ALBERT

Description: Hereditary hemolytic anemias are a heterogeneous group of disorders with consequences ranging from non-anemic hemolysis to severe life-threatening anemia. However, the late morbidity in patients without transfusions is often underappreciated because of erythropoietic compensatory stimulation inducing hematopoiesis by erythroferrone/hepcidin axis. Principal causes of hereditary hemolytic anemias are germline mutations of red cell cytoskeleton (e.g. hereditary spherocytosis and elliptocytosis/pyropoikilocytosis) or enzyme deficiencies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Routine morphological and biochemical analysis may be inconclusive and misleading particularly in transfusion-dependent infants and children. Molecular studies have not been extensively used to diagnose these disorders due to the complex genetic nature of these disorders, and multi-gene disorders. In these cases, patients may undergo multiple rounds of single gene testing, which can be very costly and time consuming. The advent of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using Sure Select kit (Agilent Technologies, Santa Clara, USA). Prior to sequencing on the Illumina Next Seq, (Illumina Inc) instrument, indexed samples are quantified using qPCR and then pooled. Samples were sequenced using 2x150 paired end sequencing. We now report the first 68 patients evaluated using our NGS panel. The age of the patients ranged from newborn to 62 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified using the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic variants in 11 patients and likely pathogenic variants in 12 others, the majority of these were novel. Many variants with unknown significance were also identified that could potentially contribute to disease. The most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits. Some complex interactions were uncovered i.e. SPTA1 mutations along with alpha LELY leading to hereditary pyropoikilocytosis; Spectrin variants along with Gilbert syndrome causing severe hyperbilirubinemia in neonates; and Spectrin variants in combination with PKLR and G6PD variants. Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes, but their clinical significance is further augmented by polymorphisms of UGT1A1 gene contributing to severe neonatal hyperbilirubinemia and its consequences. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular diagnosis, especially in instances where traditional testing failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in many cases with no prior family history. Disclosures Yaish: Octapharma: Other: Study investigator.

Description: Hereditary hemolytic anemia (HHA) are a heterogeneous group of disorders due to germline mutations of the red cell cytoskeleton (e.g. hereditary spherocytosis (HS) and hereditary elliptocytosis/pyropoikilocytosis (HE/HPP)) or enzyme deficiencies (e.g. glucose 6 phosphate dehydrogenase deficiency (G6PD) and pyruvate kinase deficiency (PKD). Routine morphological and biochemical analysis may be inconclusive in neonates due to the physiological nature of erythroid cell maturation and can also be misleading in transfusion-dependent patients. Additionally, there has been increasing awareness of inherited red cell membrane disorders that are not easily identified by routine laboratory approaches. For example, clinically insignificant defects of RBC membrane genes (e.g. alpha LELY and alpha LEPRA in SPTA1), which can be present in the parents without significant hemolysis, may result in compound heterozygosity in the offspring, causing severe morbidity or even mortality due to significant hemolysis. Awareness of these low expression alleles is important for genetic counseling purposes. Molecular studies, although becoming more mainstream, have not been used extensively to diagnose these disorders. This is most likely due to the complex genetic nature of these disorders (e.g. large genes with multiple exons involved, and multi-gene disorders (i.e. hyperbilirubinemia due to HS as well as involvement of genes involved in bilirubin metabolism). The accessibility of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our current diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for NGS are performed using a Sure Select kit (Agilent). Indexed samples are quantified using qPCR and then pooled prior to sequencing on the Illumina NextSeq or HiSeq instruments. Samples are sequenced using 150 bp paired-end sequencing. This panel includes genes responsible for RBC membrane defects, enzyme deficiencies, as well as bilirubin uridine diphosphate glucuronosyltransferase (UGT1A) genes that have a distinct role in hyperbilirubinemia. We now report the first 268 patients evaluated using our NGS panel between 2015-2018. These patients were evaluated using an Institutional Review Board Protocol (IRB - 00077285). The age of the patients ranged from newborn to 68 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified according to the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic and likely pathogenic variants in 64/268 (24%) patients that were clearly responsible for the disease phenotype (e.g. moderate to severe hemolytic anemia). Approximately half of them were novel mutations. Moreover, 29/268 (11%) of patients were homozygous for a promoter polymorphism in the UGT1A1 gene A(TA)7TAA (UGT1A1*28), which may lead to reduced expression of the UGT1A1 gene and Gilbert's syndrome. Furthermore, 4/29 UGT1A1 polymorphism cases were associated with pathogenic spectrin mutations, likely increasing the severity of the clinical phenotype in these patients. Overall, the most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits, followed by PKLR and ANK1 (Table 1). Complex interactions between variants in the SPTA1 gene and the common alpha-LELY and alpha-LEPRA alleles were predicted to be associated with HPP and autosomal recessive HS in 12/64 patients. Furthermore, 23/268 (9%) patients had mutations that were predicted to cause moderate to severe anemia if inherited with another mutation, making them important for genetic counseling purposes (data not shown). Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes. Many variants of unknown significance were also identified that could potentially contribute to disease. To conclude, the use of NGS provides a cost-effective and comprehensive method to assist in the diagnosis of hemolytic anemias, especially in instances where complex gene-gene interactions are suspected. Disclosures No relevant conflicts of interest to declare.

Description: The authors describe recurrent novel insertion/deletion mutations in the JH2 domain of JAK2 occurring in patients with eosinophilia as a prominent feature of their myeloproliferative neoplasms. Remarkably, 2 of the patients with a specific mutation (Leu583-Ala586DelInsSer) meet the criteria for both chronic eosinophilic leukemia and polycythemia vera, suggesting that this may be a distinct overlap syndrome.

Description: Introduction: Patients with Down syndrome (DS) have an increased risk of hematological disorders, including transient abnormal myelopoiesis (TAM), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Twenty percent of patients with TAM subsequently develop myeloid neoplasm in the first 4 years of life. MDS represents a clonal aberration thought to be a pre-leukemic condition characterized clinically by cytopenias and erythroid, myeloid and/or megakaryocytic dysplasia in the bone marrow with or without increase in blasts and harbors a concordant, clone-specific mutation of GATA1. WHO 2016 classification of hematopoietic neoplasms does not distinguish between MDS and AML, as their overall prognosis appears to be similar. However, due to the rarity of this disorder, limited clinical and laboratory data is available, contributing to difficulties in establishing the diagnosis. Here we describe our center's recent experience with the diagnosis and molecular findings of myeloid neoplasm associated with Down syndrome (MN-DS). Design/Method: Retrospective review of the patient's electronic medical record and review of the literature was conducted. Routine karyotype, fluorescent in-situ hybridization (FISH) and next generation sequencing (NGS) studies were reviewed where available. Results: Six patients with DS diagnosed with AML or MDS were identified over a 3-year period. Mean age of the cohort was 18.5 (range 12-24) months with a slight female predominance. Three patients had a history of TAM, all of which resolved without intervention. Three patients had asymptomatic thrombocytopenia after birth without blasts or GATA1 mutation confirmation. One of the three patients with a history of TAM presented with overt AML, while in the others diagnosis was challenging. By WHO 2008 classification of myeloid neoplasms, four patients had refractory anemia with excess blasts, one had refractory cytopenia with multilineage dysplasia, and one had AML. For two patients, in whom myeloid directed next generation sequencing was obtained, mutations were found in GATA1, EZH2, and NRAS. One of the patients in our series presented with AML with gain of MECOM, RPN1 loss and D5S23 deletion by FISH and succumbed to relapsed disease. All patients were treated per Children's Oncology Group AAML1531 arm A protocol that included 3 induction cycles and 2 intensification cycles, except for a single patient that received one cycle per AAML0431 and completed therapy per AAML1531 arm B high risk due to persistent disease following initial induction cycle. Two patients are currently receiving treatment, three have no evidence of disease recurrence on follow up ranging from 2 to 18 months, and one of the patients has died due to relapsed/refractory disease. Conclusions: We present six cases of MN-DS in patients less than four years of age. Our cohort is representative of the diversity encountered in this rare disease including patients with 1) isolated cytopenia in the absence of overt morphological findings, 2) myelodysplasia, and 3) AML. In our patient with overt AML there were karyotypic features such as gain of MECOM, which with specific translocation partners has previously been described to portend a poor prognosis. This and other cytogenetic features perhaps warrant further investigation given our patient's refractory disease. In the patient with refractory cytopenia without blasts, there was a subpopulation of cells identified by NGS panel showing mutations in GATA1, EZH2, and NRAS that led to a diagnosis of MDS/MN-DS. Four of the patients had aberrant myeloid populations and dysplasia fitting diagnostic criteria for MDS. Establishing the clonal nature of the disease either by karyotype/FISH or NGS may help with the identification, treatment and prognostication of this unique patient population, and may aid in the diagnosis of MN-DS, which may be challenging in patients with DS once they have recovered from TAM. Disclosures No relevant conflicts of interest to declare.

Description: Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status. Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84). Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation. Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis. Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. Disclosures No relevant conflicts of interest to declare.

Description: Hereditary hemolytic anemia encompasses a diverse group of genetically and phenotypically heterogeneous disorders that are characterized by increased red cell destruction, with consequences ranging from relatively harmless to severe life-threatening anemia. Moreover, red cell hemolysis leads to increased production of bilirubin, a breakdown product of hemoglobin, which in neonates places them at risk for extreme jaundice and its consequences. Two of the more common genetic causes of hereditary hemolytic anemia, excluding hemoglobinopathies, can be attributed to defects in either the red cell cytoskeleton or enzyme deficiency (e.g. G6PD, PKLR). Morphological and biochemical diagnosis of hereditary hemolytic anemia due to defects in RBC cytoskeleton or enzyme deficiency is routinely performed in many laboratories. However, routine studies can be challenging, particularly in transfusion-dependent infants and children since these patients have mostly transfused RBCs. Molecular diagnosis has also been challenging not only due to molecular heterogeneity but also due to the number and size of the genes involved. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of the molecular causes of the two major types of hereditary hemolytic anemia described above. Our diagnostic panel includes 25 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA). One hundred base-pair paired-end sequencing was done on a HiSeq 2000 system (Illumina, San Diego, CA). Bioinformatic analysis was based on an “in house” pipeline using standard open-source software. A total of 19 patients with unexplained hemolytic anemia, and 30 normal controls were tested in our assay. Mutations in the appropriate genes were identified in 17/19 patients, many of these being novel. All identified mutations were confirmed by Sanger sequencing. In silico prediction of the impact resulting from the novel mutations was performed using two web-based software packages, Sift and Polyphen. Where possible, inheritance of pathogenic mutations was determined in immediate relatives. One of the cases we investigated involved a neonate with unexplained jaundice and subsequent, significantly compensated, anemia without family history of a hemolytic disorder. Routine studies were suggestive for hereditary spherocytosis due to the presence of microspherocytes on the proband’s blood film, increased osmotic fragility, and decreased eosin-5-maleimide stained red cells. Two pathogenic mutations, in compound heterozygosity, were identified in the SPTA1 gene (α-spectrin). A previously reported mutation αLEPRA, known to be associated with recessive spectrin-deficient HS, and a novel mutation in intron 45 +1 (c.6530+1G〉A) disrupting the consensus splice site. Screening of other relevant genes failed to reveal additional mutations. Studies of his parents revealed both to be heterozygous carriers with the asymptomatic mother harboring the αLEPRA mutation and the asymptomatic father harboring the novel mutation. Our results demonstrate the clinical utility of this assay for molecular diagnosis and genetic counseling for parents at risk of having affected children. Next-generation sequencing provides a cost-effective and rapid approach to molecular diagnosis, especially in cases where traditional testing has failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in several cases with no family history. Furthermore, we have validated its clinical utility in neonates risk for hyperbillirubinemia, as well as, in patients with transfusion dependent hemolytic anemia. Disclosures: No relevant conflicts of interest to declare.

Description: Transcriptional regulation of β-globin cluster genes follows a complex, highly conserved system of gene expression with developmental and tissue-specific control. The DNase I hypersensitivity (HS) sites in the upstream locus control region (LCR) and 3' HS1 element are thought to interact with β-globin cluster genes involving long range DNA interactions mediated by various transcription factors to drive the regulation of β-like globin gene expression. The majority of studies have focused on the role of the LCR on active transcription and globin gene switching. Various in vitro and in vivo studies have shown that the LCR interacts with one gene at a time and that absence of the LCR results in a dramatic decrease (10 -100 fold) in globin transcripts (Kiefer et. al., Mutat. Res. 2008, 647:68, Noordermeer et. al., IUBMB Life 2008, 60: 824). However, the role of the 3'HS1 downstream and other distal cis -regulatory elements are not entirely understood, with recent studies in mouse and cell-culture models suggesting they play a role in the insulation of globin genes from silencing chromatin (Bender et. al., Blood 2008, 106: 1395). Our knowledge of the β-globin LCR and 3'HS1 function is still incomplete and much can still be learned from human mutations affecting these regulatory elements. We report a unique head-to-tail duplication of the β-globin cluster in a patient phenotypically expressing homozygous HbS (sickle-cell anemia, SCA) that provides insight into the regulatory role of the β-LCR and 3'HS1 on wild-type β-globin (β-A) expression in a background of SCA. The studies were driven by an apparent discrepancy between hemoglobin analysis of an infant with a SCA phenotype and no detectable HBA, and Sanger sequencing of the β-globin genes which showed a heterozygous genotype. Analyses of parents' blood samples and DNA revealed that each were carriers for sickle cell allele. Hemoglobin analysis showed the father expressed HbS fraction at 41.3% and the mother at 33.3%, while the proband had a majority of HbS, some HbF and no detectable HbA. The reduced HbS fraction in the mother could be explained by co-inheritance of α-thalassemia (αα/α-3.7). The proband did not inherit α-thalassemia from the mother. Multiplex ligation-dependent probe amplification analysis of the proband's DNA suggested duplication of the β-globin cluster, resulting in three copies of the HBB gene in the genome. Subsequent next-generation sequencing confirmed that the duplication occurred immediately adjacent to the first iteration of sequence, in head-to-tail orientation and resulted in an intact β-S cluster having both LCR and HS1 elements, followed by the duplicated β-A cluster (β-S, β-A) that excluded a part of HBE (epsilon globin) and the upstream β-LCR regions, extending through to LINE L1LBP1 ([hg19] chr11:4640332-5290168). Further analyses revealed that the duplicated β-A cluster, which encompassed approximately 650 kb sequence, lacked a DNA segment containing the 3'HS1 element (figure 1). The proband's β genotype is thus (β-S/β-S, β-A). DNA analysis showed that the father carried the duplicated β-globin cluster with genotype (β-A/β-S, β-A), and the mother, a heterozygous HbS genotype (β-A/β-S). Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) was used to assess transcription levels of β-A and β-S mRNA for each family member. Analysis of the parents' reticulocyte RNA showed that the β-globin (β-A and β-S) transcript levels were nearly balanced. RT-qPCR of proband's reticulocyte RNA showed no convincing detection of β-A transcript, but the β-A transcript was clearly detected by digitalPCR, albeit at a very low level (0.4% of total HBB transcript) (figure 2). The β-globin cluster duplication on one chromosome in a background of a phenotypically homozygous (HbSS) SCA patient has provided a unique opportunity to assess the effect of the LCR and 3'HS1 regions on the transcription of a β-A gene in an unbiased environment. Prior studies in transformed cell-lines or mouse models have shown the down regulatory effect of LCR loss and potential protective effect of the 3'HS1 element. Within the human model, the observed transcription from the duplicated, distally displaced (~650 kb) β-A cluster demonstrates that the loss of LCR and flanking HS sites does not lead to complete silencing of β-globin transcription. VD was supported by project IGA MzCR NT13587. Disclosures No relevant conflicts of interest to declare.

Description: Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.