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WO2019140517A1 - Méthode d'utilisation de ykl-40 dans la scoliose - Google Patents

Méthode d'utilisation de ykl-40 dans la scoliose Download PDF

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WO2019140517A1
WO2019140517A1 PCT/CA2019/050054 CA2019050054W WO2019140517A1 WO 2019140517 A1 WO2019140517 A1 WO 2019140517A1 CA 2019050054 W CA2019050054 W CA 2019050054W WO 2019140517 A1 WO2019140517 A1 WO 2019140517A1
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ykl
risk
developing
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Mark E. Samuels
Dina Tarek NADA
Alain Moreau
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Centre Hospitalier Universitaire Saint Justine
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Centre Hospitalier Universitaire Saint Justine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present disclosure relates to Idiopathic Scoliosis. More specifically, the present disclosure is concerned with reagents, methods, compositions and kits for the assessment and management of Idiopathic Scoliosis.
  • Idiopathic scoliosis is a prevalent spinal deformity that affects an average of 1-4% of the global pediatric population (1). It is characterized by an abnormal three-dimensional curvature of the spine with an onset that can occur between birth and sexual maturity. Thus, it has been classified as infantile, juvenile, or adolescent based on when a curve is initiated (2). Adolescent Idiopathic Scoliosis (AIS) represents the most common form of scoliosis and occurs between the ages of 10 and 15 years, with girls affected more severely than boys (3).
  • AIS Adolescent Idiopathic Scoliosis
  • Endophenotypes in complex diseases has the advantage of partitioning the genetic variation, thus providing the study with greater power to detect a genetic effect.
  • “Endophenotype” is a term that was first introduced to genetics in 1966 to describe“microscopic and internal” characteristics while phenotypes describe“obvious and external” characteristics (10, 11). Endophenotypes are heritable traits that are representative of the molecular path from genes to the phenotype (12, 13).
  • CHI3L 1 was identified as one of the genes that showed a significant differential expression among the different AIS endophenotypes
  • CHI3L 1 gene (HGNC: 1932; Entrez Gene: 1116; Ensembl: ENSG00000133048; OMIM: 601525; UniProtKB: P36222, Cytogenetic band: 1 q32 1) encodes for the secretory factor YKL-40.
  • YKL-40 is a member of the family "mammalian chitinase-like proteins", which correspond to glycoproteins that bind to heparin.
  • YKL-40 was first discovered in 1989 when it was reported to be secreted in vitro by MG63 osteosarcoma cell lines in large amounts (14). YKL-40 is expressed in many tissues and is secreted by several types of solid tumors.
  • YKL-40 acts as a growth factor in cells involved in tissue remodeling. It may have a role in cancer cell proliferation, survival and their ability to invade surrounding tissue (15). In addition, elevated serum levels of YKL-40 have also been observed in patients with non-malignant diseases of particular contexts of inflammation (16). The objectives of the present work were to determine whether the differential expression of the CW3L 1 gene initially found in AIS osteoblasts, is more systemic and plays a role in AIS pathogenesis.
  • YKL-40 levels were analyzed in a large cohort of AIS patients which are distributed among the three known biological endophenotypes (FG1, FG2 and FG3) Also, YKL-40 levels were compared between AIS patients and controls, and AIS patients were further sub-classified according to their curve severity. Moreover, the same cohort was genotyped for 12 SNPs in the CHI3L 1 gene to test the association of those SNPs to the different phenotypes and/or to the plasma levels of YKL-40 among the different sub classification of AIS patients.
  • the present disclosure concerns a method of determining whether a subject is at risk of developing Idiopathic scoliosis (IS) [e.g, AIS) comprising: (i) determining the level of YKL-40 protein in a biological sample from the subject; and/or (ii) determining the presence or absence of at least one variant in at least one allele of the CHI3L 1 gene of the subject, or a marker in linkage disequilibrium therewith.
  • IS Idiopathic scoliosis
  • the risk of developing Idiopathic scoliosis is then determined based on the level of YKL-40 protein determined in (i) and/or based on the presence or absence of the at least one variant (polymorphic marker) in the CHI3L1 gene determined in (ii).
  • Subjects with higher levels of YKL-40 protein have a lower risk of developing a scoliosis (or a severe scoliosis) than those with lower levels of YKL-40 protein.
  • the detection of a high level of YKL-protein e.g, a level above that of a control sample or reference value
  • the detection of a low level of YKL-protein e.g , a level equal to or lower than that of a control sample or reference value
  • an increased risk of developing a scoliosis e.g, a severe scoliosis
  • the present disclosure provides a method of determining whether a subject is at risk of developing Idiopathic Scoliosis (IS) (e.g, AIS) comprising (i) determining the level of YKL-40 protein in a biological sample from the subject; (ii) determining the level of ghrelin protein in a biological sample from the subject; and (iii) determining the risk of developing IS based on the ratio between the level of ghrelin and the level of YKL-40 detected. The risk of developing IS is then determined based on the ratio between ghrelin and YKL-40 in the sample.
  • IS Idiopathic Scoliosis
  • the detection of a ratio between ghrelin and YKL-40 (ghrelin:YKL-40) below that of a control sample or reference value is indicative of a lower risk of developing a scoliosis.
  • the detection of a ratio between YKL-40 and ghrelin (YKL-40: ghrelin) above that of a control sample or reference value is indicative of a lower risk of developing a scoliosis.
  • the risk of developing IS is a risk of developing a severe scoliosis. In embodiments, the risk of developing IS is a risk of scoliosis progression. In embodiments, the risk of developing IS is a risk of severe scoliosis progression.
  • the above-noted methods further comprise classifying the subject in the FG1, FG2 or FG3 endophenotype group.
  • the present disclosure also provides a method of classifying a subject (e.g., a male subject) suffering from IS or at risk of developing IS (e.g, AIS) in the FG1 , FG2 or FG3 endophenotype group comprising (i) determining the level YKL-40 protein (e.g, circulating or secreted YKL-40) in a biological sample (e.g., biological fluid sample such as blood, plasma or serum) from the subject; and/or (ii) determining the presence or absence of at least one variant (e.g, SNP) in at least one allele of the CH3L /gene of the subject, or a marker in linkage disequilibrium therewith.
  • a subject e.g., a male subject suffering from IS or at risk of developing IS (e.g, AIS) in the FG1 , FG2 or FG3 endophenotype group
  • a biological sample e.g., biological fluid sample such as blood, plasma or serum
  • the subject is classified in the FG1 , FG2 or FG3 endophenotype based on the level of circulating YKL-40 protein determined and/or based on the presence or absence of the at least one gene variant in the CH3L 1 gene, or of the marker in linkage disequilibrium therewith
  • the subject is classified in the FG3 subgroup when the level of circulating YKL-40 protein is higher than that of a control sample or reference value.
  • the subject is not classified in the FG3 subgroup when the level of YKL-40 protein is lower than that of a control sample or reference value.
  • the present disclosure also provides a method of classifying a subject having IS (e.g., AIS) in the FG1, FG2 or FG3 endophenotype group comprising (i) determining the level of ghrelin protein (e.g., circulating or secreted ghrelin) in a biological sample (e.g, biological fluid sample such as blood, plasma or serum) from the subject; and (ii) classifying the subject in the FG1 endophenotype when the level of circulating ghrelin is lower than the level in a control sample or reference value; or (ii) classifying the subject as belonging to the FG2 or FG3 endophenotype when the level of circulating ghrelin is higher than that of a control sample or reference value.
  • ghrelin protein e.g., circulating or secreted ghrelin
  • a biological sample e.g, biological fluid sample such as blood, plasma or serum
  • determining the presence or absence of at least one variant in the ⁇ 7/// Z /gene or a marker in linkage disequilibrium therewith comprises determining the genotype of the subject (i.e., the presence or absence of a given variant in both alleles of the CHI3L /gene of the subject) for the at least one variant or marker in linkage disequilibrium therewith
  • determining the presence or absence of at least one variant in the CH13L 1 gene or a marker in linkage disequilibrium therewith comprises determining the haplotype of the subject for a plurality of variants (SNPs) in the CHI3L 1 gene (i e., the presence or absence of a given set of variants in the CHI3L 1 gene of the subject, e.g. , the haplotypes listed in any of FIGs.5 to 10 or marker(s) in linkage disequilibrium therewith).
  • SNPs variants
  • determining the presence or absence of at least one variant in the CHI3L 1qene or a marker in linkage disequilibrium therewith comprises determining the presence or absence of at least one variant (e.g. , SNP) in the CH3L 1 gene in at least one allele of the subject. In embodiments, determining the presence or absence of at least one variant (e.g, SNP) in the CHI3L 1 gene or a marker in linkage disequilibrium therewith comprises determining the presence or absence of at least one variant in the CH3L 1 gene in both alleles of the subject (i.e., determining whether the subject is homozygous or heterozygous for the at least one gene variant)
  • the present disclosure concerns a method of genotyping a subject (e.g., having IS or at risk of developing IS) comprising determining the genotype of the subject for at least one variant in the CHI3L1 gene.
  • the at least one variant in the CHI3L 1 gene is at least one of the variants (SNPs) listed in Tables 1, 3, 4, 5A and/or 6.
  • the at least one variant comprises one or more of the following SNPs: rs55700740, rs946259, rs880633, rs1538372, rs4950881, rs946261, rs946262 and rs10920576.
  • the method comprises determining the presence or absence of at least two SNPs. In embodiments, the method comprises determining the presence or absence of at least three SNPs. In embodiments, the method comprises determining the presence or absence of at least four SNPs. In embodiments, the method comprises determining the presence or absence of at least five SNPs. In embodiments, the method comprises determining the presence or absence of at least six SNPs. In embodiments, the method comprises determining the presence or absence of at least seven SNPs. In embodiments, the method comprises determining the presence or absence of all SNPs identified herein.
  • the at least one variant in the C///?Z /gene comprises or consists of a combination of SNPs listed in
  • the above methods comprise the use of an oligonucleotide probe or primer.
  • the oligonucleotide probe or primer enables the specific detection of a variant sequence (allele) set forth in Tables 1, 3, 4* 5A and/or 6.
  • the biological sample is a biological fluid sample.
  • the biological fluid sample is a blood sample.
  • the biological fluid sample is plasma
  • the biological fluid sample is serum.
  • the biological sample is a cell sample.
  • the biological sample is a protein sample.
  • the biological sample is a nucleic acid sample
  • the YKL-40 is a circulating (secreted) YKL-40 polypeptide
  • the circulating YKL-40 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26, a fragment thereof, or an allelic variant thereof.
  • the subject has at least one family member diagnosed with IS (e.g., AIS) (first, second or third degree relative)
  • the subject is diagnosed with IS.
  • the subject is a subject diagnosed with IS and belonging to the FG1 endophenotype.
  • the subject is a subject diagnosed with IS and belonging to the FG2 endophenotype.
  • the subject is a subject diagnosed with IS and belonging to the FG3 endophenotype.
  • the subject is a male. In embodiments, the subject is a female In embodiments, the subject is between 6 and 26 years old. In embodiments, the subject is a pediatric subject. In embodiments, the pediatric subject is between 6 and 18 years old. In embodiments, the pediatric subject is between 10 and 15 years old. In embodiments, the subject has at least one family member diagnosed with IS [e.g, AIS). In embodiments, the subject has at least one SNP identified herein [e.g., see Tables 1, 3, 4, 5A and/or 6) in at least one allele of the CHI3L 1 gene. In embodiments, the subject has at least two SNPs identified herein in at least one allele of the CHI3L1 gene.
  • the subject has at least one SNP identified herein in both alleles of the CHI3L 1 gene (i.e., is homozygote for the SNP). In embodiments, the subject has at least two SNPs identified herein in both alleles of the CHI3L /gene (i.e., the subject is homozygote for at least two SNPs identified herein).
  • the present disclosure also concerns a method of treating or preventing IS [e.g., AIS) comprising increasing the level of secreted YKL-40 protein in the subject.
  • the method comprises administering an exogenous or recombinant YKL-40 polypeptide, or a cell expressing a YKL-40 polypeptide, to the subject.
  • the method comprises increasing the expression of the endogenous YKL-40 polypeptide or correcting a defective CHI3L 1 gene, e.g. using a genome-editing technique such as the CRISPR/Cas9 system.
  • the present disclosure also concerns the use of an exogenous or recombinant YKL-40 polypeptide for treating or preventing IS [e.g., AIS) in a subject, or for manufacture of a medicament for treating or preventing Idiopathic scoliosis in a subject.
  • the present disclosure also concerns an exogenous or recombinant YKL-40 polypeptide for treating or preventing IS [e.g., AIS) in a subject.
  • the present disclosure also provides a method [in vitro ox in vivo) of reducing the GiPCR signaling defect in a cell of a subject having IS or at risk of developing IS [e.g, AIS) comprising contacting the cell with a YKL-40 polypeptide or increasing the expression of the endogenous YKL-40 polypeptide (or correcting a defective CHI3L 1 gene) in the cell.
  • a method [in vitro ox in vivo) of reducing the GiPCR signaling defect in a cell of a subject having IS or at risk of developing IS [e.g, AIS) comprising contacting the cell with a YKL-40 polypeptide or increasing the expression of the endogenous YKL-40 polypeptide (or correcting a defective CHI3L 1 gene) in the cell.
  • the present disclosure also concerns the use of an exogenous or recombinant YKL-40 polypeptide for reducing the GiPCR signaling defect in a cell of a subject having IS [e.g, AIS) or at risk of developing IS, or for the manufacture of a medicament for reducing the GiPCR signaling defect in a cell of a subject having IS or at risk of developing IS.
  • the present disclosure also concerns an exogenous or recombinant YKL-40 polypeptide for reducing the GiPCR signaling defect in a cell of a subject having IS [e.g., AIS) or at risk of developing IS.
  • the present disclosure also provides a method of increasing GiPCR signaling in a cell of a subject having IS [e.g, AIS) or at risk of developing IS comprising contacting the cell with a YKL-40 polypeptide or increasing the expression of the endogenous YKL-40 polypeptide (or correcting a defective CHI3L 1 gene) in the cell.
  • the present disclosure also concerns the use of an exogenous or recombinant YKL-40 polypeptide for increasing GiPCR signaling in a cell of a subject having IS or at risk of developing IS, or for the manufacture of a medicament for increasing GiPCR signaling in a cell of a subject having IS or at risk of developing IS.
  • the present disclosure also concerns an exogenous or recombinant YKL-40 polypeptide for increasing GiPCR signaling in a cell of a subject having IS or at risk of developing IS.
  • the present disclosure also provides a method of reducing the effect of OPN on GiPCR signaling in a cell of a subject having IS [e.g., AIS) or at risk of developing IS comprising contacting the cells with a YKL-40 polypeptide or increasing the expression of the endogenous YKL-40 polypeptide (or correcting a defective CHI3L1 gene) in the cell.
  • IS e.g., AIS
  • the present disclosure also concerns the use of an exogenous or recombinant YKL-40 polypeptide for reducing the effect of OPN on GiPCR signaling in a cell of a subject having IS [e.g, AIS) or at risk of developing IS, or for manufacture of a medicament for reducing the effect of OPN on GiPCR signaling in a cell of a subject having IS or at risk of developing IS.
  • the present disclosure also concerns an exogenous or recombinant YKL-40 polypeptide for reducing the effect of OPN on GiPCR signaling in a cell of a subject having IS [e.g, AIS) or at risk of developing IS.
  • the above-noted method of treating or preventing, or use further comprise (i) determining the level of YKL-40 protein in a biological sample from the subject, (ii) determining the level of ghrelin protein in a biological sample from the subject (iii) classifying the subject in the FG1, FG2 or FG3 functional group; and/or (iv) determining the presence or absence of at least one variant (SNP) in at least one allele of the CHI3L /gene of the subject.
  • the present disclosure concerns a composition or kit for use in methods disclosed herein (e.g, for example, a kit for (i) detecting a variant in at least one allele of the CHI3L 1 gene in a biological sample; (ii) determining whether a subject is at risk of developing IS (e.g., AIS); (iii) treating or preventing IS (e.g., AIS) in a subject; (iv) genotyping a subject for at least one variant in the CHI3L 1 gene (e.g, SNP disclosed herein); (v) classifying a subject into a particular genetic or endophenotype group (FG1 , FG2 or FG3); (vi) reducing the GiPCR signaling defect in a cell of a subject (e.g, having IS or at risk of developing IS), (vii) reducing the effect of OPN on GiPCR signaling in a cell (e.g, of a subject having IS or at risk of developing IS), etc.).
  • a kit for for (i)
  • the kit may comprise for example one or more oligonucleotide probes or primers, one or more antibodies specific for detection of YKL-40 or C///7Z //YKL-40 gene variant, and/or soluble YKL-40 (for treating and preventing IS).
  • the composition or kit further comprises reagents for classifying the subject in the FG1 , FG2 or FG3 functional groups as disclosed for example in WO/2003/073102, WO/2010/040234, WO/2012/045176, WO/2015/032005, WO/2014/201560, WO/2014/201557.
  • the composition or kit further comprises a biological sample from the subject.
  • the present disclosure concerns a DNA chip comprising at least one oligonucleotide for detecting the presence or absence of at least one CHI3L 1 gene variant (SNP) set forth in Tables 1, 3, 4, 5A and/or 6) and a substrate on which the oligonucleotide is immobilized.
  • the variant is a variant (or combinations of variants, including haplotypes) associated with a reduced risk of developing a scoliosis disclosed herein (e.g, set forth in Tables 1, 3, , 5A and/or 6 and/or FIGs.5 to 10).
  • the present disclosure provides oligonucleotide probes or primers for use in the above described methods, compositions, kits, DNA chips, etc.
  • the oligonucleotide is for the specific detection of a variant of the present disclosure and comprises a nucleotide sequence including the desired variant nucleotide (e.g, see Table 1).
  • the variant is a variant associated with a reduced risk of developing a scoliosis disclosed herein.
  • the oligonucleotide hybridizes to a reference (ancestral) or a variant polynucleotide sequence set forth in Table 1 or its complementary sequence.
  • the oligonucleotide primer or probe further comprises a label.
  • the oligonucleotide primer or probe comprises or consists of at least 10 nucleotides of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-24 and 27, or the complement thereof, and includes the nucleotide from the ancestral or variant allele set forth in Table 1 (or its complement).
  • the oligonucleotide primer or probe comprises or consists of at least 10 nucleotides of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, and 11 or the complement thereof and includes the nucleotide from the ancestral allele set forth in Table 1 (or its complement).
  • the oligonucleotide primer or probe comprises or consists of at least 10 nucleotides of a polynucleotide sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10 and 12 or the complement thereof and includes the nucleotide from the variant allele set forth in Table 1 (or its complement)
  • the oligonucleotide primer or probe consists of 10 to 100 nucleotides, preferably 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides or 10 to 30 nucleotides.
  • the oligonucleotide primer or probe consists of at least 12 nucleotides.
  • the present disclosure relates to the use of methods, compositions, kits, oligonucleotide primers or probes, and DNA chips of the present disclosure for (i) detecting a variant in at least one allele of the CW3L 1 gene in a biological sample; (ii) determining whether a subject is at risk of developing IS; (iii) treating or preventing IS in a subject ; (iv) genotyping a subject for at least one variant in the CHI3L1 gene (e.g.
  • FIG. 1 shows plasma YKL-40 levels in function of sex and AIS biological endophenotypes. An Anova, two-sided t-test was applied and the presented p-values were after Bonferroni adjustment for pairwise comparisons;
  • FIG.2 shows linkage disequilibrium blocks of 12 SNPs identified herein
  • FIG. 3 shows that YKL-40 rescues Gi-coupled receptor signaling defect induced by rOPN.
  • Primary osteoblasts obtained from three scoliotic patients were pre-treated with purified rOPN (0.5 g/ml) with and without rYKL-40 (0.5 g/ml) for 18 h prior to the stimulation with 10 mM of oxymetazoline.
  • Error bars show SEM of independent experiments performed three times in duplicate. Data represent the percentage of the maximum impedance measured by CDS assay and was normalized to the response achieved in the absence of rOPN (vehicle only). * P ⁇ 0.01 based on one-way ANOVA followed by in post-hoc test of Dunnett;
  • FIG. 4 shows expression analysis of CHI3L 1 in primary human osteoblasts obtained from AIS patients classified in FG1 biological endophenotype vs. AIS patients classified in FG2+FG3 biological endophenotypes. Statistical analysis was performed with an unpaired T-test (two tailed p-value);
  • FIG.5 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels
  • FIG.6 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels in endophenotype FG1 ;
  • FIG.7 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels in endophenotype FG2;
  • FIG.8 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels in endophenotype FG3;
  • FIG. 9 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels in female subjects
  • FIG.10 depicts a table showing the results of the haplotype association analyses of plasma YKL-40 levels in male subjects;
  • FIG. 11 B depicts the nucleotide sequence of human YKL-40 transcript (NCBI Reference Sequence: NM_001276.2, SEQ ID NO: 27), with the coding sequence in bold.
  • Plasma YKL-40 levels were determined by ELISA and SNPs were analyzed by multiplex polymerase chain reaction and genotyping.
  • YKL-40 levels were also significantly increased in males classified in the first biological endophenotype (FG1) and associated with a non-severe scoliosis phenotype (Cobb angle ⁇ 40°). Treatmentwith YKL-40 rescued Gi-coupled receptor signaling dysfunction observed in AIS osteoblasts.
  • the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“includes” and“include”) or “containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps and are used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 18, 19 and 20 are explicitly contemplated
  • the number 6.0, 6.1 , 62, 6.3, 64, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P M. Wassarman and A. P.
  • GCID GC01 M203148; HGNC: 1932; Entrez Gene: 1116; Ensembl: ENSG00000133048; OMIM: 601525; UniProtKB: P36222; located on Chromosome 1 (1 q32 1 ); RefSeqGene on chromosome 1 : NG_013056.1
  • YKL-40 a secreted glycoprotein that is approximately 40kDa in size in humans.
  • YKL-40 is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells, vascular smooth muscle cells, and hepatic stellate cells. YKL-40 lacks chitinase activity due to mutations within the active site.
  • a non-limiting example of a human YKL-40 protein sequence include the sequence depicted in RefSeq accession No NP_001267 2 (SEQ ID NO: 25).
  • the first 21 residues of the sequence correspond to the signal peptide
  • the mature YKL-40 protein comprises residues 22-383 (SEQ ID NO: 26)
  • Residues 70-71, 97-100 and 204-207 are chitooligosaccharide-binding domains, and the region encompassing residues 324-338 is believed to be involved in AKT 1 activation and IL-8 production
  • a native YKL-40 protein ⁇ e.g., comprising the sequence of the mature form set forth in SEQ ID NO:26 or of the precursor set forth in SEQ ID NO:25) or a variant or fragment thereof having the biological activity of the native YKL- 40 protein, and more particularly the ability of rescuing (partially or completely) the a1-adrenergic receptor signaling dysfunction induced by rOPN.
  • the YKL-40 polypeptide comprises an amino acid sequence having at least 70% identity with the sequence set forth in SEQ ID NO:26 or SEQ ID NO:25, and has the biological activity of the native YKL-40 protein.
  • the YKL-40 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence set forth in SEQ ID NO:26 or SEQ ID NO:25, and has the biological activity of the native YKL-40 protein
  • the YKL-40 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:25.
  • ghrelin refers to a protein or polypeptide encoded by the ghrelin gene (GHRL: GCID:GC03M010285; HGNC: 18129; Entrez Gene: 51738; Ensembl: ENSG00000157017; OMIM: 605353; UniProtKB: Q9UBU3. Located on chromosome on chromosome 3 (3p25.3)).
  • the GHRL gene encodes the ghrelin-obestatin preproprotein that is cleaved to yield two peptides, ghrelin and obestatin.
  • ghrelin is a powerful appetite stimulant and plays an important role in energy homeostasis.
  • Non-limiting examples of ghrelin protein sequences include NP_001128413; NP_001128416; NP_001128417; NP_001128418; and NP_001289750).
  • the term“Idiopathic scoliosis” or“IS” refers to the common complex disorder of the spine. It is a three- dimensional deformity of the skeleton characterized by a lateral curvature of 310° on a standing radiograph (Cobb method), combined with vertebral rotation. It is the most common form of spinal disorder It mostly occurs at the age of adolescence and affects 1-4% (1) of the global pediatric population with higher prevalence in females who are generally more severely affected than males.
  • IS includes Infantile (age of onset ⁇ 3 years old), Juvenile (age of onset between 3 and 9 years old) and Adolescent (age of onset between 10 and 15 years old) idiopathic scoliosis.
  • a subject “diagnosed with IS” is a subject having a minimum curvature in the coronal plane of 10°, showed by for example a standing posteroanterior spinal radiograph, by the Cobb method with vertebral rotation and without any congenital or genetic disorder which could be the source of the spinal deformity observed.
  • the terms“risk of developing IS” e.g :, a subject at risk of developing IS
  • a subject at risk of developing IS refer to a genetic or metabolic predisposition of a subject to develop a scoliosis (i.e spinal deformity) and/or a more severe scoliosis at a future time (i.e , curve progression of the spine).
  • a scoliosis i.e spinal deformity
  • a more severe scoliosis at a future time i.e , curve progression of the spine.
  • an increase of the Cobb angle of a subject e.g, from 40° to 50° or from 18° to 25°
  • a“development” of a scoliosis i.e., a scoliosis progression
  • a subject at risk of developing IS includes asymptomatic subjects which are more likely than the general population to suffer in a future time of IS and includes subjects (e.g, children) having at least one parent, sibling or family member suffering from a scoliosis (either first degree, second degree or third degree relative). It also includes subjects which carry one or more known IS susceptibility markers (SNPs or other mutation/genetic variations).
  • SNPs IS susceptibility markers
  • a subject at risk of developing a scoliosis are asymptomatic subjects (i.e., subjects which do not yet have a spinal deformity of over 10°) but which have been identified as having a GiPCR signaling defect and classified in the FG1 , FG2 or FG3 endophenotype group using well known methods (e.g., cAMP measurement, cellular impedance, etc. - see, for example, WO/2003/073102, WO/2010/040234, WO/2012/045176, WO/2015/032005, WO/2014/201560, WO/2014/201557).
  • well known methods e.g., cAMP measurement, cellular impedance, etc.
  • severe scoliosis refers to a scoliosis (or scoliosis progression) with a Cobb angle of 40° or more
  • polynucleotide sample or“nucleic acid sample” is meant to refer to a DNA, or RNA (including cDNA) sample from a test subject.
  • the sample should contain sufficient amount of polynucleotides for determining the presence or absence of SNPs and/or haplotypes (i.e , for genotyping) disclosed herein according to the selected method
  • the choice of the sample type will of course depend on the specific conditions of the assay.
  • gene variants e.g., SNPs
  • the sample is a cell sample from the subject but is not so limited as long as the polynucleotide sample allows for the detection of the gene variant.
  • biological fluid sample refers to blood, saliva, tears, sweat, urine, semen and milk.
  • blood sample is meant to refer to blood, plasma or serum.
  • the term“subject” is meant to refer to any mammal including human, mouse, rat, dog, chicken, cat, pig, monkey, horse, etc.
  • the subject is a human, such as a human pediatric subject.
  • the genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome.
  • the human genome exhibits sequence variations which occur on average every 500 base pairs.
  • a “polymorphism” or“variant” refers to a variation in the sequence of nucleic acid [e.g, a gene sequence) Such variation includes insertion, deletion, and substitutions in one or more nucleotides.
  • sequence variation consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs").
  • SNPs Single Nucleotide Polymorphisms
  • SNPs that vary between paired chromosomes in an individual Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • an SNP thus refers to a variation at a single nucleotide in a given nucleic acid sequence.
  • each version of the sequence with respect to the polymorphic site represents a specific allele of the polymorphic site.
  • sequence variants can all be referred to as polymorphisms, occurring at specific polymorphic sites characteristic of the sequence variant in question.
  • polymorphisms can comprise any number of specific alleles.
  • reference is made to different alleles at a variant/polymorphic site without choosing a reference allele.
  • a reference sequence can be referred to for a particular polymorphic site.
  • the reference allele is sometimes referred to as the "wild-type” allele or“ancestral allele” and refers herein to the allele from a "non-affected" or control/reference individual [e.g., an individual that does not display a trait or disease phenotype i.e., which does not suffer from a scoliosis or which has a lower risk of (or predisposition to) developing a scoliosis).
  • A“gene variant”, “genetic marker” or “polymorphic marker”, as described herein, refers to a variation (mutation or alteration) in a gene sequence that occurs in a given population.
  • Each polymorphic marker/gene variant has at least two sequence variations characteristic of particular alleles at the polymorphic site.
  • the marker/gene variant can comprise any allele of any variant type found in the genome, including variations in a single nucleotide (SNPs, microsatellites, insertions, deletions, duplications and translocations.
  • SNPs single nucleotide
  • the polymorphic marker/gene variant if found in a transcribed region of the genome can be detected not only in genomic DNA but also in RNA.
  • the polymorphism/variant is found in the gene portion that is translated into a polypeptide or protein, the polymorphic marker/gene variant can be detected at the protein/polypeptide level.
  • the term“defective CHI3L 1 gene” as used herein refers to a CHI3L 1 gene comprising one or more mutations that affect the expression of the CHI3L1 gene and/or that results in a YKL-40 protein having reduced activity relative to the native protein
  • the defective CHI3L 1 gene comprises one or more of the SNPs (variant allele) disclosed herein [e.g., variant allele in Table 1).
  • the polymorphic marker/gene variant of the present disclosure and its specific sequence variation can be detected by various means such as by sequencing the nucleic acid or protein.
  • the biological activity can be evaluated in order to identify which allele is present in the subject's sample. For example, if a particular risk allele (comprising a risk variant or combination of risk variants) affects the enzymatic activity of the protein, then, the presence of the allele or variant(s) can be assessed by performing an enzymatic test. Alternatively, if the risk allele (comprising a gene variant or combination of variants) affects the expression level of a polypeptide or nucleic acid, then, the presence of the variants(s) can be determined by assessing the expression level (e.g.
  • a control sample e.g., sample from a subject not suffering from a scoliosis or at risk of developing a scoliosis.
  • an “allele” refers to the nucleotide sequence of a given locus (position) on a chromosome.
  • a polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.
  • Genomic DNA from an individual contains two alleles for any given polymorphic marker, representative of each copy of the marker on each chromosome.
  • A“risk allele”, a“susceptibility allele” or a“predisposition allele” or a“risk variant” is nucleic acid sequence variation that is associated with an increased risk of (i e.
  • a“protective allele” or“protective variant” is a sequence variation of a polymorphic marker that is associated with a lower risk of (i.e., compared to a control/reference) or predisposition to suffering from IS.
  • haplotype refers to a set or collection of linked single-nucleotide polymorphism (SNP) alleles that tend to occur together [e.g., that are associated statistically with a reduced risk of developing IS).
  • SNP single-nucleotide polymorphism
  • non-conservative mutation or“non-conservative substitution” in the context of polypeptides refers to a mutation in a polypeptide that changes an amino acid to a different amino acid with different biochemical properties (i e., charge, hydrophobicity and/or size).
  • a non-conservative substitution includes one that changes an amino acid of one group with another amino acid of another group [e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
  • an amino acid of one group e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
  • a“conservative substitution” or“conservative mutation” in the context of polypeptides is a mutation that changes an amino acid to a different amino acid with similar biochemical properties [e.g., charge, hydrophobicity and size)
  • biochemical properties e.g., charge, hydrophobicity and size
  • a leucine and isoleucine are both aliphatic, branched hydrophobic residues.
  • aspartic acid and glutamic acid are both small, negatively charged residues. Therefore, changing a leucine for an isoleucine (or vice versa) or changing an aspartic acid for a glutamic acid (or vice versa) are examples of conservative substitutions.
  • “Complement” or “complementary” as used herein refers to Watson-Crick [e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. "Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.
  • Homology and “homologous” refers to sequence similarity between two polypeptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences.
  • nucleic acid sequence is "substantially homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term“homologous” does not infer evolutionary relatedness, but rather refers to substantial sequence identity, and thus is interchangeable with the terms“identityTidentical”)
  • Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs.
  • sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
  • the units e.g., 66, 67. .81 , 82,...91, 92% ...) have not systematically been recited but are considered, nevertheless, within the scope of the present disclosure
  • Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul etal (Altschul et al. 1990) (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/).
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001
  • hybridize to each other under moderately stringent, or preferably stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 05 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C (Ausubel 2010).
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0 1% SDS at 68°C (Ausubel 2010).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (Tijssen 1993).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • the term“treating” or“treatment” in reference to scoliosis is meant to refer to at least one of a reduction of Cobb angle in a preexisting spinal deformity, improvement of column mobility, preservation/maintenance of column mobility, improvement of equilibrium and balance in a specific plan; maintenance/preservation of equilibrium and balance in a specific plan; improvement of functionality in a specific plan, preservation/maintenance of functionality in a specific plan, cosmetic improvement, and combination of any of the above.
  • preventing or“prevention” in reference to scoliosis is meant to refer to a at least one of a reduction in the progression of a Cobb angle in a patient having a scoliosis or in an asymptomatic patient, a complete prevention of apparition of a spinal deformity, including changes affecting the rib cage and pelvis in 3D, or a combination of any of the above.
  • follow-up schedule is meant to refer to future medical visits a subject diagnosed with a scoliosis or at risk of developing a scoliosis is prescribed once the diagnosis or risk evaluation is made.
  • a subject is identified as being at risk of developing a severe scoliosis or at risk of rapid curve progression (e.g, a subject classified as belonging to the FG2 subgroup in accordance with the present disclosure)
  • the number of medical visits e.g., to the orthopedist
  • the number of x-rays in a given period e.g, 3, 6 or 12 months
  • the number of medical visits or x-rays may be decreased to less than the average [e.g., less than 22 x-rays over a 3 year period or less than 1 visit every 3 months, 6 months or 12 months).
  • Detecting specific gene variants or polymorphic markers and/or haplotypes of the present disclosure can be accomplished by methods known in the art. Such detection can be made at the nucleic acid or amino acid (protein) level.
  • standard techniques for genotyping for the presence of gene variants can be used, such as sequencing, fluorescence-based techniques (Chen, X. eta/., Genome Res. 9(5): 492-98 (1999)), methods utilizing PCR, LCR, Nested PCR and other methods for nucleic acid amplification.
  • SNP genotyping include, but are not limited to, Restriction fragment polymorphism (RFLP), TaqManTM genotyping assays and SNPlexTM platforms (Applied Biosystems), mass spectrometry [e.g, MassARRAYTM system from SequenomTM), minisequencing methods, real-time PCR, Bio-PlexTM system (BioRad), CEQ and SNPstreamTM systems (Beckman), Molecular Inversion ProbeTM array technology [e.g., AffymethxTM GeneChip), and BeadArrayTM Technologies [e.g, Illumina GoldenGateTM and InfiniumTM assays).
  • RFLP Restriction fragment polymorphism
  • TaqManTM genotyping assays and SNPlexTM platforms Applied Biosystems
  • mass spectrometry e.g, MassARRAYTM system from SequenomTM
  • minisequencing methods minisequencing methods, real-time PCR, Bio-PlexTM system (BioRad), CEQ and
  • Linkage disequilibrium is defined as the non-random association of alleles at different loci across the genome. Alleles at two or more loci are in LD if their combination occurs more or less frequently than expected by chance in the population.
  • a particular genetic element e.g., an allele of a polymorphic marker, or a haplotype
  • another element occurs at a frequency of 0.50 (50%)
  • the predicted occurrence of a person’s having both elements is 0.25 (25%), assuming a random distribution of the elements.
  • the two elements occur together at a frequency higher than 0.25, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence [e.g, allele or haplotype frequencies) would predict
  • any SNPs in linkage disequilibrium with a given SNP involves: (a) amplifying a fragment from the gene comprising a first SNP from a plurality of individuals; (b) identifying of second SNPs in the gene comprising said first SNP; (c) conducting a linkage disequilibrium analysis between said first SNP and second SNPs; and (d) selecting said second SNPs as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated
  • Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch etai, 1996; Maniatis eta/, 2002; Reich etal, 2001). If all polymorphisms in the genome were independent at the population level (i.e., no LD), then every single one of them would need to be investigated in association studies, to assess all the different polymorphic states. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.
  • D The two metrics most commonly used to measure LD are D’ and r 2 and can be written in terms of each other and allele frequencies. Both measures range from 0 (the two alleles are independent or in equilibrium) to 1 (the two alleles are completely dependent or in complete disequilibrium), but with different interpretation. D' is equal to 1 if at most two or three of the possible haplotypes defined by two markers are present, and ⁇ 1 if all four possible haplotypes are present. r2 measures the statistical correlation between two markers and is equal to 1 if only two haplotypes are present.
  • Event like recombination may decrease LD between markers But, moderate (i.e. 0.5 £; r 2 ⁇ 0.8) to high (i.e. 0.8 £; r 2 ⁇ 1)
  • LD conserve the "surrogate" properties of markers
  • LD based association studies when LD exist between markers and an unknown pathogenic allele, then all markers show a similar association with the disease.
  • SNPs have alleles that show strong LD (or high LD, defined as r 2 3 0.80) with other nearby SNP alleles and in regions of the genome with strong LD, a selection of evenly spaced SNPs, or those chosen on the basis of their LD with other SNPs (proxy SNPs or Tag SNPs), can capture most of the genetic information of SNPs, which are not genotyped with only slight loss of statistical power.
  • this region of LD is adequately covered using few SNPs (Tag SNPs) and a statistical association between a SNP and the phenotype under study means that the SNP is a causal variant or is in LD with a causal variant.
  • a proxy (or Tag SNP) is defined as a SNP in LD (r 2 3 0.8) with one or more other SNPs.
  • the genotype of the proxy SNP could predict the genotype of the other SNP via LD and inversely.
  • any SNP in LD with one of the SNPs used herein may be replaced by one or more proxy SNPs defined according to their LD as r 2 3 0.8.
  • SNPs in linkage disequilibrium can also be used in the methods according to the present disclosure, and more particularly in the diagnostic methods according to the present disclosure.
  • the presence of SNPs in linkage disequilibrium (LD) with the above identified SNPs may be genotyped, in place of, or in addition to, said identified SNPs.
  • the SNPs in linkage disequilibrium with the above identified SNP are within the same gene of the above identified SNP. Therefore, in the present disclosure, the presence of SNPs in linkage disequilibrium (LD) with a SNP of interest and located within the same gene as the SNP of interest may be genotyped, in place of, or in addition to, said SNP of interest.
  • such an SNP and the SNP of interest have r 2 3 0.70, preferably r 2 3 075, more preferably r 2 3 0.80, and/or have D’ 3 0.60, preferably D’ 3 0.65, D' 3 0.7, D’ 3 0.75, more preferably D’ 3 0.80
  • D preferably D’ 3 0.60
  • D preferably D’ 3 0.65, D' 3 0.7, D’ 3 0.75
  • D preferably D’ 3 0.80
  • r 2 3 0.80 which is used as reference value to define "LD" between SNPs.
  • FIG.2 Exemplary markers in linkage disequilibrium are shown in FIG.2.
  • compositions and kits for use in the methods of the present disclosure may include for example (i) one or more reagents for detecting the level of YKL-40 in a biological sample; and/or (ii) one or more reagents for detecting the presence or absence of at least one CHI3L 1 gene variants (e.g, one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs.5 to 10) or a substitute marker in linkage disequilibrium therewith
  • compositions and kits can comprise oligonucleotide primers and hybridization probes (e.g, allele-specific oligonucleotide primers and hybridization probes for determining the presence or absence of a variant in the CHI3L1 gene (e.g, one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs. 5 to 10), restriction enzymes (e.g, for RFLP analysis) and/or antibodies that bind to a YKL-40 polypeptide (wild-type or variant polypeptide).
  • oligonucleotide primers and hybridization probes e.g, allele-specific oligonucleotide primers and hybridization probes for determining the presence or absence of a variant in the CHI3L1 gene (e.g, one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs. 5 to 10), restriction enzymes (e.g, for RFLP analysis) and/or antibodies
  • the kit may also include any necessary buffers, enzymes (e.g., DNA polymerase) and/or reagents necessary for performing the methods of the present disclosure.
  • the kit may comprise one or more labeled nucleic acids (or labeled antibody) capable of specific detection of YKL-40 polypeptide or at least one CHI3L /gene variant of the present disclosure (e.g., one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs. 5 to 10) or any markers in linkage disequilibrium therewith as well as reagents for the detection of the label.
  • Reagents may be provided in separate containers or premixed depending on the requirements of the method.
  • Suitable labels are well known in the art and will be chosen according to the specific method used.
  • suitable labels include a radioisotope, a fluorescent label, a magnetic label, an enzyme, etc.
  • a CHI3L1 gene variant (e.g, one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs. 5 to 10) associated with IS in accordance with the present disclosure may be determined by DNA Chip analysis
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the presence of labelled hybridized complexes is then detected.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art.
  • a composition e.g, a diagnostic composition
  • assay mixture which is generated following one or more steps of the methods describe herein and which include a biological sample (e.g, cell sample, blood sample, plasma sample, serum sample, etc ) from the subject to be tested, and means or reagents for detecting the proteins, nucleic acids, and/or SNPs described herein
  • the preparation of such composition occurs while testing a subject’s biological sample for the risk of developing a scoliosis (including the risk of developing a more severe scoliosis); for aiding in the prevention and treatment of scoliosis including for determining the best treatment regimen; for adapting an undergoing treatment regimen; for selecting a new treatment regimen, for determining the frequency of a specific treatment regimen or follow-up schedule or for classifying the subject into a particular genetic group or endophenotype (FG1 , FG2 or FG3).
  • a biological sample e.g, cell sample, blood sample, plasma sample, serum sample, etc
  • compositions and kits of the present disclosure may thus comprise one or more oligonucleotides probe or amplification primer for the detection (e.g, amplification or hybridization) of at least one CW3L 1 gene variant of the present disclosure (e.g, a variant or reference sequence defined in one or more variants listed in any of Tables 1, 3, 4, 5A and/or 6 and/or FIGs. 5 to 10).
  • oligonucleotide probes are provided in the form of a microarray or DNA chip.
  • the kit may further include instructions to use the kit in accordance with the methods of the present disclosure (e.g, for determining the risk of (or predisposition to) developing a scoliosis; for genotyping a subject; for treating a subject; for selecting the best follow-up schedule or treatment regimen; for determining the risk of future parents to have a child likely to suffer from IS (e.g, AIS) (or a severe form of IS); or for classifying a subject suffering from a scoliosis or at risk of developing a scoliosis in a specific genetic or functional group).
  • IS e.g, AIS
  • a severe form of IS or for classifying a subject suffering from a scoliosis or at risk of developing a scoliosis in a specific genetic or functional group.
  • Plasma YKL-40 levels were analyzed in 710 patients and 227 controls. As expected, there were more females in patients than in controls (Fisher’s exact test P- 0001) Plasma YKL-40 levels and genotypes for the 12 C ?Z /SNPs were analyzed for 728 patients with AIS and 216 healthy controls after ancestral and relatedness testing.
  • Stratification by scoliosis severity was determined only in the participants who have reached their skeletal maturity at the time of blood collection, which resulted in 132 AIS patients as severe cases (Cobb angle 340°) and 227 AIS patients as non-severe cases (Cobb angle 10°-39°).
  • Table 2A Clinical and biochemical characteristics of the French-Canadian cohort studied herein
  • Table 2B Demographic and clinical data for the replication cohort genotyped using multiplex PCR
  • EXAMPLE 2 ASSOCIATION BETWEEN PLASMA YKL-40 LEVELS AND AIS BIOLOGICAL ENDOPHENOTYPES
  • Plasma ghrelin levels were measured in a subset of AIS patients and matched healthy controls. The clinical and demographic summary of the participants tested is provided in Table 2C. Analysis of all AIS patients compared to matched controls did not show a significant effect of circulating ghrelin levels on plasma YKL-40 levels.
  • Table 2C Demographic and clinical data for a subset of AIS patients and controls subjects tested for unacylated ghrelin
  • Table 3 Prevalence of the studied SNPs in CtfriQZ/gene and their associations with plasma YKL-40 levels in AIS patients and healthy controls
  • the patient samples were further divided into two groups: non-severe and severe based on their scoliosis phenotype.
  • Table 4 Prevalence of the studied SNPs in CHI3L1 gene and their associations with plasma YKL-40 levels in function of scoliosis severity
  • Table 5A Prevalence of the studied SNPs in £ 7l/gene and their associations with plasma YKL-40 levels in function of AIS biological endophenotypes
  • Table 5B Demographic and clinical data for AIS patients identified as YKL-40 overproducers
  • rTIL right thoracic and left lumbar
  • rT right thoracic
  • ITrTIL left thoracic, right thoracic and left lumbar
  • haplotype A-A-G-G- G-G (rs10399805
  • EXAMPLE 8 FUNCTIONAL ASSESSMENT OF THE ROLE OF YKL-40 IN AIS PATHOGENESIS
  • YKL-40 could act as a protecting disease-modifying factor in the context of AIS and may be used in the prevention or treatment of AIS (e.g., to reduce scoliosis progression or the risk of developing a severe scoliosis).
  • EXAMPLE 9 MATERIALS AND METHODS Study populations.
  • a total of 804 French-Canadian AIS patients and 239 age- and sex-matched healthy controls were enrolled between January 2008 and December 2012 in three pediatric spine centers in Montreal and surrounding schools All participants are residents of Quebec and of European descent.
  • Each AIS patient was clinically examined by an orthopedic surgeon at the participating hospitals.
  • Full medical history of each participant was collected to assess for other conditions including YKL-40 related diseases (e g. asthma) No other disease was found at the time of sample collection
  • All healthy control subjects were screened by an orthopedic surgeon using the Adam’s forward-bending test with a scoliometer. Any children with an apparent spinal curvature or family history of scoliosis were excluded from the control cohort.
  • Osteoblasts were derived from bone specimens removed from affected vertebrae (number varied from T3 to L4), as a part of correctional surgery for severe scoliosis (AIS cases) and from trauma cases (controls). Under sterile conditions, bone was fragmented with a bone cutter, then incubated in 1x Dulbecco’s Modification Eagle’s Medium (aDMEM) (Wisent Inc, Montreal QC, Canada) supplemented with 10% HyCloneTM Fetal Bovine Serum (FBS) (Thermo Fischer Scientific, Logan UT, USA) and 1 % antibiotic (Invitrogen), at 37°C with 5% C0 2 until confluent (10-14 days). At confluence, osteoblasts were isolated by trypsinization and frozen in liquid nitrogen Patient endophenotypes were classified from osteoblast cultures using cellular dielectric spectroscopy (CDS), as previously described in (8) and (9).
  • CDS cellular dielectric spectroscopy
  • Microarray Genome-wide expression patterns were assessed on microarrays performed in triplicate using the Affymetrix, GeneChip® Fluman Exon 1.0 ST array, at Centre d’lnnovation de Genome Quebec, Montreal. All protocols were conducted as described in Affymetrix GeneChip Expression Analysis technical manuals. Analysis of microarray data was conducted using GeneSifter software (www.genesifter.net/web/DC). Raw data were normalized using Robust Multi-array Average (RMA) (35), and these derived values were log2-transformed.
  • RMA Robust Multi-array Average
  • CHI3L /gene selected from the filtered microarray dataset was further validated by real time quantitative PCR (RT-qPCR) using PerfeCTaSYBR Green SuperMixTM (Quanta Biosciences) on a 7900HT Real-Time PCR system (Applied Biosystems), according to manufacturer’s protocols.
  • CHI3L1 primers were: Forward primer: 5'-CAGGAAAGCGTCAAAAGCAAGGTG-3’ (SEQ ID NO:28) and Reverse primer: 5’- GAGTGCATCCTTGATGGCATTGGT-3’ (SEQ ID NO:29).
  • RNA was reverse transcribed into cDNA using ThermoscriptTM RT-PCR system (Invitrogen CA, USA), according to the manufacturer’s protocol A 1 in 10 dilution of cDNA was used for RT-qPCR.
  • Each cDNA sample was loaded in triplicate for every gene tested, on an optical 384 reaction plate, and beta-actin was used as an endogenous control for normalizing gene expression, based on Stephens et al, 2011 (38). The fold-change in gene expression was determined by the program RQ manager (39).
  • RQ values were log 2 transformed, and the Levene test checked for equal variances between groups If variances were equal we used either an ANOVA or a T-test, if not equal we used the Wilcoxon/Kruskal-Wallis test to examine whether average expression levels were different among clusters. Statistical analyses were performed using JMP 9 software (SAS Institute Inc 2012).
  • Plasma YKL-40 and Ghrelin levels Peripheral blood samples were collected in EDTA-treated tubes and then centrifuged. Plasma samples were collected, aliquoted and stored at -80oC until thawed and analyzed. The concentrations of plasma YKL-40 was measured by enzyme-linked immunosorbent assay (ELISA) kit (Quidel, San Diego, CA, USA) according to the protocol provided by the manufacturer. Unacylated ghrelin was measured in plasma of a subgroup of AIS patient exhibiting a severe scoliosis and matched control subjects by an EIA kit (Cayman Chemicals, Ann Arbor, Ml, USA) according to the manufacturer's specifications. Both assays were performed in duplicate and the mean values were taken for the analysis. The optical density was measured at 450 nm using a DTX880 microplate reader (Beckman Coulter, Brea, California, USA).
  • Genotyping of SNPs in the £ ⁇ 4K7Z/gene Genomic DNA samples were derived from the peripheral blood of the subjects of the same cohort using PureLink® Genomic DNA kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then they were genotyped for 12 SNPs in the region of CHI3L1 gene. Part of the cohort was genotyped by the lllumina Fluman Omni 2.5M Bead Chip as part of a GWAS study previously done by our team at the McGill University and Genome Quebec Innovation Centre (31). These SNPs were chosen due to the fact that their genotypes were already available for most of the cohort used for biochemical analysis.
  • the primers were designed using the program Primer3.
  • Sanger sequence chromatograms were analyzed using Mutation Surveyor (Soft Genetics, Inc.).
  • CDS Cellular dielectric spectroscopy
  • Phenotypic analyses To compare patients and controls and compare among different sub-classifications of patients and controls, an ANOVA test was used with the log-transformed plasma YKL-40 level as the dependent variable and the phenotype and sex as independent variables, with age ascovariate. L/alue (two-sided) ⁇ 0.05 was considered statistically significant.
  • Haplotype association analysis The linkage disequilibrium blocks of the 12 SNPs were estimated based on the genotype data using Flaploview (33) The haplotypes were inferred using UNPFIASED (34) with a sliding window of up to six SNPs. Association analyses were carried out between the inferred haplotypes and the YKL-40 level using an in-house R program Specifically, a linear regression model was performed for the haplotype associations with YKL-40 levels. The association analyses were also performed based on various subsets of the samples, such as males, females, and endophenotypes.

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Abstract

L'invention concerne de nouveaux marqueurs moléculaires dans le gène CHI3L1 associés à la scoliose idiopathique (SI). La présente invention concerne par conséquent de nouvelles méthodes permettant d'identifier des sujets présentant un risque de développer une SI ou atteint d'une SI, et de génotyper les sujets SI et de les classer dans des groupes génétiques et fonctionnels. L'invention concerne également des compositions, des puces à ADN et des kits permettant d'exécuter lesdites méthodes. L'invention concerne également de nouvelles méthodes, utilisations et compositions pour la prévention ou le traitement de l'IS par l'augmentation des niveaux du polypeptide YKL-40.
PCT/CA2019/050054 2018-01-16 2019-01-16 Méthode d'utilisation de ykl-40 dans la scoliose Ceased WO2019140517A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009070764A1 (fr) * 2007-11-28 2009-06-04 Yale University Une variation dans le gène chi3l1 affecte les teneurs sériques en ykl-40, le risque d'asthme et la fonction pulmonaire
WO2015032004A1 (fr) * 2013-09-09 2015-03-12 Chu Sainte-Justine Procédé de traitement et de pronostic de sous-groupes de patients scoliotiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009070764A1 (fr) * 2007-11-28 2009-06-04 Yale University Une variation dans le gène chi3l1 affecte les teneurs sériques en ykl-40, le risque d'asthme et la fonction pulmonaire
WO2015032004A1 (fr) * 2013-09-09 2015-03-12 Chu Sainte-Justine Procédé de traitement et de pronostic de sous-groupes de patients scoliotiques
WO2015032005A1 (fr) * 2013-09-09 2015-03-12 Chu-Sainte-Justine Nouveau marqueur pour la classification, le diagnostic et le traitement de la scoliose

Non-Patent Citations (5)

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Title
AKOUME ET AL.: "Cell -based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy", JOURNAL OF VISUALIZED EXPERIMENTS, vol. 80, 16 October 2013 (2013-10-16), pages 1 - 9, XP055323360, DOI: 10.3791/50768 *
KAZAKOVA ET AL.: "YKL-40 - a novel biomarker in clinical practice?", FOLIA MEDICA LI, vol. 51, no. 1, January 2009 (2009-01-01), pages 5 - 14, XP055625429 *
NADA ET AL.: "Association of circulating YKL-40 levels and CHI3L1 gene variants with the risk of spinal deformity progression in adolescent idiopathic scoliosis", SCOLIOSIS AND SPINAL DISORDERS, vol. 13, no. 1, 4 May 2019 (2019-05-04), XP055625421 *
SALES DE GAUZY ET AL.: "Fasting total ghrelin levels are increased in patients with adolescent idiopathic scoliosis", SCOLIOSIS, vol. 10, no. 33, 30 November 2015 (2015-11-30), pages 1 - 5, XP055625418 *
TSAI ET AL.: "CHI3L1 polymorphisms associate with asthma in a Taiwan ese population", BMC MEDICAL GENETICS, vol. 15, no. 86, 23 July 2014 (2014-07-23), pages 1 - 8, XP021194396 *

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