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WO2008018789A2 - Methods and means for diagnosing and treatment of osteoarthritis - Google Patents

Methods and means for diagnosing and treatment of osteoarthritis Download PDF

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Publication number
WO2008018789A2
WO2008018789A2 PCT/NL2007/050367 NL2007050367W WO2008018789A2 WO 2008018789 A2 WO2008018789 A2 WO 2008018789A2 NL 2007050367 W NL2007050367 W NL 2007050367W WO 2008018789 A2 WO2008018789 A2 WO 2008018789A2
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gene
polymorphism
osteoarthritis
snp
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WO2008018789A3 (en
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Pieternella Slagboom
Ingrid Meulenbelt
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Leids Universitair Medisch Centrum LUMC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the current invention relates to the field of medicine, in particular to human genetics and diagnostics for osteoarthritis.
  • Osteoarthritis is a degenerative disease of the joints characterized by degradation of the hyaline articular cartilage and remodeling of the subchondral bone with sclerosis.
  • OA Osteoarthritis
  • the currently available treatments for OA focus on symptom relief and improving function and has been based on analgesics and nonsteroidal anti-inflammatory drugs or arthroplasty (1,2).
  • analgesics and nonsteroidal anti-inflammatory drugs or arthroplasty (1,2).
  • there is a lack of an effective disease modifying treatment in which degradation of joint tissue is stabilized, retarded or halted and at the same time an improvement of symptoms is reached.
  • Results of genetic studies will contribute to the clarification of the complex genetic background of OA and the pathways involved in its occurrence and progression. Identification of genetic variation associated with OA therefore will improve our understanding of the pathogenesis and may elucidate new molecular targets for the development of new treatment.
  • the identification of relevant genetic variation within OA patients may also provide means for a better and earlier classification and diagnosis by for instance genetic analysis to identify whether a subject is at risk of developing or having OA.
  • better classification of OA patients (for example due to genetic variation that is associated with fast OA progression) may provide great opportunities in the effectiveness of clinical trials and in improving the quality and efficiency of our health-care system.
  • the current invention provides methods for the identification and use of genetic loci involved in OA phenotypes in human subjects. These genetic loci, markers and polymorphisms can be applied in diagnostic methods for determining the risk of a subject for developing osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism in a locus genetically associated with OA phenotype or pathology.
  • the invention provides the identification of genetic loci contributing to the early onset rare familial generalized OA phenotype.
  • the invention discloses genetic loci contributing to the more common generalized OA at later ages of onset and hip OA.
  • the invention achieves its aim by providing genetic loci, nucleic acid sequences and in particular polymorphisms in or near genes that are genetically and/or physiologically linked to early onset OA pathology.
  • the invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on at least one of the human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 in the genome of the subject.
  • the current inventors performed a genome wide scan in families with dominant Mendelian inherited generalized OA.
  • the inventors have further established the presence of a number of mutations and/or polymorphisms in genes within the 2q33.2 to q34 region that appear to correlate with OA in several families that were analyzed.
  • This group of genes comprises ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDHlCPO, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl,
  • the current invention provides for nucleic acid sequences which can be applied for screening, diagnosis, prognosis and treatment of osteoarthritis.
  • the invention also provides genetic loci, nucleic acid sequences and in particular polymorphisms in or near genes that are physiologically linked to the more common familial generalized (FOA) at later ages of onset.
  • FAA familial generalized
  • the inventors performed a genome wide scans in affected siblings with symptomatic OA at multiple joint sites at ages between 40-70 years (the so called GARP study). These siblings have a positive family history for OA and a complex mode of inheritance.
  • the genome wide linkage scan revealed a locus on chromosome 14q31.11 that showed significant linkage with a LOD score of 3.03 corresponding to a nominal P-value of 1.9 x 10 "4 , a global P-value for chromosome 14 of 0.0013 and a genome wide P-value of 0.0286.
  • This locus was mainly attributable to OA at multiple joint sites.
  • the location of the linkage peak revealed three candidate genes: calmodulin (CALMl, MIM 114180]), the fibronectin- leucine-rich-transmembrane protein 2 gene (FLRT2 [MIM 604807]) and the iodothyronine-deiodinase type 2 (D2) gene (DIO2 [MIM 601413]).
  • calmodulin CALMl, MIM 114180]
  • FLRT2 [MIM 604807] the iodothyronine-deiodinase type 2 (D2) gene
  • D2 [MIM 601413] iodothyronine-deiodinase type 2
  • Genotypes of a common haplotype, exclusively containing the minor allele of DIO2 rs225014, showed significant recessive association in females with symptomatic hip OA in two additional independent OA studies (OR 1.71, 95% CI 1.33-2.19, P 2.6 x 10 "5 ). These results indicate that the locus on chromosome 14q24.3-14q32.12, preferably a SNP within the DIO2 gene, is physiologically linked to the more common familial generalized (FOA) at later ages of onset, however, also concerns a susceptibility gene for even more common forms of OA such as hip OA and possibly extends to symptomatic OA features in general.
  • FAA familial generalized
  • DIO2 is a regulator of thyroid hormone metabolism in the growth plate and may confer susceptibility for OA.
  • the inventors have established for the first time that the thyroid hormone metabolism via the DIO2 gene on chromosome 14q31.1 is involved in OA.
  • the current invention provides for nucleic acid sequences which can be applied for screening, diagnosis, prognosis and treatment of osteoarthritis in humans.
  • the current invention provides nucleic acid sequences and sequence information that will aid in the classification, diagnosis, prognosis, treatment and development of effective treatment of osteoarthritis, in particular for familial osteoarthritis at both early and later ages of onset.
  • the invention achieves it's aim by the identification of genetic loci in, and in close proximity of at least one of chromosomal regions 2q33.2 to 2q34 for early onset familial generalized OA and 14q24.3 to 14q32.12 for later onset familial OA. Genes that reside within these chromosomal regions that show differential expression may associate to familial OA at multiple joint sites as well as to OA at single sites such as the hip.
  • the invention further provides sequences, in particular marker sequences and polymorphic gene sequences, which are genetically linked with OA in humans.
  • the invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on at least one of the human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 in the genome of the subject.
  • the method is preferably an in vitro or ex vivo method since material is taken from a subject and subsequently analyzed in vitro using the method of the invention.
  • the invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on the human chromosome band 2q33.2 to 2q34 in the genome of the subject.
  • the genetic markers located on or in close proximity to human chromosome bands 2q33.2 to 2q34, are particularly suitable for determining the risk of a subject for developing early onset familial generalized osteoarthritis.
  • Familial osteoarthritis may generally comprise related symptoms such as hand, discus degeneration, hip and OA at multiple sites, such as in, but not limited to OA in ankles, knees, elbows, wrists, shoulders and spine (lower back, upper back and neck areas).
  • the method of the invention teaches the use of the following group of genetic markers for human chromosome 2 in or in close proximity of the OA associated region of human chromosome 2q33.2 to 2q34; D2S326, D2S2257, D2S2314, D2S1391, D2S118, D2S315, D2S115, D2S72, D2S1384, D2S369, D2S155, D2S2358, D2S2208, D2S154, D2S2178, D2S371, D2S164, D2S126 and gata30e06 (Table 1).
  • markers D2S1384 and D2S2178 are closely linked to the genetic region on human chromosome 2 wherein the OA associated genes are located, with the highest linkage for D2S155.
  • markers can be advantageously applied for familial analysis, i.e. for determining which members of a family are likely to be affected or predisposed to developing symptoms of OA and which family members are likely to be unaffected.
  • the affection or the predisposition is preferably associated with the presence of a polymorphism, preferably a SNP.
  • the method of the invention also teaches the use of coding sequences located on human chromosomal region 2q33.2-q34 for determining the risk of a subject for developing osteoarthritis.
  • genes located on this chromosomal band may be advantageously used for determining the risk of a subject for suffering of OA or developing symptoms of OA (presence of polymorphism in); ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDHl, CPO, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl, PIP5K3, PTHR2 (Table 2).
  • the invention also teaches the use of the positional genes located on this chromosomal region 2q33.2-q34; ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDH1.CP0, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl, PIP5K3, PTHR2 (Table 2) for determining differentially expression as detected in RNA expression or protein profiles. Differentially expressed genes located within this region may explain or associate to the diagnostic or prognostic risk of developing symptomatic OA at multiple joint sites in humans.
  • the inventors found positive correlations with variants and/or mutations in the coding and non-coding sequences of the genes ADAM23, IDHl, PTHR2, FZD5 and NRP2 (Table 4). These five genes in particular appear to be closely linked with early onset OA in a number of OA affected families.
  • Mutations or polymorphisms outside the coding regions may affect splicing of messengers or may affect gene regulation, at the translational or transcriptional level, RNA stability or by any other means, thereby causing or contributing to the FOA phenotype.
  • the mutations and polymorphisms may also affect the coding sequence and thereby alter functional properties of the encoded protein, such as, but not limited to the enzymatic activity, interactions with other proteins, localization and stability or half- life.
  • the FZD 5 encodes the Wnt signaling receptor FZ5 for the Wnt5A ligand (19) and might agonist FRZB. Detectable levels of FZD 5 mRNA were present in OA tissue and the ligand receptor pair is implicated to be essential in cell fate determination and osteoarthritis.
  • the two exons of FZD 5 gene may potentially comprise a variant which co-segregates with the OA phenotypes in affected families.
  • the polymorphism is present in the PTHR2 (parathyroid hormone 2 (PTH2) receptor).
  • PTH2 parthyroid hormone 2
  • the PTHR2 is a G protein-coupled receptor selectively activated with similar potency by PTH and tuberoinfundibular peptide (TIP39) (20).
  • PTH receptor 2 Relevant functions for its possible role in OA are the expression of PTHR2 in a number of endocrine cell types that suggest a role for the PTH receptor 2 in regulating pituitary hormone secretion and specifically growth hormone. Moreover, in peripheral organs distinct cell populations express PTH receptor 2, including the calcitonin-synthesizing parafollicular C-cells of the thyroid gland involved in Ca + homeostasis and chondrocytes within cartilage growth plates of developing bone (20).
  • the PTH receptor 2 has been shown to interact with calmodulinl (CaMl), a calcium sensing protein (21), whereas a functional SNP in the core promoter region of the gene encoding calmodulinl (CALMl), was associated with hip OA in the Japanese population (13). Although the association with this functional SNP was not replicated in a UK population (15), the CaMl mediated signaling pathway is considered to be a new paradigm in the etiology and pathogenesis of OA (22). Together, these data clearly indicate a role for the PTH receptor 2 in the onset of OA, and polymorphisms and/or SNPs in PTH2 gene may be of high relevance and are therefore particularly preferred to be detected by the method of the invention.
  • the IDHl gene encodes isocitrate dehydrogenase 1, also referred to as oxalosuccinate decarboxylase.
  • IDHl supplies NADPH for antioxidant systems suggesting a regulatory role in cellular defense against oxidative stress and in senescence (25).
  • oxidative stress Although little is known about a possible role of IDHl in cartilage but increased oxidative stress would make chondrocytes more susceptible to cell death which might contribute to the onset of OA.
  • polymorphisms and/or SNPs in the IDHl gene are also preferably applied in the method of the current invention for determining the risk of a subject for developing OA.
  • the IDHl Y183C variant was a most promising variant since it co-segregated on haplotype A2 with the OA phenotype in family 2, was predicted to be damaging for the protein structure/function and concerned a very conserved amino acid. In the general population this variant was infrequent (0.02) and 36% of the carriers had generalised ROA as compared to 17% of the non carriers. Given these results and the finding that family 2 already showed a significant linkage on its own, this variant contributes to the FOA susceptibility in family 2.
  • the predicted KIAA1571 gene appears to belong, as predicted in Unigene, to the fibronectin type III and M protein repeat family in C. elegans. Fibronectin is a component of the extracellular matrix of cartilage and KIAAl 571 may therefore be an excellent candidate gene.
  • the G/T nucleotide change in the third exon of KIAAl 571 results in disruption of exonic splicer enhancer motifs which serves as binding site for Serine/ Arginine protein 40 and 55 and might be therefore a functional variant.
  • this gene is a predicted gene, little is known about other possible predicted functional effects on the protein.
  • polymorphisms and/or SNPs in the KIAA1571 gene are preferably applied in the method of the current invention for determining the risk of developing OA in a subject.
  • the polymorphism is present in the NRP2 gene.
  • the NRP2 gene encodes the vascular endothelial cell growth factor 165 receptor 2 (VEGF Receptor 2).
  • Neuropilin 2 (NRP2) is a highly relevant gene because it encodes for the co-receptor of vascular endothelial growth factories (VEGF 16 s) which is an essential factor for endochondral ossification (26,27).
  • VEGF 16 s vascular endothelial growth factories
  • MMPs extracellular cartilage matrix degrading matrix metalloproteinases
  • PIP 5KS catalyzes the phosphorylation of phosphatidylinositol-4-phosphate and has a role in endosome-related membrane trafficking (48).
  • PIP5K3 c.8429T>A and PIP5K3 c.8434insC in the 3'UTR region of PIP5K3 in family 4 which are both highly conserved residues.
  • Polymorphisms and/or SNPs in the PIP5K3 gene are preferably applied and detected in the method of the invention for determining the risk of a subject for developing OA.
  • the invention discloses a second preferred embodiment, which is a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on the human chromosome bands 14q24.3 to 14q32.12 in the genome of the subject.
  • the locus on chromosome 14q24.3-14q32.12 preferably a SNP within the DIO2 gene is physiologically linked to the more common familial generalized (FOA) at later ages of onset, however, additional replication indicated that the gene may also concerns a susceptibility gene for even more common forms of OA such as OA at the hip joint and possibly extends to symptomatic OA features in general.
  • FAA familial generalized
  • the genetic markers located between or in close proximity to human chromosome bands 14q24.3-14q32.12 are particularly suitable for determining the genes and/or polymorphisms that may contribute to the development or suffering from symptomatic OA at multiple joint sites with a familial background at later ages of onset, i.e. between 40 and 70 years.
  • Familial generalized osteoarthritis may comprise of symptomatic OA in a combination of at least two joint locations e.g. hand, knee, hip and discus degeneration.
  • the invention achieves it's aim by the identification of relevant genetic variation which may be based on identification of relevant genetic variation within or surrounding genes but also based on differentially expression of genes as detected in RNA expression or protein profiles, within the genes residing in the chromosomal region 14q24.3-14q32.12 that are linked to OA phenotype and/or pathology.
  • the invention further provides sequences, in particular marker sequences and polymorphisms in or around gene sequences in the 14q24.3-14q32.12 region which are genetically linked with OA in humans.
  • the invention teaches the use of genetic markers, D14S74, D14S1037, D14S1044 and D14S280, located on human chromosome bands 14q24.3 to 14q32.12 for determining the risk of a subject for developing symptomatic OA at multiple joint sites. Together, these markers mark the boundaries of the one LOD-drop interval of this signal which encompasses 22 cM. These markers can be advantageously applied for familial analysis, i.e. for determining which members of a family are likely to be affected or predisposed to developing symptoms of OA and which family members are likely to be unaffected.
  • the invention also teaches the use of genes and in particular polymorphisms, preferably SNP in or near the coding sequences of genes, located on human chromosome 14q24.3 to 14q32.12 region for determining the risk of a subject for developing osteoarthritis.
  • genes and in particular polymorphisms preferably SNP in or near the coding sequences of genes, located on human chromosome 14q24.3 to 14q32.12 region for determining the risk of a subject for developing osteoarthritis.
  • the list of genes as depicted in table 9 in example 2 located, on this chromosomal region may be advantageously used for determining the risk of a subject for suffering of OA or developing symptoms of OA.
  • the invention also teaches the use of the positional genes and more preferably the CALMl, FLRT2 and DI02 genes hat are located within the 14q24.3 to 14q32.12 region (table 10) for determining differential expression as detected in RNA expression or protein profiles.
  • Differentially expressed genes located within the 14q24.3 to 14q32.12 region may explain or associate to the diagnostic or prognostic risk of developing symptomatic OA at multiple joint sites in humans.
  • Differential expression may be detected on RNA or cDNA using methods known in the art such as DNA micro-array analysis and/or quantitative PCR.
  • the invention exploits the positive correlations with polymorphisms in the coding and non-coding sequences of the genes CALMl, FLRT2 and DIO2 that are located within the 14q24.3 to 14q32.12 region (table 10) with OA, preferably of later onset (40-70 years).
  • These genes and in particular polymorphisms therein appear to be closely linked with developing symptomatic familial general OA at multiple joint sites in humans.
  • the genes CALMl, FLRT2 and DIO2 mapped within the haplotype shared among affected family members and are to be considered as sequences coding for gene products that are fully, or at least in part, responsible for the OA phenotype.
  • Mutations or polymorphisms outside the coding regions may affect splicing of messengers or may affect gene regulation, at the translational or transcriptional level, RNA stability or by any other means, thereby causing or contributing to the OA phenotype. Mutations or polymorphisms in the coding regions may affect protein function, such as enzymatic activities, protein stability, interactions with other proteins or complexes of CALMl, FLRT2 and DIO2 and thereby contribute to the OA phenotype or pathology. Polymorphisms and/or SNPs in CALMl, FLRT2 and DIO2 genes are preferably detected in the method of the invention for determining the risk of a subject for developing OA.
  • a polymorphism even more preferably a SNP is present in the DIO2 gene.
  • DIO2 encodes a selenoenzyme D2 that catalyzes the conversion of thyroxin (T4) to triiodothyronine (T3) via 5-prime-deiodination and regulates the local thyroid hormone bioactivity in the growth plate (41,42).
  • T3 inhibits chondrocyte proliferation but stimulates chondrocyte differentiation and matrix synthesis (44).
  • Genetic variation in DIO2 will therefore have functional consequences for deiodinase activity but also for circulating iodothyronine levels.
  • a SNP in the DIO2 gene is rs225014 (Thr92Ala). This specific SNP may be associated with other polymorphisms and/or other SNP in the DIO2 gene and/or in other genes as mentioned in this whole application (present in 2q33.2 to 2q34 and/or in 14q24.3 to 14q32.12).
  • a polymorphism is present in the Fibronectin leucine rich transmembrane protein 2 (FLRT2) gene and/or in the Calmodulin gene CALMl.
  • FLRT2 gene encodes a small leucine-rich proteoglycan found in the extracellular matrix (49).
  • CALMl encodes Calmodulin CALMl gene previously associated with hip OA in two independent studies of Japanese patients (13).
  • a functional assay in which the associated allele modulated chondrogenic activity confirmed the relevance of the promoter SNP (rsl2885713).
  • Polymorphisms and/or SNPs in CALMl, FLRT2 and DIO2 genes are preferably detected in the method of the invention for determining the risk of a subject for developing OA.
  • the method of the invention for determining the risk of a subject to suffer from or develop OA preferably determines the presence of allelic variants and/or polymorphisms, i.e. different alleles of gene sequences comprising coding or non- coding and regulatory sequences, most preferably polymorphisms any of the genes discussed above or in combinations of these genes. Most preferably such an allele or mutation comprises a single nucleotide polymorphism (SNP), located in any of the genes located between 2q33.2-2q34, and/or on 14q24.3-14q32.12, as discussed above, for determining the risk of a subject for developing or suffering from osteoarthritis, in particular familial generalized OA with early and/or late onset.
  • SNP single nucleotide polymorphism
  • the polymorphism is preferably a polymorphism in a coding region, affecting translation and altering the encoded amino acid or RNA splicing, but it may also reside in non-coding regions affecting splicing and/or regulation of transcription or translation.
  • the invention also provides for probes and/or oligonucleotides specific for polymorphisms according to the invention. It is well within the capabilities of the skilled artisan to select sequences for making probes that can be used in specific polymorphism discriminating assays.
  • the method of the invention for determining the risk of a subject for developing or suffering from OA extends to differential levels expression of genes, which can be detected in RNA expression or protein profiles, in particular of the genes discussed above on chromosomal loci 2q33.2-2q34 and 14q24.3-14q32.12. Differential expression of the polymorphic alleles of the above discussed genes may explain the diagnostic or prognostic risk of developing OA independent of the observed variants.
  • the invention provides for specific chromosomal locations (2q33.2-2q34 and 14q24.3-14q32.12) in which the genes reside that determine the risk of a subject to develop osteoarthritis when differentially expressed.
  • Expression levels may be determined by assays customary in the art, such as quantitative PCR, Northern blotting, micro-array hybridization and the like. Detection may also take place by differential protein expression analysis on western blots, 2D gels, immunoassays and the like. Differential expression analysis on RNA or protein level is herein defined as statistically significant (p ⁇ 0.05) difference in expression (comprising up- and down- regulation) of genes in subjects diagnosed with familial generalized OA of early and/or late onset, in comparison to a 'normal' population, representative of subjects not affected by OA.
  • the invention also provides molecular probes, such as but not limited to: nucleotides, oligonucleotides, RNA, DNA, PNA or modified versions and mixtures thereof, that are specific for the polymorphisms in genes that reside within the chromosomal regions depicted.
  • the invention pertains to probes which span or flank or comprise a polymorphism in a nucleic acid sequence on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12, capable of hybridizing or annealing thereto under permissible conditions.
  • the probes anneal to polymorphisms in a coding or in a regulatory sequence that is indicative of the presence or the absence of predisposition of a subject for developing OA.
  • the polymorphism may either be linked directly, i.e. where there is a direct physiological link between the polymorphism and the OA phenotype, or the polymorphism may be linked indirectly or genetically with the OA phenotype, i.e. where the linkage of the OA phenotype with the polymorphism is to be determined in the context of familial analysis and linkage data for family members is required.
  • the invention hence also pertains to oligonucleotides which span or flank or comprise a polymorphism in a nucleic acid sequence on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12, preferably in or near a coding sequence, and which are indicative of the presence or the absence of predisposition of a subject for developing OA.
  • the method of the invention may preferably be carried out using probes and/or oligonucleotides that are capable of detecting the mRNA or cDNA of genes that resides on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12 and may quantify the amount of expression.
  • An oligonucleotide probe according to the invention capable of hybridizing to or near the polymorphism in any of the genes located on human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 discussed above, is preferably 8 to approximately 45 nucleotides in length, preferably in the range of 15 to 25 nucleotides.
  • the oligonucleotide may contain modification at the 5' or 3' ends, and may contain alternative nucleotides or analogues, or labels, such as fluorescent, enzymatic or radioactive labels or immunogenic haptens.
  • a SNP and/or allele specific nucleotide according to the invention may be physically linked to a solid support, such as but not limited to: a nylon membrane, glass slide, silica- or nitrocellulose support or on an array, preferably on a DNA microarray device.
  • a solid support such as but not limited to: a nylon membrane, glass slide, silica- or nitrocellulose support or on an array, preferably on a DNA microarray device.
  • Techniques for detection of polymorphisms in a genome are well described and known to the skilled person and may for instance be found at Techniques for detection of differentially expressed genes are well described and known to the skilled person and may for in stance be found at http://www.pro tocol- online.org/prot/Genetics_Genomics/Microarray/
  • a single nucleotide polymorphism (SNP) or gene may be detected on genomic
  • DNA, RNA or cDNA molecules and samples obtained from a subject preferably of a subject of a family wherein early onset OA has been diagnosed with at least some individuals, for instance 1, 2, 3, 5, 10, 20 or more family members, or with a frequency of at least 0.1, 1, 2, 5, 10 or more percent of all family-members examined.
  • Detection of SNP 's in a nucleic acid molecule may be performed using any standard technique customary in the art using oligonucleotides spanning or flanking the SNP, such as but not limited to: specific hybridization of an allele specific oligonucleotide, allele specific oligonucleotide ligation assays, allele specific DNA amplification (PCR or NASBA), or allele specific primer elongation and labeling techniques.
  • One of the principal methods used for the analysis of the nucleic acids of a known sequence is based on annealing two probes to a target sequence and, when the probes are hybridised adjacently to the target sequence, ligating the probes.
  • the OLA- principle (Oligonucleotide Ligation Assay) has been described, amongst others, in US 4,988,617 (31).
  • This publication discloses a method for determining the nucleic acid sequence in a region of a known nucleic acid sequence having a known possible mutation. To detect the mutation, oligonucleotides are selected to anneal to immediately adjacent segments of the sequence to be determined.
  • One of the selected oligonucleotide probes has an end region wherein one of the end region nucleotides is complementary to either the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence.
  • a ligase is provided which covalently connects the two probes when they are correctly base paired and are located immediately adjacent to each other. The presence or absence of the linked probes is an indication of the presence of the known sequence and/or mutation.
  • US 5,876,924 by Zhang et al. describes another ligation reaction using two adjacent probes wherein one of the probes is a capture probe with a binding element such as biotin.
  • WO 97/45559 (Barany et al.) describes a method for the detection of nucleic acid sequence differences by using combinations of ligase detection reactions (LDR) and polymerase chain reactions (PCR).
  • LDR ligase detection reactions
  • PCR polymerase chain reactions
  • annealing allele-specific probe sets to a target sequence and subsequent ligation with a thermostable ligase optionally followed by removal of the unligated primers with an exonuclease.
  • Amplification of the ligated products with fluorescently labelled primers results in a fluorescently labelled amplified product. Detection of the products is based on separation by size or electrophoretic mobility or on an addressable array.
  • the invention also provides for a kit of parts, comprising at least a molecular probe as described above, specific for a polymorphism in the human chromosome 2q33.2 to 2q34 and/or 14q24.3 to 14q32.12 which is directed at a diagnostic or prognostic test to establish whether a subject or a group of subjects, preferably genetically related subjects or family members, are at risk of suffering from or developing symptoms of OA, preferably familial generalized OA at later or earlier ages of onset.
  • the kit may also comprise a solid support comprising at least one and preferably more oligonucleotide probes for carrying out the method of the invention, preferably in a micro-array format.
  • the invention also provides for a kit of parts, comprising at least of molecular probe as described above, specific for the genes within the chromosomal regions 2q33.2 to 2q34 and/or 14q24.3 to 14q32.12 that, when showing differential expression of one or more genes, may be directed at a specific diagnostic or prognostic outcome which establishes whether a subject or a group of subjects, preferably genetically related subjects or family members, are at risk of having or developing symptoms of OA, preferably familial generalized OA.
  • these differentially expressed genes may direct towards therapies based on the suppression or stimulation of the expression of gene or gene product, for instance using RNA interference techniques.
  • the invention provides a nucleic acid construct comprising a nucleotide sequence encoding an RNAi or antisense agent that is capable of at least partly inhibiting the expression of a DI02 gene having the SNP rs225014
  • nucleic acid construct (Ala92, mutant allele) in a cell, wherein optionally the nucleotide sequence encoding the RNAi agent is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell.
  • This nucleic acid construct is herein referred as the inactivating nucleic acid construct.
  • SNP rs225014 (Ala92, mutant allele) is given as SEQ ID NO:1.
  • the encoded D2 enzyme is given as SEQ ID NO:2.
  • An RNAi agent is an RNA molecule that is capable of RNA interference or that is part of an RNA molecule that is capable of RNA interference. Such RNA molecules are referred to as siRNA (short interfering RNA, including e.g. a short hairpin RNA).
  • the nucleotide sequence that encodes the RNAi agent preferably has sufficient complementarities with the cellular nucleotide sequence DIO2 with SNP rs225014 to be capable of at least partly inhibiting the expression of the encoded D2.
  • the RNAi agent has at least 80% identity with SEQ ID NO:2 and has 100% identity with a fragment of at least 20 nucleotides encompassing the SNP rs 225014.
  • At least partly inhibiting the expression preferably means inhibiting at least 50% of the expression, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, 90%, 95% or more of the expresssion of the DIO2 gene having the SNP rs225014. Expression is preferably measured by Northern or by Arrays. More preferably by Arrays.
  • the antisense molecule may be provided to the cells as such or it may be provided by introducing an expression construct into the chondrocyte, whereby the expression construct comprises an antisense nucleotide sequence that is capable of at least partly inhibiting the expression of the nucleotide sequence encoding a polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving transcription of the antisense nucleotide sequence in a chondrocyte.
  • the expression level of a polypeptide may also be decreased by introducing an expression construct into a chondrocyte, whereby the expression construct comprises a nucleotide sequence encoding a factor capable of trans-repression of the endogenous nucleotide sequence encoding a polypeptide.
  • the promoter preferably is a promoter that is specific and functional for a given cell. Depending on the identity of the cell, the skilled person will know which promoter is most appropriate.
  • the promoters for use in the nucleic acid constructs of the invention are preferably of mammalian origin, more preferably of human origin.
  • the cell is a chondrocyte.
  • the chosen promoter has preferably a transcription rate that is higher in a chondrocyte than in other types of cells.
  • the promoter's transcription rate in a chondrocyte is at least 1.1, 1.5, 2.0 or 5.0 times higher than in a non-chondrocyte as measured by arrays or Northern.
  • the invention provides a nucleic acid construct comprising a nucleotide sequence, encoding a D2 polypeptide, said nucleotide sequence not comprising the SNP rs225014 (Thr92, wild type allele) and having at least 70% identity with SEQ ID NO: 3, wherein the nucleotide sequence is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell.
  • This nucleic acid construct is herein referred to as the other nucleic acid construct.
  • SEQ ID NO:3 represents a nucleotide sequence of the DIO2 gene which does not have the SNP rs225014 (Thr92, wild type allele). This sequence is called the wild type DIO2 (Thr92, wild type allele).
  • SEQ ID NO:4 is the amino acid sequence of the encoded D2 enzyme (Thr92, wild type allele).
  • both types of nucleic acid constructs herein defined are used in a chondrocyte.
  • both types of nucleic acid constructs herein defined are a viral gene therapy vector selected from gene therapy vectors based on an adenovirus, an adeno-associated virus (AAV), a herpes virus, a pox virus and a retrovirus.
  • a preferred viral gene therapy vector is an AAV or Lentiviral vector.
  • the invention provides a method for preventing, delaying and/or treating familial generalized osteoarthritis in a subject, the method comprising pharmacologically altering the activity or the steady-state level of the polypeptide encoded by the gene DIO2 gene having the SNP rs225014.
  • the presence of the SNP rs225014 has first been identified in a subject.
  • the activity or steady-state level of the polypeptide D2 having the SNP rs225014 is altered and optionally the activity or steady-state level of a polypeptide D2 not having the SNP rs225014 is restored in order to mimick the physiological situation.
  • the activity or steady-state level of D2 having the SNP rs225014 may be altered to suboptimal at the level of the polypeptide itself, e.g. by adding an antagonist or inhibitor of this polypeptide to a cell, such as e.g. an antibody against this polypeptide and/or preferably by introducing the inactivation nucleic acid construct as earlier defined herein and/or preferably by introducing the other nucleic acid construct as earlier defined herein.
  • the activity or steady-state level of a polypeptide is decreased by regulating the expression level of a nucleotide sequence encoding the polypeptide.
  • the expression level of a nucleotide sequence is regulated in the environment of a chondrocyte, preferably within a chondrocyte.
  • a D2 polypeptide may be added from an exogenous source to a cell.
  • the polypeptide may conveniently be produced by introduction of a nucleic acid construct earlier named the other nucleic acid construct and comprising a nucleic acid molecule encoding the D2 not having the SNP rs225014.
  • the method comprises the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an inactivating nucleic acid construct as earlier herein defined. More preferably, the pharmaceutical composition is administered in the environment of chondrocytes or preferably within chondrocytes.
  • the chondrocyte is preferably a chondrocyte from a subject suspected to have a high risk of developing OA, due for example to its age or its genetic background or to its diet.
  • the method of the invention is applied on chondrocytes from subjects diagnosed as already developing OA.
  • the diagnostic method used is preferably the one of the invention already earlier described herein.
  • the chondrocyte could be either from a subject being in an early stage of OA, or in a late stage of OA.
  • chondrocyte chosen to be treated may be isolated from the subject they belong to. Alternatively and according to a preferred method, the cells are not isolated from the subject they belong to.
  • a classical OA treating methods include pain relief medication such as paracetamol and/or NSAID (Non- Steroidal Anti-Inflammatory Drugs) and/or joint replacement and/or glycosamine glycans.
  • NSAID Non- Steroidal Anti-Inflammatory Drugs
  • an OA treating method comprises the addition of active T4 and/or active T3 in order to mimick the activity of D2.
  • the invention provides the use of a nucleotide sequence as present in any of the nucleic acid constructs (inactivation or the other nucleic acid construct) as earlier defined herein or of any of the defined nucleic acid constructs (inactivation or the other nucleic acid construct) for the manufacture of a medicament for preventing, delaying and/or treating familial generalized osteoarthritis in a subject.
  • a nucleotide sequence or a nucleic acid construct are used in a method as earlier herein defined.
  • Figure 1 Pedigree structures of the FOA families. Family 1-4 were used for the initial genome wide scan, additional families for replication analysis. Circles denote females, squares, males. Blackened symbols are affected individuals and symbols with diagonal lines represent diagnostic uncertainty. The co-segregating haplotypes among affected individuals are depicted as squared boxes, recombination's as crosses. Boxes that are open at the top or bottom continue. Al is the most likely affected haplotype, whereas, A2 the second likely. NAl is the most likely unaffected haplotype and NA2 the second most likely. Family 7 is not added to the Figure since it did not contribute to the linkage.
  • FIG. 4 Suggestive evidence for linkage on chromosome 14q32.11.
  • the dotted vertical lines represent the borders of the chromosomes.
  • LD blocks genomic structures surrounding the DIO2 gene and the DIO2 SNP rs225014.
  • Diamonds are the pairwise LD indices of D' between SNP rs225014 and all other SNPs in the haplotype blocks surrounding the DIO2 gene. Position of the
  • DIO2 gene with rs225014 is indicated.
  • the height of linkage disequilibrium measure D' is shown by the gradient of the red colour.
  • Example 1 Linkage of gene variants to familial early onset generalised OA
  • Rotterdam sample The Rotterdam study (33) is a population based cohort of in total 7983
  • DD spinal disc degeneration
  • microsatellite markers For the initial genome wide scan, 231 microsatellite markers, with an average spacing of 18 cM, were used which were selected from the Cooperative Human Linkage Center (CHLC) human screening set (Weber version 8.0). Fine mapping was performed using 14 additional markers in the region of positive linkage, which were selected from the Genome Database and the Marshfield Medical Research Foundation j ⁇ t ⁇ Jjr ⁇ sewc ⁇ j ⁇ h ⁇ d ⁇ d ⁇ c ⁇ orgl ⁇ e ⁇ csl. The fluorescent-labeled PCR products were electrophoretically separated with automated laser fluorescence DNA sequencer (Pharmacia Biotec). Alleles were identified with Fragment analyzer (Pharmacia Biotech).
  • Promising variants were genotyped in a sample of the Rotterdam study by using Sequenom homogenous MassExtend Massarray System (Sequenom Inc, San Diego, CA) using standard conditions. Genotypes were analyzed using Genotyper version 3.0 software (Sequenom Inc.). Successful genotypes were obtained for 1228 samples for PTHR2 variants and in 809 samples for IDHl, NRP2, KIAAl 571, ADAM23 and PIP5K3. 1.1.4 Two-point model based linkage analysis
  • Model based linkage analysis was performed using the FASTLINK 2.2 version of the linkage program MLINK (36). The disease locus was modeled as an autosomal dominant trait with a disease frequency of 0.001 since symptomatic and radiographic OA at multiple joint sites before the age of 50 is rare. Marker allele frequencies were used from the CEPH database and compared with data of the founders. Penetrances were assumed to rise linearly form 0% at age 15 to 100% at age 50 with a phenocopy penetrance of 0.001. Model based linkage analysis procedures can handle large pedigrees but only up to one or two loci at a time.
  • GENEHUNTER2 Multipoint, model free linkage analysis was performed and haplotypes were constructed using GENEHUNTER2 (37).
  • the Genehunter software does not require assumptions related to mode of inheritance or penetrance and it examines whether the allele sharing among affected relatives is greater than expected under the null hypothesis.
  • the program can handle large numbers of loci but only on a small number of family members at a time.
  • the first step was to genotype an 18-cM map of markers using 4 original FOA families. Any marker that showed a two- point LOD score of > 1 in the parametric linkage analysis was indicated as initial linkage signal.
  • the second step was to replicate the initial linkage result using 3 additional FOA families that were ascertained during the geno typing of the whole genome scan.
  • the third step consisted of refinement of the most promising linkage result by fine mapping analysis in all available FOA families.
  • multipoint model free linkage analyses was applied to increase the marker informatively and to check the robustness of the observed model based linkage.
  • Genomic DNA was isolated from EDTA blood of affected and unaffected family members. Initially, three affected family members (individual 14 from family 1, individual 10 from family 2 and individual 9 from family 4) were screened for possible mutations by direct forward and reverse sequencing from both ends ( Figure 1). If a novel variant was identified, unaffected family members of these families and family members of remaining families were sequenced. Reference sequences corresponding to all coding and 5' and 3' UTR regions of the genes were obtained from the UCSC genome browser assembly May 2004 (http://genome.ucsc.edu/) or the Ensembl Genome database v35 (www.ensembl.org), NCBI build 35. Example 1 Table 2 shows the Genbank numbers.
  • forward and reverse primer sets were designed with at least 25 bp flanking intronic sequences using Primer3 (http://www.broad.mit. edu/cgi-bin/primer/primer3_www.cgi) with the conditions described by (38).
  • Primer3 http://www.broad.mit. edu/cgi-bin/primer/primer3_www.cgi
  • 3'UTR 0 ⁇ NRP2 and exons 1-27 O ⁇ PIP5K3 have not been sequenced due to current genome browser updates.
  • PCR amplifications were carried out in a volume of 15 ⁇ l that contained 15 ng genomic DNA, 4.1 pmol of the PCR primers, 1.5 mM MgCl 2 , 0.2 mM and 0.6 units of rTaq polymerase (Amersham Biosciences) or 0.6 units of HotfirePol ® DNA polymerase and solution S (Solis Biodyne) for GC-rich regions or standard conditions of the GC-rich PCR system (Roche).
  • PCR products were purified using Multiscreen 96 well plates (Millipore) filled with Sephadex (Amersham biosciences) and quantified on 1.5% agarose gels. PCR products were sequenced for possible mutations using an ABI3730 capillary sequencer with Big Dye chemistry (Applied Biosystems).
  • PolyPhen http://tux.embl-heidelberg.de/ramensky/index.shtml
  • SIFT http://blocks.fhcrc.org/sift/SIFT.html
  • exonic variants were screened for exonic splicing enhancers sequences using http://exon.cshl.edu/ESE (42). Conservation was determined using the Multiz Alignments and Conservation track of the UCSC genome browser (http://genome.ucsc.edu/) which shows a measure of evolutionary conservation in human, chimp, mouse, rat, dog, chicken, fugu, and zebra fish.
  • Family 1 recombination's on both sides of the haplotype by individual 3 and/or 5; both affected.
  • Family 2 recombination on upper site of haplotype by proband (individual 17) and lower site of haplotype by individual 8.
  • Family 3 recombination on upper site of haplotype by individual 9 (affected) and lower site of haplotype by individual 4 (unaffected);
  • Family 36 recombination on upper site of haplotype by individual 7 (affected) and lower site of haplotype by individual 8 (affected)
  • NMJ45280 NMJ45280 hepatocellular carcinoma- 4 associated antigen (NP_660323)
  • NM_152671 NM_015040 phosphonositide d2s2178 PTH R2 Parathyroid hormone receptor 13
  • family 2 and 4 two haplotypes are shared among affected individuals which are absent in the unaffected relatives resulting in two possible haplotypes that contribute to the linkage.
  • family 2 the most likely haplotype with alleles 3-6-7-8 (Al), allocates one phenocopy (individual 9) whereas the second likely haplotype 7-6-5-8 (A2) allocates 2 phenocopies (subject 6 and 17).
  • family 4 the most likely haplotype (Al) allocates no phenocopies and the second likely haplotype (A2) allows one phenocopy (individual 7).
  • the missense variant (c.786G>T, p.A225S) was located within the second extracellular domain of the gene and was found on the second likely haplotype A2 of family 4. As a result the more distant affected subject number 7 in family 4 (niece) did not carry the mutation. The mutation, therefore, may be causal to the FOA phenotype only when the distant relative is a phenocopy.
  • the non-synonymous change was predicted to be benign by the PolyPhen software (43).
  • variants of Table 5 indicated 26 novel variants (17 SNPs and 9 insertion/deletion polymorphisms). From these 26 variants, nine promising variants co segregated with OA within one or more families as illustrated in Table 4. Three of these variants were found in coding regions and involved an amino acid change: (KIAAl 571 R2133S, IDHl Y183C and PTHR2 A225S).
  • the G/T nucleotide change in the third exon of KIAAl 571 results in disruption of exonic splicer enhancer motifs which serves as binding site for Serine/Arginine protein 40 and 55 and might be therefore a functional variant.
  • This gene is a predicted gene, little is known about other possible predicted functional effects on the protein.
  • the novel variant KIAAl 571 R2133S co segregated in family 4 and 7 with OA and showed a rare population frequency of 0.01 corresponding to nine carriers of 781 genotyped (Tables 5 and 6). Only 2 carriers of KIAAl 571 R2133S showed generalised OA and one carrier had no ROA at all which reveals this variant as an unlikely causal mutation.
  • Isocitrate dehydrogenase 1 encodes a cytoplasmic enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and has a significant role in cytoplasmic NADPH production (47).
  • IDHl two variants (Y183C and c.933- 28C>T) co segregated with the OA phenotype.
  • IDHl Y183C co segregated in affected family members in family 2 and was located in the isocitrate/isopropylmalate dehydrogenase domain (PFOO 180) of IDHl in exon 6.
  • the variant is predicted to be probably damaging for the protein structure/function by SIFT and PolyPhen, and highly conserved across all eight species investigated. Based on these results, this variant could be functional for the onset of generalised OA.
  • the Rotterdam sample we observed 14 carriers out of 785 genotyped corresponding to a frequency of 0.02.
  • IDHl c.933-28C>T Another variant in this gene, IDHl c.933-28C>T, was identified in family 2 and 4, near the intron/exon boundary of exon 7. This variant was not conserved and showed a frequency of 0.04 in the Rotterdam sample. The frequency of generalised ROA was not different between carriers and non carriers of this variant.
  • Neuropilin 2 (NRP2), is an interesting gene because it encodes for the co- receptor of vascular endothelial growth factor ⁇ (VEGF 16 s) which is an essential factor for endochondral ossification (27-28). Furthermore, VEGF and its receptors are expressed in OA cartilage and VEGF stimulates production of extracellular cartilage matrix degrading matrix metalloproteinases (MMPs) (28-29). In the NRP2 gene, two novel variants were found: c.941A>C and c.l938-21T>C.
  • MMPs extracellular cartilage matrix degrading matrix metalloproteinases
  • NRP2 c.941A>C was identified in a residue with a moderate conservation score and which showed a frequency of 0.03 in the random population and no increase of generalised ROA among carriers.
  • the second NRP2 variant, c.l938-21T>C was not conserved and co segregated in three families (1, 2, and 4) and was more frequent in the population (0.08).
  • Phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, type III catalyzes the phosphorylation of phosphatidylinositol-4-phosphate and has a role in endosome-related membrane trafficking (48)
  • PIP5K3 c.8429T>A and PIP5K3 c.8434insC novel variants in the 3'UTR region of PIP5K3 in family 4 which are both highly conserved residues.
  • A1 represents the most likely haplotype and A2 the second likely affected haplotype
  • NM_018534 (gi:41872543), NM_201266 (gi:41872561 ), NM_201264 (gi:41872556)
  • NDUFS1 5 1 UTR 1-15(C);2-7/4-8 C/G no rs4147707 (CIG) intron4 1-15/2-7/4-8[25T] 190 del no rs10560955 (23T/25T) intron4 1-15/2-7 (11 T) 4- 175 del no No 8[10T] (10T/11T)
  • CTLA4 exon 1 4-8 (G);1-14/2-7(A) 4 205 NQ yes (JIk) rs231775 np060229 exoni 1-14/2-3/4-9 1,2,4 471 del(9T/10T) utr No intron2 1-14/4-9(C/T);2- 1,4 277 C/T no rs17286674 3(TT)
  • Variant frequency carriers ' frequency carriers with
  • Example 2 Genome wide scan, identification of a haplotype containing a non- synonymous SNP in DIO2 associated with symptomatic Osteoarthritis 2.1 Subjects and methods
  • Probands (ages 40-70 years) and their siblings have OA predominantly at multiple joint sites of the hand or in two or more of the following joint sites (hand, spine (cervical or lumbar), knee or hip 63 .
  • Subjects with symptomatic OA (as defined below) in just one joint site were required to have structural abnormalities in at least one other joint site defined by the presence of ROA in any of the four joints or the presence of two or more Heberden's nodes, Bouchard's nodes, or squaring of at least one first carpometacarpal (CMCl) joint on physical examination.
  • Symptomatic OA in the knee and hip was defined according to the American College of Rheumatology (ACR) recommendations for knee and hip OA 64 ' 65 .
  • ACR American College of Rheumatology
  • Knee OA was defined as pain or stiffness for most days of the preceding month and osteophytes at the joint margins of the tibiofemoral joint (x ray spurs).
  • Hip OA was defined as pain or stiffness in the groin and hip region on most days of the preceding month in addition to femoral or acetabular osteophytes or axial joint space narrowing on radiography.
  • Prosthetic joints in the hips or knees as aresult of end stage OA were defined as OA in that particular joint.
  • Spine OA (cervical and lumbar) was defined as pain or stiffness in the spine on most days of the preceding month, in addition to a Kellgren/Lawrence score of two in at least one disc or one apophyseal joint.
  • OA in hand joints was defined according to the ACR criteria as pain or stiffness on most days of the preceding month in addition to three of the following four criteria: bony swelling of two or more of the ten selected joints (bilateral distal interphalangeal (DIP) joints 2+3, bilateral proximal interphalangeal (PIP) joints 2+3, and CMCl joints), bony swelling of two or more DIP joints, fewer than three swollen metacarpalphalangeal (MCP) joints, and deformity of at least one of the ten selected joints.
  • DIP distal interphalangeal
  • PIP bilateral proximal interphalangeal
  • CMCl joints bony swelling of two or more DIP joints
  • MCP metacarpalphalangeal
  • deformity of at least one of the ten selected joints Radiographic OA was assessed by one trained radiologist by the Kellgren/Lawrence (0-4) method.
  • Intrareader variability for the different joint sites was assessed: the intraclass correlation coefficient (ICC, with 95% confidence interval) was for the hands, 0.95 (0.92 to 0.96); for the knees (tibiofemoral), 0.92 (0.86 to 0.96); for the hips, 0.95 (0.92 to 0.98); for the cervical spine (apophyseal and disc), 0.71 (0.52 to 0.84); and for the lumbar spine (apophyseal and disc), 0.67 (0.46 to 0.81).
  • Intrareader variability was based on an examination of 40 radiographs that were selected randomly throughout the duration of the study period and were blinded for any patient characteristics. Radiographic OA was regarded as having Kellgren/Lawrence score ⁇ 2. Ethical approval for the study was obtained from the appropriate ethics committees.
  • the Rotterdam study which comprises 7,983 Caucasian participants, is a prospective, population-based cohort study of the determinants and prognosis of chronic diseases in the elderly .
  • the average age of the cases at replacement surgery was 65 years with an age range of 56-85 years.
  • the controls comprised individuals with no signs or symptoms of arthritis or joint disease (pain, swelling, tenderness or restriction of movement).
  • the average age of the controls at recruitment was 69 years with an age range of 55-89 years. Due to ethical and financial constraints the hip joints of the controls were not subjected to radiographic analysis. All cases and all controls were UK individuals of white European ethnicity.
  • a complete genome-wide scan containing 403 microsatellite markers with an average spacing of 10 cM was performed in 187 pairs and four trios with OA at multiple joint sites from the GARP study.
  • Markers and 14 additional microsatellite markers for fine mapping on chromosome 6, 10, 13 and 14 were taken from Human Linkage Set v2.5 MDlO or HD5 (Applied Biosystems), respectively and measured using an ABI Prism DNA Analyzer 3700 (Applied Biosystems). Genotyping was performed using standard conditions and reagentia with some exceptions. The amount of polymerase chain reaction (PCR) primer pairs for the markers was reduced up to 5-fold and duplex PCR reactions were designed if possible to reduce costs, time expense and amount of genomic DNA used.
  • PCR polymerase chain reaction
  • Genotypes were analysed by using Genemapper version 2.0 and 3.0 (Applied Biosystems). As quality control, approximately 8% of the samples were genotyped in duplicate and compared. In addition, 48 additional family members from 36 different sibling pairs were genotyped to improve our ability to detect genotyping errors and estimate allele sharing. Mendelian errors were checked for Mendelian inconsistencies and unlikely recombinants using Merlin 69 . These quality checks indicated that marker D6S434 and D9S158 from the Human Linkage Set v2.5 MDlO could not be genotyped reliably in our hands due to unclear one base pair differences. Subjects and markers showed an average success rate of 96% (range 77-100%) and 96% (range 83-100%), respectively. Family relationships were verified using the GRR program 70 . Eight sibling pairs showed pedigree errors and were removed for further analysis. In seven of these sibling pairs, individuals reported to be full siblings were almost certainly half siblings. The remaining siblings were monozygotic twins.
  • a locally developed SQL database was used to store genotypic data, compare repeated genotypes and generate output files for linkage analysis.
  • the location of the markers was taken from an integrated genetic map of David Duffy with interpolated genetic map positions (see URL below).
  • the position is in Decode cM, estimated via locally weighted linear regression (lo(w)ess) from the Build 35.1 (and 34.3) physical map positions and published Decode and Marshfield genetic map positions.
  • Tagging SNPs were selected from HapMap Public Release #19 applying the efficient multimarker method with r 2 > 0.8 and minor allele frequency (MAF) > 0.05 implemented at in two or more (see also URL below) 71 . Tagging SNPs or their proxies were chosen to fit efficiently in a Sequenom multiplex assay.
  • Tagging SNPs were genotyped by mass spectrometry (the homogeneous MassARRAY system; Sequenom, San Diego, CA) using standard conditions. PCR reactions were carried out in a final volume of 5 ⁇ l and contained standard reagents and 2.5 ng of genomic DNA. Genotypes were assigned by using Genotyper version 3.0 software (Sequenom, San Diego, CA). The functional SNP rsl2885713, located in CALMl, and the DIO2 SNPs rsl2885300 and rs225014 in the Rotterdam study were genotyped using a Taqman by design assay and an ABI Prism DNA Analyzer 7900 (Applied Biosystems) with standard conditions. In addition, genotype distributions of all SNPs were in agreement with Hardy Weinberg equilibrium and approximately 8% of the subjects were genotyped twice and checked. 2.1.5. Statistical analysis Linkage analysis
  • Nonparametric linkage analysis was carried out by use of the .Span- statistics implemented in the software Merlin . LOD scores were plotted on a common 1 cM grid.
  • MINX Merlin-In-X
  • the variance of the NPL test statistic was computed by Monte Carlo simulations of marker genotypes under the Housing Merlin.
  • a global p- value for chromosome 14 was computed by smoothing the likelihood with respect to a uniform prior distribution for gene location 73 .
  • Pearson's correlation coefficient between the LOD score and hip, knee or hand OA families were calculated.
  • GARP Genetics, osteoARthritis and Progression
  • BMI body mass index
  • the GARP sibling pairs comprise 183 sibships; 179 siblings and four
  • OA susceptibility loci To search for major osteoarthritis (OA) susceptibility loci we performed a genome wide linkage scan containing 403 microsatellite markers with an average spacing of 10 cM in the ongoing GARP study, which consists of 179 Caucasian sibling pairs and four trios of Dutch origin affected predominantly by symptomatic OA at multiple sites (for detailed phenotypic description Table 7) 63 .
  • Chrom chromosome
  • cM centiMorgan
  • the location of the linkage peak coincided with the CALMl gene, previously associated with symptomatic hip OA in the Japanese population 75 .
  • a search of public genome resources revealed two other attractive candidate genes within the linkage area: FLRT2, encoding a small molecule found in the extracellular matrix of cartilage , and DIO2, encoding for D2, a selenoprotein that converts intracellularly inactive thyroxine (T4) to active thyroid hormone, (T3) (Fig. 4).
  • T4 intracellularly inactive thyroxine
  • T3 active thyroid hormone
  • Thyrotropin receptor precursor TSH-R
  • NM_001018036 hormone receptor
  • FLRT2 NM_013231 Leucine-rich repeat transmembrane protein FLRT2 precursor (Fibronectin-like domain-containing leucine-rich transmembrane protein 2).
  • GPR65 NM_003608 Psychosine receptor (G-protein coupled receptor 65) (T cell- death associated protein 8).
  • GPR65 NM_003608 Psychosine receptor (G-protein coupled receptor 65) (T cell- death associated protein 8).
  • PTPN21 NM_007039 Tyrosine-protein phosphatase non-receptor type 21 (EC 3.1.3.48) (Protein-tyrosine phosphatase D1).
  • TTC8 NIVM44596 Tetratricopeptide repeat protein 8 (TPR repeat protein 8)
  • NM_198309 Bardet- Biedl syndrome 8 protein.
  • KCNK13 NM_022054 Potassium channel subfamily K member 13 (Tandem pore domain halothane- inhibited potassium channel 1 ) (THIK-1 )
  • PSMC1 NM 002802 26S protease regulatory subunit 4 (P26s4) (Proteasome 26S subunit ATPase 1).
  • GARP Genetics, osteoARthritis and Progression
  • MAF minor allele frequency aMinor allele frequency (n minor alleles/ n total alleles) b P-values using combined linkage and association with the program LAMP (see URLs provided in paper).
  • SNP single nucleotide polymorphism
  • MAF minor allele frequency
  • IBD identical by descent
  • OR odds ratio a Minor allele frequency stratified for IBD status (sum of minor alleles / sum of total alleles) b P- values observed using combined linkage and association with the program LAMP (see Web resources) 74 .
  • SNP single nucleotide polymorphism P-value of x statistics for specific case group versus the specific control sample
  • Haplotype analysis of the DIO2 SNPs rsl2588300 (OT) and rs225014 (T>C) in the combined confirmation/replication sample revealed three common haplotypes with frequencies > 0.05 (Supplementary Table 5).
  • the common haplotype CC (frequency 0.34) exclusively carried the minor allele of DIO2 SNP rs225014.
  • DIO 2 SNP rs225014 is non- synonymous, resulting in the amino acid change Thr92Ala.
  • Residue 92 is the first amino acid of the instability loop in D2 and this loop is the key determinant of D2 turnover rate 80 .
  • in vitro studies have not been able to assess a functional difference between the 92Thr and the minor 92AIa allele on the instability loop in HEK293 cells it was shown by Canani et al that D2 velocity is decreased and insulin resistance is increased in tissues of homozygous 92AIa patients with type 2 diabetes mellitus.
  • functional relevance on enzyme activity was shown by others for DIO2 SNP rsl2885300, which is localized in a short open reading frame a (ORFa) within the 5 '-untranslated region of the gene and also known as D2-ORFa- Gly3 As .
  • the ORFa has been shown to reduce the D2 translation efficiency. In vitro studies by others have shown that this inhibition was abolished by mutating the start
  • the D2 enzyme as key regulator of local T3 availability may contribute to osteoarthritis development in different ways.
  • the conversion of T4 into T3 by D2 inhibits chondrocyte proliferation but stimulates chondrocyte differentiation and subsequently bone matrix synthesis. This process of endochondral ossification is essential for the formation of the skeleton 88 .
  • Lower expression of D2 in the growth plate has been shown to contribute to the pathogenesis of tibial chondrodysplasia in chicken 89 .
  • chondrocytes In osteoarthritic cartilage, chondrocytes, in an attempt to repair damaged matrix, show increased metabolic activity. During progression of OA, chondrocytes undergo phenotypic dedifferentiation to a hypertrophic state expressing similar features as chondrocytes residing in the growth plate . As such chondrocyte hypertrophy debilitates cartilage viability by a switched expression of bone specific collagens which initiates calcification of the matrix and up-regulation of cartilage specific proteolytic enzymes . Furthermore, it was recently shown that inflammatory signals, up regulate D2 expression via a dimer combination of ReIA (p65) with nuclear factor- ⁇ B (NF- ⁇ B) . This p65/NF- KB dimer has also been shown to activate pro-inflammatory cytokines in chondrocytes mediating cartilage degeneration.
  • ReIA p65
  • NF- ⁇ B nuclear factor- ⁇ B
  • OA is characterized by formation of bony enlargements at the edges of the bone called osteophytes a process which is characterized by endochondral ossification .
  • Our findings underscore the importance of thyroid hormone in the etiology of symptomatic OA. We are confident that knowledge of genetic factors and their molecular cascades contributes to a better understanding of the pathogenesis of osteoarthritis and will lead to improved diagnosis, treatment and prevention. Supplementary Table 3 Association of DIO2 SNP rs12885300 between subjects with hip and/or knee replacement due to osteoarthritis and controls from the UK.
  • SNP single nucleotide polymorphism.
  • SNP single nucleotide polymorphism.
  • a Alleles are in the following order of SNPs rs12885300 (OT) and rs225014 (T>C).
  • Cases were females with hip replacements as compared to female controls as reference.
  • Cases were females with severe radiographic hip OA (Kellgren Lawrence grade ⁇ 3) as compared to female controls as reference.
  • dCases are siblings contributing to the linkage (identical by descent 2 status) versus subjects identical by descent 0 or 1.
  • Haplotype frequency and comparisons were adjusted for family relationship.
  • Two tailed P-values were determined by comparing the frequency of the specific haplotype compared to the summed frequency of all other haplotypes as reference.
  • Gluzman-Poltorak Z Cohen T, Herzog Y et al.
  • Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF- 145 and VEGF- 165. J.Biol. Chem. 2000; 275: 29922.
  • VEGF vascular endothelial growth factor
  • VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat.Med. 1999; 5: 623-8.
  • VEGF Vascular endothelial growth factor
  • Urinary CTX-II levels are associated with radiographic subtypes of osteoarthritis in hip, knee, hand, and facet joints in subject with familial osteoarthritis at multiple sites: the GARP study. Ann Rheum Dis 2006; 65: 360-5.
  • Hutloff A, Dittrich AM, Beier KC et al. ICOS is an inducible T-cell co- stimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6.
  • Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. J Bone Miner.Res. 2002; 17: 443-54.
  • Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. J. Bone Miner. Res. 17, 443-454 (2002).
  • Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 141, 3887-3897 (2000). 79. Bianco, A. C. & Kim, B. W. Deiodinases: implications of the local control of thyroid hormone action. J.Clin.Invest 116, 2571-2579 (2006).
  • GENEHUNTER2 http://linkage.rockefeller.edu/soft/gh/ Online Mendelian Inheritance in Man (OMIM) http://www.ncbi.nlm.nih.gov/Omim/

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Abstract

The current invention provides methods for determining the risk of a subject for developing familial generalized osteoarthritis with early or late onset, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on at least one of the human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 in the genome of a subject. The invention also pertains to gene sequences and polymorphisms therein that may be indicative of development of osteoarthritis in a subject.

Description

Title of the invention Methods and means for diagnosing and treatment of osteoarthritis.
Field of the invention
The current invention relates to the field of medicine, in particular to human genetics and diagnostics for osteoarthritis.
Background of the invention Osteoarthritis (OA [MIM 165720]) is a degenerative disease of the joints characterized by degradation of the hyaline articular cartilage and remodeling of the subchondral bone with sclerosis. Despite the major socioeconomic burden imposed by OA, the precise aetiology of this condition remains unclear. To date, there is an urgent need to improve the diagnosis and the treatment of OA. The currently available treatments for OA focus on symptom relief and improving function and has been based on analgesics and nonsteroidal anti-inflammatory drugs or arthroplasty (1,2). However, there is a lack of an effective disease modifying treatment in which degradation of joint tissue is stabilized, retarded or halted and at the same time an improvement of symptoms is reached. Results of genetic studies will contribute to the clarification of the complex genetic background of OA and the pathways involved in its occurrence and progression. Identification of genetic variation associated with OA therefore will improve our understanding of the pathogenesis and may elucidate new molecular targets for the development of new treatment. The identification of relevant genetic variation within OA patients may also provide means for a better and earlier classification and diagnosis by for instance genetic analysis to identify whether a subject is at risk of developing or having OA. Furthermore, better classification of OA patients (for example due to genetic variation that is associated with fast OA progression) may provide great opportunities in the effectiveness of clinical trials and in improving the quality and efficiency of our health-care system. Genetic factors play an important role in the etiology of OA, especially for hand, discus degeneration, hip and OA at multiple joint sites (3-7). Segregation analysis of families with early onset generalized OA indicated the presence of a major recessive OA gene with a residual multifactorial component (8). Together, it has become clear that different definitions of OA must be considered as distinct entities and may be caused by different (sets of) genes (9).
Genome wide linkage and association studies using nuclear families or sibling pairs with various subtypes of OA, including hand, hip, knee, or at multiple sites, have revealed linkage to multiple chromosome areas that may harbor OA genes (10). Successively to these linkage studies, new OA genes have emerged with variants that affected cartilage function (11-13). Investigations of these variants within other OA populations revealed confirmation in some studies (14), however, were excluded in others (15-16). Together, these studies highlight the complex and heterogeneous nature of OA genetic susceptibility. Additional genes and/or pathways may therefore be discovered to explain the genetic susceptibility to OA further. A number of other studies have focused on extended families with early onset OA phenotypes accompanied by either mild spondyloepiphyseal dysplasia or multiple epiphyseal dysplasia. In these families, several mutations in genes encoding structural proteins of the extracellular matrix (ECM) have been identified (17). Only few studies have focused on the identification of susceptibility loci and genes for familial generalised OA occurring at early ages of onset (between 20-50 years) or at later ages of onset (between 40-70 years). Given the complex heterogenous nature of OA, these studies are likely to detect both overlapping and specific OA genes. Identification of such genes may provide insight into potential pathways not yet recognized to be involved in the onset of OA. There is a need in the art for identification of genetic loci and genes and gene product involved in the pathogenesis of generalized OA occurring both at early and later ages. It is an object of the present invention to provide for nucleic acid sequences and of such genes as well as for methods of prognosis, diagnosis and therapy in which these nucleic acid sequences are applied.
Summary of the invention
The current invention provides methods for the identification and use of genetic loci involved in OA phenotypes in human subjects. These genetic loci, markers and polymorphisms can be applied in diagnostic methods for determining the risk of a subject for developing osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism in a locus genetically associated with OA phenotype or pathology. In a first aspect, the invention provides the identification of genetic loci contributing to the early onset rare familial generalized OA phenotype. In a second aspect the invention discloses genetic loci contributing to the more common generalized OA at later ages of onset and hip OA. The invention achieves its aim by providing genetic loci, nucleic acid sequences and in particular polymorphisms in or near genes that are genetically and/or physiologically linked to early onset OA pathology. The invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on at least one of the human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 in the genome of the subject. The current inventors performed a genome wide scan in families with dominant Mendelian inherited generalized OA. In these families OA occurs without detectable dysplasia and this phenotype resembles primary OA occurring at later ages in the population except for the early age of onset (20-50 years). Using a two step approach, a significant two point model based LOD score of 6.05 (theta 0.00) for marker D2S155 (chromosome 2q33.3) was observed in the second step of analysis. Multipoint model free analysis confirmed linkage in this region with a NPL score of 4.70 (Example 1 Figure 2; P-value 0.0013). Overall the linkage results appear robust since adjacent markers support the linkage in both stages and are observed both in the model based and model free approach. The inventors have further established the presence of a number of mutations and/or polymorphisms in genes within the 2q33.2 to q34 region that appear to correlate with OA in several families that were analyzed. This group of genes comprises ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDHlCPO, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl,
PIP5K3, and PTHR2. Hence the current invention provides for nucleic acid sequences which can be applied for screening, diagnosis, prognosis and treatment of osteoarthritis.
The invention also provides genetic loci, nucleic acid sequences and in particular polymorphisms in or near genes that are physiologically linked to the more common familial generalized (FOA) at later ages of onset. The inventors performed a genome wide scans in affected siblings with symptomatic OA at multiple joint sites at ages between 40-70 years (the so called GARP study). These siblings have a positive family history for OA and a complex mode of inheritance. The genome wide linkage scan revealed a locus on chromosome 14q31.11 that showed significant linkage with a LOD score of 3.03 corresponding to a nominal P-value of 1.9 x 10 "4, a global P-value for chromosome 14 of 0.0013 and a genome wide P-value of 0.0286. This locus was mainly attributable to OA at multiple joint sites. The location of the linkage peak revealed three candidate genes: calmodulin (CALMl, MIM 114180]), the fibronectin- leucine-rich-transmembrane protein 2 gene (FLRT2 [MIM 604807]) and the iodothyronine-deiodinase type 2 (D2) gene (DIO2 [MIM 601413]). By using combined linkage and association analysis, two single nucleotide variants in the DIO2 gene (SNPs rs225014 (P = 0.006) and rsl2885300 (P = 0.04)) emerged to contribute to the linkage in patients of the GARP study. Genotypes of a common haplotype, exclusively containing the minor allele of DIO2 rs225014, showed significant recessive association in females with symptomatic hip OA in two additional independent OA studies (OR 1.71, 95% CI 1.33-2.19, P = 2.6 x 10"5). These results indicate that the locus on chromosome 14q24.3-14q32.12, preferably a SNP within the DIO2 gene, is physiologically linked to the more common familial generalized (FOA) at later ages of onset, however, also concerns a susceptibility gene for even more common forms of OA such as hip OA and possibly extends to symptomatic OA features in general.
DIO2 is a regulator of thyroid hormone metabolism in the growth plate and may confer susceptibility for OA. The inventors have established for the first time that the thyroid hormone metabolism via the DIO2 gene on chromosome 14q31.1 is involved in OA. Hence the current invention provides for nucleic acid sequences which can be applied for screening, diagnosis, prognosis and treatment of osteoarthritis in humans.
Detailed description of the invention The current invention provides nucleic acid sequences and sequence information that will aid in the classification, diagnosis, prognosis, treatment and development of effective treatment of osteoarthritis, in particular for familial osteoarthritis at both early and later ages of onset. The invention achieves it's aim by the identification of genetic loci in, and in close proximity of at least one of chromosomal regions 2q33.2 to 2q34 for early onset familial generalized OA and 14q24.3 to 14q32.12 for later onset familial OA. Genes that reside within these chromosomal regions that show differential expression may associate to familial OA at multiple joint sites as well as to OA at single sites such as the hip. The invention further provides sequences, in particular marker sequences and polymorphic gene sequences, which are genetically linked with OA in humans.
In a first aspect, the invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on at least one of the human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 in the genome of the subject. The method is preferably an in vitro or ex vivo method since material is taken from a subject and subsequently analyzed in vitro using the method of the invention. In a first preferred embodiment, the invention provides a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on the human chromosome band 2q33.2 to 2q34 in the genome of the subject. The genetic markers located on or in close proximity to human chromosome bands 2q33.2 to 2q34, are particularly suitable for determining the risk of a subject for developing early onset familial generalized osteoarthritis. Familial osteoarthritis may generally comprise related symptoms such as hand, discus degeneration, hip and OA at multiple sites, such as in, but not limited to OA in ankles, knees, elbows, wrists, shoulders and spine (lower back, upper back and neck areas).
In particular the method of the invention teaches the use of the following group of genetic markers for human chromosome 2 in or in close proximity of the OA associated region of human chromosome 2q33.2 to 2q34; D2S326, D2S2257, D2S2314, D2S1391, D2S118, D2S315, D2S115, D2S72, D2S1384, D2S369, D2S155, D2S2358, D2S2208, D2S154, D2S2178, D2S371, D2S164, D2S126 and gata30e06 (Table 1). In particular the area between markers D2S1384 and D2S2178 is closely linked to the genetic region on human chromosome 2 wherein the OA associated genes are located, with the highest linkage for D2S155. These markers can be advantageously applied for familial analysis, i.e. for determining which members of a family are likely to be affected or predisposed to developing symptoms of OA and which family members are likely to be unaffected. The affection or the predisposition is preferably associated with the presence of a polymorphism, preferably a SNP. The method of the invention also teaches the use of coding sequences located on human chromosomal region 2q33.2-q34 for determining the risk of a subject for developing osteoarthritis. In particular the following list of genes located on this chromosomal band may be advantageously used for determining the risk of a subject for suffering of OA or developing symptoms of OA (presence of polymorphism in); ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDHl, CPO, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl, PIP5K3, PTHR2 (Table 2). The invention also teaches the use of the positional genes located on this chromosomal region 2q33.2-q34; ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDH1.CP0, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl, PIP5K3, PTHR2 (Table 2) for determining differentially expression as detected in RNA expression or protein profiles. Differentially expressed genes located within this region may explain or associate to the diagnostic or prognostic risk of developing symptomatic OA at multiple joint sites in humans. More in particular the inventors found positive correlations with variants and/or mutations in the coding and non-coding sequences of the genes ADAM23, IDHl, PTHR2, FZD5 and NRP2 (Table 4). These five genes in particular appear to be closely linked with early onset OA in a number of OA affected families. The genes IDHl, KIAAl 571, ADAM23, PTHR2, FZD-5 and NRP2, mapped within the haplotype shared among affected family members and may be considered as sequences coding for gene products that are fully, or at least in part, responsible for the FOA phenotype. Mutations or polymorphisms outside the coding regions may affect splicing of messengers or may affect gene regulation, at the translational or transcriptional level, RNA stability or by any other means, thereby causing or contributing to the FOA phenotype. The mutations and polymorphisms may also affect the coding sequence and thereby alter functional properties of the encoded protein, such as, but not limited to the enzymatic activity, interactions with other proteins, localization and stability or half- life.
The FZD 5, encodes the Wnt signaling receptor FZ5 for the Wnt5A ligand (19) and might agonist FRZB. Detectable levels of FZD 5 mRNA were present in OA tissue and the ligand receptor pair is implicated to be essential in cell fate determination and osteoarthritis. The two exons of FZD 5 gene may potentially comprise a variant which co-segregates with the OA phenotypes in affected families. In a preferred embodiment, the polymorphism is present in the PTHR2 (parathyroid hormone 2 (PTH2) receptor). The PTHR2 is a G protein-coupled receptor selectively activated with similar potency by PTH and tuberoinfundibular peptide (TIP39) (20). Relevant functions for its possible role in OA are the expression of PTHR2 in a number of endocrine cell types that suggest a role for the PTH receptor 2 in regulating pituitary hormone secretion and specifically growth hormone. Moreover, in peripheral organs distinct cell populations express PTH receptor 2, including the calcitonin-synthesizing parafollicular C-cells of the thyroid gland involved in Ca + homeostasis and chondrocytes within cartilage growth plates of developing bone (20). Recently, the PTH receptor 2 has been shown to interact with calmodulinl (CaMl), a calcium sensing protein (21), whereas a functional SNP in the core promoter region of the gene encoding calmodulinl (CALMl), was associated with hip OA in the Japanese population (13). Although the association with this functional SNP was not replicated in a UK population (15), the CaMl mediated signaling pathway is considered to be a new paradigm in the etiology and pathogenesis of OA (22). Together, these data clearly indicate a role for the PTH receptor 2 in the onset of OA, and polymorphisms and/or SNPs in PTH2 gene may be of high relevance and are therefore particularly preferred to be detected by the method of the invention. Exploration of the biological function of the PTH2 receptor and its agonists will determine the pathophysiological process in which the PTH2 receptor may be associated to the onset of OA. Furthermore, mutations within the same pathway of calcium sensing receptors CaR and PTHRl have been associated with premature OA (23) and metaphyseal chondrodysplasia (24), respectively, underscoring the relevance of the pathway for OA. Better understanding of the etiology of OA may provide new targets for drug discovery. Upon screening all 13 exons of the PTHR2 gene, a missense mutation was identified co segregating with the affection status in family 4 causing a substitution of Alanine to Serine at position 225 within the second extracellular (e2) domain of the gene. The variant was predicted to be benign by the PolyPhen software and it appeared to be very rare in a 55-70 year old sample (N from the Rotterdam population in the examples section (N = 1228, frequency 0.004). Among the 11 carriers we observed an increased ROA sum score (3.18) as compared to the rest of the sample (2.26; P-value = 0.05). Furthermore, a possible association towards a qualitative generalized radiographic OA phenotype with an OR of 1.7, 95% CI 0.5-6.1 exists. The low frequency of the allele, however, hampered a powerful association analysis also reflected by the large confidence interval limits. Together this data may show that the exon 6 mutation is functional, and that it plays a role in the severe familial symptomatic OA as observed in family 4 in the examples section and may play an additional role in generalized OA phenotype in the population at large.
The IDHl gene encodes isocitrate dehydrogenase 1, also referred to as oxalosuccinate decarboxylase. IDHl supplies NADPH for antioxidant systems suggesting a regulatory role in cellular defense against oxidative stress and in senescence (25). Although little is known about a possible role of IDHl in cartilage but increased oxidative stress would make chondrocytes more susceptible to cell death which might contribute to the onset of OA. Hence, polymorphisms and/or SNPs in the IDHl gene are also preferably applied in the method of the current invention for determining the risk of a subject for developing OA.
The IDHl Y183C variant was a most promising variant since it co-segregated on haplotype A2 with the OA phenotype in family 2, was predicted to be damaging for the protein structure/function and concerned a very conserved amino acid. In the general population this variant was infrequent (0.02) and 36% of the carriers had generalised ROA as compared to 17% of the non carriers. Given these results and the finding that family 2 already showed a significant linkage on its own, this variant contributes to the FOA susceptibility in family 2.
The predicted KIAA1571 gene appears to belong, as predicted in Unigene, to the fibronectin type III and M protein repeat family in C. elegans. Fibronectin is a component of the extracellular matrix of cartilage and KIAAl 571 may therefore be an excellent candidate gene. The G/T nucleotide change in the third exon of KIAAl 571 (Q9HCK1) results in disruption of exonic splicer enhancer motifs which serves as binding site for Serine/ Arginine protein 40 and 55 and might be therefore a functional variant. However, because this gene is a predicted gene, little is known about other possible predicted functional effects on the protein. The novel variant KIAAl 571 R2133S co segregated in family 4 and 7 with OA and showed a rare population frequency of 0.01 corresponding to nine carriers of 781 genotyped (Example 1; Table 6). Only 2 carriers of KIAAl 571 R2133S showed generalised OA and one carrier had no ROA at all which reveals this variant as an unlikely causal mutation. (P -value = 0.57). Hence, polymorphisms and/or SNPs in the KIAA1571 gene are preferably applied in the method of the current invention for determining the risk of developing OA in a subject.
Alternatively or in combination with other preferred embodiment, the polymorphism is present in the NRP2 gene. The NRP2 gene encodes the vascular endothelial cell growth factor 165 receptor 2 (VEGF Receptor 2). Neuropilin 2 (NRP2), is a highly relevant gene because it encodes for the co-receptor of vascular endothelial growth factories (VEGF 16s) which is an essential factor for endochondral ossification (26,27). Furthermore, VEGF and its receptors are expressed in OA cartilage and VEGF stimulates production of extracellular cartilage matrix degrading matrix metalloproteinases (MMPs) (28,29).
A promising variant, NRP2 c.l938-21T>C, emerged from our mutation analysis which co segregated in three families (1, 2, and 4) contributing most to the linkage. Carriers of this variant conferred a significant increased risk of 2.1, 95% CI, 1.1-4.1, P = 0.032, to generalized ROA. Given the low effect size and the high frequency (0.08) in the random population, this variant if not sufficient alone for the onset of FOA it may be that this variant at least partly modulates OA susceptibility and interacts genetically with other variants. Hence also polymorphisms and/or SNPs in the NRP2 gene are preferably applied and detected in the method of the invention for determining the risk of a subject for developing OA. Phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, type III
(PlP 5KS) catalyzes the phosphorylation of phosphatidylinositol-4-phosphate and has a role in endosome-related membrane trafficking (48). We found two novel variants (PIP5K3 c.8429T>A and PIP5K3 c.8434insC) in the 3'UTR region of PIP5K3 in family 4 which are both highly conserved residues. In the Rotterdam sample, we observed that PIP5K3 c.8429T>A and PIP5K3 c.8434insC were in complete LD (D'= 1, r2 = 1) with a population frequency of 0.04. Polymorphisms and/or SNPs in the PIP5K3 gene are preferably applied and detected in the method of the invention for determining the risk of a subject for developing OA.
Alternatively or in combination with the first preferred embodiment, the invention discloses a second preferred embodiment, which is a method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on the human chromosome bands 14q24.3 to 14q32.12 in the genome of the subject. The inventors discovered that the locus on chromosome 14q24.3-14q32.12, preferably a SNP within the DIO2 gene is physiologically linked to the more common familial generalized (FOA) at later ages of onset, however, additional replication indicated that the gene may also concerns a susceptibility gene for even more common forms of OA such as OA at the hip joint and possibly extends to symptomatic OA features in general.
The genetic markers located between or in close proximity to human chromosome bands 14q24.3-14q32.12, are particularly suitable for determining the genes and/or polymorphisms that may contribute to the development or suffering from symptomatic OA at multiple joint sites with a familial background at later ages of onset, i.e. between 40 and 70 years. Familial generalized osteoarthritis may comprise of symptomatic OA in a combination of at least two joint locations e.g. hand, knee, hip and discus degeneration. The invention achieves it's aim by the identification of relevant genetic variation which may be based on identification of relevant genetic variation within or surrounding genes but also based on differentially expression of genes as detected in RNA expression or protein profiles, within the genes residing in the chromosomal region 14q24.3-14q32.12 that are linked to OA phenotype and/or pathology. The invention further provides sequences, in particular marker sequences and polymorphisms in or around gene sequences in the 14q24.3-14q32.12 region which are genetically linked with OA in humans.
In one embodiment the invention teaches the use of genetic markers, D14S74, D14S1037, D14S1044 and D14S280, located on human chromosome bands 14q24.3 to 14q32.12 for determining the risk of a subject for developing symptomatic OA at multiple joint sites. Together, these markers mark the boundaries of the one LOD-drop interval of this signal which encompasses 22 cM. These markers can be advantageously applied for familial analysis, i.e. for determining which members of a family are likely to be affected or predisposed to developing symptoms of OA and which family members are likely to be unaffected. The invention also teaches the use of genes and in particular polymorphisms, preferably SNP in or near the coding sequences of genes, located on human chromosome 14q24.3 to 14q32.12 region for determining the risk of a subject for developing osteoarthritis. In particular the list of genes as depicted in table 9 in example 2 located, on this chromosomal region may be advantageously used for determining the risk of a subject for suffering of OA or developing symptoms of OA.
In a preferred embodiment, the invention also teaches the use of the positional genes and more preferably the CALMl, FLRT2 and DI02 genes hat are located within the 14q24.3 to 14q32.12 region (table 10) for determining differential expression as detected in RNA expression or protein profiles. Differentially expressed genes located within the 14q24.3 to 14q32.12 region may explain or associate to the diagnostic or prognostic risk of developing symptomatic OA at multiple joint sites in humans. Differential expression may be detected on RNA or cDNA using methods known in the art such as DNA micro-array analysis and/or quantitative PCR.
The invention exploits the positive correlations with polymorphisms in the coding and non-coding sequences of the genes CALMl, FLRT2 and DIO2 that are located within the 14q24.3 to 14q32.12 region (table 10) with OA, preferably of later onset (40-70 years). These genes and in particular polymorphisms therein appear to be closely linked with developing symptomatic familial general OA at multiple joint sites in humans. The genes CALMl, FLRT2 and DIO2, mapped within the haplotype shared among affected family members and are to be considered as sequences coding for gene products that are fully, or at least in part, responsible for the OA phenotype. Mutations or polymorphisms outside the coding regions may affect splicing of messengers or may affect gene regulation, at the translational or transcriptional level, RNA stability or by any other means, thereby causing or contributing to the OA phenotype. Mutations or polymorphisms in the coding regions may affect protein function, such as enzymatic activities, protein stability, interactions with other proteins or complexes of CALMl, FLRT2 and DIO2 and thereby contribute to the OA phenotype or pathology. Polymorphisms and/or SNPs in CALMl, FLRT2 and DIO2 genes are preferably detected in the method of the invention for determining the risk of a subject for developing OA.
In a more preferred embodiment, a polymorphism, even more preferably a SNP is present in the DIO2 gene. DIO2 encodes a selenoenzyme D2 that catalyzes the conversion of thyroxin (T4) to triiodothyronine (T3) via 5-prime-deiodination and regulates the local thyroid hormone bioactivity in the growth plate (41,42). T3 inhibits chondrocyte proliferation but stimulates chondrocyte differentiation and matrix synthesis (44). Genetic variation in DIO2 will therefore have functional consequences for deiodinase activity but also for circulating iodothyronine levels.
By using combined linkage and association analysis, two single nucleotide variants in the DIO2 gene (SNPs rs225014 (P = 0.006) and rsl2885300 (P = 0.04)) emerged to contribute to the linkage in patients of the GARP study. Genotypes of a common haplotype, exclusively containing the minor allele OΪDIO2 rs225014, showed significant recessive association in females with symptomatic hip OA in two additional independent OA studies (OR 1.71, 95% CI 1.33-2.19, P = 2.6 x 10"5). Given the low prevalence of OA at multiple joint sites and the high frequency of the associated risk alleles (0.36, 0.40 and 0.42), this allele may or may not be causal itself. The true causal variant may have a lower frequency and be in LD with this variant. Accordingly, in a most preferred embodiment, a SNP in the DIO2 gene is rs225014 (Thr92Ala). This specific SNP may be associated with other polymorphisms and/or other SNP in the DIO2 gene and/or in other genes as mentioned in this whole application (present in 2q33.2 to 2q34 and/or in 14q24.3 to 14q32.12).
In another preferred embodiment, a polymorphism is present in the Fibronectin leucine rich transmembrane protein 2 (FLRT2) gene and/or in the Calmodulin gene CALMl. The FLRT2 gene encodes a small leucine-rich proteoglycan found in the extracellular matrix (49). CALMl encodes Calmodulin CALMl gene previously associated with hip OA in two independent studies of Japanese patients (13). A functional assay in which the associated allele modulated chondrogenic activity confirmed the relevance of the promoter SNP (rsl2885713). Polymorphisms and/or SNPs in CALMl, FLRT2 and DIO2 genes are preferably detected in the method of the invention for determining the risk of a subject for developing OA.
The method of the invention for determining the risk of a subject to suffer from or develop OA preferably determines the presence of allelic variants and/or polymorphisms, i.e. different alleles of gene sequences comprising coding or non- coding and regulatory sequences, most preferably polymorphisms any of the genes discussed above or in combinations of these genes. Most preferably such an allele or mutation comprises a single nucleotide polymorphism (SNP), located in any of the genes located between 2q33.2-2q34, and/or on 14q24.3-14q32.12, as discussed above, for determining the risk of a subject for developing or suffering from osteoarthritis, in particular familial generalized OA with early and/or late onset. The polymorphism is preferably a polymorphism in a coding region, affecting translation and altering the encoded amino acid or RNA splicing, but it may also reside in non-coding regions affecting splicing and/or regulation of transcription or translation. The invention also provides for probes and/or oligonucleotides specific for polymorphisms according to the invention. It is well within the capabilities of the skilled artisan to select sequences for making probes that can be used in specific polymorphism discriminating assays.
The method of the invention for determining the risk of a subject for developing or suffering from OA extends to differential levels expression of genes, which can be detected in RNA expression or protein profiles, in particular of the genes discussed above on chromosomal loci 2q33.2-2q34 and 14q24.3-14q32.12. Differential expression of the polymorphic alleles of the above discussed genes may explain the diagnostic or prognostic risk of developing OA independent of the observed variants. Hence the invention provides for specific chromosomal locations (2q33.2-2q34 and 14q24.3-14q32.12) in which the genes reside that determine the risk of a subject to develop osteoarthritis when differentially expressed. Expression levels may be determined by assays customary in the art, such as quantitative PCR, Northern blotting, micro-array hybridization and the like. Detection may also take place by differential protein expression analysis on western blots, 2D gels, immunoassays and the like. Differential expression analysis on RNA or protein level is herein defined as statistically significant (p<0.05) difference in expression (comprising up- and down- regulation) of genes in subjects diagnosed with familial generalized OA of early and/or late onset, in comparison to a 'normal' population, representative of subjects not affected by OA. The invention also provides molecular probes, such as but not limited to: nucleotides, oligonucleotides, RNA, DNA, PNA or modified versions and mixtures thereof, that are specific for the polymorphisms in genes that reside within the chromosomal regions depicted. The invention pertains to probes which span or flank or comprise a polymorphism in a nucleic acid sequence on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12, capable of hybridizing or annealing thereto under permissible conditions. Preferably the probes anneal to polymorphisms in a coding or in a regulatory sequence that is indicative of the presence or the absence of predisposition of a subject for developing OA. The polymorphism may either be linked directly, i.e. where there is a direct physiological link between the polymorphism and the OA phenotype, or the polymorphism may be linked indirectly or genetically with the OA phenotype, i.e. where the linkage of the OA phenotype with the polymorphism is to be determined in the context of familial analysis and linkage data for family members is required.
The invention hence also pertains to oligonucleotides which span or flank or comprise a polymorphism in a nucleic acid sequence on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12, preferably in or near a coding sequence, and which are indicative of the presence or the absence of predisposition of a subject for developing OA. The method of the invention may preferably be carried out using probes and/or oligonucleotides that are capable of detecting the mRNA or cDNA of genes that resides on human chromosome 2q33.2 to 2q34 or 14q24.3 to 14q32.12 and may quantify the amount of expression. Differential expression of these genes may be indicative for the presence or the absence of predisposition of a subject for developing OA. An oligonucleotide probe according to the invention, capable of hybridizing to or near the polymorphism in any of the genes located on human chromosome bands 2q33.2 to 2q34 and 14q24.3 to 14q32.12 discussed above, is preferably 8 to approximately 45 nucleotides in length, preferably in the range of 15 to 25 nucleotides. The oligonucleotide may contain modification at the 5' or 3' ends, and may contain alternative nucleotides or analogues, or labels, such as fluorescent, enzymatic or radioactive labels or immunogenic haptens. Optionally, a SNP and/or allele specific nucleotide according to the invention may be physically linked to a solid support, such as but not limited to: a nylon membrane, glass slide, silica- or nitrocellulose support or on an array, preferably on a DNA microarray device. Techniques for detection of polymorphisms in a genome are well described and known to the skilled person and may for instance be found at
Figure imgf000015_0001
Techniques for detection of differentially expressed genes are well described and known to the skilled person and may for in stance be found at http://www.pro tocol- online.org/prot/Genetics_Genomics/Microarray/ A single nucleotide polymorphism (SNP) or gene may be detected on genomic
DNA, RNA or cDNA molecules and samples obtained from a subject, preferably of a subject of a family wherein early onset OA has been diagnosed with at least some individuals, for instance 1, 2, 3, 5, 10, 20 or more family members, or with a frequency of at least 0.1, 1, 2, 5, 10 or more percent of all family-members examined. Detection of SNP 's in a nucleic acid molecule may be performed using any standard technique customary in the art using oligonucleotides spanning or flanking the SNP, such as but not limited to: specific hybridization of an allele specific oligonucleotide, allele specific oligonucleotide ligation assays, allele specific DNA amplification (PCR or NASBA), or allele specific primer elongation and labeling techniques.
One of the principal methods used for the analysis of the nucleic acids of a known sequence is based on annealing two probes to a target sequence and, when the probes are hybridised adjacently to the target sequence, ligating the probes. The OLA- principle (Oligonucleotide Ligation Assay) has been described, amongst others, in US 4,988,617 (31). This publication discloses a method for determining the nucleic acid sequence in a region of a known nucleic acid sequence having a known possible mutation. To detect the mutation, oligonucleotides are selected to anneal to immediately adjacent segments of the sequence to be determined. One of the selected oligonucleotide probes has an end region wherein one of the end region nucleotides is complementary to either the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence. A ligase is provided which covalently connects the two probes when they are correctly base paired and are located immediately adjacent to each other. The presence or absence of the linked probes is an indication of the presence of the known sequence and/or mutation. US 5,876,924 by Zhang et al. describes another ligation reaction using two adjacent probes wherein one of the probes is a capture probe with a binding element such as biotin. After ligation, the unligated probes are removed and the ligated captured probe is detected using paramagnetic beads with a ligand (biotin) binding moiety. Abbot et al. in WO 96/15271 developed a method for a multiplex ligation amplification procedure comprising of the hybridisation and ligation of adjacent probes. These probes are provided with an additional length segment, the sequence of which, according to Abbot et al, is unimportant. The deliberate introduction of length differences intends to facilitate the discrimination on the basis of fragment length in gel-based techniques. WO 97/45559 (Barany et al.) describes a method for the detection of nucleic acid sequence differences by using combinations of ligase detection reactions (LDR) and polymerase chain reactions (PCR). Disclosed are methods that comprise of annealing allele-specific probe sets to a target sequence and subsequent ligation with a thermostable ligase, optionally followed by removal of the unligated primers with an exonuclease. Amplification of the ligated products with fluorescently labelled primers results in a fluorescently labelled amplified product. Detection of the products is based on separation by size or electrophoretic mobility or on an addressable array. The invention also provides for a kit of parts, comprising at least a molecular probe as described above, specific for a polymorphism in the human chromosome 2q33.2 to 2q34 and/or 14q24.3 to 14q32.12 which is directed at a diagnostic or prognostic test to establish whether a subject or a group of subjects, preferably genetically related subjects or family members, are at risk of suffering from or developing symptoms of OA, preferably familial generalized OA at later or earlier ages of onset. The kit may also comprise a solid support comprising at least one and preferably more oligonucleotide probes for carrying out the method of the invention, preferably in a micro-array format.
The invention also provides for a kit of parts, comprising at least of molecular probe as described above, specific for the genes within the chromosomal regions 2q33.2 to 2q34 and/or 14q24.3 to 14q32.12 that, when showing differential expression of one or more genes, may be directed at a specific diagnostic or prognostic outcome which establishes whether a subject or a group of subjects, preferably genetically related subjects or family members, are at risk of having or developing symptoms of OA, preferably familial generalized OA. Furthermore, these differentially expressed genes may direct towards therapies based on the suppression or stimulation of the expression of gene or gene product, for instance using RNA interference techniques.
Accordingly in a further aspect, the invention provides a nucleic acid construct comprising a nucleotide sequence encoding an RNAi or antisense agent that is capable of at least partly inhibiting the expression of a DI02 gene having the SNP rs225014
(Ala92, mutant allele) in a cell, wherein optionally the nucleotide sequence encoding the RNAi agent is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell. This nucleic acid construct is herein referred as the inactivating nucleic acid construct. A nucleic acid sequence of the DIO2 gene with the
SNP rs225014 (Ala92, mutant allele) is given as SEQ ID NO:1. The encoded D2 enzyme is given as SEQ ID NO:2. An RNAi agent is an RNA molecule that is capable of RNA interference or that is part of an RNA molecule that is capable of RNA interference. Such RNA molecules are referred to as siRNA (short interfering RNA, including e.g. a short hairpin RNA). The nucleotide sequence that encodes the RNAi agent preferably has sufficient complementarities with the cellular nucleotide sequence DIO2 with SNP rs225014 to be capable of at least partly inhibiting the expression of the encoded D2. More preferably, the RNAi agent has at least 80% identity with SEQ ID NO:2 and has 100% identity with a fragment of at least 20 nucleotides encompassing the SNP rs 225014. At least partly inhibiting the expression, preferably means inhibiting at least 50% of the expression, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, 90%, 95% or more of the expresssion of the DIO2 gene having the SNP rs225014. Expression is preferably measured by Northern or by Arrays. More preferably by Arrays.
Decreasing gene expression by providing antisense or interfering RNA molecules has been reviewed by Famulok et al. (2002, Trends BiotechnoL, 20(11): 462-466). The antisense molecule may be provided to the cells as such or it may be provided by introducing an expression construct into the chondrocyte, whereby the expression construct comprises an antisense nucleotide sequence that is capable of at least partly inhibiting the expression of the nucleotide sequence encoding a polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving transcription of the antisense nucleotide sequence in a chondrocyte. The expression level of a polypeptide may also be decreased by introducing an expression construct into a chondrocyte, whereby the expression construct comprises a nucleotide sequence encoding a factor capable of trans-repression of the endogenous nucleotide sequence encoding a polypeptide.
In the nucleic acid constructs of the invention, the promoter preferably is a promoter that is specific and functional for a given cell. Depending on the identity of the cell, the skilled person will know which promoter is most appropriate. The promoters for use in the nucleic acid constructs of the invention are preferably of mammalian origin, more preferably of human origin. In a preferred embodiment, the cell is a chondrocyte. The chosen promoter has preferably a transcription rate that is higher in a chondrocyte than in other types of cells. Preferably the promoter's transcription rate in a chondrocyte is at least 1.1, 1.5, 2.0 or 5.0 times higher than in a non-chondrocyte as measured by arrays or Northern.
In another aspect, the invention provides a nucleic acid construct comprising a nucleotide sequence, encoding a D2 polypeptide, said nucleotide sequence not comprising the SNP rs225014 (Thr92, wild type allele) and having at least 70% identity with SEQ ID NO: 3, wherein the nucleotide sequence is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell. This nucleic acid construct is herein referred to as the other nucleic acid construct. SEQ ID NO:3 represents a nucleotide sequence of the DIO2 gene which does not have the SNP rs225014 (Thr92, wild type allele). This sequence is called the wild type DIO2 (Thr92, wild type allele). SEQ ID NO:4 is the amino acid sequence of the encoded D2 enzyme (Thr92, wild type allele).
In a preferred embodiment, both types of nucleic acid constructs herein defined are used in a chondrocyte.
In another preferred embodiment, both types of nucleic acid constructs herein defined are a viral gene therapy vector selected from gene therapy vectors based on an adenovirus, an adeno-associated virus (AAV), a herpes virus, a pox virus and a retrovirus. A preferred viral gene therapy vector is an AAV or Lentiviral vector.
In a further aspect, the invention provides a method for preventing, delaying and/or treating familial generalized osteoarthritis in a subject, the method comprising pharmacologically altering the activity or the steady-state level of the polypeptide encoded by the gene DIO2 gene having the SNP rs225014.
In a preferred embodiment, the presence of the SNP rs225014 has first been identified in a subject. In the method of the invention, the activity or steady-state level of the polypeptide D2 having the SNP rs225014 is altered and optionally the activity or steady-state level of a polypeptide D2 not having the SNP rs225014 is restored in order to mimick the physiological situation.
The activity or steady-state level of D2 having the SNP rs225014 may be altered to suboptimal at the level of the polypeptide itself, e.g. by adding an antagonist or inhibitor of this polypeptide to a cell, such as e.g. an antibody against this polypeptide and/or preferably by introducing the inactivation nucleic acid construct as earlier defined herein and/or preferably by introducing the other nucleic acid construct as earlier defined herein. Preferably, however, the activity or steady-state level of a polypeptide is decreased by regulating the expression level of a nucleotide sequence encoding the polypeptide. Preferably, the expression level of a nucleotide sequence is regulated in the environment of a chondrocyte, preferably within a chondrocyte. For restoring D2 not having a SNP rs225014, such a D2 polypeptide may be added from an exogenous source to a cell. Preferably, the polypeptide may conveniently be produced by introduction of a nucleic acid construct earlier named the other nucleic acid construct and comprising a nucleic acid molecule encoding the D2 not having the SNP rs225014.
Preferably, the method comprises the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an inactivating nucleic acid construct as earlier herein defined. More preferably, the pharmaceutical composition is administered in the environment of chondrocytes or preferably within chondrocytes.
In the method of the invention, the chondrocyte is preferably a chondrocyte from a subject suspected to have a high risk of developing OA, due for example to its age or its genetic background or to its diet. Alternatively, the method of the invention is applied on chondrocytes from subjects diagnosed as already developing OA. The diagnostic method used is preferably the one of the invention already earlier described herein. The chondrocyte could be either from a subject being in an early stage of OA, or in a late stage of OA. In the method, chondrocyte chosen to be treated may be isolated from the subject they belong to. Alternatively and according to a preferred method, the cells are not isolated from the subject they belong to. Cells are subsequently treated by decreasing the activity or the steady state level of a D2 having the SNP rs225014. This treatment is preferably performed by infecting them with an inactivation nucleic acid construct of the invention as earlier defined herein. Finally if needed, the treated cells are placed back into the subject they belong to. In another treating method, classical OA treating methods and/or other potential treating methods are combined with any of the treating methods of the invention identified herein. A classical OA treating methods include pain relief medication such as paracetamol and/or NSAID (Non- Steroidal Anti-Inflammatory Drugs) and/or joint replacement and/or glycosamine glycans. Several classical methods of treating OA have been extensively described in M.B. Goldring (M. B. Goldring, (2006), Best practice and research Clinical Rheumatology, 20: 1003-1025).
Alternatively or in combination with any of the previous treating methods, an OA treating method comprises the addition of active T4 and/or active T3 in order to mimick the activity of D2.
In a further aspect, the invention provides the use of a nucleotide sequence as present in any of the nucleic acid constructs (inactivation or the other nucleic acid construct) as earlier defined herein or of any of the defined nucleic acid constructs (inactivation or the other nucleic acid construct) for the manufacture of a medicament for preventing, delaying and/or treating familial generalized osteoarthritis in a subject. Preferably, a nucleotide sequence or a nucleic acid construct are used in a method as earlier herein defined.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Description of the figures
Figure 1. Pedigree structures of the FOA families. Family 1-4 were used for the initial genome wide scan, additional families for replication analysis. Circles denote females, squares, males. Blackened symbols are affected individuals and symbols with diagonal lines represent diagnostic uncertainty. The co-segregating haplotypes among affected individuals are depicted as squared boxes, recombination's as crosses. Boxes that are open at the top or bottom continue. Al is the most likely affected haplotype, whereas, A2 the second likely. NAl is the most likely unaffected haplotype and NA2 the second most likely. Family 7 is not added to the Figure since it did not contribute to the linkage.
Figure 2. Multipoint model free NPL scores of 19 markers covering the 44 cM region on chromosome 2q33.2 to 2q34 using all families. Centi Morgans (cM) are provided relative to marker D2S326 which is located to the centromeric edge of the region of linkage. The dashed vertical lines indicate the one-LOD-drop interval.
Figure 3. Results from the genome-wide scan in 179 Caucasian sibling pairs and four trios from the GARP study. Solid line represents results with 14 additional fine mapping markers on chromosome 6, 10 13 and 14 (mean informativity = 0.42; range,
0.08-0.69).
Figure 4. Suggestive evidence for linkage on chromosome 14q32.11. Horizontal line represents one LOD-drop interval (75-95 cM; mean informativity = 0.50 (range, 0.46-0.56). Three vertical lines represent the genes DIO2 (78 cM; informativity =0.51), FLRT2 (82 cM; informativity = 0.54) and CALMl (89 cM; informativity = 0.47). The dotted vertical lines represent the borders of the chromosomes.
Figure 5. Supplementary figure. LD blocks, genomic structures surrounding the DIO2 gene and the DIO2 SNP rs225014. a) Diamonds are the pairwise LD indices of D' between SNP rs225014 and all other SNPs in the haplotype blocks surrounding the DIO2 gene. Position of the
DIO2 gene with rs225014 is indicated. b) Pairwise linkage disequilibrium blocks and the position of the DIO2 gene as provided by HAPMAP (http : //www . hapm ap . or g) . The height of linkage disequilibrium measure D' is shown by the gradient of the red colour. Genomic structure surrounding the DIO2 gene with physical location of the DIO2 gene. Gray boxes are locations of recombination
Examples
Example 1 : Linkage of gene variants to familial early onset generalised OA
1.1 Subjects and methods
1.1.1 Ascertainment FOA families
Families containing patients that express primary generalized OA in each generation were collected from different parts of the Netherlands. Informed consent was obtained from all patients and the Medical Ethics Committee of the Leiden University Medical Centre approved the study. Probands were recognized through Rheumatology outpatient clinics. Family members were recruited via probands. Initially, we used questionnaires to select eligible families. For eligible families, complete medical history and available radiographs were obtained from the hospitals of almost all affected family members (81%). Radiographs were re-evaluated for signs of chondrodysplasia, spinal dysplasia and abnormal development of the epiphyses of the peripheral joints. These features were absent in all families. The presence of radiographic characteristics of OA was assessed according to Kellgren criteria (32) by an experienced reader (Dr. C. Bijkerk). Some individuals had marked Heberden's nodes and ankle OA. A selection of affected and unaffected individuals of family 1, 2, 4 and 7 were additionally visited for physical examination in order to prevent misclassification. The mean age of OA onset in these patients was 33 years and ranged between 20 and 50 years. The phenotype within these families is characterized by distinct progressive OA in the absence of mild or severe chondrodysplasia. Symptoms and radiographic characteristics of OA (ROA) occur at multiple joint sites simultaneously, including involvement of the hands with noduli, knees, hips, ankle and spine. Individuals with clinical and radiographic evidence of OA in two or more joints before the age of 50 years were considered affected. Extensive description of the phenotype in family 1, which is representative for the phenotypes also of the other families included, is described elsewhere (18). All clinical diagnostic decisions were made independent to the genetic linkage analysis and homogeneity of the phenotype between different families was checked.
1.1.2 The Rotterdam sample The Rotterdam study (33) is a population based cohort of in total 7983
Caucasian participants (response rate 78%). The medical ethics committee of the Erasmus University Medical School approved the study and written informed consent was obtained from each participant. In a random sample of 1369 unrelated subjects (the Rotterdam sample), ages 55-70 years, radiographs were scored for the presence of radiographic OA (ROA) in 2 knees, 2 hips (34), 36 hand joints and 3 levels of the thoracocolumbar spine (7). All radiographs were scored according to the Kellgren criteria (32) by two independent readers, blinded to all other data of the participant. Definite ROA was defined as a Kellgren score of > 2. In the current paper we evaluated the association between generalized ROA and promising variants in a sample of the Rotterdam study using a qualitative and a quantitative ROA trait. The qualitative generalized ROA phenotype was described in detail elsewhere (14) and occurred at a frequency of 23% in the Rotterdam sample. For the quantitative trait, a ROA score, based on the number of joints with ROA, was defined equivalent to the criteria previously described (35). In short the specific ROA score (0-2) for the hip represented subjects with no (88%), uni-lateral (7%) or bi-lateral (5%) hip ROA. The knee ROA score represented subjects with no (81%), uni- (11%) or bi-lateral (8%) knee ROA. For hand and spinal disc degeneration (DD) a ROA score proportional to the hip and knee was made. Hand ROA score (0-2) represented subjects with respectively, O (40%), 1-2 (34%), and > 3 hand joint groups affected (26%). For spinal DD score (0-2) represents subjects with DD at respectively, 0 (33%), 1 (38%) and > 2 levels (30%). The quantitative total ROA score (0-8) was made by the sum of the proportioned joint-site specific ROA scores and represents a quantitative measure of ROA within each patient. The mean and median total ROA score in the Rotterdam sample was 2.3 and 2.0, respectively.
1.1.3 Genotype analysis
For the initial genome wide scan, 231 microsatellite markers, with an average spacing of 18 cM, were used which were selected from the Cooperative Human Linkage Center (CHLC) human screening set (Weber version 8.0). Fine mapping was performed using 14 additional markers in the region of positive linkage, which were selected from the Genome Database and the Marshfield Medical Research Foundation jύt^Jjr^sewc^j^^hύdάd^c^orgl^Ωeύcsl. The fluorescent-labeled PCR products were electrophoretically separated with automated laser fluorescence DNA sequencer (Pharmacia Biotec). Alleles were identified with Fragment analyzer (Pharmacia Biotech). Promising variants were genotyped in a sample of the Rotterdam study by using Sequenom homogenous MassExtend Massarray System (Sequenom Inc, San Diego, CA) using standard conditions. Genotypes were analyzed using Genotyper version 3.0 software (Sequenom Inc.). Successful genotypes were obtained for 1228 samples for PTHR2 variants and in 809 samples for IDHl, NRP2, KIAAl 571, ADAM23 and PIP5K3. 1.1.4 Two-point model based linkage analysis
Family members are affected at relatively early age in multiple joint sites simultaneously, which enabled us to give a good estimation of the parameters that describe the mode of trait inheritance. Model based linkage analysis was performed using the FASTLINK 2.2 version of the linkage program MLINK (36). The disease locus was modeled as an autosomal dominant trait with a disease frequency of 0.001 since symptomatic and radiographic OA at multiple joint sites before the age of 50 is rare. Marker allele frequencies were used from the CEPH database and compared with data of the founders. Penetrances were assumed to rise linearly form 0% at age 15 to 100% at age 50 with a phenocopy penetrance of 0.001. Model based linkage analysis procedures can handle large pedigrees but only up to one or two loci at a time.
1.1.5 Multipoint model free linkage analysis
Multipoint, model free linkage analysis was performed and haplotypes were constructed using GENEHUNTER2 (37). The Genehunter software does not require assumptions related to mode of inheritance or penetrance and it examines whether the allele sharing among affected relatives is greater than expected under the null hypothesis. The program can handle large numbers of loci but only on a small number of family members at a time.
1.1.6 Strategy of linkage analysis
We used a stepwise linkage analysis strategy. The first step was to genotype an 18-cM map of markers using 4 original FOA families. Any marker that showed a two- point LOD score of > 1 in the parametric linkage analysis was indicated as initial linkage signal. The second step was to replicate the initial linkage result using 3 additional FOA families that were ascertained during the geno typing of the whole genome scan. The third step consisted of refinement of the most promising linkage result by fine mapping analysis in all available FOA families. Finally, multipoint model free linkage analyses was applied to increase the marker informatively and to check the robustness of the observed model based linkage.
1.1.7 Mutation analysis Genomic DNA was isolated from EDTA blood of affected and unaffected family members. Initially, three affected family members (individual 14 from family 1, individual 10 from family 2 and individual 9 from family 4) were screened for possible mutations by direct forward and reverse sequencing from both ends (Figure 1). If a novel variant was identified, unaffected family members of these families and family members of remaining families were sequenced. Reference sequences corresponding to all coding and 5' and 3' UTR regions of the genes were obtained from the UCSC genome browser assembly May 2004 (http://genome.ucsc.edu/) or the Ensembl Genome database v35 (www.ensembl.org), NCBI build 35. Example 1 Table 2 shows the Genbank numbers. To amplify exons, forward and reverse primer sets (primer sequences upon request) were designed with at least 25 bp flanking intronic sequences using Primer3 (http://www.broad.mit. edu/cgi-bin/primer/primer3_www.cgi) with the conditions described by (38). 3'UTR 0ΪNRP2 and exons 1-27 OΪPIP5K3 have not been sequenced due to current genome browser updates. PCR amplifications were carried out in a volume of 15 μl that contained 15 ng genomic DNA, 4.1 pmol of the PCR primers, 1.5 mM MgCl2, 0.2 mM and 0.6 units of rTaq polymerase (Amersham Biosciences) or 0.6 units of HotfirePol® DNA polymerase and solution S (Solis Biodyne) for GC-rich regions or standard conditions of the GC-rich PCR system (Roche). Reactions were cycled at 94°C for respectively 1 min or 15 min for GC-rich regions and then cycled for 35 cycles of 94°C for 30 sec, 57°C for 1 min 15 sec, 72°C for 30 sec, and finally incubated for 6 min at 72°C on B&L primus HT cyclers. PCR products were purified using Multiscreen 96 well plates (Millipore) filled with Sephadex (Amersham biosciences) and quantified on 1.5% agarose gels. PCR products were sequenced for possible mutations using an ABI3730 capillary sequencer with Big Dye chemistry (Applied Biosystems).
1.1.8 Validation of possible mutations
To investigate whether variants were novel or common SNPs, all variants observed in affected family members were blasted for existence using National Center of Biotechnology Information (NCBI) SNP blast, build 124 (http://www.ncbi.nlm.nih.gov/SNP/snpblastByChr.html). We applied the nomenclature as describe by (39). When a novel variant was found, segregation analysis of the variant with OA within all family members of the seven families was performed. The impact of a novel variant involving an amino acid change was examined using PolyPhen (http://tux.embl-heidelberg.de/ramensky/index.shtml) or SIFT (http://blocks.fhcrc.org/sift/SIFT.html) (40). PolyPhen is a computational tool that predicts possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations (41). SIFT (sorting intolerant from tolerant) estimates tolerance indices that predict tolerated and deleterious substitutions for every position of the query protein sequence using multiple alignment information (40). To test the effect on the splicing process, exonic variants were screened for exonic splicing enhancers sequences using http://exon.cshl.edu/ESE (42). Conservation was determined using the Multiz Alignments and Conservation track of the UCSC genome browser (http://genome.ucsc.edu/) which shows a measure of evolutionary conservation in human, chimp, mouse, rat, dog, chicken, fugu, and zebra fish.
1.1.9 Occurrence of mutations and association
The variants were evaluated in the Rotterdam sample based both on its frequency and its occurrence with a generalized OA status (see above). Hardy Weinberg equilibrium (HWE) was calculated with an exact HWE test for rare alleles implemented in R version 1.9.1 (http://www.r-project.org/). A logistic regression model was fitted to measure the strength of association with the qualitative generalized OA trait, which is expressed as odds ratios (OR) with 95% confidence intervals (95% CI) adjusted for age (years), body mass index (BMI) in kg/m , and sex. Furthermore, differences of the quantitative ROA score between carriers and non-carriers were determined by using an independent T-Test and the non-parametric Mann- Whitney U test. Homozygous carriers of the risk allele were not observed. All analyses were performed with SPSS version 11 software (SPSS, Chicago, IL).
1.2 Results
1.2.1 Genome wide linkage scan and replication of positive linkage signals In the genome scan, we studied 4 FOA families (family 1-4) with generalized OA in each generation (Figure 1). Two-point model based linkage analysis revealed two loci on chromosome 2, D2S1334 (2q22.1-22.3) and D2S1391 (2q32) with a LOD score above 1. None of the other markers throughout the genome provided a likely location for an OA gene in our FOA families. Three additional FOA families, that were ascertained during the genotypings of genome scan of the first 4 families (Figure 1, family 5-7), were used to replicate the initial regions of linkage. For the 2q22.1-22.3 region, no sustained evidence of linkage emerged. ).
Table 1 Haplo sharing; Haplotype sharing affected family members
Marker Locus cM FAM 1 FAM2 FAM3 FAM4 FAM6 FAM5 d2s326 1 177.53 + d2s2257 2 181.52 3.99 + d2s2314 3 182.24 0.72 + d2s1391 4 186.21 3.97 + d2s118 5 190.00 3.79 - + d2s315 6 193.26 3.26 + + d2s115 7 195.65 2.39 + + d2s72 8 198.65 3.00 + + + d2s1384 9 200.43 1.78 + + + + d2s369 10 202.92 2.49 + + + + + + d2s155 11 202.92 0.00 + + + + + + d2s2358 12 203.46 0.54 + + + + + + d2s2208 13 205.06 1.60 + + - + + + d2s154 14 205.26 0.20 + + + + + d2s2178 15 205.59 0.33 + + + + d2s371 16 206.74 1.15 + + + gata30e06 17 210.43 3.69 - + + d2s164 18 214.71 4.3 + - d2s126 19 221.13 6.4 +
Family 1 : recombination's on both sides of the haplotype by individual 3 and/or 5; both affected.; Family 2: recombination on upper site of haplotype by proband (individual 17) and lower site of haplotype by individual 8.; Family 3: recombination on upper site of haplotype by individual 9 (affected) and lower site of haplotype by individual 4 (unaffected); Family 36: recombination on upper site of haplotype by individual 7 (affected) and lower site of haplotype by individual 8 (affected)
Table 2 Known genes on chromosome 2 between the markers D2S72 and D2S2178 based on RefSeq, mRNA, TrEMBL and Swiss-Prot prediction using Ensembl genome database v35
Marker Genbank ID HUGO ID Description No.
Exons
CTLA4 Cytotoxic T-lymphocyte protein 4 3,4
N M_001037631 , NM_005214 precursor
CD28 T-cell specific surface glycoprotein 4,5
NM_006139 CD28 precursor d2s72 ICOS Inducible T-cell co-stimulator 5
NM 012092 precursor d2s1384 NM 057177, NM 205863, ALS2CR19 Amyotrophic lateral sclerosis 2 22,23,23
NMJ52526 chromosomal region candidate gene protein 19
BC009222, NM 201264, NRP2 Neurophilin-2 precursor 5,16,16,
NM 018534, NM 201279, 16,17,17
NM_003872, NM_201266
NM_017759 NM_017759 Unknown (NP_060229) 12 d2s155 NDUFS1 NADH-ubiquinone oxidoreductase 19
NM_005006 75 kDA subunit
NM_001959, NM_021121 EEF1 B2 Elongation factor 1-beta 6,7
GPR1 Probable G protein-coupled 3
NM_005279 receptor
XM_371590 XM_371590 Unknown (Q9HCK1 ) 1
XM_496742 XM_496742 Unknown (Q9BZ60) 1 d2s369 ADAM23 A disintegrin and metalloproteinase 26
NM_003812 23 preproprotein d2s2358 MDH1 B malate dehydrogenase 1 B 10
NM_206892 (NP_996775)
NM_014929 NM_014929 Unknown (KIAA0971 ) 12
NM_173077 CPO Carboxypeptidase O 9
NM_003709 KLF7 Krueppel-like factor 7 4
CREB1 cAMP-response element binding 8,9
NM_004379, NM134442 protein
NMJ45280 NMJ45280 hepatocellular carcinoma- 4 associated antigen (NP_660323)
NMJ52523 hypothetical protein FLJ40432 11
NMJ52523 (NP_689736)
NM_030804 NM_030804 Unknown (CB031 ) 1
NM_003468 FZD5 Frizzled 5 precursor 2 d2s2208 XM_371591 XM_371591 Unknown (Q8WW68) 1
NM_006891 CRYGD Gamma crystallin D 3
NM_020989 CRYGC Gamma crystallin C 3
NM_005210 CRYGB Gamma crystallin B 3
NM_014617 CRYGA Gamma crystallin A 3
XM_371592 similar to RIKEN cDNA 9
XM_371592 D630023F18
NM_005896 IDH1 lsocitrate dehydrogenase 1 10
PIP5K3 FYVE finger containing 10,42
NM_152671 , NM_015040 phosphonositide d2s2178 PTH R2 Parathyroid hormone receptor 13
NM 005048 precursor
1.2.2 Fine mapping analysis
For the region surrounding marker D2S1391 (2q32), a selection of 14 additional markers were geno typed in all families to provide denser coverage. The highest two-point LOD score was found for marker D2S155 on chromosome locus 2q33.3 of 6.05 at recombination fraction 0.00. As shown in Table 3, family 1 and 2 contribute significantly to the height, whereas family 7 contributes negatively to this LOD score. Multipoint analysis of this region showed NPL scores above 3 for the region between marker D2S1384 and D2S371 (6 cM). The highest multipoint NPL-score was established for marker D2S2358 of 4.70 (Figure 2; P-value 0.0013). Recombinant haplotypes were determined for all families that contributed positively to the linkage. In family 2 and 4, however, two haplotypes are shared among affected individuals which are absent in the unaffected relatives resulting in two possible haplotypes that contribute to the linkage. For example in family 2, the most likely haplotype with alleles 3-6-7-8 (Al), allocates one phenocopy (individual 9) whereas the second likely haplotype 7-6-5-8 (A2) allocates 2 phenocopies (subject 6 and 17). In family 4, the most likely haplotype (Al) allocates no phenocopies and the second likely haplotype (A2) allows one phenocopy (individual 7). As also shown in Figure 1, alleles determining co-segregating haplotypes were not identical among families, indicating that different causal variants may exist contributing to the onset of the FOA phenotype. The candidate gene region was refined by combining the co-segregating haplotypes among affected individuals of families 1 and 2 since they contributed most to the linkage. A minimal candidate gene region was defined between the markers D2S1384 and D2S2178 with a, distance of 5 cM, 4.6 Mb (ENSEMBL database v35). This region completely encompassed the co-segregating haplotype of family 4.
Table 3. Two point parametric linkage analysis of chromosome 2q marker d2s155 using 7 families with early onset generalised osteoarthritis
Marker cM 0.0 0.01 0.05 0.10 0.15 d2s155 202.9
All 6.05 5.96 5.55 4.96 4.32
1 1.81 1.77 1.62 1.44 1.25
2 2.93 2.88 2.67 2.39 2.11
3 0.26 0.25 0.22 0.18 0.15
4 0.35 0.34 0.30 0.26 0.21
5 0.51 0.50 0.46 0.40 0.35
6 -0.58 -0.53 -0.39 -0.28 -0.19
7 0.76 0.75 0.67 0.56 0.46
1.2.3 Candidate genes
A query of the human genome resources within the region on chromosome 2q33.3 between markers D2S1384 and D2S2178 disclosed 18 known genes, and 9 predicted RefSeq genes (NCBI build 35; Table 2). Concerning the tissue expression pattern, function, functional domains and previous studies on these proteins, at least two genes, FZD5 and PTHR2, emerged as initial functional candidates to be involved in the onset of generalized OA.
1.2.4 Mutation analysis FZD5 and PTHR2 and occurrence in the populations
Sequence analysis was performed to identify potential DNA variations in affected and unaffected members of the OA families. In the two exons of FZD 5 gene we did not observe a variant which co-segregated with the OA phenotypes in the families. For the PTHR2 gene, we identified 3 known SNPs, a rare nonsense variant in exon 3 and a rare missense variant in exon 6, respectively. The nonsense variant (c.458C>A, p.S82X) located in the first extracellular domain of the PTHR2, however, was present in all unaffected subjects of family 2 (haplotype NAl) and does, therefore, not segregate with the disease phenotype. The missense variant (c.786G>T, p.A225S) was located within the second extracellular domain of the gene and was found on the second likely haplotype A2 of family 4. As a result the more distant affected subject number 7 in family 4 (niece) did not carry the mutation. The mutation, therefore, may be causal to the FOA phenotype only when the distant relative is a phenocopy. The non-synonymous change was predicted to be benign by the PolyPhen software (43).
1.2.5 Mutation screening of remaining positional candidate genes
On the basis of the results of PTHR2 and FZD 5 we performed extended mutation analysis of the remaining positional candidate genes on the chromosome 2 locus (Table 2). Nearby the linkage region, at marker D2S72, the ICOS-CTLA4-CD28 cluster is located which is implicated in cytokine secretion and T-cell immunity (44- 46). The entire coding region, splice sites, and 5' and 3' untranslated regions of, CTLA4, CD28, ICOS, NRP2, NM_017759, NDUFSl, EEF1B2, GPRl, XM Jl 71590, ADAM23, MDHlB, CPO, KLF7, CREBl, NM_030804, FZD 5, IDHl, and PIP5K3 were sequenced comprising 15 Refseq genes and 3 predicted genes. If the considered variant appeared novel in the current dbSNP database (build 124) and cosegregated with OA in the affected family, it was evaluated according to the criteria previously mentioned. Table 5 showes the identified variants. 1.2.6 Evaluation of novel variants and association the Rotterdam sample.
The initial screening of variants of Table 5 indicated 26 novel variants (17 SNPs and 9 insertion/deletion polymorphisms). From these 26 variants, nine promising variants co segregated with OA within one or more families as illustrated in Table 4. Three of these variants were found in coding regions and involved an amino acid change: (KIAAl 571 R2133S, IDHl Y183C and PTHR2 A225S). Six variants were located in UTR regions (PIP5K3 c.8429T>A, PIP5K3 c.8434insC and NRP2 c.941A>C) or in vicinity of exon/intron boundaries NRP2 c.l938-21T>C, ADAM23 c.2065+24C>T and IDHl C.933-28OT. Using PolyPhen, SIFT and ESE finder analysis to predict possible functional effects of these variants, two variants emerged as potential mutations: KIAA1571 R2133S and IDHl Y183C. (Tables 4 and 5) In addition, three variants (NRP2 c.941A>C, PIP5K3 c.8429T>A, PIP5K3 c.8434insC) were conserved across other species and might be of functional importance. The predicted KIAAl 571 gene probably belongs, as predicted in Unigene, to the fibronectin type III and M protein repeat family in C. elegans. Fibronectin is a component of the extracellular matrix of cartilage and KIAAl 571 may therefore be an excellent candidate gene. The G/T nucleotide change in the third exon of KIAAl 571 (Q9HCK1) results in disruption of exonic splicer enhancer motifs which serves as binding site for Serine/Arginine protein 40 and 55 and might be therefore a functional variant. However, because this gene is a predicted gene, little is known about other possible predicted functional effects on the protein. The novel variant KIAAl 571 R2133S co segregated in family 4 and 7 with OA and showed a rare population frequency of 0.01 corresponding to nine carriers of 781 genotyped (Tables 5 and 6). Only 2 carriers of KIAAl 571 R2133S showed generalised OA and one carrier had no ROA at all which reveals this variant as an unlikely causal mutation. (P -value = 0.57) Isocitrate dehydrogenase 1 (IDHl) encodes a cytoplasmic enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and has a significant role in cytoplasmic NADPH production (47). In IDHl, two variants (Y183C and c.933- 28C>T) co segregated with the OA phenotype. IDHl Y183C co segregated in affected family members in family 2 and was located in the isocitrate/isopropylmalate dehydrogenase domain (PFOO 180) of IDHl in exon 6. The variant is predicted to be probably damaging for the protein structure/function by SIFT and PolyPhen, and highly conserved across all eight species investigated. Based on these results, this variant could be functional for the onset of generalised OA. In the Rotterdam sample, we observed 14 carriers out of 785 genotyped corresponding to a frequency of 0.02. In addition, five of the 14 carriers (0.36) showed generalised ROA which is twice as much as in the population (0.17), conferring to an odds ratio, adjusted for age, BMI and sex of 2.8 (95% CI, 0.82-9.7, P-value = 0.10).
Another variant in this gene, IDHl c.933-28C>T, was identified in family 2 and 4, near the intron/exon boundary of exon 7. This variant was not conserved and showed a frequency of 0.04 in the Rotterdam sample. The frequency of generalised ROA was not different between carriers and non carriers of this variant.
Neuropilin 2 (NRP2), is an interesting gene because it encodes for the co- receptor of vascular endothelial growth factor^ (VEGF 16s) which is an essential factor for endochondral ossification (27-28). Furthermore, VEGF and its receptors are expressed in OA cartilage and VEGF stimulates production of extracellular cartilage matrix degrading matrix metalloproteinases (MMPs) (28-29). In the NRP2 gene, two novel variants were found: c.941A>C and c.l938-21T>C. In family 4, NRP2 c.941A>C was identified in a residue with a moderate conservation score and which showed a frequency of 0.03 in the random population and no increase of generalised ROA among carriers. The second NRP2 variant, c.l938-21T>C, was not conserved and co segregated in three families (1, 2, and 4) and was more frequent in the population (0.08). Carriers of at least one risk allele of the NRP2 c.l938-21T>C variant, showed a significant increased risk, adjusted for age, sex, BMI of 2.1 (95% CI, 1.1-4.1, P = 0.032), to have generalised ROA (Tables 4-6).
Phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, type III (PlP 5KS) catalyzes the phosphorylation of phosphatidylinositol-4-phosphate and has a role in endosome-related membrane trafficking (48) We found two novel variants (PIP5K3 c.8429T>A and PIP5K3 c.8434insC) in the 3'UTR region of PIP5K3 in family 4 which are both highly conserved residues. In the Rotterdam sample, we observed that PIP5K3 c.8429T>A and PIP5K3 c.8434insC were in complete LD (D'= 1, r2 = 1) with a population frequency of 0.04. The frequency of generalised ROA was not increased among carriers as compared to non carriers thereby limiting the evidence for their pathogenic role in relation to the onset of FOA. Even though these variants occurred in conserved residues, it is likely that these variants are neutral polymorphisms.
Finally, we also examined whether some novel variants were inherited together in different families to identify a possible LD pattern or genetic interaction resulting in a high LOD-score linked to OA. In the seven families, only two variants, NRP2 c.1938- 21T>C and IDHl c.933-28C>T occurred together on haplotype Al in family 2 and on hap Io type A2 in family 4. In the random population, this inheritance pattern was observed only once in 754 genotyped subjects (0.0013). This individual had spinal DD at three disc levels which has a prevalence of 0.04 in the random population.
Table 4 Possible mutations segregating in FOA families
Gene Gene DNA nt AA. Seq. Splicing J PolyPhen4 SIFTb Segregation f
Location change1 change cons.2
NRP2 31UTR' c.941A>C some F4:A1
NRP2 intron 78 c.1938-21 T>C no Possible F2:A1 , F4:A2,
KIAA1571 Exon 1 c.6368G>T R2133S no Yes benign (1.35)9 tolerated (0.00)9 F4:A1 , F7:A1
ADAM23 intron 20 C.2065+24OT no Possible F2:A2
IDH1 Exon 6 c.782A>G Y183C yes No damaging (2.89) not toler. (0.00) F2:A2
IDH1 intron 6 C.933-28OT no Possible F2:A1 , F4:A2
PIP5K3 3'UTR c.8429T>A yes F4:A2
PIP5K3 3'UTR c.8434insC yes F4:A2
PTHR2 Exon 6 c.786G>T A225S no No benign (0.12) tolerated (0.77) F4:A2
Numbers are based on positions in BC009222 (gi:33874364), NM_201267 (gi:41872566), XM_371590 (gi:42656560), NM_003812 (gi:73765550), NM_005896 (gi:28178824), NM_015040 (gi:50881947),NM_005048 (gi:39995097)
2 Sequence conservation across chimpanzee, dog, mouse and rat as predicted by the UCSC Genome browser
3 Splicing effects in exons have been predicted by ESE finder
4 PolyPhen prediction PSIC score difference
5 SIFT prediction with normalized probability
6 A1 represents the most likely haplotype and A2 the second likely affected haplotype
7 This variant is located in 1 of the 7 transcripts (BC009222, gi:33874364)
8 This variant is located in 6 of the 7 transcripts (NM_201267 (gi:41872566), NM_003872 (gi:41872532), NM_201279 (gi:41872571 ),
NM_018534 (gi:41872543), NM_201266 (gi:41872561 ), NM_201264 (gi:41872556))
9 Poor predictions due to little information
Table 5 Identification and validation of variants detected in early onset OA families by sequence analysis of coding regions on from 2q33.2-2q34
Gene exon Family family Location type codon SNP Seqscape change
ADAM23 intron 19 1-14R/4-8R 1 , 4 insertion no rs3832131 T(8T/9T) intron 20 2-7(C/T) 4-8/1-15 2, 388 C/T no no © Gene exon Family family Location type codon SNP Seqscape change exon20 1-15/4-8/2-7 (A) 3, 36, 2 A/T yes Q612L no
23 2-7 2 307 C/T no (A/A) rs3732079
CPO 3 2-7 (A/G) 2, 6, 36 240 G/A yes (M85I) rs13420911
5 2-7 2 198 T/G yes S134R rs11903403
6 1-14/2-7 1 , 2, 36 220 A/T no (A/A) rs13397039
CREB1 intron 8 1-14/2-7/4-8 319 del 9A/10A no no
FYVE intron 5 1-15/2-7(A);4-8 226 A/G before rs2304544 (A/G) exon intron 6 1-15/2-7/4-8 (T) 396 C/T after exon rs2043718
8 1-14/2-7/4-8 (A/G) 346 A/G no (TfT) rs2304545
9 1-15(A);2-7/4-8 249 A/G no (TfT) rs2118297 (A/G)
10 1-15/2-7/4-8 (G) 333 A/G yes (R/Q) rs2289170
10 1-15/2-7(G);4-8 285 A/G no (E/E) rs994697 (A/G)
31UTR 2-7/4-9 [GfT) 2, 4 536 G/T no rs10208191
31UTR 2-7/4-9 (G/C) 2, 4 617 G/C no rs10208295
31UTR 3-4R (A/C?) 3 651 A/C no no
31UTR 1-14/4-8 (A)? 917 A/G no rs6728314
31UTR 1-14 (T);4-8 (A/T) 1052 A/T no rs4675757
31UTR 1-14 (G);4-8 (A/G) 1106 A/G no rs4675758
31UTR 1-15 (G); 2-7(G/T) 1576 C/G/T no rs6710221
31UTR 1-15 (C); 2-7(C/T) 1959 C/T no rs6725932
31UTR 1-15/2-7 (T) 4- 2314 C/T no rs9646839 8(CfT)
31UTR 4-8 (insertie C) 2387 insertion no no C(C/CC)
31UTR 1-15 (12T/13T;2- 3513 insertion no rs10622340 7/4-8 (10T/13T) T/TTT
FZD5 1A 2-3/2-11 (AA) 2- 2,3,6,36 90 A/G no no 4/2-5/2-12 (AG)
1 1-15/2-7/4-8 (G) 374 A/G no (UL) rs4675711
1 1-15/2-7/4-8 (T) 587 A/T yes (SfT) rs1056614
2 4-8 [CfT) 1-14/2-7 4 970 C/T yes no ( C ) (P216L)
GPR1 1 1-15/2-7/4-8 (A) A/G yes (E/G) rs375527
1 1-15/2-7/4-8 (T) C/T no (G/G) rs1060384
1 1-15/2-7[C] 4-8 C/T yes (D/G) rs3732082 (CfT)
1 1-15/2-7/4-8[A/G] A/G no (UL) rs3732083
KLF7 51UTR 1-15/2-7/4-8 181 del 10T/11T no no
3 1-15/2-7/4-8 (A) 696 A/G no (UL) rs2700166
31UTR 1-15/2-7/4-8 ins (2*AC) no no (not (11 AC/13AC) rs3217200)
31UTR 1-15/2-7/4-8[C/T] C/T no rs1128555
NDUFS1 51UTR 1-15(C);2-7/4-8 C/G no rs4147707 (CIG) intron4 1-15/2-7/4-8[25T] 190 del no rs10560955 (23T/25T) intron4 1-15/2-7 (11 T) 4- 175 del no No 8[10T] (10T/11T)
6 1-14/1-17/36- 1 , 36 276 C/T no (D/D) rs11548670 12(C/T) 2-7/4-8 (T)
IDH1 2 36-12 (CfT) 36 315 C/T no (G/G) rs11554137 Gene exon Family family Location type codon SNP Seqscape change
4 36-10 (G/A) 36 G/A yes(V178l) No
4 2-7(NG); 1-14/4-8 2 106 NQ yes No (A) (Y183C) intron 4 2-7/4-8 (JIC); 1-14 2,4 70 T/C no No (T)
51UTR 1-14/2-7/4-8 1,2,3,4,36,6 87 del(9T/10T) no no (9T/10T) intron 2 1-14/2-7/4-8 1,2,4 449 del(9T/10T) no no (9T/10T) intron 3 1-14/2-7/4-8/3/6/36 1,2,4, 3,36, 251 del(9T/10T) no no (9T/10T) 6 intron 7 1-14/2-7 (9T); 4-8 81 del(9T/10T) no no
FRZB 4 2-7 (CIJ) 1-14/4-8 2 137 C/T yes (R/W) rs288326 ©
EEF1B1 introni 1-14 (insC/insC) 1 377 ins (3C/4C) rs17838593 intron 2 1-14/2-7/4-8(T/C) 1,2,4 332 T/C after exon rs11689362 intron 5 1-14/2-7/4- 1,2,4 335 del after exon no 8(11T/10T (11T/10T)
ICOS 51UTR 1-14/2-7/4-8 (NJ) 1,2,4 121 A/T before no exon intron 1 1-14/2-7/4-8 (QIC) 1,2,4 373 QIC after exon intron 3 1-14(T);2-7/4-8© 1 433 C/T after exon rs4264550 intron 4 1-14(GG)/2- 1,2,4 342 JIQ before rs10172036 7(TG)/4-8 (TG) exon intron 5 1-14©;2-7/4-8(A) 1 383 A/T after exon rs10183087
CD28 intron 3 1-14/2-7(C/T);4- 1,2 456 C/T after exon rs3116496 8(T) intron 3 1-14(TT);2-7/4- 1 271 A/T before 8(AA) exon
CTLA4 exon 1 4-8 (G);1-14/2-7(A) 4 205 NQ yes (JIk) rs231775 np060229 exoni 1-14/2-3/4-9 1,2,4 471 del(9T/10T) utr No intron2 1-14/4-9(C/T);2- 1,4 277 C/T no rs17286674 3(TT)
1-14/4-9(C/T);2- 1,4 316 C/T rs818009 3(TT) intronδ 1-14/2-3/4-9 del(9T/10T) No inron12 1-14/2-3/4-9 (NQ) 1,2,4 890 NQ rs1062344
MDH1B 5UTR 1-14 (QIJ) 1 86 QU rs16838839 exon4 1-14/2-3/4-9(CC) 1,2,4 560 C/T rs1990849 intronδ 1-14 (NC) 1 49 A/C rs230299 exon 8 1-14(C/T) 1 205 C/T yes(T261l) rs4673362 intronδ 1-14 (NQ) 1 323 NQ rs10209871 intronδ 1-14(C/T) 1 417 C/T rs10173707 nrp2 intron3 1-14(C/T)4-9(C/T); 1,4 C/T after exon No intronδ 1-14(C/T), 2-3 1,2,4 C/T before No (C/T);4-9 (CIJ) exon9 intron3 36-12 (del) 36 after exon No introni 1-14,2-3 (C/T) 1,2 68 C/T after exon RS849558 intron3 1-14,2-3,4-9 1,2,4 137 C/T after exon RS3771051 exon4 1-14,2-3,4-9(AA) 1,2,4 251 A/G yesK123R RS849541 exon 8 1-14,4-9 (C/T) 1,4 141 C/T no Y327Y RS849526 exon 10 1-14,2-3,4-9(CC) 1,2,4 162 C/T no V468V RS849568 exon 11 1-14,2-3,4-9(G/T) 1,2,4 98 G/T no P558P RS849563 intron12 1-14,2-3,4-9(G/T) 1,2,4 279 G/T after exon RS10932123 Table 6 Frequency of carriers of novel segregating variants in population-based sample
Variant frequency carriers' frequency carriers with
(n = 809) GOA2
Random subjects 0.17 (131/790)
NRP2 c.941A>C 0.03 (741/19) 0.11 (2/18)
NRP2 c.1938-21T>C3 0.08 (699/59) 0.26 (15/57)
KIAA1571 c.6368G>T 0.01 (772/9) 0.22 (2/9)
ADAM23 C.2065+24OT 0.03 (732/26) 0.15 (4/26)
IDH1 c.782A>G 0.02 (771/14) 0.36 (5/14)
IDH1 C.933-28OT 0.04 (763/28) 0.19 (5/27)
PIP5K3 c.8429T>A3 0.04 (744/30) 0.17 (5/29)
PIP5K3 c.8434insC3 0.04 (723/29) 0.17 (5/29)
GOA = generalised ROA
1 frequency carriers (number of noncarriers/ number of carriers)
2 Phenotypic data was not available for 19 subjects 3 Heterozygous and homozygous carriers were pooled
Example 2: Genome wide scan, identification of a haplotype containing a non- synonymous SNP in DIO2 associated with symptomatic Osteoarthritis 2.1 Subjects and methods
2.1.1 Ascertainment GARP patients
Probands (ages 40-70 years) and their siblings have OA predominantly at multiple joint sites of the hand or in two or more of the following joint sites (hand, spine (cervical or lumbar), knee or hip63. Subjects with symptomatic OA (as defined below) in just one joint site were required to have structural abnormalities in at least one other joint site defined by the presence of ROA in any of the four joints or the presence of two or more Heberden's nodes, Bouchard's nodes, or squaring of at least one first carpometacarpal (CMCl) joint on physical examination. Symptomatic OA in the knee and hip was defined according to the American College of Rheumatology (ACR) recommendations for knee and hip OA64'65. Knee OA was defined as pain or stiffness for most days of the preceding month and osteophytes at the joint margins of the tibiofemoral joint (x ray spurs). Hip OA was defined as pain or stiffness in the groin and hip region on most days of the preceding month in addition to femoral or acetabular osteophytes or axial joint space narrowing on radiography. Prosthetic joints in the hips or knees as aresult of end stage OA were defined as OA in that particular joint. Spine OA (cervical and lumbar) was defined as pain or stiffness in the spine on most days of the preceding month, in addition to a Kellgren/Lawrence score of two in at least one disc or one apophyseal joint. OA in hand joints was defined according to the ACR criteria as pain or stiffness on most days of the preceding month in addition to three of the following four criteria: bony swelling of two or more of the ten selected joints (bilateral distal interphalangeal (DIP) joints 2+3, bilateral proximal interphalangeal (PIP) joints 2+3, and CMCl joints), bony swelling of two or more DIP joints, fewer than three swollen metacarpalphalangeal (MCP) joints, and deformity of at least one of the ten selected joints. Radiographic OA was assessed by one trained radiologist by the Kellgren/Lawrence (0-4) method. Intrareader variability for the different joint sites was assessed: the intraclass correlation coefficient (ICC, with 95% confidence interval) was for the hands, 0.95 (0.92 to 0.96); for the knees (tibiofemoral), 0.92 (0.86 to 0.96); for the hips, 0.95 (0.92 to 0.98); for the cervical spine (apophyseal and disc), 0.71 (0.52 to 0.84); and for the lumbar spine (apophyseal and disc), 0.67 (0.46 to 0.81). Intrareader variability was based on an examination of 40 radiographs that were selected randomly throughout the duration of the study period and were blinded for any patient characteristics. Radiographic OA was regarded as having Kellgren/Lawrence score ≥ 2. Ethical approval for the study was obtained from the appropriate ethics committees.
2.1.2 The Rotterdam replication sample
The Rotterdam study, which comprises 7,983 Caucasian participants, is a prospective, population-based cohort study of the determinants and prognosis of chronic diseases in the elderly . The Rotterdam replication sample consists of all women aged ≥ 55 years (n = 1712) for which radiographs of the hip were scored for the presence of OA according to the Kellgren/Lawrence grading system (grades 0-4)68. Severe radiographic hip osteoarthritis cases were defined by a Kellgren and Lawrence (KL) grade of 3 or more.
2.1.3. UK replication sample The cases were ascertained through the Nuffield Orthopaedic Centre in Oxford. They had undergone total joint replacement of a hip, of a knee, or of a hip and a knee for primary OA. The cases were ascertained using the criteria of signs and symptoms of OA sufficiently severe to require joint replacement surgery. All had pain with rest and night symptoms of more than 6 months duration. The radiographic stage of the disease was a KL grade of 2 or more in all cases with over 90% being grade 3 or 4. Inflammatory arthritis (rheumatoid, polyarthritic or autoimmune disease) was excluded, as was posttraumatic or post-septic arthritis. No cases suggestive of a skeletal dysplasia or developmental dysplasia were included. The average age of the cases at replacement surgery was 65 years with an age range of 56-85 years. The controls comprised individuals with no signs or symptoms of arthritis or joint disease (pain, swelling, tenderness or restriction of movement). The average age of the controls at recruitment was 69 years with an age range of 55-89 years. Due to ethical and financial constraints the hip joints of the controls were not subjected to radiographic analysis. All cases and all controls were UK individuals of white European ethnicity.
2.1.4 Genotype measurements Short tandem repeat polymorphisms
A complete genome-wide scan containing 403 microsatellite markers with an average spacing of 10 cM was performed in 187 pairs and four trios with OA at multiple joint sites from the GARP study. Markers and 14 additional microsatellite markers for fine mapping on chromosome 6, 10, 13 and 14 were taken from Human Linkage Set v2.5 MDlO or HD5 (Applied Biosystems), respectively and measured using an ABI Prism DNA Analyzer 3700 (Applied Biosystems). Genotyping was performed using standard conditions and reagentia with some exceptions. The amount of polymerase chain reaction (PCR) primer pairs for the markers was reduced up to 5-fold and duplex PCR reactions were designed if possible to reduce costs, time expense and amount of genomic DNA used. Genotypes were analysed by using Genemapper version 2.0 and 3.0 (Applied Biosystems). As quality control, approximately 8% of the samples were genotyped in duplicate and compared. In addition, 48 additional family members from 36 different sibling pairs were genotyped to improve our ability to detect genotyping errors and estimate allele sharing. Mendelian errors were checked for Mendelian inconsistencies and unlikely recombinants using Merlin69. These quality checks indicated that marker D6S434 and D9S158 from the Human Linkage Set v2.5 MDlO could not be genotyped reliably in our hands due to unclear one base pair differences. Subjects and markers showed an average success rate of 96% (range 77-100%) and 96% (range 83-100%), respectively. Family relationships were verified using the GRR program70. Eight sibling pairs showed pedigree errors and were removed for further analysis. In seven of these sibling pairs, individuals reported to be full siblings were almost certainly half siblings. The remaining siblings were monozygotic twins.
A locally developed SQL database was used to store genotypic data, compare repeated genotypes and generate output files for linkage analysis. The location of the markers was taken from an integrated genetic map of David Duffy with interpolated genetic map positions (see URL below). The position is in Decode cM, estimated via locally weighted linear regression (lo(w)ess) from the Build 35.1 (and 34.3) physical map positions and published Decode and Marshfield genetic map positions. Single nucleotide polymorphisms
In total 179 sibling pairs and 4 trios (71 men, 301 women) of the GARP study were genotyped for highly informative tagging SNPs capturing a large fraction of all genetic variation in the CALMl, DIO2 and FLRT2. Tagging SNPs were selected from HapMap Public Release #19 applying the efficient multimarker method with r2 > 0.8 and minor allele frequency (MAF) > 0.05 implemented at in two or more (see also URL below)71. Tagging SNPs or their proxies were chosen to fit efficiently in a Sequenom multiplex assay. We genotyped the following tagging SNPs in CALMl: rs3814847, rs3814845, rs2300496, rs2300502 and rs5871. For DIO2, we measured rs225014, rs2267872, rs225011, rsl2885300 and rslO136454. For FLRT2, rs2239576, rs2057311, rsl7121375, rs 17646457 and rsl 129671 were genotyped. Multiplex genotyping assays were designed using Assay Designer software (Sequenom, San Diego, CA). Tagging SNPs were genotyped by mass spectrometry (the homogeneous MassARRAY system; Sequenom, San Diego, CA) using standard conditions. PCR reactions were carried out in a final volume of 5 μl and contained standard reagents and 2.5 ng of genomic DNA. Genotypes were assigned by using Genotyper version 3.0 software (Sequenom, San Diego, CA). The functional SNP rsl2885713, located in CALMl, and the DIO2 SNPs rsl2885300 and rs225014 in the Rotterdam study were genotyped using a Taqman by design assay and an ABI Prism DNA Analyzer 7900 (Applied Biosystems) with standard conditions. In addition, genotype distributions of all SNPs were in agreement with Hardy Weinberg equilibrium and approximately 8% of the subjects were genotyped twice and checked. 2.1.5. Statistical analysis Linkage analysis
Nonparametric linkage analysis was carried out by use of the .Span- statistics implemented in the software Merlin . LOD scores were plotted on a common 1 cM grid. For X-chromosome analyses, we used MINX (Merlin-In-X), a modified version of Merlin. The variance of the NPL test statistic was computed by Monte Carlo simulations of marker genotypes under the Housing Merlin. A global p- value for chromosome 14 was computed by smoothing the likelihood with respect to a uniform prior distribution for gene location73. We used the score statistic corresponding to this likelihood and estimated the variance by Monte Carlo simulations using Merlin. We calculated the contribution of each family to the maximum LOD score with Merlin. To analyze whether families affected with similar joint sites contributed to the maximum LOD score, we calculated Pearson's correlation coefficient between the LOD score and hip, knee or hand OA families.
Association approaches
Joint linkage and association analysis was performed with tagging SNPs to identify SNPs that fully or partly explain the observed linkage signal using the program LAMP (see URL below)74. The maximum likelihood was estimated using 50 starting points and the disease prevalence was set at 0.01. Haplotypes of individuals were estimated by the expectation maximisation algorithm implemented in SNPHAP version 1.3 (see URL below). We used the missing data option that allows us to carry out haplotype analysis in subjects with some missing genotype measurements. Posterior haplotype probabilities were used as sampling weight in all subsequent analysis.
Genotype and allele distributions in cases and controls were compared using standard chi-square analysis-of-contingency tables. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs). For stratification analysis, female cases were compared to female controls, and male cases were compared to male controls. For the distribution of genotypes, Hardy Weinberg equilibrium was tested by using the HWE program of LINKUTIL (see URL below). A logistic regression model was fitted to measure the strength of association, which is expressed as odds ratio (OR) with 95% confidence intervals (CI) and adjusted for age (years), body mass index (BMI, kg/m2) in the Rotterdam sample. All other analyses were carried out with SPSS version 14 software (SPSS, Chicago, Illinois, USA). Joint analysis of the UK and Rotterdam samples were also analysed using the Cochran-Mantel Haenszel χ 2 tests as implemented in R (see URL below). Two tailed P- values are reported for all analyses.
Table 7 Characteristics of GARP sibling pairs with symptomatic OA and subjects of the UK and Rotterdam sample
GARP study UK study Rotterdam symptomatic OA Replacements Controls populations based females
Number 370d 1535 714 1712
Age (range) 60.4 (43-80) 65 (56-85) 69 (55-89) 66 (55-89)
BMI (SD) 27.0 (4.6) - - 26.7 (3.9)
Women (%) 301 (81 ) 870 (70) 365 (30) 1712 (100)
Hand (%) 266 (72) _ _ _ Spine (%) 294 (79) -
Hip (%) 91 (25) 1099 (72) 88 (5)
Knee (%) 128 (34) 355 (23)
Hip&Knee (%) _ 81 (5)
GARP = Genetics, osteoARthritis and Progression; BMI = body mass index; The GARP sibling pairs comprise 183 sibships; 179 siblings and four
2.2 Results
2.2.1 Linkage analysis
To search for major osteoarthritis (OA) susceptibility loci we performed a genome wide linkage scan containing 403 microsatellite markers with an average spacing of 10 cM in the ongoing GARP study, which consists of 179 Caucasian sibling pairs and four trios of Dutch origin affected predominantly by symptomatic OA at multiple sites (for detailed phenotypic description Table 7)63. Initial nonparametric linkage analysis provided several suggestive linkage signals on chromosome 6, 10, 13, 14 (Table 8). Typing 14 additional markers in the four areas reduced the evidence for linkage on chromosome 6, 10 and 13. In contrast, the LOD score on chromosome 14q32.11 increased from 1.32 (P = 0.007) to
3.03 (P = 1.9 x 10 ~4) (Fig. 3), corresponding to a global P for chromosome 14 of 0.0013 and a genome wide P- value of 0.0286 (Bonferroni corrected for 22 chromosomes). The one LOD-drop interval of this signal encompasses 22 cM (75-95 cM) containing the markers D14S74, D14S1037, D14S1044 and D14S280 (Fig. 4) and the genes listed in Table 9. Among the pairs that contributed to the linkage, we observed no significant correlation between the LOD score per family and any combination of affected joint sites indicating that the linkage is based on the generalised symptomatic disease phenotype selected for in the GARP study. Table 8 lists all chromosomal regions yielding a LOD-score>1.0 in non-parametric analyses
Chrom Nearest Marker Position Interval (cM) Positio Max LOD P value
Marker (cM) n (cM)
6 D6s1574 14 0-43 0 1.13 0.011
10 D10s196 69 48-84 69 1.36 0.006
13 D13s175 1 0-12 0 2.23 0.0007
14 D14s74;D14s280 76-92 32-117 83 1.32 0.007
Chrom = chromosome; cM = centiMorgan
1X LOD -drop interval
Notably, the location of the linkage peak coincided with the CALMl gene, previously associated with symptomatic hip OA in the Japanese population75. A search of public genome resources revealed two other attractive candidate genes within the linkage area: FLRT2, encoding a small molecule found in the extracellular matrix of cartilage , and DIO2, encoding for D2, a selenoprotein that converts intracellularly inactive thyroxine (T4) to active thyroid hormone, (T3) (Fig. 4). D2 is an important provider of local bioactive T3 in target tissues such as growth plates " . To examine whether genetic variation in these 3 genes explains the observed linkage signal, tagging SNPs covering the haplotype blocks in which these genes reside were genotyped in the GARP cohort.
Table 9 Known genes within the one LOD-drop interval encompasses 26 cM (73-97 cM) containing the markers D14S74, D14S1037, D14S1044 and D14S280. on chromosome 14 based on RefSeq, mRNA, TrEMBL and Swiss-Prot prediction using Ensembl genome database v35
HUGO ID Genbank ID Description
NRXN3 NM_004796;NM_13897 Neurexin-3-alpha precursor (Neurexin Ill-alpha).
0
Full-length cDNA 5-PRIME end of clone CS0CAP002YE20 of Thymus of Homo sapiens (human). DIO2 NM 000793 Deiodinase, iodothyronine, type Il
NIVM 52446 TSHR NM_000369 Thyrotropin receptor precursor (TSH-R) (Thyroid-stimulating
NM_001018036 hormone receptor).
GTF2A1 NM 015859 Transcription initiation factor MA subunit 1
NM_201595 STON2 NM_033104 Stonin-2 (Stoned B). HUGO ID Genbank ID Description
SEL1 L NM 005065 Sel-1 homolog precursor (SeI-I L)
Full-length cDNA clone CS0DF017YB20 of Fetal brain of Homo sapiens
FLRT2 NM_013231 Leucine-rich repeat transmembrane protein FLRT2 precursor (Fibronectin-like domain-containing leucine-rich transmembrane protein 2).
GALC NM_000153 Galactocerebrosidase precursor (EC 3.2.1.46) (GALCERase)
N M_001037525 (Galactosylceramidase) (Galactosylceramide beta- galactosidase)
GPR65 NM_003608 Psychosine receptor (G-protein coupled receptor 65) (T cell- death associated protein 8). CDNA FLJ43399 fis, clone OCBBF2009926.
KCNK10 NM_021161 Potassium channel subfamily K member 10 (Outward rectifying
NIVM38317; potassium channel protein TREK-2) (TREK-2 K(+) channel
NIVM 38318 subunit).
SPATA7 NM_018418;NM_00104 Spermatogenesis-associated protein 7 (Spermatogenesis-
0428 associated protein HSD3) (HSD-3.1).
PTPN21 NM_007039 Tyrosine-protein phosphatase non-receptor type 21 (EC 3.1.3.48) (Protein-tyrosine phosphatase D1).
ZC3H14 NM_024824; nuclear protein UKp68 isoform 4
NM_207660;
NM_207661 ;
NM_207662
EML5 NM_183387 echinoderm microtubule associated protein like 5
TTC8 NIVM44596; Tetratricopeptide repeat protein 8 (TPR repeat protein 8)
NM_198309 (Bardet- Biedl syndrome 8 protein).
NM_198310
CHES1 NM_005197 Checkpoint suppressor 1 (Forkhead box protein N3). Protein PRO1768.
NM_145231
PREDICTED: similar to RAB42, member RAS homolog family
TDP1 NM_018319; Tyrosyl-DNA phosphodiesterase 1 (EC 3.1.4.-) (Tyr-DNA
N M_001008744 phosphodiesterase 1).
KCNK13 NM_022054 Potassium channel subfamily K member 13 (Tandem pore domain halothane- inhibited potassium channel 1 ) (THIK-1 )
PSMC1 NM 002802 26S protease regulatory subunit 4 (P26s4) (Proteasome 26S subunit ATPase 1).
CDNA FLJ43693 fis, clone TBAES2005543.
PREDICTED: similar to ribosomal protein L21 isoform 1
CALM1 NM 006888 Calmodulin (CaM). HUGO ID Genbank ID Description
CALM3 NM_006888 Calmodulin (CaM). TTC7B NM 001010854 Tetratricopeptide repeat protein 7B (Tetratricopeptide repeat protein 7-like-1).
CDNA FLJ37957 fis, clone CTONG2009529.
CDNA FLJ44835 fis, clone BRACE3048565.]
RPS6KA5 NM_004755; Ribosomal protein S6 kinase alpha-5 (EC 2.7.11.1) (Nuclear NM 182398 mitogen- and stress-activated protein kinase 1 ) (90 kDa ribosomal protein S6 kinase 5) (RSK-like protein kinase) (RSKL).
[Source:Uniprot/SWISSPROT;Acc:O75582]
C14orf159 NM_024952 Protein C14orf159, mitochondrial precursor. GPR68 NM 003485 Sphingosylphosphorylcholine receptor (Ovarian cancer G-protein coupled receptor 1) (OGR-1) (G-protein coupled receptor 68)
(GPR12A).
Full-length cDNA clone CS0DL007YB06 of B cells (Ramos cell line) of Homo sapiens (human) (Fragment).
C14orf161 NM 024764
2.2.2 Combined linkage and association
Joint modelling of linkage and association by using LAMP revealed no evidence for association with the putative disease locus of individual SNPs in CALMl (including the functional SNP rs 12885713) or FLRT2 (Table 11). However, a significant predisposing association (P = 0.006) with the C allele of DIO2 SNP rs225014 and a protective association with the T allele of DIO2 rs 12885300 (P = 0.04) and a putative disease locus (Table 12). The complete linkage disequilibrium (LD) test of LAMP between rs225014 and the disease locus was rejected (P = 0.002), indicating that the SNP was able to explain only part of the linkage signal. To confirm these results obtained by LAMP by a more robust method, allele frequencies in sibling pairs sharing two alleles identical by descent (IBD) at the DIO2 locus, indicating those subjects that contribute to the linkage, were compared with allele frequencies of those subjects that did not contribute to the linkage (Table 12). The frequency of the C allele of rs225014 (P = 0.0032) was again significantly increased among subjects that contributed to the linkage with this approach. The T allele of DIO2 SNP rsl2885300 did not show significant association (P = 0.13, data not shown). Combined linkage association approaches have, however, limited power for complex diseases such as OA when multiple (rare) genetic variants with small effect sizes are expected to be involved . We therefore may expect that additional relevant genetic variation may reside in the region of linkage. Table 11 Combined linkage and association analysis using LAMP
Gene SNP reference allele GARP siblings MAFd P-valueu
FLRT2 rs2057311 G>C 0.42 (303/726) 1.0 rs17121375 A>G 0.02 (14/732) 0.5 rs 17646457 G>A 0.14 (103/730) 0.2 rs2239576 T>C 0.14 (98/726) 0.09 rs1129671 OT 0.28 (208/740) 0.8
CALM1 rs3814847 OG 0.34 (243/714) 0.3 rs 12885713 T>C 0.43 (298/696) 0.3 rs2300496 A>C 0.43 (312/730) 0.3 rs2300502 G>A 0.11 (81/734) 0.8 rs5871 T>C 0.08 (60/734) 0.3 rs3814845 OG 0.07 (52/730) 0.7
GARP = Genetics, osteoARthritis and Progression, MAF = minor allele frequency aMinor allele frequency (n minor alleles/ n total alleles) bP-values using combined linkage and association with the program LAMP (see URLs provided in paper).
2.2.3 Replication by association
Next, we attempted to confirm the observed association of the DIO2 SNPs rs225014 and rsl2885300 in a UK population that consists of 360 and 1106 subjects who required replacement of knee and hip, respectively due to OA signs and symptoms and 714 controls (Table 8) . For rs225014 a significant predisposing genotypic association was revealed for cases as compared to controls (Table 13; P = 0.02). Stratified analysis established that this effect was mainly driven by female subjects with hip replacements (Table 13; P = 0.006 in genotypic frequency). Furthermore, a significant protective allelic and genotypic effect was observed among female hip replacement cases for the T allele of the DIO2 SNP rs 12885300 (P = 1.5 x 10"4 and P = 2.3 x 10"4, respectively). This latter effect was driven solely by the UK female control group, showing unexpected high frequency of the T allele (Supplementary Table 3).
Table 12 Allele frequencies of DIO2 SNPs in GARP sib-pairs stratified for IBD status.
SNP Allele IBD = O IBD =I IBD =2 P-valueu
MAFa MAFa MAFa rs12885300 OT 0.34 (48/142) 0.38 (134/354) 0.33 (77/234) 0.04 rs2267872 G>A 0.11 (15/142) 0.11 (37/344) 0.07 (16/232) 0.30 rs225011 T>C 0.44 (62/142) 0.40 (137/343) 0.48 (110/230) 0.14 rs225014 T>C 0.35 (49/142) 0.32 (111/348) 0.44 (102/232) 0.006 rs10136454 OT 0.007 (1/143) 0.014 (5/358) 0.029 (7/240) 0.60
SNP = single nucleotide polymorphism; MAF = minor allele frequency; IBD = identical by descent; OR = odds ratio aMinor allele frequency stratified for IBD status (sum of minor alleles / sum of total alleles) bP- values observed using combined linkage and association with the program LAMP (see Web resources)74.
Additional replication of DIO2 SNP rs225014 and rsl2885300, using an alternative genotyping technology, in an independent set of 1712 females from the Rotterdam study showed a significant genotypic trend test of SNP rs225014 (P = 0.038) with severe radiographic signs of OA in the hip defined by Kellgren Lawrence score ≥ 3 (Supplementary Table 4). For the rsl2885300 we did not observe an association (data not shown). Considering the confirmation and replication studies together rs225014 unequivocally confirmed significant recessive genotypic association with OR of 1.58, 95% CI 1.25-2.00 with nominal two tailed P = 1.3 x 10" ( Table 8) even after Bonferroni adjustments for the models tested (P = 5.5 x 10" ). When using the Mantel-Haenszel test an OR of 1.70, 95% CI 1.23-2.35 with P = 0.0012 (Bonferroni adjusted for the number of models P = 0.0036) was observed without evidence for genetic heterogeneity. None of the SNPs in the studies revealed a departure from Hardy- Weinberg equilibrium in controls.
Table 13 Association of DIO2 SNP rs225014 between subjects with hip and/or knee replacement due to osteoarthritis and controls from the UK.
Group Genotype P-valued Allele P-valued rs 1225014
(TT) (TC) (CC) (T) (C)
All controls Count 292 343 80 0.02 927 503 0.67
(n = 715) % 40.8 48.0 11.2 64.8 35.2
All cases Count 664 659 225 1987 1109
(n = 1548) % 42.9 42.6 14.5 64.2 35.8
Female controls Count 151 175 39 0.02 477 253 0.29
(n = 365) % 41.4 47.9 10.7 65.3 34.7
Female cases Count 371 362 142 1104 646
(n= 875) % 42.4 41.4 16.2 63.1 36.9
Male controls Count 141 168 41 0.50 450 250 0.55
(n = 344) % 40.3 48.0 11.7 64.3 35.7
Male cases Count 293 297 83 883 463
(n = 673) % 43.5 44.1 12.3 65.6 34.4
All knees Count 160 161 39 0.52 481 239 0.36
(n = 360) % 44.4 44.7 10.8 66.8 33.2
Female knees Count 88 87 25 0.57 263 137 0.89
(n = 200) % 44.0 43.5 12.5 65.8 34.3
Male knees Count 72 74 14 0.461 218 102 0.23
(n = 156) % 45.0 46.3 8.8 68.1 31.9
All hips Count 469 461 176 0.004 1399 813 0.333
(n = 1106) % 42.4 41.7 15.9 63.2 36.8
Female hips Count 263 255 111 0.006 781 477 0.146
(n = 629) % 41.8 40.5 17.6 62.1 37.9
Male hips Count 206 206 65 0.37 618 336 0.836
(n= 477) % 43.2 43.2 13.6 64.8 35.2
SNP = single nucleotide polymorphism P-value of x statistics for specific case group versus the specific control sample
Using HapMap we evaluated the LD extension of rs225014 to exclude possible other susceptibility genes or variants present in this region, in LD with SNP rs225014. Flanking recombination hotspots define an approximately 260 kb interval containing DIO2 as only known gene. Pairwise LD analysis of rs225014 with all other SNPs displayed D' scores greater than 0.90 only. It is therefore unlikely that the LD of SNP rs225014 extends beyond this haplotype block surrounding the DIO2 gene (Figure 5). Haplotype analysis of the DIO2 SNPs rsl2588300 (OT) and rs225014 (T>C) in the combined confirmation/replication sample revealed three common haplotypes with frequencies > 0.05 (Supplementary Table 5). The common haplotype CC (frequency 0.34) exclusively carried the minor allele of DIO2 SNP rs225014. This haplotype showed a significant recessive genotypic association with an OR of 1.71, 95% CI 1.33-2.19 with nominal two tailed P = 2.6 x 10"5 (Table 14) also when applying Bonferroni correction for the number of models tested P = 7.8 x 10"5. The Mantel-Haenszel test statistics with this model gave an OR of 1.94, 95%CI 1.37-2.74 with P = 2.0 x 10"4 with no evidence for genetic heterogeneity. The magnitude of the effect of the haplotype association appears higher as compared to the association of rs225014 alone. Possibly this haplotype carries functional variation in LD with the rs225014 affecting OA susceptibility.
2.3 Discussion
2.3.1. Functionality of the DIO2 SNPs
DIO 2 SNP rs225014 is non- synonymous, resulting in the amino acid change Thr92Ala.
Residue 92 is the first amino acid of the instability loop in D2 and this loop is the key determinant of D2 turnover rate80. Although in vitro studies have not been able to assess a functional difference between the 92Thr and the minor 92AIa allele on the instability loop in HEK293 cells it was shown by Canani et al that D2 velocity is decreased and insulin resistance is increased in tissues of homozygous 92AIa patients with type 2 diabetes mellitus. Furthermore, functional relevance on enzyme activity was shown by others for DIO2 SNP rsl2885300, which is localized in a short open reading frame a (ORFa) within the 5 '-untranslated region of the gene and also known as D2-ORFa- Gly3 As . The ORFa has been shown to reduce the D2 translation efficiency. In vitro studies by others have shown that this inhibition was abolished by mutating the start
Sn SV codon and weakened by the amino acid modification due to SNP rsl2885300 . The D2-92Ala allele resides exclusively on the predisposing haplotype CC, whereas, the D2- ORFa-3 Ala allele resides exclusively on haplotype TT (Supplementary Table 5). The exact (opposite) interplay and functional relevance of these two SNPs needs to be investigated further.
2.3.2. Function of D2 enzyme
The D2 enzyme as key regulator of local T3 availability may contribute to osteoarthritis development in different ways. In the growth plate, the conversion of T4 into T3 by D2, inhibits chondrocyte proliferation but stimulates chondrocyte differentiation and subsequently bone matrix synthesis. This process of endochondral ossification is essential for the formation of the skeleton88. Lower expression of D2 in the growth plate has been shown to contribute to the pathogenesis of tibial chondrodysplasia in chicken89. In the GARP study homozygous female carriers of the minor allele of rs225014 were significantly shorter (mean height 162 centimetres) as compared to other carriers (mean height 166 centimetres, P = 0.001), stressing that skeletal development and growth may be involved in osteoarthritis susceptibility in this study. Table 14 Genotypic OR of carriers of 0, 1 or 2 copies of DIO2 haplotype rs12885300-rs225014 C-C in subjects of the UK and Rotterdam study
UKa Copies of C-C P-valueD Haplotype P-valueD
(0) (1 ) (2) (others) (C-C)
Controls Count 162 169 30 0.002 494 229 0.037
% 44.9 46.8 8.3 68.3 31.7
Hip Count 269 254 99 793 452 replacement % 43.2 40.8 15.9 63.7 36.3
Model P-valuec OR 95%CI
Trend 0.045 1.22 1.01-1.47
Dominant 0.637 1.07 0.82-1.38 Recessive 0.001 2.09 1.36-3.22
Rotterdam Copies of C-C P-valueD Haplotype P-valueD
1 others C-C
Controls Count 695 111 170 0.173 2167 1117 0.078
% 42.3 47.3 10.4 66.0 34.0
Severe hip Count 31 44 14 106 72
ROA % 34.8 49.4 15.7 59.6 40.4
Model P-valuec OR 95%cr
Trend 0.045 1 .40 1. 00-1.93
Dominant6 0.318 1 .28 0. 79-2.06
Recessive6 0.035 2 .05 1. 05-3.99
Combined Copies of C-C P-valueD Haplotype P-valueD
1 others C-C
Controls Count 857 947 200 6.1x10"' 2662 1347 0.029
% 42.8 47.3 10.0 66.4 33.6
Cases Count 300 297 113 898 523
% 42.3 41.8 15.9 63.2 36.8
Model P-valuec OR 95%cr
Trend 0.028 1 .15 1. 02-1 .31
Dominant 0.817 1 .02 0. 86-1 .21
Recessive 2.6 x 105 1 .71 1. 33-2 .19
ROA radiographic signs of osteoarthritis SNP = single nucleotide polymorphism; OR = odds ratio, Cl = confidence interval aFemale cases with hip replacements versus the female controls bTwo tailed P-value of χ2 statistics logistic egression analysis. OR and P-values in Rotterdam study were adjusted for age and BMl. dFemale cases with severe radiographic hip OA (Kellgren Lawrence score > 3) versus female controls. eSince a significant trend effect is observed, the recessive and dominant model were assessed with subjects homozygous for the major allele as reference.
2.3.3 D2 and Osteoarthritis
In osteoarthritic cartilage, chondrocytes, in an attempt to repair damaged matrix, show increased metabolic activity. During progression of OA, chondrocytes undergo phenotypic dedifferentiation to a hypertrophic state expressing similar features as chondrocytes residing in the growth plate . As such chondrocyte hypertrophy debilitates cartilage viability by a switched expression of bone specific collagens which initiates calcification of the matrix and up-regulation of cartilage specific proteolytic enzymes . Furthermore, it was recently shown that inflammatory signals, up regulate D2 expression via a dimer combination of ReIA (p65) with nuclear factor-κB (NF-κB) . This p65/NF- KB dimer has also been shown to activate pro-inflammatory cytokines in chondrocytes mediating cartilage degeneration.
Finally, OA is characterized by formation of bony enlargements at the edges of the bone called osteophytes a process which is characterized by endochondral ossification . All together deficiency of D2, as key regulator of the endochondral ossification process, may predispose to the incidence of OA via its influence on skeletal formation or later in OA cartilage via its influence on viability of chondrocytes and formation of osteophytes. Our findings underscore the importance of thyroid hormone in the etiology of symptomatic OA. We are confident that knowledge of genetic factors and their molecular cascades contributes to a better understanding of the pathogenesis of osteoarthritis and will lead to improved diagnosis, treatment and prevention. Supplementary Table 3 Association of DIO2 SNP rs12885300 between subjects with hip and/or knee replacement due to osteoarthritis and controls from the UK.
Group Genotype P-valued Allele P-valued rs12885300
(CC) (CT) (TT) (C) (T)
All controls Count 263 362 101 0.17 888 564 0.16
(n = 726) % 36.2 49.9 13.9 61.2 38.8
All cases Count 625 716 211 1966 1138
(n = 1552) % 40.3 46.1 13.6 63.3 36.7
Female controls Count 116 193 63 0.001 425 319 4.5 X 10"4
(n = 372) % 31.2 51.9 16.9 57.1 42.9
Female cases Count 371 393 115 1135 623
(n= 879) % 42.2 44.7 13.1 64.6 35.4
Male controls Count 147 169 38 0.22 463 245 0.10
(n = 354) % 41.5 47.7 10.7 65.4 34.6
Male cases Count 254 323 96 831 515
(n = 673) % 37.7 48.0 14.3 61.7 38.3
All knees Count 133 175 52 0.92 441 279 0.97
(n = 360) % 36.9 48.6 14.4 61.3 38.8
Female knees Count 73 98 28 0.36 244 154 0.17
(n = 199) % 36.7 49.2 14.1 61.3 38.7
Male knees Count 60 77 24 0.35 197 125 0.19
(n = 158) % 37.3 47.8 14.9 61.2 38.8
All hips Count 463 499 148 0.06 1425 795 0.06
(n = 1110) % 41.7 45.0 13.3 64.2 35.8
Female hips Count 280 272 82 2.3 X 10"4 832 436 1.5 X 10"4
(n = 634) % 44.2 42.9 12.9 65.6 34.4
Male hips Count 183 227 66 0.36 593 359 0.19
(n= 476) % 38.4 47.7 13.9 62.3 37.7
SNP = single nucleotide polymorphism. aTwo tailed P values of χ2 statistics for specific case group versus the specific control sample Supplementary Table 4 Genotypic OR of carriers of DIO2 rs225014 in subjects of the UK and Rotterdam study
uκa Genotype Allele
(TT) (TC) (CC) (T) (C)
Controls Count 151 175 39 477 253
% 41.4 47.9 10.7 65.3 34.7
Hip Replacement Count 263 255 111 781 477
% 41.8 40.5 17.6 62.1 37.9
Model P-valueb OR 95%CI
Trend 0.160 1.14 0.95-1.37
Dominant 0.891 0.98 0.76-1.28
Recessive 0.003 1.79 1.21-2.65
Rotterdam0 Genotype Allele
(TT) (TC) (CC) (T) (C)
Controls Count 663 764 197 2090 1158
% 40.8 47.0 12.1 64.3 35.7
Severe hip ROA Count 28 45 15 101 75
% 31.8 51.1 17.0 57.4 42.6
Model P-valueb OR 95%CI
Trend 0.038 1.40 1.02-1.93
Dominant 0.182 1.40 0.86-2.28
Recess ived 0.043 1.97 1.02-3.82
Combined Genotype Allele
(TT) (TC) (CC) (T) (C)
Controls Count 814 939 236 2567 1411
% 40.9 47.2 11.9 64.5 35.5
Symptomatic OA Count 291 300 126 882 552
% 40.6 41.8 17.6 61.5 38.5
Model P-value" OR 95%CI
Trend 0.042 1.14 1.00-1.29
Dominant 0.674 1.01 0.85-1.21
Recessive 1.3 X 10"4 1.58 1.25-2.00 ROA = radiographic signs of osteoarthritis SNP = single nucleotide polymorphism; OR = odds ratio
Cl = confidence interval aFemale cases with hip replacements versus the female controls. bLogistic regression analysis, OR and P values in Rotterdam study were adjusted for age and BMI. cFemale cases with severe radiographic hip OA (Kellgren Lawrence score ≥ 3) versus female controls. dSince a significant trend effect is observed the recessive and dominant model were assessed with subjects homozygous for the major allele as reference.
Supplementary Table 5: Haplotype association analysis of Dl 02 rs12885300 (OT) and rs225014 (T>C).
Haplotypes UK study" Rotterdam study GARP"
Controls Cases P-value Controls Cases P-value Controls Cases P-value
CT 182 366 984 50 162 60
25.2 29.4 30.0 28.1 32.4 25.2
CC 229 452 0.037 1117 72 0.078 154 100 0.004
31.7 36.3 34.0 40.4 30.8 41.8
TT 292 407 6.6 x 10" 1124 52 0.169 174 74
40.3 32.7 34.2 29.2 34.8 30.9
TC 20 19 59 4 10 5
2.8 1.5 1.8 2.2 2.0 2.2
Ul
Total 723 1244 3284 178 500 240
SNP = single nucleotide polymorphism. aAlleles are in the following order of SNPs rs12885300 (OT) and rs225014 (T>C). Cases were females with hip replacements as compared to female controls as reference. cCases were females with severe radiographic hip OA (Kellgren Lawrence grade ≥ 3) as compared to female controls as reference. dCases are siblings contributing to the linkage (identical by descent 2 status) versus subjects identical by descent 0 or 1. Haplotype frequency and comparisons were adjusted for family relationship. eTwo tailed P-values were determined by comparing the frequency of the specific haplotype compared to the summed frequency of all other haplotypes as reference.
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Electronic-Database information and resources:
Accession numbers and URLs for data presented herein are as follows:
THESIAS version 2, http://ecgene.net/genecanvas/modules/news/
GENEHUNTER2, http://linkage.rockefeller.edu/soft/gh/ Online Mendelian Inheritance in Man (OMIM) http://www.ncbi.nlm.nih.gov/Omim/
(for OA and candidate genes)
Human ENSEMBL genome browser: http://www.ensembl.org/ NCBI35
Reference SNP Codes, http://www.ncbi.nlm.nih.gov/SNP/ Interpolated genetic map positions http://www2.qimr.edu.au/DavidD/ Molecular Cloning. A laboratory manual: www.molecularcloning.com Micro array protocols :http://www.protocol-online.org/prot/
Genetics Genomics/Microarray/ LAMP, http://csg.sph.umich.edu/LAMP;
Figure imgf000069_0001
LINKUTIL
Figure imgf000069_0002
HAPMAP, http ://'ww w.hapmap . org;
David Duffy, (http :// www2. qimr . cd u . au/'Dav idD/, Online Medelian inheritance in Man (OMIM) hύgjjwwwji^

Claims

Claims
1. Method for determining the risk of a subject for developing familial generalized osteoarthritis, comprising the step of determining the presence of a genetic marker indicative of a polymorphism affecting function or expression of a gene located on the human chromosome band 14q24.3 to 14q32.12 in the genome of the subject.
2. Method according to claim 1 for determining the risk of a subject for developing familial generalized osteoarthritis with late onset, comprising the step of determining the presence of a genetic marker indicative of a polymorphism located on the human chromosome band 14q24.3 to 14q32.12 in the genome of the subject.
3. The method according to claim 2, wherein the 14q24.3 to 14q32.12 genetic marker is selected from the group of markers consisting of D14S74, D14S1037, D14S1044 and D14S280.
4. The method according to claim 3, wherein the genetic marker located on human chromosome 14q24.3 to 14q32.12 for determining the risk of a subject for developing osteoarthritis is a polymorphism, preferably a single nucleotide polymorphism (SNP).
5. The method according to claim 4, wherein the polymorphism, preferably a SNP is present in a gene located on human chromosome 14q24.3 to 14q32.12 selected from the group of genes FLRT2, DIO2 and CALMl.
6. The method according to claim 5, wherein the polymorphism, preferably a SNP is in the DIO2 gene.
7. The method according to claim 5 or 6, wherein the SNP in the DIO2 gene is rs225014 (Thr92Ala).
8. The method according to any one of claims 5 to 7, wherein the polymorphism is in the FLRT2 gene.
9. The method according to any one of claims 5 to 8, wherein the polymorphism is in the CALMl gene.
10. Method preferably according to any one of claims 1 to 9 for determining the risk of a subject for developing familial generalized osteoarthritis with early onset, comprising the step of determining the presence of a genetic marker indicative of a polymorphism located on the human chromosome band 2q33.2 to 2q34 in the genome of the subject.
11. The method according to claim 10, wherein the 2q33.2 to 2q34 genetic marker is selected from the group of markers consisting of D2S326, D2S2257, D2S2314, D2S1391, D2S118, D2S315, D2S115, D2S72, D2S1384, D2S369, D2S155, D2S2358, D2S2208, D2S154, D2S2178, D2S371, D2S164, D2S126 and gata30e06.
12. The method according to claim 11, wherein the genetic marker located on human chromosome 2q33.2 to 2q34 for determining the risk of a subject for developing osteoarthritis is a polymorphism, preferably a single nucleotide polymorphism (SNP).
13. The method according to claim 12, wherein the polymorphism is present in a gene located on human chromosome 2q33.2 to 2q34 selected from the group of genes ALS2CR19, NRP2, NDUFSl, EEF1B2, GPRl, ADAM23, MDHl, CPO, KLF7, CREBl, FZD5, CRYGD, CRYGC, CRYGB, CRYGA, IDHl, PIP5K3, PTHR2.
14. The method according to claim 13, wherein the polymorphism is in the PTHR2 gene.
15. The method according to claim 13, wherein the polymorphism is in the NRP 2 gene.
16. The method according to any of claims 5 to 9 or 13 to 15, wherein the polymorphism detected by differential expression analysis.
17. The method according to any of the preceding claims, wherein osteoarthritis phenotype is early onset familial or hereditary osteoarthritis and symptomatic at multiple joints sites.
18. A probe or oligonucleotide flanking or comprising a polymorphism as defined any of claims 5 to 9 or 14 or 15..
19. The oligonucleotide according to claim 23 on a solid support and optionally on an array.
20. Kit of parts for the detection of osteoarthritis in humans comprising at least one or more oligonucleotides according to claims 23 or 24.
21. A nucleic acid construct comprising a nucleotide sequence encoding an RNAi agent that is capable of inhibiting the expression of a DIO2 gene having the SNP rs225014 (Thr92Ala) in a cell, wherein optionally the nucleotide sequence encoding the RNAi agent is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell.
22. A nucleic acid construct comprising a nucleotide sequence, encoding a D2 polypeptide, said nucleotide sequence not comprising the SNP rs225014, wherein the nucleotide sequence is operably linked to a promoter that is capable of driving expression of the nucleotide sequence in a cell.
23. The nucleic acid construct according to claim 21 or 22, wherein the cell is a chondrocyte.
24. The nucleic acid construct according to any one of claims 21 to 23, wherein the nucleic acid construct is a viral gene therapy vector selected from gene therapy vectors based on an adenovirus, an adeno-associated virus (AAV), a herpes virus, a pox virus and a retrovirus.
25. A method for preventing, delaying and/or treating familial generalized osteoarthritis in a subject, the method comprising pharmacologically altering the activity or the steady-state level of the polypeptide encoded by the gene DIO2 having the SNP rs225014 and optionally restoring the activity or the steady-state level of the polypeptide encoded by the gene DIO2 and not having the SNP rs225014.
26. The method according to claim 25, wherein the method comprises the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a nucleic acid construct as defined in any one of claims 21, 23 or 24 and optionally a nucleic acid construct as defined in any one of claims 22-24.
27. The method according to claim 26, wherein the pharmaceutical composition is administered within chondrocytes.
28. Use of a nucleotide sequence as present in the nucleic acid construct of claim
21 or 22 or of a nucleic acid construct according to any one of claims 21 to 24 for the manufacture of a medicament for preventing, delaying and/or treating familial generalized osteoarthritis in a subject , preferably in a method as defined in any one of claims 25 to 27.
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