (Ono et al., 1995). IL-8.1s a potent chemoattractant for neutrophils and there is convincing data which demonstrates that TNFa- induced transendothelial neutrophil migration is IL-8 dependent (Smart et al.. 1994).

The microvasculature of venous ulcers is characterised by pericapillary fibrin cuffs and by plugging of the capillanies by white blood cells. It has been shown that patients with venous leg, ulcers who display this pathological feature. the white blood cells express high levels of TNRt and the authors suggest that this may explain the absence of wound repair in these patients (Claudy et al., 199 1 Two groups (Stacey et al, 1995; Harris et al (1995) have measured the levels of grow---th factors and cytokines in chronic leg ulcers from human patients and found that the levels of the inflammatory cytokines (IL-1, IL-6 and TNIRt) were all significantly lower in wound fluid frorfl the healing phase, when compared with the initial non-healing phase of the venous leg ulcers. Conversely, there was no alteration in the levels of growth factors such as PDGF, FGF and tGF.

Lea ulcers are also strongly associated with diabetic pathology. In diabetics. it seems that the mechanism of ulceration may be due to heightened levels of circulating inflammatory cytokines. In this respect, Foss et al (1992) have shown that serum TNTFcc levels are significantIv hioffier in insulin-dependent type 1 diabetic patients than in nondiabetic controls. It has been postulated that raised levels of modified lipoproteins that are present in diabetics may stimulate macrophages to synthesise and release significantly higher levels of both IL-1 and TNF(x than levels that are found in healthy patients. The release of cytokines from activated macrophages into the sub-endothelial spaces may have a significant role in the promoting the interaction of endothelial cells with mononuclear cells. so causing endothelial damage (Lopes-Virella. 1996).

The role of leukocytes in tissue damage in the liposclerotic skin of venous disease has also been investigated. In a significant number of patients. lipodermatoscierosis (LDS) is the prelude to or is associated with a venous lea ulcer. It has been shown that in severe LDS in the absence of a detectable venous leg ulcer, dermal staining from both IL- 1 alpha and IL- 1 beta is increased, which is thought to be a contributing factor in the observed progression to venous disease (Wilkinson et al.. 1993).

4 Patients with severe bum injuries are another patient group in which significant effort Z:

has been made to understand the contribution of pro-inflammatory cytokines to the healing process. Plasma levels of IL-1. TNIct and 11---6have been monitored in burn 1 patients throughout the healing process. The results indicate that the systemic c-,,-tok' 1 ine response to bum injury is mainly represented by IL-6 (de Bandt et al., 1994.. Papin' 1 1 et al., 1997). A similar study by Yarnada et al (1996) measured blood levels of T-Ja. IL 6 and IL-8. The increased levels of each cytokine were found to reflect the severity of the associated bum injury. In addition. the level of IL- 1 P has been positively correlated with bum size. thereby implicating this cytokine in the pathogenesis of thermal injury, In addition to their role in orchestrating the host response to injury, cytokines such as TNF(x, IL-1 and IL-6 are kev reemlators of matrix metal loprote 1 nase and neutrophil elastase synthesis. There is a significant amount of data which suggests that raised levels of matrix metal loproteinases and neutrophil elastase are associated with poor wound healing, particularly in chronic skin wounds such as venous. diabetic, pressure ulcers and severe bum injuries. For instance. TNFa and elastase activity were found in the aranulation tissue of venous stasis ulcers although these proteins are barely detectable in acute wounds (Claudy et al., 1991 -, Wilkinson et al., 1993). Grinnell and Zhu (.1994.. 1996) have also implicated neutrophil elastase in the delayed healing of chronic skin wounds.

It has also been demonstrated (Schultz et al, 1993) that the mitogenicity of fibroblast cultures in acute wound fluid is lost if the experiment is repeated using chronic wound 1 zz- 1 215 fluid. Since the mitooen'c't., of chronic wound fluid could be restored in the presence of a protease inhibitor. this suggests that excess protease activity was responsible for the reduction in fibroblast activity.

The balance between matrix deposition and tissue turnover is fundamental in wound 3 0 healing. It is thought that the balance between proteolytic enzymes and their natural inhibitors contributes to this. A recent report (Bullen et al., 1995) has shown that chronic wounds contained si-nificantly higher levels of gelatinases and the levels of tissue inhibitors of metal loproteinase (TIMPs) were lower than in healing wounds. This data suggests that excess proteolysis in chronic wounds retards successful healing. and 1 - results from an imbalance of proteinase and inhibitors, as well as the presence of higher levels of activated metal loprote inases.

However, despite this fairly detailed knowledge that we now possess regarding the composition of the microenvironment of chronic ulcers. there is no practical test a"lable which enables a clinician to judge the severity of such wounds or the probable val 1 success with which the ulcer is likely to heal without treatment. This is considered by the present inventors to be partially due to the marked genetic variation that exists at the multiple genetic loci that control the inflammatory and other immune responses that are involved with chronic ulcers.

Furthermore. currently. there is no way by which it can be predicted whether an individual is likelv to be susceptible to chronic ulceration. There thus exists a erreat need Z for a reliable. objective test that would allow the identification of individuals who are at risk from contracting a chronic ulcer. Such a test would also be invaluable to allow the prognosis of ulcer severity and/or time to healing and would provide a clinician or nurse with an indication of what kind of treatment regime might be applicable in each case.

The inventors have noted an increased frequency of particular alleles in individuals in both population and family studies, in connection with the incidence of severe chronic ulcers that do not heal. It has been found that-there is a link between the polymorphism tylpe of various aenes that encode inflammatory cytokines in a patient and the risk that the patient may develop a chronic ulcer. Furthermore, this association can be extended to allow diagnosis of the likely severityof a chronic ulcer, if already partially developed. and the prospective efficacy with which the ulcer will heal. Measurement of these polymorphisms can be made from small samples of patient's tissue. such as blood, and compared with a database of such polymorphisms for prognosis of the ulcer.

6 Summary of the invention

According to the present invention there is provided a method of determining 'bility of a patient to developing a chronic ulcer. comprising determining the suscepti 1 ' 1 1 C5 Z" - polymorphism type in genes that encode inflammatory cytokines in the patient. According to a second aspect of the invention there is provided a method of predicting the severity of a chronic ulcer in a patient comprising determining the polymorphism type In genes that encode inflammatory cytokines in the patient. According to a still further embodiment of the present invention there is provided a method of predicting the healing response in a chronic ulcer in a patient comprising determining the polymorphism type in genes that encode inflammatory cytokines in the patient.

Preferably, the chronic ulcer is a dermal ulcer, selected from the group consisting of chronic venous ulcers., pressure sores, decubitis ulcers. diabetic ulcers and chronic ulcers of unknown aetiology.

Polymorphisms are variants in the sequence of a gene within a population. Gene 20 polymorphisms are therefore a mechanism by which individuals may exhibit variations within the range of what is considered to be biologically normal. They may be sequence alterations that are found in populations from different ethnic or geographic locations that, while having a different sequence. produce functionally equivalent gene products. A good example of such sequences are those of the major histocompatibilitv complex 2 55 (MFIC). Polymorphisms also encompass variations that can be classified as alleles and/or mutations that produce gene products which may have an altered function from that of the normal (wild type) gene product. Polymorphisms also encompass,'ariations which can be classified as alleles and,'or mutations which either produce no gene product. an inactive gene product or increased levels of gene product.

C According to the present invention. it has been found that at various loci that encode genes for inflammatory cvtok-ines. some allelic variants are o ver- represented in patients who suffer from chronic ulcers. It is these úenetic polymorphisms that give altered 7 levels or activities of inflammatory cytokines that thus lead to an- increased incidence of chronic ulcers, heiahtened severity and a decreased healing response in afflicted individuals. Such altered levels or activities may directly alter the microemaronment of an ulcer, or may exert downstream effects on molecules that themselves deteriorate the 5 condition of the wound or impair its repair.

This discovery allows the early detection of a predisposition to developing a chronic ulcer and represents a much improved opportunity for medical intervention than treatment of the disease once the symptoms have already commenced. The supervision of a patient over a period of time in which he or she is thought to be at risk from developing a chronic ulcer then allows early diagnosis that may improve prognosis and allow preventative intervention before the clinical symptoms of the disease are noticed. This also means that patients who cannot be differentiated on the basis of their clinical symptoms may be separable on the basis of their genetic disposition to the disease. such analysis allows the development and application of more individual treatments that suit patients with subtle or undetectable differences in their disease state.

In most cases, the genetic polymorp that are associated with chronic ulcers cause an increase in the activity or levels of inflammatory cytokines. As discussed above, many research groups have previously attempted to find correlations between levels of inflammatory cytokines in various inflammatory conditions. but no real consensus has emerged as to which cvtokines are causative and which are simply the result of increased levels or activities of other- cytokines. It is therefore hypothesised that this failure is due to the fact that it is subtle alterations in the activities of inflammaton cytokines that are responsible for changes in patterns of susceptibility to and prognosis of chronic ulcers.

The polymorphisms that are the subject of the present invention are present in any inflammatory cytokine whose activity. is altered in the micro eriv iro nment of chronic ulcers. Preferably. the polymorphisms are present in the inflammatory cytokines IL- 1. IL-6, IL-8 and TNF(x, although other suitable candidates will be apparent to those of skill in the an.

8 Of particular suitability for use in accordance with the present invention are the polymorphisms listed below. which are indicative of increased risk/severit-y of developing a chronic ulcer.

There are three known IL-1 genes. that form a cluster on human chromosome 'I q13. ILIA and IL-IB produce IL-1(x and IL-1p. respectively. ILAR-A binds to IL-1 receptors and acts as a receptor antagonist. The presence of allele 2 of the ILAA -889 polymorphism or allele 2 of the -3953 polymorphism of the IL-IB gene is a positive indicator of susceptibility to chronic ulcers. This is thought to be due to an elevation of 10 active levels of IL-1 produced by monocytes in individuals that possess these polymorphisms. Individuals that are heterozygous for either of these polymorphisms are at greater risk than those individuals that possess wild type ILAA or IL-IB loci. Homozygyous individuals are at even greater risk. Those individuals who possess both polymorphisms. and are either heterozygous or homozygous for either or both of these polymorphisms are at greatest risk.

-:)o 2 A further ILAB polymorphism herein linked to chronic ulcers is the IL-IB - 511 polymorphism. Details of other polymorphic sites in IL-1 genes may be found in the followina references: Laurent er al., 1997; Heresbach et al, 1997; Tarnow er al., 1997a,. Tamow er al., 1997b; Cork er al., 1996, Guasch er al., 1996; Clay er al., 1996-, Lakemore er al.. 1996. Satsangi er al., 1996-. Bioque er al., 1995.. Cruslus er al., 1995, Danis er al., 1995b.. van den Veldan er al., 1993: Baill.'. er al.. 1991, Feltes er al., 1993. Jacob et al., 1993. di Glovine er al., 1993, Todd er al.. 1993 and Richter er al., 1989.

In transgenic mice that over-produce -F-NFa, abnormal T'_-Ja production has been shown to contribute to disease initiation and progression of rheumatoid arthritis. systemic inflammatory response syndrome and diabetes (Probert er al.. 1996 J Leukocyte Biol 59(4): 518-525). TNF(x is another inflammatory cytokine for which polymorphisms that generate altered activity from normal are herein linked with chronic ulcers. particularly chronic ulcers. An example of such a polymorphism is that at position -308 in the TNFce gene. Further examples of T'NJ(x polymorphisms which the skilled man will be able to apply to the diagnosis of chronic ulcers may be found in the followini references: Abraham er al.. 1993, Wilson et al., 1992., Poclot er al., 1991, 9 Scitzer et al.. 1997; Brinkman et al., 1997. Demeter et al., 1997., Louis et al.. 1996.

Bourna et al., 1996. Chen et al., 1996; Fong, et al., 1996. Wilson et al., 1995, Dan' is et al., 1995a. Verjans et al., 1994 and Stokkers et al.. 1995.

The gene that encodes IL-6 also contains polymorphisms whose presence can be positively correlated with susceptibility to chronic ulcers. One example is the Bgill mutation (Blankenstein et al., 1989; Fugger et al., 1989a). Further examples may be found in the following references: Murray et al., 1997. Danis et al., 1995a, Stokkers et al., 1995; Toungouz et al., 1994; Shalhevet et al.. 1993: Jacob et al.. 1993: Titenko et al., 199 1; Fugger et al., 1989b and Dawson et al.. 1993).

With reference to IL-8, the HindIII polymorphism is of use in the diagnosis of susceptibility to chronic ulcers (Fey et aL, 1993).

Polymorphisms may also be present in genes that encode receptors for inflammatory cytokines, whose activity is necessary for the effective biological function of the cytokine. Examples of such olymorphisms are the promoter polymorphism of the p plasminogen activator inhibitor (PAI-1) gene that causes an altered response to IL-1 (Dawson et al.. 1993) and the polymorphisms that are responsible for alternative forms of the human granulocyte colony stimulating factor (G-CSF) that cause changes in growth signal transduction (Ziegler et al., 1991).

Typing of the genetic polvmorphisms-6f a patient are carried out ex vivo. Assessment of polymorphism type may be either thr6ugh the use of specific antibodies directed against w the antigenic determinants of the inflammatory cytokines or may be by analysis of the genotype of the patient. Preferably, typing is by genetic analysis of the inflammatory c-viokine locus.

1 In order to ascertain the genotype of a patient. a sample of the DNA of that patient must be 30 available. This sample may be obtained from any tissue of the body. CommonIv-used tissues for biopsy are the blood, buccal epithelium. skin or hair. Preferably, the DNA sample is obtained from blood samples. In a preferred embodiment. the DNA is obtained from blood cells obtained fibm a finger prick of a patient. The blood may be collected on absorbent paper, or preferably on an AmpliCard TII (University of Sheffield, Department of Medicine and Pharmacology. Royal Hallamshire Hospital, Sheffield. England SIO I.JF). also described in Tarlow JW. et al. 1994 Journal Qf Investzative Dermatology: 103: pp387389.

This embodiment has the advantage of requiring only. a small amount of blood and avoids the necessity for venipuncture or a tissue biopsy. However. other means for collectinc, DNA and determining polymorphism patterns as known in the art can be used.

Molecular DNA typing of the inflammatory cy-tokine gene locus may be carried out by detection and assignation of the DNA polymorphisms in the inflammatory cytokine gene throue, the use of various techniques that Will be well known to those of skill in the art.

h 1 There are three preferred methods. These are first the detection of restriction fragment length polymorphisms (RFLPs). second, Southern blotting of PCR-amplified DNA using specific probes; and third. direct sequencine, of PCR products. The latter method. which althoue,h more laborious is more stringent, is generally the preferred method of the present invention.

RFLPs are changes in a specific DNA (termed a polymorphism if the differences between human individuals occur more frequently than every 10 bases) that may be traced using restriction enzymes. When a polymorphism occurs in a consensus sequence that is recognised by a particular restriction enzyme so that this sequence is no longer recogruised. the DNA fragments produced by restriction enzyme digestion will be of different sizes.

The various possible fragment sizes from a given region therefore depend on the precise sequence of the DNA in the region. This variation in the fragment sizes is termed a restriction fraament length polymorphism (RFLP). and can be -\,,isualised by separating the c C DNA according to its size on an agarose (Yel 1 -- Z-- 1 The individual fragments may be visualised by annealinLY to a labelled oligonucleotide probe that is specific for the sequence of the fragment of interest. Various methods of labelling the probe will be known by those of skill in the art and will most commonly involve the use of radioactivity or fluorescent or enzymatic tags.

11 According to the present invention, the more preferred method of detection of polymorphisms is through the amplification of a DNA ftaze, Ment that is then analysed using probes that are specific for the particular polymorphism of interest. Alternatively the amplified DNA fragment may be sequenced directly. Preferably. the DNA ftagment is amplified using the polymerase chain reaction (PCR). The amplified DNA fragment W-ill of course comprise the portion of the inflammatory cytokine gene that contains the polymorphism of interest.

A diagnostic length of DNA may be amplified by PCR using primers raised to consen,.ed DNA sequence in the inflammatory cytokine gene. BY a diagnostic length is meant a fragment of sufficient length to allow discernment of the characterising polymorphisms of each inflammatory cytokine antigen type. Thus, the fragment must be of sufficient length to allow an oligonucleotide primer to hybridise specifically with this sequence. As will be apparent to those of skill in the art, this ent of DNA is of at least 50 bases. preferably 100 bases. and most preferably more than 400 bases in length.

The primers used to amplify the DNA ent may be designed by anyone of skill in the art so as to be complementary in -sequence to the gene sequence that flanks the polymorphism. Preferably the reaction conditions for PCR are as described herein or in Kimura and Sasazuki, 1992.

The PCR product can be purified -and immobilised for hybridisation by methods commonly used in the art. The fi-agm" may be purified by submarine gel electrophoresis and immobilised on membranes (Boehringer) as described in Kimura and Sasazuki. 1992.

For analysis by Southern blotting, the purified and inunobilised PCR product is challenged with labelled sequence-specific probes. Each specific probe comprises an oligonucleotide of complementary sequence to the particular defining polymorphic region of the inflammatory cytokine locus. These probes are specific for each inflammatory cnOkine polymorphism type. Under conditions of a certain stringency, each oligonucleotide will only hybridise to the polymorphic DNA sequence against which it is raised and thus will provide polymorphism typing with much more accuracy than is possible using serological l? methods. The conditions of stringency to use will be facile for the man of skill in the art to ascertain (see, for example Sambrook et al., 1989; Molecular Cloning: a laboratory manual. Ausubel et al., eds.. John Wiley & Sons. 1992). A further probe capable of 1 1 Z=k speci ic binding to all wild ty-pc loci may be used as a control.

i The method of detection of bound probes may be by any one of the methods commonly used in the art. Preferably the probes themselves are labelled. either by radiolabelling. or by. chemical modification. for example usine, digoxigenin (Kimura and Sasazuki, 1992.. Bochrineer Marinheim catalogue). Detection may be by autoradiography. or b, chemi luminescence. respectively, depending on the system chosen. Most preferably. the invention uses diúioxigenin- labelled oligonucleotides.

W-hen using di goxi genin- labelled oligonucleotides. a labelled ant'd'goxige in antibody- 1 1 1 1 ni enzyme conjuaate is used for the detection of oligonucleotide. This specific reaction can be visualised by chemiluminescent detection using an AIMPPI) substrate in accordance with the manufacturer's instructions (Boehringer Marinheirn). In the preferred embodiment of the invention, the conjugated enzyme comprises an alkaline phosphatase conjugate.

A preferred method of detection is by direct sequencing of the PCR products. This method 2 0 is commonplace and will be well-known to those of skill in the art. Briefly, the initial PCR product is subjected to a second amplification employing an Applied Biosystems sequencing kit, as described in Momison et al. 1993. The product is punified twice using phenoL,"chloroform and then precipitated using ethanol. For the sequencing reaction. the DNA is loaded onto a 6% polvacrylamide gel, before direct sequencing is performed in both forward ' and reverse directions (in triplicate) using fluorescence- label led dldeox,,, nucleotide termination on an Applied Blosystem 373A Automated DNA Sequencer. Alternative sequencing kits, PCR punification kits and automated sequencers are readily commercially available and may be employed in the present invention.

According to a further aspect of the present invention. there is provided a diagnostic kit for typing of the polymorphism type of an Uilflanunatory cytokine locus in a patient. All three detection methods described above lend themselves readily to the formulation of kits that can be used in diagnosis. Such kits will contain reagents suitable for applying the method of the invention to detect the appropriate polymorphisms and will thus provide the necessary materials to carry out the molecular biological reactions that are described above. These are packaged into suitable containers or supports useful for performing the assay.

The essential components of the assay vary depending upon which embodiment of the invention is to be utilised. Regarding the detection of RFLPs. the essential components of the assay include the restriction enzyme associated with the polymorphism and the specific probe. Additionally, packages containing concentrated forms of reaaents and buffers used for hybridisation, prehybri'disation, DNA extraction and the like may be included. In particular however, labelled probe, or reagents suitable to form conveniently labelled probe are useful in facilitating the conduct of this method of the invention. =1 In connection with the amplification of DNA fragments using PCR and

their subsequent analysis using ific probes, the essential components of the assay kit will include the 1 1 g specl 1 thermostable DNA polymerase enzyme associated with amplification of the DNA fragment and a suitable probe. For direct sequencing of PCR products, the essential components are the specific primers, a suitable thermostable DNA polymerase enzyme, ATP, the mixed nucleotide units for extension of the nucleotide chain, and fluorescent- labelled dideoxynucleotide termination products.

All documents mentioned in the text are incorporated herein by reference.

Various aspects and embodiments of the.present invention will now be described by way of example and illustrated with reference to the figures. It will be appreciated that c modification of detail may be made without departing from the scope of the invention.

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17 EXAMPLES

Analysis of genetic polymorphisms The subject's finger was cleaned with antiseptic wipes and the skin was punctured with sile lancet. Finger-stick blood samples were collected on DNAase-free blotting ted I,> paper (Tarlow et al. 1994) and analysed blind for polymorphism in the IL- IA Gene at position -889 (McDowell et al. 1995), in the IL- I B gene at positions 511 (Di Glovine et al. 1992) and +3) 953 (DI Giovine et aL 1996), the IL1 RA gene intron-Z (Tarlow et al.

1993), and the TNFA gene at position -308 (Wilson et al. 1992).

c a A reaction mix excluding Taq polymerase was prepared and 1 mm dried blood spots were added prior to. heating at 95C for 15 min. Taq polymerase (1. 25 u. GibcoBRLUK) was then added and PCR started. All reactions were carried out in 20 mM Tri'sFICI, 50 mM KCl. 0.2 mM each dNTP and 0.05% W-1 deteraent. The MCC1-, and printer concentrations varied in each type of reaction and are detailed below.

Analysis for TNFet The sinele G/A base variation polymorphism. at -308 in the TiJFa gene sequence was identified by PCR amplification of genomic templates. A single base mismatch was incorporated into one of the primers in order to complete a Ncol restriction site.

Primer 1: AGG CAA TAG G= TTG AGG GGC AT Primer 2: TCC CTG CTC CGA TTC CG PCR conditions were as follows:

Final concentration of primers: 2^ 1.5mM MaC12 was used throughout the reactions.

1 cycle [94' (3 minutes); WC (1 minute); 72C (1 minute)]; 35 cycles [94' (1 minute); 60C (1 minute); 72'C (1 m'nute)l.

18 1 cycle [94' (1 minute); WC (1 minute); 72'C (1 minute)].

Restriction enzyme digestion used 6 units per 30,ul reaction mixture of Ncol at 370C for 1 - i_ 8 hours. Sizin-o- was using 2% agarose Zc=,lels or 8% SDS-PAGE (Laemmi'. 1970).

Allele 1 yields 2 fragments of 87bp and 20bp.

1 -- Allele 2 contains no,'Col site and is thus not digested. Consequently. this allele only yields one 107bp product.

10Analysis for IL-1 IL-1A -889 Primer 1: AAG CTT GTT CTA CCA CCT GA-A CTA GGC Primer 2: TTA CAT ATG AGC CTT CCA TG Final concentration of primers: 0.8 tM; 1 m1V1 MaCL, used through out the reactions.

Z - 1 cycle [WC (2 min); 45 cycles [94'C for 1 min. 5WC for I min; 72'C for 1 min; WC for 1 min.

PCR product is digested overnight at 37C with 6 units per 3Oul reaction of Ncol. and restriction pattern visualised by electrophoresis through a 6% PAGE (1.50V for 2.5 hours). This gave products of 83bp--16bp (allele 1) and 99bp (allele 2).

IL- I P -51 l; Primer 1: TGG CAT TGA TCT GGT TCA T Primer 2: GTT TAG GAA TCT TCC CAC TT Final concentration of primers was 1 ptM.

"' i mM MgCh- was used throughout the reaction.

19 1 cycle [95'C for 2 min; 53)'C for 1 min., 74'C for 1 min] 35 cycles [95'C for 1 min; 5jC for 1 min; 74'C for 1 min] Digestion of products was with 3) units Avol per 3 041 reaction at 3 7C overnight, yields products of 190bp+ 1 14bp (allele 1) or 3) 04bp (allele 2).

IL-IB - 3953:

Primer 1: CW AGG MT CCT CGA AGA ATC AAA Primer 2: CCT TTT TTG CTG TOA GTC CW Final concentrations of primers: 2 ^ 2.5 mM MaCl-Y was used throu-hout the reactions.

t> - 0 1535 cycles [95'C for 2 min; 67.5C for 1 min; 74'C for 1 min] n ) cycles [95C for 1 min; 67.5C for 1 min; 74C for 5 min] The PCR products were digested with 10 units per -')Optl reaction of TaqI at 65C overnight. The resulting products of 12bp + 83bp - 97bp (allele 1) and 12bp+ 182bp 20 (allele 2) are diagnostic.

IL-IRN (intron 2) VNTR, Primer 1: CW AGC AM ACT CCT AT Primer 2: TCC TGG TCT GCA GGT AA Primer concentrations were 1 ptM.

1.75 mM MaCl., was used throughout the reactions.

t:l - 1 cycle [95'C for 1 min] 3 cycles [94'C for 1 min; WC for 1 min; 70C for 2 minj 1 cycle [70'C for 5 mins; 55C for 5 min] Electrophoresis in a-arose was performed at 90V for 45 min. Allele 1 (4 repeats) was 412bp: allele 2 (2 repeats). 2240bp: allele 3 (3) repeats). ')?6bp-. allele 4 (5 repeats). 498bp., and allele 5 (6 repeats), 584bp.

All PCT products were stained with ethidium bromide 0.2,uc,,'ml and visualised under ultraviolet 11-ht following electrophoresis. All PCR screening methods used in this study have been extensively validated.

Statistical methods Data analyses should be performed as follows. The presence or absence of a copy of the less frequent allele -for each DNA polymorphism or the presence or absence of a composite genotype formed by combining pairs of DNA polymorphism in the IL-1 gene cluster will be compared with the presence or absence of chronic dermal ulceration.

This will logistic regression analysis and the calculation of odds ratios with the appropriate confidence interval. The strength of association will be assessed by the X2 test or Fisher's exact test. A Bonferroni correction (Miller, 1981) will be applied to account for multiple comparisons. All analyses may be performed with the SAS 21 0 statistical package.

1 A similar analysis should be performed looking at the seventy (defined on a categorical scale) of the ulceration.

-25 21

Claims (1)

1. 4.

   5.

   6.

   7.

   8.

   2.

   3.

   A method of determining susceptibility of a patient to developing a chronic ulcer, comprising determining the polymorphism type of the patient in genes that encode inflammatory cytokines.

   A method of predicting the severity of a chronic ulcer in a patient comprising determining the polymorphism type of the patient in genes that encode inflammatory cytokines.

   A method of predicting the healing response in a chronic ulcer in a patient comprising determining the polymorphism type of the patient for inflammatory cytokines.

   A method according to any one of claims 1 to -3), wherein the chronic ulcer is a dermal ulcer.

   A method according to claim 4, wherein the dermal ulcer is selected from the Orroup consisting of venous ulcers, pressure sores and decubitis ulcers.

   A method according to any one of claims 1 to 5 wherein the method is carried out in vitro.

   A method according to any one of the previous claims wherein the inflammatory cytokine comprises any one of interleukin 1, interleukin 6, interleukin 8 and tumour necrosis factor alpha.

   The method according to claim 7, wherein the inflammatory cytokine comprises either of interleukin 1 or tumour necrosis factor alpha.

   A method according to claim 8, wherein the presence of the.t--'1953IL-IB polymorphism is diagnostic or-prognostic for chronic ulcers.

   10. A method according to claim 8, wherein the presence of the ILAA -889 polymorphism is diagnostic orprognostic for chronic ulcers.

   11. A method according to claim 8, wherein the presence of the +3953 ILAB and the IL- 1 A -889 polymorphis=is diagnostic or prognostic for chronic ulcers.

   3'0 12. The method of any precedingcciaim wherein the analysis is carried out by:

   (a) digesting genomic DNAtom a patient to a diagnostic fragment length; 1 C> (b) probing the DNA fragnwW with a probe specific for a polymorphism type, and 22 (c) detecting the bound probe.

   The method of any one of claims 1 to 11, comprising the following steps:

   (a) amplifying a diagnostic length DNA fragment of an inflammatory cvtokine from DNA samples isolated from patients, (b) probing the amplified DNA sample with a probe specific for an inflammatory cytokine polymorphism type and (c) detecting the bound probe.

   14. The method of any one of claims 1 to 11, comprising the following steps:

   (a) amplifying a diagnostic length DNA fragment of the gene encoding an inflammatory cytokine from DNA samples isolated from patients, (b) performing a second (nested) amplification to produce greater quantities of specific DNA, and (c) sequeneing the amplified DNA fracrinent in order to analyse the precise polymorphism type of the gene.

   15. The method according to any one of claims 122 to 14 wherein the patient DNA is prepared from a blood sample.

   16. The method according to either of claims 12 or 13. wherein the probe is detected 19.

   20.

   using chemiluminescence.

   The method according to either of claims 12 or 13, wherein the probe is detected by autoradiography.

   Use of polymorphism typing for inflammatory cytokines in a method of determining susceptibility to, predicting the severity of and'lor healing response of chronic ulcers in a patient.

   Use according to claim 18. wherein said patient is a human patient.

   A diagnostic kit for use in accordance with any one of the methods of previous claims 1-15 comprising a thermostable DNA polymerase enzyme, specific primers that are complementary to a gene encodina an inflammatorv cytokine.

   ATP, mixed nucleotide units for extension of the nucleotide chain. and fluorescent- label led dideoxynucleotide termination products.

   21. A diagnostic kit for use in accordance with any one of the methods of claims I comprising a thermostable DNA polymerase enzyme. specific primers that are complementary to a gene encoding an inflammatory cytokine, ATP, mixed nucleotide units for extension of the nueleotide chain, a restriction enzyme 2 3 associated with a polymorphism associated with a gene encoding an :n inflanimatorv cytokine, a specific probe and concentrated forms of reagents and buffers useful in hybridisation, pre-hybridisation and DNA extraction.