Analysis of three genes that contribute to fibrosis in South African systemic sclerosis patients

MSc (Med), Faculty of Health Sciences, University of the WitwatersrandIntroduction: Systemic sclerosis (SSc) is a complex autoimmune disease characterised by autoantibody release, leading to microvascular injury, fibroblast activation and increased production of collagen. The genetics of SSc is complex with many genes implicated in the development and maintenance of the extracellular matrix (ECM). The main aim of this study was to test for differential expression of matrix metalloproteinase 1 (MMP1), tissue inhibitor of metalloproteinase 1 (TIMP1) and hepatocyte growth factor (HGF) in SSc patients compared to healthy control individuals and to assess whether the differential expression of these genes could have an impact on clinical features of the disease. Methods: Two skin biopsies were analysed for each of 16 black SSc patients, one from clinically involved skin (lateral forearm) and one from clinically uninvolved skin (back). One skin sample was obtained from 15 ethnically matched control individuals. The differential expression of MMP1, TIMP1 and HGF in the clinically involved and uninvolved patient samples would be compared to control individuals using relative quantification polymerase chain reaction (qPCR). The gene expression profiles were then compared to specific clinical features to deduce whether any of the gene expression profiles is correlated with the manifestation of specific clinical features. Results: MMP1 gene expression was significantly decreased in SSc patients for both involved (p=0.0004) and uninvolved skin (p=0.0004) compared to controls. Conversely, TIMP1 gene expression was significantly increased in SSc patients at both sites compared to controls (p=<0.00001 for both comparisons). A trend of significance was observed for the difference in TIMP1 expression between the involved an uninvolved skin within the patients (p=0.05) with a greater increase in involved skin. HGF had increased gene expression in the patients compared to controls for involved and uninvolved skin (p=0.002 and 0.004, respectively). The difference in gene expression between the involved and uninvolved biopsies was not significant for either MMP1 or HGF (p=0.87 and 0.83, respectively). The only correlates that may have a biological significance are HGF in involved skin correlated with disease activity (r=0.60; p=0.013) and HGF in uninvolved skin vi correlated with skin score (MRSS) with r=0.50 and p=0.048. With regards to the categorical data, two marginally significant observations were found, once again with HGF, which was found to be associated with gender in involved skin (p=0.037) and renal disease in uninvolved skin (0.031). Conclusion: The relative under expression of MMP1 and over expression of TIMP1 reflect the pro-fibrotic state of scleroderma skin. The over expression of HGF suggests that HGF may play a compensatory anti-fibrotic role, although this is not sufficient to overcome the pro-fibrotic state of the skin. This study provides supporting evidence to debunk the myth of uninvolved skin in SSc patients. The altered expression of MMP1, TIMP1 and HGF in the clinically uninvolved skin of SSc patients suggests that all subcutaneous tissue is affected, although to a greater extent in the clinically involved skin of the patients

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The WNT signalling pathway in systemic sclerosis

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment for the degree of Doctor of Philosophy 2016Systemic sclerosis (SSc) is a complex autoimmune disease involving the immune system, vasculature and extracellular matrix. Dysregulation of the Wnt pathway has been implicated in the development of fibrosis in SSc and is proposed to contribute to a failure to maintain tissue homeostasis and appropriate immune response. The objective of this research study was to explore the role of altered Wnt pathway gene regulation in the development of fibrosis in black South African SSc patients with early, diffuse disease (dcSSc). The first aim was to examine differential gene expression in the Wnt pathway and the second aim to examine differential expression of microRNAs that potentially target Wnt pathway genes. Skin biopsies from eight black South African patients with dcSSc, samples from both the forearm (affected skin) and the back (unaffected skin), and eight ethnically matched healthy control skin samples were examined. The Wnt pathway RT2 Profiler qPCR Array (84 Wnt pathway genes) was used to assess differential gene expression, single gene TaqMan assays for validation and small RNA-sequencing for microRNA analysis. Data analysis was done using HTqPCR, NormqPCR and DESeq2 software. Gene expression patterns revealed five distinct differentially expressed gene clusters. Two clusters displayed genes that were upregulated in both affected and unaffected SSc skin compared to controls (one showing a more heterogeneous pattern than the other). Another showed consistently decreased gene expression and two revealed more complex patterns responsible for delineating the patients into two groups. The gene vi expression was validated for five genes. The sRNA-seq data showed differential expression of 31 miRNAs that target the Wnt pathway genes, including miR-335 and miR204 that are important regulators of normal tissue development. Other dysregulated miRNAs have been linked to fibrotic and autoimmune diseases. In this group of dcSSc patients, there is differential gene expression of several Wnt pathway genes that delineate the patients into two distinct groups. This could point to differences in disease aetiology leading to distinct clinical outcomes, such as inflammation. Together with the differentially expressed microRNAs, the findings indicate a substantial contribution of epigenetic changes to the pathogenesis, progression and diverse clinical features of dcSSc.MT201

Effect of BRCA mutations on metastatic relapse and cause-specific survival after radical treatment for localised prostate cancer

BACKGROUND:Germline BRCA mutations are associated with worse prostate cancer (PCa) outcomes; however, the most appropriate management for mutation carriers has not yet been investigated.OBJECTIVE:To evaluate the response of BRCA carriers to conventional treatments for localised PCa by analysing metastasis-free survival (MFS) and cause-specific survival (CSS) following radical prostatectomy (RP) or external-beam radiation therapy (RT).DESIGN, SETTING, AND PARTICIPANTS:Tumour features and outcomes of 1302 patients with local/locally advanced PCa (including 67 BRCA mutation carriers) were analysed. RP was undergone by 535 patients (35 BRCA); 767 received RT (32 BRCA). Median follow-up was 64 mo.OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:Median survival and 3-, 5-, and 10-yr survival rates were estimated using the Kaplan-Meier method. Generated survival curves were compared using the log-rank test. Cox regression analyses were used to assess the prognostic value of BRCA mutations.RESULTS AND LIMITATIONS:A total of 67 BRCA carriers and 1235 noncarriers were included. At 3, 5, and 10 yr after treatment, 97%, 94%, and 84% of noncarriers and 90%, 72%, and 50% of carriers were free from metastasis (p&lt;0.001). The 3-, 5- and 10-yr CSS rates were significantly better in the noncarrier cohort (99%, 97%, and 85%, respectively) than in carriers (96%, 76%, and 61%, respectively; p&lt;0.001). Multivariate analysis confirmed BRCA mutations as an independent prognostic factor for MFS (hazard ratio [HR]: 2.36; 95% confidence interval [CI], 1.38-4.03; p=0.002) and CSS (HR: 2.17; 95% CI, 1.16-4.07; p=0.016).CONCLUSIONS:BRCA carriers had worse outcomes than noncarriers when conventionally treated for local/locally advanced PCa.PATIENT SUMMARY:Prostate cancer patients with germline BRCA mutations had worse outcomes than noncarriers when conventionally treated with surgery or radiation therapy

IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus

OBJECTIVE: High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease. METHODS: 1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African–American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay. RESULTS: In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR > 2.56, p >003C; 1.9×10(−14) for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained > 70% of the genetic risk of SLE due to IRF5. In African–American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African–American subjects and absent in African patients with SLE. CONCLUSIONS: The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements. SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.(1) These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness(2) and are likely pathogenic in SLE.(34