Clinical and genetic predictors of renal dysfunctions in sickle cell anaemia in Cameroon.

Micro-albuminuria and glomerular hyperfiltration are primary indicators of renal dysfunctions in Sickle Cell Disease (SCD), with more severe manifestations previously associated with variants in APOL1 and HMOX1 among African Americans. We have investigated 413 SCD patients from Cameroon. Anthropometric variables, haematological indices, crude albuminuria, albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) were measured. Patients were genotyped for 3·7 kb alpha-globin gene (HBA1/HBA2) deletion, and for variants in APOL1 (G1/G2; rs60910145, rs73885319, rs71785313) and HMOX1 (rs3074372, rs743811). The median age was 15 years; the majority presented with micro-albuminuria (60·9%; n = 248), and approximately half with glomerular hyperfiltration (49·5%; n = 200). Age, male sex, haemoglobin level, leucocyte count, mean corpuscular volume, blood pressure, body mass index and creatinine levels significantly affected albuminuria and/or eGFR. Co-inheritance of alpha-thalassaemia was protective against macro-albuminuria (P = 0·03). APOL1 G1/G2 risk variants were significantly associated with the ACR (P = 0·01) and borderline with eGFR (P = 0·07). HMOX1 - rs743811 was borderline associated with micro-albuminuria (P = 0·07) and macro-albuminuria (P = 0·06). The results revealed a high proportion of micro-albuminuria and glomerular hyperfiltration among Cameroonian SCD patients, and support the possible use of targeted genetic biomarkers for risks assessment.

Studies of novel variants associated with Hb F in Sardinians and Tanzanians in sickle cell disease patients from Cameroon.

High level of Hb F has been shown to improve survival in sickle cell disease. Among 453 Cameroonians with sickle cell disease, we have investigated 18 selected single-nucleotide polymorphisms (SNPs) in novel and suggestive loci associated with Hb F level identified through a genomewide association study in sickle cell disease patients in Tanzania, and whole-genome sequencing of a population from Sardinia. Seven of 10 variants reported in Sardinians were either monomorphic or very rare in the Cameroonians. No associations were observed with any SNPs and Hb F levels in Cameroonians affected by sickle cell disease. The present study illustrates the complexity of replicating Hb F-promoting variants association results across populations.

Distribution of HbS Allele and Haplotypes in a Multi-Ethnic Population of Guinea Bissau, West Africa: Implications for Public Health Screening.

Background: Sickle Cell Disease (SCD) is an inherited condition that is widespread globally and especially in malaria-endemic West African countries. Limited epidemiological data on SCD are available for Guinea Bissau, where newborn screening is not yet implemented, routine diagnosis is not available, and care is case directed. Methods: Dried blood spots were collected from children accessing two hospitals managed by Italian Non-Governmental Organizations in the capital city of Bissau and sent to Padova for Hemoglobin (Hb) quantification through HPLC and molecular analysis. Beta globin gene analysis was performed in all; and Hb haplotype of the HbSS and HbSA patients was performed in South Africa. One hundred samples belonging to the most frequent ethnic groups were randomly selected for detection of G6PD mutations. Results: Samples from 848 consecutive children (498 males and 350 females, mean age 6.8 years) accessing the two hospitals were analyzed: 6.95% AS (4.42% allelic frequency), 0.94% SS, and 0.23% AC. 376G G6PD allelic frequency was 24%; 14.8% in AS individuals. The Senegal haplotype was the most prevalent (31%), and the proposition of chromosomes with the atypical haplotype was surprisingly high (56%). Conclusion: Our study demonstrates a significant frequency of the HbS allele in the population of Guinea Bissau supporting the implementation of screening strategies. The differences among ethnic groups can help guide targeted interventions for SCD awareness campaigns and determine priority areas for public health interventions. The pilot analysis on haplotypes reveals a large proportion of the atypical haplotype, which may be indicative of a genetically heterogeneous population.

Genetic modifiers of long-term survival in sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) is a clinically heterogeneous, monogenic disorder. Medical care has less-than-optimal impact on clinical outcomes in SCA in Africa due to several factors, including patient accessibility, poor access to resources, and non-availability of specific effective interventions for SCA. METHODS: Against this background, we investigated 192 African participants who underwent whole exome sequencing. Participants included 105 SCA patients spanning variable clinical expression: a "long survivor" group (age over 40 years), a "stroke" group (at least one episode of overt stroke), and a "random" group (patients younger than 40 years without overt cerebrovascular disease). Fifty-eight ethnically matched homozygous hemoglobin A controls were also studied. Findings were validated in an independently recruited sample of 29 SCA patients. Statistical significance of the mutational burden of deleterious and loss-of-function variants per gene against a null model was estimated for each group, and gene-set association tests were conducted to test differences between groups. RESULTS: In the "long survivor" group, deleterious/loss-of-function variants were enriched in genes including CLCN6 (a voltage-dependent chloride channel for which rare deleterious variants have been associated with lower blood pressure) and OGHDL (important in arginine metabolism, which is a therapeutic target in SCA). In the "stroke" group, significant genes implicated were associated with increased activity of the blood coagulation cascade and increased complement activation, for example, SERPINC1, which encodes antithrombin. Oxidative stress and glutamate biosynthesis pathways were enriched in "long survivors" group. Published transcriptomic evidence provides functional support for the role of the identified pathways. CONCLUSIONS: This study provides new gene sets that contribute to variability in clinical expression of SCA. Identified genes and pathways suggest new avenues for other interventions.

Hydroxyurea-Induced miRNA Expression in Sickle Cell Disease Patients in Africa.

Hydroxyurea (HU) is clinically beneficial in sickle cell disease (SCD) through fetal hemoglobin (HbF) induction; however, the mechanism of HU is not yet fully elucidated. Selected miRNAs have been associated with HU-induced HbF production. We have investigated differential HU-induced global miRNA expression in peripheral blood of adult SCD patients in patients from Congo, living in South Africa. We found 22 of 798 miRNAs evaluated that were differentially expressed under HU treatment, with the majority (13/22) being functionally associated with HbF-regulatory genes, including BCL11A (miR-148b-3p, miR-32-5p, miR-340-5p, and miR-29c-3p), MYB (miR-105-5p), and KLF-3 (miR-106b-5), and SP1 (miR-29b-3p, miR-625-5p, miR-324-5p, miR-125a-5p, miR-99b-5p, miR-374b-5p, and miR-145-5p). The preliminary study provides potential additional miRNA candidates for therapeutic exploration.

SAR1a promoter polymorphisms are not associated with fetal hemoglobin in patients with sickle cell disease from Cameroon.

BACKGROUND: Reactivation of adult hemoglobin (HbF) is currently a dominant therapeutic approach to sickle cell disease (SCD). In this study, we have investigated among SCD patients from Cameroon, the association of HbF level and variants in the HU-inducible small guanosine triphosphate-binding protein, secretion-associated and RAS-related (SAR1a) protein, previously shown to be associated with HbF after HU treatment in African American SCD patients. RESULTS: Only patients >5 years old were included; hemoglobin electrophoresis and a full blood count were conducted upon arrival at the hospital. RFLP-PCR was used to describe the HBB gene haplotypes and Gap PCR to investigate the 3.7 kb α-globin gene deletion. The iPLEX Gold Sequenom Mass Genotyping Array and cycle sequencing were used for the genotyping of four selected SNPs in SAR1a (rs2310991; rs4282891; rs76901216 and rs76901220). Genetic analysis was performed using an additive genetic model, under a generalized linear regression framework. 484 patients were studied. No associations were observed between any of the promoter variants and baseline HbF, clinical events or other hematological indices. CONCLUSION: The results of this study could be explained by possible population-specificity of some tagging genomic variants associated with HbF production and illustrated the complexity of replicating HbF-promoting variants association results across African populations.

Hydroxyurea down-regulates BCL11A, KLF-1 and MYB through miRNA-mediated actions to induce γ-globin expression: implications for new therapeutic approaches of sickle cell disease.

BACKGROUND: The major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to increase HbF with less toxicity. This paper investigated whether HU-induced HbF could operate through post-transcriptional miRNAs regulation of BCL11A, KLF-1 and MYB, potent negative regulators of HbF. Both ex vivo differentiated primary erythroid cells from seven unrelated individuals, and K562 cells were treated with hydroxyurea (100 µM) and changes in BCL11A, KLF-1, GATA-1, MYB, ß- and γ-globin gene expression were investigated. To explore potential mechanisms of post-transcriptional regulation, changes in expression of seven targeted miRNAs, previously associated with basal γ-globin expression were examined using miScript primer assays. In addition, K562 cells were transfected with miScript miRNA inhibitors/anti-miRNAs followed by Western Blot analysis to assess the effect on HbF protein levels. Direct interaction between miRNAs and the MYB 3'-untranslated region (UTR) was also investigated by a dual-luciferase reporter assays. RESULTS: Down-regulation of BCL11A and MYB was associated with a sevenfold increase in γ-globin expression in both primary and K562 cells (p < 0.003). Similarly, KLF-1 was down-regulated in both cell models, corresponding to the repressed expression of BCL11A and ß-globin gene (p < 0.04). HU induced differential expression of all miRNAs in both cell models, particularly miR-15a, miR-16, miR-26b and miR-151-3p. An HU-induced miRNAs-mediated mechanism of HbF regulation was illustrated with the inhibition of miR-26b and -151-3p resulting in reduced HbF protein levels. There was direct interaction between miR-26b with the MYB 3'-untranslated region (UTR). CONCLUSIONS: These experiments have shown the association between critical regulators of γ-globin expression (MYB, BCL11A and KLF-1) and specific miRNAs; in response to HU, and demonstrated a mechanism of HbF production through HU-induced miRNAs inhibition of MYB. The role of miRNAs-mediated post-transcriptional regulation of HbF provides potential targets for new treatments of SCD that may minimize alterations to the cellular transcriptome.

An Expert Review of Pharmacogenomics of Sickle Cell Disease Therapeutics: Not Yet Ready for Global Precision Medicine.

Sickle cell disease (SCD) is a blood disease caused by a single nucleotide substitution (T > A) in the beta globin gene on chromosome 11. The single point mutation (Glu6Val) promotes polymerization of hemoglobin S (HbS) and causes sickling of erythrocytes. Vaso-occlusive painful crises are associated with recurrent and long-term use of analgesics/opioids and hydroxyurea (HU) by people living with SCD. The present analysis offers a state-of-the-art expert review of the effectiveness of pharmacogenomics/genetics of pain management in SCD, with specific focus on HU and opioids. The literature search used the following keywords: SCD, pharmacogenomics, pharmacogenetics, pain, antalgics, opioids, morphine, and HU. The literature was scanned until March 2016, with specific inclusion of targeted landmark and background articles on SCD. Surprisingly, our review identified only a limited number of studies that addressed the genetic/genomic basis of variable responses to pain (e.g., variants in OPRM1, HMOX-1, GCH1, VEGFA COMT genes), and pharmacogenomics of antalgics and opioids (e.g., variants in OPRM1, STAT6, ABCB1, and COMT genes) in SCD. There has been greater progress made toward identifying the key genomic variants, mainly in BCL11A, HBS1L-MYB, or SAR1, which contribute to response to HU treatment. However, the complete picture on pharmacogenomic determinants of the above therapeutic phenotypes remains elusive. Strikingly, no study has been conducted in sub-Saharan Africa where majority of the patients with SCD live. This alerts the broader global life sciences community toward the existing disparities in optimal and ethical targeting of research and innovation investments for SCD specifically and precision medicine and pharmacology research broadly.

Genetics of Sickle Cell-Associated Cardiovascular Disease: An Expert Review with Lessons Learned in Africa.

Sickle cell disease (SCD) vastly impacts the African continent and is associated with cardiovascular diseases. Stroke, kidney disease, and pulmonary hypertension are considered as proxies of severity in SCD with several genomic loci implicated in their heritability. The present expert review examined the current data on epidemiology and genetic risk factors of stroke, pulmonary hypertension, and kidney disease associated with SCD, as indexed in PubMed® and Google Scholar®. Studies collectively show that stroke and kidney disease each affect ∼10% of SCD patients, with pulmonary hypertension displaying a higher prevalence of 30% among adults with SCD. There is some evidence that these epidemiology figures may be an underestimate in SCD patients living in Africa. A modest number of publications have identified genetic factors involved in pathways regulating inflammation, coagulation, cell adhesion, heme degradation, α-globin and γ-globin production, and others, which contribute to the development risk of targeted cardiovascular phenotypes. However, in most cases, these studies have not been validated across populations. There is therefore an urgent need for large-scale genome-wide association, whole-exome and whole-genome studies, and multiomics research on cardiovascular diseases associated with SCD, particularly in Africa, to allow for proportional investment of global research funding on diseases that greatly impact the African continent. Ultimately, this will cultivate socially responsible research investments and identification of at-risk individuals with improved preventive medicine, which should be a cornerstone of global precision medicine.

Proceedings of a Sickle Cell Disease Ontology workshop - Towards the first comprehensive ontology for Sickle Cell Disease.

Sickle cell disease (SCD) is a debilitating single gene disorder caused by a single point mutation that results in physical deformation (i.e. sickling) of erythrocytes at reduced oxygen tensions. Up to 75% of SCD in newborns world-wide occurs in sub-Saharan Africa, where neonatal and childhood mortality from sickle cell related complications is high. While SCD research across the globe is tackling the disease on multiple fronts, advances have yet to significantly impact on the health and quality of life of SCD patients, due to lack of coordination of these disparate efforts. Ensuring data across studies is directly comparable through standardization is a necessary step towards realizing this goal. Such a standardization requires the development and implementation of a disease-specific ontology for SCD that is applicable globally. Ontology development is best achieved by bringing together experts in the domain to contribute their knowledge. The SCD community and H3ABioNet members joined forces at a recent SCD Ontology workshop to develop an ontology covering aspects of SCD under the classes: phenotype, diagnostics, therapeutics, quality of life, disease modifiers and disease stage. The aim of the workshop was for participants to contribute their expertise to development of the structure and contents of the SCD ontology. Here we describe the proceedings of the Sickle Cell Disease Ontology Workshop held in Cape Town South Africa in February 2016 and its outcomes. The objective of the workshop was to bring together experts in SCD from around the world to contribute their expertise to the development of various aspects of the SCD ontology.

A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease.

AIM: To report on molecular mechanisms of fetal hemoglobin (HbF) induction by hydroxyurea (HU) for the treatment of sickle cell disease. STUDY DESIGN: Systematic review. RESULTS: Studies have provided consistent associations between genomic variations in HbF-promoting loci and variable HbF level in response to HU. Numerous signal transduction pathways have been implicated, through the identification of key genomic variants in BCL11A, HBS1L-MYB, SAR1 or XmnI polymorphism that predispose the response to the treatment, and signal transduction pathways that modulate γ-globin expression (cAMP/cGMP; Giα/c-Jun N-terminal kinase/Jun; methylation and miRNA). Three main molecular pathways have been reported: i) Epigenetic modifications, transcriptional events and signaling pathways involved in HU-mediated response, ii) Signaling pathways involving HU-mediated response and iii) Post-transcriptional pathways (regulation by miRNAs). CONCLUSIONS: The complete picture of HU-mediated mechanisms of HbF production in Sickle Cell Disease remains elusive. Research on post-transcriptional mechanisms could lead to therapeutic targets that may minimize alterations to the cellular transcriptome.

Beta-globin gene haplotypes among cameroonians and review of the global distribution: is there a case for a single sickle mutation origin in Africa?

Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n = 799) and Cameroon (19%; n = 207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation.

Association between Variants at BCL11A Erythroid-Specific Enhancer and Fetal Hemoglobin Levels among Sickle Cell Disease Patients in Cameroon: Implications for Future Therapeutic Interventions.

Variants in BCL11A were previously associated with fetal hemoglobin (HbF) levels among Cameroonian sickle cell disease (SCD) patients, however explaining only ∼2% of the variance. In the same patients, we have investigated the relationship between HbF and two SNPs in a BCL11A erythroid-specific enhancer (N=626). Minor allele frequencies in rs7606173 and rs1427407 were 0.42 and 0.24, respectively. Both variants were significantly associated with HbF levels (p=3.11e-08 and p=6.04e-06, respectively) and explained 8% and 6.2% variations, respectively. These data have confirmed a stronger effect on HbF of genomic variations at the BCL11A erythroid-specific enhancer among patients with SCD in Cameroon, the first report on a West African population. The relevance of these findings is of prime importance because the disruption of this enhancer would alter BCL11A expression in erythroid precursors and thus HbF expression, while sparing the induced functional challenges of any alterations on the expression of this transcription factor in non-erythroid lineages, thus providing an attractive approach for new treatment strategies of SCD.