Patent Application
20120003642
THIS INVENTION relates to risk factors associated with tendon and ligament injuries. More particularly, this invention relates to molecular markers useful in determining increased susceptibility or risk of a subject in developing a tendon, ligament, and/or soft tissue pathology; to an assay for use in determining an increased susceptibility or risk in a subject in developing a tendon, ligament and/or soft tissue pathology; to a method for diagnosing or determining an increased risk or susceptibility in a subject in developing a tendon, ligament and/or soft tissue pathology; and to a kit for use in determining an increased risk or susceptibility of a subject in developing tendon and/or ligament pathologies.
There is a spectrum of pathologies that can affect soft tissues, tendons and ligaments, as well as their surrounding structures'. As an example, Achilles Tendinopathy (AT) is one of these pathologies which is a painful and degenerative condition that affects subjects who participate in a range of sporting pursuits, as well as occurring in the less physically active2,3. Acute spontaneous rupture is another common pathology that can affect the Achilles tendon, particularly in the middle-aged, male athlete. Injury to the anterior cruciate ligament (ACL) is an example of a common ligament injury. It has been described as one of the most severe injuries sustained in a sporting population. Participants of sports which involve a sudden deceleration or change in direction are particularly at risk of rupturing their ACL. A number of intrinsic and extrinsic factors have been implicated in raising the risk of both tendon and ligament pathologies5.
In recent years evidence has emerged that Achilles tendon injuries, ACL injuries, as well as rotator cuff injuries and shoulder dislocations have a genetic component. As such, it has been shown that a GT repeat variant within the gene that encodes the tenascin C protein (TNC), a key constituent of tendon which is regulated by mechanical loading6,7, is associated with Achilles tendinopathy and rupture8. In addition to the TNC gene, polymorphisms within the 3′-untranslated region of the COL5A1 gene have also been associated with Achilles tendinopathy in both South African9 and, as more recently shown, Australian populations10. This polymorphism has also recently been shown to be associated with ACL injuries in female athletes. Interestingly, variations within the Sp1 binding site of another collagen gene, viz. COL1A1, does not appear to independently associate with Achilles tendon pathology11, nor do polymorphisms within the related COL14A1 and COL12A1 genes12.
In addition to the TNC and COL5A1 genes, it may well be that predisposition to tendon, ligament, and other soft tissue injuries may be associated with other genes. Such genes may encode proteins with regulatory roles in maintaining extracellular matrix (ECM) homeostasis. It is an object of the present invention to locate other genes or polymorphs which may be involved in tendon, ligament and/or other soft tissue pathologies. It is a further object of the invention to locate possible areas within so-called MMP genes which may be associated with tendon and ligament pathologies. A further object of the invention is to determine whether MMP3 may be involved in tendon and ligament pathologies by interacting with other genes or gene products, and to provide diagnostic assays to identify subjects or individuals at risk of developing tendon, ligament, and other soft tissue injuries and pathologies, including, but not limited to, Achilles tendon pathologies and ACL ruptures. A further object of the invention is to determine whether MMP genes interact with the specific markers in genes that encode other protein components, (such as collagens, proteoglycans, and glyoproteins, of connective tissue) in modulating the risk of soft tissue injuries.
Broadly, according to one aspect of the invention, there is provided a method of determining in a subject a predisposition to, or increased risk for, developing a tendon, ligament, or other soft tissue injury or pathology, the method comprising the step of screening the subject for the presence of at least one polymorphism in at least one gene family selected from the group consisting of any one or more of:
The collagen gene family may include the COL5A1 and COL12A1 genes. The glycoprotein gene family may include the TNC and COMP genes.
More specifically, the method may include the step of detecting or screening for the presence of a polymorphism in the matrix metallo-protease 3 (MMP3) gene which has modified, augmented, or mitigated interaction with a COL5A1 polymorphism product, when compared to a wild-type interaction. More particularly, the MMP3 polymorphism may be a polymorphism which has a modified, augmented, or mitigated interaction with the rs12733 COL5A1 polymorphism, and/or any other linked polymorphism, and the product encoded thereby.
According to another aspect of the invention, there is provided a molecular marker for use in diagnosing a predisposition to, or increased risk for, developing tendon, ligament, or other soft tissue pathology or injury in a subject, the molecular marker comprising at least one isolated nucleic acid fragment derived from an MMP3 gene, flanking sequences thereof, cis-regions associated therewith, 5′UTR regions, 3′UTR regions thereof, sequences complementary thereto, sequences which can hybridize under strict hybridization conditions thereto, and functional discriminatory truncations thereof. The molecular marker may be DNA-based, RNA-based, or other combinations of nucleic acids or modified bases.
The molecular marker may be a part of, or a fragment derived from, an MMP3 gene, the fragment being between 10 and 40, preferably between 15 and 35, more preferably between 20 and 30 nucleic acids in length, and which hybridizes under stringent hybridization conditions to at least a portion of the MMP3 gene. This may include sequences complementary to the marker, and sequences having substitutions, deletions or insertions, sequences which can hybridize under strict hybridization conditions thereto, and functional discriminatory truncations thereof.
In one embodiment, the molecular marker is a polymorphic marker, preferably an SNP. The SNP may be any one or more SNPs selected from the group consisting of rs591058, rs679620, and rs650108, together with any other SNP closely linked (i.e. which is in high linkage disequilibrium) with any of the three specific SNPs listed above.
More particularly, the SNPs may be selected from the group consisting of:
More particularly, the molecular marker may be, or may be detectable using, any one or more isolated oligonucleotides selected from the group comprising:
sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
Accordingly, the invention extends to a primer or oligonucleotide sets for use in detecting or diagnosing a predisposition to, or increased risk for, developing tendon, ligament, or other soft tissue pathologies or injuries in a subject, the primer or oligonucleotide sets comprising isolated nucleic acid sequences selected from the group consisting of:
sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
In addition, the molecular marker may comprise any one or more isolated nucleic acid sequences selected from the group consisting of:
fragments thereof, sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
According to a still further aspect of the invention, there is provided an isolated nucleic acid molecule for detecting at least one SNP provided hereinbefore, wherein the nucleic acid molecule comprises less than 40, less than 30, less than 20, or even preferably less than 10 contiguous nucleotides selected from the group consisting of SEQ ID NOS 1 to 12 and fragments, complementary sequences, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
The invention extends also to a detection reagent capable of detecting one or more single nucleic acid polymorphisms selected from the group consisting of the SNPs listed hereinbefore, fragments thereof, sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
The invention extends to the use of the sequences and/or markers of the invention in other assays, such as RFLPs and AFLPs.
According to another aspect of the invention, there is provided a diagnostic assay comprising any one or more of the markers described hereinbefore, fragments thereof, sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
According to yet another aspect of the invention, there is provided a method of determining a predisposition for, or increased risk of, developing a tendon, ligament and/or soft tissue pathology or injury in a subject, the method comprising the steps of screening a subject for a polymorphism in an MMP3 gene.
The polymorphism may be any one of more of the polymorphisms listed hereinbefore, polymorphisms in high linkage disequilibrium with the listed polymorphisms, or a polymorphism detectable using any one or more of the sequences listed hereinbefore, fragments thereof, sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
The method may include the additional steps of:
According to another aspect of the invention, there is provided use of a molecular marker of the invention in diagnosing a predisposition to a soft tissue pathology in a subject.
According to a still further aspect of the invention, there is provided a kit for use in diagnosing a predisposition to a soft tissue pathology in a subject, the kit comprising:
The kit may further include any one or more of reagents, such as buffers, DNases, RNAses, polymerases, instructions, and the like.
The molecular markers may be any one or more markers selected from the markers listed hereinbefore.
The soft tissue may be a connective tissue injury, and may include tendon and/or ligament injuries such as, for example, Achilles tendon, rotator cuff tendons, patellar tendon, shoulder ligament, knee ligament and ankle ligament pathologies. The sample may comprise an animal tissue or blood sample, such as a human tissue or blood sample.
Further features of the invention will now be described with reference to the following non-limiting examples and figures.
In the drawings:
For the purposes of this specification, a “polymorphism” may include a change or difference between two related nucleic acids. A “nucleotide polymorphism” refers to a nucleotide which is different in one sequence when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence. A “probe” or “molecular marker” is an RNA sequence(s) or DNA sequence(s) or analogues, modified versions, or the complement of the sequences shown. This may include a “genetic marker”, which is a region on a genomic nucleic acid mapped by a molecular marker or probe. A “probe” is a composition labelled with a detectable label. A “probe” is typically used herein to identify a marker nucleic acid. A polynucleotide probe is usually a single-stranded nucleic acid sequence that can be used to identify complementary nucleic acid sequences, or may be a double- or higher order-stranded nucleic acid sequence which can be used to bind to, or associate with, a target sequence or area, generally following denaturing. The sequence of the polynucleotide probe may or may not be known. An RNA probe may hybridize with its corresponding DNA gene, or to a complementary RNA, or to other type of nucleic acid molecules. As used herein the term “functional discriminatory truncations” mean nucleic acid sequences, modified nucleic acid sequences, or other nucleic acid variants which, although they are truncated forms of sequences presented herein or variants thereof, can still bind in a discriminatory manner to target gene or nucleic acid sequences described herein and forming part of the present invention. The terms “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. An “amplified mixture” of nucleic acids includes multiple copies of more than one (and generally several) nucleic acids. “Stringent hybridization conditions” in the context of nucleic acid hybridization are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the Tm point for a particular probe. An example of stringent wash conditions for, say, a Southern blot of such nucleic acids is a 0.2×SSC wash at 65° C. for 15 minutes. Such a high stringency wash may be preceded by a low stringency wash to remove background probe signal. An example of a low stringency wash is 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization event. For highly specific hybridization strategies such as allele-specific hybridization, an allele-specific probe is usually hybridized to a marker nucleic acid (e.g., a genomic nucleic acid, an amplicon, or the like) comprising a polymorphic nucleotide under highly stringent conditions.
One hundred and fourteen Caucasian subjects diagnosed with Achilles tendon injuries, including 75 with chronic Achilles tendinopathy (TEN) using clinical criteria and 39 with partial (N=3) or complete ruptures of the Achilles tendon (RUP), were recruited for this study from the medical practice at the Sports Science Institute of South Africa and other clinical practices within the greater Cape Town area of South Africa. Rupture of the Achilles tendon was confirmed during surgery or by imaging. Ten of the subjects in the RUP group had a history of tendinopathy. An additional, 98 apparently healthy, unrelated, Caucasian subjects without any history of symptomatic Achilles tendon injuries were recruited as controls (CON). The inclusion and exclusion criteria of the participants have been described previously8,9.
Prior to participation in this study, subjects gave informed written consent and completed medical history questionnaire forms. This study was approved by the Research Ethics Committees of the Faculty of Health Sciences within the University of Cape Town, South Africa and the University of Northampton, England.
DNA was extracted using the procedure described by Lahiri and Nurnberg20 and modified by Mokone et al.8 Single nucleotide polymorphisms (SNPs) within the MMP3 gene and its 5′-flanking sequence were identified from databases hosted by the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov) and the International HapMap project (www.hapmap.org). The MMP3 gene is transcribed in the negative orientation resulting in the mRNA sequence corresponding to the bottom DNA strand. As indicated in
DNA samples were genotyped for all three MMP3 variants using fluorescence-based Taqman® technology (Applied Biosystems, Foster City, Calif., USA). Allele specific probes and flanking primer or oligonucleotide sets (Table 1) were used along with a pre-made PCR mastermix containing ampliTaq DNA polymerase Gold (Applied Biosystems, Foster City, Calif., USA) in a total reaction volume of 25 μl. PCR consisted of a 10 minute heat activation step (95° C.) followed by 40 cycles of 15 s at 92° C. and 1 minute at 60° C. PCR was performed on an MJ Miniopticon thermocycler (BioRad, UK) and genotypes were determined by endpoint fluorescence using MJ Monitor analysis software (version 3.1).
Assuming an allele frequency of 0.43 (observed for MMP3) in the CON group, group sizes of 97 in each group would be adequate to detect an allelic odds ratio of at least 2.25 at a power of 80% and significance level of 5%. Linear and logistic regression models were used to assess differences between the characteristics of the TEN, RUP and CON groups for quantitative and categorical data, respectively. Logistic regression was used to compare the combined, as well as the separate (TEN and RUP) Achilles tendon cases to the control groups, with respect to genotype, allele and haplotype frequencies. Two different methods were used to assess gene-gene interaction between MMP3 and COL5A1 (SNP rs12733). Allele combinations consisting of the markers on the two different genes were constructed and their association with case-control status was tested. The OR MDR (odds ratio based multifactor dimensionality reduction) method23 was also used to select from the pair of SNPs (from all SNPs) with the strongest association with tendinopathy. The quantitative measure of disease risk being analysed is commonly referred to as an OR, and it represents the estimated relative risk of disease with a specific combination of genotypes. Data was analysed using the freely available programming language R (www.r-project.org), specifically packages DGC-genetics (LD, Hardy-Weinberg, genotype and allelic association), haplo.stats (inferred haplotype association) and ormdr (interaction between loci on case-control status). Pass2008 (www.ncss.com) was used for sample size calculation.
The TEN, RUP and CON groups were similarly matched for age, height, gender and country of birth (Table 2). The age of the TEN and RUP groups were the age of initial onset of symptoms of Achilles tendon injury, which were on average 7.8±8.0 and 7.5±8.9 years prior to recruitment in this study respectively. The reported weights were also as at time of recruitment, not at time of onset of injury. The TEN and RUP groups were on average significantly heavier with corresponding higher BMI than the CON group. In addition, the RUP group was also on average significantly heavier, with corresponding higher BMI, than the TEN group. There were no MMP3 single nucleotide polymorphism genotype effects on any of the subject characteristics (results not shown).
Twenty six (38.2%) and 5 (13.5%) of the subjects were diagnosed with bilateral chronic Achilles tendinopathy and Achilles rupture respectively. Multiple Achilles tendon injuries (greater than one) were documented in 16 (22.9%) and 13 (35.1%) of the TEN and RUP subjects respectively. Forty four percent of the TEN and 35% of the RUP subjects reported either a bilateral and/or multiple Achilles tendon injury.
There were no significant differences in the genotype distributions of SNPs rs679620 (P=0.064), rs591058 (P=0.117) and rs650108 (P=0.132) between the Achilles tendon pathology (combined TEN and RUP) and control groups (see
There were, however, no significant differences in the distribution of the genotype (rs679620, P=0.666; rs591058, P=0.734; and rs650108, P=0.627) (
The two SNPs rs679620 (A/G) and rs591058 (T/C) were shown to be in almost perfect LD. Allele G in the former corresponds to allele C in the latter, with only one heterozygous subject/individual for rs679620 being TT for rs591058. The D′ measure is 1 (P<0.001) and the coefficient of correlation, r, is 0.98. Both SNPs are also in high LD with rs650108, D′=1 (P<0.001). The coefficient of correlation of rs650108 is −0.57 and −0.58 with rs679620 and rs591058 respectively (
Only the AT and GC haplotypes were inferred with a frequency greater than 1% from SNPs rs679620 and rs591058. Since the GG and CC genotypes of these SNPs were over-represented in the Achilles tendinopathy group (
September et al.10 have previously shown that the CC genotype of the COL5A1 BstUI RFLP (rs12722) was under-represented in patients with chronic Achilles tendinopathy. MMP3 SNP rs679620 together with the COL5A1 SNP formed the best pair of genotypes for estimating risk for Achilles tendinoapthy. The genotype pairs together with their frequencies and estimated risk are summarised in Table 3. The MMP3 rs679620 A allele (AA or AG genotype) combined with the COL5A1 rs12722 CC genotype had the lowest risk for Achilles tendinopathy. All the possible A and C allele combinations were associated with the lowest risk. In support of this, the MMP3 rs679620 G/A and COL5A1 rs12722 C/T allele combinations (pseudo-haplotypes) were significantly associated with TEN and CON status (global-stat=10.4, df=3, P=0.016) (
All three MMP3 polymorphisms (rs679620, rs591058 and rs650108) investigated in this disclosure have been found to be associated with AT. As single loci, the rs679620 polymorphism, GG genotype, and rs591058 polymorphism, CC genotype, co-segregate with Achilles tendinopathy with odds ratios of 2.5 and 2.3 respectively. The rs650108 SNP, AA genotype has a higher odds ratio (4.9), but only 7 cases possessed this genotype due to its low allele frequency in the population. When analysed as an inferred haplotype, the ATG sequence combination of these three SNPs is significantly under-represented in AT cases compared to controls and it may be that this haplotype protects against the development of AT.
Reduction in MMP3 RNA or protein is likely to result in increased proteoglycans27 which may result in degenerative tendons. Allelic association to a trait or disease does not necessarily infer cause. However, the rs679620 variant of MMP3 is a non-synonymous polymorphism. Specifically, a glutamate residue is coded for by inclusion of the G allele (GAA codon) at the rs679620 loci, while a lysine residue is encoded for by the A allele (AAA codon). Although both residues are polar, the glutamate side chain is negatively charged compared to the positive charge on lysine31. The residue sits at position 45 from the start of the polypeptide chain32 and the first 82 residues, incorporating the propeptide,33 are cleaved by a proteinase during the processing of proMMP3 into mature MMP334. Appropriate removal of the propeptide may have some dependency on the presence of either a Lys or Glu at position 45 and hence may influence downstream function of the mature MMP3 enzyme. This association may be due to genetic linkage between the non-synonymous MMP3 polymorphism (rs679620) and other polymorphisms within the MMP3 gene and flanking sequences. In support of this, the three SNPs investigated in this study, which spanned most of the gene, were in high linkage disequilibrium (D′=1) with each other. Of the 23 MMPs in humans, 9 of their genes form a cluster on the long arm of chromosome 11. The MMP3 gene is part of this cluster and due to the nature of genetic association studies it cannot be excluded with all certainty that one of the other 8 MMP genes are involved in the pathogenesis of AT.
The data presented herein demonstrate that the MMP3 variants investigated in this study interact with the COL5A1 rs12733 polymorphism in modifying the risk of AT. Although AT is likely to be a complex condition involving a number of gene-gene and gene-environment interactions35, there have, to the Applicant's knowledge, been no such reports of a gene-gene interaction that relates to increased risk of AT. As COL5A1 is a substrate for MMP336, subjects or individuals that carry risk variants within both of these genes may have disrupted interactions between type V collagen and MMP3 during catalysis leading to a heightened risk of AT.
In one embodiment, the molecular marker is a polymorphic marker, preferably an SNP. The SNP is any one or more SNPs selected from the group consisting of rs591058, rs679620, and rs650108, together with any other SNP closely linked (i.e. which is in high linkage disequilibrium) with any of the three specific SNPs listed above.
More particularly, the SNPs is selected from the group consisting of:
More particularly, the molecular marker is, or is detectable using, any one or more isolated oligonucleotides selected from the group comprising:
sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
In certain embodiments, the invention extends to a primer or oligonucleotide sets for use in detecting or diagnosing a predisposition to, or increased risk for, developing tendon, ligament, or other soft tissue pathologies or injuries in a subject, the primer or oligonucleotide sets comprising isolated nucleic acid sequences selected from the group consisting of:
sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
In addition, the molecular marker comprises, in certain embodiments, any one or more isolated nucleic acid sequences selected from the group consisting of:
fragments thereof, sequences complementary thereto, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
Certain embodiments provide an isolated nucleic acid molecule for detecting at least one SNP provided hereinbefore, wherein the nucleic acid molecule comprises less than 40, less than 30, less than 20, or even preferably less than 10 contiguous nucleotides selected from the group consisting of SEQ ID NOS 1 to 12 and fragments, complementary sequences, sequences which can hybridize under stringent hybridization conditions thereto, and functional discriminatory truncations thereof.
The following references are incorporated herein by reference only:
aThe age of the TEN and RUP groups are the age of the onset of the initial symptoms of Achilles tendon injury, which were on average 7.8 ± 8.0 and 7.5 ± 8.9 years prior to recruitment in this study respectively.
bP value = Global, so CON vs TEN vs RUP.
cExcept for the weight difference between the TEN and RUP groups (P = 0.033), all other pairwise differences in weight and BMI were highly significant (P < 0.01).
1The CC genotype is under-represented in TEN subjects [10].
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008/09435 | Nov 2008 | ZA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB09/54892 | 11/4/2009 | WO | 00 | 9/19/2011 |
