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Methods for diagnosing osteoarthritis

Inactive Publication Date: 2010-02-04
CHU SAINTE JUSTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0035]In a specific embodiment, the selected test compound is potentially useful in preventing accumulation or retention of the pitx1 repressor protein or complex in cell nuclei or in promoting the pitx1 repressor protein or complex nuclear export.
[0077]As used herein the terms “detectably labeled” refer to a marking of a probe in accordance with the presence invention that will allow the detection of the mutation of the present invention. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods (64). Non-limiting examples of labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
[0096]The present invention relates to a kit for diagnosing OA and / or predicting whether a subject is at risk of developing OA comprising an isolated nucleic acid, a protein or a ligand such as an antibody in accordance with the present invention. For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the subject sample (DNA genomic nucleic acid, cell sample or blood samples), a container which contains in some kits of the present invention, the probes used in the methods of the present invention, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products. The present invention also relates to a kit comprising the antibodies which are specific to pitx1 repressors. Kits of the present invention may also contain instructions to use these probes and or antibodies to diagnose OA or predict whether a subject is at risk of developing OA.

Problems solved by technology

Moreover, the functional importance of these susceptibility loci has yet to be confirmed and illustrates our incomplete knowledge of the biology of OA.

Method used

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  • Methods for diagnosing osteoarthritis
  • Methods for diagnosing osteoarthritis
  • Methods for diagnosing osteoarthritis

Examples

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example 1

Comparison of Pitx1 Expression in Articular Chondrocytes of OA Subjects with that of in Articular Chondrocytes Matched Controls

[0138]To determine whether pitx1 plays a role in the genetic control of OA onset, an expression analysis of pitx1 gene using RNA prepared from articular chondrocyte cultures derived from knee cartilage of OA patients (n=7) and age- and gender-matched control subjects (n=4) was performed. Pitx1 expression was detected only in articular chondrocytes derived from matched controls, while in OA articular chondrocytes, Pitx1 expression was abrogated or barely detectable by RT-PCR (FIG. 2a). Analysis of Pitx1 protein levels and distribution in human knee joint sections showed Pitx1 proteins only in control cartilages (n=8), while Pitx1 proteins were hardly detected in OA cartilage sections (n=8) (FIG. 2bcd).

example 2

Identification of Pitx1 Promoter Mutation

[0139]To examine the mechanisms turning off pitx1 gene expression in OA patients, the 5′ regulatory region of human pitx1 gene was examined for specific mutations leading to a progressive loss of Pitx1 expression during adulthood. Sequencing analysis of genomic DNA obtained from OA, rheumatoid arthritis (RA) and matched control subjects revealed, along a 10 kb promoter region of human pitx1 gene, a single homozygous mutation (−3727 C→T) (position corresponds to distance from transcription point) affecting only OA patients (11 / 43) with a high frequency (25%) while none of the RA patients (0 / 29) and matched control subjects (0 / 11) had the homozygous mutation. The specificity, the positive predictive values and negative predictive values of the mutation were calculated for each group as reported in Table 4 below. A statistically significant association between the mutation and diagnosis was calculated (two-tailed test) by comparing OA versus RA ...

example 3

Determination of Functional Consequences of Mutation in the E2F-Like Site on Complex Binding

[0141]To determine the functional consequences of the homozygous mutation found in OA patients, it was investigated whether E2Fs were able to bind this E2F-like site using nuclear extracts prepared with OA articular chondrocytes as described above. EMSA analysis using both radiolabeled E2F-like sites (wild-type FIG. 4a versus mutant FIG. 4b) showed no supershift of the bound complex with any antibodies against E2Fs, or their dimerization partners DP-1 or DP-2 (E2F2, E2F8 and DP2 data not shown). The Sp1 and Sp3 transcription factors were also analysed since they bind GC-rich regions such as the E2F-like site found in the human Pitx1 promoter. Unfortunately, there was no supershift with either anti-Sp1 or anti-Sp3 antibodies. Addition of BCoR antibodies generated the binding of an additional lower complex bound in presence of either probes although the binding was increased with the mutant one...

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Abstract

Methods are disclosed for assessing risk of developing osteoarthritis. Said methods comprise determining the cellular localizations, blood or synovial fluid concentrations, sumoylation states and post-translational modifications of pituitary homeobox transcription factor 1 (pitx-1) repressor proteins, identifying mutations in genes which encode said proteins and identifying any mutations or post-translational modifications causing the nuclear accumulation or retention of said proteins. Said proteins include prohibitin (PHB-I), prohibitone (PHB-2) and B cell lymphoma-6 transcriptional repressor interacting co-repressor (BCoR).

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority on U.S. provisional application No. 60 / 854,077 filed on 25 Oct. 2006. All documents above are herein incorporated by reference.FIELD OF THE INVENTION[0002]The present invention relates to methods of prognosing osteoarthritis, methods of selecting compounds and kits therefore.BACKGROUND OF THE INVENTION[0003]The etiology of (OA), the most common form of arthritis, remains unclear notwithstanding the multiplicity of factors that have been considered in primary OA (1, 2). At present, it has become increasingly evident that the majority of OA genetic susceptibility loci cannot be attributed only to structural genes or genes regulating bone mass (3-5). These studies have also highlighted the great heterogeneity and differences in the degree of OA heritability between different joint sites (e.g. hand versus knee) and gender. This is also reflected by the multiplicity of loci identified in OA linkage studies and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/567C07K16/18C07K14/00
CPCC07K14/4703C12Q1/6883C12Q2600/158C12Q2600/156G01N2800/105C12Q2600/136G01N33/6893G01N2333/47G01N2800/50G01N2333/4704G01N2333/4706
Inventor MOREAU, ALAIN
Owner CHU SAINTE JUSTINE
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