The genetic dissection of fetal haemoglobin persistence in sickle cell disease in Nigeria.

The clinical severity of sickle cell disease (SCD) is strongly influenced by the level of fetal haemoglobin (HbF) persistent in each patient. Three major HbF loci (BCL11A, HBS1L-MYB, and Xmn1-HBG2) have been reported, but a considerable hidden heritability remains. We conducted a genome-wide association study for HbF levels in 1006 Nigerian patients with SCD (HbSS/HbSß0), followed by a replication and meta-analysis exercise in four independent SCD cohorts (3,582 patients). To dissect association signals at the major loci, we performed stepwise conditional and haplotype association analyses and included public functional annotation datasets. Association signals were detected for BCL11A (lead SNP rs6706648, ß = -0.39, P = 4.96 × 10-34) and HBS1L-MYB (lead SNP rs61028892, ß = 0.73, P = 1.18 × 10-9), whereas the variant allele for Xmn1-HBG2 was found to be very rare. In addition, we detected three putative new trait-associated regions. Genetically, dissecting the two major loci BCL11A and HBS1L-MYB, we defined trait-increasing haplotypes (P < 0.0001) containing so far unidentified causal variants. At BCL11A, in addition to a haplotype harbouring the putative functional variant rs1427407-'T', we identified a second haplotype, tagged by the rs7565301-'A' allele, where a yet-to-be-discovered causal DNA variant may reside. Similarly, at HBS1L-MYB, one HbF-increasing haplotype contains the likely functional small indel rs66650371, and a second tagged by rs61028892-'C' is likely to harbour a presently unknown functional allele. Together, variants at BCL11A and HBS1L-MYB SNPs explained 24.1% of the trait variance. Our findings provide a path for further investigation of the causes of variable fetal haemoglobin persistence in sickle cell disease.

Serum concentrations of IL-16 and its genetic polymorphism rs4778889 affect the susceptibility and severity of endometriosis in Nigerian women.

BACKGROUND: Endometriosis is the presence of active ectopic endometrial glands and stroma at other sites outside the uterine cavity. It is a common cause of chronic pelvic pain which is sometimes debilitating, and inflammation is one of the known triggers of endometriosis. Interleukins 6 and 16 (IL-6 and IL-16) are proinflammatory cytokines which play essential roles in inflammatory diseases. We therefore investigated the relationship between genetic polymorphisms of interleukins 6 and 16, and the development of endometriosis in Nigerian women. METHOD: One hundred and thirty (130) consenting women were consecutively enrolled, sixty-five (65) of whom had endometriosis and 65 age-matched women as reference group, surgically confirmed as not having endometriosis. Spectrophotometric determination of serum concentrations of Interleukins 6 and 16 was carried out and the genotyping of IL-6 (rs1800795) and IL-16 (rs4778889, rs11556218, rs4072111) genes were performed using TaqMan assays. RESULTS: Serum IL-16 concentration was significantly higher in women with severe chronic pelvic pain compared to those with mild pain (p = 0.023). The C allele of rs4778889 was associated with endometriosis (OR: 1.80, 95% CI: 1.08 - 3.02, p = 0.024). CONCLUSION: Serum IL-16 and IL-16 rs4778889 may be important markers for endometriosis in Nigerian, and by extension, African women. Multicentre African studies would clarify this.

The COPILOT Raw Illumina Genotyping QC Protocol.

The Illumina genotyping microarrays generate data in image format, which is processed by the platform-specific software GenomeStudio, followed by an array of complex bioinformatics analyses that rely on various software, different programming languages, and numerous dependencies to be installed and configured correctly. The entire process can be time-consuming, can lead to reproducibility errors, and can be a daunting task for bioinformaticians. To address this, we introduce the COPILOT protocol, which has been successfully used to transform raw Illumina genotype intensity data into high-quality analysis-ready data on tens of thousands of human patient samples that have been genotyped on a variety of Illumina genotyping arrays. This includes processing both mainstream and custom content genotyping chips with over 4 million markers per sample. The COPILOT QC protocol consists of two distinct tandem procedures to process raw Illumina genotyping data. The first protocol is an up-to-date process to systematically QC raw Illumina microarray genotyping data using the Illumina-specific GenomeStudio software. The second protocol takes the output from the first protocol and further processes the data through the COPILOT (Containerised wOrkflow for Processing ILlumina genOtyping daTa) containerized QC pipeline, to automate an array of complex bioinformatics analyses to improve data quality through a secondary clustering algorithm and to automatically identify typical Genome-Wide Association Study (GWAS) data issues, including gender discrepancies, heterozygosity outliers, related individuals, and population outliers, through ancestry estimation. The data is returned to the user in analysis-ready PLINK binary format and is accompanied by a comprehensive and interactive HTML summary report file which quickly helps the user understand the data and guides the user for further data analyses. The COPILOT protocol and containerized pipeline are also available at https://khp-informatics.github.io/COPILOT/index.html. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Processing raw Illumina genotyping data using GenomeStudio Basic Protocol 2: COPILOT: A containerised workflow for processing Illumina genotyping data.

Genetic polymorphisms of Vascular Endothelial Growth Factor (VEGF) associated with endometriosis in Nigerian women.

OBJECTIVE: To determine if genetic polymorphism of VEGF is associated with the development of endometriosis in Nigerian women. STUDY DESIGN: Case control study of 100 women (50 healthy controls and 50 with endometriosis). Serum VEGF concentration of participants were determined using enzyme-linked immunosorbent assay (ELISA) technique. Genomic DNAs were isolated from peripheral blood samples and quantified by nanodrop spectrophotometer one. Single nucleotide polymorphisms genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS: Mean age of participants was 32.96 ± 6.91 years for control and 32.04 ± 7.56 years for cases. VEGF levels in case and control groups were not statistically different (82.68 pg/ml [69.11-121.11 pg/ml] vs. 82.81 pg/ml [72.90-113.82 pg/ml] respectively; p = 0.967). All four genotypes examined were in Hardy-Weinberg equilibrium. Minor allele frequency of - 460T > C, - 1154G > A, + 936C > T and + 2578C > A were 24%, 8%, 6% and 10% in the control and 19%, 9%, 5% and 14% in endometriosis patients. However, allele and genotype distributions of - 460T > C, - 1154G > A, + 936C > T and + 2578C > A VEGF polymorphisms in endometriosis patients and control were not significantly different (p > 0.05). CONCLUSION: Our preliminary findings revealed no association between endometriosis and - 460T > C, - 1154G > A, + 936C > T and + 2578C > A of VEGF genes among Nigerian women.

Association of alpha-thalassemia and Glucose-6-Phosphate Dehydrogenase deficiency with transcranial Doppler ultrasonography in Nigerian children with sickle cell anemia.

BACKGROUND: Stroke is a devastating complication of sickle cell anemia (SCA) and can be predicted through abnormally high cerebral blood flow velocity using transcranial Doppler Ultrasonography (TCD). The evidence on the role of alpha-thalassemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the development of stroke in children with SCA is conflicting. Thus, this study investigated the association of alpha-thalassemia and G6PD(A- ) variant with abnormal TCD velocities among Nigerian children with SCA. METHODS: One hundred and forty-one children with SCA were recruited: 72 children presented with normal TCD (defined as the time-averaged mean of the maximum velocity: < 170 cm/s) and 69 children with abnormal TCD (TAMMV ≥ 200 cm/s). Alpha-thalassemia (the α-3.7 globin gene deletion) was determined by multiplex gap-PCR, while G6PD polymorphisms (202G > A and 376A > G) were genotyped using restriction fragment length polymorphism-polymerase chain reaction. RESULTS: The frequency of α-thalassemia trait in the children with normal TCD was higher than those with abnormal TCD: 38/72 (52.8%) [α-/ α α: 41.7%, α -/ α -: 11.1%] versus 21/69 (30.4%) [α-/ α α: 27.5%, α -/ α -: 2.9%], and the odds of abnormal TCD were reduced in the presence of the α-thalassemia trait [Odds Ratio: 0.39, 95% confidence interval: 0.20-0.78, p = 0.007]. However, the frequencies of G6PDA- variant in children with abnormal and normal TCD were similar (11.6% vs. 15.3%, p = 0.522). CONCLUSION: Our study reveals the protective role of α-thalassemia against the risk of abnormal TCD in Nigerian children with SCA.

Fetal-haemoglobin enhancing genotype at BCL11A reduces HbA2 levels in patients with sickle cell anaemia.

Understanding the interplay of genetic factors with haemoglobin expression and pathological processes in sickle cell disease is important for pharmacological and gene-therapeutic interventions. In our nascent study cohort of Nigerian patients, we found that three major disease-modifying factors, HbF levels, α-thalassaemia deletion and BCL11A genotype, had expected beneficial haematological effects. A key BCL11A variant, while improving HbF levels (5.7%-9.0%), also led to a small, but significant decrease in HbA2. We conclude that in general, interventions boosting HbF are likely to reduce HbA2 in patients' erythroid cells and that such therapeutic strategies might benefit from a parallel stimulation of HbA2 through independent mechanisms.

Barriers to the use of hydroxyurea in the management of sickle cell disease in Nigeria.

Hydroxyurea (HU) is a well-known Hb F-inducing agent with proven clinical and laboratory efficacy for patients with sickle cell disease. However, concerns about its long-term safety and toxicity have limited its prescription by physicians and acceptability by patients. Thus, this study aims to evaluate clinician's barriers to the use of HU in the management of patients with sickle cell disease in Nigeria. An online survey targeted physicians in pediatrics, hematology, medicine, family medicine and general medical practice managing sickle cell disease in Nigeria. The survey was in four sections: demographic, knowledge and experience with HU, and barriers to the use of HU. Ninety-one (73.0%) of 123 contacts completed the survey. Seventy-three percent and 74.0% of the respondents noted that HU reduced transfusion rates and improved overall quality of life (QOL) of patients, respectively. While the majority of the practitioners (55.6%) see between 10-50 patients per month, most (66.7%) write <5 prescriptions for HU per month. Lack of a national guideline for use of HU, especially in children (52.0%), concern for infertility (52.0%), and safety profile of HU in pregnancy and lactation (48.2%), top the factors considered by the respondents as major barriers to the use of HU. Hydroxyurea is grossly under prescribed in Nigeria, despite that the vast majority of physicians who attend patients with sickle cell disease know about its clinical efficacy. Evidence-based clinical practice guidelines could be explored as a way to standardize practices and improve confidence of practitioners to improve physicians' prescription of HU in the management of sickle cell disease.

Impact of training of mothers, drug shop attendants and voluntary health workers on effective diagnosis and treatment of malaria in Lagos, Nigeria.

BACKGROUND: The National Malaria Eradication Program and international agencies are keen on scaling up the use of malaria rapid diagnostic tests (mRDTs) and artemisinin-based combination therapies (ACTs) for effective diagnosis and treatment of the disease. However, poor diagnostic skills and inappropriate treatment are limiting the efforts. In Nigeria, a large proportion of infected patients self-diagnose and treat while many others seek care from informal drug attendants and voluntary health workers. AIMS: This study describes the impact of training voluntary health workers, drug shop attendants, and mothers on effective case detection and treatment of malaria in Lagos, Nigeria. METHODS: We trained mothers accessing antenatal care, drug shop attendants, and voluntary health workers selected from the three districts of Lagos, on the use of histidine-rich protein-2-based mRDTs and ACTs. Pre- and post-training assessments, focus group discussions (FGDs), and in-depth interviews (IDIs) were carried out. RESULTS: The knowledge, attitude, and skill of the participants to achieve the goal of "test, treat, and track" using mRDT and ACTs were low (11%-55%). There was a low awareness of other non-malaria fevers among mothers. Self-medication was widely practiced (31.3%). FGDs and IDIs revealed that health-care providers administered antimalarials without diagnosis. Training significantly improved participants' knowledge and expertise on the use of mRDTs and ACTs (P = 0.02). The participants' field performance on mRDT use was significantly correlated with their category (bivariate r = 0.51, P = 0.001). There was no statistically significant association between the participants' level of education or previous field experience and their field performance on mRDT (r = 0.12, P = 0.9; χ 2= 38, df = 2 and P = 0.49). CONCLUSION: These findings suggest that training of stakeholders in malaria control improves diagnosis and treatment of malaria. However, a broader scope of training in other settings may be required for an effective malaria control in Nigeria.

Current perspectives of sickle cell disease in Nigeria: changing the narratives.

Introduction: Sickle cell disease (SCD) is an inherited blood disorder characterized by clinical heterogeneity that may be influenced by environmental factors, ethnicity, race, social and economic factors as well as genetic and epigenetic factors. Areas covered: The present review was carried out to provide a comprehensive assessment of the current burden of SCD and treatments available for persons with SCD in Nigeria with the aim of identifying surveillance and treatment gaps, informing to guide the planning and implementation of better crisis prevention measures for SCD patients and set an agenda for new areas of SCD research in the country. This review assessed medical, biomedical and genetic studies on SCD patients in Nigeria and other endemic countries of the world. Expert opinion: Integration of hydroxyurea therapy into the management of SCD and surveillance via new-born screening (NBS) for early detection and management will improve the survival of persons with SCD in Nigeria. However, it will be important to carry out pilot studies, initiate strategic advocacy initiatives to educate the people about NBS benefits, develop collaborations between potential stakeholders and design sustainable financing scheme.

A survey of genetic fetal-haemoglobin modifiers in Nigerian patients with sickle cell anaemia.

Genetic variants at three quantitative trait loci (QTL) for fetal haemoglobin (HbF), BCL11A, HBS1L-MYB and the ß-globin gene cluster, have attracted interest as potential targets of therapeutic strategies for HbF reactivation in sickle cell anaemia (SCA). We carried out the first systematic evaluation of critical single nucleotide polymorphisms at these disease modifier loci in Nigerian patients with SCA. Common variants for BCL11A and HBS1L-MYB were strongly associated with HbF levels. At both loci, secondary association signals were detected, illustrating the mapping resolution attainable in this population. For BCL11A, the two independent sites of association were represented by rs1427407 (primary site, p = 7.0 x 10(-10)) and rs6545816 (secondary site, conditioned on rs1427407: p = 0.02) and for HBS1L-MYB by rs9402686 (HMIP-2B, p = 1.23 x 10(-4)) and rs66650371 (HMIP-2A, p = 0.002). Haplotype analysis revealed similarities in the genetic architecture of BCL11A and HBS1L-MYB in Nigerian patients. Variants at both loci also alleviated anaemia. The variant allele for the γ globin gene promoter polymorphism XmnI-HBG2 was too infrequent in our patients to be evaluated in this relatively small study. Studying the large and diverse SCA patient populations in African countries such as Nigeria will be key for a clearer understanding of how these loci work and for the discovery of new disease modifier genes.

Genetic Polymorphisms of Glucose-6-Phosphate Dehydrogenase in Lagos, Nigeria.

Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme in the pentose phosphate pathway that prevents oxidative damage to cells. This study determined the genotypic and allelic frequencies of G6PD G202A and A376G and also investigated correlation between G6PD polymorphisms and hemoglobin (Hb) phenotypes in children in Lagos, Nigeria. Seventy-eight children [55 with Hb AA (ßΑ/ßA) and 23 with Hb AS (ßΑ/ßS) trait] and 65 Hb SS (ßS/ßS) (HBB: c.20A>T) subjects in steady state with age range between 5-15 years were recruited for the study. Hemoglobin phenotypes of all study participants were carried out using alkaline electrophoresis and solubility tests. Genomic DNA was extracted from whole blood and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was used to determine the G202A and the A376G mutations of the G6PD gene. The genotype and allele distributions of G6PD G202A and A376G according to the Hb phenotypes were not statistically significant (p > 0.05). The minor allele frequency 202A was 0.15 (15.0%) and 0.14 (14.0%) in cases and controls, respectively. The overall frequency of 376G allele in the case group was 0.35 (35.0%) and 0.38 (38.0%) in the control group. No statistical significance was observed in the genotype and allele distributions of A376G in both the case and control groups (p > 0.05). The G6PD A- frequency in Hb SS subjects and the control group were 6.2 and 2.6%, respectively. G6PD G202A and A376G polymorphisms were not associated with Hb phenotypes and the allele distributions of 202A and 376G in this study are typical of West African populations.

Haemoglobin oxygen saturation, leucocyte count and lactate dehydrogenase are predictors of elevated cerebral blood flow velocity in Nigerian children with sickle cell anaemia.

BACKGROUND: Transcranial Doppler ultrasound (TCD) scan, which measures blood flow velocity through the time-averaged mean of maximum velocities (TAMMVs) in the internal carotid arteries and middle cerebral arteries, is a useful screening tool for predicting stroke risk in children with sickle cell anaemia (SCA). AIM: To investigate which clinical and laboratory indices predict abnormal TCD velocity in children with SCA. METHODS: Fifty-four SCA patients with normal TCD (TAMMV < 170 cm/s), classified as negative TCD (NTCD), and 93 patients with conditional and abnormal TCD velocities (TAMMV ≥ 170 cm/s) classified as positive TCD were recruited. The haemoglobin oxygen saturation, haematological variables, nitric oxide metabolites and lactate dehydrogenase activity of the patients were analysed. RESULTS: The mean (SD) age was 7.16 (3.84) years (range 2-16). The median SpO2 of the patients in the positive TCD group was significantly lower than that of the negative TCD group (p = 0.002). Multivariate logistic regression analysis indicated that the MCV [odds ratio (OR) 1.12, 95% confidence interval (CI) 1.04-1.22, p = 0.01)], MCH (OR 1.34, 95% CI 1.02-1.77, p = 0.04), leucocyte count (OR 1.26, 95% CI 1.07-1.49, p = 0.01) and lactate dehydrogenase (LDH) level (OR 1.00, 95% CI 1.00-1.01, p = 0.01) were independent predictors of high cerebral blood flow velocities. CONCLUSIONS: These clinical and laboratory indices are characteristic of chronic hypoxia and severe anaemia and are predictors of abnormal cerebral blood flow velocity. They can be used to predict stroke risk in children with SCA when access to TCD screening is limited.

Health related quality of life and perception of stigmatisation in adolescents living with sickle cell disease in Nigeria: A cross sectional study.

BACKGROUND: Sickle cell disease impacts the physical, emotional and psychological aspects of life of the affected persons, often times exposing them to disease associated stigma from the society and alters the health related quality of life (HRQoL). This study compared the HRQoL of adolescents with sickle cell disease with their healthy peers, identified socio-demographic and clinical factors impacting HRQoL, and determined the extent and effects of SCD related stigma on quality of life. PROCEDURE: We conducted a cross-sectional survey among 160 adolescents, 80 with SCD and 80 adolescents without SCD. Socio-demographic and clinical data were collected using a pre-tested questionnaire. HRQoL was investigated using the Short Form (SF-36v2) Health Survey. SCD perceived stigma was measured using an adaptation of a perceived stigma questionnaire. RESULTS: Adolescents with SCD have significantly worse HRQoL than their peers in all of the most important dimensions of HRQoL (physical functioning, physical roles limitation, emotional roles limitation, social functioning, bodily pain, vitality and general health perception) except mental health. Recent hospital admission and SCD related complication further lowered HRQoL scores. Over seventy percent of adolescents with SCD have moderate to high level of perception of stigmatisation. Hospitalisation, SCD complication, SCD stigma were inversely, and significantly associated with HRQoL. CONCLUSIONS: Adolescents living with SCD in Nigeria have lower health related quality of life compared to their healthy peers. They also experience stigma that impacts their HRQoL. Complications of SCD and hospital admissions contribute significantly to this impairment.

Evaluation of high performance liquid chromatography (HPLC) pattern and prevalence of beta-thalassaemia trait among sickle cell disease patients in Lagos, Nigeria.

INTRODUCTION: Sickle cell disease (SCD) is the most common inherited disorder of haemoglobin worldwide. This study evaluated the chromatographic patterns and red blood cell indices of sickle cell patients to determine the co-inheritance of other haemoglobin(Hb) variants and ß-thalassaemia trait. METHODS: Red cell indices, blood film, sickle solubility test, Hb electrophoresis using alkaline cellulose acetate membrane, and chromatographic patterns using Bio Rad HPLC Variant II were evaluated for 180 subjects. RESULTS: Based on low MCV <76fL and MCH<25 pg, in the presence of elevated A2 >4.0% on HPLC and Hb variants eluting outside the S and C windows, at least four haemoglobin phenotypes (SS: 87.7%; SC: 1.1%; SD Punjab: 0.6%; Sß-thalassemia: 10.6%) were identified. Mean Hb F% was 8.1±5.1 (median 7.65) for Hb SS and 6.03±5.2 (median 3.9) for Hb Sß-thalassemia trait. Majority of Hb SS (69.1%) had Hb F% less than 10 while 27.6% had 10-19.9 and 3.2% had ≥ 20. Mean Hb F% was higher in female Hb SS (9.55±5.09; mean age 7.4±3.8 years) than the males (7.63±4.80; mean age 6.9±3.8 years) (P=0.02). A borderline significant negative correlation between age and Hb F levels among Hb SS subjects (r= -0.169 P=0.038) was also observed. CONCLUSION: Our data suggests that α and ß- thalassaemia traits, and other haemoglobin variants co-exist frequently with SCD in our population.

Capacity building and stroke risk assessment in Nigerian children with sickle cell anaemia.

BACKGROUND: Transcranial Doppler ultrasonography (TCD) measures blood flow velocities in the large cerebral vessels and thus, detects the risk of stroke. This report describes a capacity building program which enabled the use of TCD for detecting stroke risk and also describes the pattern of non-imaging TCD examinations seen in Nigerian children with sickle cell anaemia (SCA). PROCEDURE: Ten university graduates were trained on the use of TCD in a 5-day capacity building workshop after which, the three best candidates were employed to provide a 5-day a week TCD screening service in Lagos. Data from TCD examination collected between March 2011 and September 2013 were analysed and reported. RESULT: Between March 2011 and September 2013, 2,331 children with SCA aged 2-16 years had TCD studies. TCD's findings were classified as normal (standard risk) in 70.4%, conditional in 19% and abnormal (high risk) in 9.3%. The majority of children (76.9%) in the high risk category were aged 2-8 years. TCD study was inadequate for risk categorisation in 1.3% of the patients. CONCLUSION: Effective capacity building of middle level manpower is feasible and can provide a credible TCD screening service to communities with a high demand and a shortage of trained professionals. The pattern of TCD abnormalities seen in Africa are comparable to those obtained in several previous worldwide reports.

Circulating adipokine levels in type 2 diabetes mellitus in Lagos, Nigeria.

BACKGROUND: Type 2 diabetes remains a global concern with its numerical increase occurring in developing countries which include Nigeria. Adipose tissue-secreted factors called "adipokines" are involved in energy homeostasis and regulation of glucose and lipid metabolism. OBJECTIVE: This study was undertaken with the aim of investigating adipokine levels in the Type 2 Diabetes Mellitus. METHODS: This is a cross sectional study conducted in Lagos University Teaching Hospital (LUTH), a-700 bed tertiary hospital centre in Lagos, Nigeria. 53 diabetic subjects and 27 non-diabetic controls with mean age (56.72 +/- 10.44) and (38.67 +/- 9.63) years respectively, were recruited into the study. Fasting blood glucose, HbA1 C, leptin and resistin levels and body mass index (BMI) were assayed. RESULTS: Mean BMI was higher but not statistically significant in diabetics than in non diabetics (diabetics 28.77 +/- 5.35; non diabetics 27.38 +/- 6.04; p > 0.05). Resistin level was significantly higher in diabetics (31.26 +/- 2.5) as compared with non diabetics (16.61 +/- 2.16) compared to non-diabetics. Leptin correlated very strongly with BMI (r = 0.620, p < 0.0001) and was significantly higher in females than males (female 9.72 +/- 1.70; male 1.79 +/- 0.54; p < 0.0001). CONCLUSION: The circulating adipokines have variable effect on the glucose and fat metabolism. BMI and resistin level were higher in diabetics. Leptin was found in this study to correlate very strongly with BMI (adiposity). There was also a strong gender dependence observed as leptin level was signficantly higher in females than males.