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Subject: search.filters.subject.Whole exome sequencing

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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
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    Exome Sequencing of individuals with vitiligo and their relatives with and without autoimmune disorders
    (University of the Witwatersrand, Johannesburg, 2023) Rabinda, Kentie Rofhiwa
    Autoimmune disorders result from the immune system attacking and damaging its healthy cells because of an acquired immune system malfunction. Vitiligo is an example of an autoimmune disorders. It is a common depigmentation skin disorder with an estimated prevalence of 0.5–2% of the population worldwide. It is shown by selective loss of melanocytes causing non- scaly, chalky-white macules. Individuals with vitiligo often have other autoimmune disorders or first-degree relatives with at least one other autoimmune disorder. This study aimed to identify genetic variants in selected genes in individuals with vitiligo and their unaffected close relatives who have other autoimmune disorders. Upon DNA extraction, whole exome sequencing was performed, and five non- major histocompatibility complex (MHC) genes were analysed. Identified variants were annotated on Ensembl Variant Effect Predictor (VEP) and compared between individuals with vitiligo and their close relatives with and without autoimmune disorders in a family-specific manner. Eight variants were identified in two families, however, the three missense variants in the NLRP1 gene (rs12150220, rs11651270, and rs2301582) were observed between and across two families in individuals with autoimmune disorders. SMOC2 rs13208776, was identified as a risk locus for vitiligo in individuals from two Romanian isolate villages with vitiligo and other autoimmune disorders. None of the genotyped individuals were homozygous for the minor allele (A) and 2/12 individuals were heterozygous therefore it is unlikely that rs13208776 has any role in the development of vitiligo or any of the autoimmune disorders present in the two families. This study showed that NLRP1 gene variants segregated with vitiligo and autoimmune disorders
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    RB1 and Beyond: Determining genetic causes of retinoblastoma in South African patients
    (University of the Witwatersrand, Johannesburg, 2024) Beukman, Danielle; Lamola, Lindie
    Retinoblastoma is the most common solid tumour of the retina, affecting mainly paediatric patients. Although loss-of-function germline variants the RB1 gene is the tumour suppressor gene most often associated with heritable retinoblastoma, two contradictory research findings complicate the assumption that a pathogenic germline variant in RB1 will lead to the development of heritable retinoblastoma. Firstly, heritable retinoblastoma has been observed in cohorts with biallelic wild-type RB1 genes, secondly, biallelic loss-of-function is not always sufficient to lead to retinoblastoma tumorigenesis. By investigating germline variants in additional cancer predisposition genes to discover candidate driver genes in heritable retinoblastoma, the full germline mutational spectrum of heritable retinoblastoma can be established. In this study we investigate sequence germline variants in cancer predisposition genes beyond RB1 in South African retinoblastoma patients with an African ancestry by performing whole exome sequencing on eight patients diagnosed with retinoblastoma. Whole exome sequencing data for genes previously associated with retinoblastoma was subjected to variant annotation by means of a variant effect predictor and filtering criteria applied manually. Variant classification was performed according to guidelines proposed by The American College of Medical Genetics and Genomics and the Association of Molecular Pathology. The highest degree of variant classification was variants of unknown significance. The absence of sequence variants capable of describing the retinoblastoma phenotype in this cohort demonstrates the necessity of looking beyond RB1, combining next generation sequencing with copy number analysis pipelines and additional genome mapping technologies capable of detecting structural variants. The detection of sequence variants previously reported to be possibly damaging but now considered polymorphisms highlights the importance of revisiting variant classification. In closing, this study adds genomic information from patients with a South African ancestry to mounting genomic data, ensuring that previously underrepresented populations can also benefit from future cancer predisposition and precision medicine research.