WO2016170513A1 - Locus à risque pour le rhumatisme psoriasique - Google Patents

Locus à risque pour le rhumatisme psoriasique Download PDF

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WO2016170513A1
WO2016170513A1 PCT/IB2016/052312 IB2016052312W WO2016170513A1 WO 2016170513 A1 WO2016170513 A1 WO 2016170513A1 IB 2016052312 W IB2016052312 W IB 2016052312W WO 2016170513 A1 WO2016170513 A1 WO 2016170513A1
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deletion
psa
intergenic
subject
copy
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PCT/IB2016/052312
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Sara Marsal BARRIL
Antonio Julià CANO
Juan DE DIOS CAÑETE CRESPILLO
Carlos FERRÁNDIZ FORASTER
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Fundació Hospital Universitari Vall D’Hebron-Institut De Recerca
Hospital Clinic De Barcelona
Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol
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Publication of WO2016170513A1 publication Critical patent/WO2016170513A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Psoriatic arthritis is a chronic musculoskeletal inflammatory disease characterized by the presence of arthritis together with psoriasis.
  • a clinical hallmark of PsA is the combination of erosive change with bone proliferation, in predominantly distal joints, such as interphalangeal joints of the hands and feet.
  • RA rheumatoid arthritis
  • PsA prevalence in psoriasis in large patient cohorts has been recently estimated to be -12-14% (Ibrahim et al., Arthritis Rheum, 61(10), 1373-1378 (2009); Julia et al., Hum Mol Genet, 21(20), 4549-4557 (2012)).
  • Psoriasis ⁇ e.g., psoriasis or psoriasis vulgaris
  • psoriasis is a chronic inflammatory disease of the skin and is one of the most prevalent autoimmune diseases in the world (Nestle et al, N Engl J Med, 361(5), 496-509 (2009)). It is a heterogeneous disease with a complex etiology, which includes the contribution of multiple risk genes.
  • PsA is a complex disease caused by the interaction of environmental and genetic risk factors (Chandran et al, Ann Rheum Dis, 68(5), 664-667 (2009)). While the environmental contribution remains largely uncharacterized, there has recently been a significant advance in the characterization of the genetic basis of PsA.
  • X-rays can also be performed to identify bony erosions.
  • bony erosions resulting from arthritis can be due to RA or PsA.
  • radiographic imaging, magnetic resonance imaging (MRI), ultrasound and CT scans may be used for differential diagnosis of RA, erosive osteoarthritis, reactive arthritis (Reiter syndrome), and PsA.
  • MRI magnetic resonance imaging
  • CT scans may be used for differential diagnosis of RA, erosive osteoarthritis, reactive arthritis (Reiter syndrome), and PsA.
  • these methods are not definitive tests for diagnosing PsA.
  • Blood tests can be performed to rule out certain types of arthritis ⁇ e.g., rheumatoid arthritis, reactive arthritis, and ankylosing spondylitis). For instance, serological tests can be performed to detect levels of rheumatoid factor (RF) and anti-citrullinated peptide antibodies, which generally indicate that the subject has rheumatoid arthritis. Blood test can also be performed to detect potentially high levels of inflammation and mild anemia which may be present in patients with PsA.
  • RF rheumatoid factor
  • PsA rheumatoid factor
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • DMARDs disease-modifying antirheumatic drugs
  • antimalarial drugs cyclosporine, imuran, lefunomide, methotrexate, retinoids, and sulfasalazine, TNFa inhibitors, PDE4 inhibitors, etc.
  • the severity of PsA at the time of diagnosis may be correlated to the subsequent course of the disease.
  • the presentation of moderate-to-severe PsA, rather than mild PsA at the time of diagnosis may be associated with a worse prognosis.
  • diagnostic methods that are not based on evaluating clinical manifestations ⁇ e.g., symptoms) of PsA.
  • novel methods that can be used to determine whether a subject has a risk of developing psoriatic arthritis. The present invention satisfies this need and provides related advantages as well.
  • the present invention provides a method for determining whether a subject has a risk of developing psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; and (b) determining that the subject has an increased risk of developing PsA based on the presence of at least one copy of the intergenic deletion.
  • the present invention provides a method for diagnosing psoriatic arthritis (PsA) in a subject.
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; and (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion.
  • the present invention provides a method for selecting a treatment for a subject suspected of having psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion; and (c) selecting a treatment for the subject to alleviate at least one symptom of PsA.
  • FIGS. 1A-1D show characterization of the ADAMTS9-MAGI1 genomic deletion.
  • the vertical lines indicate the location of the two Taqman assays (Hs03225015_cn and Hs03225295_cn, dark grey vertical lines, within deletion sequence) used to genotype the deletion.
  • FIG. IB shows the CNStream deletion calling using multiple probes within the deletion sequence.
  • FIG. 1C shows log2 intensity reads of 6 individuals for the chromosome 3 region harboring the deletion. Starting from the top, the first two individuals show the characteristic drop in intensity corresponding to hemizygous ⁇ i.e., IN) individuals. The 3 rd , 4 th and 6 th individuals show the expected log2 reads of a 2N individual, with most of the intensities centered around 0. Finally, the 5 th individual clearly shows the presence of an individual homozygous for the deletion, with no sequence reads mapping to this region of the chromosome.
  • ID provides a sequence of the reference and deletion alleles. Physical coordinates are on the reference human genome (build 37; NCBI GRCh37). The left breakpoint is located at a nucleotide from 65, 188,869-65,188,871 and the right breakpoint is at a nucleotide from 65,214,749-65,214,751 of NCBI GRCh37.
  • the intergenic deletion removes about 25,879 bp between the ADAMTS9 gene and the MAGI1 gene.
  • FIG. 2 shows a plot of the CNV GWAS results.
  • the -loglO P-values (y-axis) are plotted for each of the CNVs identified by CNSstream.
  • Each chromosome is coded in a greyscale color.
  • the probes mapping the intergenic deletion in HLA-C/-B locus in chromosome 6 were found to have a high significance; however, after correcting for HLA-C and HLA-B alleles this association disappeared.
  • chromosome 3 an intergenic deletion between genes ADAMTS9 and MAGI1 (black dot) shows a significant association that withstands multiple test correction.
  • FIG. 3 shows pairwise LD between the deletion and flanking sequence variants.
  • the 3pl4.1 chromosome regions between these three loci and that were not sequenced are left blank.
  • FIG. 4 shows the principal components of the GWAS cohort.
  • the PsA (triangles) and control patients (dots) are plotted according to their first (PCI) and second (PC2) principal components estimated in EIGENSTRAT using the genome-wide data.
  • Outlier samples i.e., samples with >6 standard deviations in any of the 10 principal components of variation were excluded from the figure.
  • FIG. 5 shows the analysis workflow of the CNV GWAS. Analysis steps used in the identification of the ADAMTS9-MAGI1 deletion association with PsA risk.
  • discovery or GWAS
  • the case-control dataset is QC-filtered and the CNV regions estimated using CNStream software. After filtering for common CNVs we found two genomic regions associated after multiple test correction.
  • validation stage we sequenced and characterized the new deletion associated with PsA risk. After confirming the accuracy of the RT-PCR assay (i.e., technical validation), we validated the association of the deletion with PsA risk in an independent sample of patients and controls. The specificity of the risk variant for PsA risk against purely cutaneous psoriasis was also tested using a sample of PsC patients.
  • PsA psoriatic arthritis
  • methods for diagnosing PsA can be used to select a treatment for a subject suspected of having PsA.
  • the methods described herein are based, in part, on the surprising discovery of an intergenic deletion in the ADAMTS9-MAGI1 locus that is present in subjects with PsA. II. Definitions
  • Guttate psoriasis with raindrop shaped lesions scattered on the trunk and limbs, is the most frequent form in children, while pustular psoriasis is usually localized to the palms and soles.
  • Erythrodermic psoriasis is an inflamed lesion of the skin with fine scales, and is frequently accompanied by severe pain, itching, and possibly swelling.
  • Plaque psoriasis the most commonly observed variety, is characterized by inflamed lesions covered with a silvery white scale. Although plaque psoriasis may occur on any skin surface, it is most commonly found on the knees, elbows, scalp, and trunk.
  • the classical inflammatory lesions vary from discrete erythematous papules and plaques covered with silvery scales, to scaly itching patches that bleed when the scales are removed.
  • Psoriasis may also present itself as pits in toenails and fingernails. The pitting may be accompanied by discoloration and thickening of the nail, and the nail may detach from the nail bed.
  • Psoriatic arthritis includes chronic inflammatory arthritic condition that affects the skin, the joints, the insertion sites of tendons, ligaments, and fascia.
  • Psoriatic arthritis is commonly associated with psoriasis. Approximately 6-42% of patients with psoriasis develop psoriatic arthritis. Patients with PsA can be seronegative for rheumatoid factor. The disease typically appears between the ages of 30-55 but it can be diagnosed during childhood. Men and women have an equal risk for developing the condition. Symptoms of psoriatic arthritis include extra bone formation, joint stiffness, dactylitis, enthesopathy, tendonitis, and spondylitis.
  • psoriasis Most patients have the classic psoriasis pattern of skin lesions. Scaly, erythematous plaques; guttate lesions, lakes of pus, and erythroderma are psoriatic skin lesions that may be seen in patients with psoriatic arthritis. Nail lesions, including pitting, Beau lines, leukonychia, onycholysis, oil spots, subungual hyperkeratosis, splinter hemorrhages, spotted lunulae, and cracking, are clinical features significantly associated with the development of psoriatic arthritis. Ocular symptoms in psoriatic arthritis include conjunctivitis, ulceris, episcleritis, keratoconjunctivitis sicca and aortic insufficiency.
  • psoriatic arthritis There are about 5 types of psoriatic arthritis: symmetric, asymmetric, distal interphlangeal predominant, spondylitis, and arthritis mutilans.
  • Symmetric PsA accounts for about 50% of the cases and affects joints on both sides of the body at the same time.
  • Asymmetric PsA affects about 35% of the patients with PsA.
  • Inflammation and stiffness at the ends of the fingers and toes are characteristic symptoms of distali interphalangeal predominant.
  • Patients with spondylitis PsA exhibit pain and stiffness in the spine and neck.
  • Arthritis mutilans is a considered the most severe form of PsA and causes deformities in the small joints at the ends of the fingers and toes.
  • PsC purely cutaneous psoriasis
  • PsC psoriasis
  • Rheumatoid factor includes an autoantibody (i.e., an antibody directed against an organism' s own tissues) that is typically directed against (i.e., binds to) the Fc (fragment crystallizable) portion of immunoglobulin G (IgG).
  • Rheumatoid factor is most often an IgM autoantibody, but may also be an IgG or IgA autoantibody.
  • anti-citrullinated protein antibody includes an autoantibody that specifically targets one or more epitopes in a peptide, polypeptide, or protein sequence where one or more arginine residues have been converted by the enzyme peptidylarginine deiminase into a citrulline residue during a post- translational modification.
  • the presence or level of anti-citrullinated protein antibodies can be detected, determined, or measured using natural or synthetic citrullinated peptides which are immunologically reactive (i.e., immunoreactive) with such antibodies.
  • Non-limiting examples of synthetic citrullinated peptides include cyclic citrullinated peptides (CCP) such as CCP1, which contains a single cyclic citrullinated peptide derived from filaggrin, and/or CCP2, which is a combination of citrullinated peptides selected from screening libraries of citrullinated peptides.
  • CCP cyclic citrullinated peptides
  • Assays for detecting anti-CCP antibodies are available from INOVA Diagnostics, Euro-Diagnostica, Axis-Shield, Phadia, and Abbott Diagnostics.
  • Anti- citrullinated protein antibodies are autoantibodies typically associated with rheumatoid arthritis.
  • ADAMTS9-MAGI1 locus refers to the genomic region on chromosome 3 that includes the ADAMTS9 gene and the MAGI1 gene, and the flanking genomic regions at the 5' end and 3' end of either genes.
  • intergenic deletion refers to a genomic deletion in an intergenic region, i.e., a genomic region located between genes.
  • an intergenic deletion in the ADAMTS9-MAGI1 locus refers to a deletion between the ADAMTS9 gene and the MAGI1 gene.
  • the presence of an intergenic deletion may produce an intergenic fusion between two genes.
  • An individual can possess one or two copies of an intergenic deletion.
  • diagnosis refers to a relative probability a subject has psoriatic arthritis. Symptoms and diagnostic criteria are summarized below. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present invention, prognosis can refer to the likelihood that an individual will develop psoriatic arthritis. Prognosis can also refer to the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, etc.). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.
  • subject typically includes humans, but can also include other animals such as, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • sample includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, synovial fluid, saliva, urine, stool, tears, hair, skin, any other bodily fluid, tissue samples (e.g., biopsy), and cellular extracts thereof (e.g., red blood cellular extract).
  • tissue samples e.g., biopsy
  • cellular extracts thereof e.g., red blood cellular extract.
  • samples such as serum, saliva, and urine is well known in the art (see, e.g., Hashida et ah, J. Clin. Lab. Anal., 11 :267-86 (1997)).
  • samples such as serum samples can be diluted prior to the analysis of marker levels.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
  • the term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • gene means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region, such as the promoter and 3 '-untranslated region, respectively, as well as intervening sequences (introns) between individual coding segments (exons).
  • genotyp refers to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous or homozygous for an intergenic deletion of interest.
  • linkage disequilibrium refers to any degree of non-random genetic association between one or more allele(s) of two different polymorphic DNA sequences and that is due to the physical proximity of the two loci.
  • the term can refer to the trend for alleles at nearby loci on haploid genomes to correlate in the population.
  • a and b alleles at close loci A and B, are said to be in linkage disequilibrium if the a b haplotype (a haplotype is defined as a set of alleles on the same chromosomal segment) has a frequency which is statistically higher than P a x P b (expected frequency if the alleles segregate independently, where P a is the frequency of allele a, and P that of allele b).
  • LD can be measured using the r 2 statistic.
  • anti-TNF therapy refers to an agent including peptides, proteins, antibodies, antibody fragments, fusion proteins, multivalent binding proteins, small molecule TNFa antagonists, similar naturally- or nonnaturally-occurring molecules, and/or recombinant and/or engineered forms thereof that inhibit/neutralize TNFa activity.
  • anti-TNF therapy or “anti-TNF drug” includes an agent that inhibits the interaction of TNFa with a cell surface receptor for TNFa, inhibits TNFa protein production, inhibits TNFa gene expression, inhibits TNFa secretion from cells, inhibits TNFa receptor signaling or any other means resulting in decreased TNFa activity in a subject.
  • Non-limiting examples of anti-TNF drugs include infliximab (REMICADETM, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRATM, Abbott Laboratories), etanercept (ENBRELTM, Amgen), certolizumab pegol (CIMZIA ® , UCB, Inc.), golimumab (SFMPONI ® ; CNTO 148), siplizumab (MEDI-507; Medlmmune), CDP 571 (Celltech), CDP 870 (Celltech), TRX 4 (TolerRx), as well as other compounds which inhibit/neutralize TNFa activity.
  • REMICADETM human anti-TNF monoclonal antibody adalimumab
  • D2E7/HUMIRATM Abbott Laboratories
  • EBRELTM etanercept
  • Amgen certolizumab pegol
  • CCMZIA ® certolizum
  • TNFa The inhibition of the biological activity of TNFa can be assessed by measuring one or more indicators of TNFa biological activity, such as TNFa- induced cytotoxicity (either in vitro or in vivo), TNFa-induced cellular activation or TNFa binding to a TNFa receptor.
  • TNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art.
  • non-anti-TNF therapy or “non-anti-TNF drug” refers to an agent (e.g., oligonucleotide, inhibitory RNA, peptide, protein, antibody, antibody fragment, fusion proteins, multivalent binding proteins, small molecule, chemical compound, and the like) that does not inhibit/neutralize TNFa activity.
  • agent e.g., oligonucleotide, inhibitory RNA, peptide, protein, antibody, antibody fragment, fusion proteins, multivalent binding proteins, small molecule, chemical compound, and the like
  • Non-limiting examples of non-anti-TNF drugs include azathioprine, cyclosporine, methotrexate, imuran, leflunomide, sulfasalazine, apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone, metformin, piclamilast, roflumilast, rolipram, a retinoid, a corticosteroid, a Cox-2 inhibitor, secukinumab, ustekinumab, brodalumab, guselkumab, ixekizumab, tofacitinib, AbGn-168H, bimekizumab, AIN457A (an anti-IL-17 monoclonal antibody), a PDE4 inhibitor, a nonsteroidal antiinflammatory drug, and combinations thereof.
  • the present invention provides a method for determining whether a subject has a risk of developing psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; and (b) determining that the subject has an increased risk of developing PsA based on the presence of at least one copy of the intergenic deletion.
  • the subject has psoriasis.
  • the intergenic deletion removes about 25,000 to about 26,000 base pairs in the genomic region between the ADAMTS9 and MAGI1 genes.
  • the genomic region comprises the nucleic acid sequence of nucleotide coordinate 64,501,330 to nucleotide coordinate 66,024,511 of NCBI Reference Sequence No. NC 000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,751 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the step of detecting the presence or absence of at least one copy ⁇ e.g., one copy or two copies) of the intergenic deletion can be performed according to any of the methods and reagents ⁇ e.g., primers and/or probes) described herein.
  • the step of detecting the presence or absence of at least one copy of the intergenic deletion comprises performing an amplification assay ⁇ e.g., polymerase chain reaction including real-time polymerase chain reaction), sequencing analysis, or a hybridization assay.
  • the sample is selected from the group consisting of whole blood, plasma, serum, synovial fluid, saliva, urine, skin, buccal swab, and hair.
  • the method further includes determining that the subject is not at risk of developing purely cutaneous psoriasis (PsC) based on the presence of at least one copy of the intergenic deletion.
  • PsC purely cutaneous psoriasis
  • the present invention provides the use of a reagent for detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in the manufacture of a kit for determining whether a subject has a risk of developing psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of the intergenic deletion in the genomic region in a sample obtained from the subject using the reagent; and (b) determining that the subject has an increased risk of developing PsA based on the presence of at least one copy of the intergenic deletion.
  • the reagent comprises one or more primers and/or probes as described herein ⁇ e.g., Table 1). Table 1. Synthetic Primers and Probes for Detecting the Presence of an Intergenic Deletion between the ADAMTS9 and MAGIl genes on human chromosome 3pl4.1
  • the present invention provides a method of detecting predisposition for an increased risk of developing psoriatic arthritis (PsA) in a subject.
  • the method comprises: (a) obtaining genomic DNA isolated from a biological sample from the subject; (b) performing a polymerase chain reaction on the isolated genomic DNA to amplify a genomic region between the ADAMTS9 and MAGIl genes on human chromosome 3pl4.1; (c) detecting the presence or absence of at least one copy of an intergenic deletion in the genomic region; and (d) determining the subject, whose genomic DNA contains at least one copy of the intergenic deletion, as having an increased risk of developing PsA.
  • the presence or absence of at least one copy ⁇ e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected based on amplicon size.
  • the genomic region is amplified using a first forward primer consisting of SEQ ID NO:9, a second forward primer consisting of SEQ ID NO: 10, and a (common) reverse primer consisting of SEQ ID NO: l 1 ⁇ see, Table 1).
  • a polymerase chain reaction comprising a forward primer consisting of SEQ ID NO:9 and a reverse primer consisting of SEQ ID NO: 1 1 can produce an amplicon that is 171 bp in length if a copy of the intergenic deletion is present.
  • a polymerase chain reaction comprising a forward primer consisting of SEQ ID NO: 10 and a reverse primer consisting of SEQ ID NO: 1 1 can produce an amplicon that is 442 bp in length if a copy of the intergenic deletion is absent.
  • a polymerase chain reaction comprising a first forward primer consisting of SEQ ID NO:9, a second forward primer consisting of SEQ ID NO: 10, and a reverse primer consisting of SEQ ID NO: 1 1 can produce a 442 bp amplicon, a 171 bp amplicon, or both a 442 bp amplicon and a 171 bp amplicon (see, Table 1). If only a 442 bp amplicon is produced, two copies of the normal amplicon are present (e.g., homozygous for the normal allele). In other words, two copies of the intergenic deletion are absent (e.g., homozygous for the absence of the deletion).
  • the intergenic deletion If only a 171 bp amplicon is produced, two copies of the intergenic deletion are present (e.g., homozygous for the presence of the deletion). If a 442 bp amplicon and a 171 bp amplicon are produced, one copy of the intergenic deletion and one copy of the normal allele are present (e.g., heterozygous for the presence of the deletion). The presence of at least one copy (e.g., one copy or two copies) of the intergenic deletion predicts that the subject has an increased risk of developing PsA.
  • the presence or absence of at least one copy (e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected using a real-time polymerase chain reaction (e.g., TaqMan® genotyping assay; ThermoFisher Scientific).
  • a real-time polymerase chain reaction e.g., TaqMan® genotyping assay; ThermoFisher Scientific.
  • Exemplary primers and probes for performing real-time polymerase chain reaction are provided in, e.g., Table 1.
  • the genomic region is amplified using a primer pair consisting of SEQ ID NO:3 and SEQ ID NO:4.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region can be detected using a probe consisting of SEQ ID NO:5.
  • the genomic region is amplified using a primer pair consisting of SEQ ID NO:6 and SEQ ID NO:7.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region can be detected using a probe consisting of SEQ ID NO:8.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region is detected using a probe consisting of SEQ ID NO: 5, and optionally, the genomic region can be amplified using a primer pair consisting of SEQ ID NO:3 and SEQ ID NO:4.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region is detected using a probe consisting of SEQ ID NO:8, and optionally, the genomic region can be amplified using a primer pair consisting of SEQ ID NO:6 and SEQ ID NO:7.
  • the probe is a fluorescent probe comprising a fluorescent reporter dye (e.g., FAM TM , VIC ® , etc.), a minor groove binder (MGB), and/or a quencher (e.g., nonfluorescent quencher (NFQ), etc.).
  • the fluorescent reporter dye may be on the 5' end of the probe and the MGB and/or NFQ may be on the 3' end.
  • an increase in fluorescence from the fluorescent reporter dye bound to the probe corresponding to the intergenic deletion indicates the presence of at least one copy of the intergenic deletion.
  • the present invention provides a method for diagnosing psoriatic arthritis (PsA) in a subject.
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; and (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion.
  • the subject has psoriasis.
  • the intergenic deletion removes about 25,000 to about 26,000 base pairs in the genomic region between the ADAMTS9 and MAGI1 genes.
  • the genomic region comprises the nucleic acid sequence of nucleotide coordinate 64,501,330 to nucleotide coordinate 66,024,511 of NCBI Reference Sequence No. NC 000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,751 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the step of detecting the presence or absence of at least one copy (e.g., one copy or two copies) of the intergenic deletion can be performed according to any of the methods and reagents (e.g., primers and/or probes) described herein.
  • the step of detecting the presence or absence of at least one copy of the intergenic deletion comprises performing an amplification assay (e.g., polymerase chain reaction including real-time polymerase chain reaction), sequencing analysis, or a hybridization assay.
  • the sample is selected from the group consisting of whole blood, plasma, serum, synovial fluid, saliva, urine, skin, buccal swab, and hair.
  • the method further includes selecting a treatment for the subject comprising an anti -tumor necrosis factor (anti-T F) therapy, a non-anti-T F therapy, or a combination thereof.
  • anti-T F anti-tumor necrosis factor
  • the anti-TNF therapy is selected from the group consisting of infliximab, adalimumab, golimumab, etanercept, certolizumab pegol, and combinations thereof.
  • the non-anti-TNF therapy is selected from the group consisting of azathioprine, cyclosporine, methotrexate, imuran, lefunomide, sulfasalazine, apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone, piclamilast, roflumilast, rolipram, a retinoid, a corticosteroid, a Cox-2 inhibitor, secukinumab, ustekinumab, brodalumab, guselkumab, ixekizumab, tofacitinib, bimekizumab, a PDE4 inhibitor, a nonsteroidal anti-inflammatory drug, and combinations thereof.
  • the method further includes determining that the subject is not at risk of developing purely cutaneous psoriasis (PsC) based on the presence of at least one copy of the intergenic deletion.
  • PsC purely cutaneous psoriasis
  • the present invention provides the use of a reagent for detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGIl genes on human chromosome 3pl4.1 in the manufacture of a kit for diagnosing psoriatic arthritis (PsA) in a subject.
  • the method comprises: (a) detecting the presence or absence of at least one copy of the intergenic deletion in the genomic region in a sample obtained from the subject using the reagent; and (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion.
  • the reagent comprises one or more primers and/or probes as described herein ⁇ e.g., Table 1).
  • the present invention provides a method for aiding in the diagnosis of psoriatic arthritis (PsA) in a subject.
  • the method comprises: (a) obtaining genomic DNA isolated from a biological sample from the subject; (b) performing a polymerase chain reaction on the isolated genomic DNA to amplify a genomic region between the ADAMTS9 and MAGIl genes on human chromosome 3pl4.1; (c) detecting the presence or absence of at least one copy of an intergenic deletion in the genomic region; and (d) determining the subject, whose genomic DNA contains at least one copy of the intergenic deletion, as having PsA.
  • PsA psoriatic arthritis
  • the presence or absence of at least one copy (e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected based on amplicon size.
  • the genomic region is amplified using a first forward primer consisting of SEQ ID NO:9, a second forward primer consisting of SEQ ID NO: 10, and a common reverse primer consisting of SEQ ID NO: l 1 (see, Table 1).
  • a polymerase chain reaction comprising a forward primer consisting of SEQ ID NO:9 and a reverse primer consisting of SEQ ID NO: 1 1 can produce an amplicon that is 171 bp in length if a copy of the intergenic deletion is present. If at least one copy (e.g., one copy or two copies) of the intergenic deletion is identified, the subject is diagnosed as having PsA. In other cases, a polymerase chain reaction comprising a forward primer consisting of SEQ ID NO: 10 and a reverse primer consisting of SEQ ID NO: 1 1 can produce an amplicon that is 442 bp in length if a copy of the intergenic deletion is absent.
  • a polymerase chain reaction comprising a first forward primer consisting of SEQ ID NO:9, a second forward primer consisting of SEQ ID NO: 10, and a reverse primer consisting of SEQ ID NO: 1 1 can produce a 442 bp amplicon, a 171 bp amplicon, or both a 442 bp amplicon and a 171 bp amplicon (see, Table 1). If only a 442 bp amplicon is produced, two copies of the normal amplicon are present (e.g., homozygous for the normal allele). In other words, two copies of the intergenic deletion are absent (e.g., homozygous for the absence of the deletion).
  • the subject is diagnosed as not having PsA. If only a 171 bp amplicon is produced, two copies of the intergenic deletion are present (e.g., homozygous for the presence of the deletion). If a 442 bp amplicon and a 171 bp amplicon are produced, one copy of the intergenic deletion and one copy of the normal allele are present (e.g., heterozygous for the presence of the deletion). The presence of at least one copy (e.g., one copy or two copies) of the intergenic deletion is indicative of the subject having PsA.
  • the presence or absence of at least one copy (e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected using a real-time polymerase chain reaction (e.g., TaqMan® genotyping assay; ThermoFisher Scientific).
  • a real-time polymerase chain reaction e.g., TaqMan® genotyping assay; ThermoFisher Scientific.
  • Exemplary primers and probes for performing real-time polymerase chain reaction are provided in, e.g., Table 1.
  • the genomic region is amplified using a primer pair consisting of SEQ ID NO:3 and SEQ ID NO:4.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region can be detected using a probe consisting of SEQ ID NO:5.
  • the genomic region is amplified using a primer pair consisting of SEQ ID NO:6 and SEQ ID NO:7.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region can be detected using a probe consisting of SEQ ID NO:8.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region is detected using a probe consisting of SEQ ID NO: 5, and optionally, the genomic region can be amplified using a primer pair consisting of SEQ ID NO:3 and SEQ ID NO:4.
  • the presence or absence of at least one copy of the intergenic deletion in the genomic region is detected using a probe consisting of SEQ ID NO:8, and optionally, the genomic region can be amplified using a primer pair consisting of SEQ ID NO:6 and SEQ ID NO:7.
  • the probe is a fluorescent probe comprising a fluorescent reporter dye (e.g., FAM TM , VIC ® , etc.), a minor groove binder (MGB), and/or a quencher (e.g., nonfluorescent quencher (NFQ), etc.).
  • the fluorescent reporter dye may be on the 5' end of the probe and the MGB and/or NFQ may be on the 3' end.
  • an increase in fluorescence from the fluorescent reporter dye bound to the probe corresponding to the intergenic deletion indicates the presence of at least one copy of the intergenic deletion.
  • the present invention provides a method for selecting a treatment for a subject suspected of having psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from the subject; (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion; and (c) selecting a treatment for the subject to alleviate at least one symptom of PsA.
  • the subject has psoriasis.
  • the intergenic deletion removes about 25,000 to about 26,000 base pairs in the genomic region between the ADAMTS9 and MAGI1 genes.
  • the genomic region comprises the nucleic acid sequence of nucleotide coordinate 64,501,330 to nucleotide coordinate 66,024,511 of NCBI Reference Sequence No. NC 000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,751 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the step of detecting the presence or absence of at least one copy (e.g., one copy or two copies) of the intergenic deletion can be performed according to any of the methods and reagents (e.g., primers and/or probes) described herein.
  • the step of detecting the presence or absence of at least one copy of the intergenic deletion comprises performing an amplification assay (e.g., polymerase chain reaction including real-time polymerase chain reaction), sequencing analysis, or a hybridization assay.
  • the sample is selected from the group consisting of whole blood, plasma, serum, synovial fluid, saliva, urine, skin, buccal swab, and hair.
  • the method further includes selecting a treatment for the subject comprising an anti -tumor necrosis factor (anti-TNF) therapy, a non-anti-TNF therapy, or a combination thereof.
  • anti-TNF anti -tumor necrosis factor
  • the anti-TNF therapy is selected from the group consisting of infliximab, adalimumab, golimumab, etanercept, certolizumab pegol, and combinations thereof.
  • the non-anti-TNF therapy is selected from the group consisting of azathioprine, cyclosporine, methotrexate, imuran, lefunomide, sulfasalazine, apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone, piclamilast, roflumilast, rolipram, a retinoid, a corticosteroid, a Cox-2 inhibitor, secukinumab, ustekinumab, brodalumab, guselkumab, ixekizumab, tofacitinib, bimekizumab, a PDE4 inhibitor, a nonsteroidal anti-inflammatory drug, and combinations thereof.
  • the method further includes determining that the subject is not at risk of developing purely cutaneous psoriasis (PsC) based on the presence of at least one copy of the intergenic deletion.
  • PsC purely cutaneous psoriasis
  • the present invention provides the use of a reagent for detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGIl genes on human chromosome 3pl4.1 in the manufacture of a kit for selecting a treatment for a subject suspected of having psoriatic arthritis (PsA).
  • the method comprises: (a) detecting the presence or absence of at least one copy of the intergenic deletion in the genomic region in a sample obtained from the subject using the reagent; (b) determining that the subject has PsA based on the presence of at least one copy of the intergenic deletion; and (c) selecting a treatment for the subject to alleviate at least one symptom of PsA.
  • the reagent can be any synthetic primer and/or probe set forth in Table 1, including one or more primers of SEQ ID NOS: 3, 4, 6, 7, 9, 10, and 11, and/or one or more probes of SEQ ID NOS: 5 and 8.
  • the present invention provides a method for treating a subject having an increased risk of developing PsA or a high risk of progression of PsA.
  • the method comprises: (a) detecting in a genomic DNA sample from the subject the presence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1, wherein the genomic DNA of the subject contains at least one copy of the intergenic deletion; and (b) administering to the subject a treatment comprising an anti -tumor necrosis factor (anti-TNF) therapy, a non-anti-TNF therapy, or a combination thereof.
  • anti-TNF anti -tumor necrosis factor
  • the presence or absence of at least one copy ⁇ e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected based on amplicon size using a polymerase chain reaction.
  • exemplary forward and reverse primers for performing polymerase chain reaction are provided in, e.g., Table 1.
  • the presence or absence of at least one copy ⁇ e.g., one copy or two copies) of the intergenic deletion in the genomic region is detected using a real-time polymerase chain reaction ⁇ e.g., TaqMan ® genotyping assay; ThermoFisher Scientific).
  • exemplary primers and probes for performing real-time polymerase chain reaction are provided in, e.g., Table 1.
  • the anti-TNF therapy is selected from the group consisting of infliximab, adalimumab, golimumab, etanercept, certolizumab pegol, and combinations thereof.
  • the non-anti-TNF therapy is selected from the group consisting of azathioprine, cyclosporine, methotrexate, imuran, lefunomide, sulfasalazine, apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone, piclamilast, roflumilast, rolipram, a retinoid, a corticosteroid, a Cox-2 inhibitor, secukinumab, ustekinumab, brodalumab, guselkumab, ixekizumab, tofacitinib, bimekizumab, a PDE4 inhibitor, a nonsteroidal anti-inflammatory drug, and combinations thereof.
  • the present invention further provides stratification methods to determine whether a subject being enrolled in a clinical trial has a risk of developing psoriatic arthritis (PsA) such that the subject can be provided an appropriate treatment ⁇ e.g., anti-TNF therapy and/or non- anti-TNF therapy) at an appropriate dose.
  • PsA psoriatic arthritis
  • the present invention provides a method for stratifying a plurality of subjects for a clinical trial, the method comprising: (a) detecting the presence or absence of at least one copy of an intergenic deletion in a genomic region between the ADAMTS9 and MAGI1 genes on human chromosome 3pl4.1 in a sample obtained from each of the plurality of subjects; and (b) enrolling or assigning each subject into a study group, recommending a treatment ⁇ e.g., anti- TNF therapy and/or non-anti-TNF therapy), and/or administering to the subject a treatment ⁇ e.g., anti-TNF therapy and/or non-anti-TNF therapy), based on the presence or absence of at least one copy of the intergenic deletion.
  • a treatment ⁇ e.g., anti- TNF therapy and/or non-anti-TNF therapy
  • the subject has psoriasis.
  • the step of detecting the presence or absence of at least one copy ⁇ e.g., one copy or two copies) of the intergenic deletion can be performed according to any of the methods and reagents ⁇ e.g., primers and/or probes) described herein.
  • ADAMTS9 ADAMTS9 gene
  • MAGI1 human MAGI1 gene
  • the methods can be used to determine the presence or absence of a genomic deletion in the intergenic region between ADAMTS9 and MAGI1.
  • the presence of a genomic deletion results in a juxtaposition of nucleic acid sequences in the genomic DNA that are normally separated by over about 25 kb at chromosome 3pl4.1.
  • the ADAMTS9 gene encodes the ADAM metallopeptidase with thrombospondin type 1, motif 9 protein.
  • the genomic sequence and location of the ADAMTS9 gene can be found in the NCBI Gene database (Gene ID: 56999).
  • the gene is located on Chromosome 3 at nucleotide coordinate 64,501,330 to nucleotide coordinate 64,673,676 of in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly, or an equivalent thereof.
  • An example of an equivalent includes NC_000003.12 of the GRCh38.p2 assembly at nucleotide coordinate 64,515,654 to nucleotide coordinate 64,644,000.
  • the ADAMTS9 gene is located on the reverse strand.
  • the MAGI1 gene encodes the membrane associated guanylate kinase, WW and PDZ domain containing 1 protein.
  • the genomic sequence and location of the MAGI1 gene can be found in the NCBI Gene database (Gene ID: 9223).
  • the gene is located on Chromosome 3 at nucleotide coordinate 65,339,200 to nucleotide coordinate 66,024,511 of in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • An example of an equivalent includes NC 000003.12 of the GRCh38.p2 assembly at nucleotide coordinate 65,330,764 to nucleotide coordinate 66,038,836.
  • the MAGIl gene is located on the reverse strand.
  • one of the chromosomal breakpoints is between about nucleotide 65,188,868 to about nucleotide 65, 188,872, e.g., about nucleotide 65,188,868, about nucleotide 65, 188,869, about nucleotide 65,188,870, about nucleotide 65,188,871 or about nucleotide 65, 188,872, in NCBI Reference Sequence No.
  • one of the chromosomal breakpoints is between about nucleotide 65,214,748 to about nucleotide 65,214,752, e.g., about nucleotide 65,214,748, about nucleotide 65,214,749, about nucleotide 65,214,750, about nucleotide 65,214,751, or about nucleotide 65,214,752, in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the two chromosomal breakpoints on a single chromosome are at a first nucleotide located at about nucleotide 65, 188,868 to about nucleotide 65,188,872, e.g., about nucleotide 65, 188,868, about nucleotide 65, 188,869, about nucleotide 65, 188,870, about nucleotide 65,188,871 or about nucleotide 65,188,872, and at a second nucleotide at about nucleotide 65,214,748 to about nucleotide 65,214,752 e.g., about nucleotide 65,214,748, about nucleotide 65,214,749, about nucleotide 65,214,750, about nucleotide 65,214,751, or about nucleotide 65,214,752, in NCBI Reference Sequence No. NC_000003.11, GRCh
  • the nucleic acid sequence immediately upstream (e.g., adjacent to the 5' end) of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: l which represents nucleotide coordinate nucleotide coordinate 65, 188,600 to nucleotide coordinate 65, 188,869 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the nucleic acid sequence immediately upstream of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the nucleic acid sequence set forth from nucleotide coordinate 65, 188,600 to nucleotide coordinate 65,188,870 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the nucleic acid sequence immediately upstream of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the nucleic acid sequence set forth from nucleotide coordinate 65,188,600 to nucleotide coordinate 65,188,871 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the nucleic acid sequence immediately downstream (e.g., adjacent to the 3' end) of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2 which represents nucleotide coordinate 65,214,749 to nucleotide coordinate 65,214,999 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the nucleic acid sequence immediately downstream of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%) or 100%), sequence identity to the nucleic acid sequence set forth from nucleotide coordinate 65,214,750 to nucleotide coordinate 65,214,999 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the nucleic acid sequence immediately downstream of the intergenic deletion has at least about 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the nucleic acid sequence set forth from nucleotide coordinate 65,214,751 to nucleotide coordinate 65,214,999 in NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the presence of the intergenic deletion in the ADAMTS9- MAGI1 locus corresponds to the absence of the nucleic acid sequence set forth in 65, 188,872 nucleotide coordinate to 65,214,748 nucleotide coordinate of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 or a fragment thereof.
  • the presence of the deletion can result in the juxtaposition of the nucleic acid sequences set forth in SEQ ID NOS: 1 and 2.
  • the presence of the intergenic deletion described herein results in the fusion of SEQ ID NOS: 1 and 2, wherein 0, 1, or 2 nucleotides at the 3' end of the sequence of SEQ ID NO: 1 and 0, 1, or 2 nucleotides at the 5' end of SEQ ID NO:2 are absent.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,748 of NCBI Reference Sequence No.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,749 of NCBI Reference Sequence No. NC_000003.112, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,870 to about nucleotide coordinate 65,214,7450 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,871 to about nucleotide coordinate 65,214,748 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof. In some instances, the intergenic deletion corresponds to about nucleotide coordinate 65,188,871 to about nucleotide coordinate 65,214,749 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof. In other instances, the intergenic deletion corresponds to about nucleotide coordinate 65, 188,871 to about nucleotide coordinate 65,214,750 of NCBI Reference Sequence No.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,872 to about nucleotide coordinate 65,214,748 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,872 to about nucleotide coordinate 65,214,749 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • the intergenic deletion corresponds to about nucleotide coordinate 65,188,872 to about nucleotide coordinate 65,214,750 of NCBI Reference Sequence No. NC_000003.11, GRCh37.pl3 Primary Assembly or an equivalent thereof.
  • An individual having one or two copies of the ADAMTS9-MAGI1 deletion has a risk for having or developing PsA ⁇ e.g., has a likelihood of having or developing PsA).
  • An individual with PsA may be heterozygous or homozygous for the deletion.
  • an individual that does not possess the ADAMTS9-MAGI1 deletion is not at risk for having or developing PsA.
  • an individual having one or two copies of the ADAMTS9-MAGI1 deletion does not have purely cutaneous psoriasis.
  • Polymorphisms e.g., single nucleotide polymorphisms (SNPs) that are physically close (adjacent to the deletion) to the ADAMTS9-MAGI1 deletion can be at moderate to high correlation to the deletion.
  • a SNP may have a linkage disequilibrium pattern that has a statistically significant correlation to the deletion.
  • the presence of the deletion and the presence of a SNP that has moderate to high correlation to the deletion indicate that an individual has a risk of having PsA.
  • the absence of the deletion and the presence/absence of a SNP that has moderate to high correlation to the deletion indicate that the individual is not at risk of having PsA.
  • the presence of the deletion and the absence of a SNP that has moderate to high correlation to the deletion indicate that the individual is at risk of having PsA. It has been determined that the ADAMTS9 and MAGI1 genes and their flanking regions ⁇ e.g., 5' and 3' untranslated regions) do not show a correlation to the intergenic deletion.
  • a variety of means can be used to genotype an individual at an intergenic region described herein to determine whether a sample ⁇ e.g., a nucleic acid sample) from the individual contains a specific genomic deletion, i.e., a deletion between the ADAMTS9 gene and MAGI1 gene.
  • the method for detecting the presence of an intergenic deletion includes performing an amplification assay, a sequencing analysis, or a hybridization assay. Detailed descriptions of useful methods for detecting genomic deletions are found, e.g., in Cantsilieris et ah, Genomics, 2013, 101(2):86-93.
  • nucleic acid means a polynucleotide such as a single- or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. This term encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular, or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.
  • nucleic acids can be unpurified, purified, or attached, for example, to a synthetic material such as a bead or column matrix.
  • Material containing nucleic acid is routinely obtained from individuals. Such material is any biological matter from which nucleic acid can be prepared. As non-limiting examples, material can be whole blood, serum, plasma, synovial fluid, saliva, buccal swab, e.g., cheek swab, sputum, skin, hair, or other bodily fluid or tissue that contains nucleic acid. In one embodiment, a method of the present invention is practiced with whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA.
  • Genotyping to detect the presence or absence of an intergenic deletion can involve amplification of an individual's nucleic acid using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Use of PCR for the amplification of nucleic acids is well known in the art ⁇ see, e.g., Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).
  • PCR amplification is performed using one or more fluorescently labeled primers.
  • PCR amplification is performed using one or more labeled or unlabeled primers that contain a DNA minor groove binder.
  • any of a variety of different primers can be used to amplify an individual's nucleic acid by PCR in order to determine the presence/absence of a deletion in the ADAMTS9-MAGI1 locus in a method of the invention.
  • the PCR primers can be used to amplify specific intergenic regions of the ADAMTS9-MAGI1 locus, such as an intergenic region between the ADAMTS9 and MAGIl genes.
  • PCR primers are used to amplify a particular genomic region that results from the presence of the deletion.
  • PCR primers are used to amplify a genomic region if the deletion is absent.
  • additional primers for PCR analysis can be designed based on the sequence flanking the intergenic region in the ADAMTS9-MAGI1 locus.
  • amplification is performed as a multiplex assay.
  • a primer can contain about 15 to about 30 nucleotides of a sequence upstream or downstream of the intergenic deletion of interest in the ADAMTS9-MAGI1 locus.
  • a primer can contain about 15 to about 30 nucleotides of a sequence within the intergenic deletion of interest in the ADAMTS9-MAGI1 locus.
  • Such primers generally are designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the amplification reaction.
  • Several computer programs, such as Primer Select, are available to aid in the design of PCR primers.
  • HRM analysis high resolution melting analysis
  • mass spectrometry can be performed in addition to an amplification reaction.
  • the resulting amplification product e.g., the amplified intergenic region with and/or without the deletion
  • FIRM analysis mass spectrometry
  • fluorescent double stranded DNA molecules can be distinguished based on their denaturation or melting behavior. For instance, the presence of a wild-type (normal) intergenic region amplicon and an amplicon corresponding to an intergenic deletion can be determined based on their melting curves.
  • a Taqman ® e.g., 5' fluorogenic real-time PCR genotyping assay
  • a Taqman ® CNV assay can be used for genotyping an individual and determining the presence or absence of a particular intergenic region in the ADAMTS9- MAGI1 locus.
  • the Taqman ® CNV assays Hs03225015_cn and Hs03225295_cn are located within the intergenic region and can be used to determine the presence or absence of the intergenic deletion.
  • specific fluorescent dye-labeled probes that recognizes either the presence or absence of the intergenic region are constructed.
  • a probe can identify an intergenic fusion resulting from the presence of the intergenic deletion.
  • another probe can identify a wild- type intergenic region that does not contain a deletion.
  • the probes contain different fluorescent reporter dyes such as FAM TM and VIC ® to differentiate amplification of the wild- type intergenic region and the intergenic fusion (e.g., intergenic region with a deletion).
  • each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonance energy transfer.
  • each probe anneals specifically to complementary sequences in the nucleic acid from the individual.
  • the 5' nuclease activity of Taq polymerase is used to cleave only probe that hybridizes to its corresponding nucleic acid region in the locus of interest. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Thus, the fluorescence signal generated by PCR amplification indicates whether the wild-type intergenic region or intergenic fusion is present in the sample. Mismatches between a probe and allele reduce the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescent signal.
  • a deletion can be detected by sequence analysis using the appropriate primers, which are designed based on the sequence flanking the deletion of interest in the ADAMTS9-MAGI1 locus.
  • Additional or alternative sequencing primers can contain from about 15 to about 30 nucleotides of a sequence that corresponds to a sequence about 40 to about 400 base pairs upstream or downstream of the deletion of interest in the ADAMTS9-MAGI1 locus.
  • Such primers are generally designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the sequencing reaction.
  • sequence analysis includes any manual or automated process by which the order of nucleotides in a nucleic acid is determined. As an example, sequence analysis can be used to determine the nucleotide sequence of a sample of DNA.
  • sequence analysis encompasses, without limitation, next generation or massively parallel sequencing methods, such as pyrosequencing on a solid support (454 sequencing, Roche), sequencing- by-synthesis with reversible terminations (Illumina ® Genome Analyzer), sequencing-by- synthesis using semiconductors (Ion Torrent TM ), sequencing-by-ligation (ABI SOLiD ® ) and sequencing-by-synthesis with virtual terminators (Heli Scope TM ), and chemical and enzymatic methods such as dideoxy enzymatic methods including, for example, Maxam-Gilbert and Sanger sequencing, as well as variations thereof.
  • next generation or massively parallel sequencing methods such as pyrosequencing on a solid support (454 sequencing, Roche), sequencing- by-synthesis with reversible terminations (Illumina ® Genome Analyzer), sequencing-by- synthesis using semiconductors (Ion Torrent TM ), sequencing-by-ligation (ABI SOLiD ® ) and sequencing-by-synthesis with virtual terminators
  • sequence analysis further encompasses, but is not limited to, capillary array DNA sequencing, which relies on capillary electrophoresis and laser-induced fluorescence detection and can be performed using instruments such as the MegaBACE 1000 or ABI 3700.
  • sequence analysis encompasses thermal cycle sequencing ⁇ see, Sears et al., Biotechniques 13 :626-633 (1992)); solid-phase sequencing ⁇ see, Zimmerman et al., Methods Mol. Cell Biol.
  • sequence analysis further includes, but is not limited to, sequencing by hybridization (SBH), which relies on an array of all possible short oligonucleotides to identify a segment of sequence (see, Chee et al, Science 274:610-614 (1996); Drmanac et al, Science 260: 1649-1652 (1993); and Drmanac et al, Nature Biotech. 16:54-58 (1998)).
  • SBH sequencing by hybridization
  • Electrophoretic analysis also can be useful in genotyping an individual according to the methods of the present invention to determine the presence or absence of a particular intergenic deletion in the ADAMTS9-MAGI1 locus.
  • Electrophoretic analysis as used herein in reference to one or more nucleic acids such as amplified fragments includes a process whereby charged molecules are moved through a stationary medium under the influence of an electric field. Electrophoretic migration separates nucleic acids primarily on the basis of their charge, which is in proportion to their size, with smaller molecules migrating more quickly.
  • electrophoretic analysis includes, without limitation, analysis using slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, or capillary electrophoresis.
  • Capillary electrophoretic analysis generally occurs inside a small-diameter (50-100 m) quartz capillary in the presence of high (kilovolt-level) separating voltages with separation times of a few minutes.
  • nucleic acids are conveniently detected by UV absorption or fluorescent labeling, and single-base resolution can be obtained on fragments up to several hundred base pairs.
  • Electrophoretic analysis is well known in the art, as described, for example, in Ausubel et al, Current Protocols in Molecular Biology Chapter 2 (Supplement 45), John Wiley & Sons, Inc. New York (1999). Electrophoresis analysis can be performed in combination with restriction enzyme digestion.
  • Restriction fragment length polymorphism (RFLP) analysis can also be useful for genotyping an individual according to the methods of the present invention to determine the presence or absence of a particular intergenic deletion in the ADAMTS9-MAGI1 locus, (see, Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York; Innis et al, (ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)).
  • restriction fragment length polymorphism analysis includes any method for distinguishing polymorphic alleles using a restriction enzyme, which is an endonuclease that catalyzes degradation of nucleic acid following recognition of a specific base sequence, generally a palindrome or inverted repeat.
  • a restriction enzyme which is an endonuclease that catalyzes degradation of nucleic acid following recognition of a specific base sequence, generally a palindrome or inverted repeat.
  • RFLP analysis depends upon an enzyme that can differentiate the presence or absence of a genomic deletion. In some instances, RFLP analysis can be used to determine whether an individual carries zero, one or two copies of a specific deletion.
  • a specific intergenic deletion is detected in an individual's genomic DNA using a hybridization assay, such as, but not limited to, a DNA (oligonucleotide) array, multiplex amplification and probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), and Southern blotting analysis.
  • a hybridization assay such as, but not limited to, a DNA (oligonucleotide) array, multiplex amplification and probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), and Southern blotting analysis.
  • PsA psoriatic arthritis
  • the individual is determined to have a risk of developing psoriatic arthritis (PsA) if one or two copies of an intergenic deletion in the ADAMTS9-MAGI1 locus is detected.
  • the intergenic deletion removes about 25 kb at chromosome 3pl4.1 between the ADAMTS9 and MAGI1 genes.
  • the risk of developing PsA is determined if the deletion at the ADAMTS9-MAGI1 locus and flanking genomic variants ⁇ e.g., neighboring S Ps and the like) that are in high LD ⁇ e.g., r 2 ⁇ 0.7, the threshold for high LD; r 2 refers to the square correlation coefficient) to the deletion are detected.
  • the method described herein can be performed in combination with other methods for diagnosing psoriatic arthritis in an individual, e.g., a human subject.
  • a test or survey such as the Psoriatic and Arthritis Questionnaire (PAQ), the Psoriatic Arthritis Screening and Evaluation (PASE), the Classification Criteria for PsA (CASPAR), a physical evaluation performed by a clinician, a blood test to detect the presence or absence of rheumatoid factor and/or anti-citrullinated peptide antibodies, a joint/synovial fluid test to detect uric acid crystals, X-rays, magnetic resonance imaging (MRI), ultrasound imaging, CT scan, and the like.
  • Standardized patient assessment questionnaires, such as PASE can be useful as a screening tool to determine whether a patient with psoriasis also has active inflammatory arthritis.
  • a diagnosis of PsA also includes performing tests and assays to determine that the individual does not have rheumatoid arthritis, gout, reactive arthritis or another indication with similar symptoms as PsA.
  • the presence or level of a serological marker such as rheumatoid factor, an anti-citrullinated protein antibody, C-reactive protein, and combinations thereof is also determined. The presence of one or more serological markers can indicate that the subject does not have PsA.
  • the presence of a clinical factor such as the age of the individual, the sex of the individual, smoking history of the individual, a diagnosis of psoriasis, erythrocyte sedimentation rate (ESR), and combinations thereof is also determined.
  • a blood test can be performed to determine if the patient suspected of having PsA has mild anemia and/or an elevated erythrocyte sedimentation rate (ESR or sed rate). ESR can provide a basic measure of inflammation.
  • the individual has been diagnosed with psoriasis. In other embodiments, the individual does not have psoriasis or has not been diagnosed with psoriasis.
  • a clinician can diagnose psoriasis by taking a medical history and examining the subject's skin, scalp and nails. The severity of psoriasis is determined by measuring the percent body surface area affected, determining the location of the lesions, and assessing the patient's quality of life and ability to function.
  • a patient can be diagnosed as having mild psoriasis which affects up to 3% of the body; moderate psoriasis which affects 3%-10% of the body's surface; and severe psoriasis which affects greater than 10% of the body.
  • Quality of life can be evaluated using clinical indices, such as the Validated Health Related Quality of Life (HRQOL) index and a psoriasis Quality of Life (PQOL-12) index.
  • HRQOL Validated Health Related Quality of Life
  • PQOL-12 psoriasis Quality of Life
  • the individual is diagnosed as having purely cutaneous psoriasis (PsC) if the individual does not have the intergenic deletion of interest (e.g., is not heterozygous or homozygous for the intergenic deletion).
  • PsC purely cutaneous psoriasis
  • a treatment for a subject suspected of having psoriatic arthritis is also a method for selecting a treatment for a subject suspected of having psoriatic arthritis.
  • the subject is diagnosed with psoriasis. In other cases, the subject has not been diagnosed with psoriasis. If it is determined that the subject carries one or two copies of an intergenic deletion in the ADAMTS9-MAGI1 locus, a treatment regimen will be recommended or selected for the subject.
  • the treatment can include an anti-tumor necrosis factor (anti-TNF) therapy, a non-anti-T F therapy, or a combination thereof.
  • anti-TNF anti-tumor necrosis factor
  • Non-limiting examples of anti-TNF therapy include infliximab, adalimumab, golimumab, etanercept, certolizumab pegol, ABT-122 (e.g., a dual variable domain (DVD)- IgTM), variants thereof, equivalents thereof, derivatives thereof, and combinations thereof.
  • ABT-122 e.g., a dual variable domain (DVD)- IgTM
  • Non-limiting examples of non-anti-TNF drugs include azathioprine, cyclosporine, methotrexate, imuran, leflunomide, sulfasalazine, apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone, metformin, piclamilast, roflumilast, rolipram, a retinoid, a corticosteroid, a Cox-2 inhibitor, secukinumab, ustekinumab, brodalumab, guselkumab, ixekizumab, tofacitinib, AbGn-168H, bimekizumab, AIN457A (an anti-IL-17 monoclonal antibody), a PDE4 inhibitor, a nonsteroidal anti-inflammatory drug, variants thereof, equivalents thereof, derivatives thereof, and combinations thereof.
  • the treatment includes one or more anti-TNF drugs. In other embodiments, the treatment includes one or more anti-TNF drugs and one or more non-anti- TNF drugs. In yet other embodiments, the treatment includes one or more non-anti-TNF drugs.
  • the treatment can be selected for a patient in order to alleviate at least one symptom (including a clinical sign) of PsA.
  • the treatment is administered to a patient at risk of having or developing PsA or a patient diagnosed as having PsA.
  • symptoms or clinical signs of PsA include synovial and adjacent soft tissue inflammation, joint stiffness, joint pain, joint swelling, tenderness of joints, ligaments and/or tendons, enthesitis, osteitis, new bone formation, osteolysis (e.g., severe osteolysis), acro- osteolysis, ankylosis, spur formation, spondylitis, joint erosion, joint space narrowing, bony proliferation, periarticular and shaft periostitis, T-cell infiltration into synovial tissue, change in synovial lymphocyte population, increased serum levels of p40 protein, IL-2, IL-15, interferon-a, EGF, VEGF, and/or macrophage inhibitory protein 1-a, increased level of TNF- a, IL-8, IL-6, IL-1, IL-10 and/or matrix metalloproteinases in joint fluid, nail lesions, and the like.
  • osteolysis e.g., severe osteolysis
  • acro- osteolysis e.g., ankylosis,
  • HAQ-DI Health Assessment Questionnaire- Disability Index
  • ACR20/50/70 American College of Rheumatology Scoring system
  • PsARC Modified Psoriatic Arthritis Response Criteria
  • MASES Maastricht Ankylosing Spondylitis Entheses Score
  • CDAI Clinical Disease Activity Index
  • DAS28 Disease Activity Score
  • BASDAI Bath Ankylosing Spondylitis Disease Activity Index
  • a clinical improvement or alleviation of at least one symptom or clinical sign of PsA corresponds to a positive change from baseline (e.g., before administration of the treatment) in score or index that assesses PsA prognosis, progression or severity.
  • Example 1 A Deletion at the ADAMTS9-MAGI1 Locus is Associated with Psoriatic Arthritis Risk.
  • This example provides data from a genome-wide scan for CNVs associated with psoriatic arthritis (PsA) risk. The study identified a new intergenic deletion at the ADAMTS9-MAGI1 locus that is associated with disease risk. Detection of the deletion can be used to differentially diagnosis PsA risk from purely cutaneous psoriasis risk.
  • PsA psoriatic arthritis
  • CNV Copy -Number Variants
  • PsA Psoriatic Arthritis
  • Psoriatic Arthritis is a chronic inflammatory arthritis that affects 10-30% of patients with psoriasis (Gladman et al, Ann Rheum Dis. 2005 Mar; 64 Suppl 2:iil4-17; Julia et al., Hum Mol Genet. 2012 Oct 15; 21(20):4549-4557).
  • GWAS genome-wide association studies
  • candidate-gene studies have shown that both diseases share a substantial genetic component.
  • sibling recurrence rates ks are much higher for PsA than psoriasis (PsA s ⁇ 37 vs.
  • GWAS Genome-wide association studies based on single-nucleotide polymorphisms (SNPs) have been highly successful in identifying >30 loci associated with psoriasis and psoriatic arthritis susceptibility (Liu et al, PLoS Genet. 2008 Mar; 4(3):el000041; Ellinghaus et al., Nat Genet. 2010 Nov; 42(11):991-995; Strange et al., Nat Genet. 2010 Nov; 42(11):985-990).
  • Copy Number Variants are fragments of DNA with sizes that range from hundreds of bases to several megabases, and that can either be absent (i.e. deletions), repeated a certain number of times (i.e., amplifications) or even rearranged (Cook and Scherer, Nature. 2008 Oct 16; 455(7215):919-923).
  • Psoriasis was one of the first chronic inflammatory diseases where CNVs were found to be associated with the disease risk.
  • the amplifications of the beta-defensin genes on 8p23.1 region Hollox et al., Nat Genet.
  • IMIDC Immune-Mediated Inflammatory Disease Consortium
  • Exclusion criteria for the present study were: i) presence of any other inflammatory joint disease ii) presence of any inflammatory bowel disease iii) positivity of Rheumatoid Factor.
  • Control individuals for the GWAS stage were recruited from blood bank donors attending at 13 hospitals from different regions in Spain in collaboration with the Spanish National DNA Bank. Eligible individuals were screened for the presence of PsA or any other autoimmune disorder, as well as for history of autoimmune disorders in first-degree relatives, and positive individuals were discarded from this study. Additionally, in order to increase the 'hypernormality' of the control cohort (Morton and Collins, Proc Natl Acad Sci USA.
  • a total of 1, 131 PsA patients and 1,831 controls were used to validate the most significant loci identified in the GWAS phase. Both cohorts were collected using the same clinical and epidemiological selection criteria as for the GWAS. Additionally, a sample of 822 patients diagnosed with psoriasis and without PsA ⁇ i.e. purely cutaneous psoriasis or PsC) was also analyzed in the validation phase. All PsC patients were diagnosed and recruited by a consultant dermatologist participating in the IMTD Consortium (Julia et al, Hum Mol Genet. 2012 Oct 15; 21(20):4549-4557).
  • Psoriasis patients with plaque psoriasis affecting torso and/or extremities and with at least one year of duration were included. Patients with a single clinical localization of plaque psoriasis ⁇ i.e. scalp, face, palmoplanar), with exclusively inverse plaque psoriasis or with an inflammatory bowel disease were excluded from the study. Finally, psoriasis patients diagnosed with PsA by a rheumatologist were excluded from this group.
  • FIG. 4 shows the PsA patient and control distributions according to the first and second principal components after excluding the outliers.
  • FIG. 5 shows a schematic representation of the global CNV analysis workflow.
  • the Illumina sequencing platform (Illumina, San Diego, US) was used to characterize the deletion sequence.
  • ADAMTS9 and MAGI1 genes were sequenced and their 5' and 3' flanking sequences.
  • DNA quality of these samples was assessed by running 1-3 ⁇ on a 1% agarose gel that contained IX Sybr Green I dye (ThermoFisher Scientific).
  • IX Sybr Green I dye ThermoFisher Scientific
  • BAC DNAs that are commercially available (ThermoFisher Scientific). BAC DNAs were pooled by percentage of the total target size (1.1 Mb) according to a 4 ⁇ g total input mass, and the pool was sheared by E220 Covaris sonication.
  • Linkers containing T7 promoter sequences were ligated to sheared BAC fragments, and biotinylated RNA probes were synthesized by in vitro transcription using a MEGAscript kit (Ambion) and biotin-11-UTP (ThermoFisher Scientific) with T7-BAC fragments as template.
  • Illumina libraries were prepared according to standard protocol using 24 inline barcoded adapters.
  • hybridization reactions were assembled with 4 barcoded libraries (125 ng of each), 20 ⁇ g of Cot-1 DNA (ThermoFisher Scientific), 236 ng probe, 20U SUPERase-In (ThermoFisher Scientific), and 2X hybridization buffer in a final volume of 26 ⁇ incubated at 65° C for 70h. Hybridization reactions were incubated with 25 ⁇ MyOne Streptavidin CI Dynabeads (ThermoFisher Scientific) for 30 min with frequent pulse vortexing.
  • Replication genotyping was performed using the TaqMan ® Genotyping System (Applied Biosystems, Foster City, CA, US). Two pre-designed TaqMan ® CNV assays Hs03225015_cn and Hs03225295_cn were found to be located within the estimated deletion boundaries. In order to validate the two assays, we genotyped the group of 200 individuals that were previously used to sequence the deletion. The correspondence between the calls of the two CNVs between the TaqMan ® and sequencing analysis was 100%. Consequently, we used the two TaqMan ® assays to genotype the ADAMTS9-MAGI1 deletion in an independent group of 1,133 PsA patients, 1,831 healthy controls and 822 PsC patients.
  • Table 2 summarizes the main features of the GWAS and replication PsA patient cohorts.
  • SD standard deviation
  • RF Rheumatoid Factor
  • BP. START Basepair of first probe (5' end) where CNV is detected.
  • BP. END Basepair of first probe (3' end) where CNV is detected.
  • MAF in control CNV minor allele frequency in GWAS control cohort.
  • CNVjype Type of CNV: Deletion/ Amplification.
  • HLA-C locus is an established risk locus for PsA we sought to estimate the association of the deletion after correcting for the HLA haplotypes associated with psoriasis risk as recently described (Okada et al., Am J Hum Genet. 2014 Aug 7; 95(2): 162-172).
  • the significance (p-value) of the regression coefficient after performing a logistic regression analysis for association with PsA risk are shown.
  • the first row shows the individual significance of the established HLA-risk alleles as well as the deletion identified in the present GWAS.
  • the second row shows the significance of the HLA-B and the deletion markers after conditioning for the strongest known risk variant for PsA (i.e., HLA-C*0602).
  • the third row shows the significance of the deletion association after conditioning for both the HLA-C allele and the more recently characterized risk allele at HLA-B (i.e., HLA-B*3801).
  • the former conditional analysis strongly supports that the deletion is a proxy genetic marker rather than a causal risk variant.
  • CHR chromosome
  • BP Start basepair of first probe where the signal is detected
  • BP End basepair of last probe where the CNV signal is detected
  • MAF minor allele frequency in controls
  • type type of CNV (-) deletion, (+) amplification
  • P p-value of association test with PsA risk
  • ADAMTS9-MAGI1 deletion sequence characterization [0133] Using a next-generation sequencing approach, we characterized the deletion region in chromosome 3ql4.2 associated with PsA risk. Using a sample of 100 PsA patients and 100 controls selected to have a higher frequency of the deletion (in total, 100 deletion carriers vs. 100 2N individuals), we determined the physical extent and molecular nature of ADAMTS9- MAGI1 deletion polymorphism. PCR capture and sequencing of the deletion breakpoints revealed that the deletion removes 25,879 nucleotides (FIG. ID).
  • ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) gene belongs to a family of enzymes specialized in the degradation of the extracellular matrix.
  • ADAMTS9 belongs to the family of the aggrecanases (which also includes ADAMTS-1, ADAMTS-4, ADAMTS-6, ADAMTS-8 and ADAMTS-15), that are specialized in the degradation of aggrecan, one of the main proteoglycan constituents of the cartilage extracellular matrix (Kevorkian et al., Arthritis Rheum. 2004 Jan; 50(1): 131-141).
  • ADAMTS9 was clearly the most highly induced amongst all the different aggrecanases (Demircan et al., Arthritis Rheum. 2005 May; 52(5): 1451-1460; Uysal et al, Cell Biochem Funct. 2013 Oct; 31(7):539-544). Consequently, genetic variants that influence the rates of matrix turnover in the cartilage and bone of the inflamed synovial joint could be crucial in determining the level of tissue degradation in PsA.
  • Membrane associated guanylate kinase WW and PDZ domain containing 1 ⁇ MAGIl is a member of the membrane-associated guanylate kinase family of genes.
  • MAGIl is known to be expressed in cell-to-cell contacts, acting as a scaffold protein to stabilize cadherin-mediated adhesions and has been found to be expressed in epithelial and endothelial tight junctions (Laura et al, Exp Cell Res. 2002 May 1; 275(2): 155-170).
  • MAGIl activity has been related to several pathological junction-associated processes, including oncogenic (Zaric et al, Oncogene.
  • MAGIl interacts with phosphatase and tensin homolog protein (PTEN), a signaling protein that participates in the negative regulation of regulatory T cells (Tregs) (Walsh et al, Clin Invest. 2006 Sep; 116(9):2521-2531) which are master inhibitors of autoimmunity.
  • PTEN phosphatase and tensin homolog protein

Abstract

La présente invention concerne des procédés de diagnostic du rhumatisme psoriasique (RPs) chez un individu. En particulier, le procédé concerne la détection de la présence ou de l'absence d'une délétion intergénique entre les gènes ADAMTS9 et MAGI1 humains. L'invention concerne également des procédés pour déterminer si un sujet présente un risque de développer le RPs. Les procédés décrits dans la description peuvent être utilisés pour sélectionner un traitement d'un sujet susceptible d'être atteint de RPs.
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