GENETIC TESTS TO GENOTYPE THE HLA-DRB1 GENE IN SCARRING

The present invention relates to methods and genetic tests for determining a patient's likelihood for developing abnormal scarring; particularly raised scarring.

Scars are areas of fibrous tissue that replace normal skin (or other tissue) after injury. A scar results from the biologic process of wound repair in the skin and other tissues of the body. Thus, scarring is a natural part of the healing process. With the exception of very minor lesions, every wound (e.g. after accident, disease, or surgery) results in some degree of scarring. Scar tissue is not identical to the tissue which it replaces and is usually of inferior functional quality. For example, scars in the skin are less resistant to ultraviolet radiation, and sweat glands and hair follicles do not grow back within scar tissue. A myocardial infarction, commonly known as a heart attack, causes scar formation in the heart muscle which leads to loss of muscular power and possibly heart failure.

Skin scars occur when the deep, thick layer of skin (the dermis) is damaged. Most skin scars are flat, pale and leave a trace of the original injury which caused them. The redness that often follows an injury to the skin is not a scar, and is generally not permanent. The time it takes for it to go away may, however, range from a few days to, in -some serious and rare cases, several years. Various treatments can speed up the process in some cases.

Scars may form differently based on the location of the injury on the body, gender and the age of the person who was injured. To mend the damage, the body has to lay down new collagen fibres (a naturally occurring protein which is produced by the body). This process results in a dermal scar. Because the body cannot re-build the tissue exactly as it was, the new scar tissue will have a different texture and quality than the surrounding normal tissue. An injury does not become a scar until the wound has completely healed.

No scar can ever be completely erased. They will always leave a trace, but their appearance can be improved by a number of means, including: laser surgery and resurfacing; steroid injections; pressure garments; radiotherapy; dermabrasion; or

collagen injections. There are also a number of gel sheets available, usually made from silicone, which can help to flatten and soften raised scars if worn regularly. Silicone, pressure, occlusion, topical cortisone and vitamin E have all been shown to decrease the collagen that forms scars. Patches and pads help but are unsightly so people tend to stop treatment. Silicone gels or sprays have also been demonstrated to improve the appearance of scars and possibly prevent abnormal or excessive scar formation.

Each year in the developed world over 100 million patients acquire scars primarily as a result of over 55 million elective operations and 25 million operations following trauma. Skin scars are the natural result of skin tissue repair following injury and all wounds except the most superficial leave a scar. However approximately 10% of individuals are predisposed to developing excess fibrous tissue at the site of the wound, resulting in an abnormal scar.

Abnormal raised scars, which result from excessive growth of repair tissue, are loosely characterised into two main types: hypertrophic scars and keloid scars. Hypertrophic scars are raised scars that remain within the boundaries of the original lesion, generally regressing spontaneously after the original injury. Hypertrophic scars are often red, inflamed, itchy, and even painful. They typically occur after burn injury on the trunk and extremities. Hypertrophic scars often improve in time, or through a course of steroid treatment. Surgical improvement of hypertrophic scars is possible, though recurrence can remain a possibility.

A keloid scar, or keloid disease, is a raised scar that spreads beyond the margins of the original wound and invade the surrounding skin in a way that is site specific. Keloid scars may be inflamed, itchy, and painful; they continue to grow over time, do not repress spontaneously, and almost invriably reoccur if removed by simple excision. A keloid scar is a benign dermal fibroproliferative tumour characterised by an excessive accumulation of extracellular mattrix proteins, leading in particular to an overabundance of colleagen formation. Keloid disease (KD) is a familial condition occuring more commonly in ethnic groups with a common pre-disposition to darker skin. Various modes of inheritance from autosomal recessive to autosomal dominant with incomplete clinical penetrance and variable expression have been proposed for

KD; however, no single disease-causing gene has so far been identified. It remains unclear whether KD is a .simple monogenic mendelian disorder or a complex oligogenic condition.

Genetically susceptible individuals form keloid scars after wounding but not at every body site. The altered tissue repair mechanism appears to be restricted to dermal wound healing, since other connective tissue abnormalities are not frequently reported in patients with KD. Therefore KD may be precipitated following disruption to the human dermis. A number of precipitating factors have been reported including chicken pox, burns, surgery, tattoos, bites, lacerations, piercing and vaccination, but not all such insults lead to a keloid scar, even in the susceptible individual. At present there is no satisfactory treatment.

Despite numerous histological and biological alterations demonstrated in abnormal scars, their pathogenesis is still poorly understood. However, a possible role of immunological mechanisms has been hypothesised on the basis of various experimental evidence. A study investigating the immunophenotypic profiles of hypertrophic scarring (HS) and keloid disease (KD) revealed that immune-cell infiltrates were detected in Keloid scars. Further studies have also reported elevated levels of HLA-DR and CDIa molecules in keloid and hypertrophic scars when compared to levels detected in normal tissue. Furthermore, investigations into the aetiopathogenesis of hypertrophic scarring have suggested a potential role of MHC molecules, in particular HLA-DR, during wound healing. This suggests there may also be an immunological aspect to keloid disease, with particular importance relating to MHC driven responses.

Several studies aimed at identifying genes associated with KD have been conducted; these include Transforming Growth Factor beta (TGF-β)l , TGF-β2, TGF-β3 and TGF-β receptor gene. In all cases the associations were negative.

In the US alone, approximately 170 million individuals have scars that can be characterized as hypertrophic or keloid; and out of the 6.2 million reconstructive procedures performed yearly over 250,000 are related to the revision of such scars. It

is therefore evident that a major healthcare challenge in scar management is to develop a method to predict an individual's response to skin trauma pre damage that can inform an individual's decision to undergo surgery and guide the healthcare practitioners wound management protocols.

Dupuytren's disease (DD; also known as Dupuytren's contracture) can also be considered to be a form of abnormal scarring affecting the palmar fascia in the hands (although similar scarring in the back of the hands is known as Garrod's pads; sole of the feet known as Ledderhose's disease and the penis known as Peyronie's disease) is associated with DD. Importantly, this common condition also has a high rate of recurrence after surgical excision.

Dupuytren's disease (DD) is a permanent nodular fibroproliferative disorder affecting the palms and digits of the hands, leading to progressive and irreversible contractures of the digits that often results in diminished function and severe deformity of the hand. The disorder is thought to be one of the most common hereditary connective tissue disorders in Caucasians. Despite the suspected genetic predisposition to the development of this disease, no susceptibility genes have yet been fully associated with DD. In addition, it is uncertain whether DD is a simple monogenic Mendelian disorder or a complex oligogenic condition.

Several studies aimed at identifying genes associated with DD have been conducted; these include Transforming Growth Factor beta 1. However, to date, there has been no report of a positive association between a specific MHC allele and the risk for development of DD.

Against this background the inventors investigated the potential association of HLA- DRBl in KD pathogenesis and DD. To their surprise they identified for the first time that a genetic association exists between the presence of a HLA-DRB l * 15 allele in a subject and an increased likelihood of that subject developing abnormal scarring; particularly raised scarring. This is of great interest as to date no case-control study has detected any particular genetic association to the development of such scarring.

Accordingly a first aspect of the invention provides a method for assessing the likelihood that a subject may develop abnormal scarring comprising determining whether the subject has a HLA-DRBl * ] 5 allele.

A preferred embodiment of the first aspect of the invention is wherein the abnormal scarring is "raised scarring", as discussed further below. Raised scarring includes hypertrophic scars, keloid disease (KD) and in the fascia as seen in Dupuytren's disease (DD).

The Human Leucocyte Antigen (HLA) system is the most polymorphic genetic system in all vertebrates. Since the discovery of the HLA system (also known as MHC system), numerous associations with a variety of disease conditions have been established. Several of these conditions are autoimmune disorders, involving cellular and humoral immune responses directed against the affected tissue. HLA involvement in a large number of disease conditions such as ankylosing spondylitis, systemic lupus erythematosus (SLE) and inflammatory bowel disease has also been documented.

The most intensely-studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPAl , HLA-DPBl , HLA-DQAl , HLA-DQB l ,

' HLA-DRA, and HLA-DRB l . In humans, the MHC is divided into three regions:

Class I, II, and III. The A, B, and C genes belong to MHC class I, whereas the six D genes belong to class II. HLA genes encode cell-surface antigen-presenting proteins.

One of the most striking features of the HLA genes, particularly in humans, is the astounding allelic diversity found therein, and especially among the nine classical genes.

HLA-DR is a major histocompatibility complex, class II, cell surface receptor encoded by the human leukocyte antigen complex on chromosome 6 region 6p21.31. HLA-DR is a aβ heterodimer, and each subunit contains 2 extracellular domains, a membrane spanning domain and a cytoplasmic tail. Both α and β chains are anchored in the membrane. The N-terminal domain of the mature protein forms an alpha-helix that forms the exposed part of the binding groove, the C-terminal cytoplasmic region

interacts with the other chain forming a beta-sheet under the binding groove spanning to the cell membrane.

The genetics of HLA-DR is complex: the α-chain polypeptide is encoded by the HLA-DRA locus; while the β-chain polypeptide is encoded by 4 loci: DRBl , DRB3, DRB4 and DRB5.

As mentioned above, there is a relatively extreme level of allelic diversity at the HLA- DRBl loci: approximately 500 alleles of HLA-DRB l have been identified to date. Much of the variation at HLA-DRBl occurs at peptide contact positions in the binding groove, as a result many of the alleles alter the way the DR binds peptide hgands and changes the repertoire each receptor can bind. This means that most of the changes are functional in nature, and therefore are under selection.

The alleles of the HLA-DRBl have been assigned into 13 classes. At the time of filing the present application, DRB 1 *01 allele group has 21 alleles; DRB 1 *03 allele group has 36 alleles; DRB 1 *04 allele group has 68 alleles; DRB 1 *07 allele group has 12 alleles; DRB 1 *08 allele group has 39 alleles; DRB 1*09 allele group has 7 alleles; DRB 1 *10 has 2 alleles; DRB1 * 1 1 has 73 alleles; DRBl * 12 allele group has 17 alleles; DRB1 * 13 allele group has 81 alleles; DRB1 *14 allele group has 68 alleles; DRB 1 * 15 allele group has 30 alleles; DRB 1 * 16 allele group has 13 alleles.

The inventors investigated whether there was any linkage between alleles of the HLA- DRBl with a likelihood of developing abnormal scarring; particularly raised scarring. The inventors investigated linkage of alleles in classes HLA-DRBl *01, 03, 04, 07, 08, 09, 1 1 , 12, 13, 14, 15 and 16. To their surprise, they identified a genetic association exists between the presence of a HLA-DRBl *15 allele and an increased likelihood of a subject having that allele developing abnormal scarring: the presence of a HLA-DRBl *15 allele was associated with a 2.4 times increased risk (odds ratio 2.41 , CI 95%) of developing keloids. Furthermore, the presence of a HLA-DRBl * 15 allele was associated with a 2.3 times increased risk (odds ratio 2.26, Cl 95%) of developing Dupuytren's disease.

It is important to point out that there are over 500 alleles of HLA-DRBl distributed into 13 different classes. Until the present invention, there has been no disclosure or suggestion that any of the HLA-DRB* 15 alleles were linked' to a likelihood for developing abnormal scarring.

HLA-DRBl *15 is a class of alleles present in exon 2 of the HLA-DRBl gene. At the time of filing the present application, known human HLA-DRBl *15 alleles include DRB1*150101, DRBl*150102, DRB1*150103, DRBl*150104, DRB1*150105, DRB1*150106, DRB1*150201, DRB 1*150202, DRBl* 150203, DRBl*150204, DRBl *1503, DRBl*1504, DRBl*1505, DRBl* 1506, DRB1*1507, DRBl*1508, DRB1*1509, DRBl*1510, DRB1*1511, DRB1*1512, DRB1*1513, DRB1*1514, DRB1*1515, DRB1*1516, DRB1*1517N, DRB1*1518, DRB1*1519, DRB1*1520, DRB 1*1521, DRB 1*1522. For the avoidance of doubt, by "determining whether the subject has a HLA-DRBl *15 allele" the method of invention may involve determining whether the subject has one particular HLA-DRBl *15 allele, for example, the HLA-DRBl* 150101 allele. Alternatively, the method of the invention can include determining whether the subject has any of the HLA-DRBl *15 alleles provided herein.

Also, the method of the invention may be performed in a serial manner: i.e. the method may determine whether the subject has the DRBl* 150101 allele, then whether the subject has the DRB 1*150102 allele, then the DRB1*150103 allele, and so on. However, the method of the invention may also be performed in a parallel manner, i.e. the method may determine whether the subject has any of the DRBl *150101 alleles in a single analysis step.

The nucleotide and amino acid sequences for each of these human alleles is provided in Annex 1 provided at the end of the specification. Here, the DRBl* 150101 allele is considered to be a "reference allele", and nucleotide or amino acid sequence variant to that are indicated. The nucleotide and amino acid sequences shown in Annex 1 are derived from the alignment of sequences shown in Annex 2. For the avoidance of any doubt, if there are any differences in the nucleotide and amino acid sequences of alleles shown in Annex 1 and 2, the sequence given in Annex 2 is the correct sequence.

The presence of a HLA-DRBl *15 allele indicates that a subject has a predisposition to developing abnormal scarring, i.e. they have a higher than average likelihood of developing abnormal scarring; preferably raised scarring. As shown in the accompanying examples, the inventors have shown that, using odds ratios (ORs), the presence of a HLA-DRB l *15 allele is associated with a 2.4 times increased risk odds of that subject developing abnormal scarring (in this case keloids); also, the presence of a HLA-DRBl * 15 allele was associated with a 2.3 times increased risk odds of that subject developing Dupuytren's disease. Therefore by "higher than average likelihood", we include that a subject having a HLA-DRBl * 15 allele has an OR of above 1.0 of developing abnormal scarring. The OR value may be, for example, 2.3, 2.4. The OR value could also be higher than 2.4.

It is envisaged that the method of the invention will find most application in predicting an individual's response to skin trauma pre damage. Hence the method of the invention can be considered to be a prognostic test that can be of use to inform the pre (before the procedure), peri (during the procedure) and post-operative (after the surgery) options available to both the surgeon and the patient. That is, the presence of a HLA-DRB* 15 allele is a prognostic indicator for predicting whether a subject may develop abnormal scarring; preferably raised scarring.

In such uses, if a subject is known to have a HLA-DRB* 15 allele, then this information can be used to help an individual's decision whether to undergo surgery; a pre-surgery measure. It can be used to avoid performing nonessential surgery in subjects at risk from abnormal scarring; a pre-surgery measure. It can also be used to inform a surgeon's surgical technique (e.g. close all surgical wounds with minimal tension); a peri-surgery measure. This information can also be used to assist healthcare practitioners wound management protocols, so as to minimise the development of abnormal scarring following trauma; a post-surgery measure.

The method according to the present invention is an in vitro method and can be performed on a sample containing nucleic acid and/or polypeptide derived from the subject.

The method of the invention is particularly suitable for being carried out on genomic DNA, particularly on isolated genomic DNA. Such genomic DNA may be isolated from blood or tissue samples (e. g. hair, oral buccal swabs, nail or skin, blood, plasma, bronchoalveolar lavage fluid, saliva, sputum, cheek-swab or other body fluid or tissue), or from other suitable sources, using conventional methods. The nucleic acid containing sample that is to be analysed can either be a treated or untreated biological sample isolated from the individual. A treated sample, may be for example, one in which the'nucleic acid contained in the original biological sample has been isolated or purified from other components in the sample (tissues, cells, proteins etc), or one where the nucleic acid in the original sample has first been amplified, for example by polymerase chain reaction. Thus, it will be appreciated that the sample may equally be a nucleic acid sequence corresponding to the sequence in the sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic variation. The accompanying examples provide a protocol for extracting genomic DNA from blood taken from a subject.

The method of the invention can also be carried out on protein samples obtained from a subject. Such protein may be isolated from blood or tissue samples as specified above, or from other suitable sources using conventional methods.

Various different approaches can be used to determine whether a subject has a HLA- DRBl * 15 allele. These include determining the nucleic acid sequence of the HLA- DRBl gene; determining whether a subject has a^ direct product of the HLA-DRB l gene; determining whether a subject has an indirect product of the HLA-DRBl gene.

An embodiment of the method of the invention is wherein the step of determining whether the subject has a HLA-DRBl *15 allele comprises genotyping the HLA- DRBl gene.

By "genotyping" we include determining whether the subject has one or more HLA- DRB l genes having a nucleic acid sequence of a HLA-DRB1 * 15 allele. As the HLA- DRB l * 15 alleles all reside in exon 2 of the HLA-DRBl gene, then the genotyping method may examine exon 2 sequence only.

Methods of genotypic analysis are well known to those skilled in the art. The genotype may preferably be determined by testing a sample from the subject. Preferably the sample contains genomic DNA. Methods of providing samples of genomic DNA from a subject are discussed above and can be routinely performed by the skilled person.

The nucleic acid sequence for each of the known HLA-DRBl *15 alleles is provided in the accompanying figures. This information can be used to design materials, such as oligonucleotide primers or probes specific for each allele that can be used when determining the genotype of the HLA-DRBl gene of a subject. The design of such oligonucleotide primers is routine in the art and can be performed by the skilled person with reference to the information provided herein without any inventive contribution. If required, the primer(s) or probe(s) may be' labelled to facilitate detection.

There are a number of commercially available systems that can be used for PCR- SSOP analysis of the HLA-DRB l gene. For example, the Dynal AutoRELI 48™ can be used. The basis of this system is as follows. Generic PCR amplification is carried out on DNA isolated from a subject using biotinylated primers that flank a known target area of a HLA locus, in this case exon 2 of HLA-DRBl gene. The resulting PCR product is then hybridised with an array of (SSO) probes that are immobilised on a nylon membrane. The immobilisation of these probes is known as Reverse Line blot and thus gives rise to the acronym 'RELI™'.

The hybridisation and strip wash process is automatic on the Dynal AutoRELI 48™ system. Using stringent wash conditions, probes will remain bound only to their complementary sequence in the amplified PCR product. The sensitivity of the system enables single nucleotide differences to be distinguished. The presence of a PCR product/probe complex is detected using a simple colorimetric reaction that appears as a blue precipitate. The pattern of positive probes are manually read and input onto Pattern Matching Programme (PMP), which will interpret the pattern of positive reactions and assign a HLA type to the sample of DNA.

Other techniques that may be used to detect polymorphisms according to the present invention include:- (1) Direct sequencing of the polymorphic region of interest (e. g. using commercially available kits such as the Cysts Thermo Sequence dye terminator kit-Amersham Pharmacia Biotech); (2) Sequence Specific Oligonucleotide Hybridization (SSO) (involving dot or slot blotting of amplified DNA molecules comprising the polymorphic region; hybridisation with labelled probes which are designed to be specific for each polymorphic variant; and detection of said labels); (3) Heteroduplex and single-stranded conformation polymorphism (SSCP) Analysis (involving analysis of electrophoresis band patterns of denatured amplified DNA molecules comprising the polymorphic region); (4) Sequence Specific Priming (SSP) [also described as Amplification Refractory Mutation System (ARMS)]; (5) Mutation Scanning [e. g. using the PASSPORT Mutation Scanning Kit (Amersham Pharmacia Biotech)]; (6) Chemical Cleavage of Mismatch Analysis; (7) Non-isotopic RNase Cleavage Assay (Ambion Ltd.); (8) Enzyme Mismatch Cleavage Assay; and (9) Single Nucleotide Extension Assay. 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 polymorphic positions in HLA-DRBl .

Another technique that can be used to genotype HLA-DRBl is an ELPHA tests (Enzyme Linked Probe Hybridization Assay). ELPHA tests can be used for the determination of HLA-DRBl alleles using a DNA hybridisation method. The hybridisation between a specific probe and target DNA is detected by a method adapted from the protein ELISA technique. According to this method, by PCR amplification of a sample of DNA isolated from a subject, together with suitable sequence specific oligonucleotides probes (SSO), it is possible to identify a large number of HLA alleles. ELPHA tests that can be used for HLA-DRB l genotyping can be obtained from, for example, Biotest

(http://www.biotest.de/ww/en/pub/diannostic/transplantati on/icaacnts/clpha.cfm).

Reference Strand mediated Conformational Analysis (RSCA) can also be used for HLA-DRB l genotyping. Here different HLA alleles can be typed according to conformation-dependent DNA mobility on a polyacrylamide gel. A PCR reaction is performed on a sample of DNA isolated from a subject using primers that flank the

Exon 2 for Class II HLA genes. The amplified product is then hybridized with fluorescent-labeled reference DNA molecules at a temperature that permits annealing to occur, even when mismatches are present. Mismatches between the reference strand and the sample DNA result in the formation of bulges or "bubbles" in the heteroduplex that is formed. The number and location of the bulges give the heteroduplex a unique mobility on a polyacrylamide gel, and can be used to type the HLA alleles.

A further method is sequence based typing (SBT). SBT combines a low-resolution SSP-PCR reaction followed by high resolution allele typing using automated DNA sequencing. In summary, DNA isolated from a subject is used as a template for a PCR reaction that amplifies the polymorphic region of a gene, in the present case exon 2 of HLA-DRBl , to create a primary amplification product. That product is then purified to remove excess reaction reagents, though there are single-tube reactions available in which this purification step is not required. The primary amplification product is then used as a template for sequencing reactions, in this case to sequence across exon 2 of HLA-DRBl . Once complete, the sequence reactions are analysed by a sequencer, and the products analysed to determine which HLA-DRBl allele is present in the subject.

Where PCR amplification is required as part of the method of HLA-DRBl genotyping, PCR primers may be designed such that they are suitable for amplifying a region around exon 2 of the HLA-DRBl gene. The design of suitable PCR primers is a routine laboratory technique.

A further embodiment of this aspect of the invention is wherein the method comprises determining whether the subject has a direct product of the HLA-DRBl * 15 allele.

By "direct product of the HLA-DRB 1 *15 allele" we include mRNA molecules encoded by the HLA-DRBl* 15 allele. Therefore the method of the invention can include determining whether the subject has a mRNA molecule encoded by a HLA- DRBl *15 allele. That is, if a sample from a subject has a mRNA molecule encoded by a HLA-DRB 1 * 15 allele, then that subject has a HLA-DRB 1 * 15 allele.

Methods of isolating mRNA molecules from a sample are routine in the art and well known to the skilled person. Once isolated, the nucleotide sequence of the mRNA molecule can be determined, preferably from a cDNA sample prepared from mRNA isolated from the subject. The sequence of cDNA molecules can be determined according to the genotyping methods set out above.

By "direct product of the HLA-DRBl * 15 allele" we also include polypeptides encoded by the HLA-DRB 1 * 15 allele. Therefore the method of the invention can include determining whether the subject has a polypeptide encoded by a HLA- DRBl * 15 allele. That is, if a sample from a subject has HLA-DRBl polypeptide encoded by a HLA-DRB1 * 15 allele (termed a HLA-DRB l * 15 protein), then that subject has a HLA-DRB 1 *15 allele.

The polypeptide sequence for each of the known HLA-DRBl * 15 alleles is provided in the accompanying figures. This information can be used to design materials, such as antibodies or further specific binding molecules, that may be required for the methods set out below.

Determining whether a subject has a HLA-DRBl * 15 protein may be conducted by isolating then sequencing HLA-DRBl protein from a sample derived from that subject. Methods of purifying proteins are well known in the art and can be readily applied to the method of the invention. For example, a molecule that selectively binds to the HLA-DRBl protein, e.g. an antibody or a fragment of an antibody, can be used to purify the HLA-DRB l protein from the sample from the subject. Then, using well known peptide sequencing methods, such as N-terminal sequencing, the amino acid sequence of the isolated HLA-DRBl protein can be determined and compared to that of the HLA-DRB 1 * 15 alleles provided herein.

Alternatively, the presence of HLA-DRB l * 15 protein in the sample can be detected using immunological methods. For example, serum can be used to detect the presence of one or more protein(s) encoded by the HLA-DRBl *15 alleles provided herein.

In a preferred embodiment, the presence of a HLA-DRB l * 15 protein in the sample can be detected using an antibody that selectively binds to a HLA-DRBl * ! 5 protein.

Antibodies which can selectively bind to individual HLA-DRBl * 15 proteins encoded by specific HLA-DRBl *15 alleles can be made, for example, using peptides that include amino acid sequences particular to that HLA-DRBl * 15 allele. The amino acid sequences of particular HLA-DRBl * 15 alleles is provided herein.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a polypeptide, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference), and are well known to those skilled in the art.

Screening assays to determine binding specificity of such an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Such methods include the use of hybridomas, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

It will be appreciated that other antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody- like antigen binding motifs.

A further embodiment of this aspect of the invention is wherein the method comprises determining whether the subject has an indirect product of the HLA-DRB 1 * 15 allele.

By "an indirect product of the HLA-DRBl * 15 allele" we include products that are indicative of the presence of a HLA-DRB 1 * 15 allele. For example, a HLA-DRB 1 * 15 protein can have specific epitope(s) that can induce the production of specific antibodies in the subject. Hence, by way of an example, an indirect product of the HLA-DRB 1 * 15 allele could be an antibody specific to a HLA-DRB 1 * 15 protein.

Methods of determining whether the subject has an indirect product of the HLA- DRB l * ] 5 allele include, for example, determining whether the serum of the subject contains an antibody to a HLA-DRBl *15 protein. Such antibodies can be detected according to methods routine in the art. For example, immunological methods in which serum from a subject is cross-reacted against immobilised HLA-DRBl *15 protein.

By "abnormal scarring" we include Keloid scarring, Dupuytren's contracture (Dupuytren's disease), breast capsular contracture, hypertrophic scarring, systemic sclerosis, scleroderma, and other fibrotic disorder (pulmonary, renal, liver, peritoneal, ocular fibrosis, heart).

Preferably the "abnormal scarring" is raised scarring. This is a well known and recognised pathological term for scarring characterised by a raised profile of the scar above the plane of the skin or fascia. Raised scarring is not systemic and occurs post trauma, for example following surgical incision. Systemic sclerosis, scleroderma, and other fibrotic disorders are not considered to be types of raised scarring, as the skilled person would appreciate. Keloid disease, hypertrophic scarring and Dupuytren's disease are types of raised scarring.

While it can be appreciated that the method of the invention can be applied to animal subjects of veterinary interest, it is preferred that the subject to be tested is a human subject.

It is noted that particular HLA-DRBl * 15 alleles are present at different frequencies in subjects derived from particular ethnic groups. For example, in the UK Caucasoid population, HLA-DRBl * 1501 accounts for nearly all alleles within the broader HLA- DRB l *15 group. In the examples provided herein, the inventors determined that all of the HLA-DRBl *15 positive Caucasoid keloid disease cases detected were specifically HLA-DRBl * 1501. As mentioned above and shown herein, there are six HLA-DRB1 * 1501 alleles: HLA-DRBl * 150101, HLA-DRB 1 * 150102, HLA- DRB 1 * 150103, HLA-DRBl * 150104, HLA-DRBl * 150105, HLA-DRB l * 150106. However, the differences in' the HLA-DRBl * 1501 alleles occur only in the nucleic acid sequences, and there is only one HLA-DRBl * 1501 amino acid allele.

In contrast the HLA-DRBl * 1501 allele is extremely rare in populations of Afro- Caribbean descent, with an allele frequency of 0.023 (2.3%). It is noted that the most common HLA-DRBl * 15 allele in Afro-Caribbean's is HLA-DRBl * 1503 with an allele frequency of 20.1 %.

Furthermore, the most common HLA-DRB 1 * 15 allele in non-Caucasians is HLA- DRB 1 * 1502. As shown herein, there are four HLA-DRB 1 * 1502 alleles: HLA- DRB l * 150201 , HLA-DRB l * 150202, HLA-DRBl * 150203, HLA-DRBl * 150204. However, the differences in the HLA-DRBl * 1502 alleles occur only in the nucleic acid sequences, and there is only one HLA-DRB 1 * 1502 amino acid allele.

Classification of a human subject according to ethnicity can be based upon a combination of categories such as: country of birth; nationality; language spoken at home; parents' country of birth in conjunction with country of birth; skin colour; national/geographical origin; racial group; and religion. On the basis of such tests, the ethnicity of a human subject can be determined. Ethnicity of a subject can be determined according to international agreed definitions of ethnicity.

The various elements required for a technician to perform the method of the invention may be incorporated in to a kit.

Thus, according to a second aspect of the invention there is provided a kit for determining whether a subject has a HLA-DRBl * 15 allele comprising:

A) means of genotyping the HLA-DRBl gene; and/or means for determining whether a subject has a direct and/or indirect product of a HLA-DRBl * 15 allele;

B) control polynucleotide and/or polypeptide sample(s) of the HLA-DRB 1 * 15 allele(s).

Since the kit of this aspect of the invention can be used to determine whether a subject has a HLA-DRBl * 15 allele, then clearly the kit can have great utility as part of the method of the first aspect of the invention, i.e. as part of the method of assessing the likelihood that a subject may develop abnormal scarring; preferably raised scarring. It is important to point out that until the present application there would have been no motivation for a kit to be devised which included means of genotyping the HLA- DRBl gene; and/or means for determining whether a subject has a direct and/or indirect product of a HLA-DRB 1 * 15 allele, as well as control polynucleotide and/or polypeptide sample(s) of the HLA-DRB1 * 15 allele(s).

The kit can be performed on a sample containing nucleic acid and/or polypeptide derived from a subject. Suitable samples and methods of preparing such samples are provided above in relation to the first aspect of the invention.

By "means of genotyping the HLA-DRB l gene" we include the oligonucleotide primers or probes that can be used in the PCR-SSOP method and SBT method discussed above. The amount of such materials supplied with the kit can be dependent on the method used to determine whether a subject has a HLA-DRB l * 15 allele; however, it is envisaged that the amount of such materials provided would be sufficient for the kit to be used at least once for the stated method.

By "means for proteotyping the HLA-DRBl protein" we include antibodies that can be used to determine whether a sample contains HLA-DRBl protein derived from a HLA-DRB l * 15 allele, as discussed above. The amount of such materials supplied with the kit can be dependent on the method used to determine whether a subject has a

HLA-DRBl *15 allele; however, it is envisaged that the amount of such materials provided would be sufficient for the kit to be used at least once for the stated method.

By "control polynucleotide and/or protein sample(s) of the HLA-DRB l *15 allele(s)" we mean that the kit can be supplied with one or more polynucleotide and/or polypeptide samples having the nucleotide or amino acid sequence of one or more of the HLA-DRBl * 15 alleles set out in the present application. The preparation of such polynucleotide and/or polypeptide samples can routinely performed by the skilled person using the information provided herein and known in the art. The amount of polynucleotide and/or polypeptide supplied can be dependent on the method used to determine whether a subject has a HLA-DRBl* 15 allele; however, it is envisaged that the amount of such samples provided would be sufficient for the kit to be used at least once for the stated method.

The kit of the second aspect of the invention may also comprise:

C) a data sheet outlining linkage between a particular HLA-DRB1 * 15 allele and the likelihood of the subject developing abnormal scarring; preferably raised scarring.

D) relevant buffers and regents for detecting the presence of a HLA- DRB1 * 15.

The buffers, and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots. Alternatively, the buffers and regents may be in concentrated (or even powder form) for dilution.

An embodiment of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1. Cutaneous wounds with fine line scars on the left forearm. Compare this to a large & symptomatic posterior ear keloid scar in Caucasian patients (post bat ear correction). This demonstrates a typical keloid scar affecting the left ear lobe in a Caucasian patient.

Figure 2. This table shows phenotype frequencies and /rvalues for HLA-DRBl alleles in KD, DD, and controls

Figure 3. This photograph of the palm and digits of the hand of a Caucasian subject demonstrates a typical patient with Advanced Dupuytren's disease (presence of nodules in the palm and digits as well as flexion contracture of the digits) that participated in the study.

Figure 4. Phenotype frequencies and p-values for HLA-DRBl alleles in DD and controls

Example 1 : Positive Association of HLA-DRB1*15 with Keloid Disease in Caucasians.

Abstract

Keloid disease (KD) is a fibroproliferative dermal tumour of unknown aetiology. The increased familial clustering in KD, its increased prevalence in certain races and concordance in identical twins suggest a strong genetic predisposition to keloid formation. To investigate the aetiology of KD, we compared the HLA-DRB l phenotype frequencies of Caucasoid patients with keloid scars against those observed in a control population (n = 537). A total number of 67 keloid cases were evaluated in the study. HLA-DRBl alleles were determined in all cases and controls using a commercially available semi-automated reverse hybridisation PCR-SSOP (sequence- specific oligonucleotide probes) typing system. HLA-DRBl * 15 phenotype frequency was higher in KD positive Caucasians (38.8%) when compared with controls (20.9%), (corrected p = 0.017): We conclude that in Caucasians of European origin, HLA- DRB 1 * 15 is associated with risk of developing KD following injury. Our data confirms the involvement of a strong immunogenic component to KD although the exact mechanisms involved in MHC-driven abnormal scarring requires further investigation. Further research is also required to determine if HLA-DRBl * 15 is also

■ involved in KD pathogenesis in other ethnic groups or if other MHC loci or genes are implicated.

Introduction

Keloid disease (KD) is an abnormal wound healing response to cutaneous injury that results in symptomatic, disfiguring and dysfunctional scars without any satisfactory treatment. It is a benign dermal fibroproliferative tumour characterised by an excessive accumulation of extracellular matrix proteins, leading in particular to an overabundance of collagen formation (Bayat, Walter et al. 2005). Abnormal skin scarring can occur post-injury in genetically susceptible individuals. (Russell, Trupin et al. 1988). KD is a familial condition (Bayat, Arscott et al. 2005), occurring more commonly in ethnic groups with a common pre-disposition to darker skin (Datubo- Brown 1990). Various modes of inheritance from autosomal recessive (Omo-Dare 1975) to autosomal dominant with incomplete clinical penetrance and variable expression (Marneros, Norris et al. 2001) have been proposed for KD, however, no single disease-causing gene has so far been identified. It remains unclear whether KD is a simple monogenic mendelian disorder or a complex oligogenic condition. Genetically susceptible individuals form keloid scars after wounding but not at every body site. The altered tissue repair mechanism appears to be restricted to dermal wound healing, since other connective tissue abnormalities are not frequently reported in patients with KD. Therefore KD may be precipitated following disruption to the human dermis. A number of precipitating factors have been reported including chicken pox, burns, surgery, tattoos, bites, lacerations, piercing and vaccination, but not all such insults lead to a keloid scar even in the susceptible individual (Bayat, Arscott et al. 2005). Identifying a polymorphic genetic marker associated with the disease could be extremely useful for identifying individuals at risk.

The most polymorphic genetic system in all vertebrates is the major histocompatibilty complex (MHC) also known as the Human Leucocyte Antigen (HLA) system. Since the discovery of the MHC, numerous associations with a variety of disease conditions have been established (Tiwari and Terasaki 1981 ). Several of these conditions are autoimmune disorders involving cellular and humoral immune responses directed against the affected tissue. A study by (Santucci, Borgognoni et al. 2001) investigating the immunophenotypic profiles of hypertrophic scarring (HS) and keloid disease (KD) revealed that immune-cell infiltrates were detected in Keloid scars. Studies by Chen et al (Chen, Bao et ai. 2001 ; Chen, Wang et al. 2003) have also

reported elevated levels of HLA-DR and CDIa molecules in keloid and hypertrophic scars when compared to levels detected in normal tissue. Furthermore, investigations into the aetiopathogenesis of hypertrophic scarring (Castagnoli, Stella et al. 1994; Castagnoli, Trombotto et al. 1997) have suggested a potential role of MHC molecules, in particular HLA-DR, during wound healing. This suggests there may also be an immunological aspect to keloid disease, with particular importance relating to MHC < driven responses.

Several studies aimed at identifying genes associated with KD have been conducted; these include Transforming Growth Factor beta (TGF-β 1 (Bayat, Bock et al. 2003), TGF-β2 (Bayat, Bock et al. 2002) TGF-β 3 (Bayat, Walter et al. 2005) and TGF-β receptor gene (Bayat, Bock et al. 2004). In all cases the associations were negative.

To date, no genes have been identified and also no association between specific MHC alleles and KD has been established. The aim of our study was therefore to investigate the potential association of HLA-DRB l in KD pathogenesis.

Materials and methods Samples Patients

All patients participating in the study were Caucasians of European origin. Stringent clinical criteria were used to identify keloid disease cases as opposed to other forms of skin scarring conditions such as hypertrophic scars. The employment of rigorous diagnostic criteria in our recruitment protocol assured us of the diagnosis of all recruited individuals. This approach was taken to limit the confounding effects of disease heterogeneity and misdiagnosis potentially inherent in other scarring studies. A keloid scar was defined as a dermal tumour that spread beyond the margin of the original wound, continued to grow over time, did not regress spontaneously, commonly recurred following excision and had been present for at least a minimum period of 1 year. In comparison, hypertrophic scars were defined as raised scars that remained within the boundaries of the original lesion, often regressed spontaneously within several months after the initial injury and had rarely recurred following

surgical excision. All cases were personally assessed by A.Bayat (n = 67). A full medical history was taken using a proforma and each scar lesion was examined in detail. Successive KD cases were identified through clinical records from the Department of Plastic and Reconstructive surgery in South Manchester University Hospital Trust, UK. The local and hospital ethical committees had given approval for the study protocol and proformas. Written consent was obtained from all individuals entered into the study.

Controls HLA-DRB l data were available on five hundred and thirty-seven UK Caucasoid controls for comparison. These originated from three sources: 1 18 were from general practice registers taken as comparative subjects for the Norfolk Arthritis Register (NOAR), the second group comprised 159 individuals from the same region of England, collected as part of a population-based survey identifying possible risk factors for cancer , and the third group was a cohort of 260 UK blood donors collected as controls for disease studies (Thomson, Barrett et al. 2002).

DNA Extraction

Each patient had a 5 ml venous blood sample taken using a standard venesection technique. Blood was collected in ethylenediaminetetraacetic acid-coated bottles and kept frozen until DNA was extracted from the peripheral blood cells, using a commercially available DNA extraction kit (Qiagen, UK). The DNA concentrations were then measured and diluted using sterile TE buffer (Qiagen) to l00ng/μl.

HLA typing

HLA-DRB l alleles were determined in all cases and controls using a commercially available semi -automated reverse hybridisation PCR-SSOP (sequence-specific oligonucleotide probes) typing system according to the manufacturer's instructions (Dynal, UK). The phenotype frequency of HLA-DRBl alleles were calculated for controls and KD cases. Phenotype frequencies were compared between all KD cases and controls using the chi square test and associations were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). The main focus of this study was to investigate HLA associations with KD. All analyses were carried out using Stata (Stata Corporation, TX, USA).

Results

The HLA-DRBl phenotype frequencies in both keloid patients and controls are summarised in table 1. For the majority of alleles, frequencies were similar between both groups and no significant differences were seen. However the frequency of DRBl *15 was significantly higher in keloid patients (38.8%) compared with controls (20.9%). This achieved statistical significance (p = 0.0013) which remained significant after correction for multiple testing (p = 0.017). HLA-DRBl * 15 status was associated with a 2.4 times increased risk (odds ratio 2.41, CI 95%) of developing keloids. Further analysis suggests that HLA-DRB 1 *03 frequency was lower in keloid cases (17.9%) compared with controls (28.3%) although this did not achieve statistical significance (p = 0.08).

Discussion We have demonstrated for the first time that a genetic association exists between HLA-DRBl *15 status and the risk of developing keloid scarring in Caucasians. This is potentially of great interest as to date no case-control study has detected any genetic association with the development of KD. The strong familial element in KD pathogenesis has been demonstrated before (Bayat et al 2005). Maneros et al Demonstrated that Keloid susceptibility loci exist on chromosome 2q23 and 7pl l in Japanese and African Americans respectively. However no specific genes were identified (Marneros, Norris et al. 2004).

Several studies have suggested an immunological component in the aetiology of hypertrophic scarring and keloid disease. A study by Santucci (et al 2001 ) investigating the immunophenotypic profiles of hypertrophic scarring (HS) and keloid disease (KD) revealed that the immune-cell infiltrates were composed of CD3+, CD45RO+, CD4+ T lymphocytes associated with CDla+/CD36+ dendritic cells. Functionally meaningful molecules were constantly expressed both by T cells (HLA- DR, LFA- 1 , CD 1 1 a, CD 18) and dendritic cells (HLA-DR, ICAM- 1 , CD54) in keloid scars. Large amounts of the cellular infiltrate were detected both in early and in long standing keloid lesions. The amount of immune cells detected in keloid lesions were consistently higher than that observed in all types of HS and normal tissue. This suggests there may be an immunological aspect to keloid disease, with particular

importance relating to MHC involvement. These results were supported by a more recent study (Chen et al 2003). Their study investigated the pathogenic mechanisms of abnormal scaring in KD and HS by comparing changes in HLA-DR and CDIa molecule density in relation to levels detected in normal tissue. Both HS and KD demonstrated increased expression of HLA-DR in dendritic cells and keratinocytes in the epidermis and fibroblasts in the dermis. In KD there was also increased expression of HLA-DR in endothelial cells in the dermis. Both of these studies demonstrate the potential significance of the MHC, in particular class II genes, in relation to KD.

HLA involvement in a large number of disease conditions such as ankylosing spondylitis (Monserrat et al 2006), systemic lupus erythematosus (SLE) (Hirose, Jiang et al. 2006) and inflammatory bowel disease (Annese, Piepoli et al. 2005) has been well documented. Although many conditions have been associated with a range of HLA polymorphisms, very few have been associated with HLA-DRBl *15. These include multiple sclerosis (Alizadeh, Babron et al. 2003; Barcellos, Sawcer et al. 2006), Goodpastures disease (Cairns, Phelps et al. 2003), narcolepsy (Roh, Park et al. 2006) and antibody production to Factor 8 in haemophiliac patients (Hay, Oilier et al. 1997). Goodpasture's disease has very strong associations with MHC class II loci, with over 80% of patients carrying DRBl * 1501 , compared with only 25% in control populations, giving an odds ratio for disease of 8.5 (Phelps, Jones et al. 2000). In contrast to Goodpasture's disease aetiology, HLA-DRBl * 15 is protective in RA (Zsilak, Gal et al. 2005).

In the UK Caucasoid population, HLA-DRBl * 1501 accounts for nearly all alleles within the broader DRB1*15 (DR2) group, and all of the HLA-DRBl * 15 positive Caucasoid KD cases detected in our study were specifically HLA-DRBl * 1501. This allele is extremely rare in populations of Afro-Caribbean descent, with an allele frequency of 0.023 (2.3%). The most common HLA-DRBl * 15 allele in Afro- Caribbean's is HLA-DRB 1 * 1503 with an allele frequency of 20.1% (www.allelefrequencies.net). It is therefore unlikely that the HLA-DRB 1 *1501 allele so strongly associated with KD in Caucasians would present the same association in Afro-Caribbean populations. Given the very high prevalence of keloids in people of Afro-Caribbean origin, it will be important to determine whether an HLA association exists in this population. In African Americans HLA-DRBl * 1503 is reported to be

involved in the aetiology of sarcoidosis, a multi-organ, granulotomous inflammatory disease resulting from exaggerated T-cell responses to airborne antigen (Rybicki, Walewski et al. 2005). Interestingly, sarcoidosis resembles KD in that in both conditions fibrosis occurs with increased levels of immune cell infiltrate.

Our study represents for the first time the association between HLA-DRBl and KD pathogenesis. Our data is in keeping with the involvement of a strong immunogenic component to KD although the exact mechanisms involved in MHC-driven abnormal scarring requires further investigation. .

References

Alizadeh, M., M. C. Babron, et al. (2003). "Genetic interaction of CTLA-4 with HLA-

DRl 5 in multiple sclerosis patients." Ann Neurol 54(1 ): 1 19-22. Annese, V., A. Piepoli, et al. (2005). "HLA-DRBl alleles may influence disease phenotype in patients with inflammatory bowel disease: a critical reappraisal with review of the literature." Pis Colon Rectum 48(1): 57-64; discussion 64- 5. ' -

Barcellos, L. F., S. Sawcer, et al. (2006). "Heterogeneity at the HLA-DRBl locus and risk for multiple sclerosis." Hum MoI Genet 15(18): 2813-24.

Bayat, A., G. Arscott, et al. (2005). "Keloid disease: clinical relevance of single versus multiple site scars." Br J Plast Surg 58(1): 28-37.

Bayat, A., O. Bock, et al. (2002). "Genetic susceptibility to keloid disease and transforming growth factor beta 2 polymorphisms." Br J Plast Surg 55(4): 283- 6.

Bayat, A., O. Bock, et al. (2003). "Genetic susceptibility to keloid disease and hypertrophic scarring: transforming growth factor betal common polymorphisms and plasma levels." Plast Reconstr Surg 111(2): 535-43; discussion 544-6. Bayat, A., O. Bock, et al. (2004). "Genetic susceptibility to keloid disease: transforming growth factor beta receptor gene polymorphisms are not associated with keloid disease." Exp Dermatol 13(2): 120-4.

Bayat, A., J. M. Walter, et al. (2005). "Genetic susceptibility to keloid disease: mutation screening of the TGFbeta3 gene." Br J Plast Surg 58(7): 914-21.

Cairns, L. S., R. G. Phelps, et al. (2003). "The fine specificity and cytokine profile of

T-helper cells responsive to the alpha3 chain of type IV collagen in

Goodpasture's disease." J Am Soc Nephrol 14(1 1): 2801-12.

Castagnoli, C, M. Stella, et al. (1994). "Similar ectopic expression of ICAM-I and HLA class II molecules in hypertrophic scars following thermal injury." Burns

20(5): 430-3. Castagnoli, C, C. Trombotto, et al. (1997). "Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues." Burns 23(7-8): 565-72. Chen, D., W. Bao, et al. (2001). "[Immunological regulations of dendritic cell in abnormal scarring tissue]." Zhonghua Zheng Xing Wai Ke Za Zhi 17(5): 282-

4. Chen, D., Q. Wang, et al. (2003). "Role of HLA-DR and CDIa molecules in pathogenesis of hypertrophic scarring and keloids." Chin Med J (Engl) 116(2):

314-5. Datubo-Brown, D. D. (1990). "Keloids: a review of the literature." Br J Plast Surg

43(1): 70-7. Greenwald, R. J., G. J. Freeman, et al. (2005). "The B7 family revisited." Annu Rev

Immunol 23: 515-48.

Hay, C. R., W. Oilier, et al. (1997). "HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO

Inhibitor Working Party." Thromb Haemost 77(2): 234-7. Hirose, S., Y. Jiang, et al. (2006). "Significance of MHC class II haplotypes and IgG

Fc receptors in SLE." Springer Semin Immunopathol 28(2): 163-74. Marneros, A. G., J. E. Norris, et al. (2001). "Clinical genetics of familial keloids." Arch Dermatol 137(1 1): 1429-34.

Marneros, A. G.,- J. E. Norris, et al. (2004). "Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7pl 1." J Invest Dermatol

122(5): 1 126-32.

Omo-Dare, P. (1975). "Genetic studies on keloid." J Natl Med Assoc 67(6): 428-32. Phelps, R. G., V. Jones, et al. (2000). "Properties of HLA class II molecules divergently associated with Goodpasture's disease." Int Immunol 12(8): 1 135-

43. Roh, E. Y., M. H. Park, et al. (2006). "Association of HLA-DR and -DQ genes with narcolepsy in Koreans: comparison with two control groups, randomly

selected subjects and DRBl *1501-DQBl *0602--positive subjects." Hum Immunol 67(9): 749-55.

Russell, S. B., K. M. Trupin, et al. (1988). "Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth." Pro c Natl Acad Sci U_S A 85(2): 587-91.

Santucci, M., L. Borgognoni, et al. (2001). "Keloids and hypertrophic scars of Caucasians' show distinctive morphologic and immunophenotypic profiles." Virchows Arch 438(5): 457-63.

Stenzel, A., T. Lu, et al. (2004). "Patterns of linkage disequilibrium in the MHC region on human chromosome 6p." Hum Genet 114(4): 377-85.

' Thomson, W., J. H. Barrett, et al. (2002). "Juvenile idiopathic arthritis classified by the ILAR criteria: HLA associations in UK patients." Rheumatology (Oxford) 41(10): 1 183-9.

Tiwari, J. L. and P. I. Terasaki (1981 ). "HLA-DR and disease associations." Prog Clin Biol Res 58: 151-63.

Zsilak, S., J. Gal, et al. (2005). "HLA-DR genotypes in familial rheumatoid arthritis: increased frequency of protective and neutral alleles in a multicase family." J Rheumatol 32( 12): 2299-302.

Example 2: Positive Association of HLA-DRB1*15 With Dupuytren's Disease in Caucasians

Abstract Dupuytren's disease (DD) is a permanent nodular fibroproliferative disorder affecting the palms and digits of the hands, leading to progressive, irreversible contractures of the digits that often results in diminished function and severe deformity of the hand. The disorder is thought to be one of the most common hereditary connective tissue disorders in Caucasians. The most polymorphic genetic system in all vertebrates is the major histocompatibilty complex (MHC) also known as the Human Leucocyte Antigens (HLA) system. Since the discovery of the MHC, numerous associations with a variety of disease conditions have been established. HLA-DRBl has been shown to be significantly associated with other fibroproliferative conditions.

To elucidate further the aetiology of DD, we compared the HLA-DRBl phenotype frequencies of patients with DD against the HLA-DRB l phenotype frequencies observed in a Caucasian control population (n = 537). A total number of 67 Caucasian DD patients were evaluated in the study. HLA-DRBl alleles were determined in all cases and controls using a commercially available semi-automated reverse hybridisation PCR-SSOP (sequence-specific oligonucleotide probes) typing system. HLA-DRB l * 15 phenotype frequency was higher in DD positive Caucasoids (37.3%) when compared to control data (20.9%), (corrected p = 0.029): We conclude that in Caucasoids of Northern. European Extraction, HLA-DRBl * 15 maybe associated with risk of developing DD. Our data suggests the potential involvement of a strong immunogenic component to DD although the exact mechanisms involving major histocompatibility complex (MHC) genes in DD require further investigation.

Introduction Dupuytren's disease (DD) is a permanent nodular fibroproliferative disorder affecting the palms and digits of the hands, leading to progressive and irreversible contractures of the digits that often results in diminished function and severe deformity of the hand (Fig 1 ). This common condition has a high rate of recurrence after surgical excision [I ]. There is an increased familial predisposition to the disease, which most often affects Northern European Caucasians. There have also been reports of the presence of DD in identical twins [2]. More than 25 percent of men older than 60 years and of Celtic ancestry show evidence of DD [3]. The disorder is thought to be one of the most common hereditary connective tissue disorders in Caucasians [4]. Autosomal dominance with variable penetrance, autosomal recessive, and maternal transmission have been proposed as likely modes of inheritance [5, 6]. Despite the suspected genetic predisposition to the development of this disease, no susceptibility genes have yet been fully associated with DD. In addition, it is uncertain whether DD is a simple monogenic Mendelian disorder or a complex oligogenic condition.

An ideal approach to unravelling the hereditary component of this common disease would be to identify susceptibility gene loci. Identification of susceptible gene loci would provide an ideal approach to discovering the hereditary component of this disease in affected individuals. Identifying a polymorphic genetic marker associated with the disease would be extremely useful for identifying individuals at risk. Several

studies aimed at identifying genes associated with DD have been conducted; these include Transforming Growth Factor beta 1 (TGF-Bl) [7], TGF-B2 [8], TGF-B receptor genes [9]. In all cases the associations were negative. Although previously a positive association was identified with Zinc Finger Protein 9 (Zf9) gene a Kruppel- like transcription actor involved in TGF-Bl regulation and a 16 subunit ribosomal ribonucleic acid (16s rRNA) mitochondrial' mutation in a maternally transmitted cohort of cases [10].

The most polymorphic genetic system in all vertebrates is the major histocompatibilty complex (MHC) also known as the Human Leucocyte Antigens (HLA) system. Since the discovery of the MHC, numerous associations with a variety of disease conditions' have been established. Several of these conditions are autoimmune disorders involving cellular and humoral immune responses directed against the affected tissue.

Neumuller et al investigated the prevalence of HLA-DR3 and auto-antibodies to connective tissue in Dupuytren's contracture [H ]. Their results strongly support the hypothesis of an immunogenic component to DD although no specific MHC alleles were identified.

To date, there has been no report of an association between a specific MHC allele and the risk for development of DD. The aim of our study was therefore to investigate the potential association of HLA-DRBl in DD pathogenesis.

Materials and methods

Samples Patients

All patients with Dupuytren's disease were assessed by the inventor(s), who took a full medical history and examined both hands in each patient. All patients had confirmed diagnoses of DD preoperatively, with the presence of characteristic Dupuytren's nodules in the palm of the hand and/or digits, and with contracture of the digits at the metacarpophalangeal or proximal interphalangeal joints. Only patients with advanced DD were selected for this study. Those having early-stage DD with only the presence of nodules and no contractures were excluded from the study. A total of 67 patients with DD were enrolled in the study (60 males; 7 females) with an age range of 37 - 81 years. A total of 28 patients had a family history of DD. All

patients were unrelated and of Caucasian origin form the Northwest of England. The successive DD cases were identified through operative records from the South Manchester University Hospital Foundation Trust, Manchester, UK. The local and hospital ethical committees gave approval for the study, and written consent was obtained from all individuals.

Controls

Five hundred and thirty-seven UK Caucasian controls were available for comparison.

These originated from three sources: 118 were from general practice registers as comparative subjects for the Norfolk Arthritis Register (NOAR), the second group comprised 159 individuals from the same region of England, collected as part of a population-based survey identifying possible risk factors for cancer, and the third group was a cohort of 260 UK blood donors collected as controls for disease studies [12]. The age range of patients in the control group was 45 - 74 years, with a gender distribution of 45.7% male and 54.3% female.

DNA Extraction

As described in Example 1.

HLA typing

As described in Example 1 , but samples were from DD cases.

Results

The HLA-DRBl phenotype frequencies in both DD patients and controls are summarised in Figure 4. For the majority of HLA-DRBl alleles, frequencies were similar between both groups and no significant differences were seen. However the frequency of DRBl * 15 was significantly higher in Dupuytren's patients (37.3%) compared with controls (20.9%). This achieved statistical significance (p = 0.0029) which remained significant after correction for multiple testing (p = 0.029). HLA- DRBl * 15 status was associated with a 2.3 times increased risk (odds ratio 2.26, CI 95%) of developing DD. Further analysis suggests that HLA-DRB l * 1 1 frequency was lower in DD cases (4.5%) compared with controls (1 1.4%) although this did not achieve statistical significance after correction for multiple testing {p - 0.56).

Interestingly, HLA-DRB 1 *08 occurred at a frequency of 6.9% in the controls and was absent from the DD cohort.

Discussion This study has demonstrated a statistically significant genetic association between HLA-DRB l * 15 status and the risk of developing Dupuytren's Disease (DD) in Caucasians of Northern European extraction. This is potentially of great interest as despite the strong familial element in DD, no genetic study has detected a positive association between an HLA gene and the pathogenesis of DD [13]. The suggestion of an autoimmune component in DD was proposed as early as 1972 [14] and subsequent studies confirmed the presence of serum antibodies to collagen in patients with DD [15, 16]. Neumiiller et al investigated the potential role of HLA class I, HLA- DR class II and autoantibodies to types I - IV collagen in Dupuytren's contracture [H]. HLA-DR3 was shown to be significantly associated with DD (p = <0.05) with a relative risk of 2.94, although no specific HLA-DR alleles were identified. Spencer and Walsh investigated MHC antigen patterns in 37 patients with DD [17]. HLA-A, B and DR locus antigens were investigated using mixed lymphocyte cytotoxicity screening! This study supported the findings of Tait (1982) by demonstrating a higher incidence of DR4 in DD patients although statistical significance was not achieved [18]. This may be a consequence of the relatively small numbers investigated. In addition, Williams et al investigated the relationship between the MHC and DD in 40 patients (34 male) compared with 229 controls for the DR locus and concluded that there was no association between the MHC and DD [19]. Our current study focussed on the HLA-DRBl locus in a larger cohort of DD patients than any of the previous studies (n = 67). The rationale for investigating the HLA system in relation to DD despite the previous negative findings was based on the fact that the HLA system has been shown to have a strong association with other fibrotic disorders such as sarcoidosis and systemic sclerosis [20, 21 ]. Sarcoidosis has been associated with the HLA-B8/DR3 haplotype [22]; [23], HLA-DR2, -DR5, -DR6 and -DR8 [24-29]. More recently, HLA-DRBl * 1 101 has been shown to be associated with sarcoidosis in both Afro-Americans and Caucasians in the USA [30]. In contrast HLATDQB I *0201 has been associated with a good prognosis in British and Dutch patients [31 ]. .These studies demonstrate the importance of the MHC in the aetiology of fibrotic disorders. Furthermore DRBl , specifically HLA-DRB 1 *03, has been shown to be implicated in

Chronic Periaortis, an autoimmune disease characterised by a fϊbro-inflammatory mass surrounding the abdominal aorta and the iliac arteries [32]. HLA-DRBl * 03 had a much higher incidence in the disease cohort than in the controls (24.28% versus 9.14%) with a corrected P value of 0.0012, odds ratio (OR) 3.187 and 95% confidence interval (95%C1) 1.74-5.83. Nevertheless, it is important to point out that, as far as the inventors are aware, the present application demonstrates for the first time that there is a significant genetic association between HLA-DRBl *15 status and the risk of developing Dupuytren's Disease (DD).

Rasmussen et al (1997) reported the association of HLA-DRBl * 15 and HLA- DRB 1 *0404 with inflammatory abdominal aortic aneurysms (IAAA). The HLA- DRB 1 * 15 and *0404 occurred more frequently in patients with inflammatory AAAs compared with control subjects (47% versus 27%, and 14% versus 3%; p < 0.05, respectively) [33]. Analysis of the functionally relevant amino acid polymorphisms encoded by the HLA-DRB l gene showed relevance at amino acid position 70. HLA- DRB l alleles overrepresented in patients with IAAAs express a glutamine substitution at position 70, whereas other alleles in the patient cohort express a negatively charged aspartic acid. These data indicate that a genetic risk determinant can be mapped to the HLA-DRBl locus in patients with IAAAs and the association suggests a potential role of antigen binding in the pathogenesis of this disease. Determination of a role for antigen binding in DD aetiology requires further investigation.

HLA-DRBl * 1501 has been shown to be associated with a susceptibility to Mycobacterium leprae infection [44]. A common characteristic of leprosy patients is deformity of the digits. The infiltration of the peripheral nerves by mycobacterium leprae initiates a series of destructive events that result in intraneural oedema and destruction of Scwann cells and axons in a CD4+ T-cell mediated granulomatous process [45]. Whether or not a CD4+ T-cell mediated response is the catalyst for DD is currently unknown. HLA-DRBl *08 occurred at a frequency of 6.9% in the control group and was absent from the DD cohort. This deviation is statistically significant (p = 0.026) and may confer a protective status against the development of DD. However HLA typing in a larger cohort is required to confirm this observation.

Within the MHC, extensive linkage disequilibrium (LD) exists between genes, thus making it difficult to determine whether HLA genes directly determine disease susceptibility/resistance, or whether the association is due to other genes within the MHC. Furthermore, the high density of immune-response genes in this region makes identifying specific gene effects difficult [46]. Our data is in keeping with the involvement of an immunogenic component to DD although the exact mechanisms involved in MHC-driven DD require further investigation.

References

1. Bayat, A. and D.A. McGrouther, Ann R Coll Surg Engl, 2006. 88(1): p. 3-8.

2. Bayat, A., E.J. Cunliffe, and D.A. McGrouther, Br J Hosp Med (Lond), 2007. 68(1 1): p. 604-9.

3. Hindocha, S., S. John, J.K. Stanley, SJ. Watson/and A. Bayat, J Hand Surg [Am], 2006. 31(2): p. 204-10.

4. Bayat, A., A. Alansar, A.H. Hajeer, et al, J Hand Surg [Br], 2002. 27(1): p. 47-9.

5. Bayat, A., J.K. Stanley, J. S. Watson, M. W. Ferguson, and W.E. Oilier, Br J Plast Surg, 2003. 56(4): p. 328-33. 6. Bayat, A., J.S. Watson, J.K. Stanley, M.W. Ferguson, and W.E. Oilier, Plast Reconstr Surg, 2003. 111(7): p. 2133-9.

Annex 1 : Nucleotide and amino acid sequences for DRBl* 15 alleles

Nucleotide and amino acid sequences for human HLA-DRB* 15 alleles. The presence of a missing nucleotide or amino acid residue is shown with a "*". Changes to the sequence are shown in bold.

(a) Nucleotide sequences

DRBl*150101

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150102

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGA GTT GTG GAG

AGC TTC ACA GTG CAG CGG *** *

DRBl*150103

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG1 GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG *** *

DRB1 *150104

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150105

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACC GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150106

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAA TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150201

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150202

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRBl*150203

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAT CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT G** ★** *** *** ★** *** *** ★** *

DRBl*150204

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAT GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1503

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA CAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

DRB1*1504

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC TTC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

DRB1*1505

** *** TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC CTC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG *** *

DRB1*1506

** *** *** CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GCG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1507

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1508

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG 'GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG AAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1509

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG 'GAG TTC CAG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

DRB1*1510

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAA ** GAC GAG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC *** *** *** *** *** *

DRB1*1511

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TAC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1512

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT AGC GCC GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1513

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CA* **G GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG *** *

DRB1*1514

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GCG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1515

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC TTC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1516

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG AGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1517N

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAA GA CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

DRB1*1518

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG ** GAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1519

CA GGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC

GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1520

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG

TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC AGG GTT GTG GAG

AGC TTC ACA GTG CAG CGG CGA G

DRB1*1521

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC

AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT

AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC

TGG AAC AGC CAG AAG GAC ATC CTG GAA ** GAC AGG CGG GCC

CTG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

DRB1*1522

CA CGT TTC CTG TGG CAG CCT AAG AGG GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAG GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CTG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG ** CAG GCG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G

(b) Amino acid sequences

DRBl*150101

GDTRPRFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRRVQPKVT

VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR WFLNGQEEKA GMVSTGLIQN GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLTVEWRA RSESAQSKML

SGVGGFVLGL LFLGAGLFIY FRNQKGHSGL QPTGFLS

DRBl*150102

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQR******* ********** ********** ********** ********** ********** ********** ********** ***** * * *** ********** ********** ********** ********** ********** *******

DRBl*150103 *****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQR******* ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** *******

DRBl*150104

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR****** ********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRBl*150105

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** ********** ********** ********** ********** ********** **********

********** ********** ********** *******

DRBl*150106

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** ********** ********** ********** ********** *******

DRBl*150201 GDTRPRFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRRVQPKVT

VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR WFLNGQEEKA GMVSTGLIQN

GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLTVEWRA RSESAQSKML

SGVGGFVLGL LFLGAGLFIY FRNQKGHSGL QPTGFLS

DRBl*150202

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRRVQPKVT

VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR -WFLNGQEEKA GMVSTGLIQN

GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLTVE*** **********

********** ********** ********** *******

DRBl*150203

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVG**** ********** ********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRBl *150204

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** ********** ********** ********** ********** *******

DRBl*1503 GDTRPRFLWQ PKRECHFFNG TERVRFLDRH FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRRVQPKVT

VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR WFLNGQEEKA GMVSTGLIQN

GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLTVEWRA RSESAQSKML

SGVGGFVLGL LFLGAGLFIY FRNQKGHSGL QPTGFLS

DRBl*1504

GDTRPRFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDFLEQ ARAAVDTYCR HNYGWESFT VQRRVQPKVT

VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR WFLNGQEEKA GMVSTGLIQN

GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLTVEWRA RSESAQSKML SGVGGFVLGL LFLGAGLFIY FRNQKGHSGL QPTGFLS

DRBl*1505 ******FLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDLLEQ ARAAVDTYCR HNYGWESFT VQR*******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1506

********WQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAA TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1507

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEYRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRRVQPKVT VYPSKTQPLQ HHNLLVCSVS GFYPGSIEVR WFLNGQEEKA GMVSTGLIQN

GDWTFQTLVM LETVPRSGEV YTCQVEHPSV TSPLT***** ********** ********** ********** ********** *******

DRB1*1508

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKNILEQ ARAAVDTYCR HNYGVGESFT VQRR****** ********** ********** ********** ********** ********** ********** ********** ********** ********** **********

********** ********** ********** *******

DRBl* 1509

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFQAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** ********** ********** ********** ********** *******

DRB1M510 *****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILED ERAAVDTYCR HNYGWESF* **********

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1511

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEYRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRR****** ********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1512

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPSAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR****** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** *******

DRB1*1513

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQ*DILEQ ARAAVDTYCR HNYGWESFT VQR*******

********** ********** ********** ********** **********

********** ********** ********** ********** ********** ********** ********** ********** *******

DRB1*1514 *****RFLWQ PKRECHFFNA TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1515

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDFLEQ ARAAVDTYCR HNYGVGESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1516

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVREFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR****** ********** ********** ********** ********** **********

********** ********** ********** ********** **•

********** ********** ********** *******

DRB1*1517N

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILED RRGPRWTPTA DTTTGLWRAS QCSGESNLRX

DRB1*1518

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILEE ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1519

*****GFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV

TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYGVGESFT VQRR****** ********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1M520

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILEQ ARAAVDTYCR HNYRWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1521

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGRPDAEY WNSQKDILED RRALVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

DRB1*1522

*****RFLWQ PKRECHFFNG TERVRFLDRY FYNQEESVRF DSDVGEFRAV TELGLPDAEY WNSQKDILEQ ARAAVDTYCR HNYGWESFT VQRR******

********** ********** ********** ********** **********

********** ********** ********** ********** **********

********** ********** ********** *******

An alignment of nucleotide and amino acid sequences for the HLA-DRB* 15 alleles in human. The presence of a missing nucleotide or amino acid residue is shown with a "*"; those identical to the * 150101 sequence are shown with a "-".

A 7