Skip Navigation



Rheumatology Advance Access published online on September 24, 2008
Rheumatology, doi:10.1093/rheumatology/ken378
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
47/11/1655    most recent
ken378v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zinovieva, E.
Right arrow Articles by Breban, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zinovieva, E.
Right arrow Articles by Breban, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Lack of association between Tenascin-C gene and spondyloarthritis

E. Zinovieva1, N. Lebrun1, F. Letourneur1, F.-X. Laurent1, R. Said-Nahal2, G. Chiocchia1 and M. Breban1,2,3

1Institut Cochin, INSERM U567, CNRS UMR8104 and Université Paris Descartes IFR116, Paris 2Rheumatology Division, Ambroise Paré Hospital, AP-HP and 3Université Versailles Saint-Quentin en Yvelines, Boulogne-Billancourt, France.

Correspondence to: M. Breban, Rheumatology Division, Hôpital Ambroise Paré, 9 ave Charles de Gaulle, 92100, Boulogne, France. E-mail: maxime.breban{at}apr.aphp.fr


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Objectives. We previously identified a new susceptibility region linked to SpA in 9q31–34. Tenascin-C (TNC) appears as one of the best positional and functional candidate genes lying within this SPA2 locus. The objectives of the present study were to identify TNC polymorphisms, and to examine their putative association with SpA.

Methods. We first performed variants screening in 20 independent SpA patients from families with high linkage score to the SPA2 locus, and three unrelated controls: TNCs coding regions (28 exons), intron–exon boundaries and 5'- and 3'-flank regions were fully re-sequenced to identify polymorphisms. Then we genotyped selected variants in 183 independent trios, and assessed their intrafamilial association with SpA by transmission disequilibrium test.

Results. Variants screening allowed us to identify 26 polymorphisms, 7 of which were selected for further study, in addition to an intronic polymorphism previously reported as associated with Achilles tendon injuries. In intrafamilial association test, none of the variants showed significant transmission disequilibrium. Results from analysis restricted to AS were not different from those obtained on the whole SpA group.

Conclusions. TNC was one of the best positional and functional candidate genes within the SPA2 locus. Nevertheless, we found no association between polymorphisms in this gene and SpA. However, we cannot exclude that variants located in intronic regions or in the vicinity of TNC, which were not tested in the present study, could be implicated in the predisposition to SpA.

KEY WORDS: Ankylosing spondylitis, Spondyloarthritis, Association study, Tenascin-C, Genetics


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
SpA is a frequent inflammatory rheumatic disorder with an estimated prevalence of 0.3% in western European adult population [1]. It refers to a spectrum of manifestations sharing anatomical grounds, and most notably enthesitis, i.e. inflammation at the insertion of tendons, ligaments and capsules to the bone [2]. Besides axial skeleton involvement, which is a hallmark of SpA, other frequent manifestations consist of peripheral arthritis, and enthesitis, dactylitis and extra-articular symptoms (anterior uveitis, psoriasis and IBD). According to its most salient presentation feature, SpA is classically split into subtypes: AS, which requires advanced radiographic sacroiliitis as a diagnostic criterion; PsA; ReA; IBD-associated arthritis (AIBD); and in the absence of any distinctive feature, uSpA. All these subsets share genetic predisposition, as shown by their tendency to familial aggregation and also by their association with HLA-B27 [3]. The HLA-B27 allele is the first genetic factor found to be associated with SpA and it is likely the most important one [2]. Nevertheless, its contribution to the overall genetic predisposition has been estimated to only 20–30%, and that of the MHC to 40–50% [2]. Thus, non-MHC genes should contribute half of the genetic susceptibility to SpA [4].

We previously performed a genome-wide linkage study in SpA multiplex families, with the objective to identify new susceptibility loci [5]. The multipoint non-parametric linkage (NPL) analysis evidenced five regions of interest (P ≤ 0.01), including the MHC. A replication study of non-MHC regions was then performed on additional families, allowing us to confirm a susceptibility region on chromosome 9q31–34 with statistical significance (NPL score = 4.87; P = 0.00002) [5]. This 23.95-cM locus that we called SPA2 overlapped with one of those identified as suggestive by linkage, in AS sibling pairs from UK [6].

In our study, the peak of linkage to the SPA2 locus was centred on microsatellite D9S1776, at 121.62 cM from p-telomere. One of the two genes mapped to the vicinity of this marker is Tenascin-Cytotactin (TNC). It appeared as one of the most attractive candidate genes located within the SPA2 locus. Indeed, it codes for an extracellular matrix glycoprotein expressed in entheses, which is a major target tissue in SpA [7]. Besides, this gene has been reported as associated with Achilles tendinopathies and ruptures [8]. In the present study, we wished to examine if variations in TNC gene were associated with SpA.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Patients
French SpA family trios, patients from multiplex families and unrelated healthy controls were recruited as previously described [3]. The study was approved by the Ethics Committee of Cochin Hospital and informed consent was obtained from participants. The diagnosis of SpA was made according to the classification criteria proposed by Amor [9] and/or the European Spondylarthropathy Study Group [10]. AS was diagnosed according to the modified New York criteria [11]. The diagnosis of psoriasis required typical lesions and/or a diagnosis established by a dermatologist. Anterior uveitis was retained after examination by an ophthalmologist. IBD diagnosis was based on endoscopic and histological examination of the gut. ReA was diagnosed according to the criteria published by Willkens [12]. Finally, uSpA was retained when SpA criteria were fulfilled, without AS, PsA, ReA or AIBD.

Variants screening by re-sequencing was performed on a panel of 20 independent patients selected from multiplex families with high linkage score to the SPA2 locus (NPL ≥ 1.34) [5] and three healthy controls. For family-based association study of polymorphisms, 183 independent trios (one SpA patient and his two parents) were analysed. Characteristics of the patients are shown in Table 1.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Description of SpA patients included in the studya

 
DNA isolation and variation screening
Genomic DNA was extracted from peripheral blood using standard methods. Coding regions, intron–exon boundaries and 5'- and 3'-flank regions of the TNC gene (28 exons) were amplified and then sequenced in 3' and 5' directions with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Primers used for PCR reaction and re-sequencing are shown in Supplementary Table 1 (see supplementary data available at Rheumatology Online). DNA sequences were analysed using SeqScape software (Applied Biosystems).

Genotyping of polymorphisms
Five single nucleotide polymorphisms (SNPs) (rs13321, rs1330361, rs2104772, rs1757095 and rs3748166) were genotyped by mean of customized Illumina BeadChip (San Diego, CA, USA) using the GoldenGate assay according to the manufacturer's recommendation, at the French National Genotyping Center (Evry, France). The rs45602433 polymorphism was genotyped using melting curve analyses (LightCycler System, Roche, Meylan, France). Ensembl database (http://www.ensembl.org/index.html) was used for the polymorphisms annotation and positioning. Primers and hybridization probes used for PCR amplification are described in Supplementary Table 2 (see supplementary data available at Rheumatology Online).

The CA-repeat containing regions (microsatellites 17 and 19) were first amplified by PCR [sequences of the primers shown in Supplementary Table 2 (see supplementary data available at Rheumatology Online)]. For analysis of repeat allele length and relative ratios, instrumentation and reagents from Applied Biosystems were used (ABI PRISM 3100 Genetic Analyzer and Gene Mapper software).

Statistical analysis
All genotypes were screened for Mendelian inheritance using FBAT (v2.0.0c) package and PedCheck version 1.1. The departure from Hardy–Weinberg equilibrium in unrelated individuals was assessed using the PEDSTATS version 0.6.11.

Family-based association analysis was carried out using the FBAT software. We used the additive model of FBAT to compute the statistic test, and gave P-value corresponding to this test statistic. Two affection traits were considered successively: SpA and AS. A P < 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Supplementary data
 Acknowledgements
 References
 
Variants screening
We started this study by re-sequencing the 28 exons, the intron–exon boundaries and the 5'- and 3'-flank regions of the TNC gene spanning 97.63 kb, in 20 independent SpA patients from families with high linkage score to the SPA2 locus [5], and three controls. Overall, we sequenced 12 462 base pairs (bp) per individual, of which 6606 bp were located in coding regions. We identified 26 polymorphisms which included 4 non-synonymous coding SNPs, 8 synonymous coding SNPs, 11 intronic SNPs and 3 intronic dinucleotide repeats. All these variants are annotated in public databases, such as Ensembl or UCSC (http://genome.ucsc.edu/cgi-bin/hgGateway). The average rate of polymorphisms was 1.82/kb of DNA in the coding regions, and 2.39/kb in the non-coding regions. Most of these polymorphisms were distributed among patients as well as controls.

Seven polymorphisms that we considered as the most relevant were selected, in order to be genotyped in the trios: the four non-synonymous coding SNPs (rs13321, rs2104772, rs45602433 and rs1757095), two intronic tag-SNPs (rs1330361 and rs3748166) and the CA-repeat polymorphism located in the beginning of the 19th intron (possibly implicated in splicing modifying). Besides, we decided to explore whether another CA-repeat polymorphism located in the middle of the 17th intron, and identified through the literature search [8], was associated with the disease.

Association study
We genotyped 549 individuals composing 183 trios for the eight selected polymorphisms. None of the genotype distributions in the founder's subjects differed significantly from those expected from Hardy–Weinberg equilibrium. Two affection traits were considered for association analyses: SpA and AS (101 out of 183 affected children presented with AS phenotype). Results of the single locus association tests under FBAT allelic additive model between each polymorphism and SpA and AS are shown in Table 2. None of the variants showed statistically significant transmission disequilibrium P-value neither with the SpA phenotype nor with the AS one. Despite two different phenotypes being analysed herein, allelic frequencies and association P-values were almost identical for SpA and AS trait. Finally, haplotype analyses performed using the hbat option of FBAT did not show any significant association either (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Genotyped TNC polymorphims, and FBAT results for the transmission disiquilibrium test (TDT) univariate analysis

 

   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Supplementary data
 Acknowledgements
 References
 
By using a genome-wide linkage approach, we have recently identified a new region of susceptibility to SpA in 9q31–34, which we called SPA2 [5]. According to our prediction, it should contain major susceptibility factor, accounting for 20–25% of the non-MHC genetic predisposition to SpA [2]. This region spans 17.44 Mb, to which about 120 genes, and a number of predicted coding sequences, have been mapped. Most interestingly, it is also one of the three regions paralogous to the MHC, which bears the most important genetic susceptibility load for SpA. Thus, one or more gene(s) in the SPA2 locus having paralogous counterpart in the MHC could be implicated in the genetic susceptibility to SpA.

One of our strategies for identifying genetic factor implicated in the predisposition to SpA at this new locus was a positional and functional candidate-gene approach. Here, we explored the implication of TNC in the predisposition to SpA and AS. Regarding location, the maximum NPL score in our study [5] was centred on marker D9S1776, in 9q33.1. TNC which is one of the two genes lying within 200 kb of this marker was one of the most attractive functional candidate gene for several reasons. First, TNC (or hexabrachion), one of five members of the tenascin family of genes, codes for 190–240 kDa extracellular matrix protein with a very restricted expression in tendons, ligaments, perichondrium and periosteum, where it is specially induced during tissue repair [13, 14]. Most interestingly, TNC expression has been shown in fibrocartilage, and more specifically in the entheses, which is a major target tissue in SpA [7]. It is increased in synovitis [15], and other conditions or tissues related to the spectrum of SpA, such as IBD, and psoriatic skin [16, 17]. In addition, TNC expression is induced during inflammation, and notably by TNF-{alpha} and IL-1, both of which have been implicated in the pathogenesis of SpA [2]. TNC could also regulate T-cell response, which is thought to be critically important in SpA pathogenesis [18, 19]. Finally, TNC has a paralogous counterpart in a MHC class III locus which may contain additional SpA susceptibility gene, i.e. TNXB [20].

We first screened the coding regions of TNC in order to identify sequence variants. In a second stage, we investigated the putative association of eight polymorphisms with SpA and AS by intrafamilial analyses. The data revealed no association between SpA and any of the polymorphisms tested. Similarly, no association was detected between AS and the variants tested herein.

The first stage of the present study consisted of re-sequencing the 28 exons, the intron–exon boundaries, and the 5'- and 3'-flank regions of the gene. This allowed us to identify 26 polymorphisms, all annotated in public databases. Some polymorphisms were of particular interest. For instance, rs45602433 located within the 10th exon, exchanges a histidine for an arginine, which may create an HLA-B27-peptide binding motif (ARVKASTAK). This was relevant for antigen-specific hypothesis of SpA mechanism, such as those suggesting an abnormal immune response towards cartilage-derived antigen, mediated by HLA-B27-restricted CD8+ T cells [18]. Overall, we have re-sequenced 12 462 bp, corresponding to 12.8% of the whole genomic size of the TNC gene. Given that the mean number of polymorphisms/kilobase pair of DNA in the non-coding regions of TNC was 2.39, we can predict that around 200 polymorphisms essentially in the intronic regions would remain to be studied.

In all, 7 of 26 polymorphisms detected through re-sequencing and one selected by a literature search [8] were genotyped in 183 trios. We have studied these variants not only for their association with the whole SpA, but also with the more restricted AS phenotype. No association was found between the selected polymorphisms and SpA, nor with AS. Noteworthy, there was no difference between analyses conducted in both phenotypes, as far as allelic frequencies or association P-values are concerned, supporting that at least for this region, the genetic behaviour of SpA and AS is the same [2]. Considering that SpA is a disease with genetic heterogeneity and complex transmission model, which underlying parameters are allegedly unknown, it was difficult to precisely calculate the power of our study to detect association of the chosen polymorphisms.

In conclusion, albeit TNC was one of the most attractive candidate genes in the SPA2 locus, we failed to identify any association between polymorphisms of this gene and SpA. However, we cannot exclude that some other variants located in intronic or intergenic regions, which were not tested herein could still be implicated in predisposition to this disease. A systematic approach using tag-SNPs covering the whole SPA2 region is under way to explore such possibility (Zinovieva et al., manuscript in preparation).

Formula


   Supplementary data
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Supplementary data
Acknowledgements
 References
 
Supplementary data are available at Rheumatology Online.


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
References
 
We thank Audrey Delaforge for excellent technical assistance, and French National Genotyping Center for some of the genotyping.

Funding: This work was supported by grants from Société Française de Rhumatologie (2007) and Arthritis Fondation Courtin (2007).

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 

  1. Saraux A, Guillemin F, Guggenbuhl P, et al. Prevalence of spondyloarthropathies in France: 2001. Ann Rheum Dis (2005) 64:1431–5.[Abstract/Free Full Text]
  2. Breban M, Miceli-Richard C, Zinovieva E, Monnet D, Said-Nahal R. The genetics of spondyloarthropathies. Joint Bone Spine (2006) 73:355–62.[CrossRef][Web of Science][Medline]
  3. Said-Nahal R, Miceli-Richard C, Berthelot J-M, et al. The familial form of spondylarthropathy: a clinical study of 115 multiplex families. Arthritis Rheum (2000) 43:1356–65.[CrossRef][Web of Science][Medline]
  4. Dernis E, Said-Nahal R, D’Agostino MA, Aegerter P, Dougados M, Breban M. Recurrence of spondylarthropathy among first-degree relatives of patients, a systematic cross-sectional study. Ann Rheum Dis. Advance access published April 15, 2008, doi: 10.1136/ard.2008.089599.
  5. Miceli-Richard C, Zouali H, Said-Nahal R, et al. Significant linkage to spondyloarthropathy on 9q31-34. Hum Mol Genet (2004) 13:1641–8.[Abstract/Free Full Text]
  6. Laval SH, Timms A, Edwards S, et al. Whole-genome screening in ankylosing spondylitis: evidence of non-MHC genetic-susceptibility loci. Am J Hum Genet (2001) 68:918–26.[CrossRef][Web of Science][Medline]
  7. de Palma L, Marinelli M, Meme L, Pavan M. Immunohistochemistry of the enthesis organ of the human Achilles tendon. Foot Ankle Int (2004) 25:414–8.[Web of Science][Medline]
  8. Mokone GG, Gajjar M, September AV, et al. The guanine-thymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with Achilles tendon injuries. Am J Sports Med (2005) 33:1016–21.[Abstract/Free Full Text]
  9. Amor B, Dougados M, Mijiyawa M. [Criteria of the classification of spondylarthropathies]. Rev Rhum Mal Osteoartic (1990) 57:85–9.[Medline]
  10. Dougados M, van der Linden S, Juhlin R, et al. The European spondylarthropathy study group preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum (1991) 34:1218–27.[Web of Science][Medline]
  11. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum (1984) 27:361–8.[Web of Science][Medline]
  12. Willkens RF, Arnett FC, Bitter T, et al. Reiter's syndrome. Evaluation of preliminary criteria for definite disease. Arthritis Rheum (1981) 24:844–9.[Web of Science][Medline]
  13. Chiquet-Ehrismann R, Chiquet M. Tenascins: regulation and putative functions during pathological stress. J Pathol (2003) 200:488–99.[CrossRef][Web of Science][Medline]
  14. Jarvinen TA, Jozsa L, Kannus P, et al. Mechanical loading regulates the expression of tenascin-C in the myotendinous junction and tendon but does not induce de novo synthesis in the skeletal muscle. J Cell Sci (2003) 116:857–66.[Abstract/Free Full Text]
  15. Hasegawa M, Nakoshi Y, Muraki M, et al. Expression of large tenascin-C splice variants in synovial fluid of patients with rheumatoid arthritis. J Orthop Res (2007) 25:563–8.[CrossRef][Web of Science][Medline]
  16. Gerritsen MJ, Elbers ME, de Jong EM, van de Kerkhof PC. Recruitment of cycling epidermal cells and expression of filaggrin, involucrin and tenascin in the margin of the active psoriatic plaque, in the uninvolved skin of psoriatic patients and in the normal healthy skin. J Dermatol Sci (1997) 14:179–88.[CrossRef][Web of Science][Medline]
  17. Riedl S, Tandara A, Reinshagen M, et al. Serum tenascin-C is an indicator of inflammatory bowel disease activity. Int J Colorectal Dis (2001) 16:285–91.[CrossRef][Web of Science][Medline]
  18. Breban M, Hacquard-Bouder C, Falgarone G. Animal models of HLA-B27-associated diseases. Curr Mol Med (2004) 4:21–40.[CrossRef][Web of Science][Medline]
  19. Kuznetsova SA, Roberts DD. Functional regulation of T lymphocytes by modulatory extracellular matrix proteins. Int J Biochem Cell Biol (2004) 36:1126–34.[CrossRef][Web of Science][Medline]
  20. Sims AM, Barnardo M, Herzberg I, et al. Non-B27 MHC associations of ankylosing spondylitis. Genes Immun (2007) 8:115–23.[CrossRef][Web of Science][Medline]
Submitted 27 June 2008; revised version accepted 19 August 2008.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
47/11/1655    most recent
ken378v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zinovieva, E.
Right arrow Articles by Breban, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zinovieva, E.
Right arrow Articles by Breban, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?