Browsing by Author "Nel, Melissa"
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- ItemOpen AccessExome sequencing identifies novel dysferlin mutation in a family with pauci-symptomatic heterozygous carriers(BioMed Central, 2018-06-07) Jalali-Sefid-Dashti, Mahjoubeh; Nel, Melissa; Heckmann, Jeannine M; Gamieldien, JunaidBackground We investigated a South African family of admixed ancestry in which the first generation (G1) developed insidious progressive distal to proximal weakness in their twenties, while their offspring (G2) experienced severe unexpected symptoms of myalgia and cramps since adolescence. Our aim was to identify deleterious mutations that segregate with the affected individuals in this family. Methods Exome sequencing was performed on five cases, which included three affected G1 siblings and two pauci-symptomatic G2 offspring. As controls we included an unaffected G1 sibling and a spouse of one of the G1 affected individuals. Homozygous or potentially compound heterozygous variants that were predicted to be functional and segregated with the affected G1 siblings, were further evaluated. Additionally, we considered variants in all genes segregating exclusively with the affected (G1) and pauci-symptomatic (G2) individuals to address the possibility of a pseudo-autosomal dominant inheritance pattern in this family. Results All affected G1 individuals were homozygous for a novel truncating p.Tyr1433Ter DYSF (dysferlin) mutation, with their asymptomatic sibling and both pauci-symptomatic G2 offspring carrying only a single mutant allele. Sanger sequencing confirmed segregation of the variant. No additional potentially contributing variant was found in the DYSF or any other relevant gene in the pauci-symptomatic carriers. Conclusion Our finding of a truncating dysferlin mutation confirmed dysferlinopathy in this family and we propose that the single mutant allele is the primary contributor to the neuromuscular symptoms seen in the second-generation pauci-symptomatic carriers.
- ItemOpen AccessInvestigating the functionality of African-specific variants in the TGFB1 regulatory region and their potential role in HIV-associated kidney complications(2016) Buys, Joy-Mari; Heckmann, Jeannine M; Prince, Sharon; Nel, MelissaTransforming growth factor beta-1 (TGF-β1) is a cytokine involved in immune modulation and tissue regeneration and has a polymorphic TGFB1 promoter. African-specific single nucleotide polymorphisms (SNPs) have been identified in the TGFB1 promoter and they occur at higher frequencies in South Africans with African-genetic ancestry (≈17%) compared with West and East African genomes (<5%). However, the functional significance of these SNPs has only been partially explored. In this study it was hypothesized that higher frequencies of immunemediated disease complications in South Africans, such as HIV-associated nephropathy (HIVAN), may be influenced by functional genetic variations in TGFB1. To address this, the African-specific TGFB1 haplotypes containing singular, or combinations of, -1287 G>A (rs11466314), -1154 C>T (rs35318502), -387 C>T (rs11466316) and -14 G>A (rs9282871) were investigated for their effect on TGFB1 promoter activity. Briefly, an extended TGFB1 regulatory region driving a luciferase reporter was used as a template to generate six TGFB1 promoter haplotypes (referred to as H-1 through H-6 in this thesis) by site-directed mutagenesis and luciferase activity was used to measure promoter activity. The functional TGFB1 -1347 C>T variant was also investigated (H-5 and H-6 containing -1347 C and T alleles, respectively) because all four of the African variants more frequently co-occurred with the previously reported "lower expressing" TGFB1 -1347 C variant (H-1 through H- 4). Transient transfection of the TGFB1 promoter reporter constructs in two renal cell lines (RCC4+VHL and Caki- 2) showed no difference between -1347 C (H-5) and T (H-6) basal promoter activity. Having at least one African variant resulted in ~5-fold loss of basal TGFB1 promoter activity in renal cells when compared to the most common haplotype (H-5) (p<0.05). The repressive effect is mainly attributed to the -387 T variant (H-1) as the addition of other African variants on this haplotype showed no additional TGFB1 promoter repression. The repressive effect of the TGFB1 -387 T variant was maintained even after in-vitro treatment of transfected renal cells with recombinant human TGF-β1 (rhTGF-β1). To determine whether the African-specific TGFB1 promoter haplotypes (H-AFR), containing TGFB1 -387 T and tested in luciferase assays, also impact on endogenous TGF-β1 protein levels, western blot analysis was performed on human dermal fibroblasts from patients who had been genotyped. Similar to the promoter studies, basal TGF- β1 protein levels in cells with the H-AFR were ~47% lower compared to cells without (p=0.04) and no difference was seen in response to hrTGF-β1. Interestingly, when western blots were screened for phosphorylated Smad3 (pSmad3) protein levels, as an indicator of the activated TGF-β1 canonical pathway, similar pSmad3 levels were observed under basal and hrTGF-β1 stimulated conditions for all haplotypes (p>0.05). The possible interaction of HIV tat on the African TGFB1 promoter variants was also assessed. Luciferase activity was measured after co-transfecting renal RCC4+VHL and HT1080 fibroblast cells with a HIV Tat expression vector and the H-6, H-5 and H-AFR promoter luciferase constructs. Results showed that the promoter activity for all TGFB1 haplotypes was upregulated in the renal cells (≥1.6-fold; p<0.001). The same result was, however, only observed for TGFB1 haplotypes H-5 and H-AFR in the fibroblast cells (≥1.4-fold; p<0.01). This is interesting because no difference was seen between TGFB1 H-5 and H-6 basal promoter activity. To investigate whether histopathological severity of HIVAN correlated with TGF-β1 staining patterns, immunohistochemistry was performed on 20 renal biopsies from patients with HIVAN. A semi-quantitative "histo" score (H-score) was calculated by multiplying the percentage of positive cells with the intensity of the stain before comparing the scores with control biopsies (HIV-negative, n=3 and HIV-positive without HIVAN, n=3). Compared to the HIV-positive controls the kidney tubules of HIVAN biopsies had higher H-scores. Strikingly, the interstitium of HIVAN samples stained much more prominently (17/18) than both control groups suggesting that TGF-β1 staining in the renal interstitium appear to be specific for HIVAN. In conclusion this study shows that the African-specific haplotypes effect basal TGFB1 promoter activity and TGF-β1 protein levels. However, they do not seem to affect the cytoplasmic TGF-β1/pSmad3 protein levels in response to rhTGF-β1 autocrine stimulation. Immunohistochemistry results suggest that TGF-β1 pathway may be prominently dysregulated in the renal interstitium of HIVAN cases.
- ItemOpen AccessProfiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis(BioMed Central, 2019-01-29) Nel, Melissa; Prince, Sharon; Heckmann, Jeannine MBackground: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific ‘myocyte’ cultures. Methods From well-characterised MG patients we developed the ‘myocyte’ culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG). Results OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p < 0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r > 0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10− 6) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway. Conclusion Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.