Whole genome sequencing approach to identifying genetic risk factors underlying anterior cruciate ligament injuries in a twin family study
Background: Predisposition to ACL rupture is multifactorial, resulting from a complex interplay of intrinsic and extrinsic risk factors. Variation in the genome is now considered a key intrinsic risk factor, but the majority of currently implicated loci have been identified through case-control genetic association studies, which are limited by a candidate gene approach and insufficient statistical power. The primary aim of this thesis was to use a whole genome sequencing (WGS) approach within the context of a twin family study to identify novel or previously implicated genetic loci contributing to ACL rupture predisposition (Chapter 2). Additionally, this research aimed to explore prioritised genetic polymorphisms previously associated with ACL rupture and functioning in key biological pathways implicated through the WGS analyses, independently and as a collective, with ACL rupture predisposition in a large combined ACL rupture dataset (Chapter 3 and 4). Methods: The complete genomes of all family members in two unrelated families, each with affected twins were sequenced. Variants with potential loss of function effect were prioritised, and explored for probable biological function in the ACL rupture risk pathway. Furthermore, identity by descent analysis (IBD) was performed to identify potential disease causing mutations, on chromosomal regions shared between family members, and across families. Enriched biological pathway analyses were further explored to prioritise potential candidate genes. Two biological networks were prioritised which highlighted the angiogenesis and proteoglycan family of proteins. Specific polymorphisms within previously investigated candidate genes were further explored in case-control genetic association studies conducted in a large collective data set, including participants from three independent (Sweden, Poland and Australia) cohorts, combined with previously published South African and Polish data. The anterior cruciate ligament (ACL) rupture group included individuals diagnosed with a clinical diagnosis of an ACL rupture based on physical examination, and confirmed by either magnetic resonance imaging or arthroscopy. Only ACL ruptures resulting from a non-contact mechanism of injury were included. The control group comprised individuals of similar age to cases with no prior history of ACL injury or other ligament and tendon injuries, and participating in regular sporting activity, which was similar to cases. Participant samples were genotyped for single nucleotide polymorphisms in the VEGFA (rs699947 C/A rs1570360 G/A, rs2010963 G/C) and KDR (rs2071559 A/G, rs1870377 T/A) genes (Sweden CON: 116 ACL: 95; Poland CON: 149 ACL: 127 and Australia CON: 83 ACL: 342). Additionally, in the ACAN (rs2351491 C/T, rs1042631 T/C, rs1516797 T/G), DCN (rs516115 T/C) and BGN (rs1126499 C/T, rs1042103 G/A) genes (Sweden and Poland). Haplotype analyses were explored (VEGFA, KDR, ACAN and BGN) using the individual genotype data. In addition, inferred allele interactions were presented for VEGFA-KDR, ACAN-BGN ACAN-DCN, BGN-DCN, and VEGFA-DCN as a proxy for gene-gene interactions within the discrete angiogenesis and proteoglycan gene families, and between genes as a proxy for pathway interactions. For association studies, frequencies were calculated for the genotype, allele, inferred haplotypes and allele interactions, and the distributions compared between the control and ACL rupture participants. The statistical programs in R were used for all the analyses, and a p value < 0.05 was accepted to be significant. Results: The WGS analyses highlighted six candidate genetic loci in three genes (COL12A1, CATSPER2, and KCNJ12) with predicted loss of function effects in all affected and unaffected family members within the two studied families. Of the three genes, polymorphisms within COL12A1 were previously associated with ACL rupture predisposition, while CATSPER2 and KCNJ12 are two novel genetic loci with no known previous association with predisposition to ACL rupture. The IBD analyses identified several regions shared in each independent family, of which a segment including a long intergenic non-protein coding RNA (lincRNA) LINC01250 gene in the telomeric region of chromosome 2p25.3 was shared between affected twins in both families, and an affected brother. Furthermore, several functional partners were highlighted. Genetic association analyses of the prioritised polymorphisms in a combined cohort identified an independent association of the VEGFA rs2010963 CC genotype and C allele with increased risk (genotype p = 0.0001, FDR p = 0.001, OR 2.16, 95% CI: 1.47-3.19; allele p = 0.0006, FDR p = 0.003, OR 1.29, 95% CI: 1.11-1.49). Furthermore, the association of the VEGFA A-A-G and A-G-G inferred haplotypes (rs699947 A/C-rs1570360 G/Ars2010963 G/C) with reduced risk (p = 0.010, haplo.score: -2.58, OR: 0.85, 95% CI: 0.69-1.05; A-G-G: p = 0.036, haplo.score: -2.09, OR: 0.81, 95% CI: 0.64-1.02) of ACL rupture. Moreover, a reduced interval (rs1570360 G/A-rs2010963 G/C) revealed an association of the VEGFA -GG and -A-G inferred haplotypes with reduced risk (-G-G: p = 0.031, haplo.score: -2.15, OR: 1.00 and -A-G: p = 0.024, haplo.score: -2.25, OR: 0.98, 95% CI: 0.82-1.18) and the -G-C inferred haplotype with increased risk p = 0.012, haplo.score: 2.50, OR: 1.18, 95% CI: 0.99- 1.40). The KDR genotype and haplotype analyses illustrated that it is highly unlikely that the investigated KDR polymorphisms are associated with modulating ACL rupture risk. Inferred allele interactions noted a significant association of the VEGFA (rs699947 A/C, rs2010963 G/C) - KDR (rs2071559 A/G, rs1870377 T/A) A-G-A-A (p = 0.005, OR: 0.51, 95% CI: 0.30- 0.87) and A-G-G-A (p = 0.018, OR: 0.93, 95% CI: 0.54-1.60) combinations with reduced ACL rupture risk. Further, a significant association of the VEGFA (rs699947 C/A, rs1570360 G/A, rs20109630 G/C) - DCN (rs516115 T/C) A-G-G-T (p = 0.010, OR: 0.53, 95% CI: 0.30-0.91), A-A-G-C (p = 0.010, OR: 0.42, 95% CI: 0.21-0.81) and A-A-G-T (p = 0.046, OR: 0.77, CI: 0.49-1.2) allele combinations with reduced risk was noted for male participants in the collective cohort. No independent or haplotype associations with ACL rupture risk were noted for any of the investigated proteoglycan polymorphisms, in the collective cohort. Conclusion: Collectively, this work has expanded current knowledge on the genetic regions contributing to ACL rupture predisposition, and further highlights the polygenic nature of multifactorial phenotypes. Employing whole genome sequencing in a twin family context, together with a pathway based approach, novel and previously implicated genetic loci were identified towards the aims of the thesis. The catalogue of candidate in silico mutations and modifier genes that clustered in pathophysiological pathways important in ACL rupture, and with implications for therapeutic intervention were identified, and need to be interrogated. Of particular interest are the novel CATSPER2, KCNJ12 and LINC01250 genetic loci. Furthermore, additional evidence to support the implication of the VEGFA gene in modulating ACL rupture risk is provided, and highlighted is the potential collaboration of members within the angiogenesis and proteoglycan gene family in modulating risk. The studies in Chapter 3 and 4 suggest genetic association studies in single populations are less informative, and instead larger collective cohorts with increased statistical power should be employed. Further to that, rather than investigating single polymorphisms, larger regions of the genome should be explored to determine the potential interacting components contributing to musculoskeletal injury risk. Going forward, characterisation of the functional biological effect of implicated loci may assist in unravelling the underlying mechanisms altering tissue homeostasis, and subsequently an individual's capacity for healing and adaptive response.
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