Browsing by Author "Esterhuizen, Alina"

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    Open Access
    A molecular approach to precision medicine in South African children with Epilepsy: towards a genetics-based diagnostic service for Epilepsy
    (2024) McIntosh, Caitlin; Esterhuizen, Alina; Fieggen Karen
    Background: Epilepsy is a neurological disorder characterised by unprovoked, recurring seizures. SubSaharan Africa carries the highest burden of epilepsy in the world, owing mainly to the increased risk factors such as infectious and parasitic disease, and traumatic brain injury. A proportion of this burden is genetic, however, genetic testing for epilepsy in South Africa is limited, currently only accessible in the private healthcare sector via international referral. Genetic testing in epilepsy is an internationally recognised diagnostic tool which can inform the diagnosis and patient management, with options for precision treatment, frequently resulting in improved outcomes. The main aim of this research project, therefore, was to design and validate a next-generation sequencing (NGS) gene panel for South African paediatric patients with drug-resistant epilepsy. The panel design was aimed primarily at the developmental and epileptic encephalopathies, where the yield of informative findings is highest, and the results often carry implications for treatment. The secondary aim of the project was to perform preliminary pharmacogenetic testing, to determine if variants previously associated with antiseizure drug metabolism in other populations are present in this study group. Materials and Methods: Forty probands with clinically complex, drug-resistant epilepsy with no identified, acquired cause were recruited from the neurology epilepsy service at the Red Cross War Memorial Children's Hospital in Cape Town. All 40 probands were tested with a panel of 78 genes selected on the basis of association with disease and clinical actionability. NGS data files were subject to variant prioritisation, followed by variant confirmation by cycle sequencing, segregation analysis, and final classification according to ACMG criteria. All 40 probands were tested with two pharmacogenomic arrays, one generalised array, the Veridose® Core Panel produced by Agena Bioscience (San Diego, USA), and one custom-designed anti-seizure medication-specific SNV array targeting eight SNVs across six different genes. Results: Three pathogenic variants were identified; two in SCN1A and one in GRIN2A, with a pickup rate of 7.5% (3/40). Two variants of uncertain significance were identified in GABRG2 and GRIN2B. The findings were aligned with the electroclinical features in each patient. The pharmacogenomic analysis revealed one variant, EPHX1 rs1051740, which appeared to be statistically significantly differently distributed in the study group to the general African population (P = 0.02). Three other variants, EPHX1 rs2234922, SCN1A rs3812718, and CYP2d6 s59421388 appear to be trending towards significance, with P-values of 0.08, 0.05, and 0.05, respectively. Conclusion: The main outcome of this project was the successful development of an NGS-based diagnostic protocol for paediatric epilepsy using the gene panel approach, now ready for implementation in the local diagnostic testing laboratory. Moreover, the genetic cause of epilepsy was identified in three study participants, with treatment implications in two (variants in SCN1A). The pharmacogenomic analysis in this project did not reveal specific insights owing to the small study group, but provided exposure to pharmacogenomic analysis, and may be used as a basis for further research into the pharmacogenomics of epilepsy in larger African cohorts.
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    Open Access
    Duchenne muscular dystrophy : mutation profiling in view of the emerging gene-based therapies
    (2010) Esterhuizen, Alina; Goliath, Rene
    Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked, recessive muscle-wasting disorder affecting 1 in 3 500 live male births worldwide, for which only palliative care is available to date. Large exonic deletions or duplications are found in approximately 70% of DMD patients, for which diagnostic testing is available. The remaining 30% carry point mutations, which go largely undetected, as no testing is currently offered due to the great size of the DMD gene and the logistical challenges involved.
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    Genetic investigation of South Africans with the Noonan Syndrome phenotype using targeted next generation sequencing
    (2017) Ngongang Tekendo, Cedrik; Wonkam, Ambroise; Esterhuizen, Alina
    Introduction: Noonan Syndrome (NS) is an autosomal dominant multisystem disorder, characterised by short stature, distinctive facial dysmorphism, cardiovascular abnormalities and developmental delay. Its estimated incidence is 1:1000 to 1:2500 live births. NS is caused by germline mutations in more than ten genes encoding proteins integral to the Ras/MAPK signaling pathway. Pathogenic variants in these genes account for 70-80% of NS cases. The clinical diagnosis of NS can be challenging in some cases, even when performed by experienced clinicians. The introduction of Next Generation Sequencing (NGS) technology in clinical practice in the Western world has tremendously facilitated the molecular diagnosis of RASopathies. Molecular testing for NS is not yet available in South Africa, nor has any study investigating NS from clinical and molecular perspectives been conducted in South Africans. Aim: The aim of this study was to investigate selected genes within a group of paediatric and adult patients with a clinical diagnosis of NS. Methods: This study was a cross-sectional descriptive study, including twenty-six familial and isolated NS patients recruited in Cape Town in the period January 2015-January 2017. Thorough phenotyping of each patient according to the international diagnostic criteria for NS was followed by targeted NGS, performed on leucocyte DNA samples from sixteen unrelated patients out of the twenty-six included. Sequencing involved all the exons and intron-exon boundaries of a predesigned panel of 14 genes, including A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1 and SPRED1. Results: Of the 26 patients included, 50% had a family history suggestive of NS. The median age at diagnosis was4.5 years (range: 1month-51years). Individuals of mixed-race ancestry were most represented (53.8%), followed by black Africans (30.8%). The clinical features identified were consistent with those reported in other populations. Compared to other series, our cohort revealed a lower frequency of Pulmonary Valve Stenosis (34.6%) and a less severe developmental phenotype. Variants predicted pathogenic were detected in 7(43.7%) DNA samples out of the 16 analysed. The genes involved were CBL in three cases (42.8%), PTPN11and MAP2K1in two cases (28.6%, for each gene). Surprisingly, the proportion of CBL variants was relatively high compared to those in the literature. Genotype-phenotype correlations showed that clinical features of NS were more typical in patients with pathogenic variants in MAP2K1, and less in those with variants in CBL. Conclusion: This is the first clinical and molecular study in South Africans with the NS phenotype. The phenotype of affected individuals with NS in South Africa is globally similar to that reported in the literature. Therefore, the use of international diagnostic criteria can effectively enable the clinical diagnosis of NS in most South African patients. These preliminary data suggest that the distribution of pathogenic variants in NS genes in South Africans may be different from that reported in other populations. Finally, this study demonstrates that Targeted NGS can be successfully applied to the molecular diagnosis of NS and related conditions in South Africa, and should be implemented in clinical practice.
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    Open Access
    Skin cells as a tool in genetic diagnosis of Duchenne muscular dystrophy
    (2016) Tyers, Lynn; Esterhuizen, Alina; Davids, Lester M
    Duchenne muscular dystrophy (DMD) is the most common and severe of the dystrophies, with an incidence of 1 in 3500 live male births, worldwide. Becker Muscular dystrophy (BMD) has a lower incidence of approximately 1 in 17500 births, a milder progression and longer life expectancy. Many advancements have been made in the development of gene-based therapies for the treatment of D/BMD, however, these treatments require genetic confirmation of the disease which continues to present a significant diagnostic challenge. The current standard for RNA-based analysis requires obtaining an invasive, often distressing, muscle biopsy. This dissertation investigated the utility of human autologous epidermal melanocyte and dermal fibroblast cell cultures for use as a tool for genetic confirmation of D/BMD from a much less invasive shave skin biopsy. Methodologies included immunohistochemical, immunocytochemical, Western blot, qPCR analysis and cDNA sequencing. The results suggest that melanocytes and fibroblasts express the full length muscle isoform of dystrophin, although at differing levels, and that melanocytes could potentially be used as an alternative for the genetic confirmation of D/BMD.