ALBERT

Description: The Hereditary Hemolytic Anemias (HHAs) are a genetically heterogeneous group of anemias characterized by decreased red blood cell (RBC) survival because of defects in hemoglobin, RBC membrane proteins or enzymes. The diagnosis of this group of disorders is complex and challenging requiring analysis of the morphology of RBCs, hemoglobin electrophoresis, and a battery of phenotypic assays. The phenotypic analysis is often problematic in transfusion dependent patients or at times of presentation with a hemolytic crisis as transfused blood or reticulocytosis confounds diagnostic testing. Molecular genetic testing has grown in popularity in the diagnosis of hereditary hemolytic anemias as it is not affected by transfusions or other clinical variables and provides additional insight into the mechanism of the disease. We have developed a Next Generation Sequencing (NGS) panel for HHA due to RBC membrane disorders and enzymopathies and congenital dyserythropoietic anemias (CDA). CDAs, although collectively rare, are included in the panel as they are occasionally misdiagnosed as hereditary spherocytosis (HS) due to their clinical characteristics of hemolysis, increased osmotic fragility, and splenomegaly albeit with inadequate reticulocytosis We reviewed the results of 282 sequential HHA/CDA panels testing for patients with suspected HHA or CDA diagnosis, performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013-5/2016. Forty-three samples were omitted from the final analysis due to diagnosis of other disorders, indicating that negative results were true-negatives. For the analysis of the remaining 239 panels, all results were reviewed and categorized based on the type of testing ordered: comprehensive HHA/CDA (32 genes), RBC membrane disorders (13 genes), RBC enzyme disorders (14 genes), or CDA (6 genes). The protein-coding exons plus 25 bases of exon-intron junction as well as promoter sequences were included in the design. Genomic DNA was isolated from blood and target regions were enriched using the Haloplex technology. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. All positive findings were confirmed by Sanger sequencing. These 239 panels included 159 (66.5%) comprehensive HHA/CDA panels, 41 (17.2%) RBC membrane disorder panels, 10 (4.2%) RBC enzyme disorder panels, and 29 (12.1%) CDA panels. Overall, a diagnosis was confirmed or identified in 135 (56.5%) patients with specific genotype of hereditary spherocytosis in 52 patients; hereditary elliptocytosis in 15 patients; hereditary pyropoikilocytosis in 7 patients; hereditary stomatocytosis/xerocytosis in 12 patients; South East Asian Ovalocytosis in 1 patient; G6PD deficiency in 15 patients; pyruvate kinase deficiency in 17 patients; other rare RBC enzymopathies in 6 patients; and CDA in 10 patients. The clinical performance of RBC membrane disorder and RBC enzyme disorder panels were comparable between 68-70% in reaching a final diagnosis, while CDA panel confirmed final diagnosis in only 20% of suspected cases. The overall low prevalence, complexity of diagnosis with findings of dyserythropoiesis in bone marrow studies in patients with severe HHA, and evidence of locus heterogeneity in CDA might explain this result. Among patients with suspected RBC membrane disorders, approximately 14% were eventually diagnosed with hereditary xerocytosis (HX). HX diagnosis is critical to make in such patients since splenectomy is contraindicated due to the high risk of life-threatening thrombophilia complications. In more than half (56.5%) of all cases with suspected hereditary hemolytic anemia, genetic testing provided or confirmed the diagnosis and optimized patients' clinical management. Further genetic counseling and testing for other at-risk family members was made possible by achieving molecular diagnosis. Genetic testing substantially altered management in approximately 14% of cases with suspected RBC membrane disorders due to the diagnosis of HX. In conclusion, genetic testing has a significant clinical utility and may facilitate and improve diagnosis, prognosis and management considerations in patients with hereditary hemolytic or dyserythropoietic anemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive 〉 memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.

Description: Background: The hemoglobinopathies are a genetically complex group of blood disorders that includes sickle cell disease (SCD), thalassemia, and other structural and/or functional hemoglobin (Hb) variants. For decades, the mainstay of diagnostic testing for hemoglobinopathies has been Hb separation techniques (e.g., electrophoresis, chromatography), sometimes paired with functional assays of Hb (e.g., solubility, instability, oxygen affinity). Such protein-based testing cannot detect or adequately differentiate a number of clinically significant hemoglobinopathies. Nevertheless, most clinicians continue to rely primarily on protein-based diagnostic methods despite the availability of genetic testing. The reasons for this are not fully understood, but include concerns about the cost of genetic testing and the perception that protein-based methods are usually sufficient for clinical care. Objective: To determine the clinical utility of genetic testing for the diagnosis of hemoglobinopathies by quantifying how often a suspected clinical diagnosis is changed, clarified or excluded by genetic testing. Methods: We reviewed the results of 500 sequential orders for clinical genetic testing for known or suspected hemoglobinopathies that were performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013 and 5/2015. This comprehensive genetic testing service is a collaboration between the divisions of Hematology and Human Genetics. For this analysis we reviewed orders for copy number variation analysis (CNV) of the α-globin gene cluster, sequencing of the α-globin genes (HBA1, HBA2), CNV of the β-globin gene cluster, and/or sequencing of the β-globin genes (HBB). An order for testing could include any combination of these 4 tests. We compared the stated indications and suspected diagnoses for testing provided by the ordering physician with the final results of the genetic testing. For each order, the results of this comparison (suspected diagnosis vs. genetic diagnosis) were classified into 1 of 4 mutually exclusive groups: (1) suspected diagnosis confirmed, (2) suspected diagnosis excluded, (3) suspected diagnosis clarified, or (4) new or unexpected diagnosis. The category "suspected diagnosis clarified" includes cases where the overall suspected diagnosis was confirmed, but additional clinically meaningful genetic data was also uncovered (e.g., homozygous sickle cell anemia was confirmed, but co-inherited α-globin gene deletions were also identified). Results: 500 serial genetic testing panels were performed on 475 unique individuals (7 specimens were from an embryo or fetus). These 500 panels included 1-4 individual tests: HBB sequencing in 423, α-globin CNV in 475, β-globin CNV in 345, and HBA1/2 sequencing in 164. The final diagnosis was a specific genotype of SCD (± α-thalassemia and/or gene-deletion HPFH) in 234; thalassemia (α, β, or δβ) in 139; Hb S trait (± α-thalassemia) in 18; Hb C trait or disease (± α-thalassemia) in 13; Hb E trait or disease (± α-thalassemia) in 9; another named or novel Hb variant in 22; and normal Hb in 65. Overall, the suspected diagnosis was confirmed in 246 (49.2%), clarified in 156 (31.2%), excluded in 77 (15.4%), and in 21 (4.2%) a new or unsuspected diagnosis was made. For patients with a suspected diagnosis of SCD, the diagnosis was mostly confirmed (66.7%) and never excluded by genetic testing, but it was clarified in 31.6%, and an unsuspected genotype of SCD was identified in 1.8%. For patients with a suspected diagnosis other than SCD, the diagnosis was confirmed in 34%, clarified in 30.6%, excluded in 29.1%, and an unsuspected genotype was identified in 6.4%. Not considering the clinical utility of the "suspected diagnosis clarified" category, a suspected diagnosis was excluded or determined to be a new or unsuspected diagnosis in 98/500 (19.6%) of all cases. Additionally, follow-up genetic testing was recommended for 31/500 (6.2%). Conclusion: In half (50.8%) of all cases, genetic testing provided new or additional diagnostic information that was not apparent on protein-based diagnostic methods. Genetic testing excluded or substantially changed a suspected diagnosis in one-quarter (19.6%) of all cases. Therefore, genetic testing for hemoglobinopathies has very high clinical utility, and we propose that it should be considered a standard of care for all patients with known or suspected hemoglobinopathies. Disclosures Quinn: Amgen: Research Funding; Eli Lilly: Research Funding; MAST Therapeutics: Research Funding; Glycomimetics: Research Funding; Silverlake Research: Consultancy. Begtrup:GeneDx: Employment.