Sickle Gene Research Articles

5613191 KNOWLEDGE, ATTITUDE, PRACTICE OF GENOTYPE SCREENING AMONG UNDERGRADUATES MEDICAL STUDENTS IN THE UNIVERSITY OF ABUJA

Background: Sickle cell disease remains the most common life threatening genetic disorder worldwide. Globally about 312,000 children are born each year with sickle cell disease (SCD), half of which are from Nigeria1,2,4,5,7,8. The prevalence of sickle cell anaemia (SS) in Nigeria is estimated to be 20 per 1000 and 25% of the population have sickle trait (AS)2,6,9,10,11. Medical students in Nigeria are key in the future management of sickle cell disease. Therefore, it is important to understand what training in SCD is given to medical students in Nigeria and to identify any important knowledge gaps which may improve training curricula. Method: A student survey was conducted at the College of Health Sciences, University of Abuja in the Federal Capital Territory of Nigeria (FCT) in the 2021/2022 academic year. At this University, all students undergo comprehensive medical tests before admission, which include genotype screening. All medical students were eligible to complete the survey and which was advertised through year group social platforms. Data were collected using a self-administered questionnaire through Google form, after the respondent indicated their consent. The questions focused on knowledge of the disease and attitude towards genotype screening, and whether the student had undergone screening and were aware of their status. Results: In the academic year of 2021/2022 there were a total number of 374 medical students from year one to year six of these 150 (40%) completed the survey. Survey respondents included similar numbers of males and females, with a mean age of 23.7 years and were from all years of study. All the respondents had heard about sickle cell disease and 85% stated that they had attended courses which covered Sickle cell disease topics (from 63% in 1st year of study rising to over 90% by year 3). A very high proportion knew that Sickle cell disease trait can be transmitted from both parents to offspring (95%), and 90% knew that SCD can affect anyone and can occur when both parents are carriers of the Sickle gene. Fewer students (79%) knew that SCD can be diagnosed at birth. Knowledge of common symptoms for sickle cell patients was variable, while most knew that one of the most common complaints was pain in the limbs (92%), only 23% were aware that yellow eyes is also a common symptom, and 10 students (7%) incorrectly identified skin rashes as a common symptom. A total of 93% of students had been genotype-screened for SCD, and attitudes towards genotype screening, was generally positive. Most agreed that genotype screening can help prevent SCD and that pre-conception genotype screening is important. Of those who had been screened and knew their status, 68.6% were AA (adult haemoglobin without any haemoglobin disorder), 28.6% were AS (had the sickle cell trait) and 2.9% were SS (sickle cell anaemia). Conclusion: Over 90% respondents knew their sickle cell status (Hb genotype) and 2.9% had SCD (HbSS), and 29% had sickle cell trait (HbAS) consistent with the national burden in Nigeria. Reassuringly, medical students had a good knowledge of and attitude towards SCD irrespective of their year of study. Although identifying symptoms, which may be important in diagnosis, could be improved. Given that almost half of the global burden of SCD is in Nigeria, this study reflects the importance of ensuring adequate health education towards sickle cell disease among Nigerian medical students.

Hydroxyurea for Children with Sickle Cell Disease in sub-Saharan Africa: A Summary of the Evidence, Opportunities, and Challenges.

Sickle cell disease (SCD) is a common and life-threatening inherited blood disorder that affects more than 300,000 newborns per year. Because of the origins of the sickle gene mutation as a protective mechanism against malaria for those with sickle cell trait, more than 90% of annual SCD births are in sub-Saharan Africa (sSA). Over the past several decades, there have been many important advances in the care of individuals with SCD, including early diagnosis through newborn screening programs (NBS), prophylactic penicillin, the development of vaccines to prevent invasive bacterial infections, and the emergence of hydroxyurea as the primary disease-modifying pharmacologic therapy. These relatively simple and inexpensive interventions have significantly reduced the morbidity and mortality of Sickle cell anemia (SCA) such that individuals with SCD can live longer and more complete lives. Unfortunately, although these interventions are relatively inexpensive and evidence-based, they are only readily available for affected individuals living in high-income settings, representing <5% of the global SCD population. In sSA, where >90% of the global SCD burden exists, SCD still results in early mortality with 50-90% of infants likely dying before reaching 5 years of age. Recently, there are an increasing number of efforts in many African countries to prioritize SCA with pilot NBS programs, improved diagnostics, and expanded SCD education for health care professionals and the general public. It is essential to include access to hydroxyurea as a core component of any SCD care program, but there are still many barriers that prevent the widespread global use of hydroxyurea. Here, we summarize what we know about SCD and hydroxyurea use in Africa and discuss a strategy to respond to what we consider to be a public health imperative to maximize access to and appropriate use of hydroxyurea for all individuals with SCD using innovative dosing and monitoring strategies.

Comparative Analysis of Genome Editing for Curing Sickle Cell Disease: Induction of Fetal Hemoglobin and Gene Correction of Sickle Gene

Known as a monogenic disease, Sickle Cell Disease (SCD) is a collective term for a series of red blood cell disorders affecting a large population in the world. Though current treatments exist and are proven effective (i.e., bone marrow transplant), accessibility severely limits their impact on most patients. Because the monogenic nature of SCD renders it simple to study, scientists are currently attempting to use gene therapy and genome editing to cure the disease. The genome editing approaches are mainly comprised of two methods: reactivating the previously suppressed fetal hemoglobin or a direct gene correction at the mutated residue in the adult beta (β) globin gene. Each approach is supported by a handful of in vitro experiments demonstrating sufficient levels of fetal hemoglobin activation or production of correct β globin in edited cells to rescue SCD. Although genome editing has shown great potential to treat this deadly disease, there remains much more work in in vivo and clinical trials to be done for further long-term evaluation before these approaches to be approved for use in humans. Safety issues and the long-term effects of the genome editing reagents are key topics that require large efforts to improve upon.

Knowledge of tribal women towards sickle cell anemia

Sickle cell anemia is wide spread among tribal communities in India. A high prevalence of the sickle gene has been demonstrated in various tribal communities of Gujarat state. Tapi is tribal dominated district. The patient suffering from sickle cell anemia develops blood related complication and sickle cell disease sufferers have also shortened lifespan and poor quality of life. So to combat against this disease we need to focus on tribal population. Knowledge plays an important role for achieving desired results. Thus, the study was carried out to assess the knowledge of tribal women towards sickle cell anemia. The data was collected purposively with 100 tribal women who have positive sickle cell status. The data was collected with the help of structured interview schedule and analyzed with appropriate statistical tools. It is indicated that majority of tribal women had young aged, sickle cell trait status, married, in joint family, more than half of tribal women had education upto higher secondary level &amp; 36.00 per cent tribal women were illiterate. Nearly half of tribal women had no any type of social participation. Majority of tribal women were not taking medical consultancy and low level of knowledge about sickle cell anemia.

Spectrum of Inherited Hemoglobin Disorder in patients presenting for Hb Electrophoresis: A Single Center Study in District Dera Ismail Khan

Background: Hemoglobinopathies remains an important public health Problem for developing countries like Pakistan as it has been associated with high morbidity and mortality rates. The objective of this study was to know the frequency distribution of various hemoglobinopathies in ethnically and geographically diverse population of Dera Ismail Khan of Pakistan referred for Hemoglobin electrophoresis. Methods: This cross sectional study was conducted at Pathology department of Gomal Medical College, Dera Ismail Khan, from January 2017 to December 2021. About 3 ml of venous anticoagulatedblood specimen were collected from suspects for Complete Blood Count, Sickling Test and hemoglobin electrophoresis. Result: Of the 538 cases, 219(40.7%) were found normal and 319(59.3%) were found positive for abnormal hemoglobin disorder. Among positive samples 317(58.9%) were male and 221(41.1%) were female. BetaThalassemia was the most common hemoglobinopathyobserved in 194(60.8%) followed by sickle cell 105(32.9%), hemoglobin E, D and C were found in 9 (2.8%), 4(1.2) and 2(0.6%) respectively. Frequency of Beta Thalassemia is quite high in Balochtribe followed by Mehsud and Marwat. Sickle gene was more prevalent in Sherani followed byBhatni and Ustrana. Conclusion: Beta Thalassemia and Sickle cell are the two most common hemoglobin disorders found in our set up. Mass screening with genetic counselingand education should be initiated to reduce disease burden. Keywords: Electrophoresis, Hemoglobinopathies, Dera Ismail Khan.

Evaluate the biomarkers of bone turnover in patients with in sickle cell anemia in Basra province

Background: Sickle cell anemia (SCD) is one of many haemoglobinopathic disorders which is probably the most common known hereditary blood disorder. In this disease, the homozygous state in which the sickle gene is inherited from the father and the mother, and patients with sickle cell disorders often suffer from chronic hemolytic anemia, which causes bone marrow hyperplasia too. The present study was designed to evaluate the biomarkers which related with the bone turnover in sickle cell anemia patients. Patients with SCA (n=120) 60 males and 60 females were on follow up in the Basra center for Hereditary Blood Disease, who were included in this study and age and sex matched healthy persons (n=60) as controls. biomarkers had important role in many biological processes. Aims: of this study to measure serum levels of different biomarkers such as; BCTx, BSAP, CRP, DPD, IGF-1, OT, TRACP-5b, FER, IGFBPT, Intact (iPTH), Ostase, PINP and serum Ca+. Using Kruskal-Wallis test as statistical test, markers in sickle cell anemia and control groups.The results: showed no significant difference of both male and female patients (HbSS, HbSC and sickle- β-Thalassemia) in compared to control group.

Near Extinction of HbS Among the Tribes in Bengal: An Effect of Epistasis?

Introduction : Tribal communities are genetically isolated populations mostly following endogamy. The large tribal population in India inhabits widely varying ecological and geo-climatic conditions in different concentration throughout the country. It is widely established that the high frequencies of genetic blood disorders (haemoglobinopathies) are the result of evolutionary selection (Haldane 1949; Flint et al. 1998). The sickle gene, which protects against malaria, provides an example of balanced polymorphism (Stuart M.J; 2004). The tribal area in India is mostly associated with high incidence of malaria. Thalassaemia is highly prevalent among the tribal communities in india; and interestingly the occurrence rate of a particular mutation/ type of thalassaemia varies to a great extent from one to another (R.Colah, 2014 ; R.Dastidar, 2007). According to a survey by ICMR the Sickle Cell gene was found amongst different tribal groups of India; which varies from 5 to 34 % (M.Kaur; 2013). The prevalence of alpha thalassaemia, which also gives protection against malaria, varies throughout the country. The frequency and distribution of thalassaemia among the tribes India is less well-documented; especially in the eastern part of the country where there have not been much thorough studies upon hemoglobinopathies among the tribes with a large number sample population. In this study the primary aim was to investigate the prevalence of anemia, Sickle Cell disease and other mutant hemoglobin amongst the tribal community in west Bengal.Method: Over a duration of 18 months, 17,369 school going children (age= 10 - 18 years), among the tribal community, from almost all the districts of West Bengal were included in this study; and were initially screened by complete haemogram and HPLC analysis. Detection for alpha 3.7 deletion & alpha 4.2 deletion, the most common form of alpha gene defect in India, were carried by GAP-PCR; and HbS mutation analysis by ARMS PCR.Result: Approximately 6 % among the total study population was found to be carrier for beta thalassaemia; and only 0.4 % of the total population with HbS. 30 % of the population were found to be absolute normal based on the HPLC parameters and RBC indices [Table-1,2 & 3; Figure 1] . Around 62% were found to have ‘normal HPLC values with abnormal RBC indices’ who were subjected to ‘further investigation’.For ‘further investigation’ we initially selected six districts; under which total number of individual screened were 5357; among which 65% were found to have ‘normal HPLC values with abnormal RBC indices’ after initial routine screening [ Table- 4, 5,] who were subjected to further investigation for the detection of alpha 3.7 deletion and alpha 4.2 deletion. Almost 83% of them were found to carry (3.7 or 4.2 or both 3.7 + 4.2) alpha gene deletion [ Table 6, 7, 8]. Though prevalence of alpha thalassaemia carrier was found to be high; HbS carrier was found to be only 0.3% by HPLC. The samples carrying alpha deletion were re-checked for the presence of HbS mutation; none of the which was found carry silent / suppressed form of HbS mutation.Discussion : Recently few studies have focused on understanding the co-association between alpha gene mutation and HbS in populations under constant pressure of malaria; which have shed light upon few aspects like- Negative Epistasis, Genetic Drift, Positive Selection etc regulating the outcome (S. Penman,2011; MB Rumaney, 2014 ). The high prevalence of alpha gene mutation found in our study may be attributed to the fact that alpha mutation is also known to have protective role against malaria. At the same time there is a genetic drift which has caused lowering the prevalence of HbS. The widely varying prevalence of alpha deletion (high) and exceptionally low prevalence of HbS (exceptionally low ) may be a consequence of epistasis and positive selection depending upon the risk factors associated with the mutations of two distinct alleles; which can be described by mathematical modeling (S. Penman;2011).Conclusion: Though asymptomatic, such high prevalence of alpha gene mutation is alarming as it may result in high occurrence of HbH disease among the tribal population where there is high rate consanguine marriage; if not intervened through proper marriage counseling and raising general awareness in the society. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stroke in Pediatric Sickle Cell Disease in Sub-Saharan Africa: Systematic Review and Estimated Prevalence

Background: Most people with sickle cell disease (SCD) live in Sub-Saharan Africa (SSA); many do not survive to adulthood (Grosse et al. Am J Prev Med 2011). Stroke is a common complication of SCD in the region (Ohene-Frempong et al.Blood 1998), yet the region's burden of pediatric SCD stroke is unknown. This systematic review aims to describe what is known, and to estimate the number of children affected by SCD stroke in SSA.Methods: Systematic reviews using Pubmed, Embase, and Web of Science were performed for all studies reporting pediatric SCD stroke prevalence data. Keyword search strategy included these or related terms: “stroke” or “brain” and “sickle cell” and “Africa” or each country in SSA available in English or French. Publication dates were selected between 1980 and December 13, 2016. Using pre-selected inclusion criteria, titles and abstracts were screened and full-text articles were reviewed. For publications meeting inclusion criteria, extracted data included: study and participant characteristics, number of patients with stroke, diagnostic criteria, imaging, management, recurrence. The number of children affected by SCD stroke in SSA was estimated based on current estimates of national population and S allele frequency (Piel et al. Lancet 2013) and the stroke prevalence in the extracted studies. The stroke prevalence in SSA was estimated based on both fixed-effect analysis and random-effect analysis.Results: After duplicates were removed, 133 individual articles remained. Ten full text articles met review selection criteria, including one in French. In all, papers reported data from seven countries in the region, including four reports from Nigeria. Cross-sectional SCD clinic-based data, with a sample size of 6,289 patients, reported pediatric stroke prevalence of 2.9-16.9%. Diagnostic criteria of stroke varied by site and were limited by access to brain imaging, which was reported in only half of the included studies. Treatment for stroke included packed red blood cell transfusion, exchange transfusion, or hyperhydration. Secondary stroke prevention with chronic blood transfusion or hydroxyurea was offered at two of 10 sites. Recurrence data was limited by variable study follow-up and limited reporting times. Using available sickle gene frequencies by country, estimated births affected by SCD per country in SSA, and a linear- or random-effects model, pediatric stroke prevalence in SSA was projected as 279,908 (95% CI: 240,335-319,481) and 561,746 (347,472-775,056), respectively. Estimates do not include stroke- or disease-associated mortality.Conclusion: This meta-analysis estimates prevalence of pediatric SCD stroke in SSA, underscoring the region's high stroke burden. Multiple clinic-based cross-sectional studies reported stroke prevalence comparable to data from high income countries prior to implementation of standardized prevention strategies. Accuracy is limited by different approaches to stroke diagnosis, paucity of population-based data on disease prevalence and mortality, regional variation in disease prevalence and severity, and overall high rates of early death in children with SCD. Available data would skew towards surviving patients, leading to possible underreporting of people at risk for SCD stroke. Improved diagnostic specificity with standardized criteria and imaging would be useful for identification of strokes. Understanding the large numbers affected by SCD stroke is critical for focusing public health efforts to introduce preventive measures to reduce stroke risk and to implement rehabilitative strategies. Adopting standardized primary and secondary stroke prevention measures in SSA may decrease the burden of SCD-associated stroke.This work was supported by funding from the National Institutes of Health (5R21HD089791, PIs Idro, Green). DisclosuresNo relevant conflicts of interest to declare.

Opioids and Sickle Cell Disease: From Opium to the Opioid Epidemic.

Sickle cell disease (SCD) is an inherited disorder of hemoglobin structure. The clinical effects of the sickle gene are pleiotropic in nature causing multiple phenotypic expressions associated with the various complications of the disease. The hallmark of the disease is pain that could be acute, chronic, nociceptive, or neuropathic that could occur singly or in various combinations. The acute vaso-occlusive painful crisis (VOC) is the most common cause of admissions to the Emergency Department and/or the hospital. Although progress has been made in understanding the pathophysiology of SCD as well as in developing preventive and curative therapies, effective pain management continues to lag behind and depend mostly on the use of opioids. This review describes the history of opioids from the ancient times of opium to the current use of the many controversial opioids. In addition, the major cause of death of patients with SCD is the complications of the disease itself and not the use of opioids. The use of opioids by patients with SCD has been stable over the years. Judicious use of opioids to treat sickle cell pain according to available guidelines could minimize the unnecessary suffering experienced by patients with SCD.

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease

93 A sheep model of sickle cell disease using CRISPR/Cas9 and somatic cell nuclear transfer

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, with more than 2 million people in the United States alone carrying the sickle gene. Approximately 100 000 of these people are homozygous and suffer from SCD. Worldwide, there are ∼4.4 million people with SCD. SCD is caused by a single A to T nucleotide replacement at the sixth codon of the β-globin gene, which results in the substitution of a valine for glutamate in the β-globin protein. This causes the resultant tetrameric haemoglobin molecule to be unstable and the red cells carrying this aberrant protein to “sickle,” decreasing the ability of these cells to carry oxygen. Sheep and humans exhibit a high degree of homology at the level of the genome. In addition, their anatomy, organ physiology, and immune system development closely parallel that of humans during fetal life. The ovine β-globin (HBB) gene shares 87.5% similarity with human HBB. Therefore, we hypothesised that the introduction of the “sickle” mutation in the sheep genome would lead to the SCD phenotype in sheep that could provide a valuable platform for evaluating prenatal and postnatal drug and gene therapies for this disease. In this study, we used a CRISPR/Cas9 gene-editing approach to introduce the SCD mutation into the sheep β-globin/HBB gene. We designed a single guide (sg)RNA targeting exon 1 of the sheep β-globin/HBB gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from IDT. Using the Lonza-4D-Nucleofector system, the Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs) along with 101-bp single-stranded oligodeoxynucleotides, flanking the sickle cell mutation to enable homology-directed repair. The transfected SFFs were then cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. After 2 days, DNA was extracted from one-third of the SFFs and the remainder were seeded individually into five 96-well plates by limited dilution. After 7 days of culture, individual colonies were expanded into 24-well plates and cultured for an additional 3 days. PCR-restriction fragment length polymorphism (RFLP) analysis using Image J software demonstrated a high rate of mutations (∼70%) by either indels or SCD mutation that led to the loss of the restriction enzyme site, which was further supported by the analysis of cell colonies. We isolated 59 single cell-derived SFF colonies and, based on PCR/RLFP assay, 31/59 (52%) of them contained biallelic mutations (either indels or point mutations) and were subsequently submitted for Sanger sequencing. The sequencing demonstrated that 3 colonies (9.6%) contained biallelic SCD mutations in the β-globin/HBB gene. These data demonstrate that we successfully introduced the SCD mutation into SFFs. These cells will be used in the production of the first large animal (sheep) SCD model by somatic cell nuclear transfer in fall of 2020. This research was supported by UAES project 1343 and by USDA/NIFA multistate research project W-4171.

Clinical and Laboratory Features of Hemoglobin La Desirade Variant in Association with Sickle Cell and Alpha Thalassemia Genes.

Abstract &#x0D; Hemoglobin La Desirade (Hb La Desirade) is an unstable hemoglobin variant characterized by amino acid Alanine (Ala) replacing Valine (Val) at position 129 (H7) in the beta chain. Hb La Desirade exhibits a decreased oxygen affinity and normal heme-heme interaction. Interestingly, on analysis by standard electrophoresis, it migrates in the same region as normal HbA, and HbA actually represents a combination of HbA and Hb La Desirade together. This variant was reported as compound heterozygous with other Hemoglobin variants such as HbS, HbC or beta thalassemia, and more recently with Southeast Asian ovalocytosis and Hb Louisville with varying clinical manifestations. Herein, we describe the clinical and laboratory findings in a number of Omani Arab families who presented to our service for various reasons, presenting with Hemoglobin La Desirade with sickle gene and alpha thalassemia. Our patients with Hb La Desirade trait, were clinically asymptomatic with no evidence of anemia. However when it is associated with other abnormal hemoglobin variants such as HbS, leading to sickle/La Desirade compound heterozygosity, there was mild anemia with significant degree of hypochromia and microcytosis. The most striking feature was that the levels of HbS and HbA were almost equal on HPLC, and these cases could be misdiagnosed as sickle cell trait (SCT). However, the levels of Hb S in these compound heterozygotes (40.4-45.9) were higher than normally seen for the diagnosis of SCT in this population.&#x0D;

Current modalities of sickle cell disease management.

Sickle cell disease (SCD) affects nearly 100,000 people in the United States of America and the sickle gene is present in approximately 8% of black Americans. Among Africans, the prevalence of sickle cell trait (heterozygosity) is as high as 30%. While SCD occurs among varying racial and ethnic groups, it is more commonly prevalent in individuals of African or African-American descent. This inherited blood disorder causes varying symptoms and complications among affected children and adults and early diagnosis and treatment are essential to help reduce mortality rates. Because there is no cure for SCD, management is vital to survival. Hence, there are different approaches in use to aid those living with SCD; thus, this paper provides insight into the current methods that are implemented in the management and maintenance of this disease.

Differential Efficacy of Anti-Sickling and Anti-Inflammatory Mechanisms in a Fluorescent Intravital Microscopy Dorsal Skinfold Vaso-Occlusion Model in Sickle Cell Disease Townes Mice

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

Malaria and the Sickle Gene: Polymorphism Balance in favour of eradication

Evolutionally, the single nucleotide mutation responsible for the sickle haemoglobin gene, (HbS gene) developed from the regions of the world where malaria is holoendemic, leading to the explanation that the mutation is in response to the presence of the malaria parasite. Studies eventually showed that individuals that are heterozygous for the HbS gene are protected from the lethal clinical effects of malaria infection. In other words, malaria confers a survival advantage to carriers of the HbS gene, and this is referred to as balanced polymorphism. On the other hand, malaria infection is associated with significant morbidity and mortality, particularly among children. Unfortunately, lack of success in the effort to eradicate the malaria parasite through the elimination of the Anopheles mosquito or efforts to limit its contact with human being has produced little success, hence the resort to roll back malaria, with the aim of reducing the morbidity and mortality associated with it. Therefore, it is attractive to consider what will happen to the sickle gene if malaria were to be eradicated. That is, the possibility is that the sickle gene may also follow suit since its evolution in the first instance was as a response to malaria., This is the hypothesis being propounded by this article. In that case, rather than shy away from the malaria eradication initiative, it should remain the goal of all malaria programs.

Geospatial Mapping of Sickle Cell Disease in Northwest Tanzania: The Tanzania Sickle Surveillance Study (TS3)

Tanzania ranks third in Africa for the estimated number of annual births with sickle cell disease, but these estimates are based on sparse data from small studies reported over the past 50 years. A recently completed surveillance study from Uganda documented substantial variation in the prevalence of sickle cell trait and disease across the country. Tanzania lacks a national newborn screening program, and no contemporary multi-regional screening of infants has been undertaken. We designed and conducted a prospective study to determine the prevalence of sickle cell trait and disease by region and district in northwest Tanzania, where the prevalence of sickle cell is thought to be highest. The study used existing public health infrastructure while building local capacity for accurate diagnosis of sickle cell disease. Secondary objectives included characterization of hemoglobin variants and exploration of associations between sickle cell trait, sickle cell disease, malaria, and HIV.The Tanzania Sickle Surveillance Study (TS3) is a prospective cross-sectional study of HIV-exposed infants born in 9 regions across the Lake Zone of northwest Tanzania. In Tanzania, the HIV early infant diagnosis (EID) program collects dried blood spots (DBS) from all children born to HIV-infected mothers. DBS are transported to a central laboratory for prompt detection of HIV vertical transmission. In northwest Tanzania, the DBS are transported to Bugando Medical Centre, a teaching and consultancy hospital in Mwanza, where they are tested for HIV and then stored on-site, and thus available for further testing. Isoelectric focusing (IEF) equipment was donated to Bugando Medical Centre along with reagents and supplies. Two laboratory staff were trained by a board certified hematologist, and then attended a two day seminar by the IEF manufacturer. One pediatrician completed a 2-month observership at Cincinnati Children's Hospital. All DBS samples were tested by IEF using appropriate controls. Completed gels were scored independently by two Tanzanian staff members as normal, disease, trait, variant, or uninterpretable. DBS samples scored as disease or variant were repeated for confirmation and preserved for later genotyping. Regular Skype calls were convened with US-based collaborators to improve quality and interpretation. HIV test results were obtained from the local EID program.Between February 2017 and May 2018, 232 IEF gels were completed by the local staff. After children >24 months of age were excluded to obtain a more accurate newborn prevalence, the median age of children tested was 52 days (IQR 41-93 days), and a total of 17,278 unique DBS samples were scored. The quality of laboratory testing was extremely high with only 20 samples scored as uninterpretable and 54 with missing results, and the primary analysis was performed on the 17,204 remaining samples. The overall prevalence of sickle cell trait and disease in the entire cohort was 20.3% and 1.2%, respectively, with a 0.1% prevalence of hemoglobin variants. This corresponds to an allelic frequency of 0.114 for the sickle gene mutation and demonstrates perfect Hardy-Weinberg equilibrium. No HbC or other common beta-globin variants were identified. Geospatial mapping revealed some variation across regions, with sickle trait ranging from 16.6% to 22.5% and disease ranging from 0.5% to 1.5%. Analysis of individual districts with >100 samples revealed wider geographic variability, with sickle trait ranging from 15.2% to 27.8% and disease ranging from 0.0% to 4.3%. Co-morbidity between HIV and sickle cell disease was analyzed to compare it with the effect on mortality previously observed in Uganda. The prevalence of sickle cell disease was the same among HIV-infected and HIV-negative children (1.2%), suggesting no difference in mortality.The prevalence of sickle cell trait and disease among infants born in northwest Tanzania is very high, exceeding 20% trait and 1.2% disease. All regions in the Lake Zone are affected possibly due to lack of immigration to the area and similar environmental exposures. Targeted newborn screening can be started in high prevalence districts, using existing public health infrastructure with minimal start-up cost and training. Future work will evaluate the correlation between historical malaria prevalence and sickle cell prevalence, and identify hemoglobin variants. [Display omitted] DisclosuresWare:Addmedica: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Agios: Other: advisory board; Global Blood Therapeutics: Other: advisory board; Biomedomics: Research Funding; Nova Laboratories: Consultancy.

Red Cell Indices and Hemoglobin Profile of Newborn Babies with Both the Sickle Gene and Alpha Thalassaemia in Central India.

This study evaluated the effect of alpha thalassemia on the red cell indices and hemoglobin profiles of normal, sickle heterozygous and sickle homozygous newborn babies in central India where the sickle gene is linked to the Arab-Indian haplotype. 265 newborn babies were analysed with complete blood count and hemoglobin analysis on high performance liquid chromatography (Variant Hb Testing System, BioRad Laboratories, Hercules, CA, USA) using the β-thal short program. The sickle genotypes was confirmed by DNA analysis. The two common alpha gene deletions (- α3.7 and - α4.2) were detected by multiplex PCR. Among the 102 normal, 106 sickle heterozygous and 57 sickle homozygous newborns, the prevalence of a single alpha gene deletion (- α/αα) was 28.3% and that of deletion of 2 alpha genes (- α/- α) was 21.5%. In all, 57 normal (55.9%), 35 (33.0%) sickle heterozygous and 41 (71.9%) sickle homozygous newborns had a normal α genotype while - α/- α was seen in 23 (22.5%) normal, 30 (28.3%) sickle heterozygous and 4 (7.0%) sickle homozygous newborns respectively. The presence of associated alpha thalassemia resulted in a reduction in the hemoglobin levels and red cell indices in normal, sickle heterozygous and sickle homozygous newborn babies, MCV and MCH being strong discriminators of alpha thalassemia with two alpha gene deletions in all the three groups. This study also helped us to know the variations in hematological parameters in normal, sickle heterozygous and sickle homozygous newborns with and without associated α thalassemia.

Control of the ethylene signaling pathway prevents plant defenses during intracellular accommodation of the rhizobia.

Massive intracellular populations of symbiotic bacteria, referred to as rhizobia, are housed in legume root nodules. Little is known about the mechanisms preventing the development of defense in these organs although genes such as SymCRK and DNF2 of the model legume Medicago truncatula are required for this control after rhizobial internalization in host nodule cells. Here we investigated the molecular basis of the symbiotic control of immunity. Proteomic analysis was performed to compare functional (wild-type) and defending nodules (symCRK). Based on the results, the control of plant immunity during the functional step of the symbiosis was further investigated by biochemical and pharmacological approaches as well as by transcript and histology analysis. Ethylene was identified as a potential signal inducing plant defenses in symCRK nodules. Involvement of this phytohormone in symCRK and dnf2-developed defenses and in the death of intracellular rhizobia was confirmed. This negative effect of ethylene depended on the M.truncatula sickle gene and was also observed in the legume Lotus japonicus. Together, these data indicate that prevention of ethylene-triggered defenses is crucial for the persistence of endosymbiosis and that the DNF2 and SymCRK genes are required for this process.

Prevalence of sickle cell disease in tribal peoples of Valsad district region in Gujarat,India

Background: High prevalence of sickle gene has been demonstrated in various tribal communities of Gujarat including Bhils and Dhodias of Panchmahal, Dublas, Naikas, Koli, Dhanka, Gamit, Vasava, Bariya, Varli, Vaghari, Kukna, Halpati, Chaudhari etc. The present study was conducted with an objective to document the prevalence of sickle cell diseasein Valsad district of Gujarat state. Methodology: The study is conducted as a retrospective survey in GMERS Medical College and hospital, Valsad between January 2015 to December 2016 were screened for sickle cell anemia by sickling test (with freshly prepared sodium metabisulphite). Those found positive for sickling test, were further analyzed by HPLC (high-performance liquid chromatography) to confirm their status as either sickle cell trait or sickle cell disease. Results: Out of 1186 cases number of male cases were 277 (23.35%) and females were 909 (76.64%) cases. The gender distribution of male to female cases is 1:3. 88.7% cases were sickle cell trait, 9.02% cases were sickle cell disease, 1.68% cases were sickle beta thalassemia, 0.25% cases were B Thalassemia Minor and 0.33% cases were B Thalassemia Major. In the present study, among the 1186 cases, 397(33.47%) were from Patel (Dhodia) community, 97 (8.17%) were from varli community, 57(4.8%) were from Halpati community and 51(4.3%) were from Dhodi community followed by other backward community. Conclusion: It was concluded that Dhodias, Varli, Halpati and Dhodi were the most common community of sickle cell disorders in tribal peoples of Valsad district.Among the Sickling test positive cases, Sickle cell trait and sickle cell disease was the most common variant.

Comorbidities in aging patients with sickle cell disease.

Sickle cell disease (SCD) in general and sickle cell anemia in particular is a highly complex disorder both at the molecular and clinical levels. Although the molecular lesion is a single-point mutation, the sickle gene is pleiotropic in nature causing multiple phenotypic expressions that constitute the various complications of the disease. Moreover, despite the fact that SCD is a chronic malady, its manifestations are both acute and chronic. The former include, among other things, the recurrent vaso-occlusive crises (its hallmark) and acute chest syndrome. The chronic complications include most commonly avascular necrosis and leg ulcers. Currently, survival of patients with SCD has improved dramatically thanks to newborn screening, antibiotic prophylaxis, better vaccine, safer blood transfusion and the use of hydroxyurea. It is the advent of these therapies that improved the survival. This improvement, however, introduced a third dimension of the disease: comorbidities that occur in aging people in the general population. There is concern that the gain in survival may be offset by the comorbidities. Thus it is the purpose of this review to identify the comorbidities in the elderly with SCD and differentiate them from the basis disease to implement proper therapies so that better survival could be maintained.