- Email: [email protected]
Molecular phenotyping of patient chondrocytes reveals genes and pathways involved in osteoarthritis
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
S209
density index (CDI). Subchondral bone plate-thickness and ectopic calcifications were also measured. Results: There was a significant genotype (strain) by procedure (surgery) interaction for all parameters studied (except for CDI and Tb.N.) (P < 0.05) indicating strain differences for susceptibility to OA. We observed that different RI strains exhibited varying degree of cartilage degeneration (Fig. 1A). We were able to segregate strains into susceptible (e.g. LGXSM-33, LGXSM-46, LGXSM-5, SM/J) and protected (e.g. LGXSM-6, LGXSM-131b, LGXSM-163, LG/J) categories in addition to some intermediate strains (e.g. LGXSM-18, LGXSM-19, LGXSM-120a) based on summed OA score (Fig. 1B), synovitis and ectopic calcification. Furthermore, we found a significant (P < 0.001) correlation (r ¼ 0.68) between summed OA score and synovitis suggesting that 47% (r2 ¼ 0.47) of variance is shared between cartilage degeneration and synovitis. Conclusions: Our study identified a number of mouse strains that are protected or susceptible to post-traumatic OA, which is explained by their genetic composition. Further investigation into gene expression profile of these strains will identify the exact genomic regions (expression quantitative trait loci) that orchestrate the phenotypic differences. These studies can further identify the pathways and mechanisms underlying OA susceptibility.
patterns (A-E) and indicated biological pathways that mediate the alterations in transcription. Functional links that connect the identified expression patterns to the PPAR signaling, adipogenesis (A); Wnt signaling (B); endochondral ossification (C); matrix metalloproteinases, ACE/RAGE pathway (D); Toll-like receptor and IL1 signaling (E) pathways were found. The dynamic profiles of transcriptional changes were assigned to cellular compartments of the knee joint. Conclusions: Our study provides evidences that the progression of cartilage damage is driven by complex but precise regulation of gene patterns that are induced or suppressed during a various stages of cartilage damage. We see a sharp temporary loss of the transcripts involved in WNT pathway as well as constant downregulation of genes related to endochondral ossification. While, the decrease of transcript abundance of genes connected to PPARG-signaling was more gradual. Transcripts related to immunological response show early, while connected to matrix metalloproteinases delayed increase in the abundance levels. We conclude that the expression of PPARG-signaling genes correlates negatively with the different stages of OA, and matrix metalloproteinases genes correlate positively with OA development. Moreover, our results indicate that the observed transcriptional alterations are located in the diverse cellular compartments of the knee cartilage. We can speculate that during OA pathogenesis synoviocytes, osteoblasts, fibroblasts epithelial and immunological cells process specific molecular complementary (diseaserelevant intracellular signaling cascades) or contrary physiological programs. The presented classification of transcriptional alterations associated with the development of cartilage degeneration provides novel insight into the OA disease process. Integration across multiple tissues holds the promise of identifying new areas of so far unrecognized molecular networks and of characterising OA processes in depth, thus leading to the development of new therapeutic interventions. Supported by National Science Centre, Poland grants: OPUS UMO-2014/13/B/NZ7/ 02311, ETIUDA UMO-2015/16/T/NZ7/00052 and statutory funds.
Figure 1. Strain differences in cartilage degeneration and OA score. A. Representative images showing cartilage degeneration on the medial tibial plateau: (a) sham, (b) no degeneration (only focal proteoglycan loss), (c) moderate degeneration, (d) severe degeneration with fibrillation (arrows show cartilage degeneration), scale bar ¼ 100 mm, B. Summed OA score. Lowercase letters in the graph indicate significant differences (P < 0.05) among RI strains. No significant differences were found in sham knees.
334 MOLECULAR PHENOTYPING OF PATIENT CHONDROCYTES REVEALS GENES AND PATHWAYS INVOLVED IN OSTEOARTHRITIS
333 THE DISSECTION OF GENE PATTERNS ALTERED IN KNEE CARTILAGE IN A RAT MODEL OF OSTEOARTHRITIS M. Korostynski, N. Malek, M. Piechota, K. Starowicz. Inst. of Pharmacology Polish Academy of Sci., KRAKOW, Poland Purpose: Osteoarthritis (OA) is a joint disease that mostly affects cartilage. It is the most common cause of musculoskeletal pain and disability in the knee joint. Gene regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage degeneration. Functional genomics is a challenging new way to address a complex disease like osteoarthritis on a molecular level. A functional genomic approach to OA focuses on measuring changes in gene expression, allowing to discover new factors involved in the disease as well as factors involved in joint tissue development or maintenance. A deeper understanding of molecular events within the tissue cells (i.e., the chondrocytes) will provide new cellular targets for therapeutic intervention. Methods: We applied transcriptomics study in order to compare data between healthy and various OA states to better understand the mechanisms underlying a disease. In our model, knee OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate (MIA) injection. Whole-genome microarrays were used to analyze gene expression alterations in a time-course of OA development (2, 14 and 28 days) in rat knee joint. We used bioinformatics tools to recognize and characterize patterns of co-expressed transcripts. Results: The identified groups of genes were analyzed for enrichment of regulatory mechanisms, functional classes and cell-type specific expression. The analysis of transcriptional alterations revealed 272 regulated transcripts (ANOVA FDR<0.1% and fold>2). Bioinformatics approaches led to the identification of five main gene expression
J. Steinberg y, G. Ritchie y, T. Roumeliotis y, R. Jayasuriya z, R. Brooks x, A. Binch k, K. Shah z, R. Coyle y, M. Pardo y, C. Le Maitre k, Y. Ramos ¶, R. Nelissen ¶, I. Meulenbelt ¶, A. McCaskie x, J. Choudhary y, M. Wilkinson z, E. Zeggini y. y Wellcome Trust Sanger Inst., Hinxton, United Kingdom,; z Univ. of Sheffield, Sheffield, United Kingdom; x Univ. of Cambridge, Cambridge, United Kingdom; k Sheffield Hallam Univ., Sheffield, United Kingdom; ¶ Leiden Univ. Med. Ctr., Leiden, Netherlands Purpose: Musculoskeletal disease is the second greatest cause of disability world-wide, affecting over 1.7 billion people. Osteoarthritis (OA) affects over 40% of individuals over the age of 70, is a leading cause of pain and loss of physical function, and is associated with higher risk of quality of life decline, comorbidity and death. The health economic burden of OA is increasing, commensurate with longevity and obesity prevalence. In the absence of a curative therapy, management strategies are shifting to disease prevention and treatment of early OA. However, the underlying mechanisms of disease pathogenesis and progression are poorly characterised. Unlike for many other diseases, the relevant tissue for OA is readily accessible at joint replacement surgery. This enables the study of molecular processes at the right tissue, both to fill a gap in our fundamental understanding of biology and to identify novel therapeutic avenues. Methods: The epigenome, transcriptome and proteome of cell types relevant to OA remain relatively uncharacterised. We have characterised isolated chondrocytes from intact and degraded cartilage samples following total knee replacement in 12 patients in terms of epigenetics (Illumina 450k methylation array), gene expression (RNAseq) and protein expression (quantitative proteomics). We have established a robust pipeline for collecting tissue, extracting high-quality DNA, RNA and protein, and processing samples through the omics platforms. The pilot has also instigated the development of systems biology approaches for the integration of multidimensional data. Results: We find a significant negative correlation between promoter region methylation and gene expression (Spearman’s rho ¼ -0.43, p < 2.2 x 10-16) and a positive correlation (rho ¼ 0.29, p < 2.2 x 10-16) between RNA and protein expression levels. We identify five discordant cases in which the gene is significantly over-expressed at the RNA level and significantly less abundant at the protein level in the degraded tissue (COL4A2, CXCL12, FGF10, HTRA3 and WNT5B). All five proteins are
S210
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
annotated as predicted secreted proteins and have potential value as biomarkers of OA. We performed an in silico DrugBank search and found ten compounds that target genes highlighted in the pilot study at stringent confidence levels, and which have FDA marketing authorisation for an existing clinical indication. Three genes are consistently differentially regulated across all 3 omics levels: AQP1, COL1A1 and CLEC3B. For example, AQP1 encodes aquaporin-1, which facilitates water transport across biological membranes. Chondrocyte swelling and increased cartilage hydration has been suggested as an important mechanism in OA. The carbonic anhydrase inhibitor Acetazolamide is a known inhibitor of aquaporin-1, indicating the potential of functional genomics to identify opportunities for drug repurposing, shortening the investigative pipeline to use in OA. We used independent datasets for knee OA and hip OA to show that the direction of change replicates for over 90% of differentially expressed genes and differentially methylated probes, and to demonstrate good agreement in the global estimates of the fold-changes. Integrated pathway analysis implicates the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Conclusions: This work provides a comprehensive view of the molecular landscape of human primary chondrocytes in OA progression. All data from these experiments are freely available as a resource for the scientific community. 335 NOVEL ASSOCIATIONS IN MITOCHONDRIA-RELATED GENES AND MITOCHONDRIA-NUCLEUS INTERACTIONS. ROLE IN OSTEOARTHRITIS s-Pereira y, M. Ferna ndez-Moreno y, I. Rego-Perez y, E. Corte zquez-Mosquera y, S. Relan ~ o y, S. Pe rtega z, A. Go nza lez x, M. Va ndez-Lo pez y, F. Blanco y. y Servicio L. Vidal-Bralo x, N. Oreiro y, C. Ferna n Biom ~ a (INIBIC). de Reumatología. Inst. de Investigacio edica de A Corun ~ a (CHUAC), Sergas. Univ.e da Complexo Hosp.ario Univ.rio de A Corun ~ a (UDC). As Xubias, 15006, A Corun ~ a, Spain; z Unidad de Corun n Biom Epidemiología Clínica y Bioestadística. Inst. de Investigacio edica ~ a (INIBIC). Complexo Hosp.ario Univ.rio de A Corun ~a de A Corun ~ a (UDC). As Xubias, 15006, A Corun ~ a, (CHUAC), Sergas. Univ.e da Corun n 10 y Unidad de Reumatología. Inst. Spain; x Laboratorio de Investigacio n Sanitaria de Santiago (IDIS), Hosp. Clínico Univ.rio de de Investigacio Santiago, Travesía Choupana s/n, 15706, Santiago de Compostela, Spain Purpose: To analyze the potential influence of specific gene polymorphisms of mitochondria-related genes involved in cellular detoxification and to explore the mitochondria-nucleus interactions in the development of hip OA Methods: For this work we used two well-characterized cohorts of ~ a and patients from Galicia (Spain) including patients from A Corun Santiago reaching a total of 944 subjects, of which 601 were hip OA with KL grade IV and 343 age-matched radiological healthy controls (KL grade 0-I). The mtDNA haplogroups were assigned to all the subjects and grouped into mtDNA clusters. In a first subset of subjects we analyzed a set of mitochondria-related gene polymorphisms involved in the cellular detoxification: CAT (rs659366), GPX1 (rs2234693), UCP2 (rs659366), SOD2 (rs4880) and NOS2A (microsatellite (CCTTT)n in the promoter region of the gene), as well as their potential interaction with mtDNA clusters. In a second set of subjects we analyzed the potential interactions between the mtDNA clusters and a set of previously proposed gene polymorphisms related to OA: GDF-5 (rs143383), CILP (rs2073711), PAI (rs2227631), MMP-1 (rs514921) and MMP-2 (rs243866). The obtained data were appropriately analyzed using PowerMarker and SPSS (v19) Results: We found that the TT genotype of rs659366 in CAT gene was significantly underrepresented in hip OA patients (OR ¼ 0.512;95% CI ¼ 0.321-0.817;p ¼ 0.004). In relation to NOS2A microsatellite, the 13repetition allele was significantly underrepresented in hip OA patients too (OR ¼ 0.675;95%CI ¼ 0.509-0.895;p ¼ 0.006) meanwhile the 11 repetition-allele was significantly associated with an increased risk of hip OA (OR ¼ 1.524;95%CI ¼ 1.133-2.049;p ¼ 0.005). The subsequent regression model adjusting for the confounder variables of gender and age confirmed the above described associations as well as the mtDNA cluster TJ as a protective factor against hip OA (OR ¼ 0.672; 95% CI ¼ 0.455-0.993;p ¼ 0.046) (Table 1). In terms of mitochondria-nucleus interactions, a significant interaction between the SNP rs2234693 in GPX1 and the mtDNA cluster TJ (p ¼ 0.003) and HV (p ¼ 0.05) was discovered; the combination of cluster TJ and CC genotype emerged as a
protective factor (OR ¼ 0.427;95%CI ¼ 0.249-0.742;p ¼ 0.002), and the combination of cluster HV and TT genotype resulted in a protective factor too (OR ¼ 0.535;95%CI ¼ 0.307-0.934;p ¼ 0.028). In addition, we found significant interactions between the mtDNA clusters HV and/or TJ and other specific nuclear polymorphisms in PAI (p < 0.001 for TJ and p ¼ 0.049 for HV) and MMP1 (p ¼ 0.016 for HV) genes. The combination of cluster TJ with TT genotype of PAI significantly associates with a decreased risk of hip OA (OR ¼ 0.183;95%CI ¼ 0.068-0.490;p ¼ 0.001), and the combination of cluster HV with no-TT genotype associated with decreased risk too (OR ¼ 0.496;95%CI ¼ 0.291-0.848;p ¼ 0.010). In relation to MMP1, the combination of cluster HV with AA genotype associates with an increased risk of hip OA (OR ¼ 1.770;95%CI ¼ 1.0243.058;p ¼ 0.041) (Table 2). Finally, the interactions involving both CILP (p ¼ 0.058 for HV) and MMP-2.1575 (p ¼ 0.062 for TJ) border-lined the statistical significance Conclusions: The associations described in this work indicate that mitochondrial genetic background determines the behavior of specific nuclear variants in the development of OA
Table 1 Variables
p-value
OR
95%CI
Gender (male) Age NOS2A-11 NOS2A-13 CAT-TT mtDNA clusters (n ¼ 944) HV (n ¼ 496) KU (n ¼ 222) TJ (n ¼ 147) Others (n ¼ 79)
0.098 0.034 0.041 0.016 0.009
1.275 0.987 1.387 0.694 0.523
0.956-1.700 0.974-0.999 1.014-1.897 0.515-0.935 0.321-0.853
0.693 0.046 0.385
1 1.073 0.672 1.277
0.756-1.522 0.455-0.993 0.735-2.216
Table 2 Cluster TJ
Cluster HV
Genotypes (TJ/HV)
p-value
OR
95% CI
p-value
OR
95% CI
GPX1. CC/TT PAI. TT/no-TT MMP1. -/AA
0.002 0.001 0.041
0.427 0.183 1.770
0.249-0.742 0.068-0.490 1.024-3.058
0.028 0.010
0.535 0.496
0.307-0.934 0.291-0.848
336 FUNCTIONAL GENETIC POLYMORPHISM OF IL-1RN ENCODING THE IL-1 RECEPTOR ANTAGONIST PREDICTS RADIOGRAPHIC SEVERITY OF SYMPTOMATIC KNEE OA M. Attur y, S. Ma y, J. Samuels y, S. Krasnokutsky Samuels y, H. Zhou y, J.T. Bencardino y, M.C. Hochberg z, B.D. Mitchell z, V.B. Kraus x, J.M. Jordan k, S.B. Abramson y. y New York Univ., New York, NY, USA; z Univ. of Maryland Sch. of Med., Baltimore, MD, USA; x Duke Univ. Med. Ctr., Durham, NC, USA; k Univ. of North Carolina, Chapel Hill, NC, USA Purpose: Growing numbers of studies show increased expression in Osteoarthritis (OA) of inflammatory cytokines, such as IL-1b and TNFa, in joint tissues and peripheral blood mononuclear (PBM) cells. The IL1 receptor antagonist (IL1RN) gene cluster region has been associated with susceptibility to knee OA, thereby further implicating inflammation in OA pathogenesis. In these studies we examined the association of IL-1RN haplotype with the radiographic severity of symptomatic knee OA (SKOA). Methods: Genomic DNA from SKOA patients from three cohorts (NYU I, NYU-II and OAI) in this prospective analysis were used to genotype three single nucleotide polymorphisms (SNPs) [rs419598, rs315952 & rs9005) based on our prior publications. Genotyping was accomplished by PCR using validated SNP primers and probes (Applied Biosystems) along with detection using allelic discrimination computation. Genotypes were scored blinded to all patients
S209
density index (CDI). Subchondral bone plate-thickness and ectopic calcifications were also measured. Results: There was a significant genotype (strain) by procedure (surgery) interaction for all parameters studied (except for CDI and Tb.N.) (P < 0.05) indicating strain differences for susceptibility to OA. We observed that different RI strains exhibited varying degree of cartilage degeneration (Fig. 1A). We were able to segregate strains into susceptible (e.g. LGXSM-33, LGXSM-46, LGXSM-5, SM/J) and protected (e.g. LGXSM-6, LGXSM-131b, LGXSM-163, LG/J) categories in addition to some intermediate strains (e.g. LGXSM-18, LGXSM-19, LGXSM-120a) based on summed OA score (Fig. 1B), synovitis and ectopic calcification. Furthermore, we found a significant (P < 0.001) correlation (r ¼ 0.68) between summed OA score and synovitis suggesting that 47% (r2 ¼ 0.47) of variance is shared between cartilage degeneration and synovitis. Conclusions: Our study identified a number of mouse strains that are protected or susceptible to post-traumatic OA, which is explained by their genetic composition. Further investigation into gene expression profile of these strains will identify the exact genomic regions (expression quantitative trait loci) that orchestrate the phenotypic differences. These studies can further identify the pathways and mechanisms underlying OA susceptibility.
patterns (A-E) and indicated biological pathways that mediate the alterations in transcription. Functional links that connect the identified expression patterns to the PPAR signaling, adipogenesis (A); Wnt signaling (B); endochondral ossification (C); matrix metalloproteinases, ACE/RAGE pathway (D); Toll-like receptor and IL1 signaling (E) pathways were found. The dynamic profiles of transcriptional changes were assigned to cellular compartments of the knee joint. Conclusions: Our study provides evidences that the progression of cartilage damage is driven by complex but precise regulation of gene patterns that are induced or suppressed during a various stages of cartilage damage. We see a sharp temporary loss of the transcripts involved in WNT pathway as well as constant downregulation of genes related to endochondral ossification. While, the decrease of transcript abundance of genes connected to PPARG-signaling was more gradual. Transcripts related to immunological response show early, while connected to matrix metalloproteinases delayed increase in the abundance levels. We conclude that the expression of PPARG-signaling genes correlates negatively with the different stages of OA, and matrix metalloproteinases genes correlate positively with OA development. Moreover, our results indicate that the observed transcriptional alterations are located in the diverse cellular compartments of the knee cartilage. We can speculate that during OA pathogenesis synoviocytes, osteoblasts, fibroblasts epithelial and immunological cells process specific molecular complementary (diseaserelevant intracellular signaling cascades) or contrary physiological programs. The presented classification of transcriptional alterations associated with the development of cartilage degeneration provides novel insight into the OA disease process. Integration across multiple tissues holds the promise of identifying new areas of so far unrecognized molecular networks and of characterising OA processes in depth, thus leading to the development of new therapeutic interventions. Supported by National Science Centre, Poland grants: OPUS UMO-2014/13/B/NZ7/ 02311, ETIUDA UMO-2015/16/T/NZ7/00052 and statutory funds.
Figure 1. Strain differences in cartilage degeneration and OA score. A. Representative images showing cartilage degeneration on the medial tibial plateau: (a) sham, (b) no degeneration (only focal proteoglycan loss), (c) moderate degeneration, (d) severe degeneration with fibrillation (arrows show cartilage degeneration), scale bar ¼ 100 mm, B. Summed OA score. Lowercase letters in the graph indicate significant differences (P < 0.05) among RI strains. No significant differences were found in sham knees.
334 MOLECULAR PHENOTYPING OF PATIENT CHONDROCYTES REVEALS GENES AND PATHWAYS INVOLVED IN OSTEOARTHRITIS
333 THE DISSECTION OF GENE PATTERNS ALTERED IN KNEE CARTILAGE IN A RAT MODEL OF OSTEOARTHRITIS M. Korostynski, N. Malek, M. Piechota, K. Starowicz. Inst. of Pharmacology Polish Academy of Sci., KRAKOW, Poland Purpose: Osteoarthritis (OA) is a joint disease that mostly affects cartilage. It is the most common cause of musculoskeletal pain and disability in the knee joint. Gene regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage degeneration. Functional genomics is a challenging new way to address a complex disease like osteoarthritis on a molecular level. A functional genomic approach to OA focuses on measuring changes in gene expression, allowing to discover new factors involved in the disease as well as factors involved in joint tissue development or maintenance. A deeper understanding of molecular events within the tissue cells (i.e., the chondrocytes) will provide new cellular targets for therapeutic intervention. Methods: We applied transcriptomics study in order to compare data between healthy and various OA states to better understand the mechanisms underlying a disease. In our model, knee OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate (MIA) injection. Whole-genome microarrays were used to analyze gene expression alterations in a time-course of OA development (2, 14 and 28 days) in rat knee joint. We used bioinformatics tools to recognize and characterize patterns of co-expressed transcripts. Results: The identified groups of genes were analyzed for enrichment of regulatory mechanisms, functional classes and cell-type specific expression. The analysis of transcriptional alterations revealed 272 regulated transcripts (ANOVA FDR<0.1% and fold>2). Bioinformatics approaches led to the identification of five main gene expression
J. Steinberg y, G. Ritchie y, T. Roumeliotis y, R. Jayasuriya z, R. Brooks x, A. Binch k, K. Shah z, R. Coyle y, M. Pardo y, C. Le Maitre k, Y. Ramos ¶, R. Nelissen ¶, I. Meulenbelt ¶, A. McCaskie x, J. Choudhary y, M. Wilkinson z, E. Zeggini y. y Wellcome Trust Sanger Inst., Hinxton, United Kingdom,; z Univ. of Sheffield, Sheffield, United Kingdom; x Univ. of Cambridge, Cambridge, United Kingdom; k Sheffield Hallam Univ., Sheffield, United Kingdom; ¶ Leiden Univ. Med. Ctr., Leiden, Netherlands Purpose: Musculoskeletal disease is the second greatest cause of disability world-wide, affecting over 1.7 billion people. Osteoarthritis (OA) affects over 40% of individuals over the age of 70, is a leading cause of pain and loss of physical function, and is associated with higher risk of quality of life decline, comorbidity and death. The health economic burden of OA is increasing, commensurate with longevity and obesity prevalence. In the absence of a curative therapy, management strategies are shifting to disease prevention and treatment of early OA. However, the underlying mechanisms of disease pathogenesis and progression are poorly characterised. Unlike for many other diseases, the relevant tissue for OA is readily accessible at joint replacement surgery. This enables the study of molecular processes at the right tissue, both to fill a gap in our fundamental understanding of biology and to identify novel therapeutic avenues. Methods: The epigenome, transcriptome and proteome of cell types relevant to OA remain relatively uncharacterised. We have characterised isolated chondrocytes from intact and degraded cartilage samples following total knee replacement in 12 patients in terms of epigenetics (Illumina 450k methylation array), gene expression (RNAseq) and protein expression (quantitative proteomics). We have established a robust pipeline for collecting tissue, extracting high-quality DNA, RNA and protein, and processing samples through the omics platforms. The pilot has also instigated the development of systems biology approaches for the integration of multidimensional data. Results: We find a significant negative correlation between promoter region methylation and gene expression (Spearman’s rho ¼ -0.43, p < 2.2 x 10-16) and a positive correlation (rho ¼ 0.29, p < 2.2 x 10-16) between RNA and protein expression levels. We identify five discordant cases in which the gene is significantly over-expressed at the RNA level and significantly less abundant at the protein level in the degraded tissue (COL4A2, CXCL12, FGF10, HTRA3 and WNT5B). All five proteins are
S210
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
annotated as predicted secreted proteins and have potential value as biomarkers of OA. We performed an in silico DrugBank search and found ten compounds that target genes highlighted in the pilot study at stringent confidence levels, and which have FDA marketing authorisation for an existing clinical indication. Three genes are consistently differentially regulated across all 3 omics levels: AQP1, COL1A1 and CLEC3B. For example, AQP1 encodes aquaporin-1, which facilitates water transport across biological membranes. Chondrocyte swelling and increased cartilage hydration has been suggested as an important mechanism in OA. The carbonic anhydrase inhibitor Acetazolamide is a known inhibitor of aquaporin-1, indicating the potential of functional genomics to identify opportunities for drug repurposing, shortening the investigative pipeline to use in OA. We used independent datasets for knee OA and hip OA to show that the direction of change replicates for over 90% of differentially expressed genes and differentially methylated probes, and to demonstrate good agreement in the global estimates of the fold-changes. Integrated pathway analysis implicates the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Conclusions: This work provides a comprehensive view of the molecular landscape of human primary chondrocytes in OA progression. All data from these experiments are freely available as a resource for the scientific community. 335 NOVEL ASSOCIATIONS IN MITOCHONDRIA-RELATED GENES AND MITOCHONDRIA-NUCLEUS INTERACTIONS. ROLE IN OSTEOARTHRITIS s-Pereira y, M. Ferna ndez-Moreno y, I. Rego-Perez y, E. Corte zquez-Mosquera y, S. Relan ~ o y, S. Pe rtega z, A. Go nza lez x, M. Va ndez-Lo pez y, F. Blanco y. y Servicio L. Vidal-Bralo x, N. Oreiro y, C. Ferna n Biom ~ a (INIBIC). de Reumatología. Inst. de Investigacio edica de A Corun ~ a (CHUAC), Sergas. Univ.e da Complexo Hosp.ario Univ.rio de A Corun ~ a (UDC). As Xubias, 15006, A Corun ~ a, Spain; z Unidad de Corun n Biom Epidemiología Clínica y Bioestadística. Inst. de Investigacio edica ~ a (INIBIC). Complexo Hosp.ario Univ.rio de A Corun ~a de A Corun ~ a (UDC). As Xubias, 15006, A Corun ~ a, (CHUAC), Sergas. Univ.e da Corun n 10 y Unidad de Reumatología. Inst. Spain; x Laboratorio de Investigacio n Sanitaria de Santiago (IDIS), Hosp. Clínico Univ.rio de de Investigacio Santiago, Travesía Choupana s/n, 15706, Santiago de Compostela, Spain Purpose: To analyze the potential influence of specific gene polymorphisms of mitochondria-related genes involved in cellular detoxification and to explore the mitochondria-nucleus interactions in the development of hip OA Methods: For this work we used two well-characterized cohorts of ~ a and patients from Galicia (Spain) including patients from A Corun Santiago reaching a total of 944 subjects, of which 601 were hip OA with KL grade IV and 343 age-matched radiological healthy controls (KL grade 0-I). The mtDNA haplogroups were assigned to all the subjects and grouped into mtDNA clusters. In a first subset of subjects we analyzed a set of mitochondria-related gene polymorphisms involved in the cellular detoxification: CAT (rs659366), GPX1 (rs2234693), UCP2 (rs659366), SOD2 (rs4880) and NOS2A (microsatellite (CCTTT)n in the promoter region of the gene), as well as their potential interaction with mtDNA clusters. In a second set of subjects we analyzed the potential interactions between the mtDNA clusters and a set of previously proposed gene polymorphisms related to OA: GDF-5 (rs143383), CILP (rs2073711), PAI (rs2227631), MMP-1 (rs514921) and MMP-2 (rs243866). The obtained data were appropriately analyzed using PowerMarker and SPSS (v19) Results: We found that the TT genotype of rs659366 in CAT gene was significantly underrepresented in hip OA patients (OR ¼ 0.512;95% CI ¼ 0.321-0.817;p ¼ 0.004). In relation to NOS2A microsatellite, the 13repetition allele was significantly underrepresented in hip OA patients too (OR ¼ 0.675;95%CI ¼ 0.509-0.895;p ¼ 0.006) meanwhile the 11 repetition-allele was significantly associated with an increased risk of hip OA (OR ¼ 1.524;95%CI ¼ 1.133-2.049;p ¼ 0.005). The subsequent regression model adjusting for the confounder variables of gender and age confirmed the above described associations as well as the mtDNA cluster TJ as a protective factor against hip OA (OR ¼ 0.672; 95% CI ¼ 0.455-0.993;p ¼ 0.046) (Table 1). In terms of mitochondria-nucleus interactions, a significant interaction between the SNP rs2234693 in GPX1 and the mtDNA cluster TJ (p ¼ 0.003) and HV (p ¼ 0.05) was discovered; the combination of cluster TJ and CC genotype emerged as a
protective factor (OR ¼ 0.427;95%CI ¼ 0.249-0.742;p ¼ 0.002), and the combination of cluster HV and TT genotype resulted in a protective factor too (OR ¼ 0.535;95%CI ¼ 0.307-0.934;p ¼ 0.028). In addition, we found significant interactions between the mtDNA clusters HV and/or TJ and other specific nuclear polymorphisms in PAI (p < 0.001 for TJ and p ¼ 0.049 for HV) and MMP1 (p ¼ 0.016 for HV) genes. The combination of cluster TJ with TT genotype of PAI significantly associates with a decreased risk of hip OA (OR ¼ 0.183;95%CI ¼ 0.068-0.490;p ¼ 0.001), and the combination of cluster HV with no-TT genotype associated with decreased risk too (OR ¼ 0.496;95%CI ¼ 0.291-0.848;p ¼ 0.010). In relation to MMP1, the combination of cluster HV with AA genotype associates with an increased risk of hip OA (OR ¼ 1.770;95%CI ¼ 1.0243.058;p ¼ 0.041) (Table 2). Finally, the interactions involving both CILP (p ¼ 0.058 for HV) and MMP-2.1575 (p ¼ 0.062 for TJ) border-lined the statistical significance Conclusions: The associations described in this work indicate that mitochondrial genetic background determines the behavior of specific nuclear variants in the development of OA
Table 1 Variables
p-value
OR
95%CI
Gender (male) Age NOS2A-11 NOS2A-13 CAT-TT mtDNA clusters (n ¼ 944) HV (n ¼ 496) KU (n ¼ 222) TJ (n ¼ 147) Others (n ¼ 79)
0.098 0.034 0.041 0.016 0.009
1.275 0.987 1.387 0.694 0.523
0.956-1.700 0.974-0.999 1.014-1.897 0.515-0.935 0.321-0.853
0.693 0.046 0.385
1 1.073 0.672 1.277
0.756-1.522 0.455-0.993 0.735-2.216
Table 2 Cluster TJ
Cluster HV
Genotypes (TJ/HV)
p-value
OR
95% CI
p-value
OR
95% CI
GPX1. CC/TT PAI. TT/no-TT MMP1. -/AA
0.002 0.001 0.041
0.427 0.183 1.770
0.249-0.742 0.068-0.490 1.024-3.058
0.028 0.010
0.535 0.496
0.307-0.934 0.291-0.848
336 FUNCTIONAL GENETIC POLYMORPHISM OF IL-1RN ENCODING THE IL-1 RECEPTOR ANTAGONIST PREDICTS RADIOGRAPHIC SEVERITY OF SYMPTOMATIC KNEE OA M. Attur y, S. Ma y, J. Samuels y, S. Krasnokutsky Samuels y, H. Zhou y, J.T. Bencardino y, M.C. Hochberg z, B.D. Mitchell z, V.B. Kraus x, J.M. Jordan k, S.B. Abramson y. y New York Univ., New York, NY, USA; z Univ. of Maryland Sch. of Med., Baltimore, MD, USA; x Duke Univ. Med. Ctr., Durham, NC, USA; k Univ. of North Carolina, Chapel Hill, NC, USA Purpose: Growing numbers of studies show increased expression in Osteoarthritis (OA) of inflammatory cytokines, such as IL-1b and TNFa, in joint tissues and peripheral blood mononuclear (PBM) cells. The IL1 receptor antagonist (IL1RN) gene cluster region has been associated with susceptibility to knee OA, thereby further implicating inflammation in OA pathogenesis. In these studies we examined the association of IL-1RN haplotype with the radiographic severity of symptomatic knee OA (SKOA). Methods: Genomic DNA from SKOA patients from three cohorts (NYU I, NYU-II and OAI) in this prospective analysis were used to genotype three single nucleotide polymorphisms (SNPs) [rs419598, rs315952 & rs9005) based on our prior publications. Genotyping was accomplished by PCR using validated SNP primers and probes (Applied Biosystems) along with detection using allelic discrimination computation. Genotypes were scored blinded to all patients