Hematological and biochemical reference values in sickle cell disease (SCD) are crucial for patient management and evaluation of interventions. This study was conducted at Mu-himbili National Hospital (MNH) in Dar es Salaam, to establish laboratory reference ranges in SCD at steady-state. Patients were grouped into five age groups with respects to their sex. Aggregate functions were used to handle repeated measures within the indi-vidual level in each age group. A nonparametric approach was used to smooth the curves and a parametric approach was used to determine SCD normal ranges. Comparison between males and females and against the general population was documented. Data from 4,422 patients collected from 2004-2015 were analyzed. The majority of the patients (35.41%) were children aged between 5-11 years. There were no significant differences (p≥0.05) in mean corpuscular hemoglobin concentration (MCHC), lymphocytes, basophils and bilirubin direct observed between males and females. Significant differences (p<0.05) were observed in all selected parameters across age groups except neutrophils and MCHC in adults, as well as platelets and alkaline phosphatase in infants when SCD estimates were compared to the general population. Laboratory reference ranges in SCD at steady-state were different from those of the general population and varied with sex and age. The established reference ranges for SCD at steady-state will be a helpful in the management and monitoring of the progress of SCD.
Abstract Background Pharmacogenomics of hydroxyurea is an important aspect in the management of sickle cell disease (SCD), especially in the era of genomic medicine. Genetic variations in loci associated with HbF induction and drug metabolism are prime targets for hydroxyurea (HU) pharmacogenomics, as these can significantly impact the therapeutic efficacy and safety of HU in SCD patients. Methods This study involved designing of a custom panel targeting BCL11A, ARG2, HBB, HBG1, WAC, HBG2, HAO2, MYB, SAR1A, KLF10, CYP2C9, CYP2E1 and NOS1 as potential HU pharmacogenomics targets. These genes were selected based on their known roles in HbF induction and HU metabolism. The panel was designed using the Illumina Design Studio (Illumina, San Diego, CA, USA) and achieved a total coverage of 96% of all genomic targets over a span of 51.6 kilobases (kb). This custom panel was then sequenced using the Illumina MiSeq platform to ensure high coverage and accuracy. Results We are reporting a successfully designed Illumina (MiSeq) HU pharmacogenomics custom panel encompassing 51.6 kilobases. The designed panel achieved greater than 1000x amplicon coverage which is sufficient for genomic analysis. Conclusions This study provides a valuable tool for research in HU pharmacogenomics, especially in Africa where SCD is highly prevalent, and personalized medicine approaches are crucial for improving patient outcomes. The custom-designed Illumina (MiSeq) panel, with its extensive coverage and high sequencing depth, provides a robust platform for studying genetic variations associated with HU response. This panel can contribute to the development of tailored therapeutic strategies, ultimately enhancing the management of SCD through more effective and safer use of hydroxyurea.
Common genetic variants residing near upstream regulatory elements for MYB, the gene encoding transcription factor cMYB, promote the persistence of fetal hemoglobin (HbF) into adulthood. While they have no consequences in healthy individuals, high HbF levels have major clinical benefits in patients with sickle cell disease (SCD) or β thalassemia. Here, we present our detailed investigation of HBS1L-MYB intergenic polymorphism block 2 (HMIP-2), the central component of the complex quantitative-trait locus upstream of MYB, in 1,022 individuals with SCD in Tanzania. We have looked at 1022 individuals with HbSS or HbS/β0 in Tanzania. In order to achieve a detailed analysis of HMIP-2, we performed targeted genotyping for a total of 10 SNPs and extracted additional 528 SNPs information from a genome wide scan involving the same population. Using MACH, we utilized the existing YRI data from 1000 genomes to impute 54 SNPs situated within HIMP-2. Seven HbF-increasing, low-frequency variants (β > 0.3, p < 10−5, f ≤ 0.05) were located in two partially-independent sub-loci, HMIP-2A and HMIP-2B. The spectrum of haplotypes carrying such alleles was diverse when compared to European and West African reference populations: we detected one such haplotype at sub-locus HMIP-2A, two at HMIP-2B, and a fourth including high-HbF alleles at both sub-loci ('Eurasian' haplotype clade). In the region of HMIP-2A a putative functional variant (a 3-bp indel) has been described previously, but no such candidate causative variant exists at HMIP-2B. Extending our dataset through imputation with 1000 Genomes, whole-genome-sequence data, we have mapped peak association at HMIP-2B to an 11-kb region around rs9494145 and rs9483788, flanked by two conserved regulatory elements for MYB. Studies in populations from the African continent provide distinct opportunities for mapping disease-modifying genetic loci, especially for conditions that are highly prevalent there, such as SCD. Population-genetic characteristics of our cohort, such as ethnic diversity and the predominance of shorter, African-type haplotypes, can add to the power of such studies.
Abstract Background: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common variants which differ across populations and hence do not fully account for HbF variation. Methods: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A , MYB , HOXA9 , HBB , HBG1 , HBG2 , CHD4 , KLF1 , MBD3 , ZBTB7A and PGLYRP1 . Results: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified deletions with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD using multiple genomic variants associated with HbF in SCD.
Hematological and biochemical reference values in sickle cell disease (SCD) are crucial for patient management and the evaluation of interventions. This study was conducted at Muhimbili National Hospital (MNH) in Dar es Salaam, Tanzania, to establish laboratory reference ranges among children and adults with SCD at steady state. Patients were grouped into five age groups and according to their sex. Aggregate functions were used to handle repeated measurements within the individual level in each age group. A nonparametric approach was used to smooth the curves, and a parametric approach was used to determine SCD normal ranges. Comparison between males and females and against the general population was documented. Data from 4422 patients collected from 2004–2015 were analyzed. The majority of the patients (35.41%) were children aged between 5–11 years. There were no significant differences (p ≥ 0.05) in mean corpuscular hemoglobin concentration (MCHC), lymphocytes, basophils, and direct bilirubin observed between males and females. Significant differences (p < 0.05) were observed in all selected parameters across age groups except with neutrophils and MCHC in adults, as well as platelets and alkaline phosphatase in infants when the SCD estimates were compared to the general population. The laboratory reference ranges in SCD at steady state were different from those of the general population and varied with sex and age. The established reference ranges for SCD at steady state will be helpful in the management and monitoring of the progress of SCD.
In Africa, sickle cell disease phenotypes' genetic contributors remain understudied due to the dearth of databases that pair biospecimens with demographic and clinical details. The absence of biorepositories in these settings can exacerbate this issue. This article documents the physical verification process of biospecimens in the biorepository, connecting them to patient clinical and demographic data and aiding in the planning of future genomic and clinical research studies' experience from the Muhimbili Sickle Cell Program in Dar es Salaam, Tanzania. The biospecimen database was updated with the current biospecimen position following the physical verification and then mapping this information to its demographic and clinical data using demographic identifiers. The biorepository stored 74,079 biospecimens in three −80°C freezers, including 63,345 from 5159 patients enrolled in the cohort between 2004 and 2016. Patients were identified by a control (first visit), entry (when confirmed sickle cell homozygous), admission (when hospitalized), and follow-up numbers (subsequent visits). Of 63,345 biospecimens, follow-ups were 46,915 (74.06%), control 8067 (12.74%), admission 5517 (8.71%), and entry 2846 (4.49%). Of these registered patients, females were 2521 (48.87%) and males were 2638 (51.13%). The age distribution was 1–59 years, with those older than 18 years being 577 (11.18%) and children 4582 (88.82%) of registered patients. The notable findings during the process include a lack of automated biospecimen checks, laboratory information management system, and tubes with volume calibration; this caused the verification process to be tedious and manual. Biospecimens not linked to clinical and demographic data, date format inconsistencies, and lack of regular updating of a database on exhausted biospecimens and updates when biospecimens are moved between positions within freezers were other findings that were found. A well-organized biorepository plays a crucial role in answering future research questions. Enforcing standard operating procedures and quality control will ensure that laboratory users adhere to the best biospecimen management procedures.
Abstract Background: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common variants which differ across populations and hence do not fully account for HbF variation. Methods: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A , MYB , HOXA9 , HBB , HBG1 , HBG2 , CHD4 , KLF1 , MBD3 , ZBTB7A and PGLYRP1 . Results: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified deletions with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD using multiple genomic variants associated with HbF in SCD.
Background: Newborn screening (NBS) for hemoglobinopathies is important for the early detection and effective management of affected children. Objectives: To determine the frequency of occurrence, types of, and factors associated with abnormal haemoglobins in newborns at Muhimbili National Hospital (MNH), Dar es Salaam. Methods: A hospital-based, descriptive cross-sectional design was used to recruit newborns at Muhimbili National Hospital in 2009. Blood specimens were analyzed by High Performance Liquid Chromatography and alkaline Hb electrophoresis to determine the type and proportion of hemoglobin variants. Complete blood counts including red cell indices were done by automated hematology analyzer. Results: Out of 2,053 samples analyzed, the prevalence of hemoglobinopathies was 18.2% (n=374). The percentages of children with defined hemoglobinopathies included 12.6% (n=258) with sickle cell trait (Hb FAS); 0.9% (n=19) as sickle cell carrier or Hb S Beta + -thalassemia (Hb FSA); 0.54% (n=11) had SCA or Hb S Beta 0 -thalassemia (Hb FS); one Hb FA-D variant and 5.3% (n=109) with possibly α -thalassemia (Hb Bart’s). The frequency of occurrence of abnormal haemoglobins were highest among participants whose parental origin were Costal Regions, 35.6% (n=133) and Lake Zone, 10.2% (n=38). Participants from the Northern Region of Tanzania had the lowest frequency of occurrence, 6.7% (n=25) (X 2 = 37.7, p < 0.01). Having abnormal haemoglobins increased the likelihood of newborns being born at low gestational age (23.8%) by 1.5 fold as compared to newborns (16.3%) born without abnormal haemoglobins (X 2 =11.7, p=0.001). Conclusions: The frequency of occurrence of abnormal hemoglobin is high and fulfills the World Health Organization (WHO) criteria of a disorder of public health significance. Therefore, newborn screening programme is highly recommended in Tanzania. The ethnic origin of the parents and the gestational age were significantly associated with occurrence of abnormal haemoglobins. Keywords: Newborn screening, neonates , abnormal haemoglobins (Hemoglobinopathies), frequency of occurrence, High Performance Liquid Chromatography
Abstract Background Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation. Methods We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A , MYB , HOXA9 , HBB , HBG1 , HBG2 , CHD4 , KLF1 , MBD3 , ZBTB7A and PGLYRP1 . Results Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.
There is increasing evidence that autonomic dysfunction in adults with homozygous sickle cell (haemoglobin SS) disease is associated with enhanced autonomic nervous system-mediated control of microvascular perfusion. However, it is unclear whether such differences are detectable in children with SS disease. We studied 65 children with SS disease [38 boys; median age 7.2 (interquartile range 5.1-10.6) years] and 20 control children without symptoms of SS disease [8 boys; 8.7 (5.5-10.8) years] and recorded mean arterial blood pressure (ABP) and daytime haemoglobin oxygen saturation (S(pO(2))). Cutaneous blood flux at rest (RBF) and during the sympathetically activated vasoconstrictor response to inspiratory breath hold (IBH) were measured in the finger pulp of the non-dominant hand using laser Doppler fluximetry. Local factors mediating flow motion were assessed by power spectral density analysis of the oscillatory components of the laser Doppler signal. The RBF measured across the two study groups was negatively associated with age (r = -0.25, P < 0.0001), ABP (r = -0.27, P = 0.02) and daytime S(pO(2)) (r = -0.30, P = 0.005). Children with SS disease had a higher RBF (P = 0.005) and enhanced vasoconstrictor response to IBH (P = 0.002) compared with control children. In children with SS disease, higher RBF was associated with an increase in the sympathetic interval (r = -0.28, P = 0.022). The SS disease status, daytime S(pO(2)) and age explained 22% of the variance in vasoconstrictor response to IBH (P < 0.0001). Our findings suggest that blood flow and blood flow responses in the skin of young African children with SS disease differ from those of healthy control children, with increased resting peripheral blood flow and increased sympathetic stimulation from a young age in SS disease. They further suggest that the laser Doppler flowmetry technique with inspiratory breath hold manoeuvre appears to be robust for use in young children with SS disease, to explore interactions between S(pO(2)), ABP and autonomic function with clinical complications, e.g. skin ulceration.
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