HK1207401B - Genetic markers for predicting responsiveness to fgf-18 compound - Google Patents

Description

Genetic markers for predicting responsiveness to FGF-18 compounds

Technical Field

The present invention relates generally to pharmacogenetics, and more particularly to genetic markers associated with clinical response to FGF-18 compounds during treatment of cartilage diseases. More particularly, the present invention relates to human genes useful in the diagnosis and treatment of cartilage disorders.

The invention also discloses specific polymorphisms or alleles of the IL1RN gene that are associated with the cartilage response to FGF-18 compound therapy, as well as diagnostic tools and kits based on these changes in sensitivity. Thus, the present invention is useful for predicting the response of FGF-18 compound therapy. Which can be used to select/determine patients to be treated by intra-articular administration of an FGF-18 compound. The use of these markers in diagnosis may provide more benefit to the patient and reduce the risk.

Background

Generally, cartilage disorders refer to disorders characterized by the deterioration of metabolic abnormalities in connective tissue, manifested by pain, stiffness and limitation of movement in the affected body part. These diseases may be caused by pathogens or by trauma or injury. Cartilage disorders include Osteoarthritis (OA), cartilage damage (including sports injuries to cartilage and joints, and surgical injuries such as microfracture), and the like. The self-repair capacity of mature cartilage is limited, mainly because mature chondrocytes have little potential for proliferation and no blood vessels. In addition, cartilage is not available in much nutrition and has a low oxygen pressure. Replacement of damaged cartilage, particularly articular cartilage, caused by injury or disease is a challenge for physicians, and existing surgical procedures are considered not to be completely predictable and effective for only a limited period of time. Therefore, most young patients do not seek treatment, or are advised to delay treatment as much as possible. Standard procedures are age-dependent when treatment is necessary, and also differ between total joint replacement, cartilage block transplantation, or bone marrow stimulation techniques (e.g., microfracture surgery). Microfracture surgery is a common procedure that involves puncturing the subchondral bone to stimulate cartilage deposition by bone marrow stem cells. However, this technique has proven to be insufficient for repairing cartilage defects, and the new cartilage formed is mainly fibrocartilage, which results in functional and biomechanical deficiencies or changes. In fact, fibrocartilage does not have the same durability and may not adhere properly to the surrounding hyaline cartilage. Therefore, newly synthesized fibrocartilage is likely to be more easily damaged (expected term: 5-10 years).

For osteoarthritis patients, non-surgical treatment mainly includes physical therapy, lifestyle changes (e.g. reduced activity), support devices, oral or injectable medications (e.g. non-steroidal anti-inflammatory drugs) and medical management. Once these treatments fail, the primary option for the patient is surgery, such as joint replacement surgery. This option may result in a reduction of symptoms that are typically present in the short term. Tibial or femoral osteotomies (cutting the bone to reestablish balance of joint wear) can alleviate symptoms, help maintain a positive lifestyle, and delay the need for total joint replacement. Total joint replacement improves the symptoms of late stage osteoarthritis, but generally requires changes in the patient's lifestyle and/or activity level.

Currently, pharmacotherapy is available on the market primarily for pain relief. There are no therapies available on the market that can repair cartilage damage (see Lotz, 2010).

Fibroblast growth factor 18(FGF-18) is a member of the FGF protein family, which is closely related to FGF-8 and FGF-17. FGF-18 has been shown to be a proliferation agent for chondrocytes and osteoblasts (Ellsworth et al, 2002; Shimoaka et al, 2002). FGF-18 has been suggested for use in the treatment of cartilage diseases such as osteoarthritis and cartilage damage, either alone (WO2008/023063) or in combination with hyaluronic acid (WO 2004/032849).

Sprifermin is a truncated form of human FGF-18, and is currently being tested clinically for the treatment of osteoarthritis and cartilage damage (see, e.g., NCT01033994, NCT00911469, and NCT01066871 for more details). Currently, the dosing regimen for Sprifermin is once a week for three weeks (one treatment cycle) by intra-articular injection. The treatment cycle may be repeated. This dosing regimen is described in WO 2008023063.

Currently, OA and cartilage injury therapies using FGF-18 provided to patients in clinical trials do not have predictive information of response, that is, it is not known whether the treatment will be very effective, moderately effective, or have little or no efficacy. Currently, many treated patient groups show moderate/high response to treatment after at least one treatment cycle with Sprifermin according to WOMAC score, but some other patients do not respond to the treatment or respond but have high WOMAC score compared to the control group.

The present specification describes for the first time genetic markers associated with the quality of clinical response to the treatment of cartilage diseases such as OA, cartilage damage or microfracture with FGF-18. Such markers can be used to identify, by pre-treatment genetic screening, a subset of patients more likely to respond specifically to FGF-18 therapy, e.g., patients who have a very good clinical response to FGF-18 therapy, or conversely, patients who may fail the therapy. Knowledge of the type of clinical response of a patient to treatment can be used to optimize therapy or to select therapy, for example, FGF-18 therapy as the first line therapy or to modify the dosing regimen. This information is clinically useful for the medical management of cartilage diseases such as OA/cartilage damage in patients. For example, if a patient with OA or cartilage damage is known to be at high risk of unresponsiveness to FGF-18 therapy, the physician may exclude the patient from FGF-18 therapy. In addition, clinically, such predictive information may also be used to guide the decision of a dosing regimen.

Disclosure of Invention

The present invention relates to a method for predicting the sensitivity of an individual suffering from a cartilage disease to a therapy with an FGF-18 compound, said method comprising the steps of:

a. determining genotypes at the loci IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample;

b. predicting from the results of step a high, medium, low or no sensitivity of said individual to a therapy with an FGF-18 compound.

According to the method, the presence of a genotype at IL-1RN rs9005 at G/G and IL-1RN rs315952 at T/T is predictive of no response or low response (i.e., insensitivity) to therapy with an FGF-18 compound. Conversely, the presence of a genotype at A/G or A/A at IL-1RNrs9005 and T/C or C/C at IL-1RNrs315952 is predictive of a high response (high sensitivity) to therapy with FGF-18 compounds. Other genotypes at these two loci are predictive of medium sensitivity (i.e., G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, or A/G or A/A at IL-1RN rs9005 and T/T at IL-1RN rs315952, or C/C is complementary to IL-1RN rs9005 and A/G or G/G is complementary to IL-1RN rs315952, or T/C or T/T is complementary to IL-1RN rs9005 and A/A is complementary to IL-1RN rs 315952).

The invention also describes a method of adding or rejecting a cartilage disease patient to a therapy with an FGF-18 compound based on its likelihood of being sensitive to said therapy or a clinical trial, the method comprising determining from a nucleic acid sample the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of said patient at said loci is predictive for the risk of said patient being sensitive or insensitive to said therapy, and selecting a sensitive patient as being suitable for said therapy. In particular, patients with genotypes at IL-1RN rs9005 with G/G and IL-1RNrs315952 with T/T would be classified as non-sensitive. Thus, these individuals may be excluded from the FGF-18 compound therapy or clinical trial. Thus, individuals with any other genotype at these loci (i.e., G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, or A/G or A/A at IL-1RN rs9005 and T/T, T/C or C/C at IL-1RN rs 315952) will be classified as being susceptible, including those that are moderately and hypersensitive (or highly sensitive), and therefore, may be enrolled in (or adapted for) therapy or clinical trials using FGF-18 compounds.

The present invention also provides a method of selecting a patient for an alternative treatment regimen using an FGF-18 compound based on the likelihood that the patient with a cartilage disease is hypersensitive to a therapy with an FGF-18 compound, the method comprising determining from a nucleic acid sample the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at said loci is predictive of the risk of the patient to be hypersensitive to the therapy with the FGF-18 compound, and selecting the patient for an alternative treatment regimen appropriate for the patient. Preferably, the total dose of FGF-18 compound administered in the alternative treatment regimen is reduced compared to the dose of FGF-18 compound administered in a patient not at risk of being hypersensitive to the FGF-18 compound therapy. In particular, patients with genotypes at IL-1RN rs9005 with a/G or a/a and IL-1RN rs315952 with T/C or C/C are classified as hypersensitizers and are selected to carry out an alternative treatment regimen of FGF-18 dosing reduction.

The invention also provides a method of selecting a patient for an alternative treatment regimen with an FGF-18 compound based on the likelihood of the patient with a cartilage disease developing an AIR event upon treatment with an FGF-18 compound, the method comprising determining from a nucleic acid sample the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at said loci is predictive of the risk of the patient developing an AIR event in response to treatment with the FGF-18 compound, and selecting the patient for an alternative treatment regimen appropriate for the patient. Preferably, the total dose of FGF-18 compound administered in the alternative treatment regimen is reduced compared to the dose of FGF-18 compound administered to a patient not at risk of developing an AIR event. In particular, patients with genotypes at IL-1RN rs9005 with a/G or a/a and IL-1RN rs315952 with T/C or C/C are classified as at risk for developing AIR events and are selected to carry out an alternative treatment regimen with reduced dosing of FGF-18.

The present invention also includes an FGF-18 compound for use in treating a patient with a cartilage disorder, wherein the patient has a genotype selected from the group consisting of: 1) G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, and 2) A/G or A/A at IL-1RN rs9005 and T/T, T/C or C/C at IL-1RN rs 315952. If the patient is classified as a hypersensitizer, i.e. having a genotype of A/G or A/A at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, the patient may be treated with a smaller dose of the FGF-18 compound than a patient having a combination of the other two genotypes.

In another aspect, the invention also describes a kit comprising a device for carrying out the above method and instructions for use. The kit comprises at least one pair of specific primers or probes for detecting the presence of the allele.

In a particular embodiment of the invention as a whole, i.e. in any of the methods or uses mentioned in the specification, the FGF-18 compound for use in therapy is Sprifermin, and said patient is suffering from a cartilage disorder selected from the group consisting of: osteoarthritis, cartilage damage, fractures affecting articular cartilage or surgical procedures affecting articular cartilage (e.g., microfracture procedures).

It will be appreciated that in any of the methods or uses referred to in this specification, a nucleic acid sample (or test sample) of the individual needs to be obtained by a method such as blood or saliva collection before the genotype at a locus is determined. Alternatively, the test sample may be selected from buccal cells, urine or feces. Preferably, the nucleic acid sample is a DNA sample. Furthermore, it is to be understood that any method or use mentioned in this specification is performed in vitro, and not in an animal or human.

It is also to be understood that in the context of the present invention as a whole, the determination may be made in complementary sequences corresponding to IL-1RN rs9005 and IL-1RNrs 315952.

Definition of

The term "FGF-18 compound" or "FGF-18" as used herein means a protein that retains at least one biological activity of a human FGF-18 protein. FGF-18 can be native, present in its mature form, or a truncated form thereof. The biological activities of the human FGF-18 protein include, inter alia, increased osteoblast activity (see W098/16644) or increased cartilage formation (see W02008/023063). Native, wild-type human FGF-18 is a protein expressed by chondrocytes of articular cartilage. W098/16644 first designated human FGF-18 as zFGF-5 and is fully described. SEQ ID NO 1 corresponds to the amino acid sequence of native human FGF-18, the signal peptide of which consists of amino acid residues 1(Met) -27 (Ala). The mature form of human FGF-18 corresponds to the amino acid sequence (180 amino acids) of residue 28(Glu) -residue 207(Ala) of SEQ ID NO: 1. The term also includes fusion proteins of an FGF-18 protein with a heterologous protein or chemical compound.

In the present invention, FGF-18 can be produced by recombinant methods, such as the method taught in patent application W02006/063362. In the present invention, FGF-18 is expressed in recombinant host cells and has an initial methionine (Met) residue or has a secretion signal sequence depending on the expression system and conditions. When expressed in prokaryotic hosts, such as E.coli, FGF-18 contains an additional Met residue at the N-terminus of its sequence. For example, in E.coli expression, the amino acid sequence of human FGF-18 begins with the Met residue at the N-terminus (position 1) followed by residue 28(Glu) -residue 207(Ala) of SEQ ID NO: 1.

As used herein, the term "truncated form" of FGF-18 refers to a protein comprising or consisting of residues 28(Glu) -196(Lys) of SEQ ID NO: 1. Preferably, the truncated form of the FGF-18 protein is the polypeptide designated "trFGF-18" (170 amino acids) which starts with the Met residue (N-terminal) and is followed by amino acid residues 28(Glu) -196(Lys) of wild-type human FGF-18. the amino acid sequence of trFGF-18 is found in SEQ ID NO:2 (amino acid residues 2-170 of SEQ ID NO:2 correspond to amino acid residues 28-196 of SEQ ID NO: 1). trFGF-18 is a truncated form of recombinant human FGF-18 produced in E.coli (see W02006/063362). The international non-proprietary name for this particular form of FGF-18 is Sprifermin. Spriformin has been shown to exhibit activity similar to that of mature human FGF-18, e.g., it increases chondrocyte proliferation and cartilage deposition, leading to repair and reconstruction of various cartilage tissues (see W02008/023063).

"cartilage disease" as used herein includes conditions arising from injury such as trauma, cartilage disease or damage due to arthritis. Examples of cartilage disorders that may be treated by administration of an FGF-18 formulation as described herein include, but are not limited to, arthritis (e.g., osteoarthritis), cartilage damage, fractures affecting articular cartilage, or surgical procedures affecting articular cartilage (e.g., microfracture surgery). The term also includes degenerative diseases/disorders of the cartilage or joints, such as chondrocalcinosis, polychondritis, relapsing polychondritis, ankylosing spondylitis, or costochondritis. The international society for cartilage repair proposes an arthroscopic rating system to assess the severity of cartilage defects: level 0: (normal) healthy cartilage, grade 1: cartilage with soft spots or blisters, grade 2: tiny clefts are visible in cartilage, grade 3: the lesions had deep fissures (more than 50% of the cartilage layer), and a 4-stage: a cartilage break exposes the underlying (subchondral) bone (see, e.g., http:// www.cartilage.org/_ files/content management/ICRS _ evaluation. pdf, page 13).

The term "osteoarthritis" is used to mean the most common form of arthritis. The term "osteoarthritis" includes primary osteoarthritis and secondary osteoarthritis (see, e.g., The Merck Manual, 17 th edition, page 449). The most common way to classify/rate osteoarthritis is using the Kellgren-Lawrence imaging grading scale (see table below). Osteoarthritis can be caused by cartilage breakdown. Small pieces of cartilage may break off and cause pain and swelling of the joints between the bones. Over time, the cartilage may wear away completely and the bones may rub against each other. Osteoarthritis can involve any joint, but usually involves the hands and weight-bearing joints, such as the hip, knee, foot, and spine. In a preferred embodiment, the osteoarthritis may be knee osteoarthritis or hip osteoarthritis. Osteoarthritis is one of the preferred cartilage diseases that can be treated by administration of the FGF-18 compounds of the present invention.

The Kellgren-Lawrence imaging scale for osteoarthritis is as follows:

osteoarthritis grading	Description of the invention
		0-has	Osteoarthritis was not found
1-suspected of being	The joint space is suspected to be narrowed and possibly has a bony spur-like protrusion
		2-slight	Clear osteophytes, clear narrowing of the joint space
3-moderate	Multiple medium osteophytes, clear narrowing of the joint space, partial sclerosis and possibly malformation of the bone contours
		4-severe	Large osteophytes, marked narrowing of the joint space, severe sclerosis and definite deformity of the bone contour

The term "cartilage damage" as used herein mainly refers to cartilage diseases or cartilage damage caused by trauma. Cartilage damage can occur primarily following traumatic mechanical disruption, particularly accidents or surgery (e.g., microfracture surgery). The term "cartilage injury" also includes cartilage or osteochondral fractures, meniscal injuries, and microfracture procedures. The definition also includes sports related injuries or sports related wear of joint tissue.

The term AIR (acute inflammatory response) as used in the present specification is defined as follows: within 1-7 days, preferably within 3 days, after intraarticular injection of the FGF-18 compound in the target knee, the following conditions must be simultaneously fulfilled:

self-reported swelling (synovial fluid exudation)

-30 mm increase in pain on a 100mm Visual Analogue Scale (VAS)

An "allele" is a particular form of a gene, genetic marker, or other locus that is distinguishable from other forms of genes, genetic markers, or other loci, such as, but not limited to, its particular nucleotide sequence. The term allele also includes, for example, but is not limited to, a form of a Single Nucleotide Polymorphism (SNP). The diploid cells of an individual may be homozygous for an allele, i.e., the alleles on both paired chromosomes are identical; or is heterozygote for the allele, i.e., the alleles on the two paired chromosomes are different.

The terms "genetic marker", "biomarker" or "marker" refer to an identifiable polymorphic (genetic) site. An example of a genetic marker is, but is not limited to, a Single Nucleotide Polymorphism (SNP).

"Single Nucleotide Polymorphism (SNP)" refers to a variation of a DNA sequence in the genome of a species (or other sequences shared between individuals of a species) -A (adenine), T (thymine), C (cytosine), or G (guanine) -a single nucleotide that does not occur simultaneously between individuals (or between paired chromosomes of an individual). Sequences that are highly conserved in the target population are typically preceded and followed by a SNP, and thus, the position of a SNP is typically determined with reference to a consensus nucleic acid sequence of thirty to sixty nucleotides in length encompassing the genetic marker site, such a consensus nucleic acid sequence sometimes being referred to as the context sequence of the SNP. SNPs analyzed by the present inventors in connection with the treatment of cartilage diseases using Sprifermin are shown in table 1.

As used herein, "genotype" refers to the combination of two alleles of a genetic marker (such as, but not limited to, a SNP) at a single locus on the paired (homologous) chromosome of an individual. As used herein, "genotype" also refers to a combination of alleles at more than one locus (such as, but not limited to, SNPs) on one or more than one pair of homologous chromosomes in an individual.

The term "haplotype" refers to variants or alleles of different markers (e.g., SNPs) located on the same chromosome. SNP genotype data measured by SNP arrays or Taqman assays are non-phased (alphased, i.e. the paternity of each allele on a chromosome is unknown). Computational methods (Browing et Browning, 2011) use trans-individual information to estimate (or infer) haplotype phase from genotype data.

The term "genotyping" refers to the process of determining the genotype of a single or multiple SNPs of an individual.

A "locus" or "genetic locus" refers to a specific location on a chromosome or other genetic material. For example, IL-1RN rs9005 is a locus, which may be referred to as "IL-1 RN rs 9005" or "locus IL-1RN rs 9005" within the framework of the present invention. The same is true for IL-1RN rs 315952. From the NCBI database of these SNPs, the skilled person will know that the genotypes to be determined at IL-1RN rs9005 and IL-1RN rs315952 are those at position 27 of each of these two loci, i.e. SEQ ID NO: 6 and position 27 of SEQ ID NO: position 27 of 7.

In the context of the present invention, the term "SNP 1" refers to SEQ ID NO: 6, identified in the NCBI database as rs 9005. SEQ ID NO: 6 is part of the genomic nucleic acid sequence of an interleukin 1 receptor antagonist (IL-1 RN). The terms "IL-1 RN rs 9005", "rs 9005" or "SNP 1" are used interchangeably.

The term "SNP 2" refers to SEQ ID NO: 7, identified in NCBI database as rs 315952. SEQ ID NO: 7 is part of the genomic nucleic acid sequence of IL-1 RN. The terms "IL-1 RN rs 315952", "rs 315952" or "SNP 2" are used interchangeably.

The term "probe" or "primer" refers to an oligonucleotide, i.e., a nucleic acid or nucleic acid derivative, including but not limited to Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), or Bridged Nucleic Acid (BNA), which is typically between 5 and 100 consecutive bases in length, most commonly between 5-40,5-35,5-30,5-25,5-20,5-15,5-10,10-50,10-40,10-30,10-25,10-20,15-50,15-40,15-30,15-25,15-20,20-50,20-40,20-30, or 20-25 consecutive bases. The sequence of the probe/primer can be designed to specifically hybridize to one of the allelic forms of the genetic marker, and such oligonucleotides are referred to as allele-specific probes. If the genetic marker is a SNP, the complementary allele of the SNP may be present at any position on the allele-specific probe. Another probe/primer useful in the practice of the present invention that specifically hybridizes to a target region adjacent to a SNP is located at the 3' end of one to less than or equal to about 10 nucleotides, preferably less than or equal to about 5 nucleotides, from the genetic marker site. Such probes/primers that hybridize adjacent to the SNPs can be used in polymerase-mediated primer extension methods, referred to herein as "primer extension oligonucleotides". In a preferred embodiment, the 3' end of the primer extension oligonucleotide is a deoxynucleotide complementary to the nucleotide immediately adjacent to the SNP.

The term "polymorphism" refers to two or more alternative forms (alleles) of a genetic locus in a population that differ in nucleotide sequence or contain a different number of repeating nucleotide units. Polymorphisms are found in coding regions (exons), non-coding regions or outside genes (intergenic regions) of genes. In general, different alleles of a polymorphism occur at different frequencies within a population, and the alleles most common in a selected population are sometimes referred to as "primary" or "wild-type" alleles. Diploid organisms may be homozygotes or heterozygotes for the different alleles present. A biallelic polymorphism has two alleles.

The term "epistatic effect" is generally used to define the interaction between genes. Bateson (Bateson et Mendel, 1909) first defined a superordinate effect as a masking effect describing a variant or allele at one locus that prevents a variant at another locus from exhibiting its effect. However, a number of different definitions are provided in the scientific literature (Phillips, 1998; Cordell, 2002). The superordinate effect is tested herein as a statistical interaction between the genotypes of two different SNPs. This is similar to the definition set forth by Fisher in 1918, namely: additive deviations in the contribution of alleles to a phenotype at different loci.

The "WOMAC total score" or "WOMAC score" ("WOMAC" stands for "sienna da and macmas university osteoarthritis index") measures pain (WOMAC pain score), function (WOMAC function score), and stiffness (WOMAC stiffness score). When used to assess pain and dysfunction associated with cartilage damage, it consists of a questionnaire containing 24 items, the 24 items being divided into 3 sub-charts (5 items for pain, 2 items for stiffness, 17 items for physical function) (see Bellamy et al, 1988; Wolfe, 1999). This well known tool has wide application, particularly in the assessment of OA severity.

To assess cartilage repair, measurements of cartilage volume were made by Magnetic Resonance Imaging (MRI) measurements, including total cartilage volume (also known as LFTC (lateral femorotibial) + MFTC (medial femorotibial)), lateral cartilage volume (also known as LFTC), medial cartilage volume (also known as MFTC), and new total average cartilage thickness.

The term "baseline" refers to clinical variables such as, but not limited to, soft body mass and WOMAC total score prior to treatment (i.e., at the start of the study), particularly at the start of the study (i.e., prior to treatment with FGF-18 compound or placebo) for a particular patient.

By "susceptible" is meant a patient who is responsive to therapy with an FGF-18 compound for the treatment of a cartilage disorder. Preferably, the increase in total cartilage volume in sensitive patients (or patients sensitive to treatment) is higher than in individuals treated with placebo, i.e. sensitive patients show cartilage repair. In addition, the improvement in WOMAC total score in sensitive patients is at least close to placebo control. The terms "hypersensitizer", "moderately sensitized" and "non-sensitized" refer to different groups of patients classified according to the increase in cartilage volume following treatment with an FGF-18 compound. Hypersensitizers have a high response to therapy with an FGF-18 compound (i.e., high cartilage repair), moderately sensitized persons have a good or moderate response to therapy with an FGF-18 compound (i.e., good or moderate cartilage repair), and non-sensitized persons have no or low response to therapy with an FGF-18 compound. The improvement in WOMAC total score for both hypersensitive and sensitive patients was close to placebo control. In contrast, the improvement in WOMAC total score for non-responders was significantly less than placebo control. The terms "supersensitor" hypersensitizer "or" hypersensitizer "are used interchangeably. It should be noted that the risk of AIR events for hypersensitizers has proven to be high.

More specifically, the terms "moderately-sensitive", "hypersensitive" and "non-sensitive" include, but are not limited to, different groups of patients classified according to an increase in cartilage volume and an improvement in the WOMAC total score after treatment with an FGF-18 compound.

The criteria for the proposed sensitizers are as follows:

1. positive increase in cartilage (+10 to +100 mm) compared to baseline³In between).

2. The change in cartilage increase was significantly higher than the placebo-controlled change (e.g., tested in a linear model adjusted for BMI, KL rating, gender, and age and alpha-5).

WOMAC score improvement, i.e., decrease compared to baseline (e.g., decrease by more than 5 points).

Changes in WOMAC score were not significantly higher than placebo-controlled changes (e.g., to test in a linear model adjusted for BMI, KL rating, gender, and age and alpha ═ 5).

The criteria for the proposed hypersensitizers were the same as those for the sensitized, but the cartilage was increased by more than 100mm compared to baseline³(Standard # 1).

A non-sensitive person may be defined as an individual who does not meet criteria #1 or #2 and does not meet criteria #3 or # 4.

Thus, moderately sensitive individuals respond well or moderately to therapy with FGF-18 compounds (or good or moderately sensitive) (see above criteria; according to the examples, median change: 84.81mm increase in total cartilage volume compared to baseline³(ii) a Median change: WOMAC total score-20 points compared to baseline; no significant difference in WOMAC total score compared to placebo control). Hypersensitizers have a high response (or sensitivity) to therapy with FGF-18 compounds (see criteria above; according to the examples, median change: 119.46mm increase in total cartilage volume compared to baseline³Increased (i.e., benefit) + 40.85% compared to sensitive patients; median change: WOMAC total score-10 points compared to baseline; no significant difference in WOMAC total score compared to placebo control). Non-sensitized persons did not respond or responded poorly (or did not or poorly) to therapy with FGF-18 compound (see above criteria; according to the examples: significantly less increase in total cartilage volume compared to placebo control (difference between median: -106.64 mm)³) (ii) a The WOMAC total score was only slightly improved compared to baseline (median change: -1 point); the WOMAC total score is significantly different compared to placebo control).

"response" or "sensitivity" to an FGF-18 compound therapy is understood to be 1 year after the first injection and is measured as follows: 1) an increase in cartilage volume, e.g. measured by MRI or X-ray, 2) a decrease in the total WOMAC score, and 3) a change in the total WOMAC score that is not significantly higher than placebo control (see definition of "sensitizers").

"prognostic biomarkers" provide information about the condition of an individual, including but not limited to disease progression, disease severity, or disease outcome, regardless of any therapy. "predictive biomarkers" provide information about the effect of a received therapy, including but not limited to efficacy and safety outcomes. The definitions of prognostic and predictive are not mutually exclusive, and thus a biomarker may be both prognostic and predictive.

The term "MAD" as used herein refers to multiple ascending doses. When this abbreviation is followed by a number, this number corresponds to the dose of FGF-18 compound injected during the treatment. For example, MAD100 may indicate that a patient receives 100mcg of FGF-18 compound per injection during a treatment period. The abbreviation "PL" (and "MADPL") refers to placebo.

The term "storage device" as used in this specification includes any suitable computing or processing device or other device arranged or adapted to store data or information. Examples of electronic devices suitable for use with the present invention include stand-alone computing devices, data telecommunications networks including Local Area Networks (LANs), Wide Area Networks (WANs), the internet, intranets and extranets, as well as local and distributed computer processing systems. Storage devices also include, but are not limited to: magnetic storage media (e.g., floppy disks, hard disk storage media, magnetic tape), optical storage media (e.g., CD-ROMs, DVDs), electronic storage media (e.g., RAMs, ROMs, EPROMs, EEPROMs, etc.), rigid disks, and hybrids of the above, such as magnetic/optical storage media.

The term "storing" as used herein refers to the process of encoding information on a storage device. One skilled in the art can readily employ any known method to record information on known media to produce a product containing expression level information.

Detailed description of the invention

There is a need to predict the clinical efficacy of FGF-18 compound therapy (particularly for cartilage repair) in the treatment of patients suffering from cartilage disorders such as osteoarthritis, cartilage damage, fractures affecting articular cartilage, or surgical procedures affecting articular cartilage such as microfracture. To optimize treatment of such patients, it is important to identify biomarkers that can be used to predict the response of a particular patient to FGF-18 compound therapy, particularly for cartilage repair. Such predictive biomarkers can be used to identify high risk groups that are not or, conversely, are hypersensitive to the therapy. For example, if an osteoarthritic patient has proven to have a high risk of being unresponsive (or insensitive) to the therapy, the physician may decide not to advise the patient to use an FGF-18 compound, such as Sprifermin. Conversely, if an osteoarthritic patient has been shown to have a high risk of hypersensitivity to the therapy, the physician may decide to adjust the dosing regimen to reduce the dose of FGF-18 given to that patient. Such predictive information is clinically useful in guiding medical decisions, particularly when joint replacement surgery is required.

The unexpected findings of the present invention are based on a study aimed at determining biomarkers associated with Sprifermin administration. Biomarkers used in this study included candidate genetic markers (see table 1) and less than 1 million SNPs covering the human genome, with the median of the spacing between markers being 680 bases. The association between genetic markers and clinical response variables was evaluated. The reason for such analysis is to identify biomarkers that can predict the clinical outcome (particularly with respect to cartilage repair) of patients treated with FGF-18 compounds such as Sprifermin. These SNPs can be used to stratify and target specific patient populations.

The inventors have unexpectedly discovered that certain biomarkers (or SNPs) are linked to the outcome (e.g., cartilage repair) and adverse effects of FGF-18 therapy. Of particular interest are the SNPs rs9005 and rs315952, both located in the IL-1RN gene (see fig. 1).

These biomarkers are documented as likely to be associated with the severity and development of disease in OA patients by haplotypes including rs419598(C), rs315952(T) and rs9005(a), the so-called C-T-a haplotypes, as described in the literature (see, e.g., WO2009/135218 or Attur et al, 2010). Interestingly, while the present invention demonstrates that two of these biomarkers, rs9005 and rs315952, are closely related to the responsiveness of FGF-18 therapy, the other biomarker, rs419598, does not appear to be further linked to the observed phenotype, although it is described in the literature to be related to the other two SNPs. In fact, the so-called C-T-A haplotype does not allow stratification of patients according to total cartilage volume (FIG. 2) or WOMAC total fraction (FIG. 3). Thus, the C-T-A haplotype is not a good predictor of FGF-18 therapy.

In contrast, the present inventors have unexpectedly found that alleles with biomarker rs9005 as a and biomarker rs315952 as C correlate with a better response to therapy with FGF-18 compounds (e.g. spifermin) in patients with cartilage damage (table 4). These patients are called hypersensitizers or hypersensitizers.

Conversely, the inventors of the present invention have also surprisingly found that the genotype of rs315952 as T/T and rs9005 as G/G in cartilage injury patients is associated with no response or low response to therapy with an FGF-18 compound, such as Sprifermin (i.e. insensitivity to therapy with an FGF-18 compound) (table 4). These patients are called non-sensitized. It follows that patients with any other genotype at these two loci (i.e., G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, or A/G or A/A at IL-1RN rs9005 and T/T at IL-1RN rs 315952) are moderately sensitive.

Thus, the present invention has found that the binding of the polymorphic loci IL-1RN rs9005 and IL-1RN rs315952 can be used as biomarkers for predicting the responsiveness of an individual to FGF-18 compound (e.g., Spriformin) therapy (Table 4). Preferably, the individual suffers from a cartilage disorder, such as osteoarthritis, cartilage damage, a fracture affecting articular cartilage, or a surgical procedure affecting articular cartilage (e.g., microfracture surgery). In a particular embodiment, if the individual has a genotype of G/G at IL-1RN rs9005 and T/T at IL-1RN rs315952, the individual will be predicted to be insensitive to FGF-18 compound therapy. Conversely, if the individual has a genotype with A/G or A/A at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, the individual will be predicted to be hypersensitive (or hypersensitive) to FGF-18 compound therapy. In any other case, the patient will be predicted to be moderately sensitive to FGF-18 compound therapy (a summary of clinical outcomes and possible therapy options is shown in table 22).

Accordingly, the present invention relates to a method of predicting the sensitivity of an individual suffering from a cartilage disease to a therapy with an FGF-18 compound, said method comprising the steps of:

a. determining genotypes at IL-1RN rs9005 and IL-1RN rs 315952;

b. predicting from the results of step a high, medium, low or no sensitivity of said individual to a therapy with an FGF-18 compound.

Prior to determining the genotype at a locus, a nucleic acid sample from the individual is obtained by methods such as blood or saliva collection. Preferably, the nucleic acid sample is a DNA sample. Accordingly, the present invention relates to a method of predicting the sensitivity of an individual suffering from a cartilage disease to a therapy with an FGF-18 compound, said method comprising the steps of:

a. obtaining a nucleic acid sample from the individual;

b. determining genotypes at IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample;

c. predicting from the results of step b a probability of having high, medium, low or no sensitivity to therapy with an FGF-18 compound.

According to the method, the presence of a genotype for G/G at IL-1RN rs9005 and T/T at IL-1RN rs315952 is predictive of no or low response to therapy with an FGF-18 compound. Therefore, the patient will be predicted to be insensitive. Conversely, the presence of a genotype at IL-1RN rs9005 at A/G or A/A and IL-1RN rs315952 at T/C or C/C is predictive of a high response to therapy with FGF-18 compounds. Therefore, the patient will be predicted to be hypersensitive. It follows that patients with any other genotype at these two loci (i.e.G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, or A/G or A/A at IL-1RN rs9005 and T/T at IL-1RN rs 315952) will be classified as having moderate sensitivity to therapy with FGF-18 compounds. Based on the prediction, the physician can easily select only patients who are predicted to be sensitive to FGF-18 compound therapy, including those who are moderately sensitive and those who are hypersensitive.

The invention also relates to a test for determining sensitivity to a therapy with an FGF-18 compound or for determining a treatment regimen with an FGF-18 compound, said test comprising: (a) performing at least one genotyping assay on a test sample from a human individual diagnosed with a cartilage disorder to determine the genotype of at least two loci, wherein the at least two loci are: (i) SNP1 and (ii) SNP2, (b) determining the genotypes of the at least two loci; (c) selecting the patient as having sensitivity to therapy with an FGF-18 compound when the presence of at least one of the following SNP combinations is determined: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7, or (iii) SNP1 genotype a/G or a/a, or in seq id NO: 6 is T/C or T/T in the complementary strand; and SNP2 genotype T/C or C/C, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or G/G in the complementary strand; and (d) optionally treating the patient selected in step (c) with an FGF-18 compound.

When the above assay is performed to determine a treatment regimen using an FGF-18 compound, step (c) is optional and preferably step (d) is performed, or step (d) is performed.

The invention also relates to a test for determining insensitivity to FGF-18 compound therapy, said test comprising: (a) performing at least one genotyping assay on a test sample from a human individual diagnosed with a cartilage disorder to determine the genotype of at least two loci, wherein the at least two loci are: (i) SNP1 and (ii) SNP2, (b) determining the genotypes of the at least two loci; (c) selecting the patient as not sensitive to therapy with an FGF-18 compound when the following combination of SNPs is determined to be present: SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; and (d) optionally treating the patient selected in step (c) with a therapeutic compound other than an FGF-18 compound.

In the above-described assays, it is necessary to obtain a nucleic acid (or test) sample of the individual by a method such as blood or saliva collection, before determining the genotype at one locus.

The present application also includes a method of adding or removing a cartilage disorder patient to a therapy or clinical trial based on the likelihood that the patient will respond to the therapy with an FGF-18 compound, the method comprising:

a. determining the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample, wherein the genotype of the patient at the loci is predictive of the risk of the patient being sensitive or insensitive to the therapy, and

b. selecting patients suitable for the therapy or clinical trial, namely: sensitive patients are selected as appropriate for the therapy or the clinical trial.

Prior to determining the genotype at a locus, a nucleic acid sample from the individual is obtained by methods such as blood or saliva collection. Preferably, the nucleic acid sample is a DNA sample. Accordingly, the present invention relates to a method of adding or removing a cartilage disorder patient to a therapy or clinical trial based on the likelihood that the patient will respond to the therapy with an FGF-18 compound, the method comprising:

a. obtaining a nucleic acid sample from said individual,

b. determining the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample, wherein the genotype of the patient at the loci is predictive of the risk of the patient being sensitive or insensitive to the therapy, and

c. selecting patients suitable for the therapy or the clinical trial, namely: sensitive patients are selected as appropriate for the therapy or the clinical trial.

According to the method, patients with genotypes with a G/G at IL-1RN rs9005 and a T/T at IL-1RN rs315952 are predicted to be non-sensitized and are preferably rejected for the FGF-18 compound therapy or clinical trials related to FGF-18 compounds. Other patients, i.e.sensitive patients (including moderately and hypersensitive patients, i.e.patients having a genotype with G/G at IL-1RNrs9005 and T/C or C/C at IL-1RN rs315952, or A/G or A/A at IL-1RN rs9005 and T/T, T/C or C/C at IL-1RN rs 315952) may optionally be suitable for therapy with FGF-18 compounds, such as Sprifermin.

Alternatively, a method of adding or rejecting a therapy or clinical trial using a FGF-18 compound based on the likelihood that a patient with a cartilage disorder is susceptible to said FGF-18 compound, comprising the steps of: (a) performing at least one genotyping assay on a test sample from a human individual diagnosed with a cartilage disorder to determine the genotype of at least two loci, wherein the at least two loci are: (i) SNP1SNP2, wherein SNP2 is the amino acid sequence of SEQ ID NO: 7, wherein SEQ ID NO: 7 is a portion of the genomic nucleic acid sequence of an interleukin 1 receptor antagonist (IL-1 RN); and (b) detecting the presence or absence of a genotype combination selected from the group consisting of: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ id no: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7, or (iii) SNP1 genotype G/G or a nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; and (c) selecting the patient for therapy or clinical trial with the FGF-18 compound when condition (i) or (ii) is detected based on the knowledge that the genotypic combination (i) and (ii) correlate with a response to the FGF-18 compound, and rejecting the patient for therapy or clinical trial with the FGF-18 compound when condition (iii) is detected based on the knowledge that the genotypic combination (iii) correlates with an inadequate response to therapy with the FGF-18 compound.

Alternatively, the method of selecting a human subject for a clinical trial testing for an FGF-18 compound may comprise the steps of: (a) testing a biological sample from a human individual diagnosed with a cartilage disorder to determine at least two of the following single nucleotide polymorphisms: (i) SNP1 and (ii) SNP2, (b) determining the genotype of said SNP; (c) selecting a human individual with one of the following genotypes among the SNPs for the clinical trial: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7, or (iii) a human individual not having the following genotype: SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand.

The present invention also describes a method of rejecting a human subject for a clinical trial testing for an FGF-18 compound, said method comprising the steps of: (a) testing a biological sample from a human individual diagnosed with a cartilage disorder to determine at least two of the following single nucleotide polymorphisms: (i) SNP1 and (ii) SNP2, (b) determining the genotype of said SNP; (c) rejecting said clinical trial from a human individual having the following genotype in said SNPs: SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; or culling out the clinical trial from human individuals who do not have any of the following genotypes: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in seq id NO: 7 is A/A in the complementary strand.

In addition to finding that an individual can be classified as hypersensitive, sensitive or non-sensitive according to his/her genotype, it has also been unexpectedly found that the genotype can also predict adverse events, such as AIR. Indeed, further studies and analyses of SNP polymorphisms have shown that binding of markers rs9005 and rs315952 is associated with clinical adverse events, based on MRI data on structural benefit and symptomatic benefit as determined by the WOMAC questionnaire. These SNPs can be used not only as a predictive tool for a patient's response to therapy with FGF-18 compounds, but also as a predictive tool for his/her risk of developing adverse events such as AIR. Thus, the "structural benefit vs. potential adverse effects" profile of FGF-18 therapy can be used to determine a better risk/benefit ratio, i.e. a risk that brings better results and lower side effects to the patient.

In fact, this is based on the following findings: hypersensitives have a higher WOMAC score and a higher likelihood of developing AIR events compared to patients treated with placebo, particularly when using FGF-18 compound, e.g. at a dose of 100 mcg. Also, non-susceptible patients also had high WOMAC scores at any dose compared to patients treated with placebo. It has also been demonstrated that hypersensitizers treated with a lower dose of an FGF-18 compound, e.g., 30mcg, have a lower WOMAC score (i.e., better WOMAC improvement) and a lower likelihood of developing AIR events, as opposed to the results at a dose of 100 mcg. Based on these results, patients can be selected based on their likelihood of responding/not responding to FGF-18 compound therapy, in combination with their level of risk of developing adverse events: non-sensitive subjects may be excluded from treatment for whom there is a high likelihood of non-effect (see selection methods above), while hypersensitive subjects may be treated with alternative treatment regimens.

Accordingly, the present invention also relates to a method of selecting a patient for an alternative treatment regimen with an FGF-18 compound based on the likelihood that the patient has a cartilage disorder that is hypersensitive to FGF-18 compound therapy, comprising: determining nucleic acids of a patient at two polymorphic loci selected from the group consisting of IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at said loci is predictive of the risk of being hypersensitive to a therapy with said FGF-18 compound and allows selection of said patient for an alternative treatment regimen appropriate for said patient in which a lower dose of the FGF-18 compound is administered than in a patient who is predicted to be sensitive but not hypersensitive to said FGF-18 compound therapy.

The present invention also describes a method for selecting patients for an improved treatment regimen using an FGF-18 compound based on the likelihood of an acute inflammatory response in patients with a cartilage disorder treated with said compound, said method comprising the steps of: (a) detecting genotypes (i) SNP1 and (ii) SNP2 from a nucleic acid sample taken from the patient; and (b) when SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/T or the nucleotide sequence set forth in SEQ ID NO: 7 is an a/a combination in the complementary strand, an improved treatment regimen is selected for the patient.

Thus, patients with the genotype IL-1RN rs9005A/G or A/A and IL-1RN rs315952T/C or C/C are predicted to be hypersensitizers and are preferably selected for a dose-reducing alternative treatment regimen of the FGF-18 compound to be administered.

The present invention also describes a method for selecting a patient for an alternative treatment regimen with an FGF-18 compound based on the likelihood of an AIR event occurring when treating a patient with a cartilage disorder with an FGF-18 compound comprising: determining from a nucleic acid sample the genotypes at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at said loci is predictive of the risk of developing an AIR event in response to therapy with said FGF-18 compound, and allowing selection of said patient for an alternative treatment regimen appropriate for said patient in which a lower dose of FGF-18 compound is administered than is administered to a patient (1) who is predicted to be sensitive and (2) who is not at risk of developing an AIR event.

Thus, patients with a genotype of A/G or A/A at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952 are predicted to be hypersensitive and are preferably selected for an alternative treatment regimen in which the dose of FGF-18 compound administered is less than the normal treatment regimen, i.e. the treatment regimen of patients predicted to be sensitive to treatment with an FGF-18 compound but not at risk of developing an AIR event.

FGF-18 compounds are typically administered intra-articularly at a dose of 100mcg per injection, once per week, 3 weeks per treatment cycle. In view of the good results obtained with 30mcg given to hypersensitizers (see examples), the proposed alternative dosing regimen for these patients predicted to be hypersensitizers is intra-articular administration at a dose of 30mcg per injection, once per week, 3 weeks per treatment cycle. It should be understood that while the presently preferred dose is 100mcg per injection, and that a hypersensitizer may reduce to 30mcg per injection, the present invention is not limited to these doses. Thus, the FGF-18 compound may be administered intra-articularly at a dose of between 50 and 300mcg per injection, preferably between 60 and 250mcg, more preferably between 100 and 200 mcg. For hypersensitive patients, the dose can be reduced to, for example, 1/2 or 1/3.

The present invention also includes an FGF-18 compound for use in treating a patient with a cartilage disorder, wherein the patient has any combination of genotypes selected from the group consisting of: (1) G/G at IL-1RN rs9005 and T/C or C/C at IL-1RN rs315952, or (2) A/G or A/A at IL-1RN rs9005 and T/T, T/C or C/C at IL-1RN rs 315952. Furthermore, one patient with at least one A allele of IL-1RN rs9005 and at least one C allele of IL-1RN rs315952T/T is eligible for lower dose therapy with FGF-18 compounds. Thus, preferably, patients who do not meet these criteria (i.e., have genotypes IL-1RN rs9005G/G and IL-1RN rs315952T/T) are rejected for FGF-18 compound therapy (see Table 22).

The invention also relates to a test for selecting a treatment regimen for a human subject suffering from a cartilage disorder, said test comprising: (a) performing at least one genotyping assay on a test sample from a human individual diagnosed with a cartilage disorder to determine the genotype of at least two loci, wherein the at least two loci are: (i) SNP1 and (ii) SNP 2; (b) detecting the presence or absence of a genotype combination selected from the group consisting of: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7, or (iii) SNP1 genotype G/G or a nucleotide sequence set forth in SEQ id no: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; and (c) selecting and optionally administering a treatment regimen comprising an effective amount of the compound based on the knowledge that the genotypic combination (i) and (ii) are associated with a response to an FGF-18 compound when condition (i) or (ii) is detected, and excluding a treatment regimen comprising the compound when condition (iii) is detected based on the knowledge that the genotypic combination (iii) is associated with an inadequate response to therapy with the compound.

The invention also describes a method of treating a human subject having a cartilage disorder comprising administering to the human subject a composition comprising an effective amount of an FGF-18 compound, the human subject being diagnosed with the cartilage disorder and determined to have a combination of Single Nucleotide Polymorphisms (SNPs) selected from: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6, wherein SNP1 is the sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 is a portion of the genomic nucleic acid sequence of an interleukin 1 receptor antagonist (IL-1 RN); and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7, wherein SNP2 is the sequence of SEQ ID NO: 7, wherein SEQ ID NO: 7 is part of the genomic nucleic acid sequence of an interleukin 1 receptor antagonist (IL-1 RN).

The present invention also discloses a method of treating a human subject suffering from a cartilage disorder comprising: (a) testing a biological sample of an individual diagnosed with a cartilage disorder to determine at least two of the following SNP loci: (i) SNP1 and (ii) SNP 2; (b) administering to the patient a therapeutic regimen comprising a composition comprising an effective amount of an FGF-18 compound if one of the following conditions is detected: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or a SNP in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand.

Alternatively, the method of treating a human subject suffering from a cartilage disorder comprises the steps of: (a) testing a biological sample of an individual diagnosed with a cartilage disorder to determine at least two of the following SNP loci: (i) SNP1 and (ii) SNP 2; (b) if SNP1 genotype G/G or the nucleotide sequence in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence shown in SEQ ID NO: 7 is a/a in the complementary strand, administering to said patient a therapeutic regimen comprising a composition comprising an effective amount of an FGF-18 compound.

Alternatively still, the method of selecting an individual having a cartilage disorder treatable with an FGF-18 compound comprises:

(a) obtaining a biological sample from said individual having a cartilage disorder to determine whether said individual's cartilage disorder is treatable with said FGF-18 compound;

(b) contacting the biological sample with at least two oligonucleotides capable of detecting whether the biological sample contains a combination of Single Nucleotide Polymorphisms (SNPs) selected from: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand;

(c) (iii) determining cartilage disease in the individual as treatable with the FGF-18 compound when the combination of (i) or (ii) is detected in the biological sample, and determining cartilage disease in the individual as hypo-responsive or non-responsive to therapy with the compound when neither (i) nor (ii) is detected in the biological sample.

The invention also describes a method of selecting a treatment regimen for an individual having a cartilage disorder comprising: (a) obtaining a test sample from a human subject diagnosed with depression; (b) performing at least one analysis on the test sample to determine parameters of at least two Single Nucleotide Polymorphisms (SNPs), wherein the at least two SNPs comprise the following: (i) SNP1 and (ii) SNP2, (c) using said SNPs to detect the presence of at least one of the following conditions or combinations thereof: SNP1 genotype G/G or in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; SNP1 genotype A/G or AA, or the amino acid sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence shown in SEQ ID NO: 7 to a/a (d) to indicate whether at least one of said conditions is detected in said test sample, and when condition (i) or (ii) is detected, a treatment regimen comprising an FGF-18 compound is selected and optionally administered to said human subject, and when condition (iii) is detected, a treatment regimen comprising said compound is not selected and not administered to said human subject.

In the above methods and trials, patients with the genotype IL-1RNrs 9007(SNP1) A/G or A/A and IL-1RNrs317972(SNP2) T/C or C/C were predicted to be hypersensitizers and preferably selected for alternative treatment regimens in which the dose of FGF-18 compound administered is lower than the normal treatment regimen, i.e. the treatment regimen of patients predicted to be sensitive to treatment with an FGF-18 compound but not at risk of developing an AIR event.

In another embodiment of the invention, a system (and computer-readable medium for a computer system) for acquiring data is also provided. The data is particularly useful for assessing whether a patient is suitable for therapy with an FGF-compound, or for assessing the risk of developing AIR when a patient is treated with an FGF-18 compound, or for monitoring the efficacy of treatment of a patient with an FGF-18 compound. The system may be used during clinical trials when therapy with FGF-18 compounds has to be considered, or when therapy with the compounds is already in progress.

Accordingly, one embodiment of the present invention includes a computer system for obtaining data from at least one test sample taken from at least one individual having a cartilage disorder, the system comprising: (a) at least one determination module configured to receive the at least one test sample and perform at least one analysis on the at least one test sample to determine whether the following conditions exist: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; or (iii) SNP1 genotype G/G or a nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; (b) at least one storage device arranged to store data output by the determination module; and (c) at least one display module for displaying content based in part on the data output by the determination module, wherein the content comprises a signal indicative of the presence of at least one of the conditions, and optionally of the absence of any of the conditions.

The invention also describes a computer system for acquiring data from at least one test sample taken from at least one individual, the system comprising: (a) a determination module configured to receive the at least one test sample and perform at least one genotyping analysis on the at least one test sample to determine genotypes at least two loci, wherein the at least two loci comprise: (i) a SNP1 and (ii) a SNP2, (b) a storage device configured to store output data from the determination module; (c) a calculation module comprising specifically programmed instructions for determining from the output data the presence of any combination of polymorphisms selected from: SNP1 genotype G/G or in SEQ ID NO: 6 is C/C in the complementary strand; and SNP2 genotype T/C or CC, or a nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; SNP1 genotype A/G or AA, or the amino acid sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; (d) a display module for displaying content based in part on the output data from the determination module, wherein the content comprises a signal indicative of the presence of the combination of SNPs (i), (ii), or (iii), and optionally the absence of any one or more of the combinations of SNPs (i), (ii), or (iii).

The computer readable medium may include computer readable instructions recorded thereon to define software modules for implementing the method on a computer. In this case, the computer-readable storage medium may include: (a) comparing data stored on the storage device with reference data to provide an indication of a result of the comparison, wherein the comparison determines whether at least one of the following conditions exists: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7, or (ii) SNP1 genotype a/G or AA, or a variant of the sequence set forth in SEQ ID NO: 6 is T/C or T/T/; and SNP2 genotype T/T or a nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; or (iii) SNP1 genotype G/G or a nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; and (b) an indication of content based in part on the data output from the determination module, wherein the content comprises a signal indicative of the presence of at least one of the conditions, and optionally of the absence of one or more of the conditions.

The computer readable storage medium may be any existing tangible medium that can be accessed by a computer. Computer-readable storage media include volatile and nonvolatile, removable and non-removable tangible media that can be used in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. Computer-readable storage media include, but are not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM (electrically erasable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVD (digital versatile disc) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and tangible media that can be used to store the desired information and that can be accessed by a computer, and any suitable combination of the foregoing.

The computer-readable data recorded on one or more computer-readable media may define instructions, e.g., as part of one or more programs that, when executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations, and combinations thereof. These indications may be written in any of a variety of programming languages, such as Java, J #, Visual Basic, C, C #, C + +, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof. The computer readable medium recording these indications may be present on one or more components of the system or the computer readable storage medium may be distributed among one or more of the components.

The computer readable medium can be removable so as to load the instructions stored therein onto any computer resource to implement aspects of the present invention described herein.

The information determined in the determination module may be read by a storage device. The storage device is configured or arranged to record expression level or protein level information thereon. This information may be provided in digital form and may be electronically transmitted and read, for example, via the internet, diskette, via USB (universal serial bus), or via any other suitable communication means.

In the context of the present invention as a whole, e.g. in the context of any of the methods, uses, assays or kits according to the present invention, preferred FGF-18 compounds are truncated FGF-18, e.g. Sprifermin, while preferred cartilage diseases are selected from the group consisting of osteoarthritis, cartilage damage, fractures affecting articular cartilage or surgical procedures affecting articular cartilage (e.g. microfracture surgery).

It is to be understood that in the context of the present invention as a whole, for example in the context of any of the methods, uses, assays, computer systems or kits according to the present invention, it is desirable to obtain a nucleic acid sample (or test sample) from an individual by a method such as blood or saliva collection prior to determining the genotype at a locus. Preferably, the nucleic acid sample is a DNA sample.

Any of the methods, uses, assays, kits and other computer systems described in accordance with the present invention will test, test and/or treated individuals having cartilage disease as candidates for therapy with FGF-18 compounds (e.g., spifermin). In a preferred embodiment, the individual is diagnosed as having, or having symptoms of, a cartilage disorder.

It is also to be understood that in the context of the present invention as a whole, the determination may be made in the complementary sequences of IL-1RN rs9005 and IL-1RNrs 315952. Thus, according to the invention as a whole, for example, in the context of any of the methods, uses, assays, computer systems or kits according to the invention, the presence of a genotype of C/C on the complementary sequence of IL-1RN rs9005 and a/a on the complementary sequence of IL-1RN rs315952 is predictive of no response or low response (i.e. insensitivity) to therapy with an FGF-18 compound. Conversely, the presence of a genotype of T/C or T/T on the complementary sequence of IL-1RN rs9005 and a/G or G/G on the complementary sequence of IL-1RN rs315952 is predictive of a high response (i.e. high sensitivity) to therapy with FGF-18 compounds. The genotype may also serve as a marker for the likelihood that a patient will develop an AIR event when treated with the FGF-18 compound. Other genotypes at these loci are predictive of moderate sensitivity (i.e., C/C on the complement of IL-1RN rs9005 and A/G or G/G on the complement of IL-1RN rs315952, or T/C or T/T on the complement of IL-1RN rs9005 and A/A on the complement of IL-1RN rs 315952).

In another embodiment, the invention includes a kit comprising a device for performing the above method and instructions for use. In particular, the kit comprises at least one pair of specific primers or probes for detecting the presence of the allele. Preferably, the kit comprises two pairs of specific primers or probes for genotyping the alleles at the loci IL-1RN rs9005 and IL-1RN rs 315952.

The kit may include an oligonucleotide array having a plurality of oligonucleotide probes immobilized thereon, the oligonucleotide probes detecting no more than 20 Single Nucleotide Polymorphisms (SNPs), the SNPs including: (i) SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; or (ii) SNP1 genotype A/G or AA, or the nucleotide sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/T or in SEQ ID NO: 7 is A/A in the complementary strand; or (iii) SNP1 genotype G/G or a nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; optionally a container containing a detectable label that can bind to a nucleotide molecule derived from a test sample from an individual diagnosed with a cartilage disorder; and at least one reagent.

Alternatively, the oligonucleotide array having a plurality of oligonucleotide probes immobilized thereon detects no more than 17 Single Nucleotide Polymorphisms (SNPs), no more than 10 Single Nucleotide Polymorphisms (SNPs), or no more than 7 Single Nucleotide Polymorphisms (SNPs).

Also described in the context of the present invention is a kit comprising: a plurality of oligonucleotide primers or primer sets, each of which binds to and detects no more than one specific allele of no more than 20 Single Nucleotide Polymorphisms (SNPs), wherein each oligonucleotide primer grouping that binds to a specific allele of a SNP is labeled with a different indicator, wherein the SNPs comprise the following SNPs: SNP1 genotype G/G or in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; SNP1 genotype A/G or AA, or the amino acid sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/T or the nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand; and at least one reagent.

Alternatively, each of the plurality of oligonucleotide primers or primer sets binds to and detects no more than one specific allele of no more than 17 Single Nucleotide Polymorphisms (SNPs), no more than one specific allele of no more than 10 Single Nucleotide Polymorphisms (SNPs), no more than one specific allele of no more than 7 Single Nucleotide Polymorphisms (SNPs).

In another embodiment, the present invention discloses a kit for selecting a treatment regimen for an individual having a cartilage disorder comprising at least one reagent for determining the presence or absence of the following SNPs in a test sample from a human individual diagnosed with the cartilage disorder: SNP1 genotype G/G or in SEQ ID NO: 6, and SNP2 genotype T/C or CC, or the nucleotide sequence set forth in SEQ ID NO: 7 is A/G or GG in the complementary strand; SNP1 genotype A/G or AA, or the amino acid sequence set forth in SEQ ID NO: 6, and SNP2 genotype T/T or the nucleotide sequence set forth in SEQ ID NO: 7 is A/A in the complementary strand; SNP1 genotype G/G or the nucleotide sequence set forth in SEQ ID NO: 6 and SNP2 genotype T/T or the nucleotide sequence in SEQ ID NO: 7 is A/A in the complementary strand.

In certain embodiments, the oligonucleotides in the kit are allele-specific probes or allele-specific primers. In other embodiments, the kit comprises a primer extension oligonucleotide. In other embodiments, the set of oligonucleotides is a combination of allele-specific probes, allele-specific primers, or primer extension oligonucleotides.

The composition and length of each oligonucleotide in the kits of the invention will depend on the nature of the genomic region containing the genetic marker of the invention, and the type of assay to be performed using the oligonucleotide, and will be readily determined by those skilled in the art. For example, the polynucleotide used in the assay may constitute an amplification product, and thus the desired specificity of the oligonucleotide is for hybridization to a target region in the amplification product, rather than genomic DNA isolated from the individual. In a preferred embodiment, each oligonucleotide in the kit is fully complementary to its target region. An oligonucleotide molecule is said to be "perfectly" or "completely" complementary to another molecule if each nucleotide of the molecule is complementary to a nucleotide at a corresponding position on the other nucleic acid molecule. While it is preferred to use fully complementary oligonucleotides to detect polymorphisms, incomplete complementarity is also contemplated, provided such incomplete complementarity does not prevent specific hybridization of the molecule to the target region described above. For example, the 5' end of one oligonucleotide primer may have a non-complementary segment, but the remainder of the primer is fully complementary to the target region. Alternatively, non-complementary nucleotides may be inserted into the probe or primer, so long as the resulting probe or primer is still capable of specifically hybridizing to the target region.

In certain preferred embodiments, each oligonucleotide in the kit specifically hybridizes to its target region under stringent hybridization conditions. Stringent hybridization conditions will be sequence dependent and will vary from case to case. Generally, stringent conditions are selected to be about 5 ℃ lower than the thermal melting point (Tm) for the specific sequence at a specific ionic strength and pH. The Tm is the temperature at equilibrium (under specified ionic strength, pH and nucleic acid concentration) at which 50% of a probe complementary to a target sequence hybridizes to the target sequence. Since the target sequence is usually in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, for short oligonucleotide probes (e.g., 10 to 50 nucleotides), stringent conditions include a salt concentration of at least about 0.01 to 1.0M sodium ion concentration (or other salt), a pH of 7.0 to 8.3, and a temperature of at least about 25 ℃. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For example, for allele-specific probe hybridization, 5 XSSPE (750mM sodium chloride, 50mM sodium phosphate, 5mM EDTA, pH7.4) and a temperature of 25-30 ℃ are suitable conditions.

The oligonucleotides in the kits of the invention may consist of any phosphorylated form of ribonucleotides, deoxyribonucleotides and acyclic nucleotide derivatives, as well as other functionally equivalent derivatives. Alternatively, the oligonucleotide may have a phosphate-free backbone, which may consist of linkages such as carboxymethyl, iminodiacetate, carbamate, polyamide [ Peptide Nucleic Acid (PNA) ]. The oligonucleotides may be prepared by chemical synthesis by any suitable method known in the art, or may be taken from a biological sample, for example by restriction digest. The oligonucleotide may comprise a detectable label according to any technique known in the art, including the use of radioactive labels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags, and the like. The oligonucleotides in the kit may be produced and sold as analyte-specific reagents (ASR) or be part of an approved diagnostic device.

In other preferred embodiments, the kit includes instructions describing various methods of using the kit to detect the presence or absence of a genetic marker of the invention. In a preferred embodiment, the set of oligonucleotides in the kit is allele-specific oligonucleotides. The term allele-specific oligonucleotide (ASO) as used herein refers to an oligonucleotide that is capable of specifically hybridizing to one allele of a genetic marker in a target region containing the genetic marker, but not to the same region containing a different allele, under sufficiently stringent conditions. One skilled in the art will appreciate that allele specificity varies with a variety of stringency conditions that are readily optimized, including salt and formamide concentrations and temperatures for hybridization and washing steps. Typically, an ASO is perfectly complementary to one allele, but contains a single mismatch to the other allele. In an ASO probe, the single mismatch is preferably located at a central position of the oligonucleotide probe (e.g., the seventh or eighth position of a pentadecamer, the eighth or ninth position of a hexadecamer, the tenth or eleventh position of an icosamer) when the oligonucleotide probe binds to a genetic marker in the target region. The single mismatch in the ASO primer is located at the 3 'terminal nucleotide, or preferably at the second nucleotide of the 3' terminal. The present invention contemplates ASO probes and primers that hybridize to the coding or non-coding strand.

In other preferred embodiments, the kit comprises a pair of allele-specific oligonucleotides for the genetic marker to be detected in the present invention, wherein one oligonucleotide has specificity for one allele and the other oligonucleotide has specificity for the other allele. In these embodiments, the oligonucleotide pairs may be of different lengths or have different detectable labels so that the user of the kit determines which allele-specific oligonucleotide specifically hybridizes to the target region and thereby determines which allele is present at the detected marker locus of the individual.

In other preferred embodiments, the oligonucleotides in the kit are primer extension oligonucleotides. The termination mixture used to terminate polymerase mediated extension of any of these oligonucleotides is selected to terminate extension of the oligonucleotide, the termination occurring at or one base after the genetic marker, depending on the different nucleotides at the marker locus.

The method and kit according to the invention can be used for clinical diagnostic applications. However, the term "diagnosis" as used herein is not limited to clinical or medical use, and the diagnostic methods and kits of the present invention may be used in any research application and clinical trial where it is desired to test an individual for the presence or absence of any genetic marker described herein.

In the context of the present invention, the presence or absence of a particular allele or pair of alleles in an individual at the locus of a genetic marker of the present invention can be detected by any technique known in the art, including sequencing, pyrosequencing, selective hybridization, selective amplification, and/or mass spectrometry including matrix-assisted laser desorption ion flight mass spectrometry. In one embodiment, the variation is detected by selective nucleic acid amplification using one or more specific primers. Variations are detected by selective hybridization using one or more specific probes.

Other techniques include gel electrophoresis based genotyping methods such as PCR in combination with Restriction Fragment Length Polymorphism (RFLP) analysis, multiplex PCR, oligonucleotide ligation analysis, and microsequencing; fluorescent dye-based genotyping techniques such as oligonucleotide ligation analysis, pyrosequencing, single base extension fluorescence detection, homogeneous liquid phase hybridization (e.g., TaqMan), and molecular beacon genotyping; sequencing-based techniques such as Sanger sequencing and next generation sequencing platforms; rolling circle amplification and infection detection, and DNA chip-based microarray and mass spectrum genotyping techniques. Protein expression analysis is known in the art and includes 2-dimensional gel electrophoresis, mass spectrometry, and antibody microarrays. Sequencing can be performed using techniques known in the art, for example using an automated sequencer. The entire gene may be sequenced, or more preferably, specific regions thereof, typically regions known or suspected to contain deleterious mutations or other variations, are sequenced. Amplification can be performed according to various techniques known in the art, such as Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), and Strand Displacement Amplification (SDA). These techniques can be carried out using reagents and protocols available on the market. The preferred technique is allele specific PCR.

Other embodiments of the invention within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. These descriptions and embodiments are to be construed as illustrative, and the scope and spirit of the invention are defined by the claims, which follow the examples.

Drawings

General description: in the figures, 1) the terms TT, CC, GG or AA are understood to be T/T, C/C, G/G or A/A, 2) the term CTA is understood to be C-T-A.

FIG. 1: tissue map of the IL1R1-IL1A-IL1B-IL1RN gene cluster. Rs315952 and Rs9005 are both located in the last 1L1RN exon. Although only 1107bp were between them, the two SNPs were not inherited simultaneously (i.e., not in linkage disequilibrium). IL1RN-rs9005 is located in the 3' UTR region and is associated with a transcription factor (ChIP-seq sequence: FOSL2) and a DNase cluster (regulatory regions and promoters are usually sensitive to DNase). IL1RN-rs315952 is a coding silent SNP (i.e. does not result in amino acid changes).

FIG. 2: patients were stratified according to the presence or absence (as determined by at least one copy) of the C-T-A haplotype. Y-axis represents total cartilage volume (unit: mm) at week 52³) A change in (c). Each point corresponds to an individual, circles indicate individuals in which AIR did not occur, and crosses indicate individuals in which AIR occurred. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 3: patients were stratified according to the presence or absence (as determined by at least one copy) of the C-T-A haplotype. The Y-axis represents the change in WOMAC total score at week 52. Each point corresponds to an individual, circles indicate individuals in which AIR did not occur, and crosses indicate individuals in which AIR occurred. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 4: total cartilage volume at week 52 (units: mm) stratified by dosing schedule and stratified by genotype at rs315952 and rs9005³) A change in (c). Each point corresponds to an individual, circles indicate individuals in which AIR did not occur, and crosses indicate individuals in which AIR occurred. MRI data from the MAD010 cohort showed abnormal variability and was therefore not included in any analysis.

FIG. 5: change in WOMAC total score at week 52 stratified by dosing schedule and genotypic stratification at rs315952 and rs 9005. Each point corresponds to an individual, circles indicate individuals in which AIR did not occur, and crosses indicate individuals in which AIR occurred.

FIG. 6: patients were stratified by the presence or absence of the "rs 9005G/G rs 315952T/T" genotype. The Y-axis represents the baseline absolute WOMAC total score. Each point corresponds to an individual, circles indicate individuals with a Kellgren-Lawrence rating equal to 2, and crosses indicate individuals with a Kellgren-Lawrence rating equal to 3. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 7: patients were stratified by the presence of the "rs 9005A vector rs315952C vector" genotype. The Y-axis represents the baseline absolute WOMAC total score. Each point corresponds to an individual, circles indicate individuals with a Kellgren-Lawrence rating equal to 2, and crosses indicate individuals with a Kellgren-Lawrence rating equal to 3. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 8: patients were stratified by the presence or absence of the "rs 9005G/G rs 315952T/T" genotype. The Y-axis represents the baseline absolute total cartilage volume (mm)³). Each point corresponds to an individual, circles indicate individuals with a Kellgren-Lawrence rating equal to 2, and crosses indicate individuals with a Kellgren-Lawrence rating equal to 3. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 9: patients were stratified by the presence of the "rs 9005A vector rs315952C vector" genotype. The Y-axis represents the baseline absolute total cartilage volume (mm)³). Each point corresponds to an individual, circles indicate individuals with a Kellgren-Lawrence rating equal to 2, and crosses indicate individuals with a Kellgren-Lawrence rating equal to 3. The indicated p-values were obtained by non-parametric one-factor tests (rank sum test).

FIG. 10: difference in WOMAC total score from baseline for all individuals who were not genotyped. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error.

FIG. 11: the difference between the WOMAC total score of individuals determined as sensitizers and super sensitizers from baseline based on the rs9005 and rs315952 genotypes. The "treatment" group corresponds to individuals from the MAD100 group having a genotype that can be determined to be sensitive individuals. Individuals from the MAD030 cohort with a genotype that can be determined to be hypersensitive individuals are also included in this "treatment" group. The "placebo" group includes placebo-controlled individuals with genotypes corresponding to either the sensitive or the hypersensitive. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error.

FIG. 12: difference from baseline in WOMAC total score for individuals with genotypes corresponding to non-susceptible. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error.

FIG. 13: all of them being non-genotypicTotal cartilage volume (mm) of an individual³) Difference from baseline. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error. MRI data from the MAD010 cohort showed abnormal variability and was therefore not included in any analysis.

FIG. 14: total cartilage volume (mm) of individuals determined as being susceptible and hypersensitive based on the rs9005 and rs315952 genotypes³) Difference from baseline. The "treatment" group corresponds to individuals from the MAD100 group having a genotype that can be determined to be a susceptible person. Individuals from the MAD030 cohort with a genotype that can be determined to be hypersensitizers are also included in this "treatment" group. The "placebo" group includes placebo-controlled individuals with genotypes corresponding to either the sensitive or the hypersensitive. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error. MRI data from the MAD010 panel failed quality control and was therefore not included in any analysis.

FIG. 15: total cartilage volume (mm) of individuals with genotypes corresponding to non-susceptible persons³) Difference from baseline. The curve corresponds to the mean difference from the baseline and the error bars correspond to the mean standard error. MRI data from the MAD010 cohort showed abnormal variability and was therefore not included in any analysis.

Fig. 16(a) - (h): full-length amino acid and nucleic acid sequences corresponding to "SEQ ID NOs" mentioned in the present application are listed.

Description of sequences：

SEQ ID NO. 1: the amino acid sequence of native human FGF-18.

SEQ ID NO. 2: the amino acid sequence of a recombinant truncated FGF-18 (trFGF-18).

SEQ ID NO. 3: IL1RN gene

SEQ ID NO. 4: IL1RN rs9005 locus

SEQ ID No. 5: IL1RN rs315952 locus

SEQ ID NO. 6: specific region of the IL1RN rs9005 locus (corresponding to nucleotides 415 to 466 of SEQ ID NO. 4), wherein N is A or G

SEQ ID NO. 7: specific region of the IL1RN rs315952 locus (corresponding to nucleotides 415 to 466 of SEQ ID NO. 5), wherein N is C or T

SEQ ID NO. 8: rs315952 primer 1

SEQ ID NO. 9: rs315952 primer 2

SEQ ID NO. 10: rs9005 primer 1

SEQ ID NO. 11: rs9005 primer 2

Examples

1. Background of genotyping:

the level of cartilage volume increase and the associated risk of developing adverse effects in response to spifermin treatment of cartilage disease may be associated with specific genetic variations in one or more genes, respectively. In this study, the relationship of genes containing variations to disease or response to therapy was explored focusing on candidate genes selected based on their physiological function of the encoded protein and their potential impact in cartilage disease or response to spifermin therapy. Table 1 lists the list of candidate SNPs that have been tested for selection.

Response to Sprifermemin therapy was measured as the difference in cartilage volume from baseline 1 year after initiation of treatment with Sprifermemin.

It should be noted that candidate and genome-wide scanning SNP markers that meet any of the following criteria were not further analyzed:

rare variant SNPs in the PGx ITT population: the Minor Allele Frequency (MAF) of the candidate SNPs and genome-wide scan SNPs was < 10%.

Suspected genotyping quality, measured as high data loss rate (. gtoreq.5%).

Significant deviation from Hardy-Weinberg equilibrium (Bonferroni adjusted p-value for candidate SNPs less than 5%, or FDR for genome-wide scanning SNPs (i.e., Benjamini-Hochberg adjusted p-value) less than 20%).

Individuals with gender differences between clinical databases and the gender predicted from genome-wide scanning SNP data (chromosome X) were excluded.

The candidate genes selected are known to be closely related to cartilage diseases such as osteoarthritis. The objective of this study was to investigate whether the level of response in cartilage disease (i.e. the increase in cartilage volume in response to spifermin therapy and/or the occurrence of adverse events) is associated with a particular DNA variation or pattern of variation. The presence of such a correlation may reveal that the gene carrying the identified variation or one or more genes located in the vicinity of the variation may be a susceptibility gene.

2 materials and methods

2.1FGF-18 Compounds

The FGF-18 compound for treatment in the examples of the invention is Sprifermin. As defined in the section "Definitions", it is a truncated form of FGF-18.

2.2 sample Collection and Dual recoding

Blood samples were collected from patients enrolled in study 28980 (randomized, double-blind, placebo, multicenter, single and multiple ascending dose studies on Sprifermin intra-articular administration to patients with primary knee osteoarthritis and who were predicted to not require knee surgery within one year).

To meet the requirements of pharmacogenomics (PGx) Informed Consent (ICF) covering DNA analysis, all samples were double-coded by Biobank (Merck Serono, Geneva) to ensure sufficient individual anonymity. Biobank provides the biomarker data management group with a double key encoding in the form of a flat file containing PGx ID and individual ID for each individual. Additional certifications were made to ensure that all DNA analyses were performed on individuals who agreed to the PGx study.

2.3DNA sample extraction, amplification, fragmentation and labelling

The analysis was performed on DNA extracted from blood. A total of 140 blood samples were collected. Wherein 3 samples were destroyed by the genomic laboratory due to patient withdrawal of their consent during the study; the remaining 137 analyzed DNAs corresponded to 137 patients. Therefore, 137 patients were genotyped and available for correlation studies.

Genomic DNA was extracted from EDTA Blood samples using Qiagen extraction Kit (QIAamp DNA Blood Maxi Kit). After extraction, the absorbance of the samples at wavelengths of 260 nm and 280 nm was measured and subjected to agarose gel electrophoresis to evaluate the quality and quantity of the genomic DNA samples.

On each plate, genomic DNA samples were digested with Nspl and Sty restriction endonucleases, ligated with specific aptamers (Nsp or Sty), and processed in parallel until Polymerase Chain Reaction (PCR). PCR triple amplifies Styl reaction products and quadruple amplifies Nspl reaction products for higher efficiency. All PCR products were pooled, purified, quantified, fragmented and labeled.

The PCR amplification step was assessed using agarose gel electrophoresis. DNA quantification step was measured using a spectrophotometer and DNA fragmentation step was assessed using agarose gel electrophoresis. The average length of the DNA fragments should be less than 180 bp.

2.4DNA microarray technology (Whole genome scanning)

Genome wide scans (without pre-set hypothesis) were performed using the Affymetrix genome wide SNP 6.0 assay. The Affymetrix technology is based on DNA chips and can genotype approximately 906600 Single Nucleotide Polymorphisms (SNPs) per patient. SNPs are randomly distributed on all chromosomes and serve as tag markers for the corresponding genomic regions. The specific steps and the scheme are carried out according to the PGX Affymetrix whole genome SNP5.6/6.0 technology.

The labeled products from each sample were hybridized on the Affymetrix whole genome SNP 6.0 gene chip. Two groups used two batches of chips.

After hybridization and staining, the Affymetrix gene chip was scanned to generate an image Data (DAT) file. The AGCC software then automatically arranges the grid on a DAT file and calculates a cell intensity data (CEL) file. The CEL data is then passed to genotyping module software to generate probe analysis (CHP) data.

Analysis Quality Control (QC) was performed using genotyping module software to evaluate dynamic model QC (dm) detection rate analysis of the 3022 SNP containing subsets after scanning the chip.

DM detection rate measures the consistency of intensity in each SNP with 4 possible genotyping states (null, AA, AB and BB). It provides an estimate of the overall quality of the data sample before performing a comprehensive aggregate analysis. It is based on QC detection rate.

QC detection rate (QC CR) is closely related to aggregation performance and is an efficient single-sample matrix for determining which samples should be used in the downstream aggregation. The fixed threshold for the genome-wide SNP 6.0 array was > 86%. In addition to QC CR, another algorithm was developed for SNP 6.0 arrays. This new algorithm is Contrast (Contrast) QC. Contrast QC is a matrix that reflects the ability of an experiment to resolve SNP signals into three genotype clusters. It measures the ability to separate allele intensities into three clusters in the "contrast space". The contrast space is a projection of the two-dimensional allele intensity space in an informative single dimension. The default threshold for each sample is > -0.4. The results of QC are automatically displayed in an intensity QC table. Samples that passed the QC threshold (detection rate > 86% and QC >0.4) were labeled "inclusion" while samples that did not pass QC (detection rate < 86% or QC <0.4) were labeled "culled". The genomic DNA samples studied passed all QCs.

2.5TaqMan SNP genotyping (candidate Gene)

TaqMan SNP genotyping was performed to detect markers picked based on literature information. A total of 19 SNPs distributed over 8 candidate genes were picked and performed in two segments (see tables 2a and 2 b). In the TaqMan SNP genotyping assay, a fragment of about 100bp is amplified using two locus specific PCR primers surrounding the SNP. Then, the two allele-specific probes hybridize to their specific SNP sequences, respectively (see, for example, Table 3). The 5' end of each probe is labeled with a fluorescent reporter dye (FAM) or VIC reporter dye. The 3' end of each probe also has a non-fluorescent quenching dye MGB. If the target sequence of the allele-specific probe is amplified during each PCR cycle, the probe will hybridize and extend to the DNA during the annealing step. When a DNA polymerase is contacted with the hybridized probe, the reporter dye of the probe is cleaved from the probe leaving the quencher dye. At each cycle of PCR, the reporter dye is cleaved from one or both of the allele-specific probes such that the fluorescence intensity increases exponentially. When the PCR was completed, the total fluorescence of each sample was measured on ABI 9700(384 well format). If fluorescence is measured from only one probe, the sample is homozygous for the allele. If fluorescence from both allele-specific probes is measured, the sample is heterozygous for both alleles. If the probe is not hybridized, the fluorescence of the dye is "quenched" or reduced by the quenching dye, and thus the measured fluorescence will be very low, indicating a failure of genotyping.

The experimental scheme is thatThe SNP genotyping data sheet is described in detail.

Section 1: by 17SNP assay DNA samples were genotyped (see Table 2 a).

Section 2: then 2 are usedSNP assay DNA samples were genotyped (see Table 2 b).

At each oneIn the SNP test, NTC clusters are specific, all NTC are undetermined, three different sample clusters exist, genotyping is automatically specified, and the detection rate is set to be higher than 85%.

19 are provided withThree fractions of each of the SNP trials met the acceptance criteria.

SNP screening

Candidate and genome-wide scan SNP markers that meet any of the following criteria were not further analyzed:

rare variant SNPs in the PGx ITT population: the Minor Allele Frequency (MAF) of the candidate SNPs and genome-wide scan SNPs was < 10%.

Suspected genotyping quality, measured as high data loss rate (. gtoreq.5%).

Significant deviation from Hardy-Weinberg equilibrium (Bonferroni adjusted p-value for candidate SNPs less than 5%, or FDR for genome-wide scanning SNPs (i.e., Benjamini-Hochberg adjusted p-value) less than 20%).

Individuals with gender differences between clinical databases and the gender predicted from genome-wide scanning SNP data (chromosome X) were excluded.

2.7. Correlation test

For correlation tests, the genotype data is encoded as the presence/absence of the SNP minor allele (i.e., the homozygote of the major allele is compared to at least one copy of the minor allele).

2.7.1. Correlation with Acute Inflammatory Response (AIR)

In these analyses, only individuals treated with 100mcg FGF-18 dose were used. In single label analysis, two methods are used: fisher's exact test and multivariate linear model (i.e., AIR status-SNP + Kellgren Lawrence ranking [ 2; 3] + gender [ female; male ] + age [ < 65; > 65] + BMI [ < 30; > 30.) in this model, the significance of each term in the model was assessed as a type III multivariate analysis.

2.7.2. Correlation with WOMAC total score and total cartilage volume

The correlation between WOMAC total score and the difference in total cartilage volume from baseline at week 52 (day of termination) was evaluated using the following linear model:

grade (change in endpoints) -branch [ placebo-controlled, individuals treated with, e.g., FGF-18100mcg dose ] + genotype group + Kellgren Lawrence ranking [ 2; 3] + gender [ female; male ] + age [ < 65; not less than 65] + BMI [ < 30; not less than 30 percent. Significance was assessed for each term in the model by type III variational analysis, with the significance threshold set at alpha-5%.

2.7.3. Correlation between assigned genotype groups and Kellgren Lawrence ranking

To test whether a given genotype group (e.g., individuals with the "IL-1 RN rs9005G/G and IL-1RN rs 315259T/T" genotypes) is abundant or deficient in individuals with severe osteoarthritis (i.e., Kellgren Lawrence grade 3), Fisher's exact test was used and independence tests were performed from the following linked list:

grade 3

Grade 3

Number of individuals in a given genotype group
			Number of individuals in the remaining genotype groups

All available patients for any dosing regimen (including placebo) were included in the analysis. P-values were calculated using a two-sided test, with significance set at alpha-5%. Odds ratios and their 95% confidence intervals were also calculated.

2.8. Haplotype analysis

The genotype data from the SNPs rs419598, rs315952, rs9005 are phased (using MACH software version 1.0.18.C, Li Y et al, 2010) to infer the presence or absence of the C-T-a haplotype in the individual. The following MACH parameters were used: "- - -rounds 50- -states 200- -phase". Fisher's exact test was used to test for correlation with AIR (significance threshold was set at alpha-5%).

2.9. Combinatorial analysis between candidate SNPs

Preliminary correlation analysis (data not shown) found that the rs9005SNP was significantly associated with AIR. A combination analysis (i.e. ectopic dominant) was performed to test whether IL1RN rs9005 in combination with another SNP selected from a list comprising about 120 candidate SNPs could better predict AIR (see table 1). This analysis was performed using logistic regression with the following model:

AIR status-rs 9005 another SNP + Kellgren Lawrence ranking [ 2; 3] + gender [ female; male ] + age [ < 65; not less than 65] + BMI [ < 30; not less than 30 percent.

Significance of each term in the model was assessed by type III variate analysis. Multiple assays were performed by adjusting the p-value of the interaction according to the Benjamini-Hochberg program (Benjamini and Hochberg,1995, J.of the Royal Statistical Society Series B (57):289), with a significance threshold set at 5% FRD. Ectopic dominant effects were confirmed according to the statistical method described in (Wirapati et al, 2011).

2.10. Expression matrix for predicting AIR

The performance matrix for the predicted AIR is from the corresponding table of ranks. These matrices include sensitivity, specificity, accuracy, precision, negative predictive value, and F1 values (i.e., the harmonic mean of precision and recall).

3. Results

3.1. Predictive analytics

Combinatorial analysis determined that only one combination (IL1RN rs9005 and IL1RN rs315259) was significantly correlated with AIR (multivariate linear model FDR 0.0187, Fisher exact test p-value 0.0018, ratiometric ratio 18.82[2.25-260.03 ]). Tables 5 and 6 show the list table and the predictive representation matrix, respectively. The combination of rs9005 and rs315259 (Table 6) predicts better performance of AIR compared to the C-T-A haplotype (Table 8, see also the tabulated column in Table 7). The combination of IL1RN rs9005 and IL1RNrs315259 has strong specificity (94.44%) and negative predictive value (89.47%), i.e. these biomarkers are strongly expressed in identifying individuals who do not develop AIR. In addition, this combination also revealed stratification of total cartilage volume (fig. 4) and WOMAC total score (fig. 5). In contrast, the C-T-A haplotype failed to achieve this stratification in clinical outcome (FIGS. 2 and 3). In fact, C-T-a cannot achieve stratification of patients by changes in total cartilage volume (fig. 2) or changes in WOMAC total score (fig. 3). It can therefore be determined that the C-T-A haplotype is not well predictive of response to drug therapy, particularly anabolic drugs such as Sprifermin.

3.2. Prognostic assay

Placebo-controlled individuals with genotypes for "IL 1RN rs9005G/G and IL1RN rs 315259T/T" were determined to have significantly higher total cartilage volume than treated individuals from the same genotype group. To follow this result, the change in WOMAC total score and the change in total cartilage volume in placebo-controlled individuals were modeled as follows:

rank (change in endpoint) -genotype group + Kellgren Lawrence ranking [ 2; 3] + gender [ female; male ] + age [ < 65; not less than 65] + BMI [ < 30; not less than 30 percent.

No significant difference was found between the WOMAC total scores of individuals of the four different genotype groups (p-value 0.63, table 10). However, a significant difference was found between the changes in total cartilage volume (p-value 0.02, table 9). Individuals of the "IL 1RN rs9005G/G and IL1RN rs 315259T/T" genotype groups had significantly higher increases in total cartilage volume compared to individuals of the remaining genotype groups.

Independence tests between the Kellgren Lawrence ranking and individuals of the indicated genotype group showed that the "IL 1RNrs9005G/G and IL1RN rs 315259T/T" genotype group significantly lacked individuals with a Kellgren Lawrence ranking of 3 (Fisher exact test p-value ═ 0.0179, table 11). The corresponding odds ratio was 0.306 (95% confidence interval [0.096, 0.885 ]). This shows that individuals of the "IL 1RN rs9005G/G and IL1RN rs 315259T/T" genotype group are classified as less severe osteoarthritis compared to individuals of other genotype groups. Supporting this result, individuals of the "IL 1RN rs9005G/G and IL1RNrs 315259T/T" genotype group had a slightly lower baseline WOMAC total score than individuals of the other genotype groups (rank and p value 0.0927, see fig. 6). Furthermore, individuals of the "IL 1RN rs9005G/G and IL1RN rs 315259T/T" genotype group had significantly higher baseline total cartilage volume compared to individuals of other genotype groups (rank and p-value ═ 0.0204, see fig. 8).

Interestingly, the proportion of individuals ranked 3 by Kellgren Lawrence did not differ between individuals in the "IL 1RN rs9005A vector and IL1RN rs315259C vector" genotype group (also referred to as hypersensitizers) and the remaining genotype group (Fisher exact test p-value 0.2736, odds ratio 1.693[0.637, 4.769], table 12). The hypersensitive group is therefore not enriched in individuals with severe osteoarthritis. Further supporting this conclusion is the fact that: hypersensitive individuals and others had similar baseline WOMAC total scores and baseline total cartilage volumes (see figures 7 and 9).

C-T-A haplotype analysis did not reveal differences in the ratios of individuals with a Kellgren Lawrence ranking of 3 and at least one copy of the C-T-A haplotype (Fisher mut mutexact test p-value ═ 1).

3.3. Clinical results Using the genetic diagnostic test proposed by the present invention

Without any genetic stratification, the clinical outcome of FGF-18 therapy is as follows: 1) the total cartilage volume of the treated individuals (MAD100) was significantly increased compared to placebo controls (i.e.: cartilage repair) (p-value 0.0157); 2) the improvement in WOMAC total score in treated individuals (MAD100) was slightly reduced compared to placebo control (p-value 0.1044); 3) AIR occurs in 20% of treated individuals. Table 13 summarizes these results, and the detailed results are listed in tables 14 and 15. Fig. 10 and 13 are also provided to visualize the data. It should be understood that FIGS. 10-15 do not correspond to the multivariate linear model used in the analysis. These figures are provided only to aid in understanding the results.

The diagnostic tests proposed by the invention (table 4) are aimed at:

1. sensitizers are identified and treated with the FGF-18 doses (e.g., 100mcg) proposed by the present invention

2. Hypersensitizers are identified and treated with lower doses of FGF-18 (e.g., 30mcg)

3. Non-sensitized persons were identified and rejected for FGF-18 therapy.

Retrospectively, the clinical outcome of individuals on selected FGF-18 therapy was:

1. total cartilage volume was significantly increased in treated individuals (sensitizers from MAD100 population + hypersensitizers from MAD030 population) compared to the corresponding placebo controls (p-value ═ 0.016 table 18, fig. 14). Simulation studies (bootstrap) showed that the increase in cartilage volume was significantly higher than that obtained without the use of diagnostic tests (p-value <1E-4)

2. The improvement in WOMAC total score was similar between treated individuals and placebo controls (p-value 0.6603, table 17, fig. 11).

3. AIR occurred in 11.43% of treated individuals (table 6).

In contrast, the clinical outcome for individuals identified as non-sensitizers is as follows:

1. the increase in total cartilage volume was significantly smaller in treated individuals (non-sensitized from the MAD100 population) compared to the corresponding placebo controls (p-value ═ 0.0289, table 21). The results for the individual MAD030 population were similar to the individual MAD100 population (fig. 15). Thus, none of the studied doses showed improvement relative to placebo control.

2. Although the p-value of the multivariate linear model was not significant (p-value 0.3068, table 20), the total WOMAC score was not improved in treated individuals (median change-1) and in placebo controls (median change-39). The results for the individual MAD010 and MAD030 populations are similar to the individual MAD100 population (fig. 12). Thus, none of the studied doses showed improvement relative to placebo control.

3. AIR occurred in 22.22% of treated individuals (table 19).

Watch (A)

Table 1: candidate SNP List

Table 2 a: detailed identification of TaqMan SNP selected in section 1

Gene symbol

Rs mark

Test mark

NCBI et al

Type of test

			Site gene
					IL1RN	rs9005	C___3133528_10	A/G	Functional testing
IL1RN	rs315952	C__11512470_10	C/T	Authentication

Table 2 b: detailed identification of TaqMan SNP selected in section 2

Table 3: taqman primer sequences

TABLE 4 genotype classes identified in the multiple ascending dose cohort (100mcg)

Table 5: list table: AIR prediction for FGF-18MAD 100-branched (n-45) individuals based on the rs9005 and rs315952 genotypes

Performance matrix	Value of
		Sensitivity of the composition	55.56％
Accuracy of	86.67％
		Specificity of	94.44％
Accuracy of	71.43％
		Negative predictive value	89.47％
Sensitivity and accuracy (F1 value)	62.50％

Table 6: AIR-PREDICTED PERFORMANCE BASED ON THE RS9005 AND RS315952 genotypes FOR FGF-18MAD100 BRANCHED (n-45) individuals

Table 7: list table: AIR prediction for FGF-18MAD 100-branched (n-48) individuals based on the presence or absence of C-T-A haplotype

Performance matrix	Value of
		Sensitivity of the composition	60％
Accuracy of	77.08％
		Specificity of	81.58％
Accuracy of	46.15％
		Negative predictive value	88.57％
Sensitivity and accuracy (F1 value)	52.17％

Table 8: mutexpression based on the presence or absence of AIR prediction by individuals with FGF-18MAD100 branches (n-48) of the C-T-A haplotype

Table 9: multivariate linear model of total cartilage volume change in placebo-only control individuals

Table 10: multivariate linear model of WOMAC total score change for placebo-only control individuals

Genotype(s)	Grade 3	Stage 2
			rs9005G/G rs315952T/T	7	15
Others	60	39

Table 11: list table: Kellgren-Lawrence ranking based on the presence or absence of the 'rs 9005G/G rs315952 TT' genotype (3 or 2) -analysis was performed on all individuals using all dosing regimens, including placebo. Fisher's exact test p-value was 0.0179, odds ratio 0.306, 95% confidence interval [0.096, 0.885 ].

Genotype(s)	Grade 3	Stage 2
			rs9005A vector rs315952C vector	17	9
Others	50	45

Table 12: list table: Kellgren-Lawrence ranking based on the presence or absence of the 'rs 9005A vector rs315952C vector' genotype (3 or 2) -analysis was performed on all individuals using all dosing regimens, including placebo. Fisher's exact test p-value was 0.2736, odds ratio 1.693, 95% confidence interval [0.637, 4.769 ].

Table 13: clinical outcome without diagnostic test (45 subjects treated with FGF-18100mcg and 27 placebo controls) -delta corresponds to the difference between the median change in placebo and the median change in treated subjects. The p-values correspond to those of a multivariate linear model adjusted in rank by gender, age, BMI, and KL.

Table 14: multivariate Linear model of Total fractional change in WOMAC for all placebo-controlled and all MAD 100-treated individuals

Table 15: multivariate Linear model of Total cartilage volume Change in all placebo-controlled and all MAD 100-treated individuals

Table 16: classified as 1) sensitizers (group B and C, n-29, treated with FGF-18100 mcg) or 2) supersensitizers (group D, n-6, at lower FGF-18 doses: 30mcg treatment). The analysis included 24 placebo-controlled-deltas with genotype B, C or group D corresponding to the difference in median placebo-controlled and median treated individual changes. The p-values correspond to those of a multivariate linear model adjusted in rank by gender, age, BMI, and KL.

Table 17: classified as 1) sensitizers (group B and C, n-29, treated with FGF-18100 mcg) or 2) supersensitizers (group D, n-6, at lower FGF-18 doses: 30mcg treatment) of the individual, a multivariate linear model of the change in WOMAC total score. The analysis included 24 placebo controls with either B, C or group D genotypes.

Table 18: classified as 1) sensitizers (group B and C, n-29, treated with FGF-18100 mcg) or 2) supersensitizers (group D, n-6, at lower FGF-18 doses: 30mcg treatment) of the subject. The analysis included 24 placebo controls with either B, C or group D genotypes.

Table 19: clinical outcome of individuals classified as non-susceptible according to the diagnostic test (MAD100n ═ 9, MADPL n ═ 3) -delta corresponds to the difference between the median change for placebo and the median change for treated individuals. The p-values correspond to those of a multivariate linear model adjusted in rank by gender, age, BMI, and KL.

Table 20: a multivariate linear model of the variation of WOMAC total score for individuals classified as non-sensitive according to diagnostic tests (MAD100n ═ 9, MADPL n ═ 3).

Table 21: multivariate linear models of the change in total cartilage volume of individuals classified as non-sensitive according to diagnostic tests (MAD100n ═ 9, MADPL n ═ 3).

Table 22: summary of clinical outcomes and possible treatment options based on the rs9005 and rs315952 genotypes

Reference to the literature

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14)http://www.cartilage.org/_files/contentmanagement/ICRS_evaluation.pdf

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16) Ellsworth et al, 2002, Osteoarthritis and Cartilage,10:308-

17) Shimoaka et al, 2002, J.Bio.chem.277(9):7493-

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Claims (13)

1. Use of one or more allele-specific probes for the manufacture of a kit for predicting the sensitivity of an individual having a cartilage disorder to therapy with an FGF-18 compound, the kit configured to be capable of:

a. determining genotypes at the loci IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample;

b. predicting from the results of step a high, medium, low or no sensitivity of said individual to a therapy with an FGF-18 compound.

2. The use of claim 1, the kit configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype for IL-1RN rs9005 at G/G and IL-1RN rs315952 at T/T, and

b. low or no sensitivity to therapy with FGF-18 compounds is predicted from the presence of said genotype.

3. The use of claim 1, the kit configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype for IL-1RN rs9005 at A/G or A/A and IL-1RN rs315952 at T/C or C/C, and

b. high sensitivity to therapy with FGF-18 compounds is predicted by the presence of said genotype.

4. The use of claim 1, the kit configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype selected from the group consisting of:

IL-1RN rs9005 at G/G and IL-1RN rs315952 at T/C or C/C, or

IL-1RN rs9005 at A/G or A/A and IL-1RN rs315952 at T/T, and

b. predicting from the presence of said genotype an intermediate sensitivity to therapy with an FGF-18 compound.

5. Use of one or more allele-specific probes in the manufacture of a kit for selecting for addition or exclusion to a therapy with an FGF-18 compound or a clinical trial based on the likelihood that a patient suffering from a cartilage disorder is susceptible to said therapy, said kit being configured to:

a. determining the genotype at the loci IL-1RN rs9005 and IL-1RN rs315952 from the nucleic acid sample, wherein the genotype of the patient at the loci is predictive of the risk of the patient being sensitive or insensitive to the therapy, and

b. sensitive patients are selected as appropriate for the therapy.

6. The use of claim 5, the kit configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype for IL-1RN rs9005 at G/G and IL-1RN rs315952 at T/T, and

b. rejecting said therapy with an FGF-18 compound from a patient having said genotype.

7. The use of claim 5, the kit configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype selected from the group consisting of:

IL-1RN rs9005 at G/G and IL-1RN rs315952 at T/C or C/C, or

IL-1RN rs9005 at A/G or A/A and IL-1RN rs315952 at T/T, T/C or C/C, and

b. adding a patient having any one of said genotypes to said therapy with an FGF-18 compound.

8. Use of one or more allele-specific probes for the manufacture of a kit for selecting a patient for an alternative treatment regimen with an FGF-18 compound based on the likelihood that the patient having a cartilage disorder will have a high sensitivity to treatment with the FGF-18 compound, the kit being configured to:

a. determining from a nucleic acid sample the genotypes at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at the loci is predictive of the risk of the patient being highly sensitive to therapy with the FGF-18 compound, and

b. selecting said patient for an alternative treatment regimen wherein the dose of the FGF-18 compound administered is lower than the dose of the FGF-18 compound administered to a patient not at risk of being highly sensitive to treatment with said FGF-18 compound.

9. Use of one or more allele-specific probes in the manufacture of a kit for selecting a patient for an alternative treatment regimen with an FGF-18 compound based on the likelihood that the patient having a cartilage disorder will develop an Acute Inflammatory Response (AIR) event upon treatment with the FGF-18 compound, the kit configured to:

a. determining from a nucleic acid sample the genotypes at the loci IL-1RN rs9005 and IL-1RN rs315952, wherein the genotype of the patient at the loci is predictive of the risk of the patient for developing an AIR event in response to treatment with the FGF-18 compound, and

b. selecting said patient for an alternative treatment regimen wherein the dose of the FGF-18 compound administered is lower than the dose of the FGF-18 compound administered to a patient not at risk of developing an AIR event.

10. The use according to claim 8 or claim 9, the kit being configured to be capable of:

a. determining from the nucleic acid sample the presence of a genotype for IL-1RN rs9005 at A/G or A/A and IL-1RN rs315952 at T/C or C/C, and

b. selecting patients with the genotype for an alternative treatment regimen that administers a lower dose of FGF-18.

11. The use according to any one of claims 1 to 10, wherein the FGF-18 compound is Sprifermin.

12. The use of any one of claims 1-10, wherein the cartilage disease is selected from the group consisting of: osteoarthritis, cartilage damage, fractures affecting articular cartilage, or surgical procedures affecting articular cartilage.

13. The use of claim 12, wherein the surgical procedure affecting articular cartilage is microfracture surgery.