ALBERT

Description: Background: Nephropathy in sickle cell anemia (SCA) begins in childhood and portends chronic kidney disease, renal failure, and early mortality among affected adults. Individuals of African descent have disproportionately higher rates of developing non-diabetic renal disease. Several candidate genetic variants have been identified, including some specific to African Americans, which are associated with the development of albuminuria and renal disease. The influence of genetic polymorphisms on albuminuria and elevated glomerular filtration rate (GFR) in children with SCA, both early signs of sickle nephropathy, has not been investigated. Objectives: To determine the influence of selected single nucleotide polymorphisms (SNPs) on the development of albuminuria and elevated GFR in children with SCA; to identify novel genetic variants influencing albuminuria and GFR by whole exome sequencing (WES). Design/Methods: Genomic DNA was collected on children with SCA enrolled in two prospective studies with pre-hydroxyurea renal assessments (n=185): (1) Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175, n=79) with no prior disease-modifying therapy; and (2) Transcranial Doppler (TCD) With Transfusions Changing to Hydroxyurea (TWiTCH, NCT 01425307, n=106) on chronic transfusions for abnormal TCD velocities. Albuminuria was defined as ≥30mg albumin/gm creatinine on the pre-hydroxyurea urine specimen. GFR was measured in HUSTLE using plasma DTPA (technetium 99m-labeled diethylenetriaminepentaacetic acid) clearance, and estimated GFR (eGFR) in TWiTCH based on serum creatinine. DNA samples were genotyped for 8 candidate SNPs previously associated with renal disease, using PCR-based allelic discrimination, bidirectional Sanger sequencing, and analysis of variable number tandem repeats (VNTR). Associations between albuminuria and genetic polymorphisms were tested using an additive model and correlation trend test. Linked WES data from the same patients were analyzed to identify other variants associated with albuminuria and GFR. Results: Albuminuria was present in 13.1% of patients, including 16.3% in HUSTLE and 11.0% in TWiTCH. APOL1 genetic variants were common (G1 allele frequency = 21.9%, G2 allele = 16.0%, Table) and similar to published cohorts. Children with two APOL1 G1 alleles had an increased risk of albuminuria that approached statistical significance (p=0.053). Conversely, the presence of the DARC SNP that confers Duffy antigen expression had a protective effect (p=.038). WES analysis did not identify additional non-synonymous APOL1 variants linked with albuminuria. However, 93 non-synonymous variants were associated with DTPA GFR (p

Description: Background: Elevated total white blood cell (WBC) count is associated with increased morbidity and mortality in sickle cell anemia (SCA), and is a biomarker for disease severity. In addition, a higher WBC and absolute neutrophil count (ANC) have been associated with a higher maximum tolerated dose (MTD) for hydroxyurea in treated patients. Several single nucleotide polymorphisms (SNPs), especially variants in the Duffy Antigen Receptor for Chemokine (DARC) gene, influence WBC and ANC in the general population; however, their effects on children with SCA have not been reported. Objectives: To determine the effects of candidate SNPs on the baseline WBC and ANC in children with SCA; to identify novel genetic variants affecting WBC and ANC; and to investigate their effects on hydroxyurea MTD. Design/Method: Genomic DNA was analyzed from children with SCA enrolled in prospective trials involving hydroxyurea treatment, including: (1) Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT 00305175); (2) Stroke With Transfusions Changing to Hydroxyurea (SWiTCH, NCT 00122980); and (3) Transcranial Doppler With Transfusions Changing to Hydroxyurea (TWiTCH, NCT 01425307). DNA samples were genotyped for 6 candidate SNPs that were previously reported to have a significant effect on the WBC and ANC in the general population, using a combination of PCR-based allelic discrimination and Sanger sequencing (Table). WBC and ANC phenotypes (n=429) were obtained at study enrollment, as well as hydroxyurea MTD values (n=224) for children receiving the drug. An additive model and correlation trend test was used to perform statistical testing. Whole exome sequencing (WES) analysis was performed after randomizing the entire cohort into a discovery (n=256) and validation group (n=127), to identify novel associations with WBC and ANC. Results: The average age (mean ± SD) of the entire cohort was 10.7 ± 4.1 years, with WBC = 13.8 ± 4.4 x 109/L and ANC = 7.6 ± 3.2 x 109/L. The DARC SNP rs2814778 regulating Duffy antigen expression had a minor allele frequency (MAF) of 15.1%, similar to published reports in African-American populations. After adjusting for age, children either homozygous or heterozygous for the A allele had significantly higher WBC and ANC, compared to G homozygotes known as "Duffy null" (14.6 vs. 13.5 x 109/L and 8.5 vs. 7.2 x 109/L, p=0.015 and p=0.001, respectively). For DARC rs12075, which distinguishes the major Duffy alleles, the G allele that results in Fya expression was associated with higher ANC than Fyb, 8.8 vs. 7.4 x 109/L, p=0.017. The hydroxyurea MTD was 25.6 ± 4.5 mg/kg/day for the treated cohort, which was associated with pre-treated WBC (p=0.009) and ANC (0.002), but with none of the DARC polymorphisms. An unbiased search using WES identified a total of 45,228 non-synonymous variants with allele frequency 〉1%, including 2,238 variants associated with WBC and ANC in the discovery group (p

Description: Introduction: Enoxaparin (low molecular weight heparin, LMWH) is the most commonly prescribed anticoagulant for pediatric patients with venous thromboembolism (VTE) (Goldenberg et al. 2015). The gold-standard LMWH activity (anti-Xa activity) assays differ in whether they add exogenous antithrombin (AT) or dextran sulfate (Ignjatovic et al. 2007). Adding AT would "standardize" results in patients with low antithrombin, such as infants and asparaginase-treated patients (Mitchell et al 2010); however, questions remain about which assay best reflects the patient's anticoagulation effect and the degree of discrepancy between assays. We assessed LMWH activity in residual plasma samples from a cohort of anticoagulated pediatric acute lymphoblastic leukemia and lymphoma (ALL) patients, with history of VTE and variable AT levels, on four platforms (two instruments and their kits +/- exogenous AT). Methods: We analyzed 60 de-identified residual plasma samples from 12 anticoagulated ALL patients (2-19 years. Mean 13.75 yr) who had AT levels obtained for clinical care. All consented to the IRB-approved Oncology Tissue Repository. LMWH activity was assessed on Siemens and Stago instruments using their recommended kits that did or did not contain exogenous AT (Table 1), according to manufacturer recommendations, by experienced laboratorians. Results: Results were interpretable on 236/240 with 4 rejected for lipemia. Mean AT activity was 80 (42-138 ng/ml, lab normal 〉81%). Correlation was acceptable for the published kit ranges of LMWH activity when comparing kits +AT (Berichrom® to Stachrom®, r=0.82, p

Description: Introduction: The Uganda Sickle Surveillance Study (US3) analyzed almost 100,000 dried blood spots (DBS) collected from infants and toddlers, with the primary objective of determining the distribution and burden of sickle cell disease and trait in the Republic of Uganda. The recently published results document a high prevalence of both sickle trait (13.3%) and disease (0.7%) with non-uniform distribution across the country (Ndeezi et al, Lancet Global Health 2016). The prevalence of established genetic modifiers of sickle cell anemia in Uganda including α-thalassemia, β-globin haplotype, and G6PD deficiency is currently unknown. In addition, isoelectric focusing electrophoresis (IEF) revealed numerous hemoglobin variants of uncertain significance, with an overall prevalence of 0.5% warranting further investigation. Methods: De-identified DBS samples identified as Disease or Variant on IEF were transported to Cincinnati Children's Hospital for analysis, with oversight from an IRB-approved protocol at both CCHMC and Makerere University. Using genomic DNA isolated from two 3mm punches, Disease samples were tested for genetic modifiers using a combination of PCR-based techniques: HbS allele (rs334) by TaqMan RT-PCR analysis; G6PD A- by 3 TaqMan RT-PCR analyses for G202A (rs1050829) to distinguish A and B isoforms followed by A376G (rs1050828) to identify the A- variant, and gender (SRY_VIC, ABCD1_CCHS0H-FAM); β-globin haplotype by PCR, restriction fragment length polymorphism (RFLP), and Taq Man RT-PCR for the classic DNA polymorphisms [XmnI (HBG2), rs7482144; HindIII Gγ (HBG2), rs2070972; HindIII Aγ (HBG1), rs28440105; HincII ψβ (HBBP1), rs10128556; HincII δ (HBD), rs968857; AvaII (HBB), rs10768683; HincII ε (ΗΒΕ1), rs3834466]; and deletional -α3.7-thalassemia using a copy-number variant RT-PCR assay with probes inside and outside the α-globin region (Hs03947236_cn, HbA_Tri_CCPAD0C). Hemoglobin variants were identified using a combination of PCR amplification of alpha, beta, gamma, and delta globin exons followed by Sanger sequencing; after missense mutations and fusion genes were documented, rapid and specific PCR-based diagnostic techniques were developed using RFLP and RT-PCR. Results: A total of 164 DBS samples with confirmed sickle cell anemia were found to have the following prevalence of genetic modifiers: G6PD A- deficiency (N=160) in 6.6% of males and 1.2% of females for an overall A-allele frequency of 15.7%; β-globin haplotypes (N=156) were Central African Republic (CAR) in 91.6% of alleles, along with a small number of Cameroon (5.1%), Benin (2.2%), or Atypical (1.0%) haplotypes; deletional α-thalassemia (N=150) had an overall prevalence of 44.6% with one-gene deletion in 38.6% and two gene-deletion in 6.0% of samples. Subsequent DNA-based investigation of 190 DBS samples with hemoglobin variants identified five common patterns: two α-globin variants (Hb Stanleyville II, Asn78Lys and Hb G-Pest, Asp74Asn), one β-globin variant (Hb O-Arab, Glu121Lys), and two fusion globin variants (Hb P-Nilotic, β31-δ50 and Hb Kenya, Aγ81Leu-β86Ala). Hb Stanleyville II was identified in 30% of the variant samples, Hb Kenya in 27%, Hb P-Nilotic in 21%, Hb G-Pest in 10% and Hb O-Arab in 1%; an additional 6% remain unidentified by sequence or IEF pattern. Compound heterozygotes with Hb S were identified with Hb O-Arab and Hb Kenya. Geospatial mapping of Hb variants document a non-uniform distribution with geographic clustering: for example Hb Kenya was concentrated in the Eastern Regions and around Lake Victoria, Hb P-Nilotic was found primarily in Northern Regions, while Hb Stanleyville II and Hb G-Pest were identified across the country. Conclusions: Genetic analyses of DBS collected in US3 reveal important findings that are relevant to the country's nascent newborn screening efforts. First, there is a high prevalence of known genetic modifiers, including 〉40% alpha thalassemia trait, 〉90% CAR haplotype, and 〉6% of males with G6PD deficiency. Second, analysis of five common IEF hemoglobin variants identified three missense mutations and two fusion proteins; recognition of their IEF patterns and development of PCR-based diagnostic algorithms will allow their correct identification. As newborn screening programs expand in Uganda and across sub-Saharan Africa, these results will be valuable adjuncts to standard IEF documentation of sickle cell trait and disease. Disclosures Ware: Nova Laboratories: Consultancy; Addmedica: Research Funding; Biomedomics: Research Funding; Bayer Pharmaceuticals: Consultancy; Bristol Myers Squibb: Research Funding; Global Blood Therapeutics: Consultancy.

Description: Sickle cell disease (SCD) is a common and life-threatening inherited disorder of hemoglobin, affecting over 400,000 newborns annually. The majority of these births occur in low-resource countries, particularly in sub-Saharan Africa, where limited access to accurate diagnostics results in early mortality. Accurate diagnosis of SCD currently relies upon analytical techniques that are relatively expensive and tedious, requiring equipment, electricity, and laboratory expertise. In Africa, outside of referral and regional hospitals, the availability of accurate SCD diagnostics is extremely scarce. A simple, rapid, accurate, and inexpensive point-of-care (POC) method for diagnosing SCD in limited resource settings would represent a tremendous advance for the management of SCD worldwide. We evaluated a novel point-of care SCD immunoassay (Sickle SCANTM, Biomedomics, Inc., Research Triangle Park, NC) that utilizes lateral flow technology and antibody-mediated detection of hemoglobin variants, with the goal of determining the accuracy, sensitivity, specificity, and ease of identifying the presence of hemoglobin A (HbA), hemoglobin S (HbS), and hemoglobin C (HbC) using blood samples from patients with a variety of hemoglobin patterns. Blood samples collected in EDTA were first tested by HPLC to determine the percentages of normal and abnormal hemoglobins and were then tested using Sickle SCAN. Mixtures of specific hemoglobin combinations were also created to determine the sensitivity of the POC assay for detecting low concentrations of HbA, HbS, and HbC. The Sickle SCAN kit includes tubes prefilled with 1.0mL of buffer, which lyses erythrocytes and releases hemoglobin. Whole blood samples were tested by adding 5µL of whole blood from the EDTA tube to the prefilled buffer container, mixing by inverting the tube three times, discarding 3 drops of the mixed solution and then applying 5 drops to the testingcartridge. Dried blood spot samples were also tested by dropping a 3mm punch into the prefilled buffer container, mixing, discarding 3 drops, and applying 5 drops to the testing cartridge. Five minutes after sample application, two independent and masked clinicians visually scored each sample for the presence/absence of each potential band (HbA, HbS, HbC, and Control). Figure 1 illustrates the visual results of common hemoglobin patterns. A total of 50 samples were evaluated using the POC device, including 32 whole blood samples, 7 dried blood spots, and 11 samples artificially created to contain known low concentrations of HbA, HbS, and HbC. Temperature and stability were evaluated using 10 additional samples that were stored at 37C for up to 30 days. In order to identify potential interference by hemoglobin variants, samples included many different types of hemoglobin (HbA2, Hb Bart's, HbD, HbE, HbF, Hb Lepore, Hb Hope, Hb I-Texas, and Hb G-Philadelphia). From whole blood samples, HbA, HbS, and HbC were easily detected in both heterozygous and homozygous samples, but the intensity of individual bands did not correlate with actual percentages. Newborn samples with high fetal hemoglobin (HbF) were also easily and accurately analyzed for the presence of HbA, HbS, and HbC, with no obvious interference from HbF. The presence of common variant hemoglobins also did not cross-react, but both observers noted a faint HbA band for a newborn sample with a HbFE pattern. For samples artificially created to contain low concentrations of HbA, HbS, or HbC, these hemoglobin were detected at concentrations of

Description: Introduction: Obesity is associated with endothelial dysfunction, hemostatic and fibrinolytic disturbances. The relationship between obesity and elevated Von Willebrand Factor (VWF) is complex and not fully elucidated. There is a significant knowledge gap regarding the impact of BMI on VWF levels. Given the proinflammatory effect associated with abdominal obesity, we hypothesized that there would be an increased prevalence of obesity among individuals with Low VWF (LVWF) compared to Type 1 Von Willebrand Disease (T1VWD) in the ATHN (American Thrombosis and Hemostasis Network) dataset. Methods: A retrospective review of de-identified patients included in the ATHN dataset as of March 2018 was performed. The dataset was queried for all patients with a diagnosis of "T1VWD," who were over 18 years of age when labs were drawn and when BMI was recorded, who had VWF Ristocetin cofactor (RCO) levels 60 years) was similar among both cohorts with the exception of a larger proportion of obese individuals over the age of 60 in the LVWF cohort (63% vs 25%). Mean FVIII level for LVWF cohort was significantly higher compared to that for T1VWD cohort (80% vs. 53%; p