Platelet Phenotype Prediction from Whole Genome Sequencing in 621 Sickle Cell Disease Patients

Introduction: Patients with sickle cell disease (SCD) are at increased risk of alloimmunization. Platelet refractoriness is a serious known complication and often seen in SCD patients who are heavily transfused and/or in the bone marrow transplantation (BMT) setting. Next generation sequencing (NGS) is an emerging and promising genotyping strategy in the context of blood typing, due its high throughput and its ability to detect both known and novel variants in the patient and donor population. Here we describe an algorithm to predict common and rare human platelet antigens (HPA) from NGS data, and its validation through Sanger sequencing. Design/Methods: Whole genome sequencing (WGS) was performed on stored blood samples from 621 SCD patients enrolled in 2 IRB-approved clinical studies. Our open source software RyLAN (Red Cell and Lymphocyte Antigen prediction from NGS) was utilized to translate WGS data into predicted RBC and platelet phenotypes. The 29 genomic variants interpreted by RyLAN in 6 HPA genes were correlated with Sanger sequencing. Results: Our study cohort consisted of 621 SCD patients (485 HbSS, 21 HbSβ0, 29 HbSβ+, 84 HbSC, 1HbS O Arab, and 1 HbSD). The mean age was 34.3 years, and 46% were male. Previous red cell transfusions were recorded in 62% of patients, and 3% were documented as never transfused. RyLAN software was executed as a singularity container in multithreaded mode, completing analysis of all 621 .bam WGS files in 18 hours. RyLAN predicted 237 unique extended platelet phenotype combinations in this cohort, with an average read depth of 33 in genomic areas of interest. Predictions for 10 platelet antigens in 26 participants, including rare phenotypes like HPA-25bw+ and HPA-13bw+, were confirmed by bidirectional Sanger. Conclusions: We describe an efficient, open-source algorithm used to interpret 6 HPA genes from WGS in a large SCD cohort. WGS, in conjunction with the RyLAN algorithm, demonstrated 100% accuracy in predicting common and rare HPA genomic variants. Future studies are needed to refine WGS algorithms in SCD, and to examine the possible value of this technology in HPA alloantibody identification workups, optimal platelet product allocation, and donor recruitment. Disclosures No relevant conflicts of interest to declare.

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Whole Genome Sequencing of Pediatric Sickle Cell Disease Patients

Oncology Times ◽

10.1097/01.cot.0000553520.51708.8c ◽

2019 ◽

Vol 41 ◽

pp. 11-12

Author(s):

Richard Simoneaux

Keyword(s):

Sickle Cell Disease ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sickle Cell ◽

Whole Genome ◽

Cell Disease ◽

Pediatric Sickle Cell Disease

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Diversity of variant alleles encoding Kidd, Duffy, and Kell antigens in individuals with sickle cell disease using whole genome sequencing data from the NHLBI TOPMed Program

Transfusion ◽

10.1111/trf.16204 ◽

2020 ◽

Author(s):

Carla L. Dinardo ◽

Theo G. M. Oliveira ◽

Shannon Kelly ◽

Allison Ashley‐Koch ◽

Marilyn Telen ◽

...

Keyword(s):

Sickle Cell Disease ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sickle Cell ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Cell Disease ◽

Sequencing Data ◽

Variant Alleles

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Red Blood Cell and Platelet Phenotype Prediction from Whole Genome Sequencing in 621 Sickle Cell Disease Patients: Correlation with Alloimmunization History, Serology and Other Genotyping Methods

Blood ◽

10.1182/blood-2018-99-115008 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2388-2388

Author(s):

Celina Montemayor Garcia ◽

Julie K. Weisman ◽

Divya Gandla ◽

James Owen Long ◽

Clifton Lee Dalgard ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Blood Group ◽

Genome Sequencing ◽

Red Blood Cell ◽

Read Depth ◽

Whole Genome ◽

Cell Disease ◽

Health Records ◽

Genomic Variants

Abstract Introduction: Red blood cell (RBC) transfusions are central in the management of sickle cell disease (SCD), an inherited hemoglobinopathy characterized by hemolysis, acute pain, and multi-systemic complications. Extended matching of patient and donor RBC antigens is an established strategy to minimize alloimmunization, which can make provision of compatible blood difficult and can result in severe, even lethal hemolytic transfusion reactions. While RBC genotype matching has proven valuable in SCD transfusion practice, current technologies are often limited in throughput and focus on selected blood groups and known variants. Limited information is available comparing whole genome sequencing (WGS) with other blood typing platforms in SCD. Design/Methods: WGS was performed on stored blood samples from 621 SCD patients recruited into two clinical studies. We utilized our open-source Python application (RyLAN), to translate WGS data into a predicted extended RBC and platelet phenotype. The 467 genomic variants interpreted by RyLAN in 41 genes were correlated with clinical and laboratory data in the immunohematology and electronic health records (Figure 1). Results: The 621 patients included 485 HbSS, 21 HbSb0, 29 HbSβ+, 84 HbSC, 1HbS O Arab, and 1 HbSD. The mean age was 34.3 ± 12.1 years, and 54% were female. Health records indicated that 383 (62%) patients had previously received RBC transfusions and 17(3%) had never been transfused; the status of the remaining 221 was unknown. RyLAN software was executed as a singularity container in multithreaded mode, completing the analysis of all 621 bam WGS files in 8.5 hours (8 CPUs and 16GB of memory per file). The average read depth for genomic positions of interest was 33 and the average QUAL value was 644. The highest variant allele frequency was detected at the Fyb, ACKR1 promoter, and the KCAM- loci (94%, 86% and 82%, respectively). Each of the 621 participants demonstrated a unique extended blood group genotype through WGS. RyLAN predicted 237 unique extended platelet phenotype combinations in this cohort, including HPA-25bw and HPA-13bw positive patients. Blood antigen WGS predictions were correlated with other typing methods in 112 individuals: 192 total serologic reactions for 8 antigens; 55 documented alloantibodies; 25 genomic variants in 71 participants by probe-elongation array; and PCR with sequence-specific primers for 8 variants in 13 individuals (Figure 1). Two instances of heterozygosity (Jka/Jkb and Doa/Dob) were undetected due to low read depth, and 8 unresolved discrepancies were identified: 2 with serology, 1 with a reported historical alloantibody, 2 with probe-elongation array determinations, and 1 with the PCR method. WGS detected multiple weak blood group variants, surpassing the sensitivity of serology in one complex case, as well as rare phenotypes including 4 Yka-, 5 Kna/Knb, 1 FORS1-, and 2 Jra- cases. The algorithm correctly predicted an Sla-negative RBC phenotype in a patient with documented anti-Sla alloantibody. Conclusion: We describe an efficient, open-source algorithm used to interpret 35 minor blood group and 6 platelet antigen genes from WGS in a large SCD cohort. Eight unresolved discrepancies were identified from 2126 correlation events with serology, alloimmunization history, and other genotyping methods in a subset of 112 individuals. WGS demonstrated higher sensitivity for weak antigen detection compared with serology, and a capacity to detect rare phenotypes not readily determined by other methods. Sanger resequencing is currently in progress to validate rare phenotype predictions and resolve remaining discrepancies. Future studies are needed to refine WGS algorithms in SCD, and determine the value of this technology for alloantibody identification, optimal blood group allocation, and donor recruitment. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Precision Medicine for Sickle Cell Disease through Whole Genome Sequencing

Blood ◽

10.1182/blood-2018-99-117606 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3641-3641 ◽

Cited By ~ 1

Author(s):

Evadnie Rampersaud ◽

Lance E. Palmer ◽

Jane S. Hankins ◽

Vivien A Sheehan ◽

Wenjian Bi ◽

...

Keyword(s):

Sickle Cell Disease ◽

Genome Sequencing ◽

Sickle Cell ◽

Research Funding ◽

Fetal Hemoglobin ◽

Whole Genome ◽

Genetic Modifiers ◽

Cell Disease ◽

Genome Wide

Abstract Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include: 1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples. 2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD. 3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care. 4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD. Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

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Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants

10.1101/316976 ◽

2018 ◽

Cited By ~ 4

Author(s):

Maxime Garcia ◽

Szilveszter Juhos ◽

Malin Larsson ◽

Pall I. Olason ◽

Marcel Martin ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Open Source ◽

Genome Sequencing ◽

Development Project ◽

Whole Genome ◽

Sequencing Analysis ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Insertion And Deletion ◽

Sample Heterogeneity

AbstractSummaryWhole-genome sequencing (WGS) is a cornerstone of precision medicine, but portable and reproducible open-source workflows for WGS analyses of germline and somatic variants are lacking. We present Sarek, a modular, comprehensive, and easy-to-install workflow, combining a range of software for the identification and annotation of single-nucleotide variants (SNVs), insertion and deletion variants (indels), structural variants, tumor sample heterogeneity, and karyotyping from germline or paired tumor/normal samples. Sarek is implemented in a bioinformatics workflow language (Nextflow) with Docker and Singularity compatible containers, ensuring easy deployment and full reproducibility at any Linux based compute cluster or cloud computing environment. Sarek supports the human reference genomes GRCh37 and GRCh38, and can readily be used both as a core production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups.AvailabilitySource code and instructions for local installation are available at GitHub (https://github.com/SciLifeLab/Sarek) under the MIT open-source license, and we invite the research community to contribute additional functionality as a collaborative open-source development project.

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Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽

10.1182/blood-2020-141603 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Yuanbin Song ◽

Rana Gbyli ◽

Liang Shan ◽

Wei Liu ◽

Yimeng Gao ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Red Cell ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

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Alkaline Phosphatase Is Associated with Red Cell Alloimmunization in the Pulmonary Hypertension and Hypoxic Response (PUSH) Sickle Cell Disease Cohort

Blood ◽

10.1182/blood-2020-143036 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-20

Author(s):

Victoria Brooks ◽

Oluwalonimi Adebowale ◽

Victor R. Gordeuk ◽

Sergei Nekhai ◽

James G. Taylor

Keyword(s):

Risk Factors ◽

Alkaline Phosphatase ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Severe Disease ◽

Case Control ◽

Red Cell ◽

Cell Disease ◽

History Of

Background: Blood transfusion is a common therapy for sickle cell disease (SCD). Although, highly effective, a major limitation is development of alloantibodies to minor blood group antigens on donor red cells. Alloimmunization has a prevalence of 2-5% for transfusions in the general population, but it is significantly higher in SCD. Risk factors for alloimmunization have been poorly characterized, although number of lifetime transfusions is an important risk factor. Alloimmunization has been clinically observed in children with a prevalence of about 7%. With development of each antibody, blood donor matching becomes increasingly difficult and expensive with an increased risk for transfusion reactions and diminished availability of compatible red cell units for treatment of SCD. The ability to identify risk factors for developing alloantibodies would be beneficial for clinicians. To identify markers for alloimmunization in SCD, we have analyzed children and adults who developed this complication. Methods: We analyzed The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study, which enrolled n=468 pediatric and n=59 adult SCD subjects. In both children and adults, alloimmunization cases were defined as a history of at least 1 alloantibody. Controls in both cohorts were defined as subjects with no history of alloantibodies and receipt of more than 10 lifetime red cell transfusions. All others within the study who did not meet these criteria were assigned to a third comparison group. To identify differences between cases, controls and all others, we performed univariate analyses (using ANOVA or Kruskal Wallace where appropriate) for clinical parameters and laboratories. Case control comparisons were also performed for selected variables and plasma levels for 11 cytokines. Results were further analyzed using regression modeling. Results: The overall prevalence of alloimmunization was 7.3% among children (34/468 subjects; median age 12, range 3-20 years) compared to 28.8% in adults (17/59 subjects; median age 37, range 18-73 years). When only considering those with >10 lifetime transfusions, the prevalence was considerably higher at 29.3% and 54.8% in children and adults, respectively. At the same time, 8 pediatric (23.5%) and 5 adult (29.4%) alloimmunization cases had received fewer than 10 transfusions. In a 3-way pediatric cohort comparison (cases, controls and all others), risk factors associated with alloimmunization included SS genotype, older age and markers of more severe disease (higher ferritin, WBCs, platelets and total bilirubin). Comparison of cases to controls showed alkaline phosphatase (P=0.05) was significantly lower in cases, whereas AST (P=0.02) was significantly higher even with adjustment for age. Levels of plasma cytokines MCP-1 (P=0.01) and IFNgamma (P=0.08) were lower in cases from a subset of the pediatric cohort. In adults, only 4/59 (6.8%) subjects had never received a lifetime transfusion (all non-SS). In the adult 3-way comparisons, only SS genotype and higher ferritin were associated with alloimmunization. The adult case control analysis showed higher absolute monocyte count (P=0.02), absolute eosinophil count (P=0.04) and absolute basophil count (P=0.008) in association with alloimmunization cases. In addition, alkaline phosphatase was again significantly lower among cases (P=0.02) as seen in the pediatric cohort. There were no significant differences in cytokine levels among adults. Conclusions: When considering only transfused SCD patients, the prevalence of alloimmunization is higher than 30%. As seen in prior studies, higher lifetime red cell transfusions are an important risk factor especially among adults where most patients have received transfusions. Children who develop alloantibodies appear to have laboratory markers of more severe disease, but this is not observed in adults. A novel association observed across both pediatric and adult subjects is a significantly lower serum alkaline phosphatase in those with alloantibodies. The results of this study suggest a need for improved tracking of red cell transfusion therapy in the US for SCD patients due to a high prevalence of alloimmunization. Further study is also needed to elucidate the significance of the alkaline phosphatase association. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Ironwood: Research Funding; Imara: Research Funding.

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