Application of an in silico approach identifies a genetic locus within ITGB2, and its interactions with HSPG2 and FGF9, to be associated with anterior cruciate ligament rupture risk.

We developed a Biomedical Knowledge Graph model that is phenotype and biological function-aware through integrating knowledge from multiple domains in a Neo4j, graph database. All known human genes were assessed through the model to identify potential new risk genes for anterior cruciate ligament (ACL) ruptures and Achilles tendinopathy (AT). Genes were prioritised and explored in a case-control study comparing participants with ACL ruptures (ACL-R), including a sub-group with non-contact mechanism injuries (ACL-NON), to uninjured control individuals (CON). After gene filtering, 3376 genes, including 411 genes identified through previous whole exome sequencing, were found to be potentially linked to AT and ACL ruptures. Four variants were prioritised: HSPG2:rs2291826A/G, HSPG2:rs2291827G/A, ITGB2:rs2230528C/T and FGF9:rs2274296C/T. The rs2230528 CC genotype was over-represented in the CON group compared to ACL-R (p < 0.001) and ACL-NON (p < 0.001) and the TT genotype and T allele were over-represented in the ACL-R group and ACL-NON compared to CON (p < 0.001) group. Several significant differences in distributions were noted for the gene-gene interactions: (HSPG2:rs2291826, rs2291827 and ITGB2:rs2230528) and (ITGB2:rs2230528 and FGF9:rs2297429). This study substantiates the efficiency of using a prior knowledge-driven in silico approach to identify candidate genes linked to tendon and ACL injuries. Our biomedical knowledge graph identified and, with further testing, highlighted novel associations of the ITGB2 gene which has not been explored in a genetic case control association study, with ACL rupture risk. We thus recommend a multistep approach including bioinformatics in conjunction with next generation sequencing technology to improve the discovery potential of genomics technologies in musculoskeletal soft tissue injuries.HighlightsA biomedical knowledge graph was modelled for musculoskeletal soft tissue injuries to efficiently identify candidate genes for genetic susceptibility analyses.The biomedical knowledge graph and sequencing data identified potential biologically relevant variants to explore susceptibility to common tendon and ligament injuries. Specifically genetic variants within the ITGB2 and FGF9 genes were associated with ACL risk.Novel allele combinations (HSPG2-ITGB2 and ITGB2-FGF9) showcase the potential effect of ITGB2 in influencing risk of ACL rupture.

Genetic variants within the COL5A1 gene are associated with ligament injuries in physically active populations from Australia, South Africa, and Japan.

Previous small-scale studies have shown an association between the COL5A1 gene and anterior cruciate ligament (ACL) injury risk. In this larger study, the genotype and allele frequency distributions of the COL5A1 rs12722 C/T and rs10628678 AGGG/deletion (AGGG/-) indel variants were compared between participants: (i) with ACL injury in independent and combined cohorts from South-Africa (SA) and Australia (AUS) vs controls (CON), and (ii) with any ligament (ALL) or only ACL injury in a Japanese (JPN) cohort vs CON. Samples were collected from SA (235 cases; 232 controls), AUS (362 cases; 80 controls) and JPN (500 cases; 1,403 controls). Genomic DNA was extracted and genotyped. Distributions were compared, and inferred haplotype analyses performed. No independent associations were noted for rs12722 or rs10628678 when the combined SA + AUS cohort was analysed. However, the C-deletion (rs12722-rs10628678) inferred haplotype was under-represented (p = 0.040, OR = 0.15, CI = 0.04-0.56), while the T-deletion inferred haplotype was over-represented in the female SA + AUS ACL participants versus controls (p < 0.001, OR = 4.74, CI = 1.66-13.55). Additionally, the rs12722 C/C genotype was under-represented in JPN CON vs ACL (p = 0.039, OR = 0.52, 0.27-1.00), while the rs10628678 -/- genotype was associated with increased risk of any ligament injuries (p = 0.035, OR = 1.31, CI = 1.02-1.68) in the JPN cohort. Collectively, these results highlight that a region within the COL5A1 3'-UTR is associated with ligament injury risk. This must be evaluated in larger cohorts and its functional relevance to the structure and capacity of ligaments and joint biomechanics be explored.Highlights The COL5A1 T-deletion inferred haplotype (rs12722-rs10628678) was associated with an increased risk of ACL rupture in the combined SA and AUS female participants.The COL5A1 C-deletion inferred haplotype (rs12722-rs10628678) was associated with a decreased risk of ACL rupture in the combined SA and AUS female participants.The COL5A1 rs12722 C/C and rs10628678 -/- genotypes were associated with increased risk of ACL rupture and of ligament injuries in JPN, respectively.A region within the COL5A1 3'-UTR is associated with risk of ligament injury, including ACL rupture, and therefore the functional significance of this region on ligament capacity and joint biomechanics requires further exploration.

Investigation of multiple populations highlight VEGFA polymorphisms to modulate anterior cruciate ligament injury.

Polymorphisms in VEGFA and KDR encoding proteins have been associated with anterior cruciate ligament (ACL) injury risk. We leveraged a collective sample from Sweden, Poland, and Australia to investigate the association of functional polymorphisms in VEGFA and KDR with susceptibility to ACL injury risk. Using a case-control genetic association approach, polymorphisms in VEGFA and KDR were genotyped and haplotypes inferred from 765 controls, and 912 cases clinically diagnosed with ACL rupture. For VEGFA, there was a significant overrepresentation of the rs2010963 CC genotype (p = 0.0001, false discovery rate [FDR]: p = 0.001, odds ratio [OR]: 2.16, 95% confidence interval [CI]: 1.47-3.19) in the combined ACL group (18%) compared to the combined control group (11%). The VEGFA (rs699947 C/A, rs1570360 G/A, rs2010963 G/C) A-A-G haplotype was significantly (p = 0.010, OR: 0.85, 95% CI: 0.69-1.05) underrepresented in the combined ACL group (23%) compared to the combined control group (28%). In addition, the A-G-G construct was significantly (p = 0.036, OR: 0.81, 95% CI: 0.64-1.02) underrepresented in the combined ACL group (12%) compared to the combined CON group (16%). Our findings support the association of the VEGFA rs2010963 CC genotype with increased risk and (ii) the VEGFA A-A-G haplotype with a reduced risk, and are in alignment with the a priori hypothesis. Collectively identifying a genetic interval within VEGFA to be implicated in ACL risk modulation and highlight further the importance of vascular regulation in ligament biology.

Exploring new genetic variants within COL5A1 intron 4-exon 5 region and TGF-ß family with risk of anterior cruciate ligament ruptures.

Variants within genes encoding structural and regulatory elements of ligaments have been associated with musculoskeletal soft tissue injury risk. The role of intron 4-exon 5 variants within the α1 chain of type V collagen (COL5A1) gene and genes of the transforming growth factor-ß (TGF-ß) family, TGFBR3 and TGFBI, was investigated on the risk of anterior cruciate ligament (ACL) ruptures. A case-control genetic association study was performed on 210 control (CON) and 249 participants with surgically diagnosed ruptures (ACL), of which 147 reported a noncontact mechanism of injury (NON). Whole-exome sequencing data were used to prioritize variants of potential functional relevance. Genotyping for COL5A1 (rs3922912 G>A, rs4841926 C>T, and rs3124299 C>T), TGFBR3 (rs1805113 G>A and rs1805117 T>C), and TGFBI (rs1442 G>C) was performed using Taqman SNP genotyping assays. Significant overrepresentation of the G allele of TGFBR3 rs1805113 was observed in CON vs ACL (P = .014) and NON groups (P = .021). Similar results were obtained in a female with the G allele (CON vs ACL: P = .029; CON vs NON: P = .016). The TGFBI rs1442 CC genotype was overrepresented in the female ACL vs CON (P = .013). Associations of inferred allele combinations were observed in line with the above results. COL5A1 intron 4-exon 5 genomic interval was not associated with the risk of ACL ruptures. Instead, this novel study is the first to use this approach to identify variants within the TGF-ß signaling pathway to be implicated in the risk of ACL ruptures. A genetic susceptibility interval was identified to be explored in the context of extracellular matrix remodeling.