Browsing by Author "Maluleke, Twananani"

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    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.