ALBERT

Description: Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF. Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD. Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively. Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p

Description: Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect 1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity 〉98% and specificity 〉99%, even in the absence of paternal DNA. Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with 〉20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is

Description: Background. Avascular necrosis (AVN) is a serious complication of sickle cell disease (SCD) that can lead to significant morbidity including chronic pain and physical impairment with treatment depending on the patient's pain severity and functional limitation. Patient age, hemoglobin levels, and abnormal rheology, particularly high blood viscosity, or thickness, and percentage of dense red blood cells, defined as cells 〉1.11 mg/mL have been identified as risk factors for the development of AVN, but their contributions to the subsequent clinical course has not been described. The natural history of AVN has been described as chronic and progressive without significant surgical or physical therapy based intervention. However, this description was established before widespread MRI use in diagnosis, or hydroxyurea (HU). A new evaluation of the natural history of AVN is needed. Objective: Review the natural history of AVN at our institution in patients for whom repeat imaging is available, and determine if patient demographics, treatment type, and laboratory data, including rheological values, predict of clinical course. Methods. We identified all patients with a diagnosis of AVN with diagnostic and follow-up imaging studies (pelvic X-rays or MRI of the hip), receiving care at Texas Children's Hospital Hematology Center 2006 to 2018, and collected demographic, treatment, rheological and clinical laboratory data. Patient radiographic images at diagnosis and follow up were reviewed and scored by a board certified pediatric radiologist using the Steinberg criteria. Laboratory values for each patient were collected as available from time of diagnosis through to the date of follow up imaging. Longitudinal patient blood samples were collected for rheological measurements under an IRB approved protocol. Whole blood viscosity was measured on a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 45 and 225 s-1 at 37oC within 4 hours of sample collection in an EDTA vacutainer tube. Percent dense red blood cells (%DRBCs) were measured by an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Statistical analysis comparing groups with and without radiographic improvement was performed using a two-tailed Student's t-test. Results. 16 patients had repeat imaging, allowing for assessment of radiographic improvement (Table1). All were diagnosed via X ray or MRI of the pelvis. Five of 16 patients had resolution or significant radiographic improvement of AVN without orthopedic intervention or prolonged physical therapy, while 11 patients had stable or worsening disease. In the group with radiographic improvement, the mean age was 9.9 years (SD=2.5) and 14.6 years (SD=2.2) in the group without radiographic improvement (p= 0.002). Five patients underwent core decompression with bone marrow aspirate concentrate injection; one progressed to complete femoral head collapse while the rest had stable or worsening disease. Mean hemoglobin (Hb), fetal hemoglobin (HbF), percent DRBCs, whole blood viscosity, and extent of AVN at diagnosis by Steinberg criteria were not statistically different between the patients that showed improvement and those that did not. Conclusions. Our results challenge the paradigm of untreated AVN in SCD as an inexorably progressive disease. Here we present 5 cases of significant radiographic improvement of AVN without surgical or significant physical therapy interventions. Conversely, 5 AVN cases treated with core decompression remained stable or worsened. Given our small cohort, we were unable to establish laboratory or rheological predictors of spontaneous resolution. However, patients in the radiographic improvement group were younger than patients with progressive disease. HU has shown promise in improving vascular complications in SCD patients however more research is needed to delineate its role as a modifier of AVN natural history as it was not correlated with the improved group in our study (all our study patients were on HU either prior to diagnosis or started during the follow up period). Given the findings that significant radiographic improvement is possible with minimal or no intervention, providers should closely weigh the risks and benefits of observation versus surgical intervention, particularly in young patients with SCD. Disclosures No relevant conflicts of interest to declare.

Description: Next-generation DNA sequencing is currently limited by an inability to accurately count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting to reconstruct the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPTs of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. The analytical sensitivity and specificity of sgNIPT was 〉98% and 〉99%, respectively. Validation of sgNIPTs was further performed with maternal blood samples collected during pregnancy, and sgNIPTs were 100% concordant with newborn follow-up.