Browsing by Author "Nembaware, Victoria"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Open Access
    Genome-wide survey of allele-specific splicing in humans
    (BioMed Central Ltd, 2008) Nembaware, Victoria; Lupindo, Bukiwe; Schouest, Katherine; Spillane, Charles; Scheffler, Konrad; Seoighe, Cathal
    BACKGROUND: Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. RESULTS: We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST) and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. CONCLUSION: We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array data, including several examples for which there is experimental evidence of polymorphisms affecting splicing in the literature. We also present a set of novel allele-specific splicing candidates and discuss the strengths and weaknesses of alternative technologies for inferring the effect of sequence variants on mRNA splicing.
  • No Thumbnail Available
    Item
    Open Access
    Investigating variants associated with quantitative red blood cell indices from TOPMed predictions in African Sickle Cell Disease populations
    (University of Cape Town, 2026) Samie, Nabeelah; Wonkam, Ambroise; Mnika, Khuthala; Esoh, Kevin; Nembaware, Victoria
    Introduction Sickle cell disease (SCD) is mostly caused, in homozygous state, by a single point mutation (c.20A>T, p.Glu7Val), which results in the formation of mutant haemoglobin S (HbS) instead of haemoglobin A. It is a life-long inherited recessive disorder that predominantly affects people of African, Indian, and Mediterranean descent. The polymerisation of HbS within red blood cells (RBCs) is the primary pathophysiological alteration in SCD, which results in the sickle shape of RBCs. Sickle-shaped RBCs are more prone to haemolysis and they block blood flow, resulting in anaemia, hypoxia painful episodes, and multiple organ damage. Haematological indices include haematocrit (HTC), haemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), RBC width, RBC count, white blood cell (WBC) count, lymphocyte count, monocyte count, platelet count, HbA2 and HbF percentage. Haematological indices indicate the size, development, and haemoglobin content of RBCs. Consequently, haematological indices are routinely measured as a means of monitoring SCD. Because these indices are potentially associated with genomic variation, extensive genome-wide association studies have been performed to determine variants associated with haematological indices. Many common variants were identified, however, non-European populations such as African and Asian populations are underrepresented in these studies. Whole genome sequencing, a method used for detecting novel variants, recently resulted in the identification of 14 novel variants associated with haematological indices in 62,653 ethnically diverse participants from the Trans-Omics for Precision Medicine (TOPMed) program, in the United States of America (USA). Namely: HBB (rs11549407 and rs34598529), RPN1 (rs112097551), ELL2 (rs116635225), 10q26 (rs986415672), MIDN (rs73494666), G6PD (rs76723693), SHANK2 (rs535577177), TMPRSS6 (rs228914), HBA1/2 (rs868351380 and rs372755452), EIF5/MARK3 (rs370308370), PIEZO1 (rs763477215) and 19q12 (rs1368500441). The association between these variants and haematological indices have not yet been investigated in African SCD patients. Aim This study aims to investigate 13 novel haematological indices associated variants, previously found in the TOPMed project, among SCD patients from Cameroon and Senegal. Methods Study participants: This study included individuals with SCD recruited from various regions of Cameroon and Senegal and ethnically matched non-SCD individuals recruited from various regions of Cameroon. Molecular methods: Genetic characterisation was performed using polymerase chain reaction (PCR) and TaqMan SNP genotyping. Capillary electrophoresis and Sanger sequencing were also performed on a subset of samples for genotype validations. Statistical analysis: Descriptive analysis was performed using IBM SPSS Statistics for Windows (version 25) on data collected from 804 SCD patients from Cameroon. The statistics performed included an analysis of sociodemographic factors, anthropometric variables and haematological indices. Association tests of SNPs were run using PLINK1.9 with the maximum permutation test procedure used to correct for multiple testing and to determine empirical significance levels. The logistic command of PLINK was used with 100,000 maximum permutations to test for significant SNPs while adjusting for sex and age. Results Both Cameroon and Senegal SCD patients had a higher percentage of females than males (53.9% and 54.1%, respectively). The median age was 16 (range: 1-66) and 21 (range: 5-45) for the Cameroon and Senegal SCD patients respectively. Among the Cameroon SCD patients, the majority did not have overt stroke (97%), kidney disease (91.2%) and pulmonary hypertension (93.4%), and 92.2% were not using hydroxyurea. Association analysis using Spearman's rank correlation test found age to be significantly negatively associated with the number of vaso-occlusive crises/year (pvalue: <0.001) and the number of hospitalisations/year (p-value: <0.001). Further analysis using Spearman's rank correlation test found a significantly positive association between the number of vaso-occlusive crises/year and MCHC (p-value: 0.039). Additionally, MCHC and HbA2 were found to be significantly positively associated with the number of hospitalisations/year (p-value: <0.001 and 0.015, respectively), while monocytes were found to be significantly negatively associated with the number of hospitalisations/year (p-value: 0.005). and Logistic regression analysis yielded five statistically significant associations between variants haematological indices. Namely, G6PD variant (NC_000023.11:g.154533025A>G) was associated with HGB, MCV and HCT; HBA1/2 variant (NC_000016.10:g.199622del) was associated with platelet count, and HBB variant (NC_000011.10:g.5227100T>C) was associated with HbF level. Eight of the 13 variants (ELL2 (rs116635225), HBB (rs11549407 and rs34598529), SHANK2 (rs535577177), HBA1/2 (rs372755452), PIEZO1 (rs763477215), MIDN (rs73494666) and 19q12 (rs1368500441)) minor allele frequencies (MAF) were found more commonly in individuals with SCD from Cameroon compared to all other populations, suggesting an enrichment among people living with SCD. Conclusions This study has revealed three variants in G6PD, alpha-thalassemia (HBA1/2), and beta-globin (HBB) genes significantly associated with various haematological indices in African SCD patients, which could have implications in SCD risk stratification models for anticipatory guidance in clinical practice. Most variants that were investigated (8/13) displayed a higher MAF in individuals with SCD, compared to other unselected global populations, in whom some variants were monomorphic i.e., European and Asian populations, probably due to the higher African genetic diversity. Alternatively, these differences in MAF, particularly when compared to control populations with the same ethnolinguistic background are suggesting an enrichment of targeted variants in people living with SCD, which are potentially due to the impact of the related genes on the pathobiology of SCD. Additional studies including specific GWAS on haematological indices in larger samples of people living with SCD from multiple African populations from diverse ethnolinguistic background, and geographical locations, will be needed to gain more insight in these SNPs, and new ones to be discovered. The outcomes of such studies will be beneficial for developing accurate predictive modelling tools to guide follow-up and treatment practices of SCD, as well as to unravelling new therapeutic targets.
  • No Thumbnail Available
    Item
    Open Access
    Risk factors associated with blood pressure variation in sickle cell disease in Cameroon
    (2024) Nguweneza, Arthemon; Wonkam, Ambroise; Nembaware, Victoria
    Background: In SCD patients, studies have shown that increased Blood Pressure (BP) is associated with higher risk of stroke and mortality, even in a range of systolic BP(SBP) and diastolic BP(DBP) that are considered relatively normal for the general population (i.e., lower than 140 mmHg). SCD patients, generally, have lower systolic, diastolic, and mean BP compared to age and sex-matched controls. Relative Systemic Hypertension (RSH) for SCD patients is defined as BP ranging from 120–139/70–89 mmHg, and Systemic Hypertension for SCD patients with BP above 140/90 mmHg. BP may be a potential modulator of clinical severity in SCD patients. BP is a heritable trait with estimates indicating that 30–70% of the trait variance is attributable to genetic variation. A recurrent deleterious and loss of function mutations in genes associated with lowering BP, e.g., variants in CLCN6, have been recently associate with long survival in SCD in Cameroon. Additionally, demographic, biological, and anthropometric factors have been reported to be associated with BP in SCD patients. Understanding the aetiology of BP variation in SCD patients and assessment of up-to-date evidence is key to early prediction of potential BP-related complications in SCD patients. Early prediction of BP-related complications in SCD patients could lead to better prevention and treatment of SCD and its associated causes of mortality. Herein, we investigated (clinical, genetic, and epidemiological) risk factors associated with RSH or Systemic Hypertension in SCD patients in Cameroon to gain insight into the pathophysiology of BP variation in this disease in an African setting. Objectives: 1) A systematic review and meta-analysis on BP variation (Normal, Relative Systemic Hypertension(RSH) and Hypertension) among SCD patients; 2) Investigate clinical and epidemiological risk factors for RSH and Systemic Hypertension in 815 SCD patients in Cameroon; 3) Identification of genetic variants associated to BP variation in SCD patients in Cameroon using a Genome-wide Association study; 4) Functional analysis on pathways and mechanisms underlying BP variation in SCD patients. Methods: Systematic review and meta-analysis: A protocol was developed and registered with the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD42020168798. A search was conducted on the following electronic databases: PubMed, Scopus, and Web of Science. The Preferred Reporting Items for Systematic Reviews and Meta- analysis (PRISMA) served as a template for reporting the review and meta-analysis. Independently, in collaboration with another reviewer, we screened studies, extracted relevant data, assessed methodological quality and risk of bias on each included study. A retrospective analysis to investigate risk factors associated with RSH or Systemic Hypertension in SCD patients in Cameroon. Participants who had incomplete/out of range BP readings were excluded from this analysis. Categorical variables were compared using Chi squared test or Fisher exact test if the expected count in a cell was less than five while continuous variables were compared according to BP category with the Kruskal–wallis test. Multivariate multinomial logistic regression modelling was used to examine the effects of the demographic, anthropometric, clinical, and laboratory factors to determine the potential independent risk factors for RSH and Systemic Hypertension. A final model was created that included all the predictors and interactions that were significantly associated at the level of P < 0.001) and gender (P = 0.022) were significantly different across the BP categories, with age increasing with BP. Weight (P < 0.001), height (P < 0.001), BMI (P < 0.001), pulse pressure (P = 0.020), history of stroke (P = 0.012), haemoglobin (P = 0.002), red blood cell count (P = 0.031), creatinine (P < 0.001), and eGFR (P= 0.002) were also significantly different across the three BP categories. Among SCD patients, univariate analyses indicated that these variables were significantly more common risk factors for higher BP values among patients with RSH than those with normal BP: Age (P < 0.001), patients >18 years (P < 0.001), weight (P < 0.001), height (P < 0.001), BMI (P < 0.001), pulse pressure (P = 0.046), creatinine (P < 0.001), eGFR (P < 0.001) and haemoglobin (P = 0.020) . Multivariate analyses found that age [OR = 1.02, (95% CI = 1.01–1.05), P = 0.021], creatinine [OR = 1.310, 95% CI = 1.05–1.63, P = 0.016], BMI [OR = 1.09, (95% CI = 1.03– 1.16), P = 0.002] were independent risk factors for high BP values in SCD patients with RSH compared with SCD patients with normal BP values. Genome-wide analysis and meta-analysis: For SBP, we found two genetic loci including independent and suggestive SNPs. The first locus was found in RP11-727A23.5 on chromosome 11 (rs146072506, P=4.88x10-08), the second locus was found on DLGAP1 gene on chromosome 18 (rs35715722, P=3.408x10-08). Two other SNPs were found in the intergenic regions. For DBP, the only significant SNP was in the intergenic region. Additionally, we found one SNP (rs186536474, p=8.90x10-09) located on GPR39 gene on chromosome 2 with a statistically significant p-value associated with BMI. Identified variants in intergenic regions may play a role in specific genes regulating BP or BMI. The meta-analysis of the Cameroon SCD cohort and Silent Infarct Transfusion Trial (SITT) cohorts, and African American cohort of SCD patients, did not find significant SNPs. After performing genetic analysis and Gene-set analysis with none of the identified genes or gene-set reached a significant threshold. Additionally, tissue expression analysis did not reach significant thresholds. Conclusion: This is the first in-depth investigation of the non-genetic and genetic risk factors associated with BP variation (RSH or Systemic Hypertension) in SCD patients in Africa, i.e., Cameroon. Age, male gender, BMI was found to be statistically significant independently associated factors of RSH and Hypertension in the SCD patients in Cameroon. The GWAS analysis identified a few SNPs which were statistically associated with BP and BMI in Cameroonian SCD patients. These SNPs require follow up studies in larger SCD cohorts. Tailored interventions that consider both genetic and non-genetic risk factors have potential to lead to better management of BP pressure in SCD patients and prevent possibly prevent development of severe SCD complications.
  • Thumbnail Image
    Item
    Open Access
    The H3ABioNet helpdesk: an online bioinformatics resource, enhancing Africa’s capacity for genomics research
    (2019-12-30) Kumuthini, Judit; Zass, Lyndon; Panji, Sumir; Salifu, Samson P; Kayondo, Jonathan K; Nembaware, Victoria; Mbiyavanga, Mamana; Olabode, Ajayi; Kishk, Ali; Wells, Gordon; Mulder, Nicola J
    Abstract Background Currently, formal mechanisms for bioinformatics support are limited. The H3Africa Bioinformatics Network has implemented a public and freely available Helpdesk (HD), which provides generic bioinformatics support to researchers through an online ticketing platform. The following article reports on the H3ABioNet HD (H3A-HD)‘s development, outlining its design, management, usage and evaluation framework, as well as the lessons learned through implementation. Results The H3A-HD evaluated using automatically generated usage logs, user feedback and qualitative ticket evaluation. Evaluation revealed that communication methods, ticketing strategies and the technical platforms used are some of the primary factors which may influence the effectivity of HD. Conclusion To continuously improve the H3A-HD services, the resource should be regularly monitored and evaluated. The H3A-HD design, implementation and evaluation framework could be easily adapted for use by interested stakeholders within the Bioinformatics community and beyond.