WO2004097045A1

WO2004097045A1 - Diagnostic assay for ankylosing spondylitis

Info

Publication number: WO2004097045A1
Application number: PCT/GB2004/001905
Authority: WO
Inventors: Matthew Arthur Brown
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2003-05-01
Filing date: 2004-05-04
Publication date: 2004-11-11
Anticipated expiration: 2005-11-01

Abstract

The present invention is to an in vitro method for detecting whether a subject has or is genetically predisposed to a seronegative spondyloarthropathy, and comprises determining the presence of a genetic polymorphism within the Interleukin 1 gene region of chromosome 2, extending from base 113621783 to base 113970711 from P-telomere.

Description

DIAGNOSTIC ASSAY FOR ANKYLOSING SPONDYLITIS

Field of the Invention

This invention relates to the identification of genetic markers associated with ankylosing spondylitis (AS). In particular, this invention relates to a diagnostic test for determining whether a patient is genetically predisposed to AS. Background of the Invention

Ankylosing spondylitis (AS) is the prototype for a variety of diseases known collectively as the seronegative spondyloarthropathies (Khan, M.A. Rheum Dis Clin Λ/ yAmer 18:1-10 (1992)). Other diseases in this group include Reiter's syndrom (RS), reactive arthritis, and other inflammatory diseases such as psoriatic arthritis, arthritis associated with inflammatory bowel disease, and acute anterior uveitis (AAU).

The seronegative spondyloarthropathies are characterised in the early stages by a chronic inflammatory infiltrate containing lymphocytes and plasma cells and later by fibroblastic proliferation, leading to formation of scar tissue as a chronic healing process ensues. The scarring results in the ankylosis of joints which may then undergo enchondral ossification of non-inflamed articular cartilage. The tendency of the fibrous tissue to ossify produces radiographic changes such as paravertebral ossification, bamboo spine, and plantar spurs. Inflammation which begins within the cartilage can involve the periosteum, ligaments, joint capsules, annulus of the interverbral disks, synovium, as well as the uveal tract and aortic wall. The main extra-skeletal manifestations include iritis and chronic aortitis leading to aortic regurgitation. In addition to lone aortic regurgitation, atrioventricular conduction abnormalities can also occur requiring valve replacement or pacemaker treatment, respectively.

Currently there is no single diagnostic test for AS and diagnosis is frequently delayed for several years after presentation of symptoms. 96% of patients with AS are HLA B27-positive; the incidence is slightly lower in other spondyloarthritides. HLA-B27 is a major histocompatibility complex (MHC) antigen strongly associated with spondyarthropathy. The role of HLA-B27 in the disease pathogenesis is yet not well understood but HLA-B27 may be a factor that influences susceptibility to Spondyarthropathy (97% of patients with Spondyarthropathy carry HLA-B27) However, conventional HLA B27 testing is not a useful screening or diagnostic test, because the antigen is present in 6-9% of the UK Caucasian population IL-1 is a primary cytokine implicated in mediating chronic and acute inflammatory diseases The IL-1 family consists of two functionally similar molecules, IL-1 α and IL-1 β, encoded by separate genes IL-1 A and IL-1 B and an IL-1 receptor antagonist IL-1 RA, encoded by IL-1 RN, which is an anti- inflammatory nonsignal ng molecule that competes for receptor binding with IL- 1 α and IL-1 β Six novel genes have been identified within the last two years that are structurally homologous to IL-1 They have recently been renamed into a family (Sims, Trends Immunol, 2001 , 22 536-537), IL-1 F5, IL-1 F6, IL-1 F7, IL- 1 F8, IL-1 F9 and IL-1 F10 with IL-1 A, IL-1 B and IL-1 RN being IL-1 F1 IL-1 F2 and IL-1 F3 respectively IL-1 F5 most closely resembles IL-1 RA competing with IL- 1 F9 as an antagonist for IL-1 RL2 receptor The functions of the other genes are yet to be elucidated although low-affinity interactions between IL-1 F7 and IL- 18R1 and between IL-1 10 and IL-1 R1 have been reported The genes are ordered from centromere to telomere IL-1 A-IL-1 B-IL-1 F7-IL-1 F9-IL-1 F6-IL-1 F8- IL-1 F5-IL-1 F10-IL-1 RN in a cluster within an ~400kb region (Nickhn et al , Genomics, 2002, 79 718-725)

McGarry et al , Rheumatology, 2001 , 40 1359-1364 discloses a link between a polymorphism within the interleukin 1 receptor antagonist (IL-1 Ra) gene and AS Polymorphic sites within the IL-1 family were examined, including the 86-base pair variable number tandem repeat within intron 2 of the IL-1 Ra gene (or chromosome 6) and polymorphisms at positions -889 in the IL1 α gene and -511 in the IL-1 β gene The authors concluded that there were no significant differences at the polymorphic alleles in the IL-1 α and IL-1 β genes, but that there was a significant association between AS and patients carrying the polymorphism in allele 2 of the IL-1 RA Van der Paardt et al, Rheumatology, 2002, 41 1419-23 studied IL1 B-511 and the IL1 RA VNTR, and reported association of the allele 2 of the IL1 RA VNTR with AS, but not of the IL1 B-511 SNP Despite these studies there still exists a need for additional diagnostic tests to determine whether a patient is suffering from or susceptible to AS Summary of the Invention

The present invention is based on the finding that polymorphisms within genes in the ιnterleukιn-1 gene region of chromosome 2 are associated with AS

According to a first aspect of the present invention, a method for detecting whether a subject has or is genetically predisposed to a seronegative spondyloarthropathy comprises determining the presence in the subject of a genetic polymorphism within the interleukin 1 gene region of chromosome 2 in the region extending from the beginning of IL-1 A to the beginning of IL-1 RN in a genetic sample obtained from a subject

According to a second aspect, an isolated polynucleotide that is useful for diagnosing whether a subject has or is predisposed to a seronegative spondyloarthropathy, comprises at least 15 contiguous bases derived from the interleukin 1 gene region of chromosome 2, extending from base 113621783 to base 11397071 1 , or its complement

According to a third aspect, a diagnostic kit comprises a polynucleotide as defined above Description of the Invention

The present invention utilises known methods of genetic analysis to determine whether a particular subject has, or is predisposed to, a seronegative spondyloarthropathy, e g AS

As used herein, "genetic predisposition" refers to an increased likelihood that a given subject has or is likely to develop a seronegative spondyloarthropathy, given the presence of a particular genomic sequence (polymorphism)

The reference to "seronegative spondyloarthropathy" is intended to refer to the related group of diseases, such as, Ankylosing Spondylitis (AS), Reiter's syndrome (RS), reactive arthritis, and other inflammatory diseases such as psoπatic arthritis, arthritis associated with inflammatory bowel disease, chronic juvinyl arthritis, Acute Anterior Uveitis (AAU), and the like The term "allele" is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms; designated herein as "allele 1 " and "allele 2". Diploid organisms may be homozygous or heterozygous for an allelic form. The term "haplotype" is used herein to refer to a combination of alleles present in a subject.

The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or subjects. A single nucleotide polymorphism (SNP) is a single base pair change. Typically, a SNP is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide, or insertion of a single nucleotide, also gives rise to single nucleotide polymorphisms.

The polymorphisms associated with a predisposition to seronegative spondyloarthropathy are found within the region from the IL-1 A gene to the beginning of the IL-1 RN gene in the interleukin 1 region of chromosome 2. This region extends from base 113621783 to 113970711 from the P-telomere of chromosome 2, numbered according to the UCSC Genome Browser July 2003 Freeze (www.genome.ucsc.edu). This region contains the genes listed in Table 1.

Table 1 IL1 Locus Genes

Single nucleotide polymorphisms (SNPs) and combinations of SNPs (haplotypes) have been found to be associated with seronegative spondyloarthropathy, in particular Ankylosing spondylitis The haplotypic associations are stronger than the associations of individual SNPs, suggesting either that there is an interaction between SNPs to cause the disease, or that the disease causing SNP lies on the haplotypes

Although any SNP found within the defined region may be used as a marker to determine the predisposition of a subject to a seronegative spondyloarthropathy disease, specific SNPs are identified in Table 2 Table 2

In Table 2, the reference to "ref snpid" refers to the database number given on the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/). The database is publicly accessible and so the specific polymorphisms can be identified for each gene. The column referred to as "allele" shows the single nucleotide substitution that characterises the SNP.

Table 3 shows preferred SNP combinations. In a preferred embodiment, one or more of these combinations of SNPs are determined, in orderto diagnose the susceptibility of a subject to a seronegative spondyloarthropathy, e.g. AS. Table 3: combination of polymorphisms

The column in Table 3 headed "Haplotype Combination" refers to the allele associated with each haplotype; 1 refers to the dominant allele, 2 refers to the recessive allele.

A further analysis of the SNPs was carried out to further investigate the association of markers independent of one another, then in combinations to determine the primary associated markers. The analysis revealed that IL-1 F10-1 and IL1 B-511 were key markers, each marking separate haplotypes that are independently associated with AS, and, when taken together have a very strong association with disease. Assuming multiplicative effects of haplotypes, in families with both parents genotyped, the combination of IL-1 B-511 and IL-1 F10- 1 is significantly associated with AS (p=0.0002). Odds ratios, significance levels and confidence intervals are given in Table 4, which shows the relative risks for combination of IL-1 B-51 1 and IL-1 F10-1 in parent-case trios, relative to the haplotype 2-2. Table 4

Once IL-1 B-51 1 was accounted for, IL-1 F10-1 remained significantly associated (p=0 002) and vice versa (p=0 0008) The combination of these two markers was also significant in the overall dataset (p=1 7x10⁸) and to a lesser extent in the case-control analysis (p=0 04)

Having identified the genetic region associated with the seronegative spondyloarthropathies, including specific SNPs, it will be apparent to the skilled person how to detect polymorphisms within this region, for a particular subject, to make a diagnosis Methods for the detection of a polymorphism are known in the art, and include polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (e g Ju et al, Proceeding of the Eleventh International Histocompatibility Workshop and Conference, Vol 2, 1992, pgs 317-319), ligase chain reaction (LCR) Abravaya et al , 1995, NAR, 23(4) 675-682), oligotyping using Sequence Specific Primers (SSP) (e g Olerup et al , Proceedings of the Eleventh International Histocompatibility Workshop and Conference" Vol 2, 1992, pgs 315-317), oligotyping using Sequence Specific Oligonucleotide Probes (SSOP) (Tiercy et al , Immunobiology of HLA, Vol II, pp 248-250, 1987, Springer Verlag, New York), Single-stranded conformation polymorphism (SSCP) (Yap et al , Feb/1992, Trends in Genetics, 8(2) 49, and Orita et al , 1989, Genomics, 5 874-879), and direct sequencing of the 3' flanking region of the HLA-B locus gene (see, e g Santamaπa et al , Proceedings of the Eleventh International Histocompatibility Workshop and Conference" Vol 2, 1992, pgs 342-345) The content of each of these publications is incorporated herein by reference The present invention is not limited to any particular method for detecting a polymorphism A preferred method for the identification of the presence of a SNP is to use the LightCycler system (Lohmann et al , Biochemica, 2000, (4) 23-28) developed by Roche Molecular Biochemicals The LightCycler system enables the amplification and real-time detection of a polynucleotide, allowing accurate quantification The system permits the detection and genotyping of single nucleotide polymorphisms by utilising a function known as melting curve analysis During the melting curve analysis the LightCycler instrument monitors the temperature-dependent hybridisation of sequence specific hybridisation probes to single stranded DNA An alternative detection method is to use what are referred to in the art as "Molecular beacons" Molecular beacons are ohgonucleotides designed for the detection and quantification of target nucleic acids The ohgonucleotides usually comprise self-hybridising portions that, in the absence of a target nucleic acid, form a stem-loop structure A fluorescent moiety and a quencher moiety are attached at each end of the oligonucleotide, and are positioned adjacently when the oligonucleotide is in the stem-loop orientation Fluorescence is effectively prevented by the quencher moiety in this orientation The loop portion of the oligonucleotide is complementary to a specific target nucleic acid and, in the presence of the target, hybridisation to the target occurs disrupting the stem loop orientation, separating the fluor and quencher, resulting in an increase in detectable fluorescence The use of the molecular beacons approach to the detection of SNPs is disclosed in US 6548254, the content of which is hereby incorporated by reference Using the known sequence information for the IL-1 gene region, including the known polymorphisms and the novel polymorphisms disclosed herein, it is possible to design hybridisation probes for use in the LightCycler system, or any hybridisation based system

The design of suitable polynucleotide/hybndisation probes will be apparent to the skilled person The probes will usually comprise the polymorphic site, e g the SNP The polynucleotides/hybπdisation probes may be detectably labelled, e g fluorescently labelled, using methods and labels known in the art, e g as used in the detection methods referred to above

The polynucleotides/hybndisation probes may be immobilised to a support surface, for use in a diagnostic assay Suitable support materials are known in the art and include, ceramics, plastics, glass and silicon materials Methods for immobilising polynucleotides to a support material are also known in the art Polynucleotide array technology (DNA chips) are suitable for use in the invention, for screening of biological samples Arrays that include the desired immobilised polynucleotides can be produced on a customised basis by various companies, including HySeq In general, the arrays employ immobilised polynucleotide probes that are complementary to target sequences from a biological sample (e g from a subject) In the context of the present invention, the target sequence will include a polymorphism as disclosed herein

The polynucleotides to be used as probes in a diagnostic method will usually complete at least an 8, 10, 15, 20 or 50 consecutive nucleic acid sequence derived from the defined IL-1 region, in particular derived from any of the genes identified in Table 1 , and most preferably derived from the polymorphic sites disclosed herein

Polynucleotides may also be designed to act as primers to amplify polynucleotides that may comprise a polymorphism

The association between the presence of polymorphisms in the IL-1 region of chromosome and AS was identified by studying the correlation between the transmission of genetic markers and the prevalence of AS throughout generations within a family (so-called linkage analysis) and within a given population (population association studies) Linkage Analysis

When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci With loci that are genetic markers, a genetic map can be established, and the strength of linkage between markers and disease states can be calculated and used to indicate the relative positions of markers and genes affecting those disease states The classical method for linkage analysis is the logarithm of odds (LOD) score method (Morton et al , Am J Hum Genet , 1955, 7 277-318) Calculation of LOD scores requires specification of the mode of inheritance for the disease (parametric method) Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb Population Association Studies

The occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls Therefore, association between the trait and any allele in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region Case-control populations can be genotyped for to identify associations that narrowly locate a trait causing allele Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms to be analysed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al , Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1982), Sambrook et al , Molecular Cloning A Laboratory Manual (2 ed ), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (19189), David et al , Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc , New York, USA (1986), or Methods in Enzymology Guide to Molecular Cloning Techniques Vol 152, S L Berger and A R Kimmerl Eds , Academic Press Inc , San Diego, USA (1987) The following Example illustrates the invention

Example

DNA was extracted by standard methods from 15ml anticoagulated blood from 227 sib-pair families and 317 parent case trios, all Caucasian AS was defined by the modified New York diagnostic criteria (van der Linden, Br J Rheumatol , 1983,22 18-19) Diagnosis of AS was confirmed by a qualified rheumatologist for all patients and followed by either examination or telephone interview In cases with atypical history or no previous radiographic evidence, pelvic and lumbosacral spine radiographs were obtained and attending general practitioners contacted to confirm diagnosis SNP genotyping

22 SNPs in the genes IL-1 F7, IL-1 F9, IL-1 F6, IL-1 F8, IL-1 F5 and IL- 1 F10 (Table 4) were genotyped Extension Reactions (hME reactions) were designed using SpectroDESIGNER, Sequenom's assay design software, amplicon size containing the polymorphic site was set at 70-150bp and the mass of the extended primer within the mass range of 5000 - 8500 Da To multiplex the hME reactions, SNPs were grouped according to termination mix, with all extension primers and possible extension products differing by a minimum of 50Da 5μl PCR reactions were generated using 16mM (NH₄)₂SO₄, 67mM Tπs-HCI (pH 8 8) 0 01 % Tween-20, 1 5mM MgCI₂ 0 4mM dNTPs, 0 5μM primers, Taq and 50ng DNA, and the PCR conditions 96°C 1 minute, 5 cycles 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 30 seconds followed by 29 cycles of 94°C 45 seconds, 65°C 45 seconds and 72°C for 30 seconds a final extension of 72°C for 15 minutes and 4°C for 15 minutes. Unincorporated dNTPs from amplification products were removed using shrimp alkaline phosphatase (SAP). The extension reaction was performed using hME Termination mix 5.4μM extension primer and 0.576 units MassEXTEND enzyme (thermosequenase). The hME cocktail was transferred into SAP-cleaned PCR plates prior to cycling at 94°C for 2 minutes followed by 55 cycles of 94°C for 5 seconds, 52°C for 5 seconds and 72°C for 5 seconds followed by a 4°C soak. SpectroCLEAN resin was used to remove excess ddNTPs and dNTPs by adding resin directly to the extension products, vortexing for 20 minutes then centrifuging for 5 minutes to pellet the resin. The products were then spotted onto a SpectroCHIP and the chip was read in a Bruker Biflex III Mass Spectrometer system. Data were analysed on SpectroTYPER. SNaPshot:

SNaPshot was used to genotype the IL-1A(-889) (rs1800587) polymorphism with forward and reverse primers 5'- GGGAACCCAAAACATTCATT-3'(SEQ ID No 1 ), 5'- CAGTGGCTAAGTTTGGGAAT-3'(SEQ ID No 2) and extension primer, 5'- CATTGAAGGCTCATATGTAAAAATCCATGGC-3'(SEQ ID No 3). An initial 20μl PCR reaction was carried out using 10mM Tris HCI pH8.3, 50mM KCI, 2.5mM MgCI₂, 50μM dNTPs, 0.2μM oligonucleotide primers, 1 unit Taq polymerase (Taq Gold, Applied Biosystems) and 30ng DNA. A touch down PCR program was used comprising 96°C for 12 minutes followed by 5 cycles, each decreasing by 2.5°C, of, 95°C for 30 seconds, 63-55.5°C for 30 seconds and 72°C for 30 seconds, followed by 20 cycles of 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 30 seconds. A final extension of 72°C for 5 minutes and 4°C for 15 minutes. Excess primers and dNTPs were removed by digestion at 37°C for 1 hour with Shrimp Alkaline Phosphatase (SAP) and Exonuclease I in a 20μl reaction mix of 20mM Tris-HCI pH8, 10mM MgCI2, 1.5 units SAP, 1 unit Exol and 10μl PCR product. The 10μl primer extension reaction consisted of 5μl SNaPshot (Applied Biosystems) reaction mix, 3μl post-digest PCR product and 2μM extension primer. Extension was carried out through 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 30 seconds followed by 4°C for 15 minutes. The unincorporated fluorescent ddNTPs were removed post-extension by SAP digestion at 37°C for 1 hour 2μl of the treated product was added to 6 5μl Hi-di-formamide and 0 5μl LIZ size standard (Applied Biosystems) and electrophoresed on an ABI 3700 capillary machine Data were analysed by Genotyper (Applied Biosystems)

RFLP and VNTR genotyping

Polymerase chain reactions were carried out for IL-1 B(+3953), IL- 1 B(+5810), IL-1 B(-51 1 ), IL-1 RN(+8006), IL-1 RN(+8061 ), IL-1 RN (+9589) and IL-1 RN(+11100) using the primers and conditions listed in Table 5, the number given for each primer is the SEQ ID No in the attached sequence listing

Table 5

PCR products were digested accordingly with the appropriate enzymes, also listed in Table 6 and resultant products separated on ethidium bromide stained agarose gels, visualised by UV light PCR was also carried out for the IL-1 RN 46bp VNTR using the assigned primers in Table 6 and products were visualised by UV imaging of ethidium bromide stained 2% agarose gels Statistical analysis

Mendelian inheritance was checked using the program GAS (version 2) MERLIN [Abecasis, Nat Gen , 2002, 31 97-101 ] and SIBMED were used to identify recombination errors Single point LOD scores were obtained through ANALYZE and single marker TDT through TRANSMIT

The results of the analyses are shown in Tables 6 and 7

Table 6

Table 7

Table 6 relates to the data on single marker case-control comparisons The P-values indicated in the shaded areas are statistically significant

Table 7 shows the within-family data In the individual columns, "Analyse" represents the single marker lod score (lod>3 6 occurs once by chance per 20 whole genome screens), "Transmit" represents the single marker test of association in the presence of linkage, where the P-value reflects associations only in this analysis, "2-marker Transmit" reflects the association of haplotypes of two adjacent markers consisting of the marker indicated on that row and the marker directly below Results are in the format giving the haplotype of alleles followed by a P-value For example, 2 1 001 means that the haplotype had allele 2 at marker 1 , allele 1 at marker 2, and P-value 0 001 The "3-marker Transmit" column is the same as for the 2-marker column except for 3 markers The "global P-value" reflects the overall association for that marker or combination of markers

Claims

1 An invitro method for detecting whether a subject has or is genetically predisposed to a seronegative spondyloarthropathy, comprising determining the presence of a genetic polymorphism within the interleukin 1 gene region of chromosome 2 extending from IL-1 A to the beginning of IL-1 RN in a genetic sample obtained from a subject

2 A method according to claim 1 , wherein the region is from base 113621783 from the P-telomere to base 113970711

3 A method according to claim 1 or claim 2, wherein the seronegative spondyloarthropathy is Ankylosing spondylitis

4 A method according to any preceding claim, wherein the polymorphism is within any of the genes IL-1 A, IL-1 B, IL-1 F5, IL-1 F6, IL-1 F7, IL-1 F8, IL-1 F9 and IL- 1 F10

5 A method according to any preceding claim, wherein the polymorphism is any of those listed in Table 2

6 A method according to any preceding claim, wherein the polymorphism is IL- 1 B-889, IL-1 B+3953, IL-1 B-511 , IL1 F8-1 , IL1 F8-3, IL1 F6-2, IL1 F5-3, IL1 F5-4 or IL- 1 F10-1

7 A method according to any preceding claim, wherein the presence of two or more polymorphisms is determined on either a single allele or both alleles

8 A method according to any preceding claim, wherein any of the combinations of polymorphisms listed on Table 3 is determined

9 A method according to claim 8, wherein the combination is IL-1 B-511 and IL- 1 F10-1 10 An isolated polynucleotide useful for diagnosing whether a subject has or is predisposed to a seronegative spondyloarthropathy, comprising at least a contiguous sequence of 15 bases derived from the interleukin 1 gene region as defined in claim 1 or claim 2, or its complement

11 Use of a polynucleotide as defined in claim 10, for determining whether a subject has or is predisposed to a seronegative spondyloarthropathy

12 A diagnostic kit comprising a polynucleotide as defined in claim 10