Molecular Biology and Human Genetics

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1000 Genomes-based metaanalysis identifies 10 novel loci for kidney function
(Nature Research, 2017) Gorski, Mathias; Van Der Most, Peter J.; Teumer, Alexander; Chu, Audrey Y.; Li, Man; Mijatovic, Vladan; Nolte, Ilja M.; Cocca, Massimiliano; Taliun, Daniel; Gomez, Felicia; Li, Yong; Tayo, Bamidele; Tin, Adrienne; Feitosa, Mary F.; Aspelund, Thor; Attia, John; Biffar, Reiner; Bochud, Murielle; Boerwinkle, Eric; Borecki, Ingrid; Bottinger, Erwin P.; Chen, Ming-Huei; Chouraki, Vincent; Ciullo, Marina; Coresh, Josef; Cornelis, Marilyn C.; Curhan, Gary C.; d’Adamo, Adamo Pio; Dehghan, Abbas; Dengler, Laura; Ding, Jingzhong; Eiriksdottir, Gudny; Endlich, Karlhans; Enroth, Stefan; Esko, Tonu; Franco, Oscar H.; Gasparini, Paolo; Gieger, Christian; Girotto, Giorgia; Gottesman, Omri; Gudnason, Vilmundur; Gyllensten, Ulf; Hancock, Stephen J.; Harris, Tamara B.; Helmer, Catherine; Hollerer, Simon; Hofer, Edith; Hofman, Albert; Holliday, Elizabeth G.; Homuth, Georg; Hu, Frank B.; Huth, Cornelia; Hutri-Kahonen, Nina; Hwang, Shih-Jen; Imboden, Medea; Johansson, Asa; Kahonen, Mika; Konig, Wolfgang; Kramer, Holly; Kramer, Bernhard K.; Kumar, Ashish; Kutalik, Zoltan; Lambert, Jean-Charles; Launer, Lenore J.; Lehtimaki, Terho; De Borst, Martin H.; Navis, Gerjan; Swertz, Morris; Liu, Yongmei; Lohman, Kurt; Loos, Ruth J. F.; Lu, Yingchang; Lyytikainen, Leo-Pekka; McEvoy, Mark A.; Meisinger, Christa; Meitinger, Thomas; Metspalu, Andres; Metzger, Marie; Mihailov, Evelin; Mitchell, Paul; Nauck, Matthias; Oldehinkel, Albertine J.; Olden, Matthias; Penninx, Brenda W. J. H.; Pistis, Giorgio; Pramstaller, Peter P.; Probst-Hensch, Nicole; Raitakari, Olli T.; Rettig, Rainer; Ridker, Paul M.; Rivadeneira, Fernando; Robino, Antonietta; Rosas, Sylvia E.; Ruderfer, Douglas; Ruggiero, Daniela; Saba, Yasaman; Sala, Cinzia; Schmidt, Helena; Schmidt, Reinhold; Scott, Rodney J.; Sedaghat, Sanaz; Smith, Albert V.; Sorice, Rossella; Stengel, Benedicte; Stracke, Sylvia; Strauch, Konstantin; Toniolo, Daniela; Uitterlinden, Andre G.; Ulivi, Sheila; Viikari, Jorma S.; Volker, Uwe; Vollenweider, Peter; Volzke, Henry; Vuckovic, Dragana; Waldenberger, Melanie; Wang, Jie Jin; Yang, Qiong; Chasman, Daniel I.; Tromp, Gerard; Snieder, Harold; Heid, Iris M.; Fox, Caroline S.; Kottgen, Anna; Pattaro, Cristian; Boger, Carsten A.; Fuchsberger, Christian
HapMap imputed genome-wide association studies (GWAS) have revealed >50 loci at which common variants with minor allele frequency >5% are associated with kidney function. GWAS using more complete reference sets for imputation, such as those from The 1000 Genomes project, promise to identify novel loci that have been missed by previous efforts. To investigate the value of such a more complete variant catalog, we conducted a GWAS meta-analysis of kidney function based on the estimated glomerular filtration rate (eGFR) in 110,517 European ancestry participants using 1000 Genomes imputed data. We identified 10 novel loci with p-value < 5 × 10−8 previously missed by HapMap-based GWAS. Six of these loci (HOXD8, ARL15, PIK3R1, EYA4, ASTN2, and EPB41L3) are tagged by common SNPs unique to the 1000 Genomes reference panel. Using pathway analysis, we identified 39 significant (FDR < 0.05) genes and 127 significantly (FDR < 0.05) enriched gene sets, which were missed by our previous analyses. Among those, the 10 identified novel genes are part of pathways of kidney development, carbohydrate metabolism, cardiac septum development and glucose metabolism. These results highlight the utility of re-imputing from denser reference panels, until whole-genome sequencing becomes feasible in large samples.
2'-5'-Oligoadenylate synthetase-like protein inhibits intracellular M. tuberculosis replication and promotes proinflammatory cytokine secretion
(Elsevier, 2019-12) Leisching, G.; Ali, A.; Cole, V.; Baker, B.
Host cytoplasmic surveillance pathways are known to elicit type I interferon (IFN) responses which are crucial to antimicrobial defense mechanisms. Oligoadenylate synthetase-like (OASL) protein has been extensively characterized as a part of the anti-viral mechanism, however a number of transcriptomic studies reveal its upregulation in response to infection with a wide variety of intracellular bacterial pathogens. To date, there is no evidence documenting the role (if any) of OASL during mycobacterium tuberculosis infection. Using two pathogenic strains differing in virulence only, as well as the non-pathogenic M. bovis BCG strain, we observed that pathogenicity and virulence strongly induced OASL expression after 24 h of infection. Further, we observed that OASL knock down led to a significant increase in M. tb CFU counts 96 h post-infection in comparison to the respective controls. Luminex revealed that OASL silencing significantly decreased IL-1β, TNF-α and MCP-1 secretion in THP-1 cells and had no effect on IL-10 secretion. We therefore postulate that OASL regulates pro-inflammatory mediators such as cytokines and chemokines which suppress intracellular mycobacterial growth and survival.
2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria
(Stellenbosch : Stellenbosch University, 2019-12) Mishra, Abhilasha Madhvi; Baker, Bienyameen; Leisching, Gina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background:Tuberculosis (TB) is a major cause of infection-related mortalityworldwide. In 2017 an estimated 1.3 million people who were HIV-negative died of TB. An estimated 5-10% of infected individual develop active TB during their lifetime, while the remaining90% (of infected population) successfully control the bacteria. Also, some of the close household contacts of TB patients remain uninfected and healthy. Studying host immune response towards Mycobacterium tuberculosis(M. tb) can unfold the reason behind this enigma. Methods:We conducted a detailed investigation of in vitrohost response from human monocyte derived macrophages(hMDMs)towards different strains of mycobacteria(grown in detergent-freemedia), i.e. pathogenic (M. tbR179) andnon-pathogenic (M. smegmatisand M. bovisBCG). The host response was measured post-infection (at mRNA and protein levels) using AmpliSeq, quantitative real time polymerase chain reaction (qRT-PCR), multiplex ELISA (Luminex), intracellular mycobacterial survivaland cytotoxicity assay. Biological network analysis (ingenuity pathway analysis IPA) was performed to understand the gene regulatory networkinvolved in the pathophysiology associated with the host-immune system.Based on false discovery rate (FDR) and biological functions, we selected an inter-related gene family of interferon induced protein with tetratricopeptides (IFIT1, IFIT2 andIFIT3) from the list of 19 potential differentially expressed genes(DEGs)for knock-up (vector-based over-expression)/down experiments. This gene family is known to form a protein complex during viral infection to act against the antigen. Studyencompassing their role against bacteria is not well established.Therefore, we performed knocking-up of IFITsvia vector-based transfection and knocking-down via small interferingRNA (siRNA) approach to investigate their effect upon mycobacteria inside the host macrophages. Results:AmpliSeqanalysis found 19 DEGs at 12 hours post-infection across all three strains. We observed lower number of mycobacterial CFUs and higher host response (at both RNA and protein level) in hMDMs infected with M. smegmatisas compared to other two strains. Biological network analysis revealed interferon-interleukin associated signalling pathways as most prominent among the 19 differentially expressed genes.We found a differed host response towardsall three strains, which mayattributeto their pathogenicity. Messenger RNA and protein level comparisons at different time points, depicted strong role of interferon and interleukin associated gene network. This network was able to successfully counter M. smegmatisbut succumb to M. bovisBCG andM. tbR179. Most importantly, across all three strains, intra-cellular bacterial growth and survival measured through colony forming units (CFUs)decreased significantly upon knocking up of IFITs(IFIT1, IFIT2 andIFIT3),while we recordedan increase in CFUs upon knocking down ofIFITsin the host macrophages. Using multiplex ELISA, we found higher expression of key pro-inflammatory cytokines (i.e. IDO1, IFN-γ, IL-6, and IL-23) during knock-up (vector-based over-expression)of IFITsresulting in reduction of mycobacteria. Conclusion:Differentially expressed IFITs showed a strong effect against mycobacteria, which can be used as a promising therapeutic targetadjunct to anti-TB therapy. This knowledge will broaden the scope of host drug targets for resistance free bacteriostatic immuno-therapy.
A 3-year cytogenetic survey of 9661 patients in South Africa
(Health and Medical Publishing Group (HMPG), 1983) Bester, Rina; Benjamin, Mercy; Retief, A. E.; Bernstein, Renee; Grace, H. J.; Nelson, Matilda M.; Jansen, S.; Benjamin, Mercy
During the period 1 January 1977 - 31 December 1979, 9661 patients underwent cytogenetic investigation at seven participating laboratories in South Africa. The chromosome data were coded using a standard protocol and the results tabulated, being listed according to the clinical signs which led to referral for investigation. Cytogenetic investigation was most commonly requested for prenatal studies, and 22% of the group's effort was directed towards this. One in 27 amniotic cell specimens was reported to have shown anomalous chromosomes, trisomy 21 being the most frequent abnormality. The majority of postnatal investigations were requested because congenital abnormalities suggested an underlying chromosomal defect. In 42.3% of 2420 patients a chromosome defect was confirmed. Results of chromosome studies are tabulated by indication for referral and the findings summarized. This collaborative study gives an indication of the nature and frequency of chromosome disorders in South Africa.
Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigens
(Stellenbosch : Stellenbosch University, 2002-12) Scriba, Thomas Jens; Van Rensburg, E. Janse; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is overwhelmingly prevalent in Botswana and South Africa and to date no interventions have been successful enough to curb the rapid spread of the virus. A number of HIV-1 vaccine strategies are being developed, however the breadth and efficacy of such candidate vaccines, many of which are based on the HIV-1 structural genes pol, gag and env, have mostly been found to be inadequate. The HIV-1 accessory genes are attractive components of HIV vaccines due to their role in viral pathogenesis, early expression and the high ratio of conserved CTl epitopes. Yet, because of undesirable properties questions regarding their safety as vaccine components are raised. In this study candidate tat, rev and nefmutants were assessed for efficient expression and inactivation of undesirable functionality. / Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat, Rev and Nef proteins were constructed. The coding sequences of the genes were codon-optimised for optimum protein expression and these synthetic genes were constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins were mutated at previously described sites by PCR-based site-directed mutagenesis to render them inactive for their respective functions. Corresponding wild-type Tat, Rev and Nef constructs were also made from viral isolates that were least dissimilar to the respective consensus amino acid sequences. tn vitro expression of the different constructs were assessed in 293 cells by Western blotting with polyclonal mouse sera, which were generated by DNA immunisation with one of the Tat, Rev and Nef constructs. The transactivation activity of Tat variants and Rev-mediated nuclear export activity of RRE-containing transcripts were studied in cotransfection experiments using reporter-gene-based assays while Nef functionality was assessed in a cotransfection assay with subsequent flow cytometric analysis of surface CD4 and MHC-I expression on 293 cells. Sequence analysis of the South African HIV-1 subtype C consensus sequences of Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence that was drawn up from a large number of viruses from southern Africa. These consensus sequences were also closer to individual viral isolate sequences than any individual sequences were, indicating that the use of a consensus sequence may serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not significantly higher than the wild-type constructs, however, the codon-optimised rev mutant exhibited markedly higher expression than the wild-type rev construct. Immunoreactivity of the protein with the mouse sera demonstrates expression and immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent CAT expression in 293T and Hela cells. Yet, this activity was significantly impaired using the single mutation, C3?, or the double mutation, C22C3? Compared to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4 downregulation was completely abrogated in one of the mutants, while both Nef mutants were entirely deficient for MHC-I downregulation. These data demonstrate the high expression levels and impaired functionality of sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens that may be used as single-standing vaccine components or form part of a multicomponent HIV-1 vaccine.
Accuracy and impact of the MTBDRplus v2 and MTBDRsl v2 line probe assays for the detection of first-line and second-line drug resistant tuberculosis
(2023-02) Pillay, Samantha; Theron, Grant; de Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Combating drug-resistant tuberculosis (DR-TB) remains a challenge globally. Treatment success rates are often derailed by under-diagnosis and under-reporting of disease. Patients remain contagious for prolonged periods prior to initiation of appropriate treatment which is further exacerbated by the amplification of drug resistance and poor treatment outcomes. Using current and new diagnostic tools effectively is key to rapid diagnosis of tuberculosis and early detection of drug resistance. Firstly, (chapter 2) line probe assays (LPAs) frequent inability to generate a resistance call in paucibacillary specimens is problematic. We showed that while MTBDRplus and MTBDRsl tests work well on smear-negative specimens for detecting drug resistance, failure rates remained high. We demonstrated with the use of routine key programmatic data how time-to-reporting of results improved with the use of molecular assays and provided evidence on how standard-of-care can be improved in a programmatic context. Secondly, (chapter 3) LPA testing on smear-negative specimens is not always performed causing diagnostic delays and hindering their role as a direct front-line diagnostic tests. Thus, by using Xpertgenerated data we determined the ratio of actionable-to-non-actionable results and the number of missed resistant cases at varying thresholds. We demonstrated that Xpert semiquantitation category is superior to informing reflex LPA testing than smear status. In short, this method provides a framework by which laboratories that currently do not test smear-negative specimens to expand testing. Thirdly (chapter 4) current pathways using Xpert MTB/RIF or Xpert Ultra as frontline tests for diagnosing TB and rifampicin resistance lack further treatment guidance. We did a systematic review and assessed the performance of Xpert MTB/XDR for the detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. Participants consisted of 1228 for pulmonary tuberculosis detection and 1141 for drug resistance. We found Xpert MTB/XDR is unlikely to test positive as a follow-up test for the detection of Mycobacterium tuberculosis in samples that test Xpert Ultra
Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis
(Nature Research, 2018-02-08) Chegou, Novel N.; Sutherland, Jayne S.; Namuganga, Anna-Ritah; Corstjens, Paul L. A. M.; Geluk, Annemieke; Gebremichael, Gebremedhin; Mendy, Joseph; Malherbe, Stephanus; Stanley, Kim; Van Der Spuy, Gian D.; Kriel, Magdalena; Loxton, Andre G.; Kriel, Belinda; Simukonda, Felanji; Bekele, Yonas; Sheehama, Jacob A.; Nelongo, Josefina; Van Der Vyver, Marieta; Gebrexabher, Atsbeha; Hailu, Habteyes; Esterhuyse, Maria M.; Rosenkrands, Ida; Aagard, Claus; Kidd, Martin; Kassa, Desta; Mihret, Adane; Howe, Rawleigh; Cliff, Jacqueline M.; Crampin, Amelia C.; Mayanja-Kizza, Harriet; Kaufmann, Stefan H. E.; Dockrell, Hazel M.; Ottenhoff, Tom H. M.; Walzl, Gerhard; AE-TBC consortium
We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1β, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76–0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7–76.5%) and specificity of 82.7%(95% CI, 72.4–89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa.
Alcohol, hospital discharge, and socioeconomic risk factors for default from multidrug resistant tuberculosis treatment in rural South Africa : a retrospective cohort study
(PLoS, 2013-12-13) Kendall, Emily A.; Theron, Danie; Franke, Molly F.; Van Helden, Paul; Victor, Thomas C.; Murray, Megan B.; Warren, Robin M.; Jacobson, Karen R.
Background: Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. Methods: We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. Results: Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age (hazard ratio [HR]= 0.97 [95% confidence interval 0.94-0.99] per year of greater age), formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. Conclusions: Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates.
Altered mitochondrial respiration and other features of mitochondrial function in parkin-mutant fibroblasts from parkinson’s disease patients
(Hindawi Publishing Corporation, 2016) Haylett, William; Swart, Chrisna; Van der Westhuizen, Francois; Van Dyk, Hayley; Van der Merwe, Lize; Van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya
Mutations in the parkin gene are the most common cause of early-onset Parkinson’s disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.
Alternative insulin mitogenic signaling pathways in immature osteoblast cell lines
(Stellenbosch : Stellenbosch University, 2002-03) Langeveldt, Carmen Ronel; Hulley, P. A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line.
Aminoglycoside-induced hearing loss : South Africans at risk
(Health and Medical Publishing Group (HMPG), 2009) Bardien, Soraya; De Jong, Greetje; Schaaf, H. Simon; Harris, Tashneem; Fagan, Johan; Petersen, Lucretia
Analysis and application of evolutionary markers in the epidemiology of Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2008-12) Van der Spuy, Gian Dreyer; Warren, R. M.; Van Helden, P. D.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.
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.
Analysis of ex vivo host biomarkers in sputum samples for diagnosis of pulmonary tuberculosis
(Stellenbosch : Stellenbosch University, 2019-12) Mendy, Joseph F.; Chegou, Novel N.; Sutherland, Jayne; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background Despite all the interventions deployed to control tuberculosis (TB), the disease still continues to be the principal cause of death from a single infectious agent in resource constrained settings. An estimated 60% of suspected TB patients do not have access to TB diagnostic tests. With the limitations of the current diagnostic tests and the importance of early diagnosis and initiation of treatment, biomarker diagnosis of TB would be an optimal option. For biomarkers are indicators of immune activity and state. Therefore, host or pathogen biomarker of TB disease would be ideal. Hence, the aim of this project was to profile a broad array of host markers for development of optimal signatures for detection of pulmonary tuberculosis from other respiratory disorders using ex vivo sputum samples Methods We recruited patients who were seeking medical attention at the MRCG at LSHTM outpatients department and Tuberculosis clinic with symptoms suggestive of TB, prior to clinical or microbiological diagnosis. All age groups were recruited. Sputa were collected at baseline from all participants and at 1 and 2 months from the confirmed TB cases. The sputa were digested with Sputolysin and the supernatant analysed using Luminex arrays while RNA extracted from the pellet were analysed with RT-qPCR. Statistical analyses and graphs were generated using R programming Language and GraphPad Prism, with a q value ≤ 0.05 considered significant. A receiver operating curve (ROC) was used to assess the diagnostic performance of individual and combination markers. Results Confirmed TB (428) and ORD (313) patients were analysed, 70 markers were assessed for diagnostic potential and treatment response. Of these, 37 were significantly different between TB and ORD. The best single marker was MMP-2 with an AUC of 0.73. An eight-marker signature (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES and VEGF) was able to diagnose smear and culture positive TB from ORD with an AUC of 0.77, sensitivity of 78% and specificity of 70%, while a threemarker signature (IL-1β, IL-7 and VEGF) classified smear negative but culture positive TB from ORD with an AUC of 0.74, sensitivity of 86% and specificity of 60%. Among children who had TB, a fourmarker signature (FGF, IL-4, MIP-1a and RANTES) differentiated those with TB from ORD, with an AUC of 0.87, sensitivity of 82% and specificity of 87% and a five-marker signature consisting of BAFF, C3L1, IL-22, MMP-3 and sTNFR1 was able to discriminate TB and HIV co-infected from ORD with an AUC of 0.90, sensitivity of 88% and specificity of 85%. We also found a four-marker signature consisting of EGF, IL-15, MIP-1β and TNF-β that could predict slow versus fast treatment responders at baseline with an AUC of 0.74, sensitivity of 75% and specificity of 80%. Conclusion We have discovered novel sputum host biomarkers and biosignatures for screening of tuberculosis and treatment response. The data is promising for potential translation into a user friendly device as a rapid screening test for pulmonary TB. However, this markers and signatures require further investigations to authenticate their usefulness.
Analysis of host determining factors in susceptibility to tuberculosis in the South African coloured population
(Stellenbosch : University of Stellenbosch, 2009-12) De Wit, Erika; Hoal, Eileen; Van Helden, Paul; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) still represents a global threat due to its devastating effect on health and the subsequent high mortality rate. Previous studies have indicated that host genetic factors are implicated in host susceptibility to TB. Since TB is a complex disease, it can be assumed that susceptibility to M. tuberculosis has multiple genetic causative factors (as well as environmental causes). The current study focussed on a number of South African Coloured (SAC) individuals, some of whom were TB cases and others controls. Population substructure was tested in the admixed SAC population as it can be a strong confounding factor for association studies. Our results using the programme STRUCTURE indicated no population substructure in the SAC population. We further investigated the population structure of the SAC group using Affymetrix 500k SNP chip data which showed that the SAC population group has 4 major ancestral components: the Khoesan, European, African and Asian (Indian). A number of candidate polymorphisms in eight genes, previously indicated to play an important role in TB susceptibility, were tested in case-control associations studies. We found statistically significant associations between IFNGR1, IL-8, IL-1Ra and NRAMP1 polymorphisms and TB susceptibility in the SAC population. It has become increasingly evident that gene-gene interactions play a far more important part in an individual’s susceptibility to a complex disease than single polymorphisms would on their own. The importance of epistasis was clearly identifiable in this study with only four associations found between the individual variants and TB susceptibility, but eight instances of statistically significant gene-gene interactions. A combined data set consisting of 106 variants constructed from our database and also used for gene-gene interaction analysis yielded numerous statistically significant interactions. The interaction between the genotype of the human host and the bacterial strain genotype was also investigated and yielded interesting results. Owing to various polymorphisms in several cytokine genes, the protein levels of the main modulators of the immune system, cytokines and chemokines, are changed in several diseases such as infectious diseases and may affect susceptibility or resistance to TB. The functional polymorphisms or haplotype patterns in some of these cytokine genes might be vital for protective immune responses and may serve as biomarkers of protection or susceptibility to TB. The present study investigated 18 cytokines including pro-inflammatory, anti-inflammatory and chemokine factors in healthy (mantoux positive or negative) children using the Linco-plex immunoassay, and investigated potential interactions. The basic research will one day contribute to personalised genetics which may benefit infectious diseases such as TB. If individuals can be identified as potentially more vulnerable, they may require different vaccination strategies, a higher index of suspicion if exposed to TB, and prophylactic treatment.
Analysis of host responses to secreted, latent and reactivation Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa
(Public Library of Science, 2013-09-10) Sutherland, Jayne S.; Lalor, Maeve K.; Black, Gillian F.; Ambrose, Lyn R.; Loxton, Andre G.; Chegou, Novel N.; Kassa, Desta; Mihret, Adane; Howe, Rawleigh; Mayanja-Kizza, Harriet; Gomez, Marie P.; Donkor, Simon; Franken, Kees; Boom, W. Henry; Thiel, Bonnie A.; Crampin, Amelia C.; Hanekom, Willem; Klein, Michel R.; Parida, Shreemanta K.; Ota, Martin; Walzl, Gerhard; Ottenhoff, Tom H. M.; Dockrell, Hazel M.; Kaufmann, Stefan H. E.
Background: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. Methods: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. Results: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. Conclusions: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.
Analysis of two mutations in the MTHFR gene associated with mild hyperhomocysteinaemia - heterogeneous distribution in the South African population
(Health and Medical Publishing Group (HMPG), 2002-06) Scholtz, Charlotte L.; Odendaal, Hein J.; Thiart, Rochelle; Loubser, Lynzie; Hillermann, Renate; Delport, Rhena; Hayward Vermaak, W. J.; Kotze, Maritha J.
Objective. The frequencies of mutations 677C→T and 1298A→C in the methylenetetrahydrofolate reductase (MTHFR) gene, previously shown to be associated with decreased enzyme activity that may lead to hyperhomocysteinaemia and consequently increased risk of cardiovascular disease (CVD), were determined in the South African population. Methods. HinfI (677C→T) and MboII (1298A→C) restriction enzyme analyses were performed on amplified DNA samples of 76 white, 73 coloured and 60 black subjects. Results. The mutant alleles of mutations 677C→T and 1298A→C were more common in the white (allele frequencies 0.36 and 0.37, respectively) than in the black population (0.04 and 0.09), while intermediate frequencies were detected ill the coloured population (0.18 and 0.30). Homozygosity for mutation 677C →T was not detected in the black cohort, while this genotype was detected in 1 coloured (1.4%) and 8white (10.5%) subjects. In the black population, 5% of the 60 subjects analysed were homozygous for mutation 1298A → C, compared with approximately 12% -in both the white and coloured populations.
Analysis of two mutations in the MTHFR gene associated with mild hyperhomocysteinaemia - heterogeneous distribution in the South African population
(Health & Medical Publishing Group, 2002-6) Scholtz, C. L.; Odendaal, H. J.; Thiart, R.; Loubser, L.; Hillermann, R.; Delport, R.; Hayward Vermaak, W. J.; Kotze, M. J.
Objective. The frequencies of mutations 677C→T and 1298A→C in the methylenetetrahydrofolate reductase (MTHFR) gene, previously shown to be associated with decreased enzyme activity that may lead to hyperhomocysteinaemia and consequently increased risk of cardiovascular disease (CVD), were determined in the South African population. Methods. HinfI (677C→T) and MboII (1298A→C) restriction enzyme analyses were performed on amplified DNA samples of 76 white, 73 coloured and 60 black subjects. Results. The mutant alleles of mutations 677C→T and 1298A→C were more common in the white (allele frequencies 0.36 and 0.37, respectively) than in the black population (0.04 and 0.09), while intermediate frequencies were detected in the coloured population (0.18 and 0.30). Homozygosity for mutation 677C→T was not detected in the black cohort, while this genotype was detected in 1 coloured (1.4%) and 8 white (10.5%) subjects. In the black population, 5% of the 60 subjects analysed were homozygous for mutation 1298A→C, compared with approximately 12% in both the white and coloured populations. Conclusions. Since hyperhomocysteinaemia is a risk factor for premature CVD, the heterogeneous distribution of the 677C→T and 1298A→C mutations across ethnic groups may partly explain ethnic differences in heart disease risk through decreased enzyme activity and hence increased homocysteine levels.
Analyzing genetic factors contributing to dysmotility in Hirschsprung’s disease and African Degenerative Leiomyopathy in a South African population
(Stellenbosch : Stellenbosch University, 2019-03) Maluleke, Twananani; Moore, Samuel; Kinnear, Craig; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Introduction Hirschsprung’s disease (HSCR) and African degenerative leiomyopathy (ADL) are rare gastrointestinal disorders affecting neonates and young children. HSCR is characterised by the absence of intrinsic ganglion cells in the distal segment of the intestine; its aetiology has been linked to cellular and molecular mechanisms associated with the enteric nervous system (ENS) development, ADL on the other hand is a distinctive form of visceral myopathy (VSCM) of uncertain aetiology affecting enteric smooth muscles (ESM) of the distal intestine. Gut motility is a result of highly coordinated contractions by muscle layers, neural network and pacemaker intestinal cells of Cajal whose development are controlled by genetic factors. The aetiology of HSCR has been associated with 15 genes linked to ENS development meanwhile ADL has been linked to environmental factors. Actin gamma 2 (ACTG2) is a gene that encodes the ACTG2 protein which is involved in ESM development. Studying the ACTG2 in HSCR patients may ascertain whether individuals affected by HSCR also display muscular dysfunction thereby providing a possible factor in the recurrence of dysmotility post-surgical resection. Additionally, ACTG2 has been identified as the genetic factor in VSCM pathology; therefore, the study may provide novel information regarding the genetic factors of ADL. Aim This project aims to study the genes associated with the development of enteric nervous system (RET, NRG1, SOX10, EDNRB) and smooth muscle cells (ACTG2) that contribute to HSCR and ADL in the South African neonate population. Methods Seventeen whole blood samples were collected from HSCR participants after informed consent; of which only 14 samples were included for genotyping and 9 samples were selected for RNA analysis based on the quality of extracted DNA and RNA respectively. Five whole blood samples were also collected from ADL patients after informed consent. RNA samples from the HSCR cohort were reverse transcribed and quantitative polymerase chain reaction was performed. DNA samples from HSCR and ADL samples were screened for variants in the ACTG2 exons through bidirectional Sanger sequencing. Novel variants were analysed in silico to ascertain their pathogenicity. Results and Discussion In both HSCR and ADL cohorts the variant K119E/R was observed in 64% (9/14) and 60% (3/5) of the study population respectively; K119E/R is likely to function as a disease modifier as it was also observed in the control samples six out nine individuals. Variants S345L and W357G in exon 10 with probable significant effect in the pathogenesis of ESM were identified in the HSCR cohort only. The ADL cohort had polymorphic intronic variants predicted to shift the exonic splice sites namely g>c -IVS12 exon 3 and c>t -IVS3 exon 5. Differential expression of ENS genes EDNRB, RET, SOX10 and NRG1 associated with ENS development in the HSCR cohort was not achieved due to experimental factors. Conclusion ACTG2 encodes an enteric smooth muscle γ-2 actin which plays a pivotal role in the contractile proteins of ESM, thus the data suggests that a muscular component may exist in HSCR aetiology that should be investigated further in vitro and provides further insights into genetic factors that may contribute to ADL pathogenesis.

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