Molecular Biology and Human Genetics

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Analysis of a neurochip array dataset to study Parkinson’s disease in a South African study collection
(Stellenbosch : Stellenbosch University,, 2023-02) Step, Kathryn; Bardien, Soraya; Vorster, Alvera; Müller-Nedebock, Amica; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is an incurable, and complex neurodegenerative disease. Both genetic and environmental factors likely contribute to disease onset. Notably, while several pathogenic variants and susceptibility factors have been described in populations of Asian and European ancestry, such variants have seldom been identified in individuals from sub-Saharan Africa (SSA). This could be due to the limited number of studies investigating the genetic etiology of PD in SSA. To address this knowledge gap, the present study undertook the largest, to date, PD-focused genomewide association study (GWAS), and pathogenic variant screening study in SSA to identify possible susceptibility variants and pathogenic variants in South African PD cases. For this, we used raw genotyping data generated from a large collaborative project known as COmprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson’s Disease (Courage-PD), whose goal was to identify PD-associated variants. The NeuroChip array, used to genotype the study participants, contained a total of 306,670 tagging variants and 179,467 custom content variants, including 349 associated with PD. The South African dataset genotyped on the array comprised 452 cases and 280 controls. We hypothesised that these individuals would harbour susceptibility and pathogenic variants. To test this hypothesis, the NeuroChip genotyping data was analysed using various bioinformatic approaches. The quality control (QC) and association analysis were completed using PLINK, and the results were visualized using R software. After excluding 15 individuals during the QC stage, population stratification analysis identified two ‘broad’ ancestral groups, designated as ‘European’ (n=497) and ‘non-European’ (n=220). For the GWAS, no variants reached the genome-wide significance threshold of 5x10-8 , however, variants were found that met the ‘suggestive significance’ criteria (1x10-5 ). A total of 17 variants of interest were identified in the European ancestral group (in the KHDRBS2, FGF14, and PDXK genes) and 2 variants of interest were identified in the non-European ancestral group (in the SYNPR and PDE10A genes). These variants highlighted possible new PD genes that are plausible candidates, but that will need to be confirmed in future, much larger GWAS. Thereafter, a Polygenic Risk Score (PRS) analysis was performed, using PRSice software, on the European ancestral group where the most predictive PRS explained 4.5% of the phenotypic variation (the phenotype being PD). Furthermore, use of the NeuroChip data as a method of pathogenic variant screening, revealed that all 12 variants detected by our group previously were also detected by the array. Moreover, an additional 16 variants in 14 individuals were prioritized as being potentially pathogenic, and warrant further study. Finally, screening of p.G2019S in the LRRK2 gene, arguably the most prevalent PD pathogenic variant, using high-resolution melt analysis, revealed a relatively low frequency of 1.2% (n= 8/689) in our entire PD study collection. Notably, this variant has not been identified in any PD individuals of African ancestry, to date. Collectively, this study highlights the importance of screening and studying underrepresented populations to uncover additional genetic-related risks for PD development. However, future largescale whole-genome sequencing and association studies, including all South African ancestral groups, will likely be needed to identify the remaining, potentially novel genetic factors contributing to PD in our local populations.
Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Celia; Bardien, Soraya; Loos, Ben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental and genetic factors. To date, a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study, and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst pharmacological and surgical interventions are available for PD, the current options exhibit adverse side effects with long term treatment. There is a great need to develop new treatments with i. less side effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One molecule is curcumin, the core component of the curry spice turmeric, which is well known for its antioxidant and anti-inflammatory properties and has already been studied for its possible neuroprotective role in Alzheimer’s disease. The aim of the present study was to create a cellular model of PD by decreasing the expression of PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained from the original cellular model. PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter, PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC- 1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV detection, MLPA was performed on 210 South African PD patients and putative mutations were verified by qRTPCR on the Lightcycler 96. PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant changes were observed in the connectivity of the mitochondrial network, but autophagic flux was significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP (p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in cells with decreased PINK1 expression. Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD patients, and fibroblasts were therefore not obtained. A number of false positive mutations were identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4. In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary regimen in most populations worldwide, further studies on elucidating its biochemical and cellular properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic agents is currently a topical and fast-growing area of research, and holds much promise for clinical application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and PD.
Development of a custom-designed targeted resequencing gene panel for Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2020-03) Mahungu, Amokelani Clementine; Bardien, Soraya; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in a part of the brain known as the substantia nigra. The selective loss of these neurons results in motor impairments also classified as cardinal PD symptoms which are tremor, rigidity, bradykinesia, and postural instability. Additionally, non-motor symptoms also occur namely loss of smell, mood disorders, cognitive decline, sleep disturbances, gastrointestinal and autonomic dysfunction. To date, no cure for PD exists and the underlying pathobiology of the disease is not fully understood. Approximately 90% of PD cases are idiopathic which is proposed to result from a complex interaction of environmental and genetic factors. The remaining 10% of PD cases are defined as monogenic and caused by genes that follow a Mendelian type of inheritance. Since the discovery of the first monogenic PD-causing gene SNCA, numerous genes have been identified and extensively studied in European and Asian populations. These include ATP13A2, CHCHD2, DJ-1, DNAJC13, DNAJC6, EIF4G1, FBXO7, GBA, GCH1, GIGYF2, HTRA2, LRRK2, PINK1, PLA2G6, PRKN, RAB39B, RIC3, SLC6A3, SNCA, SYNJ1, TMEM230, VPS13C and VPS35. However, many of these genes have not been widely studied in sub-Saharan African (SSA) populations. Furthermore, for the few that have been investigated, the studies used first-generation sequencing methods namely Sanger sequencing, that only allows screening of a single region or mutation at a time. More recently, next-generation sequencing (NGS) gene panels have been used to examine all the known PD genes in patients whose early disease onset and positive family history suggest a possible inherited genetic cause. Thus, the first aim of our study was to design a custom NGS gene panel for rapid screening of the known PD genes in South African patients. The gene panel was developed using Agilent SureSelect Target Enrichment technology and it included all of the above-mentioned 23 PD genes. Subsequently, 32 PD patients with early disease onset and family history were screened in two separate sequencing runs. Following analysis of the sequence data, we achieved a coverage of >200x for both runs. For our first run, we included positive controls with known pathogenic single nucleotide mutations, a 40bp deletion, and copy number mutations. All the mutations from the positive controls were validated except for the copy number mutations. On average, 79 variants were detected per patient of which only 15 were prioritized based on whether they were previously associated with PD, were rare (MAF <0.01), novel, and were predicted to be deleterious by the majority of the in-silico tools. These variants were further classified according to the American College Medical Genetics and Genomics (ACMG) recommendations to assess their clinical significance. Only six were found to meet the pathogenic (GBA p.L483P [p.L444P]) or likely pathogenic (GBA p.R170L [p.R131L], p.D179H [p.D140H], p.E365K [p.E326K], PINK1 p.P305A, and PRKN p.E310D) criteria. One of the major genetic risk factors for PD and a known cause of Gaucher’s disease included on the gene panel was GBA, which has a pseudogene (GBAP1) whose sequence is about 96% similar. Specialized primers that allow the amplification of only GBA are used to avoid the detection of variants in the non-functional pseudogene. Thus, the second aim of our study was to develop a method for screening and validating GBA mutations in our laboratory. Once the nested Polymerase Chain Reaction (PCR) and Sanger sequencing method was successfully optimized, 30 of our African Black PD patients were screened. To our knowledge, this is the first GBA mutation screening in PD performed within this population. Eight variants were identified of which four were predicted to be deleterious by the majority of the in-silico tools. These included three known pathogenic Gaucher’s disease-associated mutations (p.R150W [p.R120W], p.R170L [p.R131L], and p.T75del [p.T36del]) of which p.R120W is a known risk factor for PD, and one variant of uncertain significance (p.Q536* [p.Q497*]). In addition, two novel variants (p.F255L [p.F216L] and p.G517R [p.G478R]) were identified of which p.F216L was found to be common (9.9%) in controls. Furthermore, the specialized GBA primers were also used to validate all of the prioritized GBA variants identified using the gene panel. Only four of the five variants (GBA p.L483P, p.R170L, p.D179H, p.E365K) were confirmed to be in the functional gene. In conclusion, we successfully developed a method for rapid screening of the known PD genes and a technique for screening and validating GBA mutations. These methods can be used for rapid and high- throughput screening of the genetic contribution to PD in our local populations and other populations within SSA. Subsequently, these methods will enable us to identify novel candidates for validation in future functional studies. Consequently, this work will also contribute to the development of precision medicine tailored to each PD patient.
Evaluating the neuroprotective effect of curcumin on a PINK1 cell model of Parkinson's disease
(Stellebosch : Stellenbosch University, 2022-04) Chetty, Devina; Bardien, Soraya; Kenyon, Colin; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of neurons producing the neurotransmitter dopamine. Notably, despite extensive studies that have revealed numerous dysregulated processes associated with PD, fundamental gaps still exist in our knowledge of the disease pathophysiology. Our understanding of the disease includes processes such as mitochondrial dysfunction causing increased oxidative stress and energy failure, as well as misfolded protein accumulation in large inclusions. However, the spatiotemporal sequence of events leading to PD and, importantly, the initial factors that trigger disease onset remain elusive. This challenges the development of effective therapeutic strategies to prevent and cure PD. Moreover, the various side effects associated with conventional pharmaceuticals currently used to treat this disease motivate the search for a natural treatment that can avoid augmenting the suffering associated with an already debilitating disease. It is evident that mitochondrial dysfunction and oxidative stress are likely to be involved in the disease pathogenesis, therefore studying these phenomena in PD may lead to the development of more effective therapeutic strategies. Curcumin is a plant-based polyphenol that has been observed to have antioxidant properties, increase cell viability, and enhance mitochondrial function. Consequently, the aim of the present study was to create a PD cellular model and evaluate the potential protective effects of curcumin. Therefore, we sought to establish an appropriate PD model by transfecting SH-SY5Y cells with wild-type (WT) or G309D mutant PINK1 cDNA, of which the latter has been shown to upregulate dopamine and lead to cytotoxicity. Additionally, since several neurotoxins have been shown to trigger PD, the toxic herbicide paraquat was administered to complement the model with cellular damage and mitochondrial dysfunction. To confirm the model, we performed RT-qPCR to measure gene expression levels of PINK1 and tyrosine hydroxylase (TH), an enzyme in the dopamine synthesis pathway. PINK1 was significantly upregulated in the mutant, however, no significant difference in TH gene expression was observed between groups. We then sought to measure the levels of dopamine in transfected cells using liquid chromatography-mass spectrometry (LC-MS). Although no dopamine was detected using LC-MS, higher levels of phenylalanine, a precursor of dopamine, were observed in the mutant. Using this model, we sought to test the protective effects of curcumin using assays that measure cellular and mitochondrial health. A toxic paraquat concentration of 1.7 mM was chosen to elicit a 50 % decrease in cell viability for the model, while a curcumin concentration of 2.5 μM was chosen as it exhibited no toxic effects. Following the establishment of the model, four treatment groups were established for all experiments thereon: untreated control, curcumin only treatment, paraquat only treatment, and pre-treatment (curcumin treatment followed by paraquat treatment). We found that curcumin was unable to significantly rescue the paraquat-induced reduction in cell viability and mitochondrial membrane potential. The latter was significantly reduced in PINK1 transfected groups, more so in the G309D mutant, indicating the toxic effects of the mutation. Thereafter, the effects of curcumin and polycaprolactone encapsulated nanocurcumin on cell viability were compared. Formulations of curcumin including nanocurcumin are postulated to improve the stability and efficacy of curcumin. Interestingly, curcumin had a greater protective effect, whereas nanocurcumin as well as the empty nanoparticles elicited toxicity. In fact, p re-treatment with the nanocurcumin prior to paraquat treatment caused a 30 % greater loss in cell viability compared to the paraquat treatment alone. Finally, a literature review was published, exploring the potential of consistent dietary consumption of curcumin as an alternative or supplement to existing therapies. We speculate that curcumin binds to α- synuclein protein (found to accumulate in PD) and that this complex is subsequently excreted from the body via the large intestine. In this view, replacing some of the PD drugs in an individual’s treatment regime with a nutraceutical, or ‘functional food’, like curcumin may improve therapeutic benefits with fewer side effects. Considering these results and the published evidence for curcumin as a dietary ‘nutraceutical’, further studies are required to optimize curcumin treatment before advocation of its widespread use as a PD therapeutic agent. Study limitations include the use of an unverified WT plasmid and an undifferentiated cell line, which can be addressed in future work. The findings in this study are of importance as they may contribute to advancing the development of novel plant-based therapies to treat and potentially prevent this detrimental disease.
Functional characterization of sequence variants in leucine-rich repeat kinase 2 (LRRK2) and its possible interaction with the translocase of outer mitochondrial membrane (TOM) protein complex
(Stellenbosch : Stellenbosch University, 2017-03) Neethling, Annika; Bardien, Soraya; Williams, Monique Joy ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is an incurable neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the midbrain of affected individuals. Both environmental and genetic factors contribute to the aetiology of PD, with more than a dozen genes implicated in disease development. Yet, the exact mechanisms by which each gene (and mutation) contribute to the pathophysiology of PD remain to be elucidated. Mitochondrial dysfunction is a recurring theme associated with neurodegeneration and recently the translocase of outer mitochondrial membrane (TOM) complex, which plays a role in the maintenance of healthy mitochondria, has been implicated in PD pathogenesis. The TOM complex, consisting primarily of TOM20, TOM22, TOM40 and TOM70, is involved in the translocation of nuclear-encoded proteins into the mitochondria where they are needed for normal mitochondrial function. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant PD and the LRRK2 protein has been associated with numerous cellular functions including mitochondrial homeostasis, the autophagy/lysosomal pathway, cell signalling and synaptic vesicle trafficking. The most common PD-causing mutation, G2019S, is located in the kinase domain of LRRK2 and has consistently been shown by various researchers to increase kinase activity. Recently, members of our group identified a novel variant (Q2089R) in LRRK2. This variant is also located in the kinase domain of LRRK2 and requires further investigation to determine its pathogenicity. The aim of the present study was to functionally characterize wild type (WT) and mutant LRRK2 (G2019S and Q2089R) under basal and stress [Carbonyl cyanide m-chlorophenyl hydrazone (CCCP)] conditions and also to determine whether WT LRRK2 interacts with the TOM complex. The frequency of LRRK2 Q2089R in South African PD patients and controls was determined using a custom Taqman™ SNP genotyping assay. In silico analysis of the effect of the amino acid substitution from Glutamine (Q) to Arginine (R) was performed using various prediction tools. Two cellular models of PD including (1) HEK293 cells transfected with WT and mutant LRRK2 constructs and (2) patient-derived dermal fibroblasts were used for the functional studies. LRRK2 mutant constructs were generated using site-directed mutagenesis in pcDNA-DEST53, a mammalian expression vector. We obtained skin biopsies from individuals harbouring G2019S, Q2089R or WT LRRK2 and cultured dermal fibroblasts as an ex vivo model of the disorder. We investigated the kinase activity of LRRK2 using autophosphorylation of Serine 1292 and Western blot analysis. Metabolic activity was measured using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and mitochondrial membrane potential (MMP) was measured using the JC-1 fluorochrome and flow cytometric analysis. Mitochondrial and glycolytic respiration experiments were performed using the Seahorse XF Analyzer and mitochondrial DNA copy number was determined by quantitative real-time PCR (qRT-PCR). Autophagic markers, LC3 II and P62, were detected using Western blot analysis. Co-localization experiments of WT LRRK2 and the TOM complex was performed using confocal and super resolution structured illumination microscopy (SR-SIM), while protein interactions were investigated using co-immunoprecipitation and Western blot analysis. The frequency of Q2089R was found to be 0.2% (1/493) in PD patients and 0.1% (1/776) in controls. Multiple in silico tools predicted the Q to R substitution to possibly be pathogenic [‘deleterious’ (CADD score=24.1, ‘possibly damaging’ (Polyphen) and ‘disease causing’ (Mutation Taster)]. The LRRK2 constructs were successfully generated and fibroblasts were successfully cultured. Notably, in HEK293 cells, we found that Q2089R almost completely abolished autophosphorylation activity of LRRK2 (p=0.026). Q2089R-carrying cells also exhibited a decrease in metabolic activity in HEK293 cells (p=0.016) and fibroblasts (p<0.05). In addition, in both cell types a significantly decreased MMP was observed [p=0.043 and p=0.009 for HEK293 cells and fibroblasts (under stress), respectively]. Furthermore, Q2089R-carrying fibroblasts showed an increase in basal respiration (p=0.012), proton leak respiration (p=0.0001), maximal respiration (p<0.0001) and spare respiratory capacity (p<0.0001), while ATP-coupling efficiency (p=0.0014), glycolytic reserve (p=0.006) and glycolytic capacity (p=0.007) was significantly reduced. In both models, Q2089R cells exhibited an increase in autophagosome pool size (p<0.05 for LC3 II and p<0.05 for P62). In the case of G2019S, a marked increase in autophosphorylation activity (p=0.019) was observed in HEK293 cells, which is in accordance with many previous studies. Decreased metabolic activity (p=0.021) and MMP (p=0.038) were also observed in these cells. G2019S-carrying fibroblasts displayed reduced metabolic activity (p<0.05) and increased basal respiration (p=0.029), ATP-linked respiration (p=0.029), glycolysis (p=0.001) and autophagosome pool size (p=0.022 for LC3 II). The MMP of these fibroblasts showed a non-significant trend for a decrease under stress conditions (p=0.057). Interestingly, WT LRRK2 was shown to co-localize and co-immunoprecipitate with a protein complex containing subunits TOM22, TOM40 and TOM70 but not TOM20 under basal conditions. Under stress conditions, an association between LRRK2 and TOM20 was observed while the association between LRRK2 and the complex containing TOM22 and TOM70 increased. Finally, from our findings and the published literature, we propose a model for the involvement of LRRK2 (WT and Q2089R) in cellular functioning and cell death. This involves the loss of kinase activity and association with the TOM complex, which ultimately links LRRK2 with mitochondrial (dys)function, mitochondrial biogenesis and the autophagy/lysosomal pathway. In conclusion, we characterized a functional variant in the kinase domain of LRRK2 and propose additional functions for this large multi-domain protein. This study also provides evidence for a novel association between LRRK2 and the TOM complex. Interestingly, our findings challenge the notion that it is only increased LRRK2 kinase activity that is implicated in PD pathogenesis. We acknowledge, however, that our findings are preliminary and that further validation studies are necessary to validate our results and hypothesis. Future targeted experiments on LRRK2 are needed in order to unravel the complex pathobiology and to decipher the sequence of events that lead to development of PD in susceptible individuals.
Identification of components of turmeric as potential therapeutic agents to slow the progression of neurodegeneration in Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2022-04) Jansen van Rensburg, Zune; Bardien, Soraya; Kenyon, Colin; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurological disorder associated with severe loss of dopaminergic neurons in the substantia nigra. These neurons are particularly vulnerable due to increased iron and decreased antioxidant levels with aging, constant exposure to reactive oxygen species (ROS), and the presence of neurotoxic compounds. A literature review was compiled and published, in which we postulate that ROS, iron, alpha-synuclein protein (α-syn), and neuromelanin form a toxic feedback loop in individuals with PD. This feedback loop is theorised to be an early trigger culminating in neuronal death and subsequent spread of the disease to neighbouring neurons. Consequently, antioxidants and iron-chelators may be important therapeutic agents to target the accumulated ROS and iron in these neurons. Turmeric is an attractive therapeutic candidate since it has well-established antioxidant properties and may chelate iron. This study aimed to determine which components of turmeric have strong antioxidant and iron-chelating properties that could potentially protect against dopaminergic neuronal degeneration in PD. A crude extract was obtained from purchased turmeric using Soxhlet extraction. Subsequently, the crude extract was separated into different compounds using thin-layer chromatography (TLC). Mass spectrometry (MS) was used to identify the eluted compounds and a selected number of compounds were further analysed with liquid chromatography-mass spectrometry (LCMS). After identifying the compounds, four were chosen to test their total antioxidant capacity and iron chelation ability by comparing these compounds to Trolox (a known antioxidant) and ethylenediaminetetraacetic acid (EDTA) (a strong iron chelator), respectively. Finally, a human neuronal cell line, SH-SY5Y, was used to evaluate the effect of the extracted compounds on cell viability, through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We successfully obtained a crude extract from turmeric powder after Soxhlet extraction. A total of 34 bands were initially eluted from the TLC plate, with the experiment performed in duplicate. After MS, only 15 of the 34 samples had identifiable compounds. Through LCMS, four compounds were identified with confidence namely bis-demethoxycurcumin, demethoxycurcumin, curcumin, and aromatic (ar)-turmerone. The antioxidant assays showed that curcumin had the strongest antioxidant activity while ar-turmerone had the weakest. Analysis of the iron-chelating activity of these compounds found contradictory results. However, with spectrophotometric observation and a commercially validated kit, the curcuminoids were shown to have strong iron-chelation potential. Unexpectedly, ar-turmerone also showed iron-chelation potential albeit to a lesser extent compared to the curcuminoids. The exploratory analysis with the SH-SY5Y cells showed that bis-demethoxycurcumin increased cell viability by 152%, while ar-turmerone decreased it by 73.5%, versus the vehicle control. These promising findings warrant further study using more targeted techniques. Identifying new therapies for PD is of utmost importance since the current treatments only treat the symptoms and do not address the pathobiology of neuronal loss. Natural compounds extracted from plants may be particularly useful for the design of new treatment modalities since they may be more cost- effective and have fewer side effects than synthetic medicines.
Identification of novel Parkinson’s disease genes in the South African population using a whole exome sequencing approach
(Stellenbosch : Stellenbosch University, 2016-03) Glanzmann, Brigitte; Bardien, Soraya; Gamieldien, Junaid; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH SUMMARY: Parkinson’s disease (PD) is a progressive and severely debilitating neurodegenerative disorder that is characterised by a range of motor symptoms and the selective loss of dopaminergic neurons in the substantia nigra. While the aetiology of PD remains poorly understood, it is hypothesised to involve a combination of various environmental, genetic and cellular factors that independently or collectively contribute to neurodegeneration and ultimately disease. To date, a number of genes including Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2 and VPS35 that have been directly associated with disease and investigations of their functions have provided significant insights into the pathobiology of PD. However, these genes do not play a significant role in the South African PD cohort and for this reason, novel genes and pathogenic mutations must be investigated and identified. This will aid in early diagnosis of patients and also ultimately for the design of more effective therapeutic strategies to treat this debilitating and poorly understood chronic systemic disorder. The present study aimed to identify novel PD-causing mutations in the South African Afrikaner population using a genealogical and whole exome sequencing (WES) approach.. The Afrikaner are unique to South Africa and are known to have undergone a bottleneck in the 1800s which has led to genetic founder effects for a number of different disorders in this particular group. Additionally, we further aimed to determine whether the identified putative disease-causing mutation(s) could be attributed to the development of PD in other South African ethnic groups. A total of 458 patients were recruited, of which 148 were self-identified as Afrikaner. From these, a total of 48 Afrikaner probands were subjected to extensive genealogical analyses and 40 of them could be traced back to a single common couple. For this reason, it was hypothesised that the disorder in these patients may be due to a genetic founder effect. The use of a whole genome SNP array confirmed the relatedness of the individuals to varying degrees (8 to 12 generations back) and subsequently three of the probands and one affected sibling were selected for WES. The selected individuals were sequenced using the Illumina Genome Hiseq 2000TM and approximately 78 000 variants were identified for each individual. Numerous bioinformatics tools were used to scrutinize the variants but none were able to produce a candidate list of plausible disease-causing variants. All variants identified were either present at high frequency, did not co-segregate with the disorder or were artefacts. In order to facilitate and expedite the variant prioritisation process, a novel method for the filtration of WES data was designed in-house. This strategy named TAPER™ (Tool for Automated selection and Prioritisation for Efficient Retrieval of sequence variants) implements a set of logical steps by which to prioritise candidate variants that could be pathogenic. It is primarily aimed at the support of resource-constrained scientific environments with limited bioinformatics capacity. As a proof of concept various independent WES datasets for PD, severe intellectual disability and microcephaly as well as ataxia and myoclonic epilepsy were used, and TAPER™ was able to successfully prioritise and identify the causal variants in each case. Through the use of TAPER™, two putative candidate variants in SYNJ1 and USP17 were identified. The homozygous V1405I variant in SYNJ1 was found only in the affected sibling pair and in none of the 458 patients and 690 control individuals that had been screened. This variant is predicted to be deleterious across multiple platforms and has a CADD score of 29.40 and may alter synaptic vesicle recycling. The homozygous C357S variant in USP17 was found in 18/458 probands (12 Afrikaner, two white and four mixed ancestry) but was identified in 0.14% of the controls (1/184 Afrikaner, 0/160 white, 0/180 mixed ancestry and 0/160 black). This variant is also anticipated to be deleterious across multiple platforms and has a CADD score of 34.89. In summary, the results of the present study reveal that PD in the 40 South African Afrikaner patients studied is not due to a founder effect, but highlights two variants of interest for future studies. Further work is necessary to analyse both of these variants and to assess their possible effect on protein structure and function. The discovery of novel PD-causing genes is important as this allows for the generation of disease-linked protein networks, thereby facilitating identification of additional disease genes and subsequently providing insights into the underlying pathobiology. Moreover, this knowledge is critical for the development of improved treatment strategies and drug interventions that will ultimately prevent or halt neuronal cell loss in susceptible individuals. Although the present study did not conclusively identify a novel PD-causing gene, it does provide a solid foundation for future work in our laboratory in the challenging and rapidly evolving research area of WES and bioinformatics, and its application to studies on PD.
Identification of parkin interactions: implications for Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2015-12) Haylett, William Lloyd; Bardien, Soraya; Kinnear, Craig; Carr, Jonathan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a progressive and debilitating neurodegenerative disorder, characterized by a distinct motor phenotype and the selective loss of dopaminergic neurons in the substantia nigra. While the etiology of PD is not fully understood, it is thought to involve a combination of different genetic, cellular and environmental factors that independently or concurrently contribute to neurodegeneration. To date, several PD-causing genes have been identified, and investigations of their function have provided novel insights into the pathobiology of disease. Particularly interesting among the known PD genes is parkin, mutations in which are the most common genetic cause of early onset PD. Parkin is an E3 ligase that ubiquitinates protein substrates and targets such substrates for degradation via the ubiquitin proteasome system (UPS). Therefore, the loss of parkin may result in the deleterious accumulation or dysregulation of parkin substrates and neurotoxicity. Parkin’s enzymatic activity has also been implicated in the maintenance of mitochondrial health, and mitochondrial dysfunction is commonly reported in cellular and animal models of parkin deficiency. This study aimed to investigate parkin and its role in PD on various levels. Initially, genetic screening approaches were used to assess the contribution of parkin mutations to PD in a group of 229 South African patients. It was concluded that parkin mutations are rare in the South African PD population, being present in only seven (3.1%) patients in the study group. Interestingly, this study identified two of only three Black African PD patients with mutations in a known PD-causing gene to date. The low frequency of known PD genes raises the interesting possibility that the unique South African ethnic groups may harbor mutations in novel PD-causing genes. Although many parkin-interacting proteins have been identified in the literature, it is anticipated that novel, pathologically-relevant parkin substrates remain to be discovered. Hence, this study used a yeast two-hybrid (Y2H) approach to identify novel parkin interactions. This yielded 29 putative parkin interactors, of which four, namely ATPAF1, SEPT9, actin and 14-3-3η, were prioritized for verification by co-localization and co-immunoprecipitation experiments. Interestingly, two of the parkin interactors (ATPAF1 and SEPT9) were found to accumulate in the absence of parkin, supporting their role as authentic parkin substrates. The identification of these two intriguing proteins implicates parkin in the regulation of mitochondrial ATP synthase assembly and septin filament dynamics, which may be of significant relevance to our understanding of processes underlying neurodegeneration. Moreover, it was aimed to assess various markers of mitochondrial function in a parkin-deficient cellular model, as previous studies had reported conflicting results regarding mitochondrial impairments in patient-derived cells with parkin mutations. Hence, dermal fibroblasts were obtained from PD patients with homozygous parkin mutations, after which cell growth and viability, mitochondrial membrane potential, respiratory rates and the integrity of the mitochondrial network were assessed. Surprisingly, it was found that cell growth was significantly higher in the parkin-mutant fibroblasts compared to wild-type controls fibroblasts under basal conditions (p=0.0001), while exhibiting a greater inhibition of cell growth in the presence of the mitochondrial toxin CCCP (p=0.0013). Furthermore, whereas the mitochondrial networks of patient-derived fibroblasts were more fragmented than controls (p=0.0306), it was found that mitochondrial respiratory rates were paradoxically higher in the patients (p=0.0355). These unanticipated findings are suggestive of a compensatory response to the absence of parkin. The parkin-deficient cellular model was also used in a pilot study of the functional effects of vitamin K2 treatment, which has recently been identified as a promising PD therapeutic modality. It was found that treatment with vitamin K2 resulted in more interconnected mitochondrial networks (p=0.0001) and enhanced respiratory rates (p=0.0459) in both parkin-mutant and wild-type control cells. While these results need to be studied further, it suggests that vitamin K2 supplementation may be of use as a general promoter of mitochondrial integrity and function. In conclusion, this dissertation highlights some novel interactions of the parkin protein and some interesting phenotypes of parkin deficiency. It is hoped that further investigation of parkin and its role in PD will, ultimately, aid in the development of therapeutic strategies to treat this debilitating and poorly-understood disorder.
An investigation into the molecular aetiology of Parkinson's disease in South African patients
(Stellenbosch : Stellenbosch University, 2013-03) Glanzmann, Brigitte; Bardien, Soraya; Carr, Jonathan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson's disease (PD) is a severely debilitating neurodegenerative disorder that results in motor circuit dysregulation and ultimately, causes impairment of movement. This condition is due to the selective degradation of the dopaminergic neurons in the substantia nigra pars compacta in the midbrain, which subsequently results in the pathological symptoms namely bradykinesia, resting tremor, postural instability and rigidity. It was initially hypothesized that individuals who develop PD were exposed to an environmental trigger(s) that caused the onset of the disease, but more recently, a significant genetic component, coupled to environmental factors have been implicated in disease pathogenesis. Currently, there are eight genes (Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2, EIF4G1 and VPS35) that have been directly implicated in PD. Worldwide, the prevalence of neurodegenerative disorders is increasing as populations are living longer. In Europe, Canada and USA, it has been projected that the prevalence of PD may increase by a factor of two between 2010 and 2050; approximately a 92% increase. In Tanzania (the only study done in sub-Saharan Africa) an even larger increase of 184% between 2005 and 2025 is predicted, due to the fact that the speed of populations ageing in developing countries, will exceed that of developed countries. Research into the causes and risk factors underlying neurodegenerative disorders such as PD is therefore urgently needed for policy makers and governments in developing nations to take appropriate action to deal with this impending health care problem. The aim of the present study was to investigate the molecular aetiology of a group of South African PD patients. A total of 262 patients from various ethnic backgrounds were recruited for the study, and 35% had a positive family history of PD with the average age at onset (AAO) being 54.3 years of age (SD = 12.5 years). Mutation screening of the known PD genes (Parkin, PINK1, LRRK2, SNCA and DJ-1) was performed using high resolution melt and Sanger sequencing. Genotyping was done using fluorescently-labelled PCR primers followed by electrophoresis on an ABI 3130xl genetic analyser (for CTG repeats in JPH3) and with a KASP™ Genotyping Assay (for a 16bp indel in DJ-1). In order to identify a novel PD-causing gene, whole exome sequencing (WES) was conducted on three Afrikaner probands with an Illumina Genome Hiseq 2000TM and the sequences were aligned using the NCBI Human Reference Genome 37.2. The BORG (Bio-Ontological Relationship Graph) semantic database, which models the relationship of human and model organism genes to functions, pathways and phenotypes, was used to filter and prioritise genetic variants shared between the three PD exomes. It was determined that the known PD genes do not play a significant role in disease pathogenesis in the South African patients as only 15/262 (5.7%) of the patients harboured mutations: seven in Parkin, one in PINK1, six in LRRK2 and one in SNCA. Only one of the patients harboured a 16bp indel variant at the transcription start site of DJ-1. None of the Black PD patients had pathogenic repeat expansions in JPH3 thereby excluding Huntington disease-like 2 as a cause of the disease phenotype. Genealogical analysis revealed that six of the apparently unrelated Afrikaner PD probands were related to a founder couple that immigrated to South Africa in the 1600s which suggests that there is a possible founder effect for the disease. Bioinformatics analysis of WES data on three of the probands identified 21 variants in 12 genes that were present in all three PD exomes and fulfilled various criteria. Sanger sequencing was used for verification of five variants and of these, two (in CDC27 and NEDD4) were found to be artefacts. The remaining three (in HECDT1, TBCC and RNF40) were excluded based on the lack of cosegregation with disease and the high frequency of the allele in controls. Further work is necessary to verify the presence of the remaining sixteen variants and to characterise each of them for their possible pathogenicity. The discovery of novel PD-causing genes is important as this may shed light on the pathways or processes that are involved. A current hypothesis implicates the lysosome-dependent pathway as a unifying biochemical pathway that can account for the phenotypic spectrum within PD. Notably, although Mendelian forms are thought to account for only about 10- 15% of cases, the study of Mendelian inherited variants is likely to provide insight into the pathophysiology of the more common sporadic form of this condition. Dissecting the key molecular mechanisms underlying PD will provide critical information for improved treatment strategies and drug interventions that will ultimately prevent or halt neuronal cell loss in susceptible individuals.
An investigation into the role of mitochondrial dysfunction in South African Parkinson’s disease patients
(Stellenbosch : Stellenbosch University, 2012-12) Van der Merwe, Celia; Bardien, Soraya; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental (such as exposure to pesticides and neurotoxins) and genetic factors. A number of PD-causing genes have been found including SNCA, LRRK2, EIF4G1 and VPS35 (for autosomal dominant forms of PD) and parkin, PINK1, DJ-1 and ATP13A2 (for autosomal recessive forms of PD – arPD). Mutations in the parkin gene are the predominant cause of arPD. Parkin plays a role in the ubiquitin-proteasomal system which degrades damaged and unwanted proteins in the cell and it is also thought to be involved in maintaining healthy mitochondria. Numerous studies have implicated mitochondrial function in the pathogenesis of PD. Therefore the aim of the present study was to investigate the role of mitochondrial dysfunction in PD patients with parkin-null mutations. Four South African PD patients, each harbouring two parkin-null mutations, were recruited for this study. A muscle biopsy was performed for analysis of mitochondrial morphology using histology and transmission electron microscopy (TEM). Skin biopsies were taken, from which fibroblasts were cultured. These fibroblasts were used in i) mitochondrial morphological assessments using TEM, ii) mitochondrial network analysis, iii) functional studies via ROS measurement and iv) analysis of the proteome using a LTQ Orbitrap Velos mass spectrometer. In addition, RNA was isolated from peripheral blood samples for gene expression studies using the RT² Profiler PCR Array (SABiosciences, USA) and the RT² PCR Primer Assay (SABiosciences, USA). Heterozygous family members (carriers) and wild-type controls were also recruited for this study. Results from the histological and TEM analysis from the muscle biopsy observed subtle mitochondrial changes including the presence of type II fibres, atrophic fibres, the presence of lipids, and wrinkling of the sarcolemmal membrane. Enlarged mitochondria were also observed in one patient. TEM analysis on the patient’s fibroblasts observed an increase in the number of electron dense vacuoles, speculated to be autolysosomes. The mitochondrial network in two of the patients’ fibroblasts showed fragmented and dot-like networks which are indicative of damaged mitochondria. An increase in mitochondrial ROS levels was observed in three of the four patients. Expression studies found down-regulation of 14 genes from four of the five mitochondrial complexes and a total of 688 proteins were found only in the control and not in the patient fibroblasts. Some of these proteins are known to be part of the ‘mitochondrial dysfunction’ pathway. Taken together, these results indicate that the absence of parkin results in a number of mitochondrial alterations. Based on these findings, a model of PD was proposed: It is speculated that when parkin is absent, electron transport chain complex genes are down-regulated. This results in impaired oxidative phosphorylation, causing an increase in the production of mitochondrial ROS and subsequent oxidative stress. Mitochondria are then damaged; resulting in the fragmentation of the mitochondrial network. The impaired mitochondria are thus tagged for degradation, causing the recruitment of autolysosomes which engulf the mitochondria via mitophagy. Ultimately, as the compensatory mechanisms fail, this triggers the consequential cascade of cellular apoptotic events. This study has elucidated the effect of parkin on the mitochondria, and can act as a ‘stepping stone’ towards future development of therapeutic strategies and/or biochemical markers that will benefit not only patients with PD but also other neurodegenerative disorders.
Investigation of differential gene expression in Parkinson's disease patients: A whole transcriptome approach
(Stellenbosch : Stellenbosch University, 2016-12) Borrageiro, Genevie; Bardien, Soraya; Hemmings, Sian; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH SUMMARY: Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. Dopaminergic neuronal loss results in motor symptoms such as resting tremor, bradykinesia, rigidity and postural instability. Although several PD-causing genes have been identified, the process(es) that lead to progressive neuronal loss is poorly understood. Therefore, PD research efforts have turned to genome-wide next generation sequencing technologies to provide clues to the etiology. The focus of this study was to investigate the entire transcriptome in South African patients with PD (particularly in the under-studied mixed ancestry population) to identify biological pathways that may shed light on the pathobiology of PD. A total of 40 study participants (20 patients and 20 controls) were recruited for the study. The PD patients were recruited from the Movement Disorders clinic at Tygerberg Hospital in Cape Town, South Africa and had to meet the UK Parkinson’s disease Society Brain Bank Diagnostic Criteria for PD. All individuals were from the South African mixed ancestry ethnic group which is a unique admixture of Khoisan, Black, Caucasian, and Asian populations. Copy number variation (CNV) detection in known PD genes (SNCA, PARK2 (Parkin), UCHL1, PINK1, PARK7, LRRK2, GCH1 and ATP13A2 and two point mutations (SNCA A30P and LRRK2 G2019S) was determined using the Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Total RNA was extracted from whole blood samples and RNA-Sequencing was performed at NXT-Dx (Gent, Belgium) on the Illumina HiSeq® 4000. Bioinformatic analysis was performed using Partek® Flow® software. DAVID and Ingenuity Pathway Analysis (IPA) were used for enrichment analysis and to identify possible biological pathways involved. One candidate gene of interest was selected for further verification by quantitative real-time PCR (qPCR). MLPA analysis revealed a heterozygous exon 2 deletion in PARK2 in one patient however a second mutation in this gene was not identified. Therefore, all patients potentially have idiopathic PD and were analysed as one group for RNA-Seq. All samples produced good quality reads, as determined using FastQC (scores > 39) and on average 95.3% of the transcripts generated for each sample could be aligned to the reference genome (hg19). Bioinformatics analysis resulted in a candidate gene list of 132 differentially expressed genes. Analysis of these genes using IPA identified five significant dysregulated canonical pathways which included regulation of eIF4 and p70S6K signaling, EIF2 signaling, LPS/IL-1 mediated inhibition of RXR function, xenobiotic metabolism signaling and maturity onset diabetes of young (MODY) signaling which contribute to PD pathogenesis. A possible link between CEBPA, PGC-1α and PD was also highlighted as both genes were in the prioritized candidate list and IPA gene network. CEBPA has been shown to interact with known PD genes and PGC-1 is a transcriptional regulator of mitochondrial biogenesis a key pathway linked to PD. CEBPA was prioritized for replication studies using qPCR and was found to be significantly down-regulated in patients which contrasted with the RNA-Seq results. The present study revealed candidate genes, CEBPA and PGC-1α which could potentially be involved in the development of PD and a possible link to diabetes through mitochondrial mechanisms. We speculate that the increased PGC-1α levels observed is in response to loss of mitochondria, which leads to increased levels of reactive oxygen species that ultimately result in death of dopaminergic neurons. Our study illustrates that the use of RNA-Seq in combination with IPA is a powerful approach that may reveal candidate genes and biological pathways involved in PD. A better understanding of the molecular mechanisms underlying PD is critical for development of therapeutic modalities in order to prevent, stop or reverse dopaminergic neuronal loss in PD patients.
Investigation of Neurexin 2 as a candidate for Parkinson's Disease
(Stellenbosch : Stellenbosch University, 2022-09) Cuttler, Katelyn; Bardien, Soraya; Cloete, Ruben; Farrer, Matthew; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder which primarily affects movement and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). There is no cure for the disorder and current drug treatments often have severe side effects. Several pathogenic variants have been implicated in PD, in various genes including SNCA, LRRK2, PRKN, and PINK1. However, these variants have mainly been found in individuals of European ancestry. In Sub-Saharan Africa (SSA), studies done on the genetic aetiology of PD have shown that these known pathogenic variants are only minor contributors to the aetiology. Since SSA is expected to face a surge in age-related disorders, such as PD, due to the gradual improvement in quality of life and increased life expectancy, it is important to study the disorder in these populations. To this end, we have recruited individuals with PD from the South African population for genetic studies. One of the probands recruited had a family history of PD and also had several PD affected and unaffected family members. This family was designated ZA253. Therefore, we decided to perform whole exome sequencing on three of the affected individuals in an attempt to elucidate the genetic aetiology of their disorder. Variants that were novel or rare (MAF < 1%), non synonymous, heterozygous, and shared amongst the three individuals were prioritised. These were found in the CCNF, CFAP65, NRXN2, RTF1, and TEP1 genes. After screening unaffected members and ethnic-matched controls, as well as performing pathway and expression analysis and functional predictions of the effect of the variant on the translated protein, the p.G849D variant in NRXN2 (neurexin 2) was prioritised for further study. First, we performed molecular dynamic (MD) simulations after constructing a homologous model of the human NRXN2α protein. These simulations showed that the variant had a destabilizing effect on the protein structure and resulted in an extended conformation of the laminin/neurexin/sex-hormone binding domain 6 (LNS6), which is responsible for binding to other proteins. Thereafter, we performed a literature search on the neurexin gene family to determine if they are good candidate genes for PD. We found that there is a well-established role of neurexins in neuropsychiatric disorders, such as autism spectrum disorders and schizophrenia, as well as evidence of a role for neurexins in neurodegenerative disorders, such as Alzheimer’s disease and PD. Therefore, we concluded that NRXN2 is a good candidate gene for further examination using functional studies. Functional studies were then performed using a cDNA overexpression model in SH-SY5Y neuroblastoma cells to analyze the effect of the variant in an in vitro model of PD. First, we used assays to examine the effect of the mutant NRXN2α protein on cell death, mitochondrial function, and reactive oxygen species (ROS) production. We found that overexpression of the mutant protein had a negative effect on all of these aspects and therefore concluded that the mutant NRXN2α could induce a toxic feedback loop of mitochondrial dysfunction, increased ROS generation and increased neuronal cell death. Consequently, we performed proteomics analysis on the same model to determine how overexpression of NRXN2α affects cellular pathways. Interestingly, overexpression of the wild type protein led to the enrichment of proteins involved in neurodegenerative pathways, providing preliminary evidence that NRXN2α could be involved in these pathways. Overexpression of the mutant protein led to the decline in proteins involved in ribosomal functioning. Since NRXN2α is a synaptic protein, it is possible that the variant affects synaptic translation. Indeed, dysregulated synaptic translation has been linked to altered mitochondrial physiology. Therefore, we hypothesized that dysregulated synaptic translation and mitochondrial dysfunction are linked and act together to result in neuronal death. The last part of the study examined the effect of the variant on the synaptic function of NRXN2α. We first used MD simulations to examine the variant’s effect on the binding of NRXN2α to a known binding partner, neuroligin 1 (NLGN1). In synapses, neurexins bind to neuroligins to facilitate synaptic transmission and maintenance. The results of the simulations suggest that the variant may be able to disrupt this interaction. Thereafter, we stained synaptic markers in vitro, in differentiated SH-SY5Y cells, to determine whether overexpression of the mutant protein affects synapse formation and synaptic transmission. We found an increase in the levels of both markers possibly indicating that there is increased synapse formation resulting in increased transmission between synapses. Since the MD simulations showed that the variant could disrupt neurexin neuroligin signalling, we propose that this increase in transmission is a compensatory mechanism and suggest that, over time, this response would strain the synaptic maintenance systems and eventually lead to neurodegeneration. In conclusion, our findings have indicated that a variant in NRXN2α may be linked to mitochondrial and synaptic dysfunction that may eventually lead to neurodegeneration. However, further targeted experiments in other PD models are required in order to prove these findings. Nevertheless, it is important to look at the genetics of PD in understudied populations as this may lead to the discovery of new genes and disease mechanisms underlying this disorder. Therefore, studies such as these can help to shed light on this debilitating disorder.
Investigation of the genetic aetiology of aminoglycoside-induced hearing loss in South African populations
(Stellenbosch : University of Stellenbosch, 2009-12) Human, Hannique; Bardien, Soraya; De Jong, Greetje; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is permanent hearing loss and this is thought to have a significant genetic component. To date, at least six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced hearing loss. It is imperative that we investigate the frequency of these mutations in our populations and determine whether certain sub-groups are at increased risk. The aim of the present study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in the South African population. A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen for six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C. A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who were receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA sequencing, respectively. A total of 97 family members from a South African family known to harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition, mitochondrial functioning in the presence of different streptomycin drug concentrations, in transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software. We successfully developed a robust and cost-effective method that detects the presence of all six mutations simultaneously. The method worked equally well on both blood (from adults) and buccal swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high frequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5% of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation was III detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since they were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls. Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eight MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3 and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved across five species from human to frog, and the four variants in the protein coding genes resulted in missense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive (on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversible hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin were (compared to wild type) observed but this was not statistically significant (p-value: 0.615- 0.999). The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. However, future studies with larger numbers of samples are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. Our data suggests that the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the high frequencies observed in controls (>1%). The identification of the first novel variants within protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss holds great possibilities with regards to the identification of a second gene involved in drug induced hearing loss. Future studies where the possible effect of these variants on the normal functioning of these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive members of the family were given genetic reports and counseled about their risk and that of their children for developing hearing loss due to aminoglycoside use. The development of a rapid and cost-effective genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is of critical important in a low-resource country like South Africa where, despite their adverse sideeffects, aminoglycosides will be continue to be used routinely and are accompanied with very limited or no audiological monitoring. Future studies and greater public awareness is therefore needed to address this serious problem.
An investigation of the neuroprotective properties of curcumin by monitoring autophagy and apoptosis
(Stellenbosch : Stellenbosch University, 2021-03) Bekker, Minke; Bardien, Soraya; Loos, Ben; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder, with a rapidly increasing prevalence and incidence throughout the global population. As current PD therapies only rely on symptomatic treatments, there is an urgent need for the development of neuroprotective therapies, to slow or halt progressive neuronal loss. This strategy, however, is dependent on a better understanding of the pathobiology and pathways underlying PD. Multiple causative factors have been postulated to be involved in the pathobiology of PD, with defective autophagy and the subsequent upregulation of apoptotic cascades hypothesised as main contributing factors. Although the crosstalk between these pathways has become evident, the exact role of autophagy and apoptosis regarding the neuronal fate in PD remains controversial. Curcumin is a polyphenolic plant compound that has been observed to mediate autophagy and apoptosis. Consequently, the aim of the present study was to investigate the potential of curcumin as a PD therapy, and its effect on autophagy and apoptosis in a PD model. Study objectives were set out to achieve this aim, with the first objective being to better understand the interplay between autophagy and apoptosis, by performing a literature review to identify molecular components involved in both pathways. The second objective was to establish an appropriate PD model for the experimental part of the study. The cellular model selected was treatment with a neurotoxin, paraquat, in a commercially available SH-SY5Y neuroblastoma cell line. This objective was executed through performing 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide and CyQUANT assays to determine appropriate concentrations for curcumin and paraquat treatment. The third objective was to assess the effect of curcumin in the autophagic pathway, through performing western blotting to measure levels of the autophagic LC3-II, p62 and LAMP2 proteins. Lastly, the fourth objective was to assess the effect of curcumin in the apoptotic pathway in an autophagy-inhibited model. This was executed through performing western blotting to measure the levels of the autophagic Beclin-1 and apoptotic Bax proteins. The literature review pinpointed Bcl-2, JNK, p38 and Akt as proteins with the potential to mediate the activation and/or inhibition of both pathways. It was concluded that novel PD therapies could target these connecting molecular components to mediate the balance between autophagy and apoptosis. Using our PD cellular model, our findings revealed a trend for curcumin treatment to increase the overall abundance of LC3-II protein levels, while also sustaining the protein levels of LAMP2 in the presence of the autophagic inhibitor bafilomycin A1. Curcumin treatment also facilitated the efficient clearance of p62 protein, in comparison to a blunted clearance of p62 protein observed upon paraquat treatment. These findings reflect the potential of curcumin to induce autophagy while also maintaining the balance of autophagic flux. Additionally, a trend for curcumin to increase Beclin- 1 and decrease Bax protein levels in the presence of the autophagic inhibitor, 3-methyladenine, was observed. Curcumin therefore exhibited the potential to counteract autophagic inhibition while also demonstrating anti-apoptotic properties, independently from autophagy. Considering these results and published evidence of curcumin-mediated changes in the expression of Bcl-2, JNK and, Akt, it is suggested that curcumin treatment exhibits the potential to facilitate the interplay between autophagy and apoptosis. Study limitations include the challenge of interpreting autophagic flux through western blotting and the use of an undifferentiated cell line and can be addressed in future work. The findings in this study are of importance, as they may contibute to a better understanding of the pathobiology of PD which could advance the development of novel therapies, to potentially ameliorate the detrimental nature of this disorder.
A molecular investigation of a mixed ancestry family displaying dementia and movement disorders
(Stellenbosch : Stellenbosch University, 2008-12) Abrahams-Salaam, Fatima; Bardien, Soraya; Carr, Jonathan; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a movement disorder which affected a number of individuals across three generations. The initial symptoms included personality changes and tremors that escalated to severe dementia and eventually a completely bedridden state. It was determined that the mean age at onset was in the third decade of life and affected individuals died within 10-15 years after the onset of symptoms. The aim of the present study was to elucidate the genetic cause of the disorder in this family and to further investigate the patho-biology of the disease. Mutations that could possibly cause the observed phenotype in this family were screened for. These included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and direct sequencing were used to detect possible mutations while genotyping on an ABI genetic analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase chain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3) gene was performed to provide information on the expression profile of this gene. After the exclusion of several genetic loci it was shown that this family had HDL2. This is a rare disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3 gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry data suggested that the family member was most likely of South African Mixed Ancestry making this the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the repeat distribution amongst three South African sub-populations in order to determine whether there was a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease. The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeats between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to whether Black Africans harboured larger repeats that predisposes them to developing HDL2. The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly, this repeat is not present in the mouse homologue of the gene although the rest of the genomic sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR was attempted. These experiments produced inconclusive results and required further optimisation. Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained from HDL2-affected individuals would be of interest. The present study identified the first Mixed Ancestry family with HDL2. This family will now be able to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of this study provided independent confirmation of characteristics of the mutated gene. More research on HDL2 will be crucial in understanding the pathogenesis of this disease.
Screening for disease-causing genes in black South African patients with Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2015-04) Ntsapi, Claudia; Bardien, Soraya; Boolay, Sihaam; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Please see fulltext for abstract
The utilisation of whole exome sequencing to dissect the genetic aetiology of familial Parkinson’s disease in a South African Afrikaner family
(Stellenbosch : Stellenbosch University, 2019-04) Sebate, Boiketlo; Bardien, Soraya; Williams, Monique; Cloete, Ruben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a complex neurodegenerative disorder, the aetiology of which is thought to be an interaction of genetic, biological and environmental factors. Its cardinal motor features, tremor, muscular rigidity, bradykinesia and abnormal gait occur relatively late in the course of disease, as a result of over 60% loss of dopaminergic neurons in the substantia nigra pars compacta. While most reported PD cases are sporadic, 5-10% of all cases are caused by several PD-causing genes including Parkin, PINK1, LRRK2, SNCA, SYNJ1, DJ-1 and EIF4G1. These PD genes were discovered using first-generation sequencing technologies which were expensive and time consuming. The development of high-throughput next generation sequencing technologies like whole exome sequencing (WES) has fast-tracked the discovery of disease-causing genetic mutations in various Mendelian disorders like PD. WES enables the screening of only the protein coding regions of the genome, to locate mutations which can interrupt cellular processes and lead to diseases. To date, WES has identified several PD-causing genes including CHCHD2, VPS35 and LRP10, implicating the dysfunction of pathways regulating mitochondrial, lysosomal and synaptic function. The aim of the present study is to combine WES technology and functional studies to identify a novel pathogenic mutation in a gene that could be implicated in the autosomal dominant form of PD in a South African Afrikaner family. To do this, a comprehensive filtration strategy was applied, combining various bioinformatic tools, in a step-wise analysis approach to assist in the filtration, interpretation and prioritisation of the NGS results. The bioinformatic tools used included SIFT, PolyPhen-2, MutationTaster, CADD, GERP++, Allen Brain Atlas, PANTHER and SWISS-MODEL. Once a single candidate gene was selected from the computational prioritisation, its protein expression was investigated in a disease relevant cell model using Western blotting. WES was conducted on three affected individuals yielding over 20,000 variants each. Quality control (sequence alignment, alignment postprocessing, variant detection and quality evaluation) and filtering only the co-segregating non-synonymous variants through bioinformatics analysis yielded nine variants. Sanger sequencing was used to verify these variants, and four variants in the genes ACTN3, CDC27, POU2F1 and TUBB6 were found to be sequencing artefacts. Five variants in the genes RFT1, NRXN2α, TEP1, CCNF and CFAP65 were found to be present in all four affected PD individuals of the family. Only one variant, p.G849D in NRXN2α, fulfilled the various prioritisation criteria. The mutation was not present in the unaffected family members, in 671 South African PD patients and in 192 ethnically-matched controls. Multiple online population frequency databases also showed that the variant had not been previously reported in any population. Using a web-based database containing the exomes of 3000 patients with neurological disorders, 50 PD patients were identified with 24 other variants in the NRXN2α gene including indel mutations and premature stop codons. Amongst the five verified candidates, the p.G849D NRXN2α variant was predicted to be pathogenic across all four functional prediction tools with the highest Combined Annotation Dependent Depletion (CADD) score (29,50). It was also found to have a very high GERP++ score denoting that the level of evolutionary constraint acting on this site is predicted to be very high. The p.G849D NRXN2α amino acid change was the most severe, from a small, non-polar, side chain free amino acid, to Aspartic acid a larger, negatively charged amino acid. The homology modelling of the mutant vs wild-type revealed no change in the protein secondary structure but biochemically the substitution could lead to unwanted interactions with the neighbouring residues which could possibly affect the function and activity of the protein. Also, NRXN2α was found to be highly expressed in the substantia nigra which is the main region of the brain affected in PD pathogenesis. It is associated with pathways related to calcium channel regulation, transmembrane signalling receptor activity, neuronal cell adhesion, synaptic organization and neuroligin family protein binding at the synapse, which makes it a plausible candidate gene for PD. Additionally, for functional studies, SH-SY5Y Neuroblastoma cells which are commonly used in vitro model for PD, were utilised. We investigated the endogenous NRXN2α levels in the SH-SY5Y cells and found that they produced detectable levels of the NRXN2α protein and were stably expressing this protein. In summary, by integrating WES and in vitro studies, we identified p.G849D NRXN2α, a variant possibly associated with the autosomal dominant form of PD in a South African Afrikaner family. To our knowledge, this is the first report of an association between NRXN2α and PD. As a candidate, NRXN2α is well-suited for future functional mutant characterisation studies that will elucidate the impact of variants in this gene and their relative contribution to the disease phenotype. The further study of NRXN2α in PD may provide critical insight into novel disease mechanisms or genetic interactions with established PD mechanisms. Ultimately, this could potentially lead to development of improved therapeutic modalities for this debilitating disorder.

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