EP1071820A1 - Method of analysis of chronic wounds - Google Patents

Abstract

The present invention relates to methods of determining susceptibility of a patient to developing 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.

Description

Method of analysis of chronic wounds

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 severity 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 pathogenesis of the condition remains elusive.

Venous ulceration alone costs the United Kingdom Health Service about £150 to £600 million each year and affects around 150,000 patients in the United Kingdom. Much of this cost is spent on care in the community, with up to 30% of community nursing time spent on treating leg ulcers. Some chronic ulcers respond rapidly to treatment, whilst others do not; indeed, many fail 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) may be indicated. If the prognosis is poor, then a more interventional approach may be appropriate, involving surgery and skin grafting.

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 and/or dysregulation of 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 hymus- derived 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, colony-stimulating factors for granulocytes and/or macrophages, tumour necrosis factors, and interferons.

Wound fluid (the exudate from wounds) contains a mixture of serum and tissue-derived proteins, including many cytokines. Its composition is thought to reflect the microenvironment of the wound site. This environment may be different within healing and non-healing chronic wounds; it has been postulated that chronic leg ulcers do not heal because there is a deficit of growth promoting cytokines (Schultz et al., 1991). Conversely, a net excess of growth inhibiting cytokines may also be present; wound fluid from leg 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 chronicity. 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 TNFα, IL-1 and IL-8 (Silber et al., 1994).

A recent report indicates that retention fluid from blisters of partial skin thickness burns, which contain relatively large amounts of cytokines and growth factors, have a surprisingly high level of IL-8 (Ono et al, 1995). IL-8 is a potent chemoattractant for

2 neutrophils and there is convincing data which demonstrates that TNFα-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 capillaries by white blood cells. It has been shown that in patients with venous leg ulcers who display this pathological feature, the white blood cells express high levels of TNFα and the authors suggest that this may explain the absence of wound repair in these patients (Claudy et al., 1991).

Two groups (Stacey et al, 1995; Harris et al (1995) have measured the levels of growth factors and cytokines in chronic leg ulcers from human patients and found that the levels of the inflammatory cytokines (IL-1 , IL-6 and TNFα) were all significantly lower in wound fluid from 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 EGF.

Leg 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 TNFα levels are significantly higher in insulin-dependent type 1 diabetic patients than in non- diabetic 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 TNFα 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, lipodermatosclerosis (LDS) is the prelude to or is associated with a venous leg 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).

3 Patients with severe burn injuries are another patient group in which significant effort has been made to understand the contribution of pro-inflammatory cytokines to the healing process. Plasma levels of IL-1, TNFα and IL-6 have been monitored in bum patients throughout the healing process. The results indicate that the systemic cytokine response to burn injury is mainly represented by IL-6 (de Bandt et al, 1994; Papini et al, 1997). A similar study by Yamada et al (1996) measured blood levels of TNFα, IL- 6 and IL-8. The increased levels of each cytokine were found to reflect the severity of the associated burn injury. In addition, the level of IL-l β has been positively correlated with burn 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 TNFα, IL-1 and IL-6 are key regulators of matrix metalloproteinase and neutrophil elastase synthesis. There is a significant amount of data which suggests that raised levels of matrix metalloproteinases and neutrophil elastase are associated with poor wound healing, particularly in chronic skin wounds such as venous, diabetic, pressure ulcers and severe burn injuries. For instance, TNFα and elastase activity were found in the granulation 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 fluid. Since the mitogenicity 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 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 significantly higher levels of gelatinases and the levels of

4- tissue inhibitors of metalloproteinase (TIMPs) were lower than in healing wounds. This data suggests that excess proteolysis in chronic wounds retards successful healing, and results from an imbalance of proteinase and inhibitors, as well as the presence of higher levels of activated metalloproteinases.

However, despite this fairly detailed knowledge that we now possess regarding the composition of the microenvironment of chronic ulcers, there is no practical test available which enables a clinician to judge the severity of such wounds or the probable 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 likely to be susceptible to chronic ulceration. There thus exists a great need 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 type of various genes 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 severity of 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.

5 Summary of the invention

According to the present invention there is provided a method of determining susceptibility of a patient to developing a chronic ulcer, comprising determining the 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 polymoφhisms 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 histocompatibility complex (MHC). 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 variations 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.

According to the present invention, it has been found that at various loci that encode genes for inflammatory cytokines, some allelic variants are over-represented in patients who suffer from chronic ulcers. It is these genetic polymoφhisms that give altered

6 levels or activities of inflammatory cytokines that thus lead to an increased incidence of chronic ulcers, heightened severity and a decreased healing response in afflicted individuals. Such altered levels or activities may directly alter the microenvironment of an ulcer, or may exert downstream effects on molecules that themselves deteriorate the 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 polymoφhisms 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 cytokines 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 inflammatory cytokines that are responsible for changes in patterns of susceptibility to and prognosis of chronic ulcers.

The polymoφhisms that are the subject of the present invention are present in any inflammatory cytokine whose activity is altered in the microenvironment of chronic ulcers. Preferably, the polymoφhisms are present in the inflammatory cytokines IL-1, IL-6, IL-8 and TNFα, although other suitable candidates will be apparent to those of skill in the art.

7 Of particular suitability for use in accordance with the present invention are the polymoφhisms listed below, which are indicative of increased risk/severity of developing a chronic ulcer.

There are three known IL-1 genes, that form a cluster on human chromosome 2ql3. ILIA and IL-1B produce IL-lα and IL-l β, respectively. IL-1RA binds to IL-1 receptors and acts as a receptor antagonist. The presence of allele 2 of the IL-1A -889 polymoφhism or allele 2 of the +3953 polymoφhism of the IL-1B gene is a positive indicator of susceptibility to chronic ulcers. This is thought to be due to an elevation of active levels of IL-1 produced by monocytes in individuals that possess these polymoφhisms. Individuals that are heterozygous for either of these polymoφhisms are at greater risk than those individuals that possess wild type IL-1A or IL-1B loci. Homozygous individuals are at even greater risk. Those individuals who possess both polymoφhisms, and are either heterozygous or homozygous for either or both of these polymoφhisms are at greatest risk.

A further IL-1B polymoφhism herein linked to chronic ulcers is the IL-1B -51 1 polymoφhism. Details of other polymoφhic sites in IL-1 genes may be found in the following references: Laurent et al., 1997; Heresbach et al, 1997; Tarnow et al., 1997a Tarnow et al., 1997b; Cork et al., 1996; Guasch et al., 1996; Clay et al., 1996 Lakemore et al, 1996; Satsangi et al., 1996; Bioque et al., 1995; Crusius et al., 1995 Danis et al., 1995b; van den Veldan et al., 1993; Bailly et al., 1993; Feltes et al., 1993 Jacob et al., 1993; di Giovine et al., 1993; Todd et al., 1993 and Richter et al., 1989.

In transgenic mice that over-produce TNFα, abnormal TNFα production has been shown to contribute to disease initiation and progression of rheumatoid arthritis, systemic inflammatory response syndrome and diabetes (Probert et al., 1996 J Leukocyte Biol 59(4): 518-525). TNFα is another inflammatory cytokine for which polymoφhisms that generate altered activity from normal are herein linked with chronic ulcers, particularly chronic ulcers. An example of such a polymoφhism is that at position -308 in the TNFα gene. Further examples of TNFα polymoφhisms which the skilled man will be able to apply to the diagnosis of chronic ulcers may be found in the following references: Abraham et al, 1993; Wilson et al, 1992; Pociot et al, 1991;

8 Seitzer et al , 1997; Brinkman et al, 1997; Demeter et al. 1997; Louis et al . 1996; Bouma et al, 1996; Chen et al, 1996; Fong et al, 1996; Wilson et al, 1995; Danis et al, 1995a; Verjans et al, 1994 and Stokkers et al, 1995.

The gene that encodes IL-6 also contains polymoφhisms whose presence can be positively correlated with susceptibility to chronic ulcers. One example is the Bg Ll 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. , 1991 ; Fugger et al. , 1989b and Dawson et al. , 1993.

With reference to IL-8, the Hindlll polymoφhism is of use in the diagnosis of susceptibility to chronic ulcers (Fey et al, 1993).

Polymoφhisms 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 polymoφhisms are the promoter polymoφhism of the plasminogen activator inhibitor (PAI-1) gene that causes an altered response to IL-1 (Dawson et al, 1993) and the polymoφhisms 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 polymoφhisms of a patient are carried out ex vivo. Assessment of polymoφhism type may be either through the use of specific antibodies directed against 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 cytokine locus.

In order to ascertain the genotype of a patient, a sample of the DNA of that patient must be available. This sample may be obtained from any tissue of the body. Commonly-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 from a finger prick of a patient. The blood may be

9 collected on absorbent paper, or preferably on an AmpliCard™ (University of Sheffield. Department of Medicine and Pharmacology, Royal Hallamshire Hospital. Sheffield. England S 10 2JF), also described in Tarlow JW. et al. 1994 Journal of Investigative Dermatology: 103: pp387-389.

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 collecting DNA and determining polymoφhism patterns as known in the art can be used.

Molecular DNA typing of the inflammatory cytokine gene locus may be carried out by detection and assignation of the DNA polymoφhisms in the inflammatory cytokine gene through the use of various techniques that will be well known to those of skill in the art. There are three preferred methods. These are first the detection of restriction fragment length polymoφhisms (RFLPs); second, Southern blotting of PCR-amplified DNA using specific probes; and third, direct sequencing of PCR products. The latter method, which although more laborious is more stringent, is generally the preferred method of the present invention.

RFLPs are changes in a specific DNA (termed a polymoφhism if the differences between human individuals occur more frequently than every 10⁷ bases) that may be traced using restriction enzymes. When a polymoφhism occurs in a consensus sequence that is recognised by a particular restriction enzyme so that this sequence is no longer recognised, 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 fragment length polymoφhism (RFLP), and can be visualised by separating the DNA according to its size on an agarose gel.

The individual fragments may be visualised by annealing 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.

10 According to the present invention, the more preferred method of detection of polymoφhisms is through the amplification of a DNA fragment that is then analysed using probes that are specific for the particular polymoφhism of interest. Alternatively the amplified DNA fragment may be sequenced directly. Preferably, the DNA fragment is amplified using the polymerase chain reaction (PCR). The amplified DNA fragment will of course comprise the portion of the inflammatory cytokine gene that contains the polymoφhism of interest.

A diagnostic length of DNA may be amplified by PCR using primers raised to conserved DNA sequence in the inflammatory cytokine gene. By a diagnostic length is meant a fragment of sufficient length to allow discernment of the characterising polymoφhisms 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 fragment 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 fragment may be designed by anyone of skill in the art so as to be complementary in sequence to the gene sequence that flanks the polymoφhism. 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 fragment 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 immobilised PCR product is challenged with labelled sequence-specific probes. Each specific probe comprises an oligonucleotide of complementary sequence to the particular defining polymoφhic region of the inflammatory cytokine locus. These probes are specific for each inflammatory cytokine polymoφhism type. Under conditions of a certain stringency, each oligonucleotide will only hybridise to the polymoφhic DNA sequence against which it is raised and thus will provide polymoφhism typing with much more accuracy than is possible using serological 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 specific binding to all wild type loci may be used as a control.

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 using digoxigenin (Kimura and Sasazuki. 1992; Boehringer Mannheim catalogue). Detection may be by autoradiography. or by chemiluminescence, respectively, depending on the system chosen. Most preferably, the invention uses digoxigenin-labelled oligonucleotides.

When using digoxigenin-labelled oligonucleotides, a labelled anti-digoxigenin antibody- enzyme conjugate is used for the detection of oligonucleotide. This specific reaction can be visualised by chemiluminescent detection using an AMPPD substrate in accordance with the manufacturer's instructions (Boehringer Mannheim). 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 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 Morrison et al. 1993. The product is purified twice using phenol/chloroform and then precipitated using ethanol. For the sequencing reaction, the DNA is loaded onto a 6% polyacrylamide gel, before direct sequencing is performed in both forward ^' and reverse directions (in triplicate) using fluorescence-labelled dideoxynucleotide termination on an Applied Biosystem 373A Automated DNA Sequencer. Alternative sequencing kits, PCR purification 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 polymoφhism type of an inflammatory 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

12 of the invention to detect the appropriate polymoφhisms 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 polymoφhism and the specific probe. Additionally, packages containing concentrated forms of reagents and buffers used for hybridisation, prehybridisation, 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.

In connection with the amplification of DNA fragments using PCR and their subsequent analysis using specific probes, the essential components of the assay kit will include the 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 incoφorated 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 modification of detail may be made without departing from the scope of the invention.

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16 EXAMPLES

Analysis of genetic polymorphisms

The subject's finger was cleaned with antiseptic wipes and the skin was punctured with a sterile lancet. Finger-stick blood samples were collected on DNAase-free blotting paper (Tarlow et al. 1994) and analysed blind for polymoφhism in the IL-1A gene at position -889 (McDowell et al. 1995), in the IL-1B gene at positions -51 1 (Di Giovine et al 1992) and +3953 (Di Giovine et al 1996), the IL-1RA gene intron-Z (Tarlow et al. 1993), and the TNF A gene at position -308 (Wilson et al. 1992).

A reaction mix excluding Taq polymerase was prepared and 1 mm dried blood spots were added prior to heating at 95°C for 15 min. Taq polymerase (1.25 u. GibcoBRL- UK) was then added and PCR started. All reactions were carried out in 20 mM TrisHCl, 50 mM KCl. 0.2 mM each dNTP and 0.05% W-1 detergent. The MgCl₂ and printer concentrations varied in each type of reaction and are detailed below.

Analysis for TNFα

The single G/A base variation polymoφhism at -308 in the TNFα gene sequence was identified by PCR amplification of genomic templates. A single base mismatch was incoφorated into one of the primers in order to complete a Ncσl restriction site.

Primer 1 : AGG CAA TAG GTT TTG AGG GGC AT Primer 2: TCC TCC CTG CTC CGA TTC CG

PCR conditions were as follows:

Final concentration of primers: 2μM. 1.5mM MgC12 was used throughout the reactions.

1 cycle [94° (3 minutes); 60°C (1 minute); 72°C ( 1 minute)]; 35 cycles [94° (1 minute); 60°C (1 minute); 72°C (1 minute)];

17 1 cycle [94° ( 1 minute); 60°C ( 1 minute); 72°C ( 1 minute)].

Restriction enzyme digestion used 6 units per 30μl reaction mixture of Ncol at 37°C for 8 hours. Sizing was using 2% agarose gels or 8% SDS-PAGE (Laemmli. 1970).

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

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

Analysis for IL-1

IL-1A -889

Primer 1 : AAG CTT GTT CTA CCA CCT GAA CTA GGC Primer 2: TTA CAT ATG AGC CTT CCA TG

Final concentration of primers: 0.8 μM; ImM MgCL₂ used through out the reactions. 1 cycle [96'C (2 min); 45 cycles [94°C for 1 min; 50^°C for lmin; 72°C for 1 min; 50°C for 1 min.

PCR product is digested overnight at 37°C with 6 units per 30μl 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-l β -51 1 ;

Primer 1 : TGG CAT TGA TCT GGT TCA T

Primer 2: GTT TAG GAA TCT TCC CAC TT

Final concentration of primers was 1 μM. 25mM MgCl was used throughout the reaction.

18 1 cycle [95°C for 2 min: 53°C for 1 min: 74°C for 1 min] 35 cycles [95°C for 1 min: 53°C for 1 min; 74°C for 1 min]

Digestion of products was with 3 units Avol per 30μl reaction at 37°C overnight, yields products of 190bρ+ 1 14bp (allele 1) or 304bp (allele 2).

IL-1B + 3953:

Primer 1 : CTC AGG TGT CCT CGA AGA ATC AAA Primer 2: CCT TTT TTG CTG TGA GTC CCG

Final concentrations of primers: 2 μM.

2.5 mM MgCl was used throughout the reactions.

35 cycles [95°C for 2 min; 67.5^°C for 1 min; 74°C for 1 min] 3 cycles [95°C for 1 min; 67.5°C for 1 min; 74°C for 5 min]

The PCR products were digested with 10 units per 30μl reaction of Taql at 65°C overnight. The resulting products of 12bp + 83bp + 97bp (allele 1) and 12bp+ 182bp (allele 2) are diagnostic.

IL-IRN (intron 2)

VNTR; Primer 1 : CTC AGC AAC ACT CCT AT

Primer 2: TCC TGG TCT GCA GGT AA

Primer concentrations were 1 μM.

1.75 mM MgCl₂ was used throughout the reactions.

1 cycle [95 for 1 min]

35 cycles [94°C for 1 min; 60^°C for 1 min; 70°C for 2 min]

1 cycle [70°C for 5 mins; 55^°C for 5 min]

19 Electrophoresis in agarose was performed at 90V for 45 min. Allele 1 (4 repeats) was 412bp; allele 2 (2 repeats). 240bp; allele 3 (3 repeats). 326bp; allele 4 (5 repeats). 498bp; and allele 5 (6 repeats). 584bp.

All PCT products were stained with ethidium bromide 0.2μg/ml and visualised under ultraviolet light 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 polymoφhism or the presence or absence of a composite genotype formed by combining pairs of DNA polymoφhism in the IL-1 gene cluster will be compared with the presence or absence of chronic dermal ulceration. This will involve logistic regression analysis and the calculation of odds ratios with the appropriate confidence interval. The strength of association will be assessed by the χ² 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 statistical package.

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

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Claims

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

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

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

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

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

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

7. 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.

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

9. A method according to claim 8, wherein the presence of the +3953IL-1B polymoφhism is diagnostic or prognostic for chronic ulcers.

10. A method according to claim 8, wherein the presence of the IL-1A -889 polymoφhism is diagnostic or prognostic for chronic ulcers.

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

12. The method of any preceding claim wherein the analysis is carried out by:

(a) digesting genomic DNA from a patient to a diagnostic fragment length;

(b) probing the DNA fragment with a probe specific for a polymoφhism type, and

21 (c) detecting the bound probe.

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

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

14. The method of any one of claims 1 to 1 1 , 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) sequencing the amplified DNA fragment in order to analyse the precise polymoφhism type of the gene.

15. The method according to any one of claims 12 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 using chemiluminescence.

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

18. Use of polymoφhism typing for inflammatory cytokines in a method of determining susceptibility to, predicting the severity of and/or healing response of chronic ulcers in a patient.

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

20. 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 encoding an inflammatory cytokine, ATP, mixed nucleotide units for extension of the nucleotide chain, and fluorescent-labelled dideoxynucleotide termination products.

21. A diagnostic kit for use in accordance with any one of the methods of claims 1 - 15 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 nucleotide chain, a restriction enzyme

22 associated with a polymoφhism associated with a gene encoding an inflammatory cytokine. a specific probe and concentrated forms of reagents and buffers useful in hybridisation, pre-hybridisation and DNA extraction.

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