scholarly journals Individuals with Sickle Cell Disease Have a Higher Burden of Mitochondrial DNA Heteroplasmy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 954-954
Author(s):  
Laxminath Tumburu ◽  
Maliha Maryam Ahmad ◽  
Chunyu Liu ◽  
Clifton L. Dalgard ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Mitochondrial Dna ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Median Number ◽  
Sequencing Depth ◽  
Cumulative Distribution ◽  
Cell Disease ◽  
Mtdna Mutations ◽  
Hemoglobin C

Abstract Background: The simple point mutation that causes sickle cell disease (SCD) belies the extensive systemic damage it can cause. While the sickle pathology is initiated by polymerization of HbS, the multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of acquired heteroplasmy mutations in mitochondrial DNA (mtDNA). Given the underlying chronic inflammation in SCD, we hypothesized that SCD patients display increased rates of mtDNA mutations, and previously confirmed (1). Here, we further performed indepth analyses in an ethnically matched normal (HbAA) as well as sickle trait (HbAS) subjects from another independent cohort, the Jackson Heart Study (JHS). Methods: We analyzed and compared whole genome sequencing (WGS) data from the from NIH cohort of 676 SCD patients of African ancestry with that of 621 ethnic-matched indviduals from the 1000 Genomes Project (1KG), and 3,580 individuals from the JHS cohort. The NIH SCD cohort included 561 HbSS & HbSβ 0thalassemia (combined), 90 HbSC, and 25 HbSβ + thalassemia genotypes, the 1KG cohort - 516 HbAA and 105 HbAS and JHS cohort - 3,200 HbAA, 89 HbAC (hemoglobin C trait), and 291 HbAS. Additionally, to further understand any potential sequencing depth bias, as well as to compare between two patient cohorts (NIH SCD & JHS cohorts) with underlying conditons that may influence the heteroplasmy bias, we downsampled 300 NIH cohort HbSS samples to a sequencing depth similar to JHS cohort, and compared their heteroplasmy burden. Mitochondrial sequences extracted from the cleaned WGS data of these 3 cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus was ~6,671X for the NIH SCD cohort , ~2,879X for the 1KG cohort, and ~2169X for JHS cohort. We compared the quantity of heteroplasmic variants across the different NIH SCD genotype with 1KG (HbAA & HbAS), and JHS (HbAA, HbAC and HbAS) genotypic groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ +thalassemia, and HbSC with the highest median number of 118 in HbSS & HbSβ 0 (Fig 1A) in NIH SCD cohort. It is noteworthy that the median mtDNA heteroplasmy in HbAA individuals in 1KG cohort was significantly lower than those in JHS cohort (Table insert in Fig 1A) which may be related to the underlying cardiovascular disease in the JHS cohort; whereas similar heteroplasmy burden in HbAS individuals between these 2 cohorts may underscore the genotype (HbAS) as the driver of heteroplasmy in these cohorts. We compared the heteroplasmy burden of a downsampled subset (n=300) NIH HbSS with that of JHS HbAA, HbAC and HbAS genotypes (Fig 1B). Although, the 70% reduction in sequencing depth resulted in the slight reduction in heteroplasmy burden, we noticed higher heteroplasmic variability (standard deviation) in this subset of NIH HbSS patients. This variability may be attributable to extreme variation in SCD phenotypic severity. We then applied cumulative distribution function to this downsampled subset and compared with JHS genotypes. We found the NIH HbSS patients have disproportionately higher proportion of heteroplasmy variants (Fig 1D) when compared to the JHS genotypes (HbAA, HbAC, and HbAS). Conclusion: We conclude that there is an increased prevalence of heteroplasmic mtDNA variants in SCD compared to ethnic-matched normal (HbAA) populations. Normal individuals with HbAA in JHS cohort have significantly higher heteroplasmic burden compared to those in 1KG cohort, suggesting an underlying cardiovascular disease in JHS cohort as a driving factor. Within each 1KG and JHS cohorts, individuals with sickle cell trait (HbAS) have similar heteroplasmy burden and also higher than those with HbAA, highlighting the potential significance of this genotype. Reducing the sequencing depth by > 70% (downsampling) led to the filtering out of heteroplasmy variants that would have been discovered with the original deeper sequencing depth of ~7300X. Nonetheless, downsampled HbSS samples still retained disproportionately higher heteroplasmy burden compared to non-SCD subjects. We are currently investigating if there is any correlation between mtDNA heteroplasmy burden and severity of clinical phenotypes among the SCD patients. 1. Ahmad, MM et al, Blood 136 (1):11-11 (2020) Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mitochondrial DNA Variation in Individuals with Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Maliha Maryam Ahmad ◽  
Laxminath Tumburu ◽  
Chunyu Liu ◽  
Mehdi Pirooznia ◽  
Swee Lay Thein
Keyword(s):  
Mitochondrial Dna ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mutant Allele ◽  
African Ancestry ◽  
Organ Damage ◽  
Median Number ◽  
Cell Disease ◽  
Mtdna Sequences ◽  
Mitochondrial Heteroplasmy

Background: Sickle cell disease (SCD) is a complex multi-system disorder that predominantly affects individuals of African heritage. While the sickle pathology is initiated by polymerization of HbS, multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of mutations in mitochondrial DNA (mtDNA), the phenotypic effect of which will depend on the nature of the gene that harbors the mutation, the mutant allele fraction, and the pathogenicity of the mutant allele. A mutation in mtDNA is heteroplasmic when it is present in only a proportion of mtDNA, and homoplasmic, when it is present in all mtDNA molecules. Mitochondrial heteroplasmy can also occur at different tissue or cell levels, even within the same individual. Given the underlying chronic inflammation in SCD, we hypothesize that SCD patients display increased rates of mtDNA mutations. Methods: We analyzed and compared whole genome sequencing (WGS) data from the cohort of 683 SCD patients (SCD cohort) of African ancestry with that of 621 individuals of African ancestry from the 1000 Genomes Project (1KG). The SCD cohort included 561 HbSS & HbSβ0 thalassemia (combined), 90 HbSC, and 25 HbSβ+ thalassemia. The 1KG cohort included 516 HbAA and 105 sickle carriers (HbAS). mtDNA sequences of SCD and 1KG cohorts were initially aligned to the revised Cambridge Reference Sequence (rCRS NC_012920), and subsequently base recalibrated and deduplicated. Mitochondrial sequences extracted from the cleaned WGS data of both cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus is ~6,828X for the SCD cohort and ~2,879X for the 1KG cohort. We performed a locus by locus comparison between the mtDNA sequences of both cohorts. No homoplasmic variants unique to the SCD cohort were found when compared to the 1KG cohort. In contrast, there were several "hotspots" of heteroplasmic variants that were unique to the SCD cohort, and largely shared amongst the SCD patient population (Figure 1A). To identify these unique variants, we used MITOMASTER and Ensembl VEP to annotate the heteroplasmic variants that had above 40% population frequency within the SCD cohort and below 10% population frequency in the 1KG cohort. Several heteroplasmic variants were non-synonymous and the selected variants originated from the Control Region (D-loop), RNR1, RNR2, ND1, ND4, and ND5. One of the heteroplasmic variants, 2623 A>G, was found to be age linked for HbSS & HbSβ0 (Figure 1B), with its minor allele frequency (MAF) increasing with age. Further analysis needs to be done in order to determine if more variants unique to the SCD cohort are age-linked. We then compared the quantity of heteroplasmic variants across the different SCD genotypic groups with the 1KG HbAA and the 1KG HbAS groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ+ thalassemia, and HbSC with the highest median number of 119 in HbSS & HbSβ0 (Figure 1C). Mitochondrial heteroplasmy for 1KG HbAA and 1KG HbAS were statistically significant when tested against each other and against every SCD sub-group; however, the difference was not statistically significant between the different SCD genotypes (Table insert in Fig 1C). It is important to note that we did not apply a MAF threshold, thus many of the heteroplasmic variants may be present at very low levels. Conclusion: Our findings suggest that there is an increased prevalence of heteroplasmic variants in SCD compared to ethnic-matched healthy populations. Within the SCD genotypes, the heteroplasmic burden increased progressively (HbAS < HbSβ+ thalassemia < HbSC < HbSS & HbSβ0) with genotypic groups that are associated with increasing phenotypic severity. mtDNA heteroplasmic burden for one variant also increased with age in HbSS & HbSβ0 individuals, but further studies are needed to explore if mtDNA heteroplasmic burden correlates with the degree of organ damage and disease severity within the same genotypic group. Although it is not clear if the variants are a cause or effect of the sickle inflammatory pathology, our data suggest that mtDNA heteroplasmic burden is a potential biomarker of SCD severity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Is Skeletal Muscle Dysfunction a Limiting Factor of Exercise Functional Capacity in Patients with Sickle Cell Disease?

2021 ◽  
Vol 10 (11) ◽  
pp. 2250
Author(s):  
Etienne Gouraud ◽  
Philippe Connes ◽  
Alexandra Gauthier-Vasserot ◽  
Camille Faes ◽  
Salima Merazga ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sickle Cell Disease ◽  
Muscle Fatigue ◽  
Sickle Cell ◽  
Functional Capacity ◽  
Muscle Dysfunction ◽  
Electromyographic Activity ◽  
Cell Disease ◽  
Step Frequency ◽  
Hemoglobin C

Patients with sickle cell disease (SCD) have reduced functional capacity due to anemia and cardio–respiratory abnormalities. Recent studies also suggest the presence of muscle dysfunction. However, the interaction between exercise capacity and muscle function is currently unknown in SCD. The aim of this study was to explore how muscle dysfunction may explain the reduced functional capacity. Nineteen African healthy subjects (AA), and 24 sickle cell anemia (SS) and 18 sickle cell hemoglobin C (SC) patients were recruited. Maximal isometric torque (Tmax) was measured before and after a self-paced 6-min walk test (6-MWT). Electromyographic activity of the Vastus Lateralis was recorded. The 6-MWT distance was reduced in SS (p < 0.05) and SC (p < 0.01) patients compared to AA subjects. However, Tmax and root mean square value were not modified by the 6-MWT, showing no skeletal muscle fatigue in all groups. In a multiple linear regression model, genotype, step frequency and hematocrit were independent predictors of the 6-MWT distance in SCD patients. Our results suggest that the 6-MWT performance might be primarily explained by anemia and the self-paced step frequency in SCD patients attempting to limit metabolic cost and fatigue, which could explain the absence of muscle fatigue.

Sickle cell trait and the risk of venous thromboembolism among blacks

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 908-912 ◽  
Author(s):  
Harland Austin ◽  
Nigel S. Key ◽  
Jane M. Benson ◽  
Cathy Lally ◽  
Nicole F. Dowling ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Odds Ratio ◽  
Sickle Cell Trait ◽  
Coagulation System ◽  
Cell Disease ◽  
Hemoglobin C ◽  
Increased Risk ◽  
S Allele

Abstract People with sickle cell disease have a chronically activated coagulation system and display hemostatic perturbations, but it is unknown whether they experience an increased risk of venous thromboembolism. We conducted a case–control study of venous thromboembolism that included 515 hospitalized black patients and 555 black controls obtained from medical clinics. All subjects were assayed for hemoglobin S and hemoglobin C genotypes. The prevalence of the S allele was 0.070 and 0.032 for case patients and controls, respectively (P < .001). The odds that a patient had sickle cell trait were approximately twice that of a control, indicating that the risk of venous thromboembolism is increased approximately 2-fold among blacks with sickle cell trait compared with those with the wild-type genotype (odds ratio = 1.8 with 95% confidence interval, 1.2-2.9). The odds ratio for pulmonary embolism and sickle cell trait was higher, 3.9 (2.2-6.9). The prevalence of sickle cell disease was also increased among case patients compared with controls. We conclude that sickle cell trait is a risk factor for venous thromboembolism and that the proportion of venous thromboembolism among blacks attributable to the mutation is approximately 7%.

Alloimmunization Does Not Modify the Clinical Profile of Sickle Cell Disease Patients from Salvador-Brazil

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4799-4799
Author(s):  
Angela Zanette ◽  
Karla O. Mota ◽  
Marilda Souza Goncalves ◽  
Laise Vilasboas Schettini ◽  
Lais Magalhaes Aguiar ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Clinical Outcomes ◽  
Sickle Cell ◽  
Laboratory Data ◽  
Clinical Profile ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Hemoglobin S ◽  
Hemoglobin C ◽  
Significant Difference

Abstract Introduction: The hemoglobinopathies are the most common monogenic disorders known. A mutation in the gene for β globin gave origin to hemoglobin S, an abnormal hemoglobin originated in Africa. Sickle cell disease (SCD) is characterized by the presence of hemoglobin S, which results in vasoocclusion episodes and hemolytic anemia throughout patients life. Vascular occlusion leads to acute events and progressive disabling organ damage. Sickle cell anemia is the homozygous state SS, while hemoglobinopathy SC is a doubly heterozygous state, where hemoglobin S occurs in combination with hemoglobin C. Brazil has a prominent African ancestry and SCD is highly prevalent in some regions of the country. In Bahia State, for example, neonatal screening data have shown that, from every 650 children born alive, one has SCD, mostly homozygous SS. Among other therapeutic measures, packed red blood cells (RBC) play a prominent role in SCD management. In situations such as acute chest syndrome (ACS), primary and secondary prevention of stroke, splenic or hepatic sequestration crisis, severe anemia, complicated pregnancy, isquemic organ damages and others, the transfusions may save lives. Although RBC may contribute to reduce morbidity and improve quality of life in SCD patients, there still are risks. Among other risk categories, alloimmunization may result from transfusions and occurs in 5 % to 50 % of SCD patients. It is still not known whether allosensibilization significantly affects the clinical outcomes in SCD. Objecive: The purpose of this study was to compare the clinical profile of multitransfused adult SCD patients who developed alloantibodies (ALO) to patients with the same disease, coming from the same population who did not become alloimmunized (non-ALO). Methods: This is a cross sectional study where medical records of SCD patients, referred to a reference center of Salvador, the capital of Bahia State, Brazil, were reviewed. Only SCD patients 18 years of age or older were included. They had received at least 3 RBC transfusions from 2004 to 2007, or had any alloantibody identified during this period. Patient characteristics, clinical findings, number of transfusions, frequency and specificity of alloantibodies, laboratory data, and the main clinical outcomes were reviewed. Results: a hundred and eight patients were included: 105 SS and 3 SC. The pre-transfusional RBC matching was done to ABH, D,C,c,E,e and Kell antigens. 56 patients developed alloantibodies (53 SS and 3 SC). Anti-E, anti-K, and anti-C were the most prevalent alloantibodies identified (39,3 %, 21,4 % and 16,1 %, respectively). Among the variables addressed in this study, age (higher in non-ALO, .041) and antiglobulin test positivity, more prevalente in ALO (.0001), depicted statistically significant difference. A few patients developed immune hemolysis, controlled successfully with corticosteroids. Alloimmunization was more prevalent among women, although no statistically significant difference was reached between ALO and non-ALO Other variables such as number of transfusions, hematological profile, biochemical data and complications such as stroke, leg ulcers, osteonecrosis, renal disease, abnormal cardiac features, and pulmonary hypertension did not show significant difference between both groups. Conclusion: his study shows that, although alloimmunization is a potential dangerous consequence of RBC transfusions, it did not modify the clinical profile of SCD alloimmunized patients. The concomitance of allosensibilization and autoantibodies in SCD leads to additional difficulties in the RBC matching for transfusion and may exacerbate hemolysis. In order to address autoimmunity in SCD, prospective studies with larger samples are needed.

Effectiveness Of An Analgesia Protocol For The Treatment Of Painful Vaso-Occlusive Crisis Of Sickle Cell Disease Patients In Emergency Room: A Retrospective Cohort Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2222-2222
Author(s):  
Veronique Naessens ◽  
Richard Ward ◽  
Kevin H.M. Kuo
Keyword(s):  
Sickle Cell Disease ◽  
Emergency Room ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Care Center ◽  
Median Number ◽  
Cell Disease ◽  
Dose Time ◽  
Chi Squared ◽  
Opiate Administration

Background Painful vaso-occlusive crisis (VOC) is the most frequent complication of sickle cell disease (SCD) and the main reason for interacting with the emergency room (ER). Guidelines highlight the need for rapid opiate delivery (< 30 min.) via specific analgesia protocol in order to achieve rapid pain control and reduce length of stay (LOS). Objectives To determine the rate of utilization of an analgesia protocol for SCD patients presenting with painful VOC to ER. Secondary objectives are to examine the relationship between protocol use, LOS, discharge from ER, time to first opiate delivery, total amount of opiates, and time to readmission. Methods An analgesia protocol, developed according to the BCSH guideline, was deployed in the ER of a major SCD comprehensive care center in Canada since 2009. A retrospective observational study was conducted of all SCD patients who presented to the ER with painful VOC between August 2009 and September 2012. Frequent ER visitor was defined as >3 visits/year. Factors influencing protocol use and discharge from ER were examined via Chi-squared test and multivariable logistic regression. Mann-Whitney U test and multiple regression were used to examine factors related to time to first opiate administration, LOS in ER, total quantity of opiates used, and as well as time to readmission. Results 602 ER visits were included in the analysis, comprising of 116 patients (61 males, 55 females) with 82% HbSS or S/β0. The median number of visits in the observation period was 2 (1 to 90), with 12 patients responsible for 57% of visits. The protocol was used in only 51% of visits, frequent ER attenders were less likely to be treated by the protocol (OR 0.989 per additional visit, p=0.007). Higher pain score on presentation was significantly associated with protocol use (OR 1.194 per 1 point increment, p=0.001), which in turn reduced the time to first opiate administration (69 vs. 108 min., p<0.001). Visits treated by protocol were longer (457 min vs. 385 min. in ER, p<0.001), used more opiates (95 vs. 50 mg PO morphine equi-analgesic dose, p<0.001) and patients were less likely to be discharged home (OR 0.78, p=0.003), though it prolonged the time to next ER admission (22 vs. 15 days, p=0.008). The length of inpatient admission was not affected by prior use of the Protocol in ER. Conclusion In this, the largest single-center report of ER sickle cell care, the rate of protocol use was moderate and although it resulted in faster delivery of first opiate dose, time to delivery did not reach the recommended target of 30 minutes. Protocol use did not positively influence any other outcomes. The protocol was preferentially used in patients who attends the ER infrequently. Further refinement of the protocol may lead to improvements in utilization by ER staff, leading to shorter time to first analgesia administration. This in turn may result in the protocol having a greater impact on patient outcomes in ER. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TRANSFUSION PRACTICE, POST-TRANSFUSION COMPLICATIONS AND RISK FACTORS IN SICKLE CELL DISEASE IN SENEGAL, WEST AFRICA.

2022 ◽  
Vol 14 (1) ◽  
pp. e2022004
Author(s):  
Moussa Seck ◽  
Alioune Badara Senghor ◽  
Mossane Loum ◽  
Sokhna Aissatou Touré ◽  
Blaise Félix Faye ◽  
...  
Keyword(s):  
Risk Factors ◽  
Sickle Cell Disease ◽  
Iron Overload ◽  
Sickle Cell ◽  
Median Number ◽  
Transfusion Practice ◽  
Red Cell ◽  
Cell Disease ◽  
Hiv Antibodies ◽  
Transfusion Complications

Context and Objectives: Blood transfusions (BT) remain a mainstay of therapy for patients with sickle cell disease (SCD), but pose significant clinical challenges. We aim to assess infectious markers, red cell alloimmunization and iron overload secondary to BT in SCD patients. Materials and Methods: This is a case-control study included 253 SCD (153 SCD-transfused and 100 SCD non-transfused). We evaluated the transfusion practice (modalities, indications), post-transfusion complications (infections, alloimmunization, iron overload) and risk factors of these complications (socio-demographic, clinical, biological). Results: Median age was 28.5 years (5 - 59). Sex ratio was 0.86. Homozygous SCD was more common (95.3%). Simple BT was performed in 92.8% and transfusion exchange in 18.9%. Transfusion indications were dominated by acute anemia (57.06%) and vaso-occlusive crisis (VOCs) (14%). Red blood cell concentrates (RBC) were administered to 93.46%. Median number of RBC received per patient was 10 (2 - 48). The prevalence of VHC in SCD-transfused was 1.33% and 2% for VHB. Anti-HIV antibodies were not found. Red cell alloimmunization frequency was 16%. The most common alloantibodies were anti-rhesus (34.19%) and anti-Kell (23.67%). Iron overload was detected in 7.84%. The number of RBC transfused was the only risk factor for alloimmunization (p = 0.03) and iron overload (p = 0.023). BT frequency was not related to infectious transmission. Conclusion: Despite advances in blood safety, BT therapy is still a risk for SCD polytransfused patients. Although infectious transmission has rare, the risk of alloimmunization and iron overload is high in these patients.

Association of Pulmonary Hypertension with Sickle Cell Disease Subtypes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4824-4824
Author(s):  
Alice J. Cohen ◽  
Chaim Tuckman-Vernon
Keyword(s):  
Pulmonary Hypertension ◽  
Sickle Cell Disease ◽  
Hemolytic Anemia ◽  
Sickle Cell ◽  
Doppler Echocardiography ◽  
Beta Thalassemia ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Hemoglobin C ◽  
Baseline Hemoglobin

Abstract Pulmonary hypertension (PH) is a common complication of sickle cell disease (SD) and a significant cause of morbidity and mortality. PH, measured by Doppler echocardiography and defined as a tricuspid regurgitant jet velocity (TRV) &gt; 2.5 m per second (m/s), is hypothesized to be related to the chronic hemolytic anemia of SD, but causality is unproven. If so, the presence of hemoglobin C, which reduces hemolysis, would be expected to have a reduced likelihood of PH. This study reviewed the prevalence of PH in 3 categories of patients with SD: homozygous S (SS), sickle-beta thalassemia (SB), and SC. Methods: Sickle cell disease patients registered at a state funded community comprehensive care adult sickle cell center were routinely screened for PH by Doppler echocardiography. The presence of PH, the incidence of a related complication, acute chest syndrome (ACS), and baseline hemoglobin (hgb) were reviewed. Results: 16 patients with SC type, 30 with SS and 39 with SB disease underwent screening. The prevalence of PH, ACS and hgb are listed in the table below. Conclusion: SC patients have PH and ACS similar to patients with SS and SB patients. These patients have higher baseline hemoglobin and may have hyperviscosity as a cause of PH and ACS as opposed to hemolytic anemia. Further study of PH and ACS in SC patients is warranted. SC SS SB p value PH 6/16 (38%) 12/40 (40%) 11/39 (28%) p= NS ACS 7/16 (44%) 10/30 (33%) 19/39 (49%) p=NS PH + ACS 4/16 (25%) 5/30 (17%) 4/39 (10%) p=NS ACS in PH patients 4/6 (67%) 5/12 (42%) 4/11 (36%) p-=NS Hgb 10.8 7.89 8.57 p=0.000

scholarly journals Cell-Free Mitochondrial DNA Is Elevated in Sickle Cell Disease Patients, and Serve As a Potential Proinflammatory DAMP

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1068-1068
Author(s):  
Laxminath Tumburu ◽  
Shohini Ghosh-Choudhary ◽  
Emilia Alina Barbu ◽  
Simon Yang ◽  
Lauren D Harrison Ware ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Acute Pain ◽  
Organ Damage ◽  
Healthy Controls ◽  
Cell Disease ◽  
Cross Sectional ◽  
Healthy Donors

Abstract Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by hemolysis and intermittent acute pain with multi-organ damage. Previously, we showed that acute pain in SCD was associated with >10-fold increases in cell-free DNA (cfDNA) when compared to steady state, that were significantly reduced during hydroxyurea therapy. Apoptosis, necrotic cell death and lysis of intact cells in the blood stream have been proposed as sources of plasma cfDNA. Here, we explored if the cfDNA increases could have a role in inflammation, a constant pathological feature of SCD. cfDNA was extracted using QIAamp MinElute ccfDNA Kit (Qiagen), from the platelet-poor plasma processed within 30 minutes from the blood drawn in EDTA tubes, and analyzed using whole genome sequencing (WGS) and targeted quantitative PCR (qPCR). SCD patients are defined as in acute pain if there is no evident cause other than SCD, for which the patient needs hospitalization, either as in- or outpatient, and is treated with parenteral narcotics. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. A cross-sectional study of 8 healthy controls and 34 SCD patients (18 steady-state; 16 crisis) mapped WGS reads showed significantly higher proportion of cell-free mitochondrial DNA (cf-mtDNA) compared to nuclear cfDNA (cf-nDNA) in SCD patients compared with healthy controls (Fig 1A: steady-state: 14 fold; crisis: 11 fold; p = 0.0001). We used targeted qPCR to quantify both cf-nDNA and cf-mtDNA in another cross-sectional cohort of 13 healthy controls and 92 patients (72 steady-state, 20 crisis) as well as 18 paired HbSS patients (steady-state and crisis) samples with 10 healthy controls. The nuclear reference genes used were GAPDH and TERT and mitochondrial genes were MT-ND1 and MT-ND6. While cf-nDNA (TERT) was significantly increased (> 3.5 fold, p = 0.0251; Fig 1B) in SCD patients compared with healthy controls only during crises, significantly higher levels of cf-mtDNA over cf-nDNA were observed in SCD patients compared with healthy volunteers in both steady-state and crises (Fig 1C: MT-ND1/GAPDH: steady-state >19 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 7 fold; MT-ND6/GAPDH: steady-state > 7 fold, crisis > 3 fold; MT-ND6/TERT: steady-state > 4 fold; crisis > 4 fold; p < 0.05). In the paired samples, cf-nDNA (GAPDH andTERT) was significantly increased (> 3 fold; Fig 1D-E) in crisis compared to steady-state (p < 0.05). The differential increase in cf-mtDNA (cf-mtDNA:cf-nDNA ratio) levels in these patients during crises, were significantly higher compared with healthy controls (Fig 1F: MT-ND1/GAPDH: steady-state >9 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 9 fold; MT-ND6/GAPDH: steady-state > 8 fold, crisis > 8 fold; MT-ND6/TERT: steady-state > 8 fold; crisis > 7 fold; p < 0.005). Using confocal microscopy and mitochondrial-specific dyes (MitoTracker Green and TMRM), we show that substantial numbers of red blood cells from SCD patients retain their mitochondria in the circulation. We next explored if the elevated cf-mtDNA in SCD could contribute to its pathophysiology, via activating neutrophils to form neutrophil extracellular traps (NETs), a recognized immunological response in inflammation. Initially, we confirmed that mtDNA can induce NETosis by treating neutrophils from healthy donors with mtDNA isolated from human platelets. mtDNA consistently induced a robust NETs response (N=8) while genomic nuclear DNA did not cause any NETosis. SCD plasma containing high levels of cf-mtDNA also caused a strong NETosis response while plasma from healthy donors did not (N=11). Cytosolic adaptor STING has a central role in sensing of cytosolic double stranded DNA. We sought to determine if the downstream STING-TBK1-IRF3 pathway is associated with the mtDNA-mediated formation of NETs. We inhibited the catalytic activity of the STING downstream effector TBK1 with BX795 prior to treating neutrophils with cf-mtDNA-containing plasma (N=5). The TBK1 inhibition consistently reduced the NETs response by at least 70% confirming that cytosolic DNA sensors are involved in promoting mtDNA-mediated formation of NETs. Our findings suggest that cf-mtDNA induces NETosis contributing to the pathological sterile inflammation in SCD patients. Continual release of these mitochondrial DAMPs in hemolysis may serve as key link between inflammation and organ damage in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Use of an automated pyrosequencing technique for confirmation of Sickle Cell Disease

2019 ◽  
Author(s):  
CC Martino ◽  
CS Alencar ◽  
P Loureiro ◽  
AB Carneiro-Proietti ◽  
CA Máximo ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Performance ◽  
Large Scale ◽  
Clinical Care ◽  
Beta Thalassemia ◽  
Cell Disease ◽  
Hemoglobin S ◽  
Hemoglobin C ◽  
Confirmatory Assay

ABSTRACTBackgroundThe diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.ObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene.MethodsWe developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.ResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant HPLC and PyS= heterozygous S, which would suggest Sβ-thalassemia or other hemoglobin S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.DiscussionThe PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil.

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