scholarly journals Role of Next Generation Sequencing in the Diagnosis of Unexplained Thrombocytopenias- an Experience at a Tertiary Care Hospital

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1017-1017
Author(s):  
Karthika K V ◽  
Mobin Paul ◽  
Anjana K V ◽  
Ullas Mony ◽  
Ganeshprasad Arunkumar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Family History ◽  
Bone Marrow Failure ◽  
Positive Family History ◽  
Bone Marrow Examination ◽  
Care Hospital ◽  
Next Generation ◽  
Inherited Disorders ◽  
Generation Sequencing

Abstract BACKGROUND Next Generation Sequencing (NGS) has been enormously rewarding in the field of diagnostic hematology. In particular, the diagnosis of inherited disorders has progressed in leaps and bounds. These patients tend to remain undiagnosed for a long period of time not only because of unavailability of molecular diagnostics but also due to lack of cognizance and atypical presentations. Thrombocytopenia (TCP) is a common hematological presentation and can lead to chronic hospital visits to life-threatening bleeds. Most of these patients have acquired disorders such as immune TCP, malignancies, liver disease etc. However, some of them are likely to have unidentified inherited causes. We thus intended to study the utility of NGS in the definitive diagnosis of unexplained TCPs with or without other cytopenias to understand the clinicopathologic characteristics of these patients. METHODOLOGY This was a retrospective descriptive study done at two centres over three years from May 2018 to May 2021. Patients with TCP with one of the following: (a) positive family history (b) clinical/ laboratory clues to an inherited cause (c) chronic TCP with no response to conventional therapies and sent for clinical exome sequencing done by NGS were included in the study. Patients who were negative for germline mutations were excluded. Sequencing of targeted genes was performed on the Illumina platform with a mean coverage of >80-100X. Mutations annotated as pathogenic, likely pathogenic and variant of uncertain significance (VUS) were considered clinically significant. VUS are mutations that are difficult to classify as pathogenic and require clinical validation and family testing. RESULTS Our cohort included 18 patients and were divided into two groups- cases of isolated TCP and cases of TCP with anemia and/or neutropenia. Patients presenting with isolated thrombocytopenia We had nine cases of isolated TCP out of which there were three cases of X-linked macrothrombocytopenia with MYH9 mutation, two cases of Wiskott Aldrich syndrome (WAS) and one case each of congenital thrombotic thrombocytopenic purpura (TTP), atypical Hemolytic Uremic Syndrome (HUS), Fanconi anemia (FA) and grey platelet syndrome. The demographic and mutational characteristics are described in Table 1. Clinical and laboratory clues were present in 7 cases, such as chronic kidney disease, micro/ macrothrombocytopenia, neutrophil inclusions etc. Bone marrow examination was carried out in 4 cases- the significant dyspoiesis in FA and myelofibrosis in grey platelet syndrome mislead to a diagnosis of MDS and myelofibrosis respectively. Seven patients had received treatment with steroids, immunosuppressants, splenectomy, danazol and TPO mimetics before the NGS diagnosis. Patients presenting with thrombocytopenia and other cytopenias This group consisted of nine cases with two cases each of Dyskeratosis Congenita (DKC) and WAS and one case each of TTP, Ghosal hematodiaphyseal dysplasia, Congenital Amegakaryocytic Thrombocytopenia (CAMT), B-cell immunodeficiency with hypogammaglobulinemia type-25 and double homozygous for FA and DKC. The majority of patients in this group were young and had lower platelet counts (Table 2). The most common associated cytopenia was anemia. Phenotypic clues to diagnosis were present in cases of DKC and WAS. The common differentials considered in this group were inherited bone marrow failure syndromes (IBMFS), congenital immunodeficiency syndromes etc. Bone marrow examination was done more frequently in these patients and showed hypocellular marrow in IBMFS and absent megakaryocytes in CAMT. These patients have also been treated with steroids, IVIg, danazol and TPO mimetics. CONCLUSION Patients with inherited isolated TCPs have a chronic course and heterogenous causes therefore tend to be diagnosed later in life. However, patients with TCPs and other cytopenias tend to present at a younger age with infrequent family history. IBMFS was the most common disorder identified in this latter group of patients. Positive family history, clinical and laboratory clues and absence of response to conventional therapies should prompt workup of inherited causes by NGS to avoid long term ineffectual treatment. Further, NGS mutations, in particular VUS have to be interpreted with caution with the help of parental study, clinical presentation, in-silico analysis and inputs from molecular and genetic experts. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Recurrent and Refractory Hemophagocytic Lymphohistiocytosis in an Elderly. Role of Immune Aberration Due to Myeloid Gene Mutation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-26
Author(s):  
Suthanthira kannan Ramamoorthy ◽  
Tina Noutsos ◽  
David Wei ◽  
Alexandra Yasmin Laidman ◽  
Ferenc Szabo
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Next Generation Sequencing ◽  
T Cell ◽  
Myelodysplastic Syndrome ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Bone Marrow Examination ◽  
Next Generation ◽  
Tet2 Mutation ◽  
Generation Sequencing

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyper-inflammatory disease induced by aberrant immune activation and subsequent proliferation of macrophages, histiocytes and T-helper cells. In this abstract we present a case of HLH, which relapsed twice despite ongoing treatment, and we hypothesize on possible causes and mechanisms. A 77 year old female presented to our hospital with ongoing fevers and worsening cytopenia. Blood counts from three years before the current presentation showed Hb 120g/L, WBC 4.0 x 10^9/L, Neutrophil count 1.8 x 10^9/L, Lymphocytes count 1.8 x 10^9/L and Platelet count 104 x 10^9/L. A bone marrow examination at that time revealed a normocellular marrow with 28% lymphocytes of which70% were CD 4+, CD 3+, CD5+ and CD 7-. Molecular studies confirmed T cell receptor (TCR) gamma gene rearrangement. The karyotype on the bone marrow was normal. In the absence of clinical symptoms, the patient was regularly followed up without specific therapy. During the current admission, however, the patient was febrile, had progressive pancytopenia and biochemistry suggestive of HLH (Fig 1). She was extensively evaluated which ruled out infective and malignant causes. A bone marrow aspirate and biopsy was performed and treatment initiated as per HLH-94 protocol. The bone marrow examination showed marked features of haemophagocytosis on a normocellular background. A small clone of T-lymphocytes was again noted with similar features as in the first biopsy. In addition, a prominent population (10%) of promonocytes was apparent with an uncertain significance. Karyotype was normal. Next Generation Sequencing showed TET2 frame shift mutation at low variant allele frequency (5%). Patient responded well to treatment. While on tapering dose of steroids, the disease flared up (Fig 1) and the patient was restarted on high dose steroids with etoposide. After a quick initial response, while still on active treatment, she again relapsed within 3 weeks, coupled with sepsis and acute myocardial ischemia, followed by sudden death. We were unable to identify a cause for HLH. There were 10% promonocytes in bone marrow and evidence of aberrant T-cells on flow cytometry. Although there was no obvious evidence of dysplasia on microscopy, the flow cytometry showed up- regulation of CD 64 and CD 14, down regulation of CD13 and 11b, and CD 34 expression in granulocytes possible indicating dysplasia as per the Wells criteria. (Wells et al., Blood 2003; 102(1):393) The Next generation sequencing showed TET2 mutation as mentioned above. Mutations in TET2 have been found to have overrepresented in chronic myelomonocytic leukemia in as much as 50% of patients and around 20-35% of patients with myelodysplastic syndrome (MDS). These patients with TET2 mutation have been found to have altered methylation. Recently TET2 has been implicated in immune regulation with evidence of abnormal CD 4 T cell proliferation (present in our patient) and disruption of T cell homeostasis. In addition, patients with TET2 mutation associated myelodysplastic syndrome are known to have auto-immune manifestations (Yimei Feng et al., Frontiers in Oncology, 2019 (9):1). Alyssa H et al have shown that TET2 mutant in patients with MDS (Myelodysplasia) leads to alteration of immune environment in the macrophage differentiation (Alyssa et al., Experimental Hematology, 2017:55; 56). Whether these immune aberration caused recurrent florid relapse of hemophagocytosis in our patient within a span of 2-3 months remained unclear and it could be considered in future research. Even though the occurrence of hemophagocytic syndrome has been described in acute leukemias, the association of the same with myeloid gene aberrations with or without overt myelodysplastic features is unknown. However since hemophagocytosis itself is not common, a careful evaluation to look for uncommon associations which may be a triggering factor may pave the way for identifying their possible role in the pathogenesis. And therapeutic options such as hypo methylating agents can evolve when such associations are confirmed in future studies. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals New monogenic disorders identify more pathways to neutropenia: from the clinic to next-generation sequencing

Hematology ◽  
2017 ◽  
Vol 2017 (1) ◽  
pp. 172-180 ◽  
Author(s):  
Seth J. Corey ◽  
Usua Oyarbide
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Molecular Mechanisms ◽  
Bone Marrow Failure ◽  
Next Generation ◽  
Bone Marrow Failure Syndrome ◽  
Monogenic Disorders ◽  
Distribution Of Values ◽  
Life Threatening ◽  
Generation Sequencing

Abstract Neutrophils are the most common type of leukocyte in human circulating blood and constitute one of the chief mediators for innate immunity. Defined as a reduction from a normal distribution of values, neutropenia results from a number of congenital and acquired conditions. Neutropenia may be insignificant, temporary, or associated with a chronic condition with or without a vulnerability to life-threatening infections. As an inherited bone marrow failure syndrome, neutropenia may be associated with transformation to myeloid malignancy. Recognition of an inherited bone marrow failure syndrome may be delayed into adulthood. The list of monogenic neutropenia disorders is growing, heterogeneous, and bewildering. Furthermore, greater knowledge of immune-mediated and drug-related causes makes the diagnosis and management of neutropenia challenging. Recognition of syndromic presentations and especially the introduction of next-generation sequencing are improving the accuracy and expediency of diagnosis as well as their clinical management. Furthermore, identification of monogenic neutropenia disorders is shedding light on the molecular mechanisms of granulopoiesis and myeloid malignancies.

scholarly journals Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes

2017 ◽  
Vol 19 (7) ◽  
pp. 796-802 ◽  
Author(s):  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Kenichi Yoshida ◽  
Yuichi Shiraishi ◽  
Sayoko Doisaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Clinical Utility ◽  
Bone Marrow Failure ◽  
Next Generation ◽  
Bone Marrow Failure Syndromes ◽  
Generation Sequencing

scholarly journals Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias: Experience of the Spanish Network on Bone Marrow Failure Syndromes

HemaSphere ◽  
2021 ◽  
Vol 5 (4) ◽  
pp. e539
Author(s):  
Eva Gálvez ◽  
Elena Vallespín ◽  
Elena G. Arias-Salgado ◽  
Carmen Sánchez-Valdepeñas ◽  
Yari Giménez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Bone Marrow Failure ◽  
Next Generation ◽  
Bone Marrow Failure Syndromes ◽  
Generation Sequencing

scholarly journals Next Generation Sequencing For Inherited Bone Marrow Failure Syndromes (Ibmfs) In Children: A Pilot Study

2019 ◽  
Vol 4 (2) ◽  
pp. S2-S3
Author(s):  
Prashant Chhabra ◽  
Prateek Bhatia ◽  
Aditya Singh ◽  
Deepak Bansal ◽  
Richa Jain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Pilot Study ◽  
Bone Marrow Failure ◽  
Next Generation ◽  
Bone Marrow Failure Syndromes ◽  
Generation Sequencing

scholarly journals A landscape of germ line mutations in a cohort of inherited bone marrow failure patients

Blood ◽  
2018 ◽  
Vol 131 (7) ◽  
pp. 717-732 ◽  
Author(s):  
Olivier Bluteau ◽  
Marie Sebert ◽  
Thierry Leblanc ◽  
Régis Peffault de Latour ◽  
Samuel Quentin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Clinical Features ◽  
Germ Line ◽  
Bone Marrow Failure ◽  
Next Generation ◽  
Key Points ◽  
Germ Line Mutations ◽  
Natural Histories ◽  
Generation Sequencing

Key Points Next-generation sequencing broadens the spectrum of germ line mutations in a cohort of patients with likely-inherited BMF. Salient clinical features and distinct natural histories are consistently found in SAMD9L and SAMD9, MECOM/EVI1, and ERCC6L2 disorders.

scholarly journals Improved Molecular Diagnosis of Aplastic Anemia/Bone Marrow Failure Syndromes Using a Novel Comprehensive Next Generation Sequencing Gene Panel

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4790-4790
Author(s):  
Majed Dasouki ◽  
Syed Osman Ahmed ◽  
Ali Alahmari ◽  
Amal Jabr ◽  
Moheeb Ali Alawwami ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Aplastic Anemia ◽  
Molecular Diagnosis ◽  
Bone Marrow Failure ◽  
Gene Panel ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Bone Marrow Failure Syndromes ◽  
Generation Sequencing

Abstract Introduction: Various forms of aplastic anemia (AA)/bone marrow failure syndromes (IBMFs) show significant clinical and molecular heterogeneity with significant clinical overlap and are often diagnosed based on established clinical and pathological criteria. While >70 genes have been identified in patients with AA/IBMFs, most cases have are labelled as idiopathic with no identifiable genetic abnormlaity found. Precise detection of genetic abnormalities in these patients may assist in more accurate molecular diagnosis in these patients, proper counseling, cancer surveillance and personalized clinical intervention. Method: As part of the Saudi Human Genome Project, we developed a comprehensive 405 gene panel encompassing all known Mendelian hematological disorders (hemolytic anemias, aplastic anemias/bone marrow failure syndromes, coagulation disorders) using the Ion Torrent AmpliSeq technology. Patients who met the clinical diagnosis of aplastic anemia/bone marrow failure syndrome were enrolled into this study. Peripheral blood samples were subjected to this next-generation sequencing analysis. Results: We validated the Saudi Mendeliome assay using 642 samples with known mutations across various medical specialties. We then tested 37 patients with AA/IBMFS using this Proton-Ion sequencing platform. Mutations were identified in 7/37 (19%) of patients, followed by whole exome sequencing (WES) in those patients without identifiable mutations. Conclusion: Compared with clinical WES and/or whole genome sequencing (WGS), which are still expensive, time consuming and difficult to interpret, this novel and comprehensive targeted gene panel is more economical (< $150), faster (3-4 weeks), upgradable (by spiking in newly identified AA/IBMFs genes) and can be used to genotype patients with acquired aplastic anemia/bone marrow failure syndromes and guide their management. Second tier testing using WES/WGS is recommended for cases without identifiable mutations. Disclosures No relevant conflicts of interest to declare.

The Patient With Transfusion-Dependent Anemia: Diagnosis and Directed Management With Targeted Next Generation Sequencing and Copy Number Analysis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3419-3419
Author(s):  
Vincent P Schulz ◽  
Yelena Maksimova ◽  
Kimberly Lezon-Geyda ◽  
Patrick G Gallagher
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Functional Studies ◽  
Bone Marrow Failure Syndromes ◽  
In Trans ◽  
Generation Sequencing

Abstract Management of the patient with transfusion-dependent anemia (TDA) is complex. Diagnosis is frequently difficult as numerous disorders may lead to TDA, including bone marrow failure syndromes, congenital dyserythropoietic anemias, or inherited hemolytic anemias. Assigning the diagnosis may be problematic as transfused blood or reticulocytosis confound diagnostic testing, or, mutant erythrocytes are so unstable, they are rapidly destroyed. Complications of chronic transfusion include iron overload, infection risk, alloimmunization, cost, and inconvenience. TDA is an excellent candidate for targeted next generation sequencing. There is significant genotypic variability and many of the associated genetic loci are very large, making traditional sequencing strategies cumbersome. We studied 21 patients with TDA using genome-wide targeted exon capture followed by high-throughput next generation DNA sequencing (whole-exome sequencing, WES) using a NimbleGen SeqCap EZ Exome v2.0 solution-based capture system followed by next-generation sequencing on a HiSeq 2000 with paired-end sequencing at 75bp read length. The male:female ratio was 13:8. Age at referral ranged from 2 months to 14 years. All patients were transfusion dependent by 6 months of age. Working diagnoses included possible marrow failure syndrome, congenital dyserythropoietic anemia, and possible enzyme or membrane defect. Variant analyses were performed using the GATK pipeline. Targeted filtering and annotation of protein changing variants in 154 erythrocyte disease genes were performed using the ANNOVAR algorithm. Variants were assessed by mutation prediction and conservation programs including PolyPhen2, Sift, LRT, and Mutation Taster. Variants were also assessed for occurrence and frequency Thousand Genomes, Exome Sequencing Project, dbSNP, on line and local mutation databases, and PubMed. Copy number variants were assessed by ExomeCount and visual inspection. Potential disease-associated variants were validated by Sanger sequencing of DNA from the proband and parents. Interpretation was made using historical, clinical, laboratory and genetic data. The most common diagnosis was hereditary spherocytosis due to alpha spectrin gene (SPTA1) mutations, found in 7 patients. Two patients had deleterious mutations in both SPTA1 alleles; one with nonsense mutations in trans died of liver failure associated with iron overload, the other with nonsense and splicing mutations in trans remains transfusion dependent. One patient homozygous for an SPTA1 missense mutation in a highly conserved, functionally important amino acid had a sibling homozygous for the same mutation die in the perinatal period due to complications of anemia. Finally, one patient with SPTA1 nonsense and missense mutations in trans became transfusion independent post splenectomy. Ten patients had defects in erythrocyte metabolism. Mutations in the pyruvate kinase gene PKLR were found in 6 patients; two of these patients had deletions in the PKLR gene locus suggested by WES and confirmed by Gap PCR and MLPA. Three patients had bi-allelic mutations in the glucose phosphate isomerase gene and one had bi-allelic mutations in the hexokinase gene. Homozygosity was found in 4 of 10 patients with metabolic gene mutations. A single patient had beta thalassemia major with homozygous beta-globin gene mutations. Confirmatory functional studies are underway in three patients. Two TD patients had bone marrow findings suggestive of hypoplastic anemia; one had a missense mutation in a highly conserved residue of RPS7, recently associated with Diamond Blackfan anemia; the other had a deleterious mutation in FANCI predicted to function as a dominant negative. Functional studies are underway in a third patient with likely deleterious, membrane-linked variants. Application of targeted WES to TDA allows precise diagnosis to guide appropriate therapy, e.g. splenectomy or transplant; it allows genetic counseling of associated family members, and permits diagnosis and expectant management of future pregnancies. Targeted WES is an excellent tool for application to monogenic hematologic diseases where genotypic variability, i.e. mutations in numerous genes, leads to the same clinical phenotype. Examples include bone marrow failure syndromes, hemolytic anemia, congenital neutropenia, and immunodeficiency syndromes. Disclosures: No relevant conflicts of interest to declare.

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