Browsing by Author "Molatoli, Mhlekazi Cathrine"

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    Detection of antiretroviral drug resistant minority variants in patients failing second line antiretroviral therapy in South Africa
    (2019) Molatoli, Mhlekazi Cathrine
    The development of antiretroviral (ARV) drug resistance to Protease (PR) Inhibitors (PIs) requires the emergence of both primary and secondary/ compensatory mutations in the PR enzyme. Several studies have shown that most patients virologically failing a PI based ARV drug regimen do not harbour PI drug resistant mutations (DRMs). The presence of minority HIV-1 PI variants undetected by population-based Sanger sequencing (sensitivity limitation of ≥ 15- 20%), have been hypothesised to possibily contribute to the development of PI resistance in these patients. A total of 188 participant samples previously collected from six provinces across South Africa and virologically failing a PI based second-line regimen were available for this study. Population based Sanger sequences and associated DRMs were available for all the participants. Following viral RNA extraction using the EasyMag Extraction Kit (Biomerieux, INC, France) and the NucliSeNS® EasyMag instrument (Biomerieux INC, France), RT-PCR amplification and sequencing-by-synthesis on the next generation sequencing (NGS) platform, Illumina Miseq (California, USA), was successful for 158 of the participants. Identification of all HIV-1 DRM variants was performed using both Deepchek® and Geneious® analysis tools. Following a comparison of DR variants obtained using Deepchek® and Geneious®, a minimal cut-off of ≥ 4.5% was used for the remainder of the study. NGS detected virus containing PI DRMs in 26 of the participants versus 24 participants detected by Sanger sequencing. Minority HIV-1 variants as defined at ≥ 4.5- 20.0%, were identified in viral PR for five of the 26 participants sequences by NGS and resulted in PI regimen drug resistance for only two of the five participants. Overall, NGS detected PI DRMs resulting in PI resistance for 20 participants versus 18 participants by Sanger sequencing. Thus, using NGS based genotyping results, an additional two participants would be referred to switch to a third-line ART regimen. Furthermore, the detection of minority RT inhibitor variants in three of the abovementioned 20 participant sequences resulted in a further change in the recommended third-line RT backbone. Our findings confirm previous studies and show that the majority (83.5%) of study participants that were failing a PI based regimen do so in the absence of detectable, known PI DRMs. It is likely that non-adherence to the PI based regimen may explain the observed virological failures in the absence of relevant DRMs. Alternatively; an as of yet unidentified mechanism of DR may contribute to changes in PI drug resistance or susceptibility. Future work must focus on elucidating whether additional viral mechanisms or patient non-adherence are responsible for virologic failure to a PI based regimen in the absence of PI DRMs.
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    The role of small genetic variants in the aetiology of developmental disorders in South Africa - a whole exome sequencing study
    (University of the Witwatersrand, Johannesburg, 2024) Molatoli, Mhlekazi Cathrine; Lombard, Zané
    Developmental disorders (DD) are a diverse group of chronic conditions characterized by significant limitations to both mental and physical development. Genetic variants have been identified as the underlying aetiology in about 40-50% of DD cases. Whole exome sequencing (WES) is the recommended first-line genetic test in this group of patients and is associated with diagnostic yields of 16-45%. However, in South Africa and other resource-poor settings, karyotype testing and MLPA analysis (offering low diagnostic rates of 3% and ~9% respectively) are still being utilized for genetic testing. Thus, a higher proportion of patients remain with unexplained DD due to the limitations of these diagnostic tools and limited genetic services. The main challenge facing the clinical implementation of WES in African settings is the complex data analysis and interpretation associated with the large amount of variant data produced. This is especially challenging as African ancestry individuals have been demonstrated to have a high level of genetic diversity resulting in a higher number of novel variants reported compared to European ancestry individuals. This study seeks to investigate whether the clinical utility of WES can be replicated in an African setting. Additionally, we seek to make recommendations for variant filtering and prioritization, thus making the process of WES data analysis for DD patients more efficient. To achieve these, WES was performed in 117 patients with unexplained DD and their 180 unrelated parents. Variant data was filtered and prioritized using two in-house semi-automated pipelines. The first pipeline, prioritized variants overlapping known DD genes, as identified using the G2P-DDG2P bioinformatics analysis tool. The second pipeline identified de novo variants in trio families using the trio-dnm bioinformatics analysis tool. Sanger sequencing was used to validate low-quality prioritized variants prior to in-house interpretation and curation, and all subsequently identified putative disease-causing variants prior to reporting. Of the 117 patients from 115 families analysed, a positive molecular diagnosis was achieved for 29 families, resulting in a diagnostic yield of 25.2% (29/115). Leveraging currently available DD data, our findings demonstrate the diagnostic and clinical utility of WES which resulted in recommendations for improving patient clinical management and surveillance. This study has also developed and made recommendations for variant filtering and prioritization strategies, which can be implemented in both research and diagnostic settings to streamline and aid in the identification of putative disease-causing variants in DD patients