Browsing by Author "Neethling, Annika"
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- ItemExome sequencing identifies a novel TTC37 mutation in the first reported case of Trichohepatoenteric syndrome (THE-S) in South Africa(BioMed Central, 2017-03-14) Kinnear, Craig; Glanzmann, Brigitte; Banda, Eric; Schlechter, Nikola; Durrheim, Glenda; Neethling, Annika; Nel, Etienne; Schoeman, Mardelle; Johnson, Glynis; Van Helden, Paul D.; Hoal, Eileen G; Esser, Monika; Urban, Michael; Moller, MarloBackground Trichohepatoenteric syndrome (THE-S) or phenotypic diarrhoea of infancy is a rare autosomal recessive disorder characterised by severe infantile diarrhoea, facial dysmorphism, immunodeficiency and woolly hair. It was first described in 1982 in two infants with intractable diarrhoea, liver cirrhosis and abnormal hair structure on microscopy. We report on two siblings from a consanguineous family of Somali descent who, despite extensive clinical investigation, remained undiagnosed until their demise. The index patient died of fulminant cytomegalovirus pneumonitis at 3 months of age. Methods Whole exome sequencing (WES) was performed on a premortem DNA sample from the index case. Variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants using TAPER™. Sanger sequencing was done to genotype the parents, unaffected sibling and a deceased sibling for the variant of interest. Results Exome sequencing identified a novel homozygous mutation (c.4507C > T, rs200067423) in TTC37 which was confirmed by Sanger sequencing in the index case. The identification of this mutation led to the diagnosis of THE-S in the proband and the same homozygous variant was confirmed in a male sibling who died 4 years earlier with severe chronic diarrhoea of infancy. The unaffected parents and sister were heterozygous for the identified variant. Conclusions WES permitted definitive genetic diagnosis despite an atypical presentation in the index case and suggests that severe infection, likely secondary to immunodeficiency, may be a presenting feature. In addition definitive molecular diagnosis allows for genetic counseling and future prenatal diagnosis, and demonstrates the value of WES for post-mortem diagnosis of disorders with a non-specific clinical presentation in which a Mendelian cause is suspected.
- ItemFunctional 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; 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.
- ItemLong QT syndrome : the identification and verification of putative KCNE2-interacting proteins(Stellenbosch : Stellenbosch University, 2013-12) Neethling, Annika; Kinnear, Craig; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Long QT syndrome (LQTS) is a cardiac repolarization disorder affecting every 1:2000-1:3000 individuals. This disease is characterized by a prolonged QT interval on the surface electrocardiogram (ECG) of patients. Symptoms of LQTS range from dizziness and syncope to more severe symptoms such as seizures and sudden cardiac death (SCD). Clinical features of LQTS are a result of the precipitations of Torsades de Pointes, which is a polymorphic form of ventricular tachycardia. A number of genetic forms of LQTS have been identified with more than 700 mutations in 12 different genes leading to disease pathogenesis. However it has been estimated that approximately 25% of patients with compelling LQTS have no mutations within the known LQT genes. This proves to be problematic since treatment regimens depend on the genetic diagnosis of affected individuals. Of the known mutated genes, KCNE2 is associated with LQT6. KCNE2 encodes the beta-subunit of potassium ion channel proteins. These proteins contain cytoplasmic C-terminal domains in which many mutations have been identified. We hypothesize that genes encoding KCNE2-interacting proteins might be identified as disease-causing or modifying genes. The present study aimed to use yeast two-hybrid (Y2H) methodology to screen a pre-transformed cardiac cDNA library in order to identify putative interactors of the C-terminal of KCNE2. Through specific selection methods the number of KCNE2 ligands was reduced from 296 to 83. These interactors were sequenced and 14 were identified as putative interacting proteins. False positive ligands were excluded based on their function and subcellular location. Ultimately three strong candidate ligands were selected for further analysis: Alpha-B crystallin (CRYAB), Filamin C (FLNC) and voltage-dependent anion-selective channel protein 1 (VDAC1). Three-dimensional (3D) co-localization and co-immunoprecipitation were used to verify these proposed interactions and succeeded in doing so. The genes encoding verified interactors will be screened in our SA panel of LQT patients, to potentially identify novel LQT causative or modifying genes. Furthermore, the interactions verified in the present study may shed some light on the mechanism of pathogenesis of LQT causative mutations in KCNE2.