Browsing by Author "September, Alison V"

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    Open Access
    Tendon and Ligament Genetics: How Do They Contribute to Disease and Injury? A Narrative Review
    (2022-04-29) Ribbans, William J; September, Alison V; Collins, Malcolm
    A significant proportion of patients requiring musculoskeletal management present with tendon and ligament pathology. Our understanding of the intrinsic and extrinsic mechanisms that lead to such disabilities is increasing. However, the complexity underpinning these interactive multifactorial elements is still not fully characterised. Evidence highlighting the genetic components, either reducing or increasing susceptibility to injury, is increasing. This review examines the present understanding of the role genetic variations contribute to tendon and ligament injury risk. It examines the different elements of tendon and ligament structure and considers our knowledge of genetic influence on form, function, ability to withstand load, and undertake repair or regeneration. The role of epigenetic factors in modifying gene expression in these structures is also explored. It considers the challenges to interpreting present knowledge, the requirements, and likely pathways for future research, and whether such information has reached the point of clinical utility.
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    The COL5A1 gene and musculoskeletal soft-tissue injuries
    (2010) Posthumus, Michael; September, Alison V; Schwellnus, Martin P; Collins, Malcolm
    Background. It has been shown that there is an association between various genetic variants and Achilles tendon injuries as well as anterior cruciate ligament (ACL) ruptures. Among other variants the BstUI restriction fragment length polymorphism (RFLP) within the COL5A1 gene has been shown to be over-represented in asymptomatic participants when compared with those with chronic Achilles tendinopathy, and in asymptomatic female participants when compared with those with ACL ruptures. The male asymptomatic control participants in the ACL study, which were 10 years younger than previously investigated cohorts, had a distinctly different genotype frequency. Aim. The aim of this study was therefore to determine whether the distribution of the COL5A1 BstUI RFLP in the combined asymptomatic participants without any known history of tendon injuries is age dependent, particularly among males. Results. When the 265 male asymptomatic participants from all studies were pooled and divided into age-group tertiles, there was a significant linear increase in the CC genotype frequency (p=0.032) among the male age groups, with the youngest group having the lowest frequency (CC genotype frequency, 13%) and the oldest group having the highest (CC genotype frequency, 27%) frequency. There was however a similar CC genotype content in all three female (N=231) age groups (CC genotype frequency, 24 - 27%; p=0.795). Conclusion. The practical implication is that the selection of asymptomatic groups is of critical importance when future studies of this nature are designed. Future research investigating this genetic variant as a risk factor for soft-tissue injuries should consider these findings when selecting asymptomatic participants.
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    The relationship between genes associated with the pain pathways and the development of chronic shoulder pain and disability in South African breast cancer survivors
    (2024) Firfirey, Firzana; Shamley, Delva; September, Alison V
    Background A growing challenge in South Africa (SA) healthcare is the frequency of chronic shoulder pain and disability among breast cancer survivors (BCS). Compounding this issue is that a significant number of BCS in South Africa are low-income individuals, exacerbating the economic burden associated with managing chronic pain and disability. These sequelae can last for as much as 6 years post-surgery and thereby negatively impact the overall quality of life. The current standard treatment in SA for acute and chronic pain include opioids and opioid derivatives. Several risk factors have been highlighted to increase susceptibility to developing the sequelae which include severe acute post-operative pain and genetics. Polymorphisms within genes functioning within the opioid signalling and pain pathways have been implicated in variability in opioid use and the development of chronic musculoskeletal pain and disability conditions. The SA population has a diverse genetic background and an increasing BCS cohort that are of mixed ancestry. This thesis therefore sought to identify potential genetic contributors to variability in pain response and to understand the development of chronic pain and disability in SA BCS of mixed ancestry background. The study aimed to (i) assess chronic shoulder pain and disability symptoms using self reported outcome measures in a unique SA BCS cohort; (ii) investigate non-genetic and genetic risk factors associated with chronic pain and disability; (iii) explore the genetic variability in key genes within the opioid signalling and pain pathways and (iv) characterise the potential biological and functional networks related to the opioid signalling and pain pathways. The objectives included : i. Describing the prevalence of chronic pain and disability in BCS using the Shoulder Pain and Disability Index (SPADI) in the SA cohort of mixed ancestry ii. Examine the genetic association between eight prioritised single nucleotide polymorphisms in three candidate genes: (I) ATP-Binding Cassette- Subfamily B, Member 1 Gene (ABCB1); (rs1045642 G>A; rs1128503 G>A), (II) Opioid Receptor, Mu 1 Gene (OPRM1); (rs1799971 A>G; rs540825 T>A) and (III) Catechol-O-Methyl Transferase Gene (COMT); (rs6269 A>G; rs4633 C>T; rs4818 C>G; rs4680 G>A) and the prevalence of chronic pain and disability. iii. Evaluate the gene-gene interaction and association between prioritised SNPs for ABCB1 (rs1045642 G>A; rs1128503 G>A), OPRM1 (rs1799971 A>G; rs540825 T>A) and COMT (rs6269 A>G; rs4633 C>T; rs4818 C>G; rs4680 G>A), and the prevalence of chronic pain and disability in the SA BCS. iv. Conduct bioinformatic analyses to comprehensively examine the functional effects of the prioritised SNPs, identify associated networks and identify potential protein network partners relative to the opioid signalling and pain pathway. Methods A cross-sectional retrospective study was followed (Chapter Error! Reference source not found.), that enrolled two hundred and fifty-two SA BCS. This study was a sub-study of a larger project approved by the Human Research Ethics Committee of the Faculty of Health Sciences within the University of Cape Town (HREC: 312/2012, 125/2017). Qualifying participants were BCS that were diagnosed with unilateral breast cancer ( >1yr before study), older than 18yrs, had no history of neck or shoulder pathology and self-identified as mixed ancestry. Patient-reported pain, disability, and combined symptoms associated with shoulder pathologies were evaluated using the Shoulder Pain and Disability Index (SPADI). Participants scores were calculated and categorized into no-low (A), OPRM1 (rs1799971 A>G), and COMT (rs4680 G>A) . Statistical analysis was performed to describe the relationship between a convenient sample's clinical variables and total drug doses. Moreover, analyses were performed to examine differences in outcome measure scores between the three time points, T1 (pre-operative), T2 ( 3-month post-operative), and T3 (1- year post-operative). Evaluation of the individual genotype, and allele frequencies of each SNPs for each gene between groups were also examined. Haplotype frequencies were statistically inferred for all SNPs within each gene and evaluated between groups. As a proxy for gene-gene interaction, inferred allele–allele combination frequencies were evaluated using the individual genotype data for each SNP. Nonparametric and parametric statistical tests were employed where appropriate, with statistical significance accepted at pA-rs1045642 G>A) haplotype analysis, the inferred G-A (p=0.029, OR: 0.00, 95% CI: 0.00-0.00) haplotype was significantly associated with reduced likelihoods of reporting moderate-high disability. In addition, the inferred A-A (p=0.029, OR:0.63, 95% CI: 0.37-1.06) haplotype was also significantly associated with reduced likelihood of reporting moderate-high combined (pain and disability). The OPRM1 (rs1799971 A>G – rs540825 T>A) inferred G-T (p=0.019, OR:0.33, 95% CI: 0.14-0.75) haplotype was significantly associated with reduced likelihoods of reporting moderate-high pain. Inferred haplotype analysis of five COMT haplotypes noted significant associations for H2-H5, the most notable associations being for the rs6269 A>G -rs4680 G>A genetic interval. This analysis revealed the G-G (p=0.026, OR: 0.67, 95% CI: 0.38-1.18) and A-A (p=0.007, OR: 2.09, 95% CI: 0.89-4.88) haplotypes were associated with reduced and increased likelihood of reporting moderate-high pain, respectively. Gene–gene interaction analyses demonstrated significant associations between the ABCB1 (rs1045642 G>A) – OPRM1 (rs1799971 A>G – rs540825 T>A) and the ABCB1 (rs1045642 G>A) – OPRM1 (rs1799971 A>G). The inferred A-A-T (p=0.029, OR: 0.58, 95% CI: 0.18-1.45) and A-A (p=0.008, OR: 0.44, 95% CI:0.24-0.80) allele-allele combinations were associated with reduced likelihoods of reporting moderated-high combined (pain and disability). ABCB1 (rs1045642 G>A) – OPRM1 (rs540825 T>A) combination analyses demonstrated that the A-T (p=0.019, OR: 0.62, 95% CI: 0.33- 1.16/p=0.014, OR:0.62, 95% CI:0.35-1.10) combination was associated with reduced likelihood of reporting moderate-high disability/combined (pain and disability) symptoms. While the alternate G-A (p=0.021, OR: 1.57, 95% CI: 0.30-3.10/p=0.030, OR: 1.50, 95% CI: 0.78-2.86) combination was associated with increased likelihood of reporting moderate high disability/combined (pain and disability) symptoms. The OPRM1 (rs1799971- rs540825) - COMT (rs4680) combination analyses demonstrated that the A-T-A (p=0.008, OR: 1.36, 95% CI: 0.77-2.41) and G-T-G (p=0.004, OR: 0.00, 95% CI: 0.00-0.00) were associated with increased, and reduced likelihoods of reporting moderate-high pain. Similarly, the OPRM1 (rs1799971 A>G)-COMT (rs4680 G>A) allele-allele combinations A-A (p=0.004, OR: 1.35, 95% CI: 0.85-2.15), and G-G (p=0.010, OR: 0.23, 95% CI: 0.05- 1.03) combinations were associated increased and reduced likelihoods of reporting moderate-high pain and combined (pain and disability). The OPRM1 (rs540825 T>A) - COMT (rs4680 G>A) A-A (p=0.012, OR: 1.89, 95% CI: 0.81-4.38) allele-allele combination was associated increased likelihoods of reporting moderate-high combined (pain and disability). Analyses of the ABCB1 (rs1128503 - rs1045642) - COMT (rs4680) combination demonstrated that the A-A-G (p=0.006, OR:0.68, 95% CI: 0.27-1.71) were significantly associated with reduced likelihood of reporting combined (pain and disability). For the 2-SNP pairing, the ABCB1-COMT (rs4680 G>A) G-A allele combinations were associated with increased likelihoods of reporting moderate-high pain (p=0.005, OR: 2.08, 95% CI: 1.12-3.84), disability (p=0.018, OR: 1.16, 95% CI: 0.62-2.15), and combined (p=0.008, OR: 1.94, 95% CI: 1.02-3.69) groups, pA, and the prevalence of disability, indicating movement-related pain. Despite the absence of an independent association for the functional variant rs1799971 A>G, and rs540825 T>A, for OPRM1, haplotype analyses showed a correlation, supporting the relationship of pain treated with opioids being impacted by this gene. In addition to the independent association observed for COMT rs4680 G>A, gene interactions were observed highlighting the role of collective modulation in pain and disability in the present cohort. Additionally, in-silico analyses revealed strong relationships between the genes and important pathways and mechanisms, further strengthening and supporting the hypotheses presented in the aims of this study. The clinical implications of this study aimed to assist in the understanding of the pain mechanisms and opioid pathways towards the development of novel and innovative therapeutics for pain, in personalized medicine.
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    To investigate whether VEGF and KDR polymorphisms are associated with chronic Achilles tendinopathy and self-reported measurements of tendon pain
    (2024) Brazier, Christina Daniela; Collins, Malcolm; September, Alison V; Laguette, Mary-Jessica Nancy
    Background: Chronic Achilles tendinopathy (AT) is prevalent in the sporting population, specifically in sporting codes with a large running component, and presents as swelling, impaired lower limb function, and pain of insidious onset. Although the mechanisms are unclear, current theories implicate structural changes and neovascularisation in tendinopathy. Vascular endothelial growth factor (VEGF) and its receptor referred to as kinase domain receptor (KDR) are key regulators of neovascularisation and can be associated with pain. Common DNA sequence variants within the VEGFA and KDR genes have previously been associated with musculoskeletal soft tissue injuries, including AT. The primary aim of this dissertation was to identify whether VEGF and KDR variants were associated with (i) the severity of AT, (ii) AT ultrasound findings and (iii) self-reported measurements of Achilles tendon pain using multidimensional pain scales. Methods: One hundred and eighty-five recreational athletes with clinically confirmed Achilles tendinopathy for at least 3 months were recruited from Cape Town, South Africa. The injured and uninjured Achilles tendons were examined using conventional grayscale ultrasound. Tendinopathy pain was rated by completing the Victorian Institute Sports Assessment - Achilles (VISA-A), Short-form McGill Pain Questionnaire (sf- MPQ), and Short-form Brief Pain Inventory (sf-BPI) questionnaires. One hundred and ninety-four asymptomatic healthy appropriately matched individuals with no history of tendon injuries were also recruited for this study. Participants were genotyped for VEGFA rs699947 (C/A), VEGFA rs2010963 (G/A), KDR rs2071559 (G/C) and KDR rs1870377 (T/A). Results: Although the VEGFA and KDR variants were not associated with AT, either independently, as inferred haplotypes or via allele interactions, (i) the VEGFA rs699947 CC genotype was significantly associated with decreased risk of bilateral AT, (ii) the A-G VEGFA inferred haplotype constructed from rs699947 and rs2010963 was associated with increased risk of bilateral AT, (iii) the KDR rs2071559 AA genotype was significantly associated with increased risk of a history of multiple (two or more) AT, (iv) the G-T and A-A KDR inferred haplotypes constructed from rs2071559 and rs1870377 were associated with decreased risk and increased risk of multiple and/or bilateral AT, respectively, and (v) the C-G and A-A VEGFA rs699947 and KDR rs2071559 allele-allele interactions were significantly associated with decreased and increased risk of bilateral or multiple injuries respectively. There were no significant differences in the diameters or the relative number of abnormal ultrasound findings of the injured and uninjured Achilles tendons between the VEGFA and KDR genotype groups. Finally, there were no significant differences in VISA-A, sf-MPQ and sf-BPI scores, as well the subscale scores between the VEGFA and KDR genotype groups. Conclusion: The novel findings of this dissertation implicate the VEGF and KDR genes, and by implication the potential biological role of the angiogenesis signalling pathway, with bilateral and/or multiple Achilles tendinopathy risk. The investigated variants within these genes however were not associated with tendon diameters, the relative number of abnormal ultrasound findings or self-reported Achilles tendon pain measured using multidimensional pain scales.
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    Whole exome sequencing: a customised approach to exploring the genetic basis of musculoskeletal soft tissue injuries
    (2018) Gibbon, Andrea; September, Alison V; Collins, Malcolm
    Several DNA sequence variants have previously been associated with the risk of musculoskeletal soft tissue injuries, suggesting a role for genetics in the aetiology of common sporting injuries such as chronic Achilles tendinopathy (AT) and anterior cruciate ligament (ACL) ruptures. Genetic risk modifiers have primarily been identified using a hypothesis driven candidate gene approach. However, the ability to identify all risk-conferring variants using this approach alone is limited. Therefore, the primary aim of this thesis was to further define the molecular signatures of musculoskeletal soft tissue injuries mapping to specific genomic intervals encoding several structural and regulatory components of the extracellular matrix (ECM). Genes encoding the tenascin-C (TNC) glycoprotein (9q33.1) and the α1 chain of type XXVII collagen (COL27A1, 9q32), as well as matrix metallopeptidase 3 (MMP3, 11q22) and the α1 chain of type I collagen (COL1A1, 17q21.33), have previously been associated with the risk of injury and were therefore prioritised for further interrogation. Previously associated variants within these regions and/or new candidate variants identified by whole exome sequencing (WES) and prioritised through the application of a customised, tiered filtering strategy, were genotyped in several previously recruited, self-identified White Achilles tendon injury and ACL rupture cohorts. The second aim of this study was to determine whether the observed risk-associated signatures in the self-identified White cohorts were similar to those underpinning injury in an ancestrally admixed sample, using ACL ruptures in a South African Coloured cohort as the phenotypic model.
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    Whole genome sequencing approach to identifying genetic risk factors underlying anterior cruciate ligament injuries in a twin family study
    (2022) Feldmann, Daneil; September, Alison V; Collins, Malcolm; Chimusa, Emile
    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.